Tumour proliferation is promoted by an intratumoral metabolic symbiosis in which lactate from stromal cells fuels energy generation in the oxygenated domain of the tumour. Furthermore, empirical data show that tumour cells adopt an intermediate metabolic state between lactate respiration and glycolysis. This study models the metabolic symbiosis in the tumour through the formalism of evolutionary game theory. Our game model of metabolic symbiosis in cancer considers two types of tumour cells, hypoxic and oxygenated, while glucose and lactate are considered as the two main sources of energy within the tumour. The model confirms the presence of multiple intermediate stable states and hybrid energy strategies in the tumour. It predicts that nonlinear interaction between two subpopulations leads to tumour metabolic critical transitions and that tumours can obtain different intermediate states between glycolysis and respiration which can be regulated by the genomic mutation rate. The model can apply in the epithelial–stromal metabolic decoupling therapy. Metabolic dynamics within a tumour build on these population interactions –5. TumouTumour cells alter their metabolic patterns compared with those of normal cells and use both glycolysis and respiration . This dylactate-shuttle interaction between epithelial cancer cells and stromal fibroblast cells and proposes an evolving tumour ecosystem based on the notion of interacting adaptive cells.Furthermore, it has been suggested that separating intracellular pathways and intercellular interaction fails to capture a realistic pattern of tumour metabolism. Indeed, in most tumours both the cancer-producing pathways  and intercellular communication network evolve in tandem. However, intercellular signalling such as lactate secretion in some tumour microenvironments still needs more careful study and thus has attracted significant interest in recent years ,10–14. OAdenosine triphosphate (ATP) production pathways have been a subject of game theoretical approaches –18, and We use a framework defined by replicator dynamics equations ,22 which2.et al. [Warburg effect [In 1926, Warburg et al.  addresseg effect ,25.reverse Warburg effect [However, recent studies have demonstrated that the Warburg effect only characterizes a portion of the tumour metabolism happening in the hypoxic domain of the tumour. Some hypoxic cells use glycolysis and produce lactate as a metabolic product whereas oxygenated cells use the lactate to generate ATP . Thus, ag effect .Figure This metabolic symbiosis, which promotes tumour progression, is often observed between cancer cells and stroma –32. For 3.Evolutionary game theory provides a systematic explanation for tumour structure as an outcome of the cellular decision process ,34. StraTo apply this theory to tumour metabolism coupling requires definition of a game that includes players and actions in tumour metabolism. The metabolic symbiosis between the two subpopulations can be put in the framework of a two-player, two-action normal game. The tumour microenvironment roughly consists of two main domains, one domain is adjacent to the vascular system (the oxygenated cell population) and the other domain has less available oxygen (the hypoxic cell population). Hypoxic and oxygenated cell populations are the two players in a normal game.It is known that cells can use different sources of energy such as fatty acids, glutamine , monocarL. The energy production by oxygenated or hypoxic cells using glucose is indicated by Go or as Gh, respectively. If two oxygenated or hypoxic cells meet while both are using glucose, then the glucose energy source divides between each of them as Go/2 or Gh/2, respectively. It is also assumed that the only source of the lactate in the tumour is provided by the hypoxic cells' glycolysis process.The reward matrix of the game is based on the ATP per mole production, which reflects the cellular metabolic rate. It is known that the oxygenated cells—not the hypoxic cells—can use lactate for energy generation, the value of which is defined as The general reward matrix for the two-player, two-action tumour metabolism game is shown in unilaterally deviating from the equilibrium. This means that neither of the two subpopulations can gain more energy by cheating and changing their strategy unilaterally.The Nash equilibrium is a central concept in game theory. A strategy profile forms a Nash equilibrium if no player can increase its expected reward by L > Go/2 [L for oxygenated cells and Gh for hypoxic cells , whereas for T → ∞ the cell's strategy is completely random (pure exploration). The tumour cells demonstrate a wide range of exploration in phenotypic states. The T-value changes during different stages of cancer development or at different stages of therapy. Genetic and epigenetic mutations are among the possible cellular biological factors which can determine the T-value.The crosstalk between cell proliferation signal transduction and cellular energy metabolism is critical for cell survival in a changing environment. Cell energy metabolism alterations usually come with changes in cellular proliferation and vice versa ,38. To cApplying the genomic mutation rate in the evolutionary modelling. Tumorigenesis can promote by abnormality in mutation rates within the genome. For example, in hereditary cancers genomic instability is already present in precancerous tissues [T. tissues , which lT-value reflects the rate of function-altering mutations and genomic instability during tumour progression. There is usually an increase in the genetic/epigenetic mutation rate during the progression of carcinomas [T is to assess tumour MSI and length distribution of the microsatellite.To address this cancer hallmark, our model assumes that the rcinomas . Many of4.1.i be the phenotype with the energy pathway that uses energy source i where i = 1,2, … ,n, and ir is the observed metabolic rate that has a direct proportional relation with ATP production through pathway i and frequency of population or strategy i is ix, then it has been proved [Here, we are specifically focused on the temporal evolution of tumour states. Let n proved  that thei increases with a rate proportional to the overall efficiency of that strategy, whereas the second term describes the cells' tendency to explore across possible energy pathways.The dynamics explains how strategic optimization is reached in an ecosystem through cell proliferation success. The first term in equation (4.2) demonstrates that the probability of selecting energy metabolism X and Y that are adapting concurrently. Let A and B be the two reward matrices: ija and ijb are the rewards of the X and Y population when one selects i and the other population selects j. This reward can be in the form of biological pay-off, such as the cellular metabolic rate. Furthermore, let x = , y = , Now assume there are two types of populations ir with the expected pay-off using the reward matrix, which yieldsAy)i and (Bx)i are the i element of the vectors Ay and Bx that determine the expected rewards of the population X and Y, respectively. Tx and Ty represent the exploration rates in the metabolic pathways of population X and Y, respectively.The learning dynamics in a two-player scenario case is obtained from equation (4.2) by replacing xT = yT = 0 are also the Nash equilibrium of a game [T-value. We study the outcomes of the dynamical system equations (4.3) and (4.4) when xT and yT are not zero.It is important to note that the stable rest points for replicator system equations (4.3) and (4.4) at f a game . One canTo examine a typical metabolic environment, empirical data were used from studies that report typical values of ATP production in the tumour oxygenated and hypoxic cells, using either lactate or glucose for energy metabolism ,36,37.A isB isSubstituting these empirical values in the reward matrix in 5.Critical transitions describe sudden changes in the outcome of dynamical systems when an underlying process parameter slightly changes. Many natural ecosystems may suddenly switch to different stable states  as a resxT and yT values. The bifurcation graphs in figures Equations 4.3) and (4.4) show critical transitions with regard to  and , which corresponds to high glycolysis.urcation b, which Critical transitions in oxygenated tumour cells.xT (a), oxygenated cancer cells are in the lower branch with high lactate respiration. In a certain range of Tx the dynamics have bistable equilibrium .ells.Tx a, oxygenilibrium b,c.d, oxygenated cancer cells mainly use glucose respiration. In this case, the hypoxic cells might lose their survival advantage in the tumour.Finally, in figure 45.1.xT and yT are not independent. A domain of parameters (xT and yT) demonstrate bistability in the dynamics equilibrium. This leads to different zones in the parameter space of xT and yT. xT and yT.The model shows that metabolic transitions lead to different classes of tumour with possibly different therapeutic plans. The critical values of strong metabolic coupling, if at least half of the tumour cells participate in metabolic symbiosis, and we consider it a weak metabolic coupling if less than half of the tumour cells participate in metabolic symbiosis. In the bistable domain of We consider a tumour metabolic state as a xT, yT) in There is a domain of  and activation of the lactate respiration pathway are common aberrations in adenocarcinomas. Epithelial cancer cells induce the surrounding stroma to express MCT4 for lactate efflux. The level of metabolic cooperation can be tested by measuring the lactate transportation between cells that is mediated by MCTs, especially MCT1 and MCT4, which are highly expressed in cancer cells. For example, in both oestrogen receptor-positive and oestrogen receptor-negative breast cancer tumours, the high MCT4 expression is associated with more aggressive tumours. Furthermore, cancer epithelial cells upregulate the expression of MCT1 as a transporter for lactate uptake .The lactate dehydrogenase-A (LDH-A) enzyme catalyses the conversion of pyruvate and lactate, and its upregulation is associated with aggressive tumour outcomes. Emperical results show that tumour cells are susceptible to LDH-A inhibition . Thus, Lc, where the cancer cell population can alter from a high lactate uptake state to a high glucose respiration state, thus the therapeutic outcome would be a sudden decrease in tumour cancer cell metabolic coupling which can lead to decreased tumorigenesis. Using the bifurcation graphs in Any method of lactate downregulation such as MCT1,4 or LDH-A inhibition therapies can be designed for the tumour state similar to the case in figure 66.This study applied the mathematical formalism of evolutionary game theory to demonstrate that cancer metabolism might show critical transitions over certain ranges of biological conditions. We used the Warburg effect and reverse Warburg effect to define a tumour metabolic game between oxygenated and hypoxic tumour cells. Dynamic modelling results show the coexistence of two stable states (bistability) in tumour ecology. Our model shows that a simple intercellular signal such as lactate secretion in some tumour microenvironments can induce a critical transition between high and low levels of tumour glucose consumption. Our co-evolving dynamics also can classify different tumour cells, which provides useful hints during stromal–epithelial cancer cell metabolic decoupling therapy.a priori. In addition, we assumed that the glucose level, lactose level and blood vasculature remain constant and the hypoxic–oxygenated population changes only by intramural interactions. Therefore, one can extend this work by investigating the metabolic changes as a result of glucose/lactate level variations, considering lipid metabolism and also blood vasculature development—angiogenesis—in the tumour.We acknowledge that a Boltzmann-like mechanism is a simplified version of the cell behaviour, but it can be incorporated into experimental designs for future studies on the interaction between cellular energy and proliferation pathways. One of our model limitations is the assumption of the ecosystem with two different phenotypic cells—hypoxic and oxygenated—as In addition, lactic acid can also be detrimental to the cancer cells. In this study, we consider the lactate acid contribution to the oxygenated cells as an energy fuel. One can also consider the detrimental role of the lactate, as the lactate acid concentration passes a threshold value and becomes toxic for the neighbouring cells. On the other hand, a threshold proportion of lactate secretion might be needed before the emergence of functional symbiosis.therapeutic window for sensitive tumour cells [This study shows that tumour cells in certain ranges of metabolic exploration rate can obtain alternative stable states. Our model prediction is consistent with empirical data on the mixed energy metabolism of cancer cells. Breaking the metabolic coupling in tumours may have therapeutic benefits. However, one of the major challenges is to find a ur cells ,48. To t


In a previous study we observed that a fluid challenge of 4 ml/Kg is adequate to raise the mean systemic filling pressure (Pmsf) and to increase venous return in fluid responsive patients.The objective of this study is to describe the changes on microcirculation after a fluid challenge and describe the relationship with the changes on Pmsf. Our hypothesis is that changes in Pmsf could induce changes on the number of perfused vessels (PVD).plus monitor . Pmsf was measured using the stop-flow arterial venous equilibrium pressure method (1). Sublingual microcirculation was recorded with CytoCam . Data was recorded at baseline, immediately after the fluid infusion, 5 and 10 minutes after the end of 4 ml/Kg of crystalloids infused in 5 minutes. At each time at least three sublingual microcirculation videos were acquired to assess total vessel density (TVD), microcirculatory flow index (MFI), proportion of perfused vessel (PPV), perfused vessel density (PVD) and microcirculatory heterogeneity index (MHI). Analysis was performed with AVA software v. 3.2 . Responders are defined by an increase in CO greater than 10%.24 post-operative cardiac patients were prospectively enrolled. Arterial pressure and central venous pressure was measured invasively. Cardiac output (CO) was measured with a non-calibrated LiDCOp= 0.59) or Pmsf .Changes on haemodynamics and microcirculatory indices are presented in table Despite the changes in Pmsf and macrohaemodynamics, a fluid challenge does not improve microcirculation in patients after cardiac surgery.


