Canine distemper virus : persistence and pathology in the central nervous system

Résumé : Le virus de la maladie de Carré (en anglais: canine distemper virus, CDV) qui est pathogène pour les chiens et autres carnivores, est très semblable au virus de la rougeole humaine (en anglais MV). Ces deux virus font partie du genre des Morbillivirus qui appartient à la famille des Paramyxoviridae. Ils induisent des complications dans le système nerveux central (SNC). Au stade précoce et aigu de l'infection du SNC, le CDV induit une démyélinisation (1). Ce stade évolue dans certains cas vers une infection chronique avec progression de la démyélinisation. Pendant le stade précoce, qui suit en général de trois semaines les premiers symptômes, le processus de démyélinisation est associé à la réplication du virus et n'est pas considéré comme inflammatoire (1). Par contre, au stade chronique, la progression des plaques de démyélinisation semble être plutôt liée à des processus immunogènes caractéristiques (2), retrouvés également dans la sclérose en plaques (SEP) chez les humains. Pour cette raison, le CDV est considéré comme un modèle pour la SEP humaine et aussi pour l'étude des maladies et complications induites par les Morbillivirus en général (3). Dans notre laboratoire, nous avons utilisé la souche A75/17-CDV, qui est considérée comme le modèle des souches neurovirulentes de CDV. Nous avons cherché en premier lieu à établir un système robuste pour infecter des cultures neuronales avec le CDV. Nous avons choisi les cultures primaires de l'hippocampe du nouveau-né de rat (4), que nous avons ensuite infecté avec une version modifiée du A75/17, appelée rgA75/17-V (5). Dans ces cultures, nous avons prouvé que le CDV infecte des neurones et des astrocytes. Malgré une infection qui se diffuse lentement entre les cellules, cette infection cause une mort massive aussi bien des neurones infectés que non infectés. En parallèle, les astrocytes perdent leur morphologie de type étoilé pour un type polygonal. Finalment, nous avons trouvé une augmentation importante de la concentration en glutamate dans le milieu de culture, qui laisse présumer une sécrétion de glutamate par les cultures infectées (6). Nous avons ensuite étudié le mécanisme des effets cytopathiques induits par le CDV. Nous avons d'abord démontré que les glycoprotéines de surface F et H du CDV s'accumulent massivement dans le réticulum endoplasmique (RE). Cette accumulation déclenche un stress du RE, qui est caractérisé par une forte expression du facteur de transcription proapoptotique CHOP/GADD 153 et de le la calreticuline (CRT). La CRT est une protéine chaperonne localisée dans le RE et impliquée dans l'homéostasie du calcium (Ca2+) et dans le repliement des protéines. En transfectant des cellules de Vero avec des plasmides codant pour plusieurs mutants de la glycoprotéine F de CDV, nous avons démontré une corrélation entre l'accumulation des protéines virales dans le RE et l'augmentation de l'expression de CRT, le stress du RE et la perte de l'homéostasie du Ca2+. Nous avons obtenu des résultats semblables avec des cultures de cellules primaires de cerveau de rat. Ces résultats suggèrent que la CRT joue un rôle crucial dans les phénomènes neurodégénératifs pendant l'infection du SNC, notamment par le relazgage du glutamate via le Ca2+. De manière intéressante, nous démontrons également que l'infection de CDV induit une fragmentation atypique de la CRT. Cette fragmentation induit une re-localisation et une exposition sélective de fragments amino-terminaux de la CRT, connus pour êtres fortement immunogènes à la surface des cellules infectées et non infectées. A partir de ce résultat et des résultats précédents, nous proposons le mécanisme suivant: après l'infection par le CDV, la rétention dans le RE des protéines F et H provoque un stress du RE et une perte de l'homéostasie du Ca2+. Ceci induit la libération du glutamate, qui cause une dégénération rapide du SNC (sur plusieurs jours ou semaines) correspondant à la phase