Algorithmic assessment of cellular senescence in experimental and clinical specimens.

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies-pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells-that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies.

Mitochondrial dysfunction increases pro-inflammatory cytokine production and impairs repair and corticosteroid responsiveness in lung epithelium.

COPD is characterized by chronic lung inflammation and irreversible lung tissue damage. Inhaled noxious gases, including cigarette smoke, are the major risk factor for COPD. Inhaled smoke first encounters the epithelial lining of the lungs, causing oxidative stress and mitochondrial dysfunction. We investigated whether a mitochondrial defect may contribute to increased lung epithelial pro-inflammatory responses, impaired epithelial repair and reduced corticosteroid sensitivity as observed in COPD. We used wild-type alveolar epithelial cells A549 and mitochondrial DNA-depleted A549 cells (A549 Rho-0) and studied pro-inflammatory responses using (multiplex) ELISA as well as epithelial barrier function and repair (real-time impedance measurements), in the presence and absence of the inhaled corticosteroid budesonide. We observed that A549 Rho-0 cells secrete higher levels of pro-inflammatory cytokines than wild-type A549 cells and display impaired repair upon wounding. Budesonide strongly suppressed the production of neutrophil attractant CXCL8, and promoted epithelial integrity in A549 wild-type cells, while A549 Rho-0 cells displayed reduced corticosteroid sensitivity compared to wild-type cells. The reduced corticosteroid responsiveness may be mediated by glycolytic reprogramming, specifically glycolysis-associated PI3K signaling, as PI3K inhibitor LY294002 restored the sensitivity of CXCL8 secretion to corticosteroids in A549 Rho-0 cells. In conclusion, mitochondrial defects may lead to increased lung epithelial pro-inflammatory responses, reduced epithelial repair and reduced corticosteroid responsiveness in lung epithelium, thus potentially contributing to the pathogenesis of COPD.

The optimal balance between quality and efficiency in proton radiography imaging technique at various proton beam energies: A Monte Carlo study.

Proton radiography is a novel imaging modality that allows direct measurement of the proton energy loss in various tissues. Currently, due to the conversion of so-called Hounsfield units from X-ray Computed Tomography (CT) into relative proton stopping powers (RPSP), the uncertainties of RPSP are 3-5% or higher, which need to be minimized down to 1% to make the proton treatment plans more accurate. In this work, we simulated a proton radiography system, with position-sensitive detectors (PSDs) and a residual energy detector (RED). The simulations were built using Geant4, a Monte Carlo simulation toolkit. A phantom, consisting of several materials was placed between the PSDs of various Water Equivalent Thicknesses (WET), corresponding to an ideal detector, a gaseous detector, silicon and plastic scintillator detectors. The energy loss radiograph and the scattering angle distributions of the protons were studied for proton beam energies of 150MeV, 190MeV and 230MeV. To improve the image quality deteriorated by the multiple Coulomb scattering (MCS), protons with small angles were selected. Two ways of calculating a scattering angle were considered using the proton's direction and position. A scattering angle cut of 8.7mrad was applied giving an optimal balance between quality and efficiency of the radiographic image. For the three proton beam energies, the number of protons used in image reconstruction with the direction method was half the number of protons kept using the position method.

Beam-on imaging of short-lived positron emitters during proton therapy.

In vivo dose delivery verification in proton therapy can be performed by positron emission tomography (PET) of the positron-emitting nuclei produced by the proton beam in the patient. A PET scanner installed in the treatment position of a proton therapy facility that takes data with the beam on will see very short-lived nuclides as well as longer-lived nuclides. The most important short-lived nuclide for proton therapy is 12N (Dendooven et al 2015 Phys. Med. Biol. 60 8923-47), which has a half-life of 11 ms. The results of a proof-of-principle experiment of beam-on PET imaging of short-lived 12N nuclei are presented. The Philips Digital Photon Counting Module TEK PET system was used, which is based on LYSO scintillators mounted on digital SiPM photosensors. A 90 MeV proton beam from the cyclotron at KVI-CART was used to investigate the energy and time spectra of PET coincidences during beam-on. Events coinciding with proton bunches, such as prompt gamma rays, were removed from the data via an anti-coincidence filter with the cyclotron RF. The resulting energy spectrum allowed good identification of the 511 keV PET counts during beam-on. A method was developed to subtract the long-lived background from the 12N image by introducing a beam-off period into the cyclotron beam time structure. We measured 2D images and 1D profiles of the 12N distribution. A range shift of 5 mm was measured as 6 ± 3 mm using the 12N profile. A larger, more efficient, PET system with a higher data throughput capability will allow beam-on 12N PET imaging of single spots in the distal layer of an irradiation with an increased signal-to-background ratio and thus better accuracy. A simulation shows that a large dual panel scanner, which images a single spot directly after it is delivered, can measure a 5 mm range shift with millimeter accuracy: 5.5 ± 1.1 mm for 1 × 108 protons and 5.2 ± 0.5 mm for 5 × 108 protons. This makes fast and accurate feedback on the dose delivery during treatment possible.

