Blebs produced by actin-myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs.

Apoptosis is a fundamental homeostatic mechanism essential for the normal growth, development and maintenance of every tissue and organ. Dying cells have been defined as apoptotic by distinguishing features, including cell contraction, nuclear fragmentation, blebbing, apoptotic body formation and maintenance of intact cellular membranes to prevent massive protein release and consequent inflammation. We now show that during early apoptosis limited membrane permeabilization occurs in blebs and apoptotic bodies, which allows release of proteins that may affect the proximal microenvironment before the catastrophic loss of membrane integrity during secondary necrosis. Blebbing, apoptotic body formation and protein release during early apoptosis are dependent on ROCK and myosin ATPase activity to drive actomyosin contraction. We identified 231 proteins released from actomyosin contraction-dependent blebs and apoptotic bodies by adapted SILAC (stable isotope labeling with amino acids in cell culture) combined with mass spectrometry analysis. The most enriched proteins released were the nucleosomal histones, which have previously been identified as damage-associated molecular pattern proteins (DAMPs) that can initiate sterile inflammatory responses. These results indicate that limited membrane permeabilization occurs in blebs and apoptotic bodies before secondary necrosis, leading to acute and localized release of immunomodulatory proteins during the early phase of active apoptotic membrane blebbing. Therefore, the shift from apoptosis to secondary necrosis is more graded than a simple binary switch, with the membrane permeabilization of apoptotic bodies and consequent limited release of DAMPs contributing to the transition between these states.

How apoptotic cells aid in the removal of their own cold dead bodies.

Apoptotic cell clearance facilitates the removal of aged, damaged, infected or dangerous cells although minimizing perturbation of surrounding tissues, and is a vital process in the development and homeostasis of multicellular organisms. Importantly, failure to correctly execute programmed cell death and subsequent corpse clearance is broadly associated with chronic inflammatory and/or autoimmune diseases such as systemic lupus erythematosus. Apoptotic cells develop dramatic morphological changes including contraction, membrane blebbing and apoptotic body formation, which were among the first and most readily identifiable features of cellular suicide. However, understanding the purpose of apoptotic cell morphological changes has proven to be elusive, and recent studies have made somewhat surprising, and occasionally opposing, conclusions about the contribution of blebbing to phagocytic clearance and prevention of inflammatory/autoimmune disease. We review the evidence indicating how apoptotic blebs actively promote corpse recognition, uptake, and generation of auto-reactive antibodies.

Activating ROCK1 somatic mutations in human cancer.

Cancer cells acquire characteristics of deregulated growth, survival and increased metastatic potential. Genetic mutations that provide a selective advantage by promoting these characteristics have been termed 'drivers,' whereas mutations that do not contribute to disease initiation/progression are termed 'passengers.' The advent of high-throughput methodologies has facilitated large-scale screening of cancer genomes and the subsequent identification of novel somatic mutations. Although this approach has generated valuable results, the data remain incomplete until the functional consequences of these mutations are determined to differentiate potential drivers from passengers. ROCK1 is an essential effector kinase downstream of Rho GTPases, an important pathway involved in cell migration. The Cancer Genome Project identified three nonsynonymous mutations in the ROCK1 gene. We now show that these somatic ROCK1 mutations lead to elevated kinase activity and drive actin cytoskeleton rearrangements that promote increased motility and decreased adhesion, characteristics of cancer progression. Mapping of the kinase-interacting regions of the carboxy terminus combined with structural modeling provides an insight into how these mutations likely affect the regulation of ROCK1. Consistent with the frequency of ROCK1 mutations in human cancer, these results support the conclusion that there is selective pressure for the ROCK1 gene to acquire 'driver' mutations that result in kinase activation.

Assessment of correlated dose and sensitivity profiles on a multi-slice CT scanner.