aiguë de la maladie chez le chien. En revanche, les fragments amino-terminaux de la CRT libérés à la surface des cellules infectées peuvent avoir un rôle important dans l'établissement d'une démyélinisation d'origine immunogène, typique de la phase chronique de l'infection de CDV. Summary : The dog pathogen canine distemper virus (CDV), closely related to the human pathogen measles virus (MV), belongs to the Morbillivirus genus of the Paramyxoviridae family. Both CDV and NIV induce complications in the central nervous system (CNS). In the acute early stage of the infection in CNS, the CDV infection induces demyelination. This stage is sometimes followed by a late persistent stage of infection with a progression of the demyelinating lesions (1). The acute early stage occurs around three weeks after the infection and demyelinating processes are associated with active virus replication and are not associated to inflammation (1). In contrast during late persistent stage, the demyelination plaque progression seems to be mainly due to an immunopathological process (2), which characteristics are shared in many aspects with the human disease multiple sclerosis (MS). For these reasons, CDV is considered as a model for human multiple sclerosis, as well as for the study of Morbillivirus-mediated pathogenesis (3). In our laboratory, we used the A75/17-CDV strain that is considered to be the prototype of neurovirulent CDV strain. We first sought to establish a well characterized and robust model for CDV infection of a neuronal culture. We chose primary cultures from newborn rat hippocampes (4) that we infected with a modified version of A75/17, called rgA75/17-V (5). In these cultures, we showed that CDV infects both neurons and astrocytes. While the infection spreads only slowly to neighbouring cells, it causes a massive death of neurons, which includes also non-infected neurons. In parallel, astrocytes undergo morphological changes from the stellate type to the polygonal type. The pharmacological blocking of the glutamate receptors revealed an implication of glutamatergic signalling in the virus-mediated cytopathic effect. Finally, we found a drastic increase concentration of glutamate in the culture medium, suggesting that glutamate was released from the cultured cells (6). We further studied the mechanism of the CDV-induced cytopathic effects. We first demonstrated that the CDV surface glycoprotein F and H markedly accumulate in the endoplasmic reticulum (ER). This accumulation triggers an ER stress, which is characterized by increased expression of the proapoptotic transcription factor CHOP/GADD 153 and calreticulin (CRT). CRT is an ER resident chaperon involved in the Ca2+ homeostasis and in the response to misfolded proteins. Transfections of Vero cells with plasmids encoding various CDV glycoprotein mutants reveal a correlation between accumulation of viral proteins in the ER, CRT overexpression, ER stress and alteration of ER Ca2+ homeostasis. Importantly, similar results are also obtained in primary cell cultures from rat brain. These results suggest that CRT plays a crucial role in CNS infection, particularly due to CRT involvement in Ca2+ mediated glutamate releases, and subsequent neurodegenerative disorders. Very intriguingly, we also demonstrated that CDV infection induces an atypical CRT fragmentation, with relocalisation and selective exposure of the highly immunogenic CRT N-terminal fragments at the surface of infected and neighbouring non-infected cells. Altogether our results combined with previous findings suggest the following scenario. After CDV infection, F and H retention alter Ca2+ homeostasis, and induce glutamate release, which in turn causes rapid CNS degeneration (within days or a week) corresponding to the acute phase of the disease in dogs. In contrast, the CRT N-terminal fragments released at the surface of infected cells may rather have an important role in the establishment of the autoimmune demyelination in the late stage of CDV infection.

