Asymmetric pore distribution and loss of membrane lipid in electroporated DOPC vesicles.

An externally applied electric field across vesicles leads to transient perforation of the membrane. The distribution and lifetime of these pores was examined using 1,2-di-oleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid vesicles using a standard fluorescent microscope. The vesicle membrane was stained with a fluorescent membrane dye, and upon field application, a single membrane pore as large as approximately 7 microm in diameter was observed at the vesicle membrane facing the negative electrode. At the anode-facing hemisphere, large and visible pores are seldom found, but formation of many small pores is implicated by the data. Analysis of pre- and post-field fluorescent vesicle images, as well as images from negatively stained electron micrographs, indicate that pore formation is associated with a partial loss of the phospholipid bilayer from the vesicle membrane. Up to approximately 14% of the membrane surface could be lost due to pore formation. Interestingly, despite a clear difference in the size distribution of the pores observed, the effective porous areas at both hemispheres was approximately equal. Ca(2+) influx measurements into perforated vesicles further showed that pores are essentially resealed within approximately 165 ms after the pulse. The pore distribution found in this study is in line with an earlier hypothesis (E. Tekle, R. D. Astumian, and P. B. Chock, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:11512--11516) of asymmetric pore distribution based on selective transport of various fluorescent markers across electroporated membranes.

Phase I trial of photodynamic therapy in the treatment of recurrent superficial transitional cell carcinoma of the bladder.

OBJECTIVES: A Phase I trial of photodynamic therapy (PDT) in the treatment of superficial transitional cell carcinoma (TCC) of the bladder was performed. METHODS: Twenty patients with recurrent superficial TCC of the bladder after receiving a mean of 2.6 (range 1 to 6) courses of intravesical therapy were treated with PDT. The photosensitizer Photofrin II dose was 1.5 or 2.0 mg/kg. A 630-nm intravesical red laser was used to activate the photosensitizer 2 days after administration of Photofrin II. A 0.01% intralipid solution was used as a bladder-filling medium to scatter light and achieve more homogeneous light distribution. Light doses from 5.1 to 25.6 J/cm2 (total dosage 1500 to 5032 J) were used to illuminate the bladder. RESULTS: Twenty patients underwent 21 treatments with PDT. Complications included asymptomatic reflux in 4 patients. One other patient, treated at the highest total light dose, experienced bladder contraction and fibrosis. Nine patients (45%) had no tumor evident at cystoscopy, on random biopsies, or in urinary cytology at the 3-month evaluation after treatment. Four patients remained without recurrent disease for 23 to 56 months. Sixteen of 20 (80%) patients experienced recurrence, and 8 of the 16 underwent cystectomy. CONCLUSIONS: An intravenous photosensitizer dose of 1.5 mg/kg Photofrin II followed by light energy in the range of 13 J/cm2 (total light dose 2500 to 3250 J) was defined as a safe treatment parameter and resulted in tumor responses. With present technologies, administration of PDT requires careful dosimetry.

A high speed optical multichannel analyzer.

An optical multichannel analyzer capable of recording spectra at sampling rates up to 100 kHz is described. The instrument, designed to gather data on the kinetic reaction mechanisms of biological preparations such as cytochrome oxidase and bacteriorhodopsin, features a massively parallel approach in which each photosensing element of the detector array has a dedicated amplifier, integrator, analog to digital converter, and sample buffer. The design has 92 such elements divided in two separate arrays, each of which sits at the focal plane of a 1/4 m Ebert spectrometer. The spectrometers may be tuned to cover independent, 130 nm wide, regions of the spectrum from 350 nm to 900 nm with a dispersion of 2.8 nm per element. Each detection channel has 12-bit resolution with an electronic dark count of 1 count and may be sampled 1024 times during a single experiment with dynamically variable sampling intervals from 10 microseconds to several seconds. Time averaging of up to thousands of consecutive laser-initiated kinetic cycles allows analyses of spectral changes < 0.001 optical density units. A personal computer with custom software provides a number of features: entry of experiment parameters; transfer of data from temporary buffers to permanent files; real time display; multiple spectrum averaging; and control and synchronization of associated system hardware. Optical fibers or lenses provide coupling from a parabolic reflector Xenon arc monitoring light source, through the sample chamber, to the entry slit of the monochromator. The instrument has been used for extensive studies on the rapid kinetics and definition of reaction sequences of the energy-transducing enzymes cytochrome oxidase and bacteriorhodopsin. Some results from these studies are discussed.