Using simulated data, we show that using dTAV to optimize the parameter values for an established parametric realignment algorithm improved its efficacy and, therefore, reduced the jitter of neuronal responses. By removing the jitter more effectively and, therefore, enabling more accurate estimation of neuronal responses, dTAV can improve analysis and interpretation of the neural responses.Neuronal responses to sensory stimuli or neuronal responses related to behaviour are often extracted by averaging neuronal activity over large number of experimental trials. Such trial-averaging is carried out to reduce noise and to diminish the influence of other signals unrelated to the corresponding stimulus or behaviour. However, if the recorded neuronal responses are jittered in time with respect to the corresponding stimulus or behaviour, averaging over trials may distort the estimation of the underlying neuronal response. Temporal jitter between single trial neural responses can be partially or completely removed using realignment algorithms. Here, we present a measure, named difference of time-averaged variance ( Many neurophysiological studies are investigating neuronal responses to external events. These studies range from simple stimulus evoked neuronal responses in the corresponding primary sensory areas, e.g. neuronal responses to light flashes in the primary visual cortex , to neurSeveral attempts have been made to realign the jittered neural responses using realignment algorithms , 10. By Typically, parametric realignment algorithms offer a selection of different parameter values, potentially providing greater flexibility in term of neural responses for which the algorithm can be applied to reduce the jitter. However, selecting parameter values suitable for a particular neural response without having accurate knowledge of the response itself can be difficult. Specifically, in situations where the neural recordings are noisy and jittered, accurately estimating the shape of the neural response may not be possible.dTAV) can be used as a measure of jitter reduction. This property is crucial for the operation of a parametric realignment algorithm as it can be used to identify a set of parameter values for which the algorithm performed an accurate realignment. We demonstrate this property by comparing the jitter reduction obtained by a parametric algorithm for a range of parameter values versus the jitter reduction obtained using parameter values selected by dTAV from the same range. This analysis was performed using an established parametric realignment algorithm [Here, we demonstrate that the reduction of variability across trials . Second, we present the motivation behind the design of the lgorithm , 10. Finr(t) evoked at response onset times ti plus the Gaussian white noise signal η:ση is the standard deviation of the Gaussian white noise process and N is a normal distribution with a mean of μ and a standard deviation of σ. After recording the neuronal signal and if internal neuronal response triggers are known, one can estimate the neuronal response by calculating the response-triggered average M is the number of responses used to calculate the average; ηi(t) is the noise in i-th trial; and E for expectation, V for variance, r(t) in the presence of the noise is given by:χ2 distribution [We assume that the neuronal signal is a superposition of neuronal responses ribution :(M−1)V^M being large, the χ2 distribution can be approximated by a normal distribution using the following transformation [For a large number of degrees of freedom, i.e. ormation :a∼χk2⇒aUsing this transformation, tEi and the onset of the neuronal response ti may not be constant and can be regarded as a stochastic process. In our model, the difference between these two time points is modelled by a Gaussian distribution:μJ and σJ are the mean and the standard deviation of the distribution. If one has access to event times only, as it is the case in a real experiment where the time points ti when the brain triggers a response are unknown, one can estimate the neuronal response by calculating the event-triggered sample mean ση= 0). The sample variance of Experiments are usually designed in such a way that the studied neuronal responses are elicited by events, e.g. sensory stimuli or certain types of behaviour. However, the onset times of the neuronal responses are not known, since these are triggered internally by the brain. The time shift between an event ηi(t) in a way given by The first term of ti—tEi, we can use a Taylor series expansion to express For normally distributed and small jitters M, we can use transformation (6) and obtain:For large r(t + tEi—ti) and the noise η are not correlated, the expectation of Since Using Eqs σJ and the sample variance of the event-triggered response σJ values, e.g. σJ’ and σJ”, is larger than the other. If σJ’ > σJ”, the corresponding expectations of the Our intention was to design a quantitative measure of jitter that could be calculated without knowing the internal triggers of neural responses. ti. A possible approach minimizes the variance of the stimulus triggered response TAV:To optimize the alignment, it is necessary to reduce the amount of jitter without the knowledge of the real neuronal response triggers ^(rJ(t))  at one pvariance . HoweverTAV, thereby increasing the reliability of the measure. On the other hand, integrating over periods of time where the neuronal response is small compared to the noise or completely absent may not increase the reliability of TAV but instead increase the variance of TAV. This trade-off means that the integration window across which the variance is averaged should neither be too long nor too short.As discussed before, averaging over time can reduce the variance of the TAV, dTAV, as a measure of jitter reduction:In this study, we used the difference of We used dTAV to optimize parameters of our realignment algorithm by assuming the following is true:dTAV is largest. While Eqs If dTAV is a reliable measure of the reduction in jitter, we performed a set of simulations, each composed of 2000 repetitions of an experiment composed of 100 trials. The neuronal responses were simulated as mono-phasic and bi-phasic functions composed from Gaussian functions , defined as the ratio of the maximum absolute value of the neuronal response and the standard deviation of the noise ση. We used ση values that yielded SNR values of 0.03, 0.05, 0.08, 0.13, 0.20, 0.32, 0.50, 0.79, 1.26 and 2.00 for both mono and bi-phasic responses.Each simulation was performed by selecting a combination of σJ’ = 60ms before the realignment. This initial dataset was compared to datasets with jitter standard deviations σJ” ranging from 0ms (no jitter) to 120ms (doubled jitter) which represented the dataset after the realignment. dTAV was then calculated for each combination of k, σJ”, ση and TI.We used these simulations to emulate an experiment where the jitter standard deviation of the dataset was dTAV values varied across different orders of magnitude for different ση and TI values. We therefore normalized dTAV values by dividing them by the maximum of the absolute value of the 1st and 99th percentile.X-th percentile operator acting over variables a and b; and max is the maximum value operator. The normalized dTAV (ndTAV) was binned in 50 equally wide bins spanning the space from -1 to 1. Binned values were used to calculate the probability of jitter reduction, ndTAV values, while keeping ση and TI constant. To show how the reliability of dTAV as a measure of jitter reduction depends on SNR and TI, we calculated the ndTAV and TI for each value of SNR separately. We also calculated the joint probabilities for each combination of ση and TI values, Ranges of dTAV-based optimization can improve realignment results obtained using previously published MaxCorr algorithm [N trials, N(N-1)/2 crosscorrelations CXij for all possible trial pairs i and j and time lags (λi- λj) up to half of the trial length are calculated. Second, a parabolic function Fij(λi- λj) is fitted to the crosscorrelation between the i-th and the j-th trial around the time lag (λi- λj)MAX for which the crosscorrelation is at maximum.We tested whether lgorithm , 10. TheFij(λi- λj) are fitted using a neighbourhood (λi- λj)MAX of several tens of milliseconds. In the last step, all parabolic functions are summed up to derive a new function F, which is quadratic in all of its variables and, therefore, has a unique global maximum  MAX. The values of  MAX are calculated by solving the system of linear equations obtained by applying partial derivatives to the parabolic function F.  are used to realign the trials relative to the first trial. MaxCorr algorithm was applied to the same signal used to evaluate the realignment of the dTAV algorithm and we evaluated jitter reduction metrics (σJ’—σJ”) / σJ’ for both algorithms for comparison.Parabolic functions The implementation of the MaxCorr algorithm in the FIND Matlab toolbox  providesλMAX). Cross-correletaion is calculated only up to the provided value of the parameter thereby enforcing that all (λi- λj)MAX values stay within the range of the parameter.Maximum allowed correlation time lag (ΔFij(λi- λj).Processing of the cross-correlation coefficients. The user can choose whether or not to apply a natural logarithm to the cross-correlation coefficients before fitting the parabolic functions Normalization of cross-correlation coefficients. The user can chose the type of cross-correlation coefficient normalization between: (i) no normalization, which leads to a linear drop of coefficients for larger time lags; (ii) unbiased normalization, which corrects for the linear drop of coeffiecints; and (iii) normalization to autocorrelation, which divides the coefficients with a squared root of a product of autocorrelations of both signals.NREP), each time reducing ΔλMAX by half.Iterative jitter reduction. The user can chose to repeat the whole MaxCorr realignment process multiple times  / σJ’. After all permutations of the selected parameter values and option settings were used, we selected the (σJ’—σJ”) / σJ’ for which the dTAV was the highest.Different settings of these parameters and options can improve the jitter reduction of a MaxCorr algorithm when applied to a specific dataset. However, a principal way of determining which parameter settings should be used for a given dataset has not yet been investigated. We tested whether dTAV–based optimization of parameters can improve its efficacy when performing realignment of simulated single trial neural responses.We used previously published MaxCorr realignment algorithm , 10 to irM) and bi-phasic response (rB); and (iii) the distribution of the temporal jitters between