Variability of a peripheral dose among various linac geometries for second cancer risk assessment.

Second cancer risk assessment for radiotherapy is controversial due to the large uncertainties of the dose-response relationship. This could be improved by a better assessment of the peripheral doses to healthy organs in future epidemiological studies. In this framework, we developed a simple Monte Carlo (MC) model of the Siemens Primus 6 MV linac for both open and wedged fields that we then validated with dose profiles measured in a water tank up to 30 cm from the central axis. The differences between the measured and calculated doses were comparable to other more complex MC models and never exceeded 50%. We then compared our simple MC model with the peripheral dose profiles of five different linacs with different collimation systems. We found that the peripheral dose between two linacs could differ up to a factor of 9 for small fields (5 × 5 cm(2)) and up to a factor of 10 for wedged fields. Considering that an uncertainty of 50% in dose estimation could be acceptable in the context of risk assessment, the MC model can be used as a generic model for large open fields (≥10 × 10 cm(2)) only. The uncertainties in peripheral doses should be considered in future epidemiological studies when designing the width of the dose bins to stratify the risk as a function of the dose.

A novel dominant mutation of the Nav1.4 alpha-subunit domain I leading to sodium channel myotonia.

BACKGROUND: Mutations in SCN4A may lead to myotonia. METHODS: Presentation of a large family with myotonia, including molecular studies and patch clamp experiments using human embryonic kidney 293 cells expressing wild-type and mutated channels. RESULTS: In a large family with historic data on seven generations and a clear phenotype, including myotonia at movement onset, with worsening by cold temperature, pregnancy, mental stress, and especially after rest after intense physical activity, but without weakness, the phenotype was linked with the muscle sodium channel gene (SCN4A) locus, in which a novel p.I141V mutation was found. This modification is located within the first transmembrane segment of domain I of the Na(v)1.4 alpha subunit, a region where no mutation has been reported so far. Patch clamp experiments revealed a mutation-induced hyperpolarizing shift (-12.9 mV) of the voltage dependence of activation, leading to a significant increase (approximately twofold) of the window current amplitude. In addition, the mutation shifted the voltage dependence of slow inactivation by -8.7 mV and accelerated the entry to this state. CONCLUSIONS: We propose that the gain-of-function alteration in activation leads to the observed myotonic phenotype, whereas the enhanced slow inactivation may prevent depolarization-induced paralysis.

Skeletal muscle mitochondria in the elderly: effects of physical fitness and exercise training.

Context: Sarcopenia is thought to be associated with mitochondrial (M) loss. It is unclear whether the decrease in M content is consequent to aging per se or to decreased physical activity. Objectives: To examine the influence of fitness on M content and function, and to assess whether exercise could improve M function in older adults. Design and subjects: Three distinct studies were conducted: 1) a cross-sectional observation comparing M content and fitness in a large heterogeneous cohort of older adults; 2) a case-control study comparing chronically endurance-trained older adults (A) and sedentary (S) subjects matched for age and gender; 3) a 4-month exercise intervention in S. Setting: University-based clinical research center Outcomes: M volume density (Mv) was assessed by electron microscopy from vastus lateralis biopsies, electron transport chain proteins (ETC) by western blotting, mRNAs for transcription factors involved in M biogenesis by qRT-PCR and in-vivo oxidative capacity (ATPmax) by (31)P-MR spectroscopy. Peak oxygen uptake (VO2peak) was measured by GXT. Results: VO2peak was strongly correlated with Mv in eighty 60-80 yo adults. Comparison of A vs. S revealed differences in Mv, ATPmax and some ETC complexes. Finally, exercise intervention confirmed that S are able to recover Mv, ATPmax and specific transcription factors. Conclusions: These data suggest that 1) aging per se is not the primary culprit leading to M dysfunction, 2) an aerobic exercise program, even at an older age, can ameliorate the loss in skeletal muscle M content and may prevent aging muscle comorbidities and 3) the improvement of M function is all about content.

Timing effects of combined radioimmunotherapy and radiotherapy on a human solid tumor in nude mice.

Timing effects of radioimmunotherapy (RIT) combined with external-beam radiotherapy (RT) were assessed in human colon carcinoma xenografts. Initially, dose effects of fractionated RT and RIT were evaluated separately. Then, 30 Gy RT (10 fractions over 12 days) were combined with three weekly i.v. injections of 200 microCi of 131I-labeled anti-carcinoembryonic antigen monoclonal antibodies in four different treatment schedules. RIT was given either prior to, concurrently, immediately after, or 2 weeks after RT administration. The longest regrowth delay (RD) of 105 days was observed in mice treated by concurrent administration of RT and RIT, whereas the RDs of RT and RIT alone were 34 and 20 days, respectively. The three sequential combination treatments produced significantly shorter RDs ranging from 62 to 70 days. The tumor response represented by the minimal volume (MV) also showed that concurrent administration of RT and RIT gave the best result, with a mean MV of 4.5% as compared to MVs from 26 to 53% for the three sequential treatments. The results were confirmed in a second experiment, in which a RT of 40 Gy was combined with an identical RIT as above (three injections of 200 microCi of 131I-labeled monoclonal antibodies). At comparable toxicity levels, the maximum tolerated RT or RIT alone gave shorter RDs and less tumor shrinkage compared to simultaneous RT+ RIT. These results may be useful for designing clinical protocols of combined RIT and RT.

Safety of combined heat and moisture exchanger filters in long-term mechanical ventilation.