In the case of computed tomography (CT) scanners as well as other imaging techniques utilising ionising radiation, it is imperative that radiation is confined to the sensitive part of the image detector. Assuring this for a CT scanner requires detailed information about the scanner dose and sensitivity profiles and their spatial correlation. The profiles should ideally be co-centric and tightly fit to each other. Ensuring this inherent performance of the scanner can be seen as one of the fundamental steps in optimising diagnostic examinations with CT. A measurement device using a dedicated liquid ionisation chamber is employed to investigate the performance of a Toshiba Aquilion 16 scanner in this aspect. Dose profile and sensitivity profile pairs for four collimations are presented where each pair of profiles are spatially correlated to each other. The measurement device can be applied to any scanner for fast and accurate assessment of dose and sensitivity profiles and their spatial correlation.

The polycationic aminoglycosides modulate the vasoconstrictive effects of endothelin: relevance to cerebral vasospasm.

1. The vasoactive peptide endothelin (ET) has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid haemorrhage. In these studies we investigated the involvement of protein kinase C (PKC) in sustained vasoconstriction induced by ET-1 in canine cerebral arteries. We also examined the ability of the aminoglycoside antibiotics to reverse the effects mediated by ET-1 in canine cerebrovascular smooth muscle cells (CVSMC). 2. The ET(A) receptor antagonist, BQ-123, showed a competitive inhibition of the ET-1 responses. 3. The vasoconstrictor action of both ET-1 (0.5 nM) and phorbol myristate acetate (PMA) (160 nM) was reversed by a selective PKC inhibitor, Ro-32-0432. 4. In cerebral arteries precontracted with ET-1 the aminoglycosides caused a concentration-dependent relaxation. The EC(50s) for the relaxation were as follows: 0.54+/-0.05, 0.63+/-0.01, 1.88+/-0.46 and 2.3+/-0.92 mM for gentamicin, neomycin, streptomycin and kanamycin, respectively. 5. Gentamicin caused a concentration-dependent decrease of the PMA-induced responses in calcium free medium. 6. PKC activity was elevated in CVSMC exposed to ET-1 (170%) and PMA (167%) for a period of time (60 min) corresponding to maximum tonic contraction induced by these agents in arterial rings. 7. The administration of the aminoglycosides to CVSMC, in concentrations corresponding to the EC(50s) from contractility studies, reduced the effects of both ET-1 and PMA on PKC activity to the levels not different from controls. 8. These results show that the aminoglycosides are able to inhibit sustained vasoconstriction induced by ET-1, an effect which is due, at least in part, to the inhibition of PKC.

Long-term stability of liquid ionization chambers with regard to their qualification as local reference dosimeters for low dose-rate absorbed dose measurements in water.

The long-term sensitivity and calibration stability of liquid ionization chambers (LICs) has been studied at a local and a secondary standards dosimetry laboratory over a period of 3 years. The chambers were transported several times by mail between the two laboratories for measurements. The LICs used in this work are designed for absorbed dose measurements in the dose rate region of 0.1-100 mGy min(-1) and have a liquid layer thickness of 1 mm and a sensitive volume of 16.2 mm3. The liquids used as sensitive media in the chambers are mixtures of isooctane (C8H18) and tetramethylsilane (Si(CH3)4) in different proportions (about 2 to 1). Operating at a polarizing voltage of 300 V the leakage current of the chambers was stable and never exceeded 3% of the observable current at a dose rate of about 1 mGy min(-1). The volume sensitivity of the chambers was measured to be of the order of 10(-9) C Gy(-1) mm3. No systematic changes in the absorbed dose to water calibration was observed for any of the chambers during the test period (sigma < 0.2%). Variations in chamber dose response with small changes in the polarizing voltage as well as sensitivity changes with accumulated absorbed dose were also investigated. Measurements showed that the LIC response varies by 0.15% per 1% change in applied voltage around 300 V. No significant change could be observed in the LIC sensitivity after a single absorbed dose of 15 kGy. The results indicate that the LIC can be made to serve as a calibration transfer instrument and a reference detector for absorbed dose to water determinations providing good precision and long-term reproducibility.

Effects of tamoxifen on oxyhemoglobin-induced cerebral vasoconstriction.

