Mechanical load-induced intraosseous pressure gradients may result in some fluid stimuli effects, such as fluid flow and fluid shear stress (FSS), which may enable bone cells to detect external mechanical signals. Interstitial bone fluid flow is known to occur in lacunar–canalicular porosity (PLC).In order to characterize lacunar–canalicular fluid flow behavior, a hierarchical osteon system is developed. The osteon is modeled as a poroelastic annular cylinder with two types of impermeable boundary cases considered on its outer wall: one is elastic restrained (Case I), whereas the other is displacement confined (Case II). Analytical solutions such as canalicular fluid velocity, pressure, fluid flow rate (FFR), and shear stress are obtained.Results show that the amplitudes of FFR and FSS are proportional to strain amplitude and frequency. However, the key loading factor governing canalicular fluid flow behavior is the strain rate. The larger canalicular radius is, the larger amplitudes of FFR and FSS generalized, especially, the FSS amplitude is proportional to canalicular radius. In addition, both FFR and FSS amplitudes produced in case II are larger than those of case I.Strain rate can be acted as a representative loading parameter governing the canalicular fluid flow behavior under a physiological state. This model can facilitate better understanding the load induced the fluid permeation in the PLC. The approach can also be used to analyze the structure of the proteoglycan matrix in the fluid space surrounding the osteocytic process in the canaliculus. As a poroelastic material, bone often bears cyclic loads that might come from walking, running, or other daily activities . These aResearch for the intraosseous fluid stimuli should be conducted at the osteon level . Osteon The PLC dimension is characterized by the radius of the canaliculus (order = 0.1 μm) . The canFSS plays an important role in modulating cell functions, and several laboratory systems have applied FSS to cells . TheoretMany theoretical models , 24–26 hTherefore, we will establish a hierarchical model to directly evaluate the first physical effects of canalicular fluid transport behavior, which allows the linking of cyclic mechanical loading on osteon to the fluid flow in the canaliculi. The lacunar–canalicular primary porosity scale is proposed to relax the pore pressure associated with FSS because of mechanical loading. A homogenization canaliculated porosity approach may avoid multiscale parameters of permeability. We specifically develop a canaliculi model on the basis of our previous osteon model –36 to liBiot’s poroelastic theory is used to account for fluid–solid interactions in osteon tissue. According to Wu et al. , the ostpm = pm0(r)eiωt (m = 1 is for case I and m = 2 is for case II) in the osteon wall is given by:Er and Ez are radial and longitudinal Young modulus, μr and μz are radial and longitudinal drained Poisson’s ratio, In and Kn are the first kind and the second kind modified Bessel function of order n respectively, and cm is constant and can be found in Ref. \documentWe present a theoretical model to describe the fluid flow and shear stress in the canaliculi of an osteon under axial harmonic loading. The current model links the mechanical loading at the osteon scale to the scale of canalicular fluid pressure, velocity, flow rates, and shear stress, which may have a significant stimulus to bone mechanotransduction.εz0∙ω (strain rate amplitude), is reasonable and crucial, and it can be considered a representative loading parameter under a physiological state.We have developed the analytical solutions of the FFR and FSS as theoretical basis, and we aim to see the effects of the variation in boundary conditions and material parameters on the FFR and FSS. Loading boundary and the material parameters are the two major groups of factors that influence the FFR and FSS induced in the canaliculi. The linear increase of loading factors—strain amplitude and frequency—leads to the linear increase of FFRA and FSSA see Fig. . Howeverk mainly differ in terms of the bone scale. For heterogeneous structures, the local permeability value is related to a particular location on the osteon sample, and several experiments are required to obtain a representative or average value [−18 m2 as a reference case for the osteon associated to microscopic lacuno–canalicular.Canalicular fluid flow behavior depends not only on loading conditions, but also on geometric characteristics and material parameters. Figure ge value . We seleThe boundary conditions of case I allow fluid passage from the inner osteon wall and none across the outer elastic constrained wall. It can be assumed that the environmental liquid around the osteon can automatically produce physiological pressure on the cement surface to balance the pore pressure . This boIn the model of Literature , the porStrain-derived interstitial fluid stimuli, such as FSS  and fluiThe Hierarchical modeling processes have several limitations: (1) The proposed osteon boundary cases are the idealization of physiological conditions. (2) Every canaliculus is assumed to straightly run across the osteon wall and neglect the effect of lacuna. (3) A homogenized distribution of the canaliculus leads the permeability to be a constant. (4) One-dimensional  canalicular fluid flow is considered.I.The amplitudes of FFR and FSS are proportional to the strain amplitude and frequency. However, the key loading factor governing the FFR and FSS is the strain rate and it can be considered as a representative loading parameter in a physiological state.II.The larger canalicular radius is, the larger FFRA and FSSA generalized, and the FSSA is proportional to the canalicular radius.III.At the osteon scale, both the FFRAs and FSSAs experience a monotonic decrease as permeability increases.IV.Both the FFR and FSS produced in case II  are larger (approximately 6.7 times) than those of case I .This hierarchical model firstly provides the analytical estimate of canalicular fluid flows in an axial loaded osteon. At the canaliculus scale, the fluid flow is described by a Stokes equation and its directly stimuli effect—FSS is focused on. This approach will enhance the evaluation of canalicular fluid flow related to bone mechanotransduction, and could also potentially account for other geological engineering problems. Despite the limitations of this study, the following conclusions are drawn:


However, individuals undertaking subsequent activity may have limited time to consume fluid. Within this context, the impact of fluid intake practices is unclear. This systematic review investigated the effect of fluid consumption following a period of dehydration on subsequent athletic and cognitive performance.The deleterious effects of dehydration on athletic and cognitive performance have been well documented. As such, dehydrated individuals are advised to consume fluid in volumes equivalent to 1.25 to 1.5 L kgcontinuous, intermittent, resistance, sport-specific and balance exercise) or cognitive performance following dehydration of participants under control (no fluid) and intervention (fluid intake) conditions. Meta-analytic procedures determined intervention efficacy for continuous exercise performance.PubMed (MEDLINE), Web of Science (via Thomas Reuters) and Scopus databases were searched for articles reporting on athletic (categorized as: n = 643 participants) derived from 42 publications were reviewed. Dehydration decreased BM by 1.3–4.2%, and fluid intake was equivalent to 0.4–1.55 L kg−1 BM lost. Fluid intake significantly improved continuous exercise performance , Hedges’ g = 0.46, 95% CI 0.32, 0.61. Improvement was greatest when exercise was performed in hotter environments and over longer durations. The volume or timing of fluid consumption did not influence the magnitude of this effect. Evidence indicating a benefit of fluid intake on intermittent , resistance , sport-specific  and balance  exercise and on cognitive performance  was less apparent and requires further elucidation.Sixty-four trials  contains supplementary material, which is available to authorized users. Evidence indicating a beneficial effect for fluid intake on athletic performance was strongest when a continuous exercise task was employed; whereas limited research has examined the effect of fluid intake on intermittent, resistance and sport-specific exercise performance.The magnitude of improvement was greater when the continuous exercise was performed at elevated environmental temperatures and over longer exercise durations.−1) are yet to be thoroughly investigated.Whilst the volume of fluid consumed (relative to BM lost) did not appear to influence the size of the treatment effect, fluid intake at levels that comply with current recommendations for restoring body water content (1.25–1.50 L kg BM lostThe deleterious effects of dehydration (fluid loss) on athletic and cognitive performance have been extensively researched. Recent meta-analyses detected meaningful decrements in aerobic  and anaeThe American College of Sports Medicine (ACSM) Guidelines on Exercise and Fluid Replacement  and the Ingesting large volumes of fluid may cause gastrointestinal (GI) discomfort, impeding performance. Particularly if the amount of time available to consume fluid is limited or fluids with higher calorie loads (e.g. milk-based beverages) and hence slower rates of gastric emptying are ingested , 19. ThePreferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols PRISMA-P 2015 Statement [The following research protocol was devised in accordance with specifications outlined in the tatement . The metPotential research studies were identified by searching the online databases PubMed (MEDLINE), Web of Science (via Thomas Reuters) and Scopus from inception until April 2016 using the terms exercise, athletic, performance, mood and cognit* , each in combination with “fluid replacement” (the enclosed quotation marks were used to search for an exact phrase), “fluid ingestion”, “fluid intake”, “fluid consumption”, “fluid administration”, rehydrat* and euhydrat*. Records that contained irrelevant terms  were excluded from the literature search using the Boolean search operator ‘NOT’. Two investigators (D.M. and C.I.) independently screened potential research studies to identify relevant texts. Full details of the screening process are presented in Fig. Repeated measures experimental design.Human studies on adult (≥18 years of age) male or female participants with no known medical conditions or co morbidities.An athletic or cognitive performance outcome  was used to indicate the level of dehydration attained.Accessible full text articles written in English.Research studies containing a control-arm and one or