STUDY OBJECTIVE: To evaluate the safety of a combined heat and moisture exchanger filter (HMEF) for the conditioning of inspired gas in long-term mechanical ventilation (MV). DESIGN: Randomized controlled trial. SETTING: Medical ICU in a large teaching hospital. PATIENTS: One hundred fifteen consecutive patients who required > or = 48 h of MV. INTERVENTIONS: Patients were randomized at intubation time (day 1) to receive inspired gas conditioned either by a water-bath humidifier heated at 32 degrees C (HWBH) or by an HMEF (Hygroster; DAR; Mirandola, Italy). MEASUREMENTS AND MAIN RESULTS: The two study groups were comparable in terms of primary pathologic condition at the time of hospital admission, disease severity as measured by the Simplified Acute Physiology Score, and ICU mortality. They did not differ with respect to ventilator days per patient (mean +/- SD: HMEF, 7.6 +/- 6.5; HWBH, 7.8 +/- 5.8), incidence of endotracheal tube obstruction (HMEF, 0/59; HWBH, 1/56), and incidence of hypothermic episodes (HMEF, five; HWBH, two). In 41 patients receiving MV for > or = 5 days, the morphologic integrity of respiratory epithelium was evaluated on day 1 and day 5, using a cytologic examination of tracheal aspirate smears. The state of ciliated epithelium was scored on a scale from 0 (poorest integrity) to 1,200 (maximum integrity), according to a well-described method. In both patient groups, the scores slightly but significantly decreased from day 1 to day 5 (mean +/- SD: HWBH, from 787 +/- 104 to 745 +/- 88; HMEF, from 813 +/- 79 to 739 +/- 62; p < 0.01 for both groups); there were no statistically significant differences between groups. CONCLUSIONS: These data indicate acceptable safety of HMEFs of the type used in the present study for long-term mechanical ventilation.

Respiration, metabolic balance, and attention in affective picture processing

The respiratory behavior during affective states is not completely understood. We studied breathing pattern responses to picture series in 37 participants. We also measured end-tidal pCO2 (EtCO2) to determine if ventilation is in balance with metabolic demands and spontaneous eye-blinking to investigate the link between respiration and attention. Minute ventilation (MV) and inspiratory drive increased with self-rated arousal. These relationships reflected increases in inspiratory volume rather than shortening of the time parameters. EtCO2 covaried with pleasantness but not arousal. Eye-blink rate decreased with increasing unpleasantness in line with a negativity bias in attention. This study confirms that respiratory responses to affective stimuli are organized to a certain degree along the dimensions of valence and arousal. It shows, for the first time, that during picture viewing, ventilatory increases with increasing arousal are in balance with metabolic activity and that inspiratory volume is modulated by arousal. MV emerges as the most reliable respiratory index of self-perceived arousal

Competition between beta-subunits of cardiac L-type calcium channels

Cardiac L-type Ca (CaV1.2) channels are composed of a pore forming CaV1.2-α1 subunit and auxiliary β- and α2δ-subunits. β-subunits are important not only for surface expression of the channel pore but also for modulation of channel gating properties. Different β-subunits differentially modulate channel activity (Hullin et al., PLOSone, 2007) and thus L-type Ca2+ channel gating is altered when β-subunit expression pattern is changed. In human heart failure increased activity of single ventricular L-type Ca2+-channels is associated with an increased expression of β2-subunits. Interestingly, induction of β2-subunit over-expression in hearts of transgenic mice resembled this heart failure phenotype of hyperactive single L-type Ca2+-channel channels (Beetz et al., Cardiovasc Res. 2009). We hypothesised that competition of less stimulating β-subunits (e.g. β1) with β-subunits causing strong channel stimulation (e.g. β2) might be a means to treat dysfunctional L-type Ca2+-channel activity. To test this hypothesis, we performed whole-cell and single-channel measurements employing recombinant CaV1.2 channels expressed in HEK293 cells together with both β- and β1a2b-subunits. Whole-cell analysis revealed no differences of maximum L-type Ca2+-current densities [pA/pF] with coexpression of either β1a-subunits (-52±3.8), β2b-subunits (-61.5±6.6) or the mixtures of β- and β1a2b-subunits with the plasmid transfection ratio of 2:1 (-60.2±1.6) and 1:1 (-56.7±2.6) respectively. However, steady state inactivation kinetics differed between particular β-subunit and the relative amount of β-subunit presence in the mixtures (β1a1a-subunit (-41.1±1.0), β2b-subunits (-35.1±1.1), mixture 2:1 (-40.3±1.5), and mixture 1:1 (-38.4±2.0); [mV]; p<0.05, students t-test). Using a novel single-channel analysis, switching of gating between β1-like and β2-like modes was monitored on a minute time-scale when both β-subunits were co-expressed in the same cells, but the larger amount of β1a-subunits is required for the effective switching of gating. Our results indicate a model of mutually exclusive binding and effective competition between several β-subunits suggesting that hyperactive channel gating mediated e.g. by β2-subunits can be normalized by β1-subunits. Therefore, competitive replacement between different L-type Ca2+-channel β-subunits might serve as a novel therapeutic strategy for e.g. heart failure.