a Gaussian distribution with zero mean and 0.1s standard deviation and an uniform distribution spanning -0.2s and 0.2s. For each type of simulated experiments, we performed 100 simulations. In each trial, the single channel neuronal response to an arbitrary stimulus was recorded at 1KHz.We performed 16 types of simulated experiments that differed according to: (i) the number of trials in each experiment between 20, 50, 100 and 200 trials; (ii) the type of the neural response between mono-phasic (rM) and bi-phasic neural responses (rB) were modelled to resemble reported neurophysiological responses as follows (Mono-phasic ( follows :rM(t)={Neural responses were simulated in the following way:ση: 31.62, 19.95, 12.59, 7.94, 5.01, 3.16, 2.00, 1.26, 0.79 and 0.50, which corresponded to SNRs of 0.03, 0.05, 0.08, 0.13, 0.20, 0.32, 0.50, 0.79, 1.26 and 2.00. Temporal distances between the stimulus times were drawn from a Gaussian distribution with a mean of 10s and a standard deviation of 10s. To keep neuronal responses from overlapping, temporal distances below 3s were redrawn. To avoid occasional very large jitters, all jitters with an absolute value above 0.3s were redrawn. The range of the uniform distribution was chosen to roughly contain 95% of the jitters of the Gaussian distribution.Noise in the recordings was simulated as additive Gaussian noise with zero mean and different standard deviations λMAX = 50ms, 100ms, 200ms, 400ms and 800ms; (ii) processing of the cross-correlation coefficients: “lin” (no processing) and “log” ; (iii) normalization of cross-correlation coefficients: “none”, “unbiased” and “coeff” ; (iv) NREP = 1 and 3. dTAV was calculated using an integration time TI of 350ms.We optimized parameters and options over the following range of parameter values / range of options: (i) Δnd order symmetric Savitzky-Golay filters [To correctly simulate the outcome of the experiment, we assumed that the person analysing the data would filter the data using a low-pass filter, given that low-frequencies dominate the simulated neuronal responses. We filtered the simulated recordings using 2 filters , 18 withdTAV on the reduction of the jitter for different SNR levels and integration times TI by simulating 100-trial experiments 2000 times. Results are summarized in Using the model of neuronal responses described in section 2.1, we calculated the dependence of TI = 300ms, selected for presentation in Figs dTAV increased monotonously with the amount of jitter reduction and showed little or no dependence on SNR , the relation between dTAV and jitter reduction became less variable and dTAV became an increasingly good predictor of the amount of jitter reduction. For high SNRs (1.26 and higher), even small differences in dTAV indicated increased jitter reduction with high certainty. This suggests that dTAV is a good predictor of the amount of jitter reduction for sufficiently high SNRs (of about 0.2 and higher) and can be used to optimize the parameters of our realignment algorithm in such cases. The parameter selection based on dTAV will improve with increasing SNR.So far we have computed the probability of jitter reduction given a reduction in response . For SNRdTAV, which was calculated using an integration window starting at TS = 0s and ending at TE = 1s, both in respect to the stimulus times tE. We intentionally did not want to use the results of the dTAV reliability analysis  MaxCorr increased the amount of jitter, rather than decreasing it, except in 200-trial simulations with mono-phasic responses and SNR of 0.20, where the jitter was slightly reduced. For intermediate SNRs  MaxCorr reduced the jitter of responses, with its efficacy improving with the number of trials used for realignment. For high SNRs (SNR of 0.79 and higher), MaxCorr removed more than 83% of jitter from the recorded signal, regardless of the number of trials used, distribution of the jitter and the type of response used in the simulations.dTAV optimization improves the efficacy of MaxCorr algorithm, we compared the jitter reduction obtained by dTAV-optimized MaxCorr with the maximum and the median of jitter reductions obtained using MaxCorr with all parameter sets used for dTAV-optimization, as specified in section 2.3. dTAV will contribute to the selection of parameter values (i) only if the jitter reduction is higher than the median jitter reduction, which is in some way analogous to random selection of parameter values, and (ii) only if the selected parameters can be used to obtain a large portion of the maximum jitter reduction value.To demonstrate that the dTAV optimization of MaxCorr’s options and parameters reduced the jitter, jitter reduction was higher than the median jitter reduction . For SNR of 0.32 and a monophasic response, the recovery was 42% and 50% for 20-trial simulations for Gaussian and uniform distributions, respectively, and then increased with the increasing number of trials in the experiment up to 84% and 85% for 200-trial simulations for Gaussian and uniform distributions. For a biphasic response and SNR of 0.32 and for lower SNRs for both response types, the recovery is either low or there is even an increase of the jitter standard deviation after the realignment.For all SNRs for which eduction . The prodTAV optimization selects efficient sets of parameter values, we counted the percentage of parameter value combinations for which the jitter reduction was lower than that obtained by using dTAV optimization, and called it dTAV percentile. For all SNRs for which dTAV optimization of MaxCorr’s options and parameters reduced the jitter, dTAV percentile was higher than 62% , confirming the property that more trials improve the robustness of the dTAV optimization. In addition, dTAV percentiles for SNR of 0.50 and higher were higher for biphasic neural response , implying that dTAV optimization improves realignment to a larger extent for a more complex neural responses.To further show that than 62%  insets. dTAV), that can be calculated without knowing the internal triggers of neural responses. We derived the relationship between dTAV and the precise amount of misalignment, as measured by the standard deviation of the temporal jitter between the trials. Using simulations, we showed that dTAV can be used as a measure of misalignment for a wide range of signal-to-noise ratios (SNRs). In addition, we showed that dTAV can be used to optimize the parameters of an established MaxCorr realignment algorithm designed to increase the temporal alignment of single-trial neural responses. MaxCorr realignment algorithm was applied to simulated sets of mono and bi-phasic neural responses. We showed that dTAV optimization of parameter values of the MaxCorr realignment algorithm leads to substantial efficacy improvements for noise levels up to two and three times larger than the neuronal response amplitudes for mono and biphasic neural response, respectively.We presented and evaluated a measure for the amount of temporal misalignment between the set of single trial neural responses, called difference of time-averaged variance  and for both mono and bi-phasic neuronal responses, suggests that the dTAV by exchanging the time variable by the corresponding variable , thus enabling the effective use of parametric realignment algorithms.Our simulations assumed that the recorded neuronal signal is a continuously modulated signal, which is valid for example for local field potentials, electro-corticographic signals (ECoG), electro-/magnetoencephalographic recordings (EEG/MEG) as well as for optical calcium imaging, near-infrared spectroscopy (NIRS) and functional magnetic resonance imaging (fMRI) signals. Furthermore, our measure can also be applied to spike trains by estimating instantaneous neuronal firing from the spike times , 20 and dTAV was defined for one channel of neural signals only. Nonetheless, the measure can easily be extended to multiple channels by summing up dTAV of individual channels. MaxCorr algorithm has already been implemented for multiple channels [dTAV for MaxCorr parameter optimization in the case of multi-channel neural recordings can be easily achieved.In our study, channels , 10. ThedTAV. This can be achieved by defining admissible values for each parameter  and scan all possible combinations. If the number of admissible values for each parameter is very high, this process becomes computationally demanding and, therefore, may be time consuming. In our simulations we obtained good realignments within a reasonable timeframe (less than an hour), by optimizing the parameters across a limited number of values. Indeed, our results show (e.g. Figs We show that the performance of MaxCorr algorithm can be improved by testing different parameter sets and calculating In general, realignment algorithms can be used to improve the analysis of neurophysiological experiments by improving the estimation of neuronal responses. The obvious case is the estimation of the neuronal response by calculating trial averages. As shown in our example , even ifAdditionally, brain-machine interface systems that detect events based on neuronal recordings –33 may bdTAV measure of misalignment can be used to improve the performance of realignment algorithms when applied on simulated neuronal responses for response waveforms commonly observed in neurophysiological recordings and noise levels higher than the neuronal response itself. Hence, the application of the dTAV measure and the realignment algorithms that use it to optimize their parameter values can improve analysis and interpretation of neuronal responses and improve the performance of asynchronous detection of events from neuronal recordings.In summary, we showed that the