more intervention-arms fulfilling the following criteria were eligible for inclusion:l-alanyl-l-glutamine or external cooling); (4) subjects ingested >200 mL of fluid or an unspecified volume of fluid on control trials  dehydration involved restriction of food intake; (2) fluids were not administered orally (e.g. intravenous infusions) or (3) were co-administered with another experimental treatment ; (5) matrials’. Separate trials derived from a single research study are denoted by additional letters (i.e. a–d) in the citation.For the purpose of this systematic review, research studies containing multiple intervention-arms that were eligible for inclusion (each paired against a suitable ‘no fluid’ control group)  objective indicators of athletic performance; subjective measurements of performance (e.g. ratings of perceived exertion) were not examined in this review. The types of athletic performances studied were broadly classified as follows: (a) continuous exercise; (b) intermittent exercise; (c) resistance exercise; (d) sport-specific exercise and (e) balance tasks. Performances that incorporated a coordinated motor-movement resembling some skill involved in a particular sporting event were categorized as ‘sport-specific’ exercises, whereas non-specific sporting activities (e.g. sprint running) were categorized into one of the remaining groups (where possible). Where more than one type of athletic performance was measured within a single experimental trial Participant characteristics: description, age, euhydrated body mass (BM) and maximal oxygen consumption  and BM mass loss (kg) using the following formula:−1). If the quantity of fluid consumed was not expressed as a proportion of BM loss, values were calculated from fluid intake (L) and percentage of BM loss using the following formula:The volume of fluid consumed was expressed as a proportion of BM loss, i.e. relative fluid intake  and time from commencing fluid ingestion to commencing the subsequent performance task (fluid assimilation time) were approximated from the experimental protocol, where adequate information was provided.Time from completing the dehydration protocol to commencing the subsequent performance task  intermittent and sport-specific exercise performance trials were methodologically heterogeneous, particularly in regards to the exercise protocol and outcomes used to determine a treatment effect; (2) few authors responded to an email request for raw data regarding resistance exercise performance, preventing computation of the correlation coefficient and (3) cognitive performance data was rarely quantified (descriptive terms only).g [g value indicates a beneficial effect of fluid intake on continuous exercise performance. Where the SD of the performance change was not reported, the missing value was imputed using a correlation coefficient [All statistical procedures were performed using IBM SPSS Statistical Software, Version 22.0 and Comprehensive Meta-Analysis, Version 3.0. Repeated measures intervention effect sizes were calculated as Hedges’ g , where tg : ES ≤0.2fficient  calculat∆ is the missing standard deviation of change and R is the correlation coefficient. R was approximated as the mean correlation coefficient (R = 0.84) calculated using raw performance data from nine continuous exercise trials (derived from four separate publications). Sensitivity analysis was performed using R = 0.50, 0.74 and 0.94 to test the robustness of the imputed correlation coefficient. The weighted mean treatment effect was calculated using random-effect models, where trials were weighted by the inverse variance for the standardized performance change. Statistical significance was attained if the 95% CI did not include zero. Data are described as mean ± SD, unless otherwise indicated; articles that reported SEM had their values multiplied by the square root of the sample size to convert to SD. All research studies measuring performance on a continuous exercise task used a single objective measurement to demonstrate the presence or absence of a treatment effect (e.g. time to exhaustion or power output). Hence, no additional precautions were taken to limit data dependency.Where SDI2 index. Low, moderate and high heterogeneity was indicated by an I2 value of 25, 50 and 75%, respectively [p value <0.10 for Cochran’s Q was used to indicate significant heterogeneity [Heterogeneity was assessed using Cochran’s Q and the ectively . A p valogeneity . Sensiti−1 BM lost) and fluid assimilation time as variables that might moderate the effect of fluid intake on athletic performance. However, prior research indicates that environmental temperature, exercise duration and the ecological validity of the exercise protocol employed may influence the effect of dehydration on athletic performance [g) using a restricted maximum likelihood multiple meta-regression (random effects) model that controlled for potential confounders. Restricted maximum likelihood simple meta-regression was also performed to explore the influence of environmental temperature on Hedges’ g values. The ecological validity of each continuous exercise protocol was defined in accordance with Goulet [A priori, we identified the volume of fluid ingested  [n = 47), conducted in a thermoneutral laboratory ≤25 °C (n = 16) [n = 28) [n = 3) [n = 2) [n = 2) [presumed to be water) [−1 BM lost [−1) [Characteristics of included studies are summarized in Tables n = 11) , 50–56 on = 16) , 57–61, [n = 28) , 62–71 o [n = 3) , 73. The [n = 2) , 75 or d [n = 2) , 77. In  [n = 2) , 74–77.  [n = 2)  to 4.2%  [n = 2) . Ingestee water) , 56, ande water) . The vole water)  to 1.55  BM lost . In 20 t BM lost , 76, 77. BM lost , 65 prov , 57–61,ost [−1) . In 14 tost [−1) , 66, 71 ost [−1) .Table 1Cn = 461, 95% male, excluding Del Coso et al. [Forty-nine trials  measured the effect of fluid intake on continuous exercise performance (Table 2 max 57.5–68.4 mL kg−1 min−1) [n = 13 trials). In n = 11 trials, exercise was performed in a warm or hot environment (30–40 °C), by acclimated (n = 2) [n = 5) [n = 7) or cold (2–10 °C) [n = 2) conditions, where ambient temperature was specified. Fluid intake significantly improved continuous exercise performance in 13 out of the 22 trials reviewed.Twenty-two trials (1 min−1) , 76, 77  (n = 2) , 77 and  [n = 5) , 62, 76  [n = 5) , 41, 42 n = 139 subjects, 97% male) were included in the meta-analysis examining the effect of fluid consumption on continuous exercise performance. Four continuous exercise trials included in the systematic review were omitted from the meta-analysis on the basis of: (1) duration of the TTE performance test was capped (n = 2) [n = 1) [n = 1) [2 max, without any recovery. All other investigations completed on participants with a VO2 max less than 50.0 mL kg−1 min−1, i.e. untrained or physically active, employed passive methods of dehydration or allocated ~2 h recovery post-active dehydration [g = 0.48, 95% CI 0.33, 0.63), thus it was removed.Eighteen trials ( (n = 2) , 62; (2) [n = 1)  and (3)  [n = 1) , with th [n = 1) , untrainydration , 42. Excg = 0.46, 95% CI 0.32, 0.61) . High heterogeneity was evident between trials . Subsequent analyses (see below) determined that 82% of variation between trials is due to differences in the ambient environmental temperature at which the exercise was performed. Thus, sensitivity analysis was completed with trials sub-grouped by environmental temperature, where cold-thermoneutral = ≤25 °C and warm-hot = >25 °C. The magnitude and statistical significance of the treatment effect was stable during sensitivity analysis where trials were sequentially removed, with Hedges’ g ranging between 0.29–0.35 and 0.70–0.81 for cold-thermoneutral and warm-hot ambient temperature subgroups, respectively . Findings are comparable across different levels of correlation , therefore the meta-analysis  are robust to the imputed R = 0.84  detected a strong significant correlation  between these parameters  and the magnitude of the weighted mean effect  (Table p = 0.071). The majority of trials (n = 12) measured continuous exercise performance on an ecologically valid exercise protocol, e.g. total work or power output completed within a predefine timeframe (n = 10) [n = 2) [n = 5) employed a fixed-power TTE exercise protocol with low ecological validity [p = 0.188), or the level of dehydration and the magnitude of the weighted mean effect (p = 0.845).The influence of environmental temperature, exercise duration, ecological validity of the exercise protocol and the level of dehydration were controlled in the modelling of the relationship between the volume of fluid consumed  Table . Mean exs p = 0.6 (Table p(n = 10) , 41, 42  [n = 2) , 63. Thevalidity , 76, 77.g. Modelling of this relationship corrected for the influence of environmental temperature and type of exercise protocol. Exercise duration was omitted from the model due to collinearity with fluid assimilation time . Fluid assimilation time ranged between 45 and 390 min. Analyses of the 16 eligible trials  did not detect a significant influence of fluid assimilation time on the weighted mean effect (p = 0.11).One trial  failed tn = 95 male subjects) evaluated intermittent exercise performance (Table −1) significantly improved intermittent exercise performance on 4 out of the 10 trials [g = 0.19–0.97).Ten trials  [−1) significantly improved performance on 7 of 22 resistance exercise tasks completed across 5 trials (Hedges’ g = 0.22–5.57) [Nine trials  , 57, 66,8 tasks) , 74. Ind8 tasks) , 74. Flu22–5.57) , 66, 75,n = 64 subjects, 84% male) evaluated athletic performance on exercise tasks that were specific to either cricket (n = 1) [n = 3) [n = 1) [n = 1) [−1).Six trials ( (n = 1) , soccer  [n = 3) , 59, squ [n = 1)  or raceh [n = 1)   examined balance performance  examined the effect of fluid intake on cognitive performance and/or mood. Major findings are summarized in Table Fifteen trials (−1) [Individuals prone to dehydration  may have limited opportunity to adequately rehydrate prior to performing physically or cognitively demanding activities. The present systematic review and meta-analysis examines evidence for the effects of fluid intake on subsequent athletic and cognitive performance following dehydrating sweat loss. A beneficial effect for fluid intake was strongest when athletic performance involved continuous exercise tasks. Further, the magnitude of improvement appeared greater when the continuous exercise was performed at elevated environmental temperatures and over longer exercise durations. Whilst the volume of fluid consumed (relative to BM lost) did not appear to influence the size of the treatment effect, fluid intake at levels complying with current recommendations for completely replacing lost