3D dose reconstruction for narrow beams using ion chamber array measurements.

3D dose reconstruction is a verification of the delivered absorbed dose. Our aim was to describe and evaluate a 3D dose reconstruction method applied to phantoms in the context of narrow beams. A solid water phantom and a phantom containing a bone-equivalent material were irradiated on a 6 MV linac. The transmitted dose was measured by using one array of a 2D ion chamber detector. The dose reconstruction was obtained by an iterative algorithm. A phantom set-up error and organ interfraction motion were simulated to test the algorithm sensitivity. In all configurations convergence was obtained within three iterations. A local reconstructed dose agreement of at least 3% / 3mm with respect to the planned dose was obtained, except in a few points of the penumbra. The reconstructed primary fluences were consistent with the planned ones, which validates the whole reconstruction process. The results validate our method in a simple geometry and for narrow beams. The method is sensitive to a set-up error of a heterogeneous phantom and interfraction heterogeneous organ motion.

Acute multivessel revascularization improves 1-year outcome in ST-elevation myocardial infarction: a nationwide study cohort from the AMIS Plus registry.

BACKGROUND: The optimal strategy for percutaneous coronary intervention (PCI) of ST-segment elevation myocardial infarction (STEMI) in multi-vessel disease (MVD), i.e., multi-vessel PCI (MV-PCI) vs. PCI of the infarct-related artery only (IRA-PCI), still remains unknown. METHODS: Patients of the AMIS Plus registry admitted with an acute coronary syndrome were contacted after a median of 378 days (interquartile range 371-409). The primary end-point was all-cause death. The secondary end-point included all major adverse cardiovascular and cerebrovascular events (MACCE) including death, re-infarction, re-hospitalization for cardiac causes, any cardiac re-intervention, and stroke. RESULTS: Between 2005 and 2012, 8330 STEMI patients were identified, of whom 1909 (24%) had MVD. Of these, 442 (23%) received MV-PCI and 1467 (77%) IRA-PCI. While all-cause mortality was similar in both groups (2.7% both, p>0.99), MACCE was significantly lower after MV-PCI vs. IRA-PCI (15.6% vs. 20.0%, p=0.038), mainly driven by lower rates of cardiac re-hospitalization and cardiac re-intervention. Patients undergoing MV-PCI with drug-eluting stents had lower rates of all-cause mortality (2.1% vs. 7.4%, p=0.026) and MACCE (14.1% vs. 25.9%, p=0.042) compared with those receiving bare metal stents (BMS). In multivariate analysis, MV-PCI (odds ratio, OR 0.69, 95% CI 0.51-0.93, p=0.017) and comorbidities (Charlson index ≥ 2; OR 1.42, 95% CI 1.05-1.92, p=0.025) were independent predictors for 1-year MACCE. CONCLUSION: In an unselected nationwide real-world cohort, an approach using immediate complete revascularization may be beneficial in STEMI patients with MVD regarding MACCE, specifically when drug-eluting stents are used, but not regarding mortality. This has to be tested in a randomized controlled trial.

Influence of scatter reduction method and monochromatic beams on image quality and dose in mammography.