Purpose. To describe the enhanced depth imaging optical coherence tomography (EDI-OCT) findings in a patient with an extra macula choroidal melanoma before and after treatment. Methods. Observational case report. Results. A 45 year-old Caucasian male patient was referred to retina clinic for management of choroidal melanoma. Examination revealed a nasal choroidal melanoma while EDI-OCT illustrated subfoveal fluid pocket with elongated shaggy photoreceptors distant and separate from the tumor. The patient was treated with plaque brachytherapy and intravitreal bevacizumab. One week after plaque removal, there was a dramatic reduction in the shaggy photoreceptors. Conclusion. Choroidal melanomas have effects that are not localized to the area of the tumor. This loculated pocket of subretinal fluid and coinciding changes to photoreceptor morphology may be related to global changes in choroidal function or release of tumor related cytokines. The term “shaggy photoreceptors” has been used to describe distinct morphological changes, specifically swelling and elongation of the outer retina as seen on enhanced depth imaging-optical coherence tomography (EDI-OCT). This finding was first described in central serous chorioretinopathy in 2008  and in sA 45 year-old Caucasian man presented with a one-month history of painless decrease in vision, photopsia, and metamorphopsia. The patient's best-corrected visual acuity was 20/300 OD and 20/20 OS. On dilated exam, he was found to have a medium sized nasal choroidal melanoma and large inferior serous retinal detachment in his right eye . The patThree weeks after diagnosis, an 18 mm radioactive plaque was placed on the right eye and was removed 5 days later. Intravitreal bevacizumab  was given at the time of plaque placement. At the one-week postoperative office visit following plaque removal, best-corrected visual acuity had improved to 20/125 OD. EDI-OCT of the macula showed persistent subfoveal fluid, resolution of subretinal lipofuscin precipitates, and dramatic volume reduction in shaggy photoreceptors . At the µm in the area of subretinal fluid versus the norm of 30 µm in unaffected eyes [We report a case of a peripheral nasal choroidal melanoma not involving the macula that produced distant subfoveal fluid and shaggy photoreceptors demonstrated by EDI-OCT. Shaggy photoreceptors appear elongated and irregular. It has been hypothesized that morphological change is due to an incompletely understood effect that the subretinal fluid has on the photoreceptors. This feature was initially described in central serous chorioretinopathy, where the mean length of photoreceptor outer segments measuring 50 ted eyes . More reIn this case, the subretinal fluid and the shaggy photoreceptors are observed in a localized pocket under the fovea and distant from the tumor itself. The etiology of this fluid is unclear although its presence is potentially indicative of some combination of changes to choroidal permeability, changes to retinal pigment epithelial function, and changes to circulating cytokines related to or produced by the tumor. As there was a serous retinal detachment adjacent to the choroidal melanoma, the subretinal fluid may be explained by tracking from the tumor itself. During I-125 plaque placement, intravitreal bevacizumab was administered, resulting in resolution of the subretinal fluid and improvement in the appearance of the photoreceptors at the two-week postoperative assessment. After four months the shaggy photoreceptors were again present, in association with subfoveal fluid.The reduction in volume of the shaggy photoreceptors may be related either to the effect of intravitreal bevacizumab administration or to the brachytherapy. Both treatments can result in reduction of subretinal fluid associated with melanoma, and the resolution of the subretinal fluid may be correlated with normalization of photoreceptor appearance. There appears to be a spectrum of photoreceptor changes in association with subretinal fluid. The acute presence of subretinal fluid in this setting results in photoreceptor cellular edema which leads to the appearance of shaggy photoreceptors. When the subretinal fluid is chronically present, the photoreceptor layer atrophies. The loss of the this layer evident on spectral domain OCT, and it has been thought that the shaggy photoreceptors became more granulated and eventually shed , 4.Improvement of shaggy photoreceptors has been reported in patients treated for choroidal metastases  and has


Diamond aggregates were added to the experimental capsule set-up in order to separate the fluid from the solid residue and enable in situ analysis of the quenched solute by LA-ICP-MS. The chlorine content of forsterite and enstatite was measured by electron microprobe, and the nature of hydrous defects was investigated by infrared spectroscopy. Partition coefficients show similar incompatibility for Cl in forsterite and enstatite, with DClfo/fl = 0.0012 ± 0.0006, DClen/fl = 0.0018 ± 0.0008 and DClfo/en = 1.43 ± 0.71. The values determined for mineral/fluid partitioning are very similar to previously determined values for mineral/melt. Applying the new mineral/fluid partition coefficients to fluids in subduction zones, a contribution between 0.15% and 20% of the total chlorine from the nominally anhydrous minerals is estimated.Cl partition coefficients between forsterite, enstatite and coexisting Cl-bearing aqueous fluids were determined in a series of high pressure and temperature piston cylinder experiments at 2 GPa between 900 and 1300 °C in the system MgO–SiO−1 that are characteristic for hydroxyl point defects associated with trace Ti substitutions, and strongly suggest that the TiO2 content of the system can influence the chlorine and OH incorporation via the stabilization of Ti-clinohumite-like point defects. The water contents for coexisting forsterite and enstatite in some runs were determined using unpolarized IR spectra and calculated water partition coefficients Infrared spectra of experimental forsterite show absorption bands at 3525 and 3572 cm The transfer of chlorine in subduction zones will depend on its relative partitioning between mineral–fluid, mineral–melt, and fluid–melt. At high pressure aqueous fluids and hydrous melts tend to be completely miscible and a distinction between them cannot be made. The critical pressure in the simple system MgO–SiO>10 GPa, , but in >10 GPa,  it range>10 GPa, .2O–MgCl2 at 1100 °C and 2.6 GPa are in the range 10−3–10−5 . A combination of electron microprobe and LA-ICP-MS was used to analyze Cl in experimental phases and FTIR to characterize OH bearing defects in forsterite and enstatite. Furthermore, the water contents for coexisting forsterite and enstatite were determined and the influence of Ti and F on Cl and H22.12–H2O–NaCl–BaO–C±TiO2±Al2O3±CaCl2±MgF2 powders with varying chlorine content from 0.45 to 11 wt.%  kindly provided by Servsix GmbH , and with 20–35 mg of the starting material powder. The capsule was welded while cooled in water, and the weight of the capsule was compared before and after welding to ensure that no water was lost during welding. The sealed capsule was squeezed to a final cylindrical shape with a length of 5.5–6.5 mm and then held at 120 °C for 24 h, in order to check whether the capsule was sealed. Care has to be taken that: (1) the volume of the fluid or the melt exceeds the volume of the pore space in the diamond layer to ensure homogeneous pressure distribution within the capsule; (2) the experimental charge with its diamond layer is placed in the hotspot of the experimental assembly to prevent unwanted crystal growth within the diamond trap  at a rate of 15 °C/min and the pressure was increased during heating until the final pressure was reached. To grow relatively large, high quality crystals necessary for FTIR analyses, synthesis experiments involved different thermal regimes (fT) and held for the duration of the run. (b) The sample was held to iT for 1 h and then slowly cooled to fT at a rate between 6 and 50 °C/h and held at this temperature for the requested time. (c) Olivine and enstatite seeds were added to the starting mixture and then run was performed using the cooling strategy (b) described above. (d) The sample was cooled from iT to fT and then immediately quenched. (e) The sample was held at iT for 1 h and then slowly cooled to an intermediate temperature (mT), held at this temperature for 24–33 h, and then cooled at fT and held for the requested time. Using method (b) and (e), crystals in excess of 300 μm in length were synthesized. The aim of the different strategies was to understand if the growth rate influences the chlorine incorporation into the crystals. Strategy (d) implies a total experimental time 4–5 times shorter than that of most of the other experiments, thus providing a proof for equilibrium conditions. The runs were terminated by switching off the power and the experiments were quenched rapidly (∼50 °C/s). After gradual decompression, the charge was removed from the assembly. To check for potential leakage the recovered capsule was weighed and pierced. When pierced the pH of the extruding fluid was checked with indicator paper, the checked fluid was in all cases neutral (pH 7), indicating that the possibly dissolved alkalis are balanced by chlorine. Subsequently, the pierced capsule was dried in an oven at 120 °C  and weighed again. Usually, the weight loss upon drying corresponds to 90–100% of the initial water content. Experimental run times were 6–100 h at or above 1100 °C and 1 week for runs at 900 °C. Some experiments were conducted without a diamond trap in the Pt-capsule in order to leave a larger volume for the run products, which were subsequently used for FTIR measurements.Experiments were carried out in an end-loaded piston–cylinder apparatus at 2 GPa and temperatures in the range of 900–1300 °C for durations between 6 h and 1 week at Innsbruck University. From the outside to the inside, concentric cylindrical assemblies consist of a lead foil wrapped around a talc cell, a Pyrex cylinder, a graphite furnace, and inner pieces of crushable alumina sleeves. Run temperature was controlled to within ∼1 °C of the set point using Pt/Pt regimes . (a) TheAll recovered capsules were embedded in epoxy and ground by hand. As soon as the trap became visible, the sample was impregnated with epoxy to prevent destruction during preparation. Later, the capsule was ground until its maximum cross section was exposed.2.32.3.1The electron microprobe measurement on forsterite and enstatite were carried out using a Jeol Superprobe 8100 at Innsbruck University. The crystals were analyzed with an electron beam energy of 15 keV, a beam current of 10 nA, a focused beam, and counting times of 20 s , 120 s (Ca in forsterite). Subsequently, Ba, Ti, Na, Cl, Al, and F were measured with a beam current of 400 nA, and 240 s counting time. BSE images of the samples are reported in 2.3.223Na, 24Mg, 25Mg, 27Al, 28Si, 29Si, 42Ca, 44Ca, 47Ti, 48Ti and 137Ba. Internal standardization required for element concentration calculations was based on the Ba concentrations (the most incompatible element in this study). A synthetic Cl-bearing basaltic glass was synthesized at 1 atm and 1400 °C and used as a calibration standard for Cl. The composition of the synthetic glass was analyzed by microprobe  molar ratio increases from 0 to 0.1, and ∼200–250 °C for an increase from 0 to 0.2 (2O) molar ratio in the range 0.10–0.26 most of the experiments reported here can be considered being subsolidus. For a few runs (Cl-14 to Cl-17) that have a Cl/(Cl + H2O) molar ratio in the range 0.01–0.05, it cannot be excluded that melting occurred in an early stage of the run. More constraints for the potential presence of a melt in the experiments with the lowest Cl/(Cl + H2O) molar ratios can be placed comparing the textures of the experimental charges. Textures of representative run products are shown in 2O) molar ratios (⩽0.05) cooled from 1400 °C or held directly at the final temperature (2O) molar ratios (⩾0.1)  a–d, g, i2O) molar ratios was observed, which is interpreted to indicate the absence of a melt in all performed experiments.The small amount of quench phase i is inte3.2100) coexists with enstatite. Forsterite shows a maximum Cl content of 133 ± 63 ppm and coexisting enstatite has a Cl content of 119 ± 38 ppm.Run products were mainly composed of forsterite, enstatite, salt and aqueous fluid. Individual phase assemblages are listed in 3.32O) molar ratio of the fluid. Calculated chlorine partition coefficients  are listed in The composition of the aqueous fluid coexisting with forsterite and enstatite was determined with LA-ICP-MS . The aqu2 molar ratios of the fluids vs. the Cl/(Cl + H2O) molar ratios of the starting materials  are reported in 2 content of the fluid is around 2000 ppm  and the coexisting fluid and the calculated partition coefficients for chlorine  DClforsterite/fluid is close to ∼10−4 and ∼10−3 for systems with Al and F. The monotonous decreases of DClforsterite/fluid with increasing salinity could indicate that experiments with more than 5 wt.