fluid (1.25–1.50 L kg BM lost−1) , 17 are I2 = 80.5%) which may reflect differences in the methodologies employed between studies. Simple meta-regression determined that 82% of variation between trials can be attributed to differences in the ambient environmental temperature at which subsequent exercise was performed, with fluid intake demonstrating greater efficacy under heat stress conditions. The decline in aerobic performance that occurs with hypohydration has largely been attributed to circulatory strain, whereby reductions in blood volume limit oxygen transport to the exercising muscle [The weighted mean effect suggests that fluid ingestion following a period of dehydration significantly improves continuous exercise performance, compared to control conditions (no fluid or negligible fluid intake). Individual estimates all indicated a beneficial effect from fluid intake; however, the magnitude of the improvement was heterogeneous . Hence, the performance effects associated with ingesting a comparably small volume of fluid (e.g. ≤0.75 L kg BM lost−1) or an amount consistent with recommended guidelines (e.g. 1.25–1.50 L kg BM lost−1) remains uncertain. Three experimental investigations have examined the dose-response effect of ingested fluid volume on continuous exercise performance following a period of dehydration with the results demonstrating inconsistent findings [−1 [Results of the meta-regression failed to indicate a statistically significant relationship between the volume of fluid consumed and continuous exercise performance improvements. However, the majority of trials tested a quantity of fluid that was within a narrow fluid intake range  did notings [−1 . However−1) were described as mild to moderate and moderate to high on each trial, respectively. This suggests that larger fluid volumes are likely to induce some degree of participant discomfort which may compromise performance. However, research examining continuous exercise performance following two volumes of fluid intake (i.e. 0.75 vs. 1.50 L kg BM lost−1) demonstrated significantly faster (~3.0%) running performance associated with the larger bolus [If relatively small and large fluid intakes elicit comparable treatment effects, individuals who have limited time to rehydrate prior to performing aerobic activities may opt to consume smaller fluid boluses, delaying complete rehydration until circumstances permit (e.g. overnight). This strategy may reduce the probability of the drinker experiencing volume-induced GI discomfort during subsequent activity, which may occur when larger fluid volumes are ingested . Only oner bolus . ImportaThe effect of fluid intake on intermittent, resistance, sport-specific and balance exercise types remains unclear. It appears that fluid ingestion following a period of dehydration may improve performance on subsequent intermittent, resistance and sport-specific exercise tasks. However, methodological differences make comparison of results across trials challenging.n = 95 male subjects) observed a significant positive effect of fluid intake on performance, whilst no trial reported a significant performance decrement. Similarly to the results from continuous exercise, beneficial effects of fluid intake are apparent when intermittent exercise tasks have been completed in warm environments [In regards to intermittent exercise, 4 of 10 trials  observed a significant positive effect of fluid intake on performance. One trial reported that fluid intake was detrimental to performance [Concerning resistance exercise, 6 of 9 trials  that evaluated the effect of fluid intake on sport-specific exercise performance exhibited considerable heterogeneity, with tests of cricket [The 6 trials  examining the effect of fluid intake following a period of dehydration on cognitive function and mood state. Evidence indicating a beneficial effect of fluid intake on cognitive performance was only observed in some studies [The present systematic review identified 14 trials , rather than following dehydration (i.e. subsequent fluid intake), different physiologic responses to the dehydration protocol may occur on control and intervention trials, e.g. decreased core temperature leading to reduced central fatigue. This could have implications for subsequent athletic performance, and consequently, the magnitude of the overall treatment effect. Sixth, fluid intake ≤200 mL was considered ‘negligible’ and included within the definition of control conditions. However, one study has reported that ingesting 100 mL of fluid  increased TTE following exercise-induced dehydration [This review does contain a number of limitations. Firstly, only studies with accessible full text articles written in English were included. Second, three of the studies reviewed , 71, 77 ydration . Thus, t−1). The influence of fluid intake for those individuals performing intermittent, resistance and sport-specific exercise or undertaking cognitively demanding tasks is not as well understood, and this review serves to highlight areas for future research.Collectively, the results of the present review suggest that individuals who have limited opportunity to adequately rehydrate prior to performing continuous exercise in a heated environment should consume fluid, even if the body water deficit is modest (1.3% reduction in BM) and fluid intake is inadequate for complete rehydration (0.5 L kg BM lost


The tumour-associated antigen 5T4 is an attractive target for cancer immunotherapy. However to date, reported 5T4-specific cellular immune responses induced by various immunisation platforms have been largely weak or non-existent. In the present study, we have evaluated a heterologous prime boost regime based on the simian adenovirus ChAdOx1 and modified vaccinia virus Ankara (MVA) expressing 5T4 for immunogenicity and tumour protective efficacy in a mouse cancer model. Vaccination-induced immune responses were strong, durable and attributable primarily to CD8+ T cells. By comparison, homologous MVA vaccination regimen did not induce detectable 5T4-specific T cell responses. ChAdOx1-MVA vaccinated mice were completely protected against subsequent B16 melanoma challenge, but in therapeutic settings this regime was only modestly effective in delaying tumour outgrowth. Concomitant delivery of the vaccine with monoclonal antibodies (mAbs) targeting immune checkpoint regulators LAG-3, PD-1 or PD-L1 demonstrated that the combination of vaccine with anti PD-1 mAb could significantly delay tumour growth and increase overall survival of tumour-bearing mice. Our findings support a translation of the combinatorial approach based on the heterologous ChAdOx1-MVA vaccination platform with immune checkpoint blockade into the clinic for the treatment of 5T4-positive tumours such as prostate, renal, colorectal, gastric, ovarian, lung cancer and mesothelioma. It was identified in 1990 by searching for shared surface molecules of human trophoblast and cancer cells, with the rationale that they may have a function in survival of the foetus as a semi-allograft  and an aThe first clinical testing of the 5T4-targeting vaccine started more than a decade ago, with the 5T4 protein expressed from the modified vaccinia Ankara virus (MVA). This vaccine was administered to late stage colorectal cancer patients as a homologous prime-boost vaccine known under the trade name of TroVax . To dateHowever, T cells are known to be important in immune control of cancer, and a significant body of evidence accumulated over the last two decades have shown that prime-boost protocols involving sequential administration of different vectors encoding the same antigen(s) yield considerably higher immune responses with protective capability in several animal models and clinical trials. In fact, a vaccination strategy based on the simian adenovirus prime and MVA boost (pioneered in our laboratories over a decade ago) proved to be the most powerful approach for the induction of polyfunctional protective T cell responses against some human pathogens in clinical trials –16.We have previously extended this approach to test the ability of the simian adenovirus and MVA vaccination regime to break tolerance to the tumour associated self-antigen STEAP1 in a mouse model of prostate cancer. In that study, we demonstrated that protection against tumour outgrowth was mediated by the induced T cell responses to the vaccine transgene . EncouraThe experimental findings presented here demonstrate that although ChAdOx1-MVA 5T4 vaccine as a monotherapy completely protects mice against subsequent tumour challenge, it is modestly effective in preventing outgrowth of established tumours. However, a combination of the vaccine with anti PD-1 antibody significantly delays tumour growth of aggressive B16 melanoma cells even in therapeutic settings and increases overall survival. In this study, we have demonstrated for the first time that ChAdOx1-MVA vaccination regime is much more effective in inducing strong 5T4-specific T cell responses compared to the previously tested vaccines targeting 5T4 , 19. Ourex vivo IFN-γ ELIspot assay was performed on PBMCs using a pool of h5T4 peptides covering the entire protein. As shown in Figure It has been shown in the past that cellular immune responses against h5T4 antigen following a homologous MVA vaccination regime have been weak  or non-eWe have also measured immune responses following the ChAdOx1.5T4 prime and MVA.5T4 boost in the BALB/c mouse strain, and found that they were detectable only after the boosting immunization and were of a lower magnitude compared to C57BL/6 mice. We have also used the ChAdOx1-MVA vaccination regime to immunise mice against the murine 5T4 antigen in its native and Ii-fused forms, and, unsurprisingly, only sporadic responses were detected, likely because of self tolerance. On average these responses were one log lower than the responses against the human antigen (data not shown).Specific responses to h5T4 in the ChAdOx1-MVA vaccinated groups were further analysed by flow cytometry in order to assess the relative contribution of CD4+ and CD8+ T cells. Representative results are shown in Figure 5T4-specific antibody responses after vaccination were also measured. To this end, mice were primed either with ChAdOx1 or MVA vectors and boosted with MVA twice at 3-week intervals. As shown in Figure In order to investigate whether h5T4-specific T cell responses detected after the heterologous vaccination could protect against tumour outgrowth, a challenge study was carried out using the syngeneic TRAMP-C1 cell line. This prostate cancer cell line typically expresses a number of murine prostate-associated antigens, such as PAP, PSCA and STEAP1, and has been used for testing protective efficacy of cancer vaccines