In mammography, the image contrast and dose delivered to the patient are determined by the x-ray spectrum and the scatter to primary ratio S/P. Thus the quality of the mammographic procedure is highly dependent on the choice of anode and filter material and on the method used to reduce the amount of scattered radiation reaching the detector. Synchrotron radiation is a useful tool to study the effect of beam energy on the optimization of the mammographic process because it delivers a high flux of monochromatic photons. Moreover, because the beam is naturally flat collimated in one direction, a slot can be used instead of a grid for scatter reduction. We have measured the ratio S/P and the transmission factors for grids and slots for monoenergetic synchrotron radiation. In this way the effect of beam energy and scatter rejection method were separated, and their respective importance for image quality and dose analyzed. Our results show that conventional mammographic spectra are not far from optimum and that the use of a slot instead of a grid has an important effect on the optimization of the mammographic process. We propose a simple numerical model to quantify this effect.

Reproducibility of Fatmax and Fat Oxidation Rates during Exercise in Recreationally Trained Males.

Aerobic exercise training performed at the intensity eliciting maximal fat oxidation (Fatmax) has been shown to improve the metabolic profile of obese patients. However, limited information is available on the reproducibility of Fatmax and related physiological measures. The aim of this study was to assess the intra-individual variability of: a) Fatmax measurements determined using three different data analysis approaches and b) fat and carbohydrate oxidation rates at rest and at each stage of an individualized graded test. Fifteen healthy males [body mass index 23.1±0.6 kg/m2, maximal oxygen consumption ([Formula: see text]) 52.0±2.0 ml/kg/min] completed a maximal test and two identical submaximal incremental tests on ergocycle (30-min rest followed by 5-min stages with increments of 7.5% of the maximal power output). Fat and carbohydrate oxidation rates were determined using indirect calorimetry. Fatmax was determined with three approaches: the sine model (SIN), measured values (MV) and 3rd polynomial curve (P3). Intra-individual coefficients of variation (CVs) and limits of agreement were calculated. CV for Fatmax determined with SIN was 16.4% and tended to be lower than with P3 and MV (18.6% and 20.8%, respectively). Limits of agreement for Fatmax were -2±27% of [Formula: see text] with SIN, -4±32 with P3 and -4±28 with MV. CVs of oxygen uptake, carbon dioxide production and respiratory exchange rate were <10% at rest and <5% during exercise. Conversely, CVs of fat oxidation rates (20% at rest and 24-49% during exercise) and carbohydrate oxidation rates (33.5% at rest, 8.5-12.9% during exercise) were higher. The intra-individual variability of Fatmax and fat oxidation rates was high (CV>15%), regardless of the data analysis approach employed. Further research on the determinants of the variability of Fatmax and fat oxidation rates is required.

Hypofractionated radiotherapy in the treatment of diffuse intrinsic pontine glioma in children: a single institution's experience

Objective: To demonstrate our institutional experience in the treatment ofdiffuse intrinsic pontine glioma (DIPG) with an hypofractionated external beam radiotherapy schedule.Materials and Methods: Between April 1996 and January 2004, 22 patients, ages 2.9-12.5 years, with newly diagnosed DIPG were treated by hypofractionated radiation therapy delivering a total dose of 45 Gy in daily fraction of 3 Gy, given over 3 weeks. No other treatment was applied concomittently.Results: Fourteen of the 22 patients received the prescribed dose of 45 Gy in 15 fractions of 3 Gy, two patients received a total dose of 60 and 45 Gy with a combination of two different beams (photons and neutrons), in 5 cases the daily fraction was modified to 2 Gy because of bad tolerance and one patient died due to serious intracranial hypertension after 2 fractions of 3 Gy and one of 2 Gy. Fourteen patients of 22 patients/of the total showed a clinical improvement, usually starting in the second week of treatment. No grade 3 or 4 acute toxicity from radiotherapy was observed. No treatment interruption was needed. In six patients, steroids could be discontinued within one month after the end of radiotherapy. The median time to progression and the median overall survival were 5.7 months and 7.6 months, respectively.Conclusion: External radiotherapy with a radical hypofractionated regimen is feasible and well tolerated in children with newly diagnosed DIPG. This regimen does not seem however to change the overall survival in this setting. It could represent an alternative option of short duration to more protracted regimens.

Accuracy of out-of-field dose calculation of tomotherapy and cyberknife treatment planning systems: A dosimetric study.