% chlorine in the fluid were outside the region of application of Henry’s law. Therefore only partition coefficients derived from experiments with less than 5 wt.% chlorine in the fluid are considered for further interpretation. The lack of a significant change in DClforsterite/fluid with salinity higher than 5 wt.% for run times varying from 6 to 39 h at 1200 °C is interpreted to reflect equilibrium among minerals and the aqueous fluid. No significant effect of temperature is recognized in the calculated DClforsterite/fluid from experiments at different temperatures. However, at 1100 °C DClforsterite/fluid increases for Ti-bearing forsterite by a factor of ∼1.3 with respect to Ti-free forsterite, and at 900 °C DClforsterite/fluid increases by a factor of ∼4.5 with respect to the value calculated for the run at 1300 °C. The possible reason is discussed in Section The results for forsterite/fluid partitioning at 1200 °C a show a −3 if the chlorine content of the fluid is less than 5 wt.%. With salinity of the fluid higher than 5 wt.% DClenstatite/fluid tend to decrease to ∼10−4. No recognizable dependence on temperature is observed. Enstatites synthesized at lower temperature (900 °C) show DClenstatite/fluid values (5 × 10−4) comparable to those synthesized in presence of aluminum or fluorine. In general, both for forsterite and enstatite the DClmineral/fluid values calculated in systems with Ca, Al or F are a factor 3–5 higher compared to values derived from the system free in Ca, Al and F.The chlorine content of enstatite at a temperature of 1200 °C is shown in DClforsterite/enstatite values. The average DClforsterite/enstatite is 1.43 ± 0.71 implying no significant preference for chlorine in forsterite compared to enstatite.DClforsterite/enstatite values similar to those of most of the experiments performed with the duration of 24–36 h. These observations suggest that chemical equilibrium was attained even at shortest run times.The partition coefficients between forsterite and enstatite are also plotted in 3.5Unpolarized IR spectra collected on forsterite and enstatite from selected runs are shown in −1. Minor absorption bands are observed at 3612 cm−1 and at 3325 and 3352 cm−1. Ti-free forsterite displays also the absorption band at 3612 cm−1, but the band at 3572 cm−1 tends to disappear and is substituted by two weak peaks at ∼3568 and ∼3580 cm−1. The thickness and the average orientation of the samples are comparable and hence the intensity of absorption is related to the amount of OH. The observed spectra indicate that Ti-bearing forsterite incorporates significantly higher amounts of OH compared to Ti-free forsterite. Furthermore, Ti-forsterite from the runs at 900 and 1100 °C exhibits stronger OH-bands than the Ti-forsterite derived from the run at 1300 °C.Ti-bearing forsterite crystals  exhibit −1  show enhanced OH-contents relative to those performed with minor amounts  or without (Cl-20) TiO2. For example, samples with the Ti-rich composition have stronger absorption bands at 3525 and 3572 cm−1 than other samples at comparable temperature  show no significant difference in OH defect population confirming that crystal growth rate does not have a significant effect on the concentration of OH defects incorporated into synthetic enstatite .The relative uniformity of the spectra for enstatite  over the entire range of investigated temperature and composition, strongly suggest that lamellae of monoclinic pyroxene  similar 2O that is considerably lower than the ∼210 wt ppm for the run carried out at 1300 °C  are higher than those for Ti-free olivine (DH2Ofo/en = 0.08 at 1100 °C and DH2Ofo/en ∼ 0.01 at 900 °C), suggesting that the effect of Ti incorporation into forsterite significantly increases the solubility of water in forsterite giving a large increase in DH2Ofo/en.Water partition coefficients between coexisting forsterite and enstatite are between 0.01 and 0.5. The data provide information on the effect of temperature and Ti impurities on DH2Ool/opx that vary significantly depending on pressure, temperature, major and trace elements composition. A DH2Ool/opx value = 0.6 was calculated from xenocrysts in a kimberlite , and for DClen/fl is 0.0018 ± 0.0008 (n = 18).Partition coefficients of chlorine between the coexisting phases are presented in DClol/opx of 0.72 ± 0.15 is in close agreement with our results.If we compare our results with experiments from chlorine partition coefficients between basaltic melt and lherzolite minerals for pressures from 0.8 to 2.5 GPa and temperatures from 1265 to 1430 °C by 2O–MgCl2 at 1100 °C and 2.6–3 GPa .Experiments performed in the system forsterite–enstatite–pyrope–H.6–3 GPa  give par2–H2O–NaCl, DClen/startmix at 2.5 GPa and 1150 °C is estimated to be 0.0026  by 4.6n = 4) ppm Cl were measured in synthetic enstatite  ppm were detected in synthetic forsterite , the abundance of chlorine in the mantle is estimated to approximately 1 ppm (DClperidotite/fluid is very similar to DClfluid/melt = 2.0 ± 0.9 (this study) and DClfluid/melt = 3.0 ± 0.6 calculated from the data of From geochemical considerations  at shallow levels in the Loihi magma chamber . The chl


Mixed adenoneuroendocrine carcinoma (MANEC) is a rare tumour of the gastrointestinal tract that consists of a dual adenocarcinomatous and neuroendocrine differentiation, each component representing at least 30% of the tumour. We report a case of a 68-year-old man who presented with two-month history of postprandial pain and vomiting. Gastric endoscopy revealed a polypoid mass in the lower part of the oesophagus. In contrast to the majority of these tumours, this biopsy was immunohistochemically positive for chromogranin A, and synaptophysin and Ki-67 index was 50% and the tumour was diagnosed as poorly differentiated neuroendocrine carcinoma of the oesophagus. The patient underwent surgery and lower oesophagus resection was performed. Based on the histopathology and immunohistochemistry of the tumour in the oesophagogastrectomy specimen, a mixed adenoneuroendocrine carcinoma (MANEC) was diagnosed. The objective of this case report is to advocate for the focus on the MANEC diagnosis as such patients need to be referred to a centre of excellence with expertise in NET tumours, to have the correct diagnostic work-up, treatment, and secondary diagnostic procedures performed at progression, as this will have paramount influence of the choice of treatment. Mixed adenoneuroendocrine carcinoma (MANEC) in the oesophagus is an extremely rare cancer diagnosis. However, correct diagnosis is important for guiding the choice of treatment and prognosis.Planocellular and adenocarcinomas are the most common tumours of the oesophagus constituting 95% of primary epithelial carcinomas. Other rare tumours of the oesophagus include lymphomas, sarcomas, melanomas, and neuroendocrine tumours.The purpose of this case report is to raise awareness of this diagnosis because it affects the treatment strategy and follow-up.A 68-year-old man was referred to our department for endoscopy with a 2-month history of postprandial pain and vomiting. The patient had a medical history of hypertension, type II diabetes, and hypercholesterolemia.Oesophagogastroduodenoscopy revealed a polypoid mass in the distal oesophagus, which was approximately 35 cm from the dental arch and extended to the gastrooesophageal junction (GEJ) . The hisA PET-CT scan showed increased fluorodeoxyglucose (FDG) uptake, corresponding to the tumour in the distal oesophagus. Somatostatin receptor imaging using Gallium DOTANOC PET/CT revealed slightly positive uptake above liver, corresponding to a primary tumour in the oesophagus.The patient underwent macroradical transthoracic oesophagus resection without oncological pretreatment. Histopathological examination of the specimen revealed a highly differentiated adenocarcinoma and neuroendocrine carcinoma (NEN G3) , alternaMANEC is a tumour that consists of two components, an adenocarcinoma and neuroendocrine tumour of the oesophagus, which is most often a neuroendocrine carcinoma. Referring to the WHO criteria, each component may represent at least 30% of the tumour . MANEC iThe use of somatostatin receptor scintigraphy (SRI) is appropriate for diagnosis and in follow-up of these tumours, but the use of SRI is not established. According to Ilett et al.  37–71% oThe evidence for determining MANEC prognosis and treatment is limited , 4, 5. MSurgical resection is the primary treatment strategy in localized disease. Preoperative oncological treatment is experimental and may be used as a downstaging strategy for primarily nonresectable tumours. Adjuvant oncological treatment is poorly understood. According to the “Nordic guidelines for neuroendocrine neoplasms in 2014” , 7, cispFor patients with metastatic GEP NEC (gastroenteropancreatic-neuroendocrine neoplasms) and MANEC, rapid initiation treatment with palliative chemotherapy is important . If the In the case of recurrence or progression, it is important to assess patients with both FDG PET/CT and Gallium DOTANOC PET/CT. Furthermore, rebiopsy may be relevant because MANEC is a 2-component tumour with a potentially visible mixed response. The conclusion is that a diagnosis of MANEC should be considered when examining patients with a suspected tumour in the upper GI because a MANEC diagnosis has consequences for the treatment strategy and follow-up.