targeting those antigens , 24; howex vivo IFN-γ Elispot . Representative images of ELISpot plate wells shown in Figure Having demonstrated a remarkable, though surprising, superiority of h5T4 over h5T4Ii vaccination in preventing tumour development, subsequent experiments to assess vaccine therapeutic efficacy were performed with vectors expressing the native h5T4 antigen. Due to aggressiveness of the B16 tumour model, it was necessary to optimise our vaccination regime for therapeutic efficacy testing. We have previously shown for the STEAP1 tumour antigen that a one log reduced dose of the vaccines given at one week intervals was able to stimulate IFN-γ responses comparable with the ones generated by the standard dose immunisations. Also, this accelerated reduced-dose regime translated into superior protection against tumour challenge in the TRAMP-C1 tumour model . TherefoIn order to improve therapeutic efficacy of the heterologous h5T4-targeting vaccination regime in the B16.h5T4 model, we next investigated whether we could achieve synergistic anti-tumour effects by combining the vaccine with immune checkpoint inhibitors. Blockade of immune suppressive signals using CPIs proved to be very successful as a monotherapy, having provided clinical benefit for some patients with several cancer types , and it We have previously demonstrated that blockade of PD-1 receptor in combination with STEAP1 heterologous vaccination enhances efficacy and significantly improves survival of mice in the TRAMP-C1 subcutaneous tumour model . TherefoFurthermore, we aimed to investigate whether local administration of PD-1 antibody at the tumour site was more efficacious than systemic delivery. If proved true, such a finding could potentially have important implications for the clinic, in that much lower doses of the drug should be required, thus diminishing the severity and incidence of adverse events in cancer patients. To this end, mice were randomised into two groups and were immunised with ChAdOx1-MVA encoding h5T4 following the experimental settings mentioned above. At the boost vaccination, one group received PD-1 mAb intraperitoneally, while mice in the second group were given the antibody intratumourally. Pooled data from two independent experiments show that the local administration of anti PD-1 therapy did not offer an overall survival advantage over the systemic delivery, although there is a trend for a slight delay in tumour growth following intratumoural administration compared with intraperitoneal PD-1 mAb injection Figure .Finally, we extended the panel of CPIs to test PD-L1 and LAG-3 mAbs alongside anti PD-1. C57BL/6 mice were challenged with B16.h5T4 cells and randomised to receive ChAdOx1.h5T4 or no vaccine. One week later, vaccinated mice were boosted with MVA.h5T4 as a single agent, MVA.h5T4 concurrently with one of the antibodies , or injected with a mixture of all three mAbs. ChAdOx1-MVA monotherapy, as well as each combinatorial treatment, delayed tumour progression compared to naïve controls Figure . Tumour Results presented in this study describe the advantage of targeting the 5T4 tumour antigen through a heterologous ChAdOx1-MVA vaccination platform over homologous MVA regime in that it induces strong tumour protective CD8+ T cell responses in preclinical settings. In addition, we have shown an enhanced efficacy of ChAdOx1-MVA based cancer vaccines when delivered in combination with anti PD-1 treatment.It has been widely accepted that CD8+ T cells are essential for controlling tumour growth, with numerous studies demonstrating that intratumoral T-cell infiltrates, and CD8+ T cells in particular, correlate with better prognosis and clinical outcome in diverse tumour types –38. TherIn the past, heterologous prime-boost vaccination regimens based on ChAdOx and MVA viruses have been shown to generate strong, durable and protective antigen-specific T cell responses in infectious disease clinical settings , 16, 39.By linking h5T4 antigen to the MHC class II invariant chain (Ii), we were able to significantly enhance antigen-specific immune responses. Our results are consistent with several studies demonstrating that fusion with Ii improves antigen presentation and is able to accelerate, broaden and increase CD8+ T cell responses against pathogens –44 and tEncouraged by the high immunogenicity of the vaccine, we moved on to efficacy studies using the TRAMP-C1 tumour cell line in challenge experiments. We have demonstrated that TRAMP-C1 cells, originally derived from the mouse adenocarcinoma of the prostate, express 5T4 along with other defined prostate-associated antigens. As mouse and human 5T4 proteins share ~80% homology  and we hin vitro killing of h5T4-expressing tumour cells by h5T4-reactive splenocytes was completely abrogated by incubation of the splenocytes with CD8-specific antibody prior to the assay. Importantly, the heterologous vaccination used, based on adenovirus and DC cells, was only effective in therapeutic settings when Ad was used as a priming agent [In contrast, ChAdOx1-MVA vaccines expressing h5T4 and h5T4Ii showed a prominent prophylactic efficacy in the mouse B16 melanoma model, where tumour cells were stably transfected to express the human 5T4 protein. Importantly, all but one of the mice that had been completely protected against tumour development remained tumour-free after re-exposure to the tumour cells, pointing to strong immunological memory induced by the vaccines. Moreover, these mice manifested very high IFN-γ responses that were attributable primarily to CD8+ T cells. Similarly, Ali and colleagues showed that ng agent . Thus, wng agent , 47, couSurprisingly, the higher frequency of IFN-γ-secreting T cells following h5T4Ii vaccination did not translate into a better efficacy in the B16 melanoma model, when vaccines were given as prophylactic treatment. We had previously observed a similar effect in the TRAMP-C1 tumour model using the same vectored vaccines expressing a different prostate cancer associated antigen in its native or Ii-fused forms (unpublished data). Our results are in contrast with the data published by Sorensen and colleagues showing superiority of the Ii fusion with LCMV glycoprotein as the vaccine target antigen, delivered by Ad5 in a B16 melanoma model expressing an immunodominant peptide from LCMV glycoprotein as a tumour neoantigen . The reaDespite its high efficacy as a preventive cancer vaccine, ChAdOx1-MVA vaccination against h5T4 only modestly delayed tumour growth and marginally improved survival of B16.h5T4 tumour-bearing mice. In agreement with our data, MVA.h5T4 homologous vaccination has been previously shown to protect from B16 melanoma tumours, but has only marginally increased survival of mice with established tumours . These rTo improve the ChAdOx1-MVA h5T4 vaccine efficacy in therapeutic settings, we set out to evaluate a combination immunotherapy with monoclonal antibodies against checkpoint targets to counteract the mechanisms blunting the induced immune responses. PD-1 up-regulation on activated T cells in circulation and in the tumour microenvironment has been well documented, as has the upregulation of its ligand, PD-L1, by tumour cells and several immune cell subsets (reviewed by Ostrand-Rosenberg ). More eAiming at the translation of our experimental work into the clinic, we attempted to optimise the combination therapy by delivering checkpoint blockers intratumourally at a reduced dose, in order to minimize potential immune-related adverse effects attributable to the systemic administration. After an initial slight delay of the tumour growth in mice given anti PD-1 intratumorally, eventually the survival rates evened out between the mice given the antibody intratumourally and intraperitoneally. However, we believe that intratumoural administration of CPIs can be more efficacious than systemic delivery in other less aggressive mouse tumour models, and we will continue to evaluate this possibility. If proved correct in mouse models, this route of administration should be considered for clinical testing in suitable cancer types.Although we could significantly improve survival of mice using the combination treatment, tumour protection was not complete. Despite the success of immune checkpoint blockade across several cancer types in clinic , resistaIn the present study, we have chosen to test a triple blockade of PD-1, PD-L1 and LAG-3 alongside vaccination. In our hands, the combination of ChAdOx1-MVA regime with the blockade of PD-1 alone had the same effect as the triple blockade of PD-1, PD-L1 and LAG-3 in terms of growth inhibition and survival. A previous study has shown that anti LAG-3/anti PD-1 combinatorial immunotherapy was not effective against established B16 melanoma tumours, as opposed to fibrosarcoma and colorectal adenocarcinoma tumour models . In compIn conclusion, preclinical data presented herein demonstrate the high translational potential of the vaccination platform based on ChAdOx1 and MVA viral vectors expressing h5T4 in combination with PD-1 blockade for cancer immunotherapy and provide a foundation for further clinical development.6 week old male C57BL/6 mice used in this study were purchased from Harlan, UK. Mouse care and experimental procedures were carried out in accordance with the terms of the UK Animals (Scientific Procedures) Act Project License (PPL 30/2947) and approved by the University of Oxford Animal Care and Ethical Review Committee.TRAMP-C1 cell line was purchased from the American Type Culture Collection (ATCC) and maintained in high glucose DMEM supplemented with 5% fetal bovine serum, 5% NuSerum (Becton Dickinson), 4 mM L-Gln, 1% Pen-Strep, 10 nmol/L dihydrotestosterone (Sigma), and 5 μg/mL bovine insulin (Sigma).B16.h5T4 cell line was a kind gift from Peter Stern  and maintained in DMEM supplemented with 10% fetal bovine serum, 4 mM L-Gln and 1 mg/ml G418.in vitro site-specific recombination (GatewayTM cloning). The constructs were then used to transfect HEK293A cells to generate the recombinant adenoviruses expressing the antigens. The MVA.GFP shuttle vector drives the expression of h5T4 and m5T4 under the P7.5 early/late promoter inserted at the thymidine kinase locus of MVA and the GFP from the fowlpox FP4b late promoter. The plasmids were transfected into MVA-infected primary chick embryo fibroblasts  and recombinant viruses were isolated by selection of GFP-positive plaques, amplified, purified over sucrose cushions and titred in CEFs according to standard practice. The integrity, identity and purity of the viruses were confirmed by PCR analysis.The DNA sequences encoding human 5T4 antigen (h5T4) (NCBI RefSeq NM_001166392.1), its mouse counterpart m5T4 (NCBI RefSeq NM_011627.4) and a fusion construct of h5T4 