Redundancies and correlations in the responses of sensory neurons may seem to waste neural resources, but they can also carry cues about structured stimuli and may help the brain to correct for response errors. To investigate the effect of stimulus structure on redundancy in retina, we measured simultaneous responses from populations of retinal ganglion cells presented with natural and artificial stimuli that varied greatly in correlation structure; these stimuli and recordings are publicly available online. Responding to spatio-temporally structured stimuli such as natural movies, pairs of ganglion cells were modestly more correlated than in response to white noise checkerboards, but they were much less correlated than predicted by a non-adapting functional model of retinal response. Meanwhile, responding to stimuli with purely spatial correlations, pairs of ganglion cells showed increased correlations consistent with a static, non-adapting receptive field and nonlinearity. We found that in response to spatio-temporally correlated stimuli, ganglion cells had faster temporal kernels and tended to have stronger surrounds. These properties of individual cells, along with gain changes that opposed changes in effective contrast at the ganglion cell input, largely explained the pattern of pairwise correlations across stimuli where receptive field measurements were possible. An influential theory of early sensory processing argues that sensory circuits should conserve scarce resources in their outputs by reducing correlations present in their inputs. Measuring simultaneous responses from large numbers of retinal ganglion cells responding to widely different classes of visual stimuli, we find that output correlations increase when we present stimuli with spatial, but not temporal, correlations. On the other hand, we find evidence that retina adjusts to spatio-temporal structure so that retinal output correlations change less than input correlations would predict. Changes in the receptive field properties of individual cells, along with gain changes, largely explain this relative constancy of correlations over the population. An influential theory of early sensory processing argues that sensory circuits should conserve scarce resources in their outputs by reducing correlations present in their inputs Are there mechanisms that might allow the retina to adjust its functional properties when stimulus correlations change? Traditionally, retinal ganglion cells (RGCs) have been described by a fixed linear receptive field followed by a static nonlinearity To test these ideas, we performed a series of experiments in which we presented the retina with several stimuli with varying degrees of spatial and temporal correlations. The retina never fully decorrelated its input; even for the least correlated white noise checkerboard stimuli, some correlations were present between pairs of retinal ganglion cell spike trains. Responding to natural movies, however, output correlations were only moderately increased compared to correlations in responses to white noise checkerboards, despite the dramatic difference in input-induced correlations. Specifically, the differences in output correlations were much less than those predicted by a non-adapting linear-nonlinear functional model responding to these stimuli. We found a similar result for spatio-temporal exponentially correlated stimuli, with an even smaller change in output correlations. In this way, pairwise output correlations change by a relatively small amount as compared to the expected input-induced change in response to stimuli that span a broad range of spatio-temporal correlations. Stimuli with only spatial correlations, on the other hand, produced increases in output correlations similar to the input-induced changes predicted by a static, non-adapting functional model. In the extreme case, for temporally uncorrelated full-field flicker, the output correlation increased substantially. These results suggest a key role for temporal processing in maintaining the level of output correlations. Indeed, we observed a robustly faster response timecourse and a modest skew towards stronger inhibitory surrounds in response to spatio-temporally correlated stimuli. These changes were sufficient to largely explain the observed relative suppression of pairwise correlations in the retinal output for those experimental conditions where receptive field measurements could be made.We used a multi-electrode array to measure simultaneous responses from groups of For preliminary analyses, we measured the spike-triggered average (STA) from each ganglion cell's response to white noise. The resulting receptive fields typically gave good coverage of the sampled visual field  and clusTo probe the effect of stimulus correlation on ganglion cell response properties in detail, we applied a standard functional model, the linear-nonlinear (LN) model. In this model, the visual stimulus is filtered with a linear kernel that represents the spatio-temporal receptive field (STRF) of the cell. The filter output is then passed through a nonlinear transfer function to generate a predicted firing rate. The nonlinearity encompasses thresholding and saturation, as well as any gain on the linear response. For white noise stimuli, the STA is a good estimator of the STRF We computed the correlation coefficient between spike trains (binned at 33 ms) for all pairs of simultaneously recorded neurons. In response to natural movies, correlations between most pairs of cells increased in magnitude when compared with the correlations between the same pairs when viewing white noise . We quanBecause the retinal ganglion cell output is a highly transformed representation of its input, it is not trivial to formulate a naïve expectation for the magnitude of output correlation given an input correlation. In particular, simply evaluating the input correlation between stimuli at the receptive field centers of a pair of cells provides a misleading picture, since it neglects the spatial extent and possible overlap of receptive fields. We therefore chose to quantify the output correlation expected for a given input in a simple null model: the LN model fit to the white noise responses. This model captures correlation due to receptive field overlap and simple nonlinear processing, while neglecting correlations due to shared circuitry and more complex nonlinear behavior, such as adaptation. For cells which had sufficiently well-estimated white noise LN model parameters . Thus the receptive fields are less well estimated and further studies are needed to verify with confidence the predictive performance of a non-adapting model.For further analysis, we focused on the subset of stimuli shown in As discussed above, we were able to identify the cell classes for many of our recorded neurons. In response to spatio-temporally exponentially correlated noise and natural movies, cell class had a modest effect on output correlations . Cells wWe next sought to determine whether receptive fields adapt to stimulus correlations and whether this adaptation can explain the observed pattern of output correlations. As noted above, we were able to obtain STAs from responses to white noise. STAs computed in response to correlated stimuli, however, will be artificially blurred by the stimulus correlations. To obtain a better estimate of the spatio-temporal receptive field (STRF), we used maximum likelihood estimation to fit a LN model separately for the white and exponentially correlated stimuli Spike trains with sparse, transient firing events tend to be more decorrelated Next, we found separate temporal profiles for the center and surround and computed the latency, measured as time to peak, of each. Surround latencies did not differ between white noise and spatio-temporally exponentially correlated noise , we also measured receptive fields from a separate population of ganglion cells responding to white noise and to a stimulus that was exponentially correlated in space but not in time. In this case, filters did not show a systematic change in power spectra , bottom,ng model .The conventional view of retinal circuitry suggests that adaptive decorrelation arises from stronger or wider surround inhibition during viewing of correlated stimuli. We thus computed the amplitudes of the surround and center components of each neuron's STRFs in both white noise and spatio-temporally exponentially correlated noise. Defining the relative surround strength, emselves . In addiFinally, we examined the gain onse see . Since tFinally, we assessed whether the receptive field changes reported above could account for the observed modest increase in output correlations between white noise and the spatio-temporal exponential stimulus. For experiments using spatio-temporally exponential noise, as discussed above, we measured the adaptation in LN model parameters fit to each stimulus. We then separately examined the effect of adaptation in different parameters on the excess correlations predicted by the LN models. Including adaptation of the linear filters, but not the gain, produced a significantly improved match between the model and the data . Low conOur principal finding is that the mammalian retina maintains a moderate level of output correlation across a wide range of spatio-temporally correlated stimuli ranging from white noise checkerboards (with limited correlations) to natural movies . While the amount of output correlation varies between stimuli, the changes are much less than predicted by a non-adapting linear-nonlinear functional model. Our data also suggest a differential effect of spatial versus temporal correlations on the functional properties of the retinal output. We focused here on spatial variations in our stimuli, but it would be interesting to design future studies to explore the space-time differences more systematically. In response to spatio-temporal exponential noise, where the receptive fields could be estimated, we showed that the relative invariance of output correlations is largely accounted for by the observed changes in the linear receptive field  and by changes in the nonlinear gain (anti-correlated to changes in effective contrast). The latter findings give an interpretation of the results in terms of a conventional functional model (here a linear-nonlinear cascade), but the measurement of output correlations is model-independent.within a channel, with residual correlations across classes signaling to downstream areas relevant relationships between the information in different channels.Classifying cells into classes revealed a slight dependence of excess correlation on cell class: most robustly, opposite polarity ON-OFF pairs showed the greatest increase in correlation magnitude when stimulus correlation increased. Indeed, if the retinal output is split across parallel functional channels, redundancy is likely to be highest within a channel due to shared circuit inputs. It may thus be advantageous, from an information encoding perspective, for decorrelation to act Pitkow and Meister We also found that the gain of retinal ganglion cells responding to correlated stimuli changes with “effective contrast” change in noise correlation with stimulus would provide a different candidate mechanism to account for our data