Short-lived positron emitters in beam-on PET imaging during proton therapy.

The only method for in vivo dose delivery verification in proton beam radiotherapy in clinical use today is positron emission tomography (PET) of the positron emitters produced in the patient during irradiation. PET imaging while the beam is on (so called beam-on PET) is an attractive option, providing the largest number of counts, the least biological washout and the fastest feedback. In this implementation, all nuclides, independent of their half-life, will contribute. As a first step towards assessing the relevance of short-lived nuclides (half-life shorter than that of (10)C, T1/2 = 19 s) for in vivo dose delivery verification using beam-on PET, we measured their production in the stopping of 55 MeV protons in water, carbon, phosphorus and calcium The most copiously produced short-lived nuclides and their production rates relative to the relevant long-lived nuclides are: (12)N (T1/2 = 11 ms) on carbon (9% of (11)C), (29)P (T1/2 = 4.1 s) on phosphorus (20% of (30)P) and (38m)K (T1/2 = 0.92 s) on calcium (113% of (38g)K). No short-lived nuclides are produced on oxygen. The number of decays integrated from the start of an irradiation as a function of time during the irradiation of PMMA and 4 tissue materials has been determined. For (carbon-rich) adipose tissue, (12)N dominates up to 70 s. On bone tissue, (12)N dominates over (15)O during the first 8-15 s (depending on carbon-to-oxygen ratio). The short-lived nuclides created on phosphorus and calcium provide 2.5 times more beam-on PET counts than the long-lived ones produced on these elements during a 70 s irradiation. From the estimated number of (12)N PET counts, we conclude that, for any tissue, (12)N PET imaging potentially provides equal to superior proton range information compared to prompt gamma imaging with an optimized knife-edge slit camera. The practical implementation of (12)N PET imaging is discussed.

Spectra of clinical CT scanners using a portable Compton spectrometer.

PURPOSE: Spectral information of the output of x-ray tubes in (dual source) computer tomography (CT) scanners can be used to improve the conversion of CT numbers to proton stopping power and can be used to advantage in CT scanner quality assurance. The purpose of this study is to design, validate, and apply a compact portable Compton spectrometer that was constructed to accurately measure x-ray spectra of CT scanners. METHODS: In the design of the Compton spectrometer, the shielding materials were carefully chosen and positioned to reduce background by x-ray fluorescence from the materials used. The spectrum of Compton scattered x-rays alters from the original source spectrum due to various physical processes. Reconstruction of the original x-ray spectrum from the Compton scattered spectrum is based on Monte Carlo simulations of the processes involved. This reconstruction is validated by comparing directly and indirectly measured spectra of a mobile x-ray tube. The Compton spectrometer is assessed in a clinical setting by measuring x-ray spectra at various tube voltages of three different medical CT scanner x-ray tubes. RESULTS: The directly and indirectly measured spectra are in good agreement (their ratio being 0.99) thereby validating the reconstruction method. The measured spectra of the medical CT scanners are consistent with theoretical spectra and spectra obtained from the x-ray tube manufacturer. CONCLUSIONS: A Compton spectrometer has been successfully designed, constructed, validated, and applied in the measurement of x-ray spectra of CT scanners. These measurements show that our compact Compton spectrometer can be rapidly set-up using the alignment lasers of the CT scanner, thereby enabling its use in commissioning, troubleshooting, and, e.g., annual performance check-ups of CT scanners.

Using standard calibrated geometries to characterize a coaxial high purity germanium gamma detector for Monte Carlo simulations.

A detector model optimization procedure based on matching Monte Carlo simulations with measurements for two experimentally calibrated sample geometries which are frequently used in radioactivity measurement laboratories results in relative agreement within 5% between simulated and measured efficiencies for a high purity germanium detector. The optimization procedure indicated that the increase in dead layer thickness is largely responsible for a detector efficiency decrease in time. The optimized detector model allows Monte Carlo efficiency calibration for all other samples of which the geometry and bulk composition is known. The presented method is a competitive and economic alternative to more elaborate detector scanning methods and results in a comparable accuracy.

["With all suitable means". Off-label-use and public statutory employers' liability insurance]. / "Mit allen geeigneten Mitteln". Off-label-Use und Berufsgenossenschaften.