The effect of tamoxifen on oxyhemoglobin-mediated cerebral vasoconstriction was examined. Tamoxifen caused a concentration-dependent relaxation of cerebral artery preparations contracted with oxyhemoglobin and phorbol myristate acetate with the IC(50) values 0.66+/-0.1 and 1.1+/-0.1 microM, respectively. In cerebrovascular smooth muscle cells, oxyhemoglobin and phorbol myristate acetate induced protein kinase C activation, which was 220+/-7% and 203+/-8% of control, respectively. The increase in protein kinase C activity was prevented by tamoxifen. The results suggest that the ability of tamoxifen to reverse vasoconstriction is mediated, at least in part, via inhibition of protein kinase C.

Assessment of the relative dose distribution around an 192Ir line source using a liquid ionization chamber.

The relative absorbed dose distribution in water around an 192Ir line source of 50 mm length and 0.3 mm diam has been measured using a liquid ionization chamber (LIC). The sensitive volume of the chamber is a cylinder with 3.0 mm diam and a thickness of 1.0 mm. The sensitive medium in the LIC consists of a mixture of two dielectric liquids, tetramethylsilane and isooctane. The mixture has been optimized so that the LIC provides an almost energy-independent response for the radiation qualities present at different distances from the source in water. The measurements were carried out at distances of 2.5-50.0 mm along the source bisector and at distances of 0.0 to +/-40.0 mm along the source axis. The results were compared with measurements made with LiF chips in Solid Water and with calculated data based on an analytical solution to the Sievert integral as well as with Monte Carlo calculations of absolute dose rate in water. Considering the uncertainties involved, the dose distribution measured by the LIC is in reasonably good agreement with the theoretical data as well as the results held with LiF. In comparison with the Monte Carlo calculations the discrepancies range from 0.1% to 18% with the largest differences at points close to the source. This work demonstrates the ability of the LIC for mapping the dose distribution around a low-dose-rate brachytherapy source emitting photons of intermediate energies.

General collection efficiency in liquid iso-octane and tetramethylsilane used as sensitive media in a thimble ionization chamber.

The general collection efficiency in the dielectric liquids iso-octane (CaH18; 2-2-4 trimethylpentane) and tetramethylsilane (Si(CH3)4), used as sensitive media in a thimble liquid ionization chamber (LIC) with a liquid layer thickness of 1 mm, has been studied. Measurements were made for continuous radiation at varying dose rates using 140 keV photons from the decay of 99mTc for chamber polarizing voltages of 50, 100 and 500 V. The maximum dose rate in each measurement session was about 150 mGy min(-1). The experimental results were compared with theoretical general collection efficiencies calculated by the equation for the general collection efficiency in gases. The results show that the general collection efficiency in a thimble LIC for continuous radiation can be calculated with the equation for the general collection efficiency in gas ionization chambers, using the same chamber geometry correction factors and analogous characteristic ion recombination parameters for the dielectric liquids.

Liquid ionization chambers for absorbed dose measurements in water at low dose rates and intermediate photon energies.

Two new liquid ionization chamber (LIC) designs, consisting of cylindrical and plane-parallel configurations, are presented. They are designed to be suitable for high-precision measurements of absorbed dose-to-water at dose rates and photon energies typical for LDR intermediate photon energy brachytherapy sources. The chambers have a sensitive liquid layer thickness of 1 mm and sensitive volumes of 7 mm3 (plane-parallel) and 20 mm3 (cylindrical). The liquids used as sensitive media in the chambers are either isooctane (C8H18), tetramethylsilane (Si(CH3)4) or mixtures of these two liquids in the approximate proportions 2 to 1. A chamber filled with such a liquid mixture and with a polarizing voltage of 300 V, provides a volume sensitivity of about 10(-9)C Gy(-1) mm(-3) for absorbed dose measurements in water in an x-ray radiation field with an effective photon energy of 120 keV. In the interval 30 to 140 keV, the relative change in sensitivity is less than +/- 2.5%. The leakage current of the chambers is low and stable, which implies that absorbed dose measurements can be done with good reproducibility at dose-rates as low as 50 microGy min-1 (sigma < 3%). The long-term calibration stability was tested for a set of five chambers over a period of more than 1 year. No systematic change in their sensitivity could be observed. The general recombination at a polarizing voltage of 300 V is less than 2% for dose-rates up to about 100 mGy min-1. The temperature dependence at room temperature is 0.5% per degree C. The response is almost independent of the direction of the radiation for the plane-parallel LIC.