We have focused in the present work on the failure of a non-adapting linear-nonlinear model to capture the relatively small scale of observed excess correlations and have seen that adaptation in the linear filter might remedy this discrepancy. Alternatively, shared circuitry in the population of neurons may be engaged by correlated inputs and require explicit inclusion in any functional model of retinal responses to different classes of correlated stimuli We have focused here on the effects of spatial correlations in an experimental design where we could compare receptive fields computed from responses to two different stimuli. Thus, we used relatively weak exponential correlations to ensure that we were not measuring artifacts of the stimulus correlations themselves. Recovering receptive fields from strongly correlated stimuli can require long recording times. Because our experimental design involved comparisons between several different stimuli, we were only able to recover receptive fields for moderately correlated stimuli. Future work could simply present each stimulus for a longer duration to assess receptive field changes at a population level rather than analyzing multiple stimuli in one experiment. In such experiments with more data from each cell, alternative methods of receptive field estimation such as Maximally Informative Dimensions Finally, it would be interesting to determine the timecourse of the adaptations observed here. Knowing whether a change in stimulus correlations induces changes in receptive fields and output correlations within seconds, tens of seconds, or longer would help to clarify the relationship between processing of correlations and adaptation to other stimulus features such as contrast. Again, the design of our experiments precluded making these measurements – we focused on long segments to measure steady-state processing of correlations, whereas assessing the timecourse of changes requires finer and more systematic sampling of transitions between stimuli.Why would the retina need to adapt, in the behaving animal, to variations in spatial correlations? While natural scenes are scale-invariant on average, the specific correlations do vary depending on the scene and the viewing distance see . Barlow This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, as well as the guidelines of the American Veterinary Medical Association. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania (Permit Number 803091). All surgery was performed under anesthesia, and every effort was made to minimize suffering.We recorded retinal ganglion cells from Hartley guinea pig using a 30-electrode array . After anesthesia with ketamine/xylazine (100/20 mg/kg) and pentobarbital (100 mg/kg), the eye was enucleated and the animal was euthanized by pentobarbital overdose. The eye was hemisected and dark adapted. The retina was separated from the pigment epithelium, mounted on filter paper, and placed onto the electrode array, ganglion cells closest to the electrodes. Extracellular signals were recorded at 10 kHz. The retina was maintained in well-oxygenated bath of Ames' medium at a temperature of http://dx.doi.org/10.5061/dryad.246qg.Data are available from the Dryad Digital Repository: We displayed checkerboard stimuli see  at 30 Hzl scenes . This strelation ; thus, wWe used reverse correlation to compute the spike-triggered average (STA) for each cell responding to white noise. We performed principal component analysis (PCA) on the best-fitting temporal kernels and split cells into two clusters based on the sign of the first component; the clusters were identified as ON and OFF classes based on the sign of their temporal kernels.  for both white and exponentially correlated stimuli, we used publicly available code  and then used gradient ascent to separately fit the gain and offset of a logistic nonlinearity. Since the likelihood function in this case is convex, there was no possibility of local maxima. While we were able to compute unbiased estimates of STRFs from responses to stimuli with exponential correlations, our multiscale and natural movie stimuli were too correlated to estimate unbiased receptive fields with the number of spikes we were able to obtain in a single recording. Maximally Informative Dimensions, an important alternative receptive field estimation method Correlations were measured as the correlation coefficient between pairs of simultaneously recorded neurons. Spike trains were divided into 33 ms bins; we assigned a bin a 1 if it had one or more spikes and a zero otherwise. The results reported above did not change if we used spike counts in each bin rather than binarizing. Indeed, 98% of timebins had one or fewer spikes and less than 0.05% had more than three spikes. We summarized the results by finding the best fit line of the form Such instantaneous correlations are thought to combine slow stimulus-driven effects with fast intrinsic effects due to shared noise We also computed the power spectra of the stimuli, the best-fitting temporal kernels, and the filter outputs (i.e. stimulus power spectra multiplied by filter power spectra). We summarized each power spectrum by computing the total power above 5 Hz divided by the total power below 5 Hz.Given a STRF estimated for one cell under one of the stimulus conditions, we first performed principal component analysis on the timecourses of the individual pixels. From the resulting set of “principal timecourses” we selected the one most similar to the timecourse of the pixel that achieves the peak value in the full STRF. The complete linear filter was collapsed into a single frame by finding the projection of each pixel onto this principal timecourse. This procedure is equivalent to finding the best (least squares) spatio-temporally separable approximation to the STRF: Taking the center defined in this way as a mask for the full STRFs, we summed all pixel values within the center at each time point to generate a temporal profile for the central receptive field. To obtain temporal kernels with greater precision than the 30 Hz time scale of our STRFs, we used cubic spline interpolation with knots spaced every 33 ms. From the interpolated timecourses, we measured the time to peak under each stimulus for the center. In addition, the peak value of this temporal profile was taken to be the center weight of the receptive field. Similar computations yielded the surround time to peak and surround weight. We then quantified the relative surround strength as the ratio of surround weight to center weight.In addition, we measured the gain We used the analysis method described here because it gave the most robustly unbiased results in our simulations (see below), but we also wanted to verify that our results did not change dramatically with slightly different methods  model. We then applied STRF extraction and analysis methods identical to those applied to real data to check that the known LN parameters were extracted in an unbiased fashion. The linear filter was chosen to be spatio-temporally separable, with the temporal component taken from measured ganglion cell responses. The spatial filter was modeled as a difference-of-Gaussians, where the size and strength of the surround Gaussian relative to the center Gaussian were chosen to mimic receptive fields of real neurons. In each simulation, parameters for 100 model neurons were chosen independently. The results are summarized in In our first simulation, the surround radius (relative to center radius) was chosen from a Gaussian distribution with mean 2 and standard deviation 0.3, the relative surround strength from a Gaussian distribution with mean 1 and standard deviation 0.1, and the offset coordinates from Gaussian distributions with mean 0 and standard deviation 2  Center time to peak for a population of non-adapting model neurons, plotted as in B) Model neurons described in (A) show a slight bias toward smaller recovered relative surround strength under correlated noise compared to white noise. (C) Center time to peak is longer for white noise than for correlated noise when the surround only includes non-center pixels whose time courses are positively correlated with the time course of the peak negative pixel. (D) Relative surround strengths is marginally higher for correlated noise than for white noise under the same analysis as in (C). (E) Center time to peak is longer for white noise than for correlated noise when the center is not required to form a single contiguous component. The figure omits a few outliers that lie outside the range of the horizontal axis and have longer time courses under correlated noise. (F) Relative surround strengths is marginally higher for correlated noise than for white noise under the same analysis as in (E).(TIF)Click here for additional data file.Figure S2Correlation measurement controls. (A) As a control on the quality of the non-adapting LN model, we examined the difference between its predicted pairwise correlations under the white noise stimulus (the stimulus to which the model was fit) and the observed correlations. Since the model is a single-neuron model that does not attempt to capture pairwise correlations, we do not expect it to reproduce these correlations perfectly. Nevertheless, many cell pairs are well-predicted, indicating that their correlation is largely due to receptive field overlap. There is, however, a slight tendency for the model to underestimate correlations: this is likely due to its neglect of noise correlations. We note that such a bias will not effect the model's predicted excess correlation, unless it is very different in the two stimulus conditions. But such an effect would represent a form of non-trivial processing in its own right. (B) Our correlation measurements were based on binned spike trains. We measured excess correlation, in the spatiotemporal exponential dataset, for a variety of bin sizes. Its value is largely independent of bin size. Error bars represent (TIF)Click here for additional data file.Figure S3Firing rates are within a physiologically normal range. (A) Firing rates are similar across stimuli. Each cell's average white noise firing rate was subtracted to aid in comparison across experiments. Red line indicates the median for each stimulus. Blue boxes show the middle 50% of values; black boxes, the middle 90%. Gray crosses represent the outlying 10% of cells. Numbers below each box show the number of cells included. (B) Comparison of the excess correlation for each correlated stimulus to the average response (relative to the white noise response) shows that excess correlation is not simply a reflection of firing rate.(TIF)Click here for additional data file.Figure S4Dependence of excess correlation on response polarity and type of scene.  