In Germany, responsibility for treatment of occupational diseases lies with the public statutory employers' liability insurances (SELI). According to their legal obligation SELI have to ascertain cure--wherever possible--"with all suitable means". Thus, dermatologists treating patients with occupational dermatoses are required to deliver the best possible therapy, which, according to the current scientific knowledge, may in some cases be off-label. For example, in occupational contact dermatitis a number of scientifically promising topical and systemic drugs are not yet licensed for this indication. Off-label prescribing is not prohibited and there are no laws limiting physician flexibility in such prescribing. SELI also allows the use of off-label drugs. The goals of optimal treatment or effective prevention of occupational disease determine the measures which can be employed. Off-label use is approved for occupational skin diseases when the medication is required for cure or prevention and its use meets generally accepted medical standards. In these cases, SELI will cover off-label drug prescriptions. However, detailed patient counseling and informed consent are mandatory.

A pepper-pot emittance meter for low-energy heavy-ion beams.

A novel emittance meter has been developed to measure the four-dimensional, transverse phase-space distribution of a low-energy ion beam using the pepper-pot technique. A characteristic feature of this instrument is that the pepper-pot plate, which has a linear array of holes in the vertical direction, is scanned horizontally through the ion beam. This has the advantage that the emittance can also be measured at locations along the beam line where the beam has a large horizontal divergence. A set of multi-channel plates, scintillation screen, and ccd camera is used as a position-sensitive ion detector allowing a large range of beam intensities that can be handled. This paper describes the design, construction, and operation of the instrument as well as the data analysis used to reconstruct the four-dimensional phase-space distribution of an ion beam. Measurements on a 15 keV He(+) beam are used as an example.

Optimization of a charge-state analyzer for electron cyclotron resonance ion source beams.

A detailed experimental and simulation study of the extraction of a 24 keV He(+) beam from an ECR ion source and the subsequent beam transport through an analyzing magnet is presented. We find that such a slow ion beam is very sensitive to space-charge forces, but also that the neutralization of the beam's space charge by secondary electrons is virtually complete for beam currents up to at least 0.5 mA. The beam emittance directly behind the extraction system is 65 π mm mrad and is determined by the fact that the ion beam is extracted in the strong magnetic fringe field of the ion source. The relatively large emittance of the beam and its non-paraxiality lead, in combination with a relatively small magnet gap, to significant beam losses and a five-fold increase of the effective beam emittance during its transport through the analyzing magnet. The calculated beam profile and phase-space distributions in the image plane of the analyzing magnet agree well with measurements. The kinematic and magnet aberrations have been studied using the calculated second-order transfer map of the analyzing magnet, with which we can reproduce the phase-space distributions of the ion beam behind the analyzing magnet. Using the transfer map and trajectory calculations we have worked out an aberration compensation scheme based on the addition of compensating hexapole components to the main dipole field by modifying the shape of the poles. The simulations predict that by compensating the kinematic and geometric aberrations in this way and enlarging the pole gap the overall beam transport efficiency can be increased from 16% to 45%.

Cigarette smoke impairs airway epithelial barrier function and cell-cell contact recovery.

Cigarette smoking, the major cause of chronic obstructive pulmonary disease (COPD), induces aberrant airway epithelial structure and function. The underlying mechanisms are unresolved so far. We studied effects of cigarette smoke extract (CSE) on epithelial barrier function and wound regeneration in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from COPD patients, nonsmokers and healthy smokers. We demonstrate that CSE rapidly and transiently impairs 16HBE barrier function, largely due to disruption of cell-cell contacts. CSE induced a similar, but stronger and more sustained, defect in PBECs. Application of the specific epidermal growth factor receptor (EGFR) inhibitor AG1478 showed that EGFR activation contributes to the CSE-induced defects in both 16HBE cells and PBECs. Furthermore, our data indicate that the endogenous protease calpain mediates these defects through tight junction protein degradation. CSE also delayed the reconstitution of 16HBE intercellular contacts during wound healing and attenuated PBEC barrier function upon wound regeneration. These findings were comparable between PBECs from smokers, healthy smokers and COPD patients. In conclusion, we demonstrate for the first time that CSE reduces epithelial integrity, probably by EGFR and calpain-dependent disruption of intercellular contacts. This may increase susceptibility to environmental insults, e.g. inhaled pathogens. Thus, EGFR may be a promising target for therapeutic strategies to improve mucosal barrier function in cigarette smoking-related disease.

SU-E-T-294: Maximizing the Availability of Positron Emitting Nuclei for Proton Therapy Verification Using Different Beam Irradiation Sequences.