Objectives. Biologically guided radiotherapy needs an understanding of how different functional imaging techniques interact and link together. We analyse three functional imaging techniques that can be useful tools for achieving this objective. Materials and Methods. The three different imaging modalities from one selected patient are ADC maps, DCE-MRI, and 18F-FDG PET/CT, because they are widely used and give a great amount of complementary information. We show the relationship between these three datasets and evaluate them as markers for tumour response or hypoxia marker. Thus, vascularization measured using DCE-MRI parameters can determine tumour hypoxia, and ADC maps can be used for evaluating tumour response. Results. ADC and DCE-MRI include information from 18F-FDG, as glucose metabolism is associated with hypoxia and tumour cell density, although 18F-FDG includes more information about the malignancy of the tumour. The main disadvantage of ADC maps is the distortion, and we used only low distorted regions, and extracellular volume calculated from DCE-MRI can be considered equivalent to ADC in well-vascularized areas. Conclusion. A dataset for achieving the biologically guided radiotherapy must include a tumour density study and a hypoxia marker. This information can be achieved using only MRI data or only PET/CT studies or mixing both datasets. To this project –36.18F-FDG PET images the glucose consumption of each region. Tumour cells use glycolysis rather than lipolysis as the metabolic process to produce ATP and they use more glucose than normal cells. Glycolysis is a rather inefficient process and therefore large amounts of glucose are needed for cell survival and tumour growth. The PET enhancement  in tumours is due to three different mechanisms: (i) cancer cells produce more ATP outside the mitochondria, even in well-oxygenated conditions (Warburg effect ); (ii) cDW-MRI measures the diffusion of protons in a medium. Its principle is based on the attenuation of the signal according to Stejskal and Tanner's model . Tumour DCE-MRI has been proposed by several authors for treatment monitoring –46 and fktrans parameter in DCE-MRI is related to vascularization and then to hypoxia [ve is related to extracellular volume and in heterogeneously vascularized areas to tumour density [In order to characterize the tumour and to implement new predictive models based on functional imaging data, we must ensure we can extract as much information as possible from the available data. Some of the main parameters to characterize tumour behaviour, along with radiotherapy treatment, must be initial tumour density, hypoxia, malignancy/proliferation, dose to each voxel, and timing of the dose. In this work, attention is focused on showing the relationship between ADC maps, DCE-MRI parameters, dose, and 18-F-FDG PET/CT SUV . Many other types of images can show the main parameters we are interested in modelling  is to create a network for sharing information and for developing predictive individualized models of the tumour response to radiotherapy in patients with head and neck cancer based on in vivo functional data. For this purpose, several studies of MRI and PET/CT were performed. Patients within the ARTFIBio project [pretreatment: MRI study (DCE-MRI + ADC) and PET/CT study (18F-FDG),first control (10–30 Gy): MRI study (DCE-MRI + ADC),second control (30 Gy–60 Gy): MRI study (DCE-MRI + ADC),three months after the treatment: PET/CT and MRI study (DCE-MRI + ADC).For all imaging studies the patient is positioned using the RT immobilisation devices. The geometrical distortion on MRI images and registration process (rigid registration and deformable registration) were checked with an MRI phantom. Regardless, only central slices showing low distortion were analysed. For each patient and each set of images the ADC values, contrast exchange coefficients (Ktrans), SUV, dose, and Hounsfield units (HU) per voxel were recorded of each volume.The aim of ARTFIBio project  with the patients in supine position. Routine T2-weighted, T1, DW-MRI, and DCE-MRI were obtained using the parameters showed in A nonlinear model  was utilktrans, kep, and vb from the modified Tofts model. This method also provided semiquantitative metrics such as area under the curve (AUC) and time to max enhancement.The relationship between all these parameters can be obtained byts model . A 3D vots model , 52 was 0 of the GTV (gross tumour volume) of 3 different patients. The average T10 of these patients (800 ms) was applied when calculating the concentration of the analyzed patient which unfortunately did not have VFA scans themselves.Variable flip angle (VFA) spoiled gradient recalled echo scans at three flip angles variations  were utilized to calculate the voxel by voxel T1The arterial input function (AIF) was chosen in the carotid artery near the base of skull.Whole-body PET/CT scan was carried out from head to thigh, 60 min after intravenous administration of approximately 370 MBq (±10%) of 18F-FDG on a PET/CT scanner  with a 70 cm axial FOV, a 218 × 218 matrix. Study was acquired in 3D mode. The pixel spacing was 5.47 mm with a slice thickness of 3.27 mm. The spatial resolution to 1 cm varies from 3.99 mm to 4.56 mm. PET images were corrected for attenuation, scatter, decay, dead time, random coincidences, and slice sensitivity.To calculate the SUV  for the To reduce image noise a 3 × 3 nearest-neighbour smoothing filter was applied to the DCE-MRI, PET-CT, and ADC images. Deformable registration of the images, with the CT of treatment as reference, was performed using tailored in-house software specifically developed for the ARTFIBio project  and baseKtrans), surrounding necrotic volume , and heterogeneously vascularized tumour  have each been considered separately  needs to be selected.0 in the carotid at the level of the neck , a clear relationship between both datasets is found, indicating that a smaller extracellular volume corresponds to a higher tumour cell density in well-vascularized or heterogeneously vascularized areas but not hypoxic areas.For values of ktrans is the most related to vascularization. Vascularization must be related to oxygenation [Ktrans values, that is, increasing perfusion, SUV values decrease because of the reduction of the Pasteur effect (green dots, Of all the analyzed parameters, genation –59, as Fktrans values, although ADC values appear to be rather constant (blue dots, On the other hand, no clear relationship has been found between ADC map and We have generated the ADC values during the treatment for a heterogeneously vascularized tumour volume. In this case, the delivered dose to achieve an ADC value corresponding to normal tissue is much lower than for badly vascularized voxels. The influence of vascularization/oxygenation in the ADC response can be observed with the DCE-MRI studies, as shown in The results presented have some similarities to those obtained by Atuegwu et al.  and indiThe polarographic electrode has been considered by some authors as the gold standard for measuring tumour hypoxia in vivo , althoug18F-FDG shows different aspects of the tumour behaviour, mainly associated with tumour cell density, malignancy, and oxygenation, and the quotient between ADC and SUV has been proposed as a measurement of malignancy in breast tumours  and in iUsing biomechanical models  that conADC maps appear to be a good option for evaluating tumour response; however their disadvantage is image distortion. Unfortunately, this cannot be corrected using standard deformable registration algorithm, but reversed gradients method looks like a very promising algorithm to solve this problem . It is pMultimodality imaging offers much more information about tumour behaviour than the individual datasets on their own. The relationship between different types of images must be studied in detail in order to establish a minimum set of data required to personalize the radiotherapy treatment and to optimize the treatment for each patient. This could comprise not only a gradient of dose along the treatment, but also different fractionation for each voxel.Multicentre studies can be useful for recruitment of a large number of patients and increase the statistical power of the results, if imaging standards and protocol compliance are followed .Voxel by voxel analysis seems possible if we consider small volumes and undistorted regions from ADC maps or corrected data.