Excess correlation values for natural movie (A) and spatio-temporal exponential (B) stimuli separated by polarity of the recorded pair. Correlated stimuli evoked a greater increase in output correlations for ON-OFF pairs than for ON-ON and OFF-OFF pairs (white bars). This increase was consistent with correlation changes predicted by a non-adapting model for opposite-polarity pairs responding to natural scenes  but was smaller than the expected correlation change in all other cases (gray bars). For the spatio-temporal exponential stimulus, where we could compute white noise and correlated noise receptive fields, the observed changes filter and gain were able to reproduce the smaller change in output correlations relative to input correlations . (C) Excess correlation computed by comparing responses to different natural movies directly. Our natural stimulus ensemble was composed of movies taken in three outdoor settings. During an experiment, movies from these scenes were interspersed. Here we see that output correlation are similar for all three; thus, in the main text we group all natural movie data together.(TIF)Click here for additional data file.Figure S5Dependence of correlation on linear filter overlap for opposite-polarity cell pairs. Correlation coefficient between opposite-polarity cell pairs recorded under a white noise stimulus. The dot product of the two cells' spatiotemporal linear filters is used as a measure of the overlap between areas with the same polarity; a pair of cells whose receptive fields are identical in shape but have opposite polarity will have a strong negative filter dot product. The fact that positive correlation between opposite-polarity pairs' spike trains is strongly associated with positive filter dot products may be due to one cell's receptive field center lying in the other's surround or to differences in the temporal filtering of the two cells such that the RF centers do not overlap temporally.(TIF)Click here for additional data file.Table S1Variants on receptive field analysis method. Adaptation index (AI) in surround strength and center latency measured by variations in our analysis method. Columns labeled “mean,” “std,” and “skew” show the mean, standard deviation, and skewness of the adaptation indices for the indicated analysis; columns labeled “p” show the Standard analysis: Receptive fields were analyzed as presented in the main text. Disconnected center: The center was not required to form a contiguous region. Surround threshold: A threshold criterion was used to find the surround so that only pixels positively correlated with the peak surround pixel were included. No mean subtraction: The mean of the frames after each spike was not subtracted from the STRFs. First principal component: The full STRF was collapsed onto a single frame by projecting onto the first principal timecourse rather than the principal timecourse most similar to the peak pixel. Masks from WN: Center and surround regions measured from white noise were used to analyze STRFs from both stimuli. Masks from CN: Center and surround regions measured from spatio-temporal exponentially correlated noise were used to analyze STRFs from both stimuli.(PDF)Click here for additional data file.Text S1Dependence of output correlation on gain and firing rate. In the main text we found that gain adaptation contributes to decorrelation in a population of LN neurons. In (PDF)Click here for additional data file.Text S2Tests of robustness. To verify that our receptive field results were not an artifact of our specific analysis method, we repeated the analysis with a number of modifications. In (PDF)Click here for additional data file.


We contend that hypertension management research trials (including drug studies) need to elucidate the benefits and risks of drug-lifestyle interaction, to support the priority of lifestyle modification, and that lifestyle modification, rather than drugs, is seen by patients and the public as a priority for health professionals. The inclusion of lifestyle modification strategies in research designs for hypertension drug trials could enhance current research, from trial efficacy to clinical outcome effectiveness, and align hypertension best practices of a range of health professionals with evidence-based knowledge translation.Established standards for first-line hypertension management include lifestyle modification and behavior change. The degree to which and how lifestyle modification is systematically integrated into studies of first-line drug management for hypertension is of methodological and clinical relevance. This study systematically reviewed the methodology of articles from a recent Cochrane review that had been designed to inform first-line medical treatment of hypertension and was representative of high quality established clinical trials in the field. Source articles ( Large-scale randomized controlled clinical trials (RCCTs) have long been considered the gold standard to evaluate treatment approaches to control blood pressure. Two streams of research have emerged: pharmacological and behavioral. Pharmacological research has generally investigated five classes of pharmaceuticals for blood pressure control: ACE inhibitors, angiotensin II receptor blockers, beta-receptor blockers, calcium channel blockers, and thiazide diuretics , 2. BehaAlthough there are several established first-line practice guidelines for the detection and management of hypertension, lifestyle behavior change is considered first line across levels of disease severity and concurrent comorbid risk factors –5. EducaStudies that investigate the outcomes of the interaction of the two research streams, that is, pharmacological and behavioral, are lacking. Given the unequivocal relationships among health behaviors and blood pressure, interactions between health behavior and drug efficacy are conceivable. Pharmacokinetics of hypertension drugs have been reported to be impacted by lifestyle factors . If confThe purpose of this systematic review was to establish how lifestyle modification is integrated into leading established antihypertension drug trials. Of interest were factors including smoking and stress reduction whose links to hypertension have been less well established compared with body weight and inactivity. Based on first-line clinical practice guidelines, the inclusion of lifestyle modification can not only be justified but also argued to be imperative, if related research is to be practice-informed. Specifically, we investigated to what extent and how lifestyle behaviors are systematically integrated into hypertension research paradigms of established hypertension drug trials and the extent to which lifestyle modification is masked by the use of nonspecific terms such as usual care. We contend that attention to lifestyle in the research paradigm of pharmacological studies related to common lifestyle-related conditions such as hypertension would enhance the practical implications and outcomes of such studies in terms of knowledge translation and outcome effectiveness versus simply efficacy and align them with real-world best practice recommendations.The Cochrane Collaboration is an internationally recognized benchmark for high quality information based on best available evidence. Cochrane enables healthcare providers, policy makers, patients, and their advocates to make well-informed decisions about health care –31. TherFor each large clinical drug trial included in this review, Wright and Musini  identifiEach source article was reviewed for “mention of lifestyle interventions” including smoking cessation, diet, weight loss, physical activity and exercise, stress feduction, and moderate alcohol consumption. These lifestyle intervention categories were selected based on the 2012 Canadian Hypertension Education Program recommendations that are reported to be evidence based . “MentioWithin the selected Cochrane review , 24 majoOf the 11 articles that mentioned lifestyle modification (46%), description of the interventions, their specific details and how they were systematically implemented, was lacking  did so only within their secondary sources; however, this methodology was not detailed in the corresponding major publication. In publications from the OSLO drug trial , for exaThe Cochrane review by Wright and Musini  that we A common feature of the source studies was a lack of details of the lifestyle modification interventions. Descriptions within the methods sections were nonspecific, for example, “avoidance or reduction of obesity will be advised” , 15, “enThe lack of consistency observed for prescriptions of lifestyle modification across trials presents further challenges to interpreting the consistency and degree of emphasis that was placed on lifestyle and the degree to which modifiable factors confounded the study outcomes . For example, SHEP  providedAnother factor that could affect the consistency of the lifestyle behavior change prescriptions was the way in which they were administered. Most source studies that provided lifestyle modification advice described it as either “general” or “standardized.” Due to the range of publication dates and the diversity of “standardized” protocols, these terms were ambiguous. Other studies however reported having had doctors or the investigators provide lifestyle modification advice on an individual basis. Given the powerful effects of lifestyle modification on normalizing blood pressure given its inclusion in best hypertension practice, researchers created potential confounding variables within the trials by not standardizing their lifestyle modification prescriptions. Although the MRC-TMH  drug triOnly one drug trial, MRC-TMFH , reporteOne noteworthy source article that mentioned lifestyle factors was the UKPDS  drug triOf interest is the dearth of attention given to stress and its role in hypertension, in hypertension drug studies. Although stress is viewed as contributing to high blood pressure indirectly, autonomic activity is increased with activation of the sympathetically mediated fight/flight/fright mechanism, often the object of hypertensive medication. It could be argued that reducing this backdrop of elevated sympathetic activity might lower a person's threshold for clinical hypertension.Several counterarguments could be posed given this analysis of RCCTs and the inclusion of lifestyle modification. One could argue that by design a RCCT homogenizes variation from known and unknown variables including lifestyle behaviors of drug trial participants thereby negating the need to consider lifestyle factors. Lifestyle modification has been long established as first-line intervention across all classifications of hypertension and concurrent comorbid risk factors, and thus, we believe drug trials need to shift to the next level of methodological sophistication and factor in lifestyle into their base designs. Given the power of lifestyle in preventing and reversing as well as managing hypertension , 2, presA possible limitation of our analysis of the review by Wright and Musini  is that Although we have no reason to believe that comparable review of other trials would result in different findings, replication of other trials may have some benefit.It is worth noting that, conversely, RCCTs examining the effects of lifestyle behavior change on blood pressure would be viewed as methodologically deficient if the confounding effects of medications were not controlled in some respect or built into the design of the trial.Our findings have implications for the design of future studies. Foremost, we recommend that best practice hypertension guidelines need be reflected in hypertension studies; specifically, lifestyle modification strategies need to be a fundamental component of the research design of pharmacological studies on hypertension.