Phase III randomized trial of surgery with or without intraoperative photodynamic therapy and postoperative immunochemotherapy for malignant pleural mesothelioma.

BACKGROUND: Patients with malignant pleural mesothelioma (MPM) usually die of progressive local disease. This report describes the results of a Phase III trial comparing maximum debulking surgery and postoperative cisplatin, interferon alpha-2b, and tamoxifen (CIT) immunochemotherapy with and without intraoperative photodynamic therapy (PDT) to determine (1) whether such a multimodal approach can be performed with minimum morbidity and mortality in malignant pleural mesothelioma (MPM), and (2) whether first-generation (i.e., 630-nm laser light, Photofrin II) intrapleural PDT impacts on local recurrence of survival. METHODS: From July 1993 to June 1996, 63 patients with localized MPM were randomized to either PDT or no PDT. The tumors of 15 patients could not be debulked to 5 mm. Patients assigned to PDT (n = 25) and no PDT (n = 23) were similar with respect to age, sex, tumor volume, and histology. RESULTS: The type of resection (11 pleurectomies and 14 pneumonectomies vs. 12 pleurectomies and 11 pneumonectomies), length postoperative stay, and ICU time were comparable (PDT vs. no PDT). There was one operative death (hemorrhage), and each group had two bronchopleural fistulas. Postoperative staging divided patients into the following categories: stage I: PDT, 2, no PDT, 2; stage II: PDT, 2, no PDT, 2; stage III, PDT, 21; no PDT, 17; stage IV, PDT, 0; no PDT, 2. Comparable numbers of CIT cycles were delivered. Median survival for the 15 non-debulked patients was 7.2 months, compared to 14 months for the 48 patients on protocol. There were no differences in median survival (14.4 vs. 14.1 months) or median progression-free time (8.5 vs. 7.7 months), and sites of first recurrence were similar. CONCLUSIONS: Aggressive multimodal therapy can be delivered for patients with higher stage MPM. First-generation PDT does not prolong survival or increase local control for MPM.

Determination of the Na permeability of the tight junctions of MDCK cells by fluorescence microscopy.

The kinetics of Na movement across the tight junctions of MDCK cells, grown on coverslips and perfused with HEPES or bicarbonate Ringer at 37 degrees C, were investigated after filling the lateral intercellular spaces (LIS) of the epithelium with SBFO, an Na-sensitive fluorescent dye. Dilution and bi-ionic potential measurements showed that MDCK cell tight junctions, although cation-selective, were poorly permeable to N-methyl-D-glucamine Cl (NMDG) but freely permeable to Li. In previous experiments in which Na was replaced by NMDG, a very slow decrease in LIS Na concentration (time constant = 4.8 min) resulted. In the present study, reduction of perfusate Na from 142 to 14 or 24 mM with Na replaced by Li caused LIS Na concentration to decrease with a time constant of 0.43 min. The time constant for Na increase of the LIS was 0.28 min, significantly shorter than that for Na decrease because of the additional component of transcellular Na influx. Ouabain eliminated the transcellular component and equalized the time constants for Na influx and efflux. These results were incorporated into a mathematical model which enabled calculation of the transcellular and paracellular Na fluxes during fluid reabsorption. Regulation of the Na permeability of individual tight junctions by protein kinase A (PKA) was evaluated by treating the monolayers with the Sp-cAMPS, a cAMP substitute, or Rp-cAMPS, a specific inhibitor of PKA. Stimulation of PKA strikingly increased tight junctional permeability while PKA inhibition diminished junctional Na permeability.

Combination of potentiometry and resonance Raman spectroscopy for the analysis of a redox protein.

This paper describes apparatus and procedures for combining resonance Raman and optical absorption spectroscopies with potentiometry for the study of redox-active heme proteins. A specially designed anaerobic titration cell is described which allows for the laser excitation of the sample and the monitoring of both Raman scattered light and directly transmitted light from an optical source. New procedures for utilization of A/D and D/A converters on a standard I/O computer card are described, which allow for computer-controlled potentiometry and coulometry. The system was tested with cytochrome c, a well-characterized respiratory protein. The correct values for the midpoint potential and electron number of the Nernst equation were obtained both by the optical absorption and resonance Raman measurements.