In this cohort study, we explored the relationship between fluid balance, intradialytic hypotension and outcomes in critically ill patients with acute kidney injury (AKI) who received renal replacement therapy (RRT).We analysed prospectively collected registry data on patients older than 16 years who received RRT for at least two days in an intensive care unit at two university-affiliated hospitals. We used multivariable logistic regression to determine the relationship between mean daily fluid balance and intradialytic hypotension, both over seven days following RRT initiation, and the outcomes of hospital mortality and RRT dependence in survivors.In total, 492 patients were included , mean (standard deviation (SD)) age 62.9 (16.3) years); 251 (51.0%) died in hospital. Independent risk factors for mortality were mean daily fluid balance (odds ratio (OR) 1.36 per 1000 mL positive  1.18 to 1.57), intradialytic hypotension ), age (OR 1.15 per five-year increase (95% CI 1.07 to 1.25)), maximum sequential organ failure assessment score on days 1 to 7 (OR 1.21 (95% CI 1.13 to 1.29)), and Charlson comorbidity index (OR 1.28 (95% CI 1.14 to 1.44)); higher baseline creatinine (OR 0.98 per 10 μmol/L (95% CI 0.97 to 0.996)) was associated with lower risk of death. Of 241 hospital survivors, 61 (25.3%) were RRT dependent at discharge. The only independent risk factor for RRT dependence was pre-existing heart failure (OR 3.13 (95% CI 1.46 to 6.74)). Neither mean daily fluid balance nor intradialytic hypotension was associated with RRT dependence in survivors. Associations between these exposures and mortality were similar in sensitivity analyses accounting for immortal time bias and dichotomising mean daily fluid balance as positive or negative. In the subgroup of patients with data on pre-RRT fluid balance, fluid overload at RRT initiation did not modify the association of mean daily fluid balance with mortality.In this cohort of patients with AKI requiring RRT, a more positive mean daily fluid balance and intradialytic hypotension were associated with hospital mortality but not with RRT dependence at hospital discharge in survivors.The online version of this article (doi:10.1186/s13054-014-0624-8) contains supplementary material, which is available to authorized users. Acute kidney injury (AKI) occurs commonly in critical illness, with 4 to 10% of patients admitted to the intensive care unit (ICU) requiring renal replacement therapy (RRT) . In receWe conducted a cohort study to investigate the relationship between fluid balance and outcome in critically ill patients with AKI requiring RRT. We hypothesised that positive fluid balance and intradialytic hypotension during the first seven days after RRT initiation would be associated with both mortality and RRT dependence at hospital discharge.This cohort study used data from the Toronto Acute Kidney Injury registry. The registry is a prospectively collected database of patients who started RRT for AKI at two university-affiliated hospitals  and includes information such as patient demographics, baseline admission characteristics, comorbidities identified in medical records, fluid balance, RRT variables, vital signs during RRT, and outcomes. In both centres, RRT is initiated by the nephrology team in collaboration with the critical care service, according to standard indications; we did not collect data on the specific indication(s) for each patient. Both teams agreed on modality  and fluid management goals.We considered patients aged 16 years or older who received RRT for ≥2 days in an ICU at the two study hospitals  during the time period noted above, and for whom fluid balance data were available for ≥1 day of their ICU stay. Patients were excluded from the registry if RRT was initiated to treat a toxic ingestion, if they had pre-existing end-stage renal disease or had received a renal transplant in the year prior to hospital admission.We defined day 1 as the 24-hour period, or part thereof, on which RRT was initiated. Daily fluid balance was defined as total fluid intake from all sources  minus output . We calculated mean daily fluid balance for the first seven days following RRT initiation, using the number of days with data available if less than seven. Cumulative fluid balance was the total fluid balance between ICU admission and the day of RRT initiation. Fluid overload at RRT commencement was defined as positive cumulative fluid balance >10% of admission weight, as in previous studies ,11. We dOne session of RRT was defined as continuous renal replacement therapy (CRRT) for a 24-hour period or part thereof or one session of either intermittent hemodialysis or sustained low-efficiency dialysis (SLED). Baseline creatinine was defined as premorbid creatinine or the first creatinine during the index hospital admission if premorbid creatinine was unavailable. Intradialytic hypotension was defined for each session of RRT as either the occurrence of mean arterial pressure (MAP) <65 mmHg or a reduction of ≥20% from the starting MAP, consistent with widely used definitions of absolute  and relaFor eligible patients, we abstracted registry data on primary diagnoses, maximum sequential organ failure assessment (SOFA) scores ,16 on daContinuous variables were summarized as mean  or median (quartile 1 (Q1), quartile 3 (Q3)) as appropriate, and discrete variables are presented as frequencies and percentages.P ≤0.2, clinical plausibility, and absence of important multicollinearity (P <0.8). We did not consider interaction terms in the absence of clinically compelling reasons to believe that any covariate would modify the effect of the main exposure variables. We did not consider fluid overload at RRT commencement as an independent variable in the main analyses due to the extent of missing data. Results are expressed as odds ratios (ORs) with 95% confidence intervals (CIs). We assessed the relationship between continuous covariates and log odds of the outcomes graphically and detected no departures from the assumption of linearity.We constructed logistic regression models for the outcomes of hospital mortality and RRT dependence at hospital discharge in survivors, with mean daily fluid balance, intradialytic hypotension and sex forced into both models. Other covariates were assessed using univariable logistic regression models; selection criteria for covariate inclusion in the final models were univariable We anticipated that the problem of immortal time bias would lead to an overestimation of the strength of the associations between the exposures of mean daily fluid balance and intradialytic hypotension and the outcome of hospital mortality. This bias arises because a patient who survives longer has more time to develop a negative fluid balance and intradialytic normotension ,19. We apost hoc logistic regression model in the subgroup of patients with data available to calculate fluid overload. We used the same adjustment variables as in the main model but also adjusted for fluid overload prior to RRT, dichotomised as >10% vs. ≤10%, and the interaction term between mean daily fluid balance on RRT and fluid overload. We did not consider a similar logistic regression model for RRT dependence in hospital survivors in fluid overloaded patients due to few outcome events.We conducted two additional sensitivity analyses. First, we analysed the same two outcomes with the mean daily fluid balance dichotomised as positive or negative. Second, to investigate whether fluid overload at commencement of RRT modified the relationship between mean daily fluid balance and mortality, we constructed a P <0.05 to be statistically significant.Data were recorded in a Microsoft  Access database. All analyses were done using SAS version 9.3  or R version 2.15.3 . All tests are two-sided; we considered The Research Ethics Boards of St Michael’s Hospital and Sunnybrook Health Sciences Centre approved this study. In view of the observational nature of the study and measures to maintain confidentiality of patient data, the requirement for individual patient consent was waived.P <0.01), but not between survivors who were RRT dependent at hospital discharge  mL) and those who were not  mL; P = 0.36). Figures A total of 492 patients, of whom 299 (60.8%) were male, met criteria for inclusion in the study Figure . The mearge . In our final multivariable logistic regression model . The associations between mortality and the main exposures of mean daily fluid balance  and intradialytic hypotension  remained similar to the original model (Table Three hundred and eight 62.6%) patients in the cohort had sufficient data to calculate fluid overload at the start of RRT; of those, 125 40.6%) had >10% fluid overload. Compared to patients without fluid overload, those with >10% fluid overload , premorbid health (Charlson comorbidity index), cardiovascular instability , and age, we lacked data on fluid overload at the time of RRT initiation in 40% of the cohort and therefore could not adjust for this covariate without a substantial loss of statistical power. However, sensitivity analysis in patients in whom fluid overload was calculable produced similar results to that in the whole cohort. It is also possible that other unknown confounders influenced both fluid requirements and the outcomes of mortality and RRT dependence in survivors. Immortal time bias was also a potential problem; however, a sensitivity analysis that accounted for the number of days with data available did not materially alter our findings. Residual confounding may have contributed to the surprising findings that higher baseline creatinine was associated with lower risk of death, but not with RRT dependence at discharge. These associations may reflect the occurrence of RRT-dependent AKI with less severe systemic illness among patients with pre-existing chronic kidney disease.Other important limitations relating to the observational nature of this study include the lack of standardised criteria for the initiation of and management of RRT, leading to fluid overload as the primary indication for RRT in some patients, the lack of data on the relative contribution of intake and output to overall fluid balance, and missing data. Other limitations of data restricted the calculation of fluid balance to the first seven days following RRT initiation. Although the endpoints of mortality and RRT dependence at hospital discharge are clinically important, some patients may have been transferred to other acute care hospitals, with less certainly regarding final disposition. We defined intradialytic hypotension to include both absolute and relative hypotension and considered the frequency as a percentage of the total number of RRT sessions during which it occurred. However, similar to other AKI studies that reported details of hypotension , we lackIn summary, our study provides supportive evidence for reduced mortality with a less positive fluid balance after critically ill patients commence RRT. A less positive fluid management strategy does not appear to influence RRT dependence at hospital discharge. Further multicentre cohort investigations, if confirming these findings, would support the design of a randomized trial of an aggressive vs. less aggressive approach to fluid removal in this population.A more positive fluid balance and intradialytic hypotension are associated with increased risk of death in a cohort of critically ill patients undergoing renal replacement therapyFluid balance and intradialytic hypotension are not associated with requirement for renal replacement therapy at the time of hospital dischargeClinicians may consider using ultrafiltration to minimise fluid overload in critically ill patients with acute kidney injury receiving renal replacement therapy


Varicella zoster virus (VZV) causes mild self limiting illness in children. Older children and adults may present with atypical manifestations. At present VZV vaccine is not included in national immunization schedule. The study was undertaken to determine the presence of VZV infection in patients with acute febrile illness with rash.Blood samples were collected from patients (n=36) with acute febrile illness with rash in a tertiary care hospital. IgM ELISA was performed according to manufacturer’s instructions . Kit standards were used to obtain a standard curve. The results of the samples were interpreted from the standard curve.All enrolled patients had fever with rash without evidence of vesicles. Majority, (n=29) were adults and 18 were male. Of the 14 (38.9%) that were positive by IgM ELISA for VZV, 5 (35.7%) belonged to the age group of 19-30 years, 4 (28.5%) were < 18 years and 2 (14.3%) in age group 51-60 years. All patients had normal platelet count except 9, who had mildly deranged platelet count (>1.5 to < 2 lakh /cumm). All sera that were tested were negative for Dengue NS1 antigen, IgM and IgG antibody capture ELISA .VZV may present with atypical manifestation. Non progression of rash to vesicle may warrant testing for VZV and other viruses in the absence of positive Dengue serology.