Liquid ionization chamber measurements of dose distributions in small 6 MV photon beams.

A new liquid ionization chamber (LIC) design optimized for high spatial resolution was used for measurements of dose distributions in radiation fields intended for stereotactic radiosurgery (SRS). This work was mainly focused on the properties of this detector in radiation fields from linear accelerators for clinical radiotherapy (pulsed radiation with dose rates from approximately 0.5 to 5 Gy min-1 and beam diameters down to 8 mm). The narrow beams used in stereotactic radiosurgery require detectors with small sizes in order to provide a good spatial resolution. The LIC is investigated to see whether it can be used as a detector for dose measurements in beams currently used for stereotactic radiosurgery. Its properties are compared with those of silicon diodes. The comparisons include output factor (OF), depth dose and profile measurements in 6 MV photon fields of different sizes. For OF measurements, an NACP air ionization chamber was also used in the comparison. The dependence of the response on the detector orientation in the photon beam is also investigated for the diodes and the LIC. The results suggest that LICs can provide better properties than diodes for measuring dose distributions in narrow photon beams.

General collection efficiency for liquid isooctane and tetramethylsilane in pulsed radiation.

The general collection efficiency in pulsed radiation was studied for isooctane (C8H18) and tetramethylsilane (Si(CH3)4). These two liquids were used as sensitive media in a parallel-plate liquid ionization chamber with a 1 mm sensitive layer. Measurements were carried out using 20 MV photon radiation from a linear accelerator with a pulse repetition frequency of 30 pulses/second and a pulse length of 3.5 microseconds. The general collection efficiency was determined for polarizing voltages in the interval 1000-2000 V for isooctane and 500-2000 V for tetramethylsilane and for pulse doses in the interval 0.06-1.9 mGy/pulse. An air ionization chamber was used as a pulse dose reference monitor. The experimental results were compared with those predicted by the equation for the general collection efficiency for gases in pulsed radiation, using the permittivity of each of the liquids. It was found that for general collection efficiencies down to 80% the differences between the predicted and experimental general collection efficiencies in the two liquids were within +/- 1% at electric field strengths exceeding 10(6) V m-1.

General collection efficiency for liquid isooctane and tetramethylsilane used as sensitive media in a parallel-plate ionization chamber.

The general collection efficiency has been measured in liquid isooctane (C8H18) and tetramethylsilane (Si(CH3)4) used as the sensitive media in a parallel-plate ionization chamber, with an electrode distance of 1 mm, intended for photon and electron dosimetry applications. The liquid ionization chamber was irradiated at different dose rates by 140 keV photons from the decay of radioactive 99mTc. The measurements were made at potential differences of 50, 100, 200 and 500 V. Measurements were performed for each liquid and electric field strength, with the decay rate of 99mTc used as the dose-rate reference. The maximum dose rate was about 150 mGy min-1 in each experiment. When the measured general collection efficiency values are compared with the theoretical predictions for collection efficiency in gases, it is found that the latter also describe the general collection efficiency in the two liquids within 1% of the saturation current for collection efficiencies down to 60% when using experimentally determined recombination rate constants and on mobilities characteristic of each of the liquids.

Properties of liquid ionization chambers at LDR brachytherapy dose rates.

Properties such as sensitivity, general recombination and reproducibility of liquid-filled parallel-plate ionization chambers for dosimetry in low-dose-rate brachytherapy radiation fields have been evaluated. Two different dielectric liquids, isooctane (C8H(1)8) and tetramethylsilane (Si(CH3)4), have been used as sensitive media in chambers having a coin-shaped sensitive volume of 3 mm in diameter and 1 mm thickness. An electric field strength of 300 kV m-1 was found to be optimal with respect to sensitivity, leakage current and general recombination. At absorbed dose rates from 0.1-100 mGy min-1 the ionization charge measurements at an irradiation time of 1 min showed a reproducibility better than 1%, and a general recombination not exceeding 0.5%. The calibration--absorbed dose to water against ionization charge at a 60Co reference source--did not show any significant change over an observation time of one year for any of the chambers.