Intrapleural photodynamic therapy: results of a phase I trial.

BACKGROUND: The management of pleural neoplasms, specifically mesothelioma, remains difficult. We performed a phase I trial in 54 patients with isolated hemithorax pleural malignancy to determine (a) the feasibility of intraoperative, intrapleural photodynamic therapy after debulking surgery; (b) the influence of light dose/sensitizer interval on postoperative morbidity in order to define the photodynamic therapy (PDT) maximal tolerated dose (MTD); and (c) whether first order dosimetry could be applied to this complex geometry. METHODS: Cohorts of three patients were given escalating intraoperative light doses of 15-35 J/cm2 48 h after i.v. delivery of 2.0 mg/kg Photofrin II (Quadra Logic Technologies, Vancouver, British Columbia, Canada), and then escalating light doses of 30-32.5 J/cm2 after a 24-h sensitizer/operation interval. Twelve patients could not be debulked to the prerequisite 5 mm residual tumor thickness. The remaining 42 patients underwent 19 modified pleuropneumonectomies, five lobectomy-pleurectomies, and 18 pleurectomies. Intrapleural PDT was delivered using 630 nm light from two argon pump-dye lasers, and real-time and cumulative light doses were monitored using seven uniquely designed, computer-interfaced photodiodes. RESULTS: There was one 30-day mortality from intraoperative hemorrhage. In the 48-h sensitizer/operation group (n = 33), possible PDT-related complications included an empyema with late hemorrhage in one of three patients at 17.5 J/cm2 and a bronchopleural fistula at 35 J/cm2. At each of these light doses, three additional patients were treated without complication. Two patients subjected to 24-h sensitizer dosing and 32.5 J/cm2 developed esophageal perforations after pleuropneumonectomy at identical sites. The MTD was declared as 30 J/cm2 light with a 24-h dosing interval when none of the six patients (three original, three repeat) at that level developed toxicity. CONCLUSIONS: These data demonstrate that resection and intrapleural PDT can be performed safely with currently available sensitizers and lasers. Phase II and III trials are now warranted at this MTD in a homogeneous population of patients with pleural malignancies.

Phase I study of debulking surgery and photodynamic therapy for disseminated intraperitoneal tumors.

PURPOSE: Phase I study designed to determine the maximum tolerated dose of intraoperative photodynamic therapy (PDT) at laparotomy/debulking surgery in patients with refractory or recurrent, disseminated intraperitoneal tumors. METHODS AND MATERIALS: Patients received dihematoporphyrin ethers (DHE) 1.5-2.5 mg/kg by i.v. injection prior to surgery. Patients resected to < or = 5 mm of residual disease underwent laser light delivery to all peritoneal surfaces. RESULTS: Fifty-four patients entered the study. Thirty-nine underwent resection and light delivery/PDT. PDT dose was escalated by increasing DHE from 1.5 to 2.5 mg/kg, shortening the interval between DHE injection and surgery from 72 to 48 hr, and increasing the light dose. Initially, 630 nm red light alone was used. In this group, PDT of 2.8-3.0 J/cm2 induced small bowel edema and resulted in 3 small bowel perforations after bowel resection or enterotomy. Further light dose escalation, however, was achieved by switching to less penetrating 514 nm green light to the bowel/mesentery. In later patients, whole peritoneal PDT was supplemented with boost doses of 10-15 J/cm2 red light or 5-7.5 J/cm2 green light to high risk areas. Small bowel complications were not seen after switching to less penetrating green light. Dose limiting toxicities occurred in 2 of 3 patients at the highest light dose of 5.0 J/cm2 green light with boost. These patients had pleural effusions that required thoracentesis and postoperative respiratory support for 7-9 days, while one had a gastric perforation. At potential follow-up times of 3.8-43.1 months (median 22.1 months), 30/39 patients are alive and 9/39 are free of disease. CONCLUSION: The maximum tolerated dose of intraoperative PDT following debulking surgery performed 48 hr after intravenous administration 2.5 mg/kg DHE is 3.75 J/cm2 of 514 nm green light to the entire peritoneal surface with boosts to 5.0-7.5 J/cm2 of 514 nm green light or 10-15 J/cm2 of 630 nm red light to sites of gross disease encountered at surgery.

Transcutaneous determination of tissue dihematoporphyrin ether content. A device to optimize photodynamic therapy.