Studies suggest that endogenous field effects may play a role in neuronal oscillations and communication. Non-invasive transcranial electrical stimulation with low-intensity currents can also have direct effects on the underlying cortex as well as distant network effects. While Parkinson’s disease (PD) is amenable to invasive neuromodulation in the basal ganglia by deep brain stimulation (DBS), techniques of non-invasive neuromodulation like transcranial direct current stimulation (tDCS) and transcranial alternating current stimulation (tACS) are being investigated as possible therapies. tDCS and tACS have the potential to influence the abnormal cortical-subcortical network activity that occurs in PD through sub-threshold changes in cortical excitability or through entrainment or disruption of ongoing rhythmic cortical activity. This may allow for the targeting of specific features of the disease involving abnormal oscillatory activity, as well as the enhancement of potential cortical compensation for basal ganglia dysfunction and modulation of cortical plasticity in neurorehabilitation. However, little is currently known about how cortical stimulation will affect subcortical structures, the size of any effect, and the factors of stimulation that will influence these effects. Are transcranial direct current stimulation (tDCS) and transcranial alternating current stimulation (tACS) potential treatment modalities for Parkinson’s disease (PD)?While the foremost treatment for PD continues to be dopaminergic medications, invasive neuromodulation through deep brain stimulation (DBS) has become a mainstay of therapy in selected patients Okun, . As in aAlthough classically considered a disease of the basal ganglia, functional imaging and EEG studies have shown altered cortical activity in the supplementary motor area (SMA), dorsolateral prefrontal cortex (DLPFC), and primary motor cortex (M1) in patients with PD  or sham stimulation (n = 12) while on medication, with stimulation in the tDCS group alternating between the premotor/motor area and prefrontal cortex stimulation. tDCS decreased walking time (the primary outcome) compared to sham one day after stimulation, but only when tested off medications and after exclusion of an outlier in the sham group. Though motor Unified Parkinson’s Disease Rating Scale (UPDRS) and reaction time changes did not differ between groups, upper extremity bradykinesia was significantly improved at all evaluations periods up to three months after stimulation. In addition to motor symptoms, a wide variety of non-motor symptoms occur in PD that are not responsive to levodopa therapy and could potentially be treated with tDCS (Wu et al., The study of the therapeutic potential of tDCS in PD is still largely preliminary, yet with some promising findings (Rothwell, As in tDCS, the literature related to PD using tACS is sparse, though intriguing. tACS at varying frequencies was shown to modulate the rate of force development and peak force in handgrip response to a go/no-go task (Joundi et al., The application of non-invasive applied electric fields provides a potential window through which the dysfunctional subcortical-cortical networks in PD can be accessed and influenced. However, it remains largely speculative how cortical stimulation will affect subcortical structures, what the effect size will be, and the factors of stimulation that will influence these effects. Given the progressive neurodegenerative nature of the disease and the increasing recognition of the full range of symptoms associated with it, the utility of these techniques may be more as adjuncts to other therapies Chen, . This beA better understanding of the mechanisms by which non-invasive electrical stimulation affect neural networks would likely streamline the discovery of any potential therapeutic applications in PD. Yet as we have seen with DBS in PD, our mechanistic understanding can sometimes lag behind successful therapeutic implementation. Further studies will help to clarify factors such as the optimal montages, sites, and intervals of stimulation Paulus, , as wellThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Research on calibration remains a popular line of inquiry. Calibration is the degree of fit between a person’s judgment of performance and his or her actual performance. Given the continued interest in this topic, the questions posed in this article are fruitful directions to pursue to help address gaps in calibration research. In this article, we have identified six research directions that if productively pursued, could greatly expand our knowledge of calibration. The six research directions are: (a) what are the effects of varying the anchoring mechanisms from which calibration judgments are made, (b) how does calibration accuracy differ as a function of incentives and task authenticity, (c) how do students self-report the basis of their calibration judgments, (d) how do group interactions and social comparisons affect calibration accuracy, (e) what is the relation between absolute and relative accuracy, and (f) to what extent does calibration accuracy predict achievement? To help point the way to where we go from here in calibration research, we provide these research questions, propose research methods designed to address them, and identify prior, related studies that have shown promise in leading the way to fill these gaps in the literature. Calibration has been defined as the degree of fit between a person’s judgment of performance and his or her actual performance Keren, . As suchThe broad research literature in educational psychology and the more specific literature on self-regulated learning reveal a growing interest in calibration that is well-warranted. For instance, students studying for a test need to be accurate in monitoring their knowledge acquisition and retention if they hope to successfully control further study. On one hand, students may develop a false sense of their mastery of studied material and overestimate how well they will perform. These students’ positive biases could lead to premature termination of study and place them at risk for failure . In addition, because studies on the effects of incentives on motivation to achieve greater accuracy have been mixed, greater attention needs to be focused on how motivation can be manipulated (research question b).During the performance phase, the learners gain feedback concerning self-control and self-observation, processes that are essential for continued attention to and action on a task. Learners self-explanations about how and why they use their self-observations to self-control and whether those self-explanations are mediated or moderated by other factors such as attributional style (research question c) or social influences (research question d) are critical areas of investigation. Maintaining attention and action on a task also requires that ongoing performance is being judged accurately. Performance can be judged at a global level  and at local levels . Knowing whether there is a relation between these global and local levels of judgment can provide insights into the psychological mechanisms upon which they are made (research question e).Finally, during the self-reflection phase, the learner makes self-judgments and self-reactions on their performance. This feedback on whether actual achievement, the end product of self-regulation, is high or low, satisfactory or unsatisfactory, or simply just good enough, then exerts an influence on whether further action will be taken in the forethought phase. Therefore, knowing whether there is a payoff to self-regulation is instrumental to continued self-regulation (research question f).Our attention now turns more specifically to our proposed research agenda on how knowledge of calibration can be promoted in further lines of inquiry. Table Judgment under Uncertainty: Heuristics and Biases, Tversky and Kahneman  or by oneself  provides estimates of overall memory retrieval (Lichtenstein et al., Accuracy of both global and item-level calibration judgments plays an important role in current and future study efforts because poor judgments can lead to either premature or protracted termination of study of general and specific content. Current research of metacomprehension judgments has shown that there is little or no relation between absolute and relative accuracy (Maki et al., Therefore, future correlational research could examine whether there a relation between absolute accuracy at the test-level and relative accuracy at the item-level. If such a relation exists, whether it varies by item difficulty or type would be important to examine further. Moreover, if this relation exists, developing interventions that could capitalize on it and provide students with ways of better judging item-level and test-level knowledge to prepare for tests would be important contributions to self-regulated learning.Although widespread acceptance has been given to the theoretical argument that accurate metacognitive monitoring is essential to self-regulated learning, the empirical question of whether achievement is enhanced because of accurate monitoring has received surprisingly scant attention. Correlational and experimental studies have established that monitoring can positively impact decisions about what to study (e.g., Metcalfe and Finn, Dunlosky and Rawson  experimeSimilar experimental manipulations of accuracy need to be conducted to more firmly establish the link between calibration accuracy and achievement. Empirical findings provide multiple ways to manipulate accuracy: study guidelines used in group settings (Bol et al., We do not assume that these are the only fruitful research directions to pursue in order to more thoroughly understand calibration and its role in promoting self-regulated learning. However, they represent a good start. The research agenda outlined is based on the social cognitive model of self-regulation developed by Zimmerman and his colleagues (Schunk and Zimmerman, Similarly, the methods we propose are not exhaustive and reflect examples of how these questions may be pursued. The methods we propose range from qualitative approaches investigating how students explain their calibrations judgments, comparing motivational factors linked to task characteristics, correlating absolute and relative accuracy, predicting achievement based on calibration accuracy, and manipulating anchoring and adjustment effects as well as social interactions in controlled experiments.Calibration research will be further advanced when we identify patterns of findings guided by sound theoretical models and based on precise descriptions of terms, measures, contexts, tasks, and populations. As we have argued previously (Hacker et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The conventional view of neurons is that synaptic inputs are integrated on a timescale of milliseconds to seconds in the dendrites, with action potential initiation occurring in the axon initial segment. In a subset of rodent hippocampal and neocortical interneurons in acute slices prepared from serotonin 5b receptor (Htr5b) BAC transgenic mice , we founPersistent firing was observed in response to step current injections, synaptic stimulation, sine wave current injections or in response to stimulation with natural spike trains . With alIn some recordings (n = 11), partial spikes (spikelets) were observed during persistent firing. These spikelets overlapped the first component of the full-amplitude spikes, with the peak of the spikelets corresponding to an inflection on the rising phase seen in the full-amplitude spikes. These observations suggest that the first component of each action potential during persistent firing is an axonal spike, which sometimes fails to evoke a somato-dendritic spike. Furthermore, in some cells (n=3), spikelets were observed if the soma was hyperpolarized during persistent firing. These spikelets were smaller than those observed without hyperpolarization, suggesting that they are caused by propagation failures at a more distal axonal location.Using a stylized computational model constructed with the NEURON simulation environment  of a braAdditionally, in paired recordings, persistent firing was not restricted to the stimulated neuron; it could also be produced in the unstimulated cell (n=3). None of these pairs exhibited direct electrical coupling, and both glutamate and GABA receptors were blocked.Consolidating these results suggests the existence of a previously unknown operational mode for some mammalian neurons. These interneurons can slowly integrate spiking, share the output across a coupled network of axons and respond with persistent firing even in the absence of input to the soma or dendrites.