PURPOSE: Late toxicities such as second cancer induction become more important as treatment outcome improves. Often the dose distribution calculated with a commercial treatment planning system (TPS) is used to estimate radiation carcinogenesis for the radiotherapy patient. However, for locations beyond the treatment field borders, the accuracy is not well known. The aim of this study was to perform detailed out-of-field-measurements for a typical radiotherapy treatment plan administered with a Cyberknife and a Tomotherapy machine and to compare the measurements to the predictions of the TPS. MATERIALS AND METHODS: Individually calibrated thermoluminescent dosimeters were used to measure absorbed dose in an anthropomorphic phantom at 184 locations. The measured dose distributions from 6 MV intensity-modulated treatment beams for CyberKnife and TomoTherapy machines were compared to the dose calculations from the TPS. RESULTS: The TPS are underestimating the dose far away from the target volume. Quantitatively the Cyberknife underestimates the dose at 40cm from the PTV border by a factor of 60, the Tomotherapy TPS by a factor of two. If a 50% dose uncertainty is accepted, the Cyberknife TPS can predict doses down to approximately 10 mGy/treatment Gy, the Tomotherapy-TPS down to 0.75 mGy/treatment Gy. The Cyberknife TPS can then be used up to 10cm from the PTV border the Tomotherapy up to 35cm. CONCLUSIONS: We determined that the Cyberknife and Tomotherapy TPS underestimate substantially the doses far away from the treated volume. It is recommended not to use out-of-field doses from the Cyberknife TPS for applications like modeling of second cancer induction. The Tomotherapy TPS can be used up to 35cm from the PTV border (for a 390 cm(3) large PTV).

Nanomedicines for active targeting: physico-chemical characterization of paclitaxel-loaded anti-HER2 immunonanoparticles and in vitro functional studies on target cells.

Paclitaxel (Tx)-loaded anti-HER2 immunonanoparticles (NPs-Tx-HER) were prepared by the covalent coupling of humanized monoclonal anti-HER2 antibodies (trastuzumab, Herceptin) to Tx-loaded poly (dl-lactic acid) nanoparticles (NPs-Tx) for the active targeting of tumor cells that overexpress HER2 receptors. The physico-chemical properties of NPs-Tx-HER were compared to unloaded immunonanoparticles (NPs-HER) to assess the influence of the drug on anti-HER2 coupling to the NP surface. The immunoreactivity of sulfo-MBS activated anti-HER2 mAbs and the in vitro efficacy of NPs-Tx-HER were tested on SKOV-3 ovarian cancer cells that overexpress HER2 antigens. Tx-loaded nanoparticles (NPs-Tx) obtained by a salting-out method had a size of 171+/-22 nm (P.I.=0.1) and an encapsulation efficiency of about of 78+/-10%, which corresponded to a drug loading of 7.8+/-0.8% (w/w). NPs-Tx were then thiolated and conjugated to activated anti-HER2 mAbs to obtain immunonanoparticles of 237+/-43 nm (P.I.=0.2). The influence of the activation step on the immunoreactivity of the mAbs was tested on SKOV-3 cells using 125I-radiolabeled mAbs, and the activity of the anti-HER2 mAbs was minimally affected after sulfo-MBS functionalization. Approximately 270 molecules of anti-HER2 mAbs were bound per nanoparticle. NPs-Tx-HER exhibited a zeta potential of 0.2+/-0.1 mV. The physico-chemical properties of the Tx-loaded immunonanoparticles were very similar to unloaded immunonanoparticles, suggesting that the encapsulation of the drug did not influence the coupling of the mAbs to the NPs. No drug loss was observed during the preparation process. DSC analysis showed that encapsulated Tx is in an amorphous or disordered-crystalline phase. These results suggest that Tx is entrapped in the polymeric matrix and not adsorbed to the surface of the NPs. In vitro studies on SKOV-3 ovarian cancer cells demonstrated the greater cytotoxic effect of NPs-Tx-HER compared to other Tx formulations. The results showed that at 1 ng Tx/ml, the viability of cells incubated with drug encapsulated in NP-Tx-HER was lower (77.32+/-5.48%) than the viability of cells incubated in NPs-Tx (97.4+/-12%), immunonanoparticles coated with Mabthera, as irrelevant mAb (NPs-Tx-RIT) (93.8+/-12%) or free drug (92.3+/-9.3%).