to full length human invariant chain (Ii) (NCBI RefSeq NP_004346.1) were obtained from GeneArt . The h5T4.Ii construct was designed as an in-frame fusion of the N terminus of the h5T4 cDNA sequence, with the signal peptide deleted to the C terminus of the full length human invariant chain. The construction of ChAdOx1 vectors was described earlier . Briefly8 infectious units (IU) of ChAdOx1 or 107 plaque forming units (PFU) of MVA expressing m5T4, h5T4, or h5T4Ii were diluted in DPBS and injected intramuscularly  in C57BL/6 mice. In alternative experiments, virus doses were reduced by one log. Immunisations were performed weekly or alternatively at 3–4 weeks intervals, and immune responses in blood or spleen were measured at indicated time points. Total number of vaccinations ranged between 2 and 4.Human and mouse native 5T4 antigen and h5T4Ii fusion were tested in immunogenicity and efficacy studies as follows. A dose of 106 TRAMP-C1 or 0.5 × 106 B16.h5T4 cells were injected subcutaneously (s.c.) into the right flank of C57BL/6 male mice in a total volume of 100 μl DPBS. Tumour growth was monitored 3 times weekly and mice were sacrificed when tumour size reached 10mm in any direction. Tumour volume was calculated by the following formula: length (mm) × width2 (mm) × 0.52.To assess vaccine tumour-protective efficacy in a transplantable tumour model, 2 × 10In combination studies, the monoclonal antibodies against mouse PD-1 (clone RMP1-14), PD-L1 (clone 10F.9G2), and LAG3 (clone C9B7W) or Rat IgG2a isotype control antibody (clone 2A3) were administered intraperitoneally (i.p.) or intratumourally (i.t.) at the dose of 50 μg per mouse at indicated time points.ex vivo IFN-γ Elispot assay was performed using Multiscreen IP ELISPOT plates (Millipore) and mIFN-γ Elispot kit (ALP) (Mabtech). Mouse and human 5T4 peptide libraries were synthesized by Mimotopes (UK) and consisted of 15-mer peptides overlapping by 10 amino acids spanning the whole protein length. Individual peptide pools or a mix of all pools at a final concentration of 5 μg/ml were used to stimulate PBMCs or splenocytes for 18–22 hours prior to detection of spot-forming cells (SFCs). Enrichment of splenocytes for CD8 positive T cells was performed by negative selection using a mouse CD8a+ T Cell Isolation Kit (Miltenyi Biotec). ELISpot plates were read using an AID automated ELISPOT counter (AID Diagnostika GmbH) using identical settings for all plates.An Anti-5T4 antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) against recombinant 5T4 protein at 2.5 ug/ml  as described earlier . Endpoinex vivo with 5T4 peptide pools (5 μg/ml of each peptide) and 1 μg/ml Golgi-Plug (BD) for 6 hours. Following stimulation, Fc receptors were blocked with anti mouse CD16/32 and cells were labelled with anti-mouse CD4-AlexaFluor700, and CD8-PerCPCy5.5 (eBioscience Ltd), fixed-permeabilized in CytofixCytoperm buffer (BD) and incubated with IFN-γ-PE, IL-2-APC and TNF-α-FITC antibodies (eBioscience Ltd).Mouse PBMCs or splenocytes were stimulated To confirm 5T4 expression on the surface of the B16.h5T4 cell line, cells were gently dissociated and incubated with anti 5T4 antibody  or mouse IgG isotype control antibody. Anti mouse IgG-FITC was used as secondary antibody.All sample acquisitions were performed on a BD LSRII™ analyzer and data analysed with FlowJo software (Treestar).5T4 mRNA expression in TRAMP-C1 cells and thymi of C57BL/6 mice was detected by semi-quantitative reverse transcription PCR. Total RNA was extracted from cells and tissue samples with RNeasy Plus minikit (Qiagen) and a total of 1–2 μg of RNA was used to synthesize the first single-strand cDNA using QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturers’ guidelines. The primers for RT-PCR amplification have been reported earlier .p values < 0.05. Statistical analysis was performed using the GraphPad PRISM software (v6).Data presented in this manuscript are representative of at least 2 independent experiments using a minimum of 6 animals per group. Unless stated otherwise, median values ± SEM are shown. Comparisons of ELISpot and ICS results among groups were analysed using unpaired Student's t test. Area under curve (AUC) analysis was performed to compare tumour growth kinetics in naïve versus 5T4-vaccinated mice. For comparisons between groups, a one-way ANOVA with Tukey's post-test comparison was performed. Statistical significance in survival experiments was evaluated by the log-rank test. Differences were considered statistically significant for two-sided


Angiotensin II (Ang II), the primary effector hormone of therenin-angiotensin system (RAS), acts systemically or locally, being producedby the action of angiotensin-converting-enzyme (ACE) on angiotensin I.Although several tissue RASs, such as cardiac RAS, have been described,little is known about the presence of an RAS in the pericardial fluid andits possible sources. Locally produced Ang II has paracrine and autocrineeffects, inducing left ventricular hypertrophy, fibrosis, heart failure andcardiac dysfunction. Because of the difficulties inherent in humanpericardial fluid collection, appropriate experimental models are useful toobtain data regarding the characteristics of the pericardial fluid andsurrounding tissues.To evidence the presence of constituents of the Ang II production paths inbovine pericardial fluid and parietal pericardium.Albumin-free crude extracts of bovine pericardial fluid, immunoprecipitatedwith anti-ACE antibody, were submitted to electrophoresis (SDS-PAGE) andgels stained with coomassie blue. Duplicates of gels were probed withanti-ACE antibody. In the pericardial membranes, ACE was detected by use ofimmunofluorescence.Immunodetection on nitrocellulose membranes showed a 146-KDa ACE isoform inthe bovine pericardial fluid. On the pericardial membrane sections, ACE wasimmunolocalized in the mesothelial layer.The ACE isoform in the bovine pericardial fluid and parietal pericardiumshould account at least partially for the production of Ang II in thepericardial space, and should be considered when assessing the cardiacRAS. Tissue Ang II has paracrine and autocrineactions, promoting cell growth, apoptosis, inflammation, oxidative stress and tissuedamage, leading to hypertrophy, fibrosis, heart failure and cardiacdysfunction.7 Local tissue8 and intracellular9 RASs, such as cardiac RAS, have been described, although littleis known about the presence of an RAS in the pericardial fluid and its possiblesources. Angiotensin II, some growth factors and enzymes have been identified inthat fluid.10 Gomes etal.11 have shown ACEactivity in the human pericardial fluid, and Bechtloff et al.12 have shown the presence of theprotein fraction of ACE in the pericardial fluid of patients with coronary arterydisease. However, the source of that enzyme in the pericardial fluid remainsunknown.Cardiovascular diseases are the major cause of morbidity and mortalityworldwide.14Because of the difficulties inherent in pericardial fluid collection, the use ofappropriate experimental models is essential. The heart is contained inside afibroserous sac, the pericardial sac, which has an inner layer, the serouspericardium, which delimits the pericardial cavity. Serous pericardium has avisceral membrane, inseparable from the heart, and a parietal membrane, continuouswith the visceral one. The pericardial fluid is found inside that cavity.Therefore, characterization in animal models of the pericardial fluid and surroundingtissues, including the source of the macromolecules of that fluid, is essential sothat the results can be translated to human beings. This study aimed at collectingevidence in the bovine pericardial parietal membrane and pericardial fluid of thepresence of constituents of the Ang II production paths.Bos indicus, 1758)collected in Delta slaughterhouse (Delta-MG), subject to authorization by theveterinarians in charge. The fragments of pericardial membranes collected werewashed and conditioned in saline solution at 4ºC. The pericardial fluids,aspirated from the pericardial cavities with 20-mL sterile syringes, weremaintained at 4ºC and, with the membranes, transported to the laboratory.This study used fragments of pericardial parietal membranes, as well aspericardial fluid, of six Nelore cattle  UFTM, according to the InnerRegulation of the CEUA/UFTM, article 2, subsection I, §2º.The fragments of the pericardial parietal membranes were washed in salinesolution and dissected in horizontal laminar flow , in nutrientDMEM medium, to remove the adipose tissue of the epipericardial layer of theparietal pericardium. Then they were washed in TBS and sliced into fragments ofapproximately 1.0x0.3 cm, which were embedded in a cryoprotective medium (OCT)and submitted to frozen fixation with liquid nitrogen. After fixation, thefragments were sectioned in a cryostat (Leica Microsystems), and the 2-µmsections obtained were mounted in glass slides, fixed in acetone for 10 minutes,and stored at -20ºC.The pericardial fluid was transferred to microcentrifuge tubes and centrifuged at14000 rpm and 4ºC for 10 minutes . The supernatantswere collected, and the clear ones, with no visual blood contamination, wereused, constituting a pericardial fluid pool.The high concentrations of albumin in the pericardial fluid were reduced by usingblue agarose resin (Bio-Rad). Samples of 250 µL of crude extract ofpericardial fluid were incubated with 1 mL of blue agarose, balanced with sodiumphosphate buffer 0.05 M, pH 6. They were incubated for 2 hours, at roomtemperature, under agitation. Then, the pericardial fluid extracts werecentrifuged at 14000 rpm and 4ºC, and the supernatants were collected forfurther use.15 Theimmunocomplexes obtained were analyzed with SDS polyacrylamide gelelectrophoresis (SDS-PAGE).Samples of 500 µL of pericardial fluid, obtained after removing albumin,were incubated with 2.5 µL of anti-ACE antibody , during the night. Then, 25 µL of CL-4B Sepharose spheres (AmershamBiosciences) conjugated with G protein were added to the samples and incubatedfor 2 hours. The suspensions were centrifuged for 5 minutes at 14000 rpm. Allprocedures were performed under agitation at 4ºC. The supernatants werediscarded and the precipitates collected were diluted with 20 µL of thesample solution.16 under agitation, during thenight, at 4ºC, with a constant 200-mA current. The membranes obtained werestained with Ponceau S to assess the presence of polypeptide fractions, bleachedwith distilled water, dried in filter paper and submitted to ACEimmunodetection.The immunocomplexes obtained were diluted with the sample solution under reducingconditions, at 70ºC, centrifuged and applied to gels at 7.5% concentration. Thepolypeptide bands were separated by use of SDS-PAGE  at constant 25-mA