Conventional glucocorticoid (GC) replacement for patients with adrenal insufficiency (AI) is inadequate. Patients with AI continue to have increased mortality and morbidity and compromised quality of life despite treatment and monitoring.i) To review current management of AI and the unmet medical need based on literature and treatment experience and ii) to offer practical advice for managing AI in specific clinical situations.The review considers the most urgent questions endocrinologists face in managing AI and presents generalised patient cases with suggested strategies for treatment.Optimisation and individualisation of GC replacement remain a challenge because available therapies do not mimic physiological cortisol patterns. While increased mortality and morbidity appear related to inadequate GC replacement, there are no objective measures to guide dose selection and optimisation. Physicians must rely on experience to recognise the clinical signs, which are not unique to AI, of inadequate treatment. The increased demand for corticosteroids during periods of stress can result in a life-threatening adrenal crisis (AC) in a patient with AI. Education is paramount for patients and their caregivers to anticipate, recognise and provide proper early treatment to prevent or reduce the occurrence of ACs.This review highlights and offers suggestions to address the challenges endocrinologists encounter in treating patients with AI. New preparations are being developed to better mimic normal physiological cortisol levels with convenient, once-daily dosing which may improve treatment outcomes. If left untreated, adrenal insufficiency (AI) is a lethal condition and before the availability of glucocorticoids (GCs), the majority of patients with primary AI died within 2 years of diagnosis For several years, the life expectancy of patients with AI who received conventional GC replacement therapy and adequate follow-up was considered normal In healthy individuals cortisol secretion follows a circadian rhythm The challenge of management is to tailor the GC replacement therapy to the needs of each patient. To prevent adrenal crisis (AC) there are three areas to be considered: i) the daily maintenance dose, ii) the daily dose/exposure of cortisol required to mimic normal physiology and iii) the need for extra cortisol during an intercurrent illness or non-illness-related events, such as exercise, in order to prevent AC.This review considers the current management of AI and unmet medical needs based on the literature and our experience of treating patients with AI. We provide practical advice for endocrinologists and other physicians who face the challenges of managing AI in diverse situations.An electronic survey sponsored by ViroPharma  was distributed by the learned endocrine societies of Great Britain and Germany to the participants of the German Endocrine Society Meeting and of the British Endocrine Society both in March 2013. The three most important short-term goals of AI treatment identified were the same in the UK and Germany a: i) to Achieving these short-term goals is difficult because GC needs can change during the day as well as during times of physiological and psychological stress. Developing GC therapy to perfectly parallel the physiological cortisol circadian rhythm remains a challenge Furthermore, monitoring of replacement therapy to optimise the individual replacement dose or make dose adjustments is largely based on clinical signs and symptoms, rather than objective biological serum markers assessing the tissue activity of cortisol. Furthermore, the signs and symptoms indicating overtreatment  or under-treatment  The top three long-term goals were also similar between Germany and UK participants in the previously mentioned survey b and in None of the conventional GC treatments can perfectly imitate the physiological cortisol rhythm. The fall in cortisol around midnight seems to be especially important to guarantee normal physiology. GC administration in the evening seems to predispose individuals to metabolic changes and sleep disorders. Interestingly, a lesser decrease in cortisol is observed in patients with depression. In addition, increased cortisol levels at night are significantly associated with light sleep and wakefulness The main challenges with conventional treatments are shown in HC is the most commonly used GC for AI therapy. Dose replacement with HC should be customised on an individual basis and may be guided by weight-related regimens. HC has high oral bioavailability, but has a short half-life between 60 and 120 min Administration of multiple daily doses of HC poses several problems, in particular during GC replacement. There are peaks and troughs resulting in overexposure followed by underexposure to GC. Also, the timing of the GC dose is likely to be different on a day-to-day basis resulting in different time-exposure profiles. Consequently, patients with AI on replacement therapy report impaired health-related QoL and often experience fatigue during the day before their next HC dose. Such patients report subsequent socioeconomic health problems such as hospitalisation, absenteeism from work and need for disability pensions In small, uncontrolled studies in patients with AI, HC i.v. infusions by a programmable pump have been reported to restore a normal cortisol circadian rhythm and improve all subscales of QoL. However, HC infusions are cumbersome and clearly not practical in general clinical practice. In addition, metabolic and QoL outcomes resulting from infusion treatments have not been systematically reported In summary, the non-physiological circadian profile is believed to be the major explanation for worse outcome of AI, although the level of evidence for this is still poor due a lack of randomised, placebo-controlled studies. Other issues such as actual dose and management of interrcurrent illness can negatively impact outcomes.in vivo and, as such, has a lower cortisol peak, slower onset of action and slower decline to trough than HC In some European countries, cortisone acetate is used as GC replacement. Cortisone acetate can be administered up to three times daily . It requPrednisolone, administered as a single morning dose, has also been used as GC replacement  (16). PrPrimary AI is considered to be an incurable disease with a need for lifelong GC  replacement therapy. Apart from one exceptional case in which partial recovery of autoimmune Addison's disease primary AI was documented Many patients with hypopituitarism develop secondary AI Cushing's syndrome caused by a corticotroph pituitary adenoma (Cushing's disease) or by ectopic ACTH secretion leads to AI after successful treatment of the source of excessive ACTH secretion. This is caused by suppression of normal pituitary corticotroph cells as a result of long-term hypercortisolaemia. The AI in this particular situation can last for several months or years until full recovery of adrenocortical function occurs after gradually diminishing the dose of GC replacement. The same is true for the AI following unilateral adrenalectomy for a cortisol-producing adenoma or carcinoma.Secondary AI caused by HPA axis suppression by long-term, high-dose, exogenous GC therapy  is also reversible if gradual withdrawal of the exogenous steroids is feasible DHEAS are androgen precursors secreted primarily by the adrenal cortex, which in peripheral tissues are converted to more potent androgens such as testosterone and oestrogens. In both primary and secondary AI, DHEA secretion is clearly decreased and often even absent in both men and women with hypoadrenalism. DHEA deficiency with reduced vitality and libido is clinically more evident in women due to the usually preserved gonadal androgen production in men Apart from its mild androgenic effects, DHEA may also act as a neurosteroid with possible effects on mood, cognition and well-being. The first randomised controlled trial of DHEA in 24 female patients with primary or secondary AI showed that DHEA 50 mg daily for 4 months led to improvement in sexual function, decreased depression scores and improved well-being when compared with placebo The side effects of DHEA replacement include acne, hirsutism , alopecia, itching scalp/skin and particularly increased sweat odour when the dose is too high, which may occur more frequently in elderly women.The dose of DHEA used in most studies is between 25 and 50 mg once daily. Clinical assessment of well-being, skin and axillary and pubic hair as well as plasma DHEAS trough levels and plasma testosterone can be measured as monitoring tools of the correct DHEA dose Based on the evidence currently available, DHEA replacement should not be undertaken routinely in clinical practice in patients with AI. Women with hypopituitarism with concomitant AI have a more severe androgen deficiency than those with primary AI. In this case, DHEA replacement could be tried on an individual basis for patients with persistent and seriously impaired QoL and reduced libido despite optimised conventional GC and mineralocorticoid replacement  Under-replacement with mineralocorticoids has been reported in patients with primary AI In patients with primary AI who develop hypertension, the first step should be to assess possible mineralocorticoid  over-replacement and lower the fludrocortisone dose. Even without clear mineralocorticoid over-replacement, the fludrocortisone dose could be mildly decreased while carefully monitoring signs of mineralocorticoid under-replacement.The secretion of aldosterone is primarily stimulated by the renin–angiotensin system with only a minor component under ACTH control. As a consequence, there is no need for mineralocorticoid