Photodynamic therapy involves the use of light of appropriate wavelength to excite a photosensitizer resulting in tissue destruction. The photosensitizer dihematoporphyrin ether is selectively retained in tumors allowing for tumor destruction while sparing normal structures. Accessibility of skin tumors makes them well suited for photodynamic therapy. Tissue and tumor dihematoporphyrin ether content is estimated based on the amount of dihematoporphyrin ether administered. In our study, skin dihematoporphyrin ether content was measured in guinea pigs transcutaneously by a hand-held fluorometer and compared with dihematoporphyrin ether determinations done on skin biopsy specimens. Fluorometry was performed on guinea pigs receiving 0, 2.5, 5, 10, and 25 mg/kg of dihematoporphyrin ether. Transcutaneous measurements of skin fluorescence increased with increasing dihematoporphyrin ether dose and correlated well with skin dihematoporphyrin ether content as determined by extracting dihematoporphyrin ether from skin samples. Transcutaneous fluorescent measurements of guinea pigs given 0 and 2.5, 2.5 and 5, 5 and 10, and 10 and 25 mg/kg of dihematoporphyrin ether differed in a statistically significant manner. Transcutaneous fluorometric determination of dihematoporphyrin ether content and extraction of dihematoporphyrin ether from skin samples were able to reflect differences in dihematoporphyrin ether dosing and presumably skin dihematoporphyrin ether content. However, transcutaneous fluorometry provides an instantaneous estimate of tissue dihematoporphyrin ether without the need for a tissue sample. This may provide a clinical tool to predict more accurately the optimal light dose necessary to maximize photodynamic therapy.

Technique of photodynamic therapy for disseminated intraperitoneal malignant neoplasms. Phase I study.

Patients with disseminated intraperitoneal malignant neoplasms were given intra-abdominal photodynamic therapy. Patients received dihematoporphyrin ethers intravenously 48 to 72 hours before laparotomy at doses of 1.5 to 3.0 mg/kg. At operation, as much tumor as possible was resected. Red light (630 nm) was delivered to all peritoneal surfaces from an argon-pumped dye laser at doses ranging from 0.2 to 3.0 J/cm2 in an escalating fashion. Viscera and peritoneal surfaces were anatomically isolated and exposed to light for intervals calculated to deliver the prescribed energy. Light was delivered to mesentery and bowel by a flat-cut optical fiber, while other areas, including diaphragm, viscera, omental bursa, gutters, and pelvis, were delivered light through a diffusing wand. Twenty-three patients (13 with ovarian cancer, eight with sarcoma, and two with pseudomyxoma peritoneii) underwent photodynamic therapy. Five of eight patients cleared positive peritoneal cytologies after treatment. Six patients remained clinically free of disease for up to 18 months, and five patients had treatment-related complications. Intraperitoneal phototherapy is technically feasible and deserving of clinical evaluation.

A theoretical calculation of the electric field induced by magnetic stimulation of a peripheral nerve.

A mathematical model is presented that predicts the electric field induced in the arm during magnetic stimulation of a peripheral nerve. The arm is represented as a homogeneous, cylindrical volume conductor. The electric field arises from two sources: the time-varying magnetic field and the accumulation of charge on the arm surface. In magnetic stimulation both of these contributions are significant. The magnitude of the electric field is greatest near the surface of the arm, and is well localized. Various coil orientations are examined; the smallest electric fields are induced when the coil is perpendicular to the arm surface, the largest when the coil is parallel. These results are consistent with many experimental observations in the literature, and aid in the basic understanding of magnetic stimulation of the peripheral nervous system.

Effects of coil design on delivery of focal magnetic stimulation. Technical considerations.

The localization of effects from magnetic coil stimulation is not immediately obvious. We measured the magnetic fields produced by several different coils and compared the results with theoretical calculations. Magnetic stimuli were delivered from a Cadwell MES-10 magnetic stimulator using 3 circular coils (one 9 cm in diameter; two with an angulated extension, 5 and 9 cm in diameter) and twin oval coils arranged in a butterfly shape (each coil approximately 4 cm in diameter) and from a Novametrix Magstim 200 using two circular flat-spiral coils (6.7 and 14 cm in diameter). Peak-induced strength of the magnetic field was recorded with a measuring loop (1 cm in diameter) at different distances from the center of the coil. When the measuring loop was moved in the same plane laterally from the center of the coil, for all coils except the butterfly-shaped coil, the field was highest in the center and fell off near the circumference of the coil. The field dropped progressively when measurements were made more distant from the plane of the coils. The electric field induced from the magnetic coil could be calculated from the coil geometry. For all coils except the butterfly-shaped coil, the largest electric field was at the circumference of the coils. The 6.7 cm flat-spiral coil induced currents similar to those induced by the larger coils but more focally. The butterfly-shaped coil induced the largest currents under its center, where the circumferences of the two component coils come together. The component of the electric field parallel to the wire in the center of this coil was the largest and most localized.