Selective salpingography and fluoroscopic transcervical salpingoplasty for diagnosis and treatment of proximal fallopian tube occlusions.

In 25 women with primary or secondary infertility, primary hysterosalpingography (HSG) or laparoscopy with chromopertubation indicated 33 proximal and 2 distal tubal obstructions. Four salpingectomies had been performed earlier. All patients were considered for transcervical catheter salpingoplasty (TCSP) to reconstruct tubal patency. Secondary examination with repeat HSG or selective osteal salpingography confirmed 26 proximal and 3 distal tubal occlusions while 17 tubes were patent. Selective osteal salpingography was performed successfully in 32 of 33 (97%) tubes. Sixteen of 26 (61.5%) proximally occluded tubes were successfully recanalized by coaxial catheter and guidewire technique while two of three distally occluded tubes were reopened by forceful flushing of contrast medium. So far, one ectopic and five intrauterine pregnancies were achieved in ten patients with observation time more than four months. Three patients have had normal deliveries. The favorable results, lack of complications and low costs seem to justify the recommendation to use selective salpingography and fallopian tube recanalization as the first intervention in patients with obstruction of the proximal fallopian tube.

Polarity effect in plane-parallel ionization chambers using air or a dielectric liquid as ionization medium.

A plane-parallel ionization chamber having a sensitive volume of 2 mm3 and using the dielectric liquid tetramethylsilane as the sensitive medium instead of air is described. In the design of the chamber special attention was given to the factors that can cause unwanted currents in the cable, stem, or the chamber dielectric material. The chamber has been tested with respect to the polarity effect in regions of radiation fields where ordinary plane-parallel ionization chambers will often fail. These regions are the build-up region in photon fields, and the region close to the practical range for electrons where nonelectronic equilibrium is significant. Experimental results show that, despite the extremely small ionization volume in the liquid ionization chamber, the polarity effect never exceeds a few tenths of a percent in field positions where well-known commercially available chambers with much less spatial resolution designed for measurements in radiation therapy fields can show polarity effects of 5% to 30%. The origin of spurious currents and how they must be minimized in the design of either a liquid- or gas-filled ionization chamber is discussed.

Absorbed dose in water. Comparison of several methods using a liquid ionization chamber.

In the present investigation a liquid ionization chamber has been used as a transfer instrument for the quantity absorbed dose in water in a cobalt-60 gamma-ray beam. The characteristics of the liquid ionization chamber are described. The transferred dosimetric information has been compared with absorbed-dose determination using air-ionization-chamber dosimetry, water calorimetry and ferrous-sulphate dosimetry. The agreement between the different measured absorbed-dose values is very good, i.e. within 0.2%. This is an indication that the consistency in the methods used to determine absorbed dose in water is good. The impact of the new standard for air kerma in air, introduced in 1986 by the BIPM, on the air-ionization-chamber dosimetry is investigated. It is shown that any differences in the dosimetry when using the old or the new set of data cancel out for the cobalt-60 beam. The investigation also shows that the value of epsilon mG for the ferrous-sulphate dosimeter recommended in ICRU 35 for electrons can be used also in cobalt-60 beams.

Low field (0.02 T) MR imaging of the whole body.

The diagnostic utility of extremely low field magnetic resonance (MR) (0.02 T) was evaluated in various parts of the body outside the head. The areas investigated included the spine (104 patients), kidneys (19 patients), female pelvis (21 patients), and hips (15 patients). The results were compared with those of other imaging modalities, e.g., ultrasound, CT, and scintigraphy. Available pulse sequences and typical examination times for different types of studies are reported. Poor spatial resolution and long imaging times limit the application of this technique to the whole body. Due to poor signal-to-noise ratio, image quality is not comparable with that obtained at higher field strengths. In spite of these restrictions it was possible to demonstrate lumbar disk herniations, to differentiate renal and ovarian cysts from tumors, and to detect early avascular necrosis of the femoral head. Extremely low field MR imaging may eventually be used in screening spinal pathology and in the diagnosis of early avascular necrosis of the femoral head. At present, this technique can not replace ultrasound or CT in abdominal examinations.