PURPOSE: To demonstrate that the amount of nuclei available for post- irradiation proton treatment verification using positron emission tomography (PET) can be enhanced by reversing the beam delivery sequence in proton scanning beam irradiations. METHODS: A time-dependent analytical model is used to calculate the distributions of positron emitting nuclei for three different irradiation sequences: a scattered beam and a scanning beam in both the conventional sequence, distal edge first, and reverse sequence, distal edge last. The simulated geometry emulates reference dosimetry measurements conducted at the Paul Scherrer Institute (PSI). The reference measurements irradiate a 10 ×10 cm2 field, delivering about 1 Gy to a 10 cm wide spread-out Bragg peak (SOBP). Positron emitter availability with different beam sequence and imaging times and the impact of the different irradiation sequences on the statistical error on a range extrapolation were investigated. RESULTS: The ratio of the amount of positron emitters from the distal last beam sequence to that from the distal first sequence was 2.22 in the last centimeter of the SOBP. The comparison between distal last and a scattered beam gave a ratio of about 1.7 in the same region. In the distal last irradiation, more isotopes decay within a 120 second window, than in a 240 second window using a distal first irradiation. The statistical fluctuation on a range extrapolation was also smallest in the distal last beam sequence. CONCLUSIONS: We demonstrated the effect of the irradiation beam sequence on the isotope production relevant for the verification of proton spot scanning therapy with PET. The largest amount of isotopes is available by irradiating the distal edge last. This new beam sequence reduces the PETmeasurement time while still offering higher counts and accuracy compared with both the conventional beam sequence and the scattering method. This project was supported by JSPS Core-to-Core Program.

Lung irradiation induces pulmonary vascular remodelling resembling pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a commonly fatal pulmonary vascular disease that is often diagnosed late and is characterised by a progressive rise in pulmonary vascular resistance resulting from typical vascular remodelling. Recent data suggest that vascular damage plays an important role in the development of radiation-induced pulmonary toxicity. Therefore, the authors investigated whether irradiation of the lung also induces pulmonary hypertension. METHODS: Different sub-volumes of the rat lung were irradiated with protons known to induce different levels of pulmonary vascular damage. RESULTS: Early loss of endothelial cells and vascular oedema were observed in the irradiation field and in shielded parts of the lung, even before the onset of clinical symptoms. 8 weeks after irradiation, irradiated volume-dependent vascular remodelling was observed, correlating perfectly with pulmonary artery pressure, right ventricle hypertrophy and pulmonary dysfunction. CONCLUSIONS: The findings indicate that partial lung irradiation induces pulmonary vascular remodelling resulting from acute pulmonary endothelial cell loss and consequential pulmonary hypertension. Moreover, the close resemblance of the observed vascular remodelling with vascular lesions in PAH makes partial lung irradiation a promising new model for studying PAH.

Study of ion beam extraction and transport from an electron cyclotron resonance ion source.

We have started an experimental and theoretical program to better understand the extraction and transport of intense multiply charged ion beams from an electron cyclotron resonance ion source (ECRIS). In this paper we present the first results of this program concerning a simple, monocomponent He(+) beam extracted from an ECRIS. We have calculated the ion trajectories starting from the ECRIS plasma electrode up to the image plane of the analyzing magnet taking into account space-charge effects and fringe fields. The initial phase-space distribution of the He(+) beam at the extraction aperture has been calculated with a particle-in-cell code. To check the simulations we have measured beam profiles with a viewing screen both before and after the analyzing magnet. In addition also measurements with a pepperpot emittance meter located behind the analyzing magnet have been performed. We find good agreement between these measurements and simulations showing that (i) there is a significant compensation of the space charge and that (ii) our analyzing magnet causes a severe increase in effective beam emittance.

Characterisation of cell adhesion in airway epithelial cell types using electric cell-substrate impedance sensing.

Research on epithelial cell lines and primary epithelium is required to dissect the mechanisms underlying the structural abnormalities in airway epithelium observed for respiratory diseases, including asthma and chronic obstructive pulmonary disease. The novel electric cell-substrate impedance sensing technique was used to monitor cell adhesion/spreading, barrier function and wound healing. Primary bronchial epithelium was compared with airway epithelial cell lines 16HBE14o-, BEAS-2B, NCI-H292 and A549. BEAS-2B, A549 and primary cells form a confluent monolayer more rapidly than do 16HBE14o- cells. In contrast, 16HBE14o- cells form stronger intercellular contacts, with a 10-fold higher resistance than BEAS-2B, A549 and NCI-H292 cells and a five-fold increase over primary cells. Accordingly, expression of the adhesion molecules zona occludens-1 and E-cadherin was highest in 16HBE14o- cells. These molecules were localised in intercellular junctions in both 16HBE14o- and primary cells. Finally, restoration of barrier function upon injury was impaired in BEAS-2B compared to 16HBE14o- cells. In conclusion, epithelial cell types display remarkable phenotypic differences and should, accordingly, be used to address specific research questions. 16HBE14o- cells appear most suitable for studies on barrier formation, whereas resemblance in attachment of primary and BEAS-2B and A549 cells makes the latter more important for translational research on cell-matrix contact.