replacement in secondary and tertiary AI.New estimates of the cortisol production rate in normal subjects indicate that a total dose of HC 15–20 mg is required daily et al.AC is a life-threatening complication in patients suffering from chronic AI and is usually defined as acute impairment of general health with the need for parenteral GC administration and probable hospital admission All patients and their partners/caregivers should be educated such that they are able to recognise an imminent AC and understand how to adjust the dose of GC appropriately, including the use of parenteral GC . While aIn general, it is important to cover the whole day by the increased dosing, and not to just double the dose in the morning. When modified-release HC (PLENADREN) is the replacement therapy, doubling or tripling the daily dose should be accomplished by taking the additional doses after 6–8 h. To complicate dose determinations, there is an apparent lack of dose proportionality that may be related to reduced drug availability or altered distribution with increasing dose The clinical features of an approaching AC may not be immediately recognised resulting in a delay of administration of emergency GC. We believe it is essential that all patients with AI should receive a ‘steroid emergency card’, which provides information as to the necessity for treatment, the current replacement regimen and any relevant contact information for the responsible clinician. The card should be recognised by other healthcare personnel such as paramedics and emergency responders. An example of an emergency card used in Sweden is shown in All patients should also be provided with an emergency kit and be trained in the appropriate use of all components. This kit may contain:Rectal suppositories (prednisolone-suppository (Rectodelt 100)), which are easily administered. One suppository contains prednisolone 100 mg . In the case of diarrhoea, rectal administration is not regarded as sufficient. HC suppositories (200 mg), which result in a peak in plasma HC levels between 1 and 2 h that persist for ≥8 h, have also been used to prevent ACs An ampoule of 100 mg HC-21-hydrogensuccinate for i.m. or s.c. injection, injection devices and an instruction leaflet on self-administration in emergency situations (e.g. diarrhoea and vomiting) and in situations in which the increased oral HC dose failed to sufficiently improve the patient's symptoms. The patient or their regular caregiver or partner should be instructed in its use.In one survey of patients with AI, 94% of respondents carried an emergency card, but only 30% possessed a GC emergency kit and 10% reported that they had never increased their GC dose A number of drugs interfere with hepatic HC metabolism by influencing the CYP3A4 hydroxylating enzyme activity as illustrated in Women with known AI who become pregnant in most cases do not need adjustment of the GC replacement dose during the first two trimesters of pregnancy. An increased dose of HC  may be needed during the third trimester because of increased free cortisol during this period In some pregnant women with primary AI, the mineralocorticoid dose may also need a small increase during the last trimester because of the anti-mineralocorticoid activity of progesterone New timed-release GC preparations may allow improved mimicking of the early morning cortisol rise before awakening in patients with AI and may hold the potential to positively affect QoL in patients with AI A modified-released HC tablet, formerly known as Duocort In a recent study in patients with rheumatoid arthritis, a timed-release prednisone tablet providing GC release before waking was able to markedly reduce disease activity when compared with a standard preparation of prednisolone at the same dose A new, oral, timed-released HC preparation  imitates the physiological release of cortisol better than conventional preparations Although immediate-release GC therapy has enabled patients with life-threatening AI to survive, management of AI continues to be challenging. Current therapies are inadequate and result in an increased mortality risk and compromised QoL. In addition, the failure of current treatment regimens to individualise total cortisol exposure and to mimic the normal physiological rhythm of cortisol release results in comorbidities such as cardiovascular disease and osteoporosis. Serum cortisol levels differ greatly between patients, and requirements change over the course of the day and in response to stressful situations and intercurrent illness. This variation necessitates the tailoring of therapy to individual patients and often results in the division of an overall GC dose into two, three or even four doses throughout the day. The need to take several doses each day interrupts activities and can be a reminder of illness, often resulting in poor patient adherence. Critically, patients, their relatives and caregivers must be taught to recognise signs of ACs and to be prepared to administer life-saving treatment. Hopefully, new HC preparations that are available and those in development will better mimic normal physiological cortisol levels with once-daily dosing and improve disease control and outcomes.


Pseudomyxoma peritonei (PMP) is a rare clinical condition, where copious mucinous ascites accumulate in the peritoneal cavity due to dissemination of mucin-producing tumor. Because of this disseminating, yet nonmetastasizing, behavior, PMP attracts much interest from surgical oncologists in that aggressive locoregional therapy can give the opportunity of long survival and even cure. Although extra-abdominal metastasis is exceptionally rare, the lung is the most likely site in such a case. In this paper, the clinical findings and treatment of eleven cases with pulmonary metastasis from PMP were reviewed, including ten cases in the literature and one case which we experienced. The clinical features of PMP cases with pulmonary metastasis were similar to cases without pulmonary metastasis. The histological type was low-grade mucinous neoplasm in most cases. Pulmonary lesions were resected in seven cases in which abdominal lesions were controlled by cytoreductive surgery and hyperthermic intraperitoneal chemotherapy or another therapeutic modality. Disease-free state was maintained in five cases at the end of the follow-up period. However, it should be noted that rapid progression after resection was seen in two cases, suggesting that biological features may have changed by surgical intervention. Pseudomyxoma peritonei (PMP) is a rare clinical condition, where copious mucinous ascites accumulate in the peritoneal cavity due to dissemination of mucin-producing tumor . It is iAlthough extra-abdominal metastasis is very rare, the lung is one of the most probable sites in such a case. Once pulmonary metastasis occurs, it is important to consider that the biological features of PMP with metastasis may be different from those without metastasis. It is not clear whether surgical resection of metastatic lesions improves prognosis. In addition, if the biological features of PMP cases with pulmonary metastasis are more aggressive than those without pulmonary metastasis, CRS and HIPEC may not be indicated for these cases. In this paper, the clinical findings and treatment of eleven cases with pulmonary metastasis from PMP were reviewed, including ten cases reported in the previous literature and one case which we experienced.A 60-year-old female was referred to us for the treatment of PMP. She had undergone palliative resection and HIPEC for PMP one year before. Histological diagnosis was low-grade appendiceal mucinous neoplasm with peritoneal dissemination, classified as disseminated adenomucinosis (DPAM) according to the criteria by Ronnett. At the time of referral, tumor was diffusely spread in the peritoneal cavity and single nodule was observed in the right lower lung . CRS andEleven cases of pulmonary metastasis from PMP have been reported including the present case –10 Tabl. PatientIn the three cases which were reported earlier by Berge, Chevillotte et al., and Kreissig et al. –6, patieExtra-abdominal metastasis of PMP is exceptionally rare, but the lung and pleura are the most likely sites in such a case . The majResection of metastatic lesions was indicated, when abdominal lesions were controlled by CRS and HIPEC. Wedge resection would be enough except such occasions where the possibility of primary lung cancer cannot be excluded. Prognosis was fairly good, although the follow-up period was rather short. It is noteworthy that long survival was achieved in one case in which multistep pulmonary resection was performed for multiple and bilateral lung metastases. However, it should be cautiously noted that rapid progression after pulmonary resection was seen in two cases. It was suspected that inflammatory reaction caused by CRS and abdominal infection may have changed biological features towards rapid deterioration , 19.A more recent retrospective study showed that 42 cases of intrathoracic metastases were found out of 626 cases of appendiceal adenocarcinoma , which iPulmonary resection for pulmonary metastasis of colon cancer is recommended by NCCN guidelines , but fewExtra-abdominal metastasis from PMP was exceptionally rare, but the lung was the most likely site in such a case. Clinical findings of PMP cases with pulmonary metastasis were similar to those without pulmonary metastasis. Resection of pulmonary lesions was indicated, and long survival may be expected when abdominal lesions were controlled by CRS and HIPEC. However, it should be cautiously noted that rapid progression after resection was seen in some cases, where biological features may be changed.


There is an urgent need to improve our understanding of the mucosal immuno-pathogenesis of HIV acquisition in the female genital tract, particularly in high-risk women such as female sex workers (FSWs). Cervical biopsy samples offer technical advantages over cytobrush sampling, but there are concerns that this might increase HIV acquisition, particularly if healing is slow and/or women do not abstain from sex during healing.Cervical biopsy samples and cervico-vaginal swabs for co-infection diagnostics, prostate specific antigen (PSA) and immune studies were collected from 59 women, including HIV seropositive and HIV-exposed seronegative (HESN) FSWs as well as lower risk women from Nairobi, Kenya. A clinical-demographic questionnaire was administered and women were instructed to avoid sexual intercourse, douching and the insertion of tampons for 14 days. All participants underwent a repeat exam to assess healing within the 14 days, and had HIV diagnostics at six months. Cervical sampling was well tolerated, and 82% of participants had healed macroscopically by 5 days. Both self-report and PSA screening suggested high levels of compliance with pre- and post-procedure abstinence. Delayed healing was associated with vulvovaginal candidiasis (VVC) and HESN status. At six-month follow up all low-risk and HESN participants remained HIV seronegative.Cervical biopsy sampling is a safe and well-tolerated method to obtain cervical biopsies in this context, particularly if participants with VVC are excluded. As healing could be delayed up to 11 days, it is important to support  a period of post-procedure abstinence to minimize HIV risk. While the human immunodeficiency virus (HIV) is generally acquired across the mucosal lining of the genital tract or rectum during sex, occasional individuals appear to be relatively resistant to HIV acquisition despite repeated mucosal exposure. Defining the mucosal immune correlates of protection in these subjects could provide important clues for the HIV vaccine and microbicide fields Immune studies in HESN cohorts initially focused on blood-derived lymphocytes, but later studies emphasized the importance of cervicovaginal samples when investigating possible correlates of protection against HIV HESN individuals are generally under intense HIV infection pressure, and so one reason for the lack of mucosal biopsy studies is concern regarding the potential for enhanced HIV susceptibility after cervical/rectal biopsy. However, if such studies could be performed safely according to a well-controlled protocol, then they might provide the field with urgently needed information regarding the mucosal immune correlates of HIV protection. Therefore, the objective of this study was to assess the acceptability and feasibility of obtaining cervical biopsies by using a well tolerated methodology in a cohort of female sex workers with multiple clients. Since HIV is considered endemic in this area it was important to reach a high compliance with the requested period of post-procedure sexual abstinence to minimize HIV exposure.The study was reviewed and approved by the regional ethical boards at Kenyatta National Hospital, Nairobi, Kenya; the Karolinska Institutet, Stockholm, Sweden; and the University of Manitoba, Winnipeg, Canada. Written informed consent was obtained from all study participants. All ethical committees approved the consent procedure.HIV-uninfected and infected FSW participants were recruited at the Majengo Sex Worker Clinic 2) from the superior portion of the ectocervix were collected with Schubert biopsy forceps . A polycresulin gel was applied after sampling to induce vasoconstriction and subsequent homeostasis and all participants were observed for up to one hour to ensure that no active bleeding occurred prior to clinic discharge.Cervical biopsies were collected using a protocol previously shown to be safe and well-tolerated in Swedish participants at low risk of HIV exposure Chlamydia trachomatis and Neisseria gonorrhoeae ; syphilis serology ; Gram stain for bacterial vaginosis  and lactobacillus colonization and vulvovaginal candidiasis (VVC) . All participants were provided with HIV/STI prevention counseling, male and female condoms, family planning services, treatment of STIs, medical care for acute and chronic illnesses, access to adequate diagnostic testing and referral for specialist consultant and/or hospitalization at Kenyatta National Hospital if needed.Data regarding demographical, reproductive, sexual and clinical characteristics were collected using a questionnaire. All participants were provided with both written and verbal information about the potential for cervical biopsy to increase the risk of HIV acquisition and/or transmission, this information was repeated at two separate clinic visits prior to enrolment. Participants were asked to abstain from unprotected vaginal sex for a period of one day prior to the procedure and to refrain from any vaginal sex, vaginal douching or tampon insertion for fourteen days after the procedure. Since the study protocol precluded income from sex work, FSW participants received monetary compensation equivalent to the expected lost income; lower risk controls received the same compensation. Genital infection diagnostics included: HIV  and Herpes Simplex Virus type 2 (HSV2) serology ; urine for Study participants were asked to return for clinical follow-up 3–5 days post-procedure; those who were actively menstruating were requested to come back as soon as menses had ceased. Participants were clinically evaluated and asked about any bleeding or discomfort, and whether they had had vaginal sex since the procedure. A gynecological exam was performed, including clinical assessment of biopsy healing. The biopsy site was considered as healed when no bleeding from biopsy site, no hyperemia and no abnormalities were detected during the macroscopic evaluation. Vaginal lavages (VagL) were collected by gently aspirating 2 mL of PBS without getting in contact with the cervix. Participants were again informed of the importance of sexual abstinence for a full two weeks post-procedure. as a marker of recent unprotected sex PSA levels in enrolment CVS and follow up VagL were assayed using a Chemiluminescent microparticle immunoassay Univariate analyses were performed using Fisher’s exact test when comparing categorical variables, and non-parametric Mann-Whitney test when comparing continuous variables. Forward conditional binary logistic regression analyses were performed with healing status of biopsy site as the dependent variable and all variables associated with non-healing of the biopsy site in univariate analysis as covariates. P-value of <0.05 was considered significant. Software products used were Prism 5.00  for Windows and PASW Statistics 18 .Participants, including HESN sex workers (n = 18), HIV-infected sex workers (n = 20) and HIV seronegative lower risk individuals  were similar, except for HSV2 seropositivity and douching practices which was lower in the low-risk participants as compared to FSW  . FurtherIn junction with a previous study on low risk Swedish control participants Biopsy site healing was assessed for all participants, with complete macroscopic visual healing at clinical follow-up date in 48/59 (81%). Healing status was not associated with age, hormonal contraceptive use, stage of the menstrual cycle, cervical ectopy, douching practices, or the time interval to follow-up . NeitherStudy participants who were initially HIV seronegative  were reassessed at six months for serological HIV testing. All participants remained HIV uninfected. No gynecological exam was performed at this time.Elucidating the mucosal immune correlates of HIV susceptibility and protection is an important research focus for the vaccine and microbicide field, but genital immune studies are hampered by limitations in current sampling techniques. Cervical biopsy sampling might improve research capacity, but little is known about the safety and tolerability of such sampling within HIV endemic regions, particularly within HIV high-risk populations such as female sex workers. To our knowledge we have performed the first methodological evaluation of cervical biopsy sampling within an HIV-endemic area and an HIV high-risk cohort. The ectocervical biopsy protocol was well tolerated among study participants, with no reported adverse effects beyond minor same-day bleeding and discomfort. Healing was generally rapid, and importantly no HIV acquisition was observed among initially seronegative participants. The 3–5 day period between biopsy and follow up appeared reasonable on the basis of the presented data, and was well-accepted among the participants.Although healing was generally rapid, it was incomplete at five days for 15% of participants. Incomplete healing was not associated with HIV status, hormonal contraception, stage of menstrual cycle or presence of cervical ectopy, although those factors would be important to include as potential confounders in any immune studies. Classical STIs such as chlamydia and gonorrhea were infrequent or absent, and so their effect could not be assessed; HSV2 and syphilis seropositivity was not associated with healing. However, VVC was common and was associated with impaired healing, suggesting that screening and treatment of VVC in advance of cervical biopsy, in addition to general STI screening, might be advisable. As the specificity and sensitivity of clinical VVC diagnosis may be low we advise this diagnosis to be verified microscopically.Further, we observed that incomplete healing was more common within HESN participants. The basis for this was not clear, but was not associated with co-infections such as HSV2 that were increased in this group. HESN individuals have recently been shown to demonstrate relative “immune quiescence”, based on low resting levels of proinflammatory cytokine expression, higher levels of T regulatory cells and down regulation of genes in key signaling pathways of HIV infection In general, a high degree of compliance with the instructions from the clinical staff regarding the fifteen-day period of abstinence from recent (within 48 hours) unprotected vaginal sex was confirmed at the clinical follow up visit by the objective assessment of PSA levels. Although self-reported information may be limited in assessing the underlying reason for delayed healing,  82% of participants had healed macroscopically by 5 days. Nevertheless, since delayed healing was observed in a minority of participants (15%) at five days, recommending a longer period of sexual abstinence (two weeks) will remain as the standard procedure for these studies at our site to maximize safety precautions. In order to support compliance with these recommendations, it will be important to include financial compensation at an appropriate level for female sex workers who would otherwise be without income for a substantial period. However, the finding of PSA in two lower risk women reminds us that abstinence from sexual intercourse that is not related to sex-work may be more difficult to achieve and that rigorous HIV/STI prevention counseling and the provision of male and female condoms is as important as financial compensation. In summary, ectocervical biopsy as a means to obtain genital samples for immunology studies in high-risk populations such as female sex workers from an HIV endemic area appeared to be safe and well tolerated when a high compliance with the requested period of post-procedure sexual abstinence was achieved Despite macroscopic healing by five days in most participants, supporting a more prolonged period of sexual abstinence will be important for safety reasons; promoting a restricted use of NSAID/acetylsalicylic acid components as well as pre-screening and treatment for VVC, in addition to general STI screening, should also be considered.