current. The gels obtained were fixed,stained with coomassie blue and bleached. Duplicates of non-fixed gels weretransferred to the nitrocellulose membrane (Invitrogen), in TE 22 (AmershamBiosciences) transfer unit, containing modified Towbin buffer,2], rabbit anti-IgG,conjugated with peroxidase (Amersham), diluted at 1:1000, for 2 hours. Themembranes were washed again, and the immunoreactive bands were revealed in asolution containing diaminobenzidine . The revelation was inactivatedin distilled water. All antibodies were diluted in a solution of 1% bovine serumalbumin and 0.05% Tween 20 in TBS, the incubation with antibodies beingperformed at room temperature under agitation. To determine the specificity ofthe reaction, the membranes were incubated without the primary antibody.The nitrocellulose membranes were incubated with 10% skim milk and 2% serumbovine albumin in Tris-buffered saline (TBS), during the night, under agitation,at 4ºC, to block nonspecific bindings. Then, that solution was replaced withanother containing the primary anti-ACE antibody ,diluted at 1:100, and the membranes were incubated for 2 hours. After incubationwith the primary antibody, the membranes were extensively washed with TBS andincubated with the secondary antibody [F(ab')The pericardial parietal sections obtained with the cryomicrotome were washed inTBS and incubated with anti-ACE antibody , for 1hour, at room temperature, in a dark humid chamber. After incubation withprimary antibody, the sections were washed in TBS plus 0.05% Tween 20 severaltimes and incubated with rabbit anti-IgG secondary antibody conjugated withrhodamine (Alexa Fluor Molecular Probes 568). After being extensively washed,the sections mounted with Fluoromount G (Southern Biotech) were observed anddocumented under a fluorescence microscope Olympus, with a 568-nm wave length.To determine the immunostaining specificity, control sections were incubatedwithout the primary antibody.When the crude extracts of the pericardial fluid underwent immunoprecipitationwith anti-ACE antibody and were analyzed with SDS-PAGE under reducingconditions, a band with molecular mass of approximately 146 kDa, similar to theACE mass ; arrow, The histological sections of the parietal pericardium submitted to ACE detectionby use of immunofluorescence showed unequivocal positivity for ACE in themesothelial cells , right. The present study evidences the presence of an ACE isoform in bovine pericardialfluid, and establishes the ACE location in mesothelial cells of the bovinepericardial parietal membrane, for the first time, indicating that membrane as apossible source of the pericardial fluid ACE.The RAS, originally characterized as a circulating endocrine system, comprisesseveral enzymatic paths and bioactive components that have several functions.20 Local RASs have been shown in many tissues/organs, such asheart, kidneys, adrenal glands, blood vessels, pancreas, liver, brain, and adiposetissue.24 Regarding the cardiac RAS, severalof its constituents, such as angiotensinogen, renin, ACE, Ang I and Ang II, and AT1and AT2 receptors, were detected in different regions of the heart, such as theatria, conduction system, heart valves, coronary arteries and ventricles, beingsynthesized by different cell types, such as fibroblasts and myocytes.26Currently, there is plenty of evidence of the presence of tissue RASs that influencelocal cell actions, with intracellular and subcellular components.28 Thus, characterizing the bovinepericardium, mainly the macromolecules and mediators produced by the cells thatdelimit the pericardial cavity, is paramount to the better understanding of thebiology and importance of that membrane and of the pericardial fluid underphysiological conditions or in association with any disease.The importance of the pericardium and pericardial fluid to control cardiac functionhas been established in past years. The quiescent nature of the visceral andparietal pericardium has been questioned, resulting in evidence for an importantrole in the production of substances that could have paracrine actions on the heart.When the human parietal pericardium is compared with that of other species, weobserve that bovine parietal pericardium has the closest histological constitutionto that of the human species.14 Substances detected in the humanor animal pericardial fluid, such as endothelin-1, beta fibroblast growth factor(bFGF), Ang II, renin, atrial natriuretic factor, vascular endothelial growth factor(VEGF), interleukin-6, and cell adhesion molecules, could act upon theheart.31Modulation of growth and survival of cardiac myocytes,10 endothelial cells and smooth musclecells33 are some biological effects of mediators existingin the pericardial fluid of patients with ischemic and non-ischemic cardiacdiseases. Limana et al.34 haveshown that, in response to myocardial infarction, epicardial c-kit+ cells reactivatean embryonic program, in which soluble factors of the pericardial fluid play afundamental role. Thus, the knowledge about that fluid composition haspathophysiological importance and diagnostic significance.30The pericardial fluid is considered an ultrafiltrate of plasma, added by somecomponents of the myocardial interstitial fluid. Its protein concentration is lowerthan that of the plasma, but with a relatively high albumin concentration.35 preventsparacellular transport of macromolecules with molecular mass above 40 KDa.36 Because the ACE isoform present inthe bovine pericardial fluid has molecular mass of approximately 146 KDa, similar tothat predicted for human ACE,5 theparacellular route would not be an access way to the pericardial cavity.Our results evidence the presence of an ACE isoform in the bovine pericardial fluid,showing the existence of a part of the RAS in the pericardial cavity, probably oflocal origin. Although the pericardial fluid is a plasma ultrafiltrate and plasmamediators, such as Ang II, can spread to the pericardial fluid with no restriction,the same does not happen with ACE. The structural organization of the mesoteliallayer of the pericardium, both parietal and visceral ones, prevent that freecirculation. The presence of tight junctions between the mesothelial cells37 However, in mesothelial cells, its immunolocalization is astrong evidence that those cells are the source of pericardial fluid ACE, because:i) they have a close anatomical relationship with thepericardial cavity, because they delimit it; ii) ACE is an integralprotein of the membrane, being, thus, produced by mesothelial cells, with itsextracellular domain directed to the pericardial cavity;5iii) the ability of mesothelial cells to synthesize ACE has beendemonstrated by the presence of that enzyme's mRNA in cultured human peritonealmesothelial cells, by use of RT-PCR;38iv) they have abundant endoplasmic reticulum and developed Golgicomplex,35consistent with the profile of cells capable of active protein synthesis.In addition to the above cited factors, which partially support the local synthesisof ACE, ACE localization should be considered. Immunofluorescence evidencedpositivity in parietal pericardial mesothelial cells and in the blood vessels of thepericardial membrane. Immunolocalization of ACE in blood vessels was expected,because ACE has ubiquitous distribution in the endothelium.39 They play a critical role in homeostasis controlof the serous membranes in response to injury, inflammation andimmunoregulation.39 Inaddition, mesothelial cells play a central role in the repair of serous membranes,secretion of inflammatory mediators, chemokines, growth factors and extracellularmatrix components. They have different phenotypes, which, depending on theirlocation and activation status, reflect functional differences.39Corroborating with those arguments, several studies have shown the metabolic profileof mesothelial cells. Mesothelial cells synthesize and secrete lubricants, such asglycosaminoglycans and surfactant, to prevent friction and the formation ofadhesions between the parietal and visceral surfaces.40 Our resultsshowed both the presence of an ACE isoform in the pericardial fluid, and, for thefirst time, the immunolocalization of that protein in parietal pericardialmesothelial cells, suggesting that the parietal pericardial mesothelial layer is onepossible source of the pericardial fluid ACE. Thus, the Ang II produced locallycould act on its own pericardial mesothelial cells, both parietal and visceral, oreven directly on the myocardium, promoting inflammation, oxidative stress and celldeath, contributing to cardiac hypertrophy and fibrosis. In addition, it could actdirectly on the myocardial microcirculation promoting important vasomotor effects.In that context, the pericardial fluid would be an important reservoir of mediatorsthat could modulate the functions of cardiac cells.The importance of local RASs has not been totally clarified. Higher concentrations ofactive cardiovascular mediators in the pericardial fluid than in the plasma raises aquestion about their origin and possible actions upon the surrounding tissues. Thepericardial fluid of patients with coronary artery disease trigger substantialarterial contractions in isolated carotid arteries of rats, which are mediatedprimarily by ET-1.The use of experimental models with tissues similar to the human ones, both regardingstructural organization and cell constitution, would be more suitable for studyingcertain human conditions. In addition to structural organization, biochemical andmolecular features are fundamental to achieving optimal balance between quantity andquality of the data produced and their relevance for the condition investigated.28 suggest our results can be extended to humanpericardial mesothelial cells, which could be the partial source of the humanpericardial fluid ACE.12 A better knowledge of both thepericardial fluid constituents and the mesothelial cells in proper animal modelscould help understanding the paracrine or autocrine effects of mediators produced bythe pericardium on the heart.The structural features of the bovine pericardial mesotelial layer, similar to thehuman ones,One limitation of our study was the volume of the pericardial fluid obtained from theanimals in the sample. Because of the difficulties inherent in collecting bovinepericardial fluid, the volume was relatively low. Thus, further research is requiredto clarify how mesothelial cells interact with their local environment and what istheir contribution to the production of mediators in the pericardial fluid that canmodulate cell actions essential to maintain cardiac homeostasis.The Ang II present in the bovine pericardial fluid is partially produced by theaction of the ACE existing in that fluid, pericardial parietal mesothelial cellsbeing a source of that ACE.