Based upon promising preclinical studies, a clinical trial was performed in which encapsulated cells overexpressing cytochrome P450 enzyme isoform 2B1 were implanted around malignant mammary tumours arising spontaneously in dogs. The dogs were then given cyclophosphamide, one of the standard chemotherapeutic agents used for the treatment of mammary tumours. The dogs were assessed for a number of clinical parameters as well as for reduction in tumour size. The treatment was well tolerated with no evidence of adverse reactions or side effects being associated with the administration of the encapsulated cells. Reductions in tumour size of more than 50% were observed for 6 out of the 11 tumours analysed while 5 tumours showing minor responses, i.e. stable disease. In contrast, the tumours that received cyclophosphamide alone showed only stable disease. Taken together, this data suggests that encapsulated cytochrome P450 expressing cells combined with chemotherapy may be useful in the local treatment of a number of dog mammary tumours and support the performance of further clinical studies to evaluate this new treatment. The development and pathological features of spontaneously occurring mammary tumours in dogs are remarkably similar to breast cancer in women Cyclophosphamide is a standard chemotherapeutic agent that is commonly used to treat breast cancer in women We have developed a system for the local conversion of chemotherapeutic agents such as cyclophosphamide (and the related agent ifosfamide) to their toxic metabolites, rather than the usual conversion that occurs primarily in the liver The DNA of all cells including the cells in the capsule are alkylated by the activated toxic prodrug metabolite phosphoramide mustard, but the cells are only killed when they try to divide. The cells in the capsule, and also non-tumour cells, are not dividing so they are not killed. We have shown that forced growth arrest of non-encapsulated cells indeed protects them from a self-killing effect in vitro Promising data have been obtained from this treatment protocol in both animal models of pancreatic adenocarcinoma The current paper for the first time presents the primary data from the clinical protocol that was previously published A total of 16 female dogs were enrolled in the study. The dogs were divided into two groups: a study group and a control group. The control group included 6 female dogs marked as: I, II, III, IV, V, and VI. The study group included 10 female dogs marked as: VII, VIII, IX, X, XI, XII, XIII, XIV, XV, and XVI. The mean age of the dogs that took part in the clinical trial was 8.8 years (range: 5–14 years) and the median age was 9 years. Due to the different breed of dogs included in the study, the body weight varied in the range: 9–43 kg with a mean of 18.8 kg and median of 17.8 kg. None of the 16 dogs included in the trial were spayed. Although most of the dogs (n = 13) had only a single detectable mammary tumour, four animals  had two tumours. Three of these dogs  were in the study group; whereas the dog marked IV was included in the control group.3 and not more than 250 cm3, 3) absence of regional metastatic lymphadenopathy, 4) absence of radiographic and ultrasonographic signs compatible with metastatic spread. The studies were reviewed and approved by the Ethics Committee of the University of Agriculture in Lublin (Poland) (Permission Number 53/2004).The suitability of the animals for testing was based on the following criteria: 1) a cytological diagnosis confirming malignancy, 2) mammary tumour sizes being not less than 1 cmA cell clone of the human embryonic kidney cell line, 293, transfected with a cytochrome P450 2B1 expression construct was used for encapsulation (CapCell) as previously described and characterised 3 tumour, and the encapsulated cells were injected at five sites around the tumour using a G17 butterfly needle and 2 ml syringe Encapsulation of the cells was achieved as previously described Although the protocol stipulated that the encapsulated cells should be applied to only one tumour in those animals with two or more tumours, the owner of one dog (XII) only agreed to the treatment if both tumours received the encapsulated cells. The other dogs with more than one tumour and not belonging to the control group  were only treated with encapsulated cells at one tumour site, in line with the clinical trial protocol The dosage and administration schedule for cyclophosphamide was based on the scheme proposed by Löscher and co-workers 3. Measurements were taken before treatment on the day of implanting encapsulated cells (baseline) and on days 9, 22, 29 and 57.Measurements of tumour dimensions were performed with a ruler and the largest three dimensional extensions of the tumour were used to calculate the tumour volume in cmThe general and preliminary criteria used to assess the safety of the treatment were haematological investigation, blood chemistry and urinalysis. The haematological investigation included determination of haemoglobin level, leucocyte and trombocyte count. Blood chemistry comprised determination of aspartate aminotransferase (AST), alanine aminotransferase (ALT) activity and blood urea nitrogen (BUN), creatinine, bilirubin and total protein levels. Urine was analysed by means of strips and, if necessary, confirmed by routine methods.The final result of the treatment efficacy was evaluated based on the dynamics of tumour size change according to one of the following categories: 1. Complete remission (CR), 2. Partial remission (PR) – regression of the cancer by at least 50% of the initial tumour mass, 3. Stabilization of the disease (SD) – regression of the tumour by less than 50% or its enlargement by less than 25% of the initial tumour mass, 4. Progression of the disease (PD) – increase of the tumour size by at least 25% or new neoplastic foci.Biopsies, using a 0.9 mm diameter needle with a 20 ml syringe, were obtained from several sites from all tumours  and transferred to slides. The cell smear was immediately fixed with Fixocyt  and stained with haematoxylin and eosin (H&E) and Papanicolaou stain. Resected tumour samples  destined for histopathology were fixed in 10% neutral buffered formalin, followed by embedding in paraffin. Sections 5 µm thick were prepared and stained with H&E. Tumours were classified after routine histopathological examination according to the WHO classification system 10 dogs (13 tumours) formed the study group and were treated with both capsules and cyclophosphamide.6 dogs formed the control group and were treated only with cyclophosphamide. The control group included additionally tumours in the XIII, and IX dogs without the CapCell implantation, considered an internal control site for the other tumour in these females; these tumours were taken into consideration in assessing treatment efficiency.Each dog in the study group received injections of the encapsulated cells around the tumour. The peri-tumour injections and the administration of the capsules were well tolerated, with no study medication related serious adverse events being observed in any of the dogs treated with the encapsulated cells over the entire observation period (56 days).9/L [reference ranges 200–500×109/L]). In the following female dogs: V, VI, IX, XI, XII, and XVI a lower platelet count was found during the 2nd and 3rd visit, and in female dogs: III, VIII, XIII, XIV and XV during the 2nd, 3rd and 4th visit. In 5 female dogs  thrombocytopenia persisted throughout the therapy.After administration of the encapsulated cells, the dogs received cyclophosphamide at a dose of 7 mg/kg. Some of the dogs  showed reduced appetite for 2–3 days after infusion of cyclophosphamide, a common side-effect of treatment with this chemotherapeutic agent Some animals  showed urine abnormalities such as the presence of erythrocytes and/or traces of protein. Serum creatinine and BUN levels were slightly elevated in one dog, XV . AST levels rose in 6 to 10 hours and remained above the physiological range (1–37 U) for about 4 days in all dogs of the study group. The activity of ALT was also slightly elevated in most animals (reference ranges 4–91 U), although in all cases the abnormalities for all of these laboratory parameters subsided within a few days after treatment, and were self resolving.Thus, in summary, the application of encapsulated cells in these 10 dogs (13 tumours) was well tolerated with no obvious side-effects. The subsequent treatment with cyclophosphamide resulted in mild side effects including thrombocytopenia which has been described previously as a side effect of cyclophosphamide treatment in dogs 3 to 244.8 cm3   participating in this clinical trial. The baseline tumour measurement (i.e. before treatment) ranged from 1.0 cm44.8 cm3  and 4 wi growth) . This da growth) . The larMore than one tumour was present in three dogs from the study treatment group . Both tumours from dog XII were treated with encapsulated cells followed by cyclophosphamide and, interestingly, both tumours showed a similar reduction in tumour size (62 and 65%). It is also worthy of note that the encapsulated cell treated XIII tumour showed a reduction in size of 70%, whereas the second tumour in this dog, which did not receive encapsulated cells, only showed a 14% reduction, comparable to the reduction of 19% in tumour size in dogs from the control group.In each of the 16 female dogs that qualified for inclusion in the study, the cytological picture of biopsy specimens obtained from all the tumours indicated malignancy of the neoplastic process. At the microscopic level, the most frequently registered were the presence of necrotic masses, neutrophilic granulocytes, deformed epithelial cells and epithelial cells with a varying degree of nuclear atypia , 3.Twenty tumours  were histopathologically tested.Malignant tumours were found in all dogs. In 9 cases the diagnosis was complex carcinoma, in 4 cases simple tubulopapillary carcinoma, in 3 cases simple tubulopapillary carcinoma, partially solid, in 3 cases spindle cell carcinoma, and in one case the diagnosis was in situ carcinoma of the comedo type. An overview is given in The encapsulated cells could be found in the tissue surrounding the resected tumours. This tissue did not show any evidence of an inflammatory reaction, although there was a slight fibrosis as demonstrated by the presence of fibroblasts and thin fibres of connective tissue. A weak infiltration of monocytes and macrophages could be observed at the border between a group of cell filled capsules and the surrounding tissue . Thus, tIn this study, the safety of introducing encapsulated cells capable of converting the chemotherapeutic agent, cyclophosphamide, to tumour toxic metabolites was examined. A secondary objective was to determine whether this local conversion had a better therapeutic effect than the usual cyclophosphamide treatment that relies on conversion of the chemotherapeutic agent by cytochrome P450 enzymes expressed in the liver.Although side effects of the treatment could be observed, none of these were due to the implantation of encapsulated cells but rather to the administration of cyclophosphamide at the standard dose, since they were also experienced by the control animals receiving cyclophosphamide only. Histological sections of material resected from the tumour and surrounding areas showed that the encapsulated cells were well tolerated, with no or only mild immune reactions. These findings are in line with those from previous studies, both from a clinical phase I/II trial for pancreatic cancer in human patients In studies performed in patients with inoperable pancreatic cancer, the microcapsules were angiographically placed in the blood vessels leading to the tumour. In the mammary cancer study reported here the microcapsules were injected peri-tumorally. Since similar tumour shrinkages were observed in both studies, this argues strongly that the results observed in the human clinical trial were not caused by vessels being blocked by microcapsules, but rather from the focusing of the toxic effect of cyclophosphamide on the tumour. In follow-up studies, similarly to the study reported here, no allergic reactions or signs of pancreatitis were observed in these patients. These results demonstrate that microcapsules effectively protect the encapsulated cells against attack by the host's immune system whilst allowing free diffusion of nutrients from the external environment into, and the release of cellular metabolites out of, the microcapsule. In this study, peri-tumoral injection of encapsulated cells, followed by systemic application of cyclophosphamide, was used in a neo-adjuvant setting to shrink tumours before they were surgically removed. This improves the success of the surgical excision since less skin has to be removed, hence improving wound closing and thus the healing process The use of encapsulated drug converting cells for improved, local chemotherapy is a safe treatment strategy. The use of this strategy results in improved anti-tumour effects as measured by tumour reductions, suggesting that it may be useful for the treatment of mammary tumours in human and animal patients. Thus, further clinical studies are warranted.