[Cooperation among clinics and practices. Integrated medical care in occupational dermatology]. / Neue Formen der Zusammenarbeit zwischen Praxis und Klinik. Integrierte Versorgung in der Berufsdermatologie.

Preventive measures in occupational dermatology have proven to be very effective in recent years, especially measures of primary and secondary prevention as components of a complex hierarchical prevention concept. For those cases of occupational dermatoses in which these outpatient prevention measures are not successful, interdisciplinary inpatient rehabilitation measures have been developed ("tertiary individual prevention" [TIP]). TIP comprises 3 weeks inpatient treatment including intensive disease-oriented teaching and psychological counseling, followed by outpatient treatment by the local dermatologist. In 2005, a German prospective cohort multicenter study ("Medizinisch-Berufliches Rehabilitationsverfahren Haut--Optimierung und Qualitätssicherung des Heilverfahrens" [ROQ]) started which will further standardize TIP and evaluate long-term success and scientific sustainability in depth. This integrated concept of an inpatient/outpatient disease management reveals remarkable pertinent options for patients with severe occupational dermatoses in all high-risk professions.

[Optimal care of patients with occupational hand dermatitis: considerations of German occupational health insurance]. / Optimierte Versorgung von Patienten mit berufsbedingten Handekzemen: Hautarztverfahren und Stufenverfahren Haut der gesetzlichen Unfallversicherung.

Occupational dermatoses (OD) have been at the top of all occupational diseases in Germany for years (>25% of all occupational diseases officially reported annually to the public statutory employers' liability insurance bodies). More than 90% of OD-cases are hand eczema. Several pilot initiatives to improve prevention of occupational skin diseases have been developed and funded since 2005 by the statutory employers' liability insurance schemes. The concept underpinning these initiatives is a nationwide multi-step intervention approach recently implemented by the public statutory insurance system administration ("step- wise procedure") which aims at offering quick and specific preventive help at all levels of severity of OD. The dermatologist has a pivotal function in this concept; for early secondary prevention, the so-called dermatologist's procedure was recently updated in order to provide more rapid and concise dermatological consultations and care. Additionally, combined outpatient dermatological and educational intervention seminars ("secondary individual prevention", SIP) and interdisciplinary inpatient prevention measures have been developed ("tertiary individual prevention", [TIP]) and are currently being further evaluated in multicenter studies.

Ion source development at KVI.

Ion source development at KVI is focused on increasing the beam intensity from the electron cyclotron resonance ion source injector and optimizing the beam transport and injection into the superconducting AGOR cyclotron. We describe several modifications that have resulted in a significant performance increase of the ion source. We also present the first results of ion transport simulations that have been performed to better understand beam losses in the extraction region and in the low-energy beam transport system. Finally, a new emittance meter based on a combination of the pepperpot and scanning techniques will be described, which will be used to benchmark the simulation studies of ion extraction and transport in detail.

A status report of the multipurpose superconducting electron cyclotron resonance ion source.

Intense heavy ion beam production with electron cyclotron resonance (ECR) ion sources is a common requirement for many of the accelerators under construction in Europe and elsewhere. An average increase of about one order of magnitude per decade in the performance of ECR ion sources was obtained up to now since the time of pioneering experiment of R. Geller at CEA, Grenoble, and this trend is not deemed to get the saturation at least in the next decade, according to the increased availability of powerful magnets and microwave generators. Electron density above 10(13) cm(-3) and very high current of multiply charged ions are expected with the use of 28 GHz microwave heating and of an adequate plasma trap, with a B-minimum shape, according to the high B mode concept [S. Gammino and G. Ciavola, Plasma Sources Sci. Technol. 5, 19 (1996)]. The MS-ECRIS ion source has been designed following this concept and its construction is underway at GSI, Darmstadt. The project is the result of the cooperation of nine European institutions with the partial funding of EU through the sixth Framework Programme. The contribution of different institutions has permitted to build in 2006-2007 each component at high level of expertise. The description of the major components will be given in the following with a view on the planning of the assembly and commissioning phase to be carried out in fall 2007. An outline of the experiments to be done with the MS-ECRIS source in the next two years will be presented.