In the Funding section and the Acknowledgments, the grant number from the funder Yonsei University College of Medicine is listed incorrectly. The correct grant number is: 6-2013-0187.


Restrictive intraoperative fluid management is increasingly recommended for patients undergoing esophagectomy. Controversy still exists about the impact of postoperative fluid management on perioperative outcome.We retrospectively examined 335 patients who had undergone esophagectomy for esophageal cancer at the University Hospital Freiburg between 1996 and 2014 to investigate the relation between intra- and postoperative fluid management and postoperative morbidity after esophagectomy.p < 0.001) and morbidity . Univariate analysis for risk factors for adverse surgical outcome (Clavien ≥ III) identified ASA-score (p = 0.002), smoking (p = 0.036), reconstruction by colonic interposition (p = 0.036), cervical anastomosis (p = 0.017), blood transfusion (p = 0.038) and total fluid balance on the operation day and on POD 4 (p = 0.001) as risk factors.Perioperative morbidity was 75%, the in-hospital mortality 8%. A fluid balance above average on the operation day was strongly associated with a higher rate of postoperative mortality  and total fluid balance (p = 0.001) as independent predictors of adverse surgical outcome.Multivariate analysis confirmed only ASA-score (Intra- and postoperative fluid overload is strongly associated with increased postoperative morbidity. Our results suggest restrictive intra- and especially postoperative fluid management to optimize the outcome after esophagectomy. Surgery for esophageal cancer has traditionally been associated with high postoperative morbidity and mortality and poor long-term survival, but results have improved dramatically over the last decade , 2. ImprAmong other factors, restrictive intraoperative fluid management has proven to reduce perioperative morbidity in colorectal and pancreatic surgery . While nTo date no study provides sufficient evidence supporting restrictive intra- and postoperative fluid management after esophagectomy. The aim of this study was thus to evaluate risk factors for surgical and pulmonary morbidity focusing on the impact of intraoperative and especially postoperative fluid management.This study evaluates the outcome of 335 consecutive patients with esophageal cancer undergoing esophagectomy between 01/1996 and 03/2014 at a high-volume tertiary referral centre. Our primary Hypothesis was that restrictive postoperative fluid management is associated with a reduced postoperative morbidity. Informed consent was obtained from all patients before their inclusion in the cancer registry. The Medical Ethics Committee of the University of Freiburg approved the study.Diagnostic work-up included endoscopy with biopsies and thoraco-abdominal computerized tomography (CT) in all patients, as well as cardiac and pulmonary work-up. Endoscopic ultrasound was used routinely for locoregional staging if technically possible. In general, lymph nodes were preoperatively classified as malignant if >1 cm by computerized tomography or endoscopic ultrasound. PET-CT was reserved for diagnostic nondestinctive cases.Neoadjuvant chemoradiation has been performed since 1994 when  the T stage was T3 or T4 and/or lymph nodes were suspected to be positive, and patients had no other medical contraindication for neoadjuvant chemoradiation. Neoadjuvant chemoradiation was performed according to a protocol suggested by Naunheim et al.  or the CNeoadjuvant chemotherapy has been performed since 2006 in patients with Adenocarcinoma (AC) of the distal esophagus and esophagogastric junction if the T stage was T3 or T4 and/or lymph nodes were suspected to be positive, and patients had no other medical contraindication for neoadjuvant chemotherapy. Neoadjuvant chemotherapy was performed according to the protocol suggested by Cunningham et al.  or the FAfter neoadjuvant chemoradiation or neoadjuvant chemotherapy, the patients were restaged by endoscopy and CT, and resection was performed approximately 6 weeks after the end of neoadjuvant treatment.The operative procedure was chosen according to tumor location. Patients were mostly operated by an Ivor-Lewis thoraco-abdominal approach , a few underwent esophagectomy by a transmediastinal approach . Reconstruction was routinely performed by a gastric tube formation and pull-up, continuity was established only in individual cases by a colonic interposition. We routinely performed two-field lymphadenectomy in patients with a thoraco-abdominal approach. In patients with a transmediastinal approach, lymphadenectomy was limited to the lower mediastinum and abdominal compartment. The majority of patients received an intraoperatively placed jejunostomy catheter for postoperative enteral feeding.Neither the anesthetic approach, nor postoperative care processes were strictly standardized at our institution. Whenever possible, patients received an epidural catheter for intra-and postoperative pain management. Patients were kept on a surgical intermediate care unit for at least 4 days after surgery. Patients were restricted to oral fluids until postoperative day (POD) 5 and started on solids after a routine contrast esophagogram ruled out anastomotic leak or stenosis. Patients usually received 1ml/kg/h of Normofundin ® G-5 solution  on the day of operation after admission to the intensive care unit. Enteral feeding via jejunostomy catheter was started 6 h after surgery and was continued till patients were able to consume sufficient solid food. Patients without a jejunostomy catheter received total parenteral nutrition instead. In the operating room and on the intermediate care unit, all patients were monitored according to standard parameters regarding blood pressure , and urine output (targeting 0.3–0.5 ml/kg/h). Noradrenalin was the standard catecholamine used intra- and postoperatively in almost all cases.Blood product transfusions were generally initiated when hemoglobin levels were less than 8 g/dl in patients without coronary artery disease or less than 10 g/dl in patients with coronary artery disease. The crystalloid fluid used was a balanced electrolyte solution . The colloidal solutions were based on hydroxyethyl starch (HES) 6%  with less sodium and chloride and an acetate buffer.Intraoperative fluid administration was extracted from the anesthetic protocols, the postoperative amount of administered fluid from the electronic health records of each patient. The type and amount of fluids given during and after esophagectomy was evaluated. In further detail, the amount and type of fluids administered during the operation was assessed as well as the sum (of each type) of fluids given on the operation day and until the end of POD 4. Thus, not only the specific amount of fluids given during the operation, but also the fluids given postoperatively on the intensive care unit are displayed and evaluated. Not considered were perspiratio insensibilis and additional fluid loss not recorded in the charts. Perioperative blood transfusion was defined as any number of erythrocyte concentrates administered intraoperatively or until POD 4.Furthermore, the correlation of the intra- and postoperative fluid administration and surgical outcome was investigated. Postoperative complications were noted and graded according to the Clavien-Dindo classification . PrimaryU-test or Kruskal-Wallis-test as appropriate. Multivariate logistic regression analysis with forward stepwise including was used to identify independent risk factors for adverse surgical outcome. Because overlapping variables  were used in our analyses, multivariate analysis was performed in different models with the inclusion of only one of these variables each time to prevent multicollinearity. Inclusion p for multivariate analysis was 0.10. A p-value <0.05 was considered statistically significant.The results of our study were gained by retrospective analysis of our prospectively maintained esophagogastric database. IBM SPSS Statistics for Windows,  was used for statistical analysis. Categorical variables were put in absolute and relative frequencies; differences were evaluated by one-tailed Fisher’s exact test. Quantitative values were expressed as medians with range, and differences were measured using the Mann-Whitney-Between 1996 and 2014, 335 patients underwent esophagectomy at our institution for esophageal cancer. Eighty-seven percent of the patients were male and the mean age was 62 years. The majority of tumors were adenocarcinomas (59%) and located in the lower third of the esophagus (75%). Neoadjuvant chemoradiation was performed prior to the operation in the majority of cases (56%), while 22% received neoadjuvant chemotherapy and 22% unimodal surgical treatment , Stage II in 42%, Stage III in 19% and Stage IV in 6%. Nineteen patients (6%) had a margin-positive resection  at the end of the operation day (POD 0) and 5500 ml (range 4000 – 37200 ml) at the end of POD 4. The actual fluid balance has to be estimated lower considering the additional fluid loss like perspiratio insensibilis, which would be very hard to quantify. Data regarding fluid input and output were not available for 2 patients intraoperatively, one additional patient on the operation day and for another three additional patients for POD 1 – 4. Patients were divided into groups according to their fluid balance on POD 0 and again on POD 4: Fluid balance above the median was defined as fluid overload, below median as fluid restrictive. The majority of patients (77%) were identical in the fluid groups on POD 0 and POD 4, but 11% with intraoperative fluid overload were below average on POD 4, while 12% with restrictive fluid management during the operation showed an above-average fluid balance on POD 4. Figure n = 122) with a median amount of 900 ml (range 300 – 16200 ml), while fresh frozen plasma was given in only 12% of the cases .Erythrocyte concentrates were administered intraoperatively in 36% of the cases , administered was a median of 900 ml (range 300 – 16200 ml). Two hundred four patients received at least one unit of fresh frozen plasma . Sixty-seven percent of all patients required catecholamine therapy during the operation and directly after, while only 52% needed further catecholamine therapy in the postoperative phase. During the first four postoperative days, 61% of the patients received