Stereotactic radiation approaches are gaining more popularity for the treatment of intracranial as well as extracranial tumors in organs such as the liver and lung. Technology, rather than biology, is driving the rapid adoption of stereotactic body radiation therapy (SBRT), also known as stereotactic ablative radiotherapy (SABR), in the clinic due to advances in precise positioning and targeting. Dramatic improvements in tumor control have been demonstrated; however, our knowledge of normal tissue biology response mechanisms to large fraction sizes is lacking. Herein, we will discuss how SABR can induce cellular expression of MHC I, adhesion molecules, costimulatory molecules, heat shock proteins, inflammatory mediators, immunomodulatory cytokines, and death receptors to enhance antitumor immune responses. Stereotactic radiosurgery (SRS) was originally developed for the treatment of intracranial tumors and has demonstrated clinical effectiveness in treating a variety of benign and malignant conditions. Its extracranial counterpart, stereotactic body radiation therapy (SBRT), also known as stereotactic ablative radiotherapy (SABR), has more recently shown efficacy for the treatment of tumors in organs such as the liver and lung. The potential for using SABR is likely greater than for SRS given the larger volume of potential indications outside the central nervous system. Technology, rather than biology, is driving the rapid adoption of SABR in the clinic due to advances in precise positioning, motion control, dosimetry, and precise targeting with image guidance. Dramatic improvements in tumor control have been demonstrated in several studies due to the demonstration that very potent dose can be delivered by use of the mentioned technology. However, our knowledge of normal tissue biology response mechanisms to large fraction sizes is relatively lacking compared to conventional fractionation.in vitro and in vivo at doses up to 10 Gy. This conclusion is based on the premise that cell killing is the dominant process mediating the radiotherapeutic response for both early and late effects including vascular effects. Brenner argued that, to date, there is no evidence of problems when LQ has been applied in the clinic. A fundamental issue in SABR is whether classical radiobiologic modeling with the linear-quadratic (LQ) model is a valid method to assess the biologically effective dose at the high doses typically encountered in radiosurgery. This point was debated in back-to-back papers in seminars in radiation oncology , 2, wherin vivo had a much greater effect than predicted by the LQ model; they cited several examples including Leith et al. [in vitro cell survival data and observed clinical data which suggests that there is more than one mechanism of radiation damage and that these operate differentially at low and high doses. In addition, Kirkpatrick argues that the LQ model does not effectively address the potential existence of radioresistant cancer stem cells, which may require a threshold dose to be crossed before their death is triggered. However, this was the crux of Kirkpatrick and colleagues' argument. They noted multiple studies demonstrating that the administration of a single high dose of radiation h et al.  who calcin vitro survival curve has goodness of fit in all clinically significant ranges including the ablative range characteristic of SABR. Admittedly, none of the proposed mathematical models properly account for in vivo effects including vascular and immune contribution to cell death.Several authors have proposed alternate models to the LQ. In all cases, they argue that the LQ model was intended as a low dose mathematical representation of the data constituting the survival curve , 5. As mAs stated above, the accepted rationale for radiotherapy (RT) is based on causing lethal DNA damage to tumor cells and the tumor-associated stroma. There is unequivocal evidence which has been presented by Fuks and colleagues that the tumor stroma plays an important role in the response to high dose per fraction radiation treatment. They demonstrated that vascular endothelial cell apoptosis is rapidly activated above 10 Gy per fraction , and thaDamage to vascular/stromal elements in tumors has also been observed around 2 weeks after radiation exposure that was less dependent on size of dose per fraction . PatholoIn terms of the infiltrating immune cell component of tumor stroma, conventional RT has traditionally been viewed as immunosuppressive , but theSeveral lines of evidence have suggested direct immune modulation of immune cells by RT , 18–22. The early report of Stone  that theThe possibility that there may be a certain dose per fraction that is optimal for stimulating radiation adjuvanticity is of relevance to mechanism of radiation-induced immune stimulation and clinical practice. Conventional RT has already been shown to enhance tumor-specific T-cell responses , but sucRadiation can not only kill tumor cells releasing tumor antigens and molecules with what are collectively called damage-associated molecular patterns (DAMPs) that exert various immunomodulatory effects including induction of the expression of cytokines, chemokines, and release of inflammatory mediators –30 Figu. AlthougAdvances in the understanding of the mechanisms that regulate the development of antitumor immunity, as well as improved knowledge of the complex effects of radiation on tissues , have reThis situation changed with the molecular cloning of human tumor-associated antigens that could be recognized by T cells, the ability to culture powerful antigen presenting cells (APCs) in the form of dendritic cells (DCs), and to assess immune responses to specific tumor epitopes using tetramer and ELISPOT assays . These aThe recognition of the fact that the host can break a state of tolerance that has developed to its own tumor offers many possibly effective immunotherapeutic strategies, some being currently tested in clinical trials. The “danger” model of immunity suggests that pathogens with associated molecular patterns (PAMPs) and DAMPS engender an inflammatory milieu that promotes the development of antigen-specific immunity through DC maturation that allows internalization of apoptotic and necrotic cellular debris and presentation of processed antigen to T cells. Thus, administration of radiation may therefore be considered to create an inflammatory setting via DC maturation, induction of apoptosis, necrosis, cell surface molecules, and secretory molecules. As with many other challenges, radiation upregulates expression of immunomodulatory surface molecules  and secretory molecules  in tumor, stromal, and vascular endothelial cells. Important amongst these may be the upregulation of TNF family members that could promote cell killing, not only by TNF in the microenvironment but also by radiation-induced TNF.in vivo data are sparse, the dose-response may be linear above 12 Gy [Therefore, the evidence would seem to suggest that there are several potential immunologic mechanisms for cell killing in the high-dose range. The LQ model has long been considered to overestimate radiation cell killing at these doses as a consequence of the model's prediction of a continuous downward bend (ßd2) in the survival curve. While ve 12 Gy , and twove 12 Gy  describeve 12 Gy . Other mve 12 Gy . HoweverIt is clear from what has been said that localized cancer has systemic immune effects as does RT. It is also clear that the outcome of cancer and cancer therapy depends heavily upon the nature of the cells that are generated, in particular with respect to metastasis and overall survival. It seems likely that unexpected discrepancies in the relative efficacies of different anticancer regimens and divergence or convergence between regional and distant failures could be due to such systemic influences, for example, of local tumor control on the incidence of distance metastasis. Future studies aimed at assessing the predictive value of systemic responses in the response of cancer to different dose schedules of RT are likely to be very informative, and strategies that target systemic innate and cancer and radiation-induced regulatory mechanism hold great promise. These strategies, together with DC-based and other forms of antitumor vaccination, can greatly modify the total radiation dose required to achieve local control as well as influencing distant disease, and RT should adapt to optimally integrate with such approaches. While most chemotherapy regimens are thought to compromise the immune system, they also can have immunomodulatory effects that require study.Searching for references on PubMed that contain “SBRT” or “SABR” and “biology” reveals very few hits emphasizing that this is an area of modern radiotherapy where detailed understanding biology needs to catch up with the clinic . Small aAt present, SABR represents an exciting, effective, yet empirically designed radiation therapy. Increasing our knowledge of the underlying biology associated with modern high-dose delivery will only serve to improve the therapeutic benefit of this modality. In addition, we believe that SABR could be optimized for use with immunotherapeutic approaches so as to better generate tumor antigen-specific cellular immunity.