Subdural electrode as a dipole source for magnetoencephalography.

A subdural electrode was designed and constructed with 3 pairs of contacts to make 3 dipoles. The dipoles well approximate the expected magnetic behavior for a current dipole and can be used clinically to test localization capabilities of magnetoencephalography.

Localization of magnetic interictal discharges in temporal lobe epilepsy.

Three young adults with intractable complex partial seizures were studied by electroencephalography, magnetoencephalography, and electrocorticography. Interictal electroencephalographic (EEG) spikes for each patient were grouped according to their morphological characteristics and distribution across channels. Mapping of simultaneously recorded magnetoencephalographic signals produced dipolar patterns from which the three-dimensional locations of equivalent current dipoles were calculated, whereas the mapping of EEG spikes showed single regions of electronegativity. The magnetic spikes were localized to the anterotemporal lobe, and the EEG spikes were localized somewhat anterior or posterior to the magnetic spikes. The magnetoencephalographic findings corresponded well with intraoperative electrocorticographic and depth-electrode findings of discharging areas located over the lateral temporal lobe and on the basal and mesial surfaces of the temporal cortex.

An optimized differential heat conduction solution microcalorimeter for thermal kinetic measurements.

Heat conduction calorimeters are widely used in the biological sciences, but baseline instability, low resolution, electrical noise and motion artifacts have limited their utility. Two main sources of noise, baseline fluctuation or drift and a motion artifact, were traced to amplifier drift, a small (0.015 degrees C) gradient within the constant temperature cylinder, and the method of installing the thermopiles. The addition of heaters to the top and bottom of the cylinder reduced the gradient to approximately 0.003 degrees C and greatly reduced the slow component of the motion artifact. The drift error was reduced by proper mounting of the amplifier and its external components and the enclosure of the calorimeter in a temperature-controlled box. An R-C model of the heat flow in the calorimeter was developed which was employed to discover several means of increasing sensitivity without increasing the rise-time of the calorimeter. Analysis, also based on the model, showed that variations in the air gap between the cell and cell holder can be a major source of error when the calorimeter is used to investigate the kinetics of a chemical reaction. This analysis also showed that the time for the heat to flow through the solution in the cell can be the dominant factor in determining the rise-time of the instrument. The heat conduction calorimeter described here has improved characteristics: a baseline stability of 200 nJ x s-1 (peak-to-peak) over a 48 h period; a resolution of 200 nJ x s-1; a sensitivity of 6.504 +/- 0.045 J x V-1 x s-1 referred to the sensor output; and a rise-time of 122 s for the 10-90% response.

Design of a high resolution positron emission tomograph: the Neuro-PET.

The design of a high resolution positron emission tomograph is described. The scanner has four rings of detectors with an inside diameter of 38 cm and produces seven simultaneous slices, including three cross-slices. Each ring contains 128 bismuth germanate scintillation detectors with dimensions of 8.25 x 20 x 35 mm; adjacent crystals are separated by tapered tungsten septa that extend to within 7.5 mm of the front faces. The anticipated geometrical spatial resolution of the scanner is 4.5 mm full width at half maximum (FWHM) at the center of the image, and the sensitivity is 44,000 true counts/sec/ring (390,000 counts/sec total) for a uniform phantom 20 cm in diameter containing 1 muCi/cc activity. There are interchangeable collimators for use in high count rate studies, for narrowing the slice width from 1 cm FWHM to 5 mm FWHM, and for ultra-high resolution studies with a 2.5 mm FWHM geometrical point spread function. The electronic circuitry has separate timing and energy verification channels and can detect 95% of the coincidences with a timing window (twice the maximum time between two coincident pulses) of 14 nsec. The count rate capability of the electronics is 150,000 counts/sec/ring and 1.5 million counts/sec total.