diuretics.p < 0.001) resulting in a more distinct volume overload .Patients operated before 2006 received significantly more intraoperative fluids  and complications , especially severe complications (Grade III or higher). Higher rates of pneumonia, pleural effusion, need for tracheostomy, anastomotic leakage, and even cardiac incidents were observed. Interestingly, pulmonary embolism was the only complication registered almost exclusively in the group with restricted fluid balance .Seventy-five percent of our patients developed at least one postoperative complication according to the Clavien-Dindo-Classification . Twenty-p < 0.001), wound infection (p = 0.007) and renal complications (p = 0.01). Not surprisingly, neither fluid balance on POD 0 nor POD 4 had any influence on the rates of postoperative pneumo- and chylothorax, pleural empyema, anastomotic stricture, postoperative ileus and deep vein thrombosis.Statistical analysis showed an even more distinct correlation for the above-average fluid balance on POD 4, with significant values even for the rate of reintubations (p < 0.001). Figure The total fluid balance on POD 0 and 4 correlated significantly with the severity of postoperative complications: Patients with no complication had a median fluid balance of 5300 ml on POD 0 and 3900 ml on POD 4, while patients with a complication Grade II-IV had a fluid overload of 6100 ml on POD 0 and 6000 ml on POD 4. Patients who died subsequent to the operation had received even higher amounts with 8000 ml on POD 0 and 10000 ml on POD 4 (p = 0.002), recent smoking (p = 0.036), reconstruction by colonic interposition (p = 0.036), cervical anastomosis (p = 0.017) and perioperative blood transfusion (p = 0.038) as risk factors. Sex, age, BMI, alcohol abuse, type of multimodal therapy, surgical approach, tumour localisation, histological type and stage were not associated with adverse surgical outcome. Most importantly, both, total fluid balance on the operation day as well as on POD 4 were strongly associated with adverse surgical outcome (p = 0.001), while the sole rate of intraoperative fluid administration could not be identified as a risk factor (p = 0.479).Patients with a complication grade III or higher were defined as having an adverse surgical outcome (52%) and underwent further risk factor analysis for adverse surgical outcome. Univariate analysis Table  identifip = 0.001) and fluid balance on POD 0 (p = 0.001) as independent predictors of adverse surgical outcome. Fluid balance on POD 4 was analysed in a separate model to prevent multicollinearity and was likewise confirmed as an independent risk factor(p = 0.001).Multivariate analysis Table  confirmep = 0.015). In multivariate analysis treatment period was eliminated (p = 0.741). Therefore the effect has to be attributed to the changes made in perioperative management and surgical technique.We noticed a shift of fluid management towards a more restrictive regime over time. To analyse the correlation between year of treatment and postoperative morbidity, patients were divided by treatment period in two groups (1996–2005 and 2006–14). Patients treated after 2006 had a significantly lower postoperative morbidity  and are supported by sufficient evidence provided by randomized trials and systemic reviews . These sThe pathophysiology of our findings may mainly be related to fluid impact on perioperative lung physiology. The postoperative morbidity of esophagectomy strongly consists of the pulmonary component . RestricImpaired wound healing may also contribute to morbidity and mortality after esophagectomy. In experimental settings, intraoperative fluid overload impairs gastrointestinal wound healing –24, anotOur study analyses fluid administration over 4 postoperative days and correlates the results with perioperative outcome. Most other previous studies focused on intraoperative fluid administration. Postoperative fluid management even has been analyzed over the course of 2 days by only one study . Our resThe retrospective design of our study certainly allows only limited conclusions. Confounding factors leading to intra- and postoperative fluid overload and increased morbidity and mortality can only be partially eliminated by multivariate analysis. Possibly this study overvalues the negative effect of intra- and postoperative fluid overload on the postoperative morbidity. Our analysis shows that intraoperative infusion have been reduced during the study period and that a restrictive fluid management has already been implemented in our hospital and is likely to be an essential contributor to the reduced postoperative morbidity after esophagectomy , 2. On tA further limitation of our study is its non-randomized character. Patients were not randomized to restrictive or liberal fluid management, but the decision was made to some extent arbitrarily by the attending anesthesiologist and surgeon. Operating time and intraoperative blood loss might effect both postoperative fluid balanace and morbidity. Blood transfusions were given in 61% of the cases intra- or postoperatively and contributed to the observed fluid overload. Perioperative blood transfusion itself has been identified as a risk factor for adverse surgical outcome in other analyses , 27. TheOur study identifies postoperative fluid overload alongside high ASA-score as the strongest risk factor for adverse surgical outcome after esophagectomy for esophageal cancer. In the context of previous studies from different surgical specialties our results suggest that a restrictive fluid management can contribute to a further reduction of postoperative complications after esophagectomy.


In patients with acute circulatory failure, the decision to give fluids or not should not be taken lightly. The risk of overzealous fluid administration has been clearly established. Moreover, volume expansion does not always increase cardiac output as one expects. Thus, after the very initial phase and/or if fluid losses are not obvious, predicting fluid responsiveness should be the first step of fluid strategy. For this purpose, the central venous pressure as well as other “static” markers of preload has been used for decades, but they are not reliable. Robust evidence suggests that this traditional use should be abandoned. Over the last 15 years, a number of dynamic tests have been developed. These tests are based on the principle of inducing short-term changes in cardiac preload, using heart–lung interactions, the passive leg raise or by the infusion of small volumes of fluid, and to observe the resulting effect on cardiac output. Pulse pressure and stroke volume variations were first developed, but they are reliable only under strict conditions. The variations in vena caval diameters share many limitations of pulse pressure variations. The passive leg-raising test is now supported by solid evidence and is more frequently used. More recently, the end-expiratory occlusion test has been described, which is easily performed in ventilated patients. Unlike the traditional fluid challenge, these dynamic tests do not lead to fluid overload. The dynamic tests are complementary, and clinicians should choose between them based on the status of the patient and the cardiac output monitoring technique. Several methods and tests are currently available to identify preload responsiveness. All have some limitations, but they are frequently complementary. Along with elements indicating the risk of fluid administration, they should help clinicians to take the decision to administer fluids or not in a reasoned way. Volume expansion, the first-line treatment of acute circulatory failure, can be the source of a crucial therapeutic dilemma. On the one hand, the severity of the disease incites one to initiate treatment rapidly and massively. In line with this, the pivotal study by Rivers et al.  showed tThe response to a fluid challenge is complicated by cardiovascular physiology . Due to The question of predicting fluid responsiveness with CVP use is quite controversial and somewhat perplexing. On the one hand, there is a tremendous amount of evidence that a given value of CVP does not predict fluid responsiveness. This has been established by a number of studies and meta-analyses , 16. On This inconsistency is even more difficult to understand since the inability of CVP to reflect preload responsiveness comes from simple physiology. A static value of CVP could correspond to preload responsiveness as well as preload unresponsiveness, depending on the shape of the Frank–Starling curve, which varies from one patient to another and, in a patient, from one time to another Fig. . This isThe variations of stroke volume (SVV), and of surrogates, that are induced by mechanical ventilation were the first methods to be developed for the dynamic assessment of preload responsiveness. The rationale is that, during positive pressure ventilation, insufflation decreases preload of the right ventricle. When transmitted to the left side, this induces a decrease in preload of the left ventricle. If left ventricular stroke volume changes in response to cyclic positive pressure ventilation, this indicates that both ventricles are preload dependent. Mechanical ventilation can be used as a provocative test to challenge the slope of the Frank–Starling curve at the bedside Fig. . The ampThe diagnostic accuracy of PPV has been analysed through the prism of the “grey zone analysis”. Using complex statistical methods, a study demonstrated that there is a grey zone of PPV values, between 9 and 13%, where the sensitivity or the specificity is lower than 90% . It was Following invasive arterial pulse pressure, many other surrogates of stroke volume have been investigated to assess SVV during mechanical ventilation. In recent years, research has focused on less-invasive and non-invasive techniques. These techniques may be particularly useful when no arterial line is in place, typically in the operating room. The ventilation-induced variations in arterial pulse pressure estimated by volume-clamp photoplethysmography , stroke While the utility of the PPV and SVV has become better established, those conditions in which they become unreliable have been more clearly defined. These are summarised in Table In practice, the conditions where the reliability of PPV and SVV is decreased are quite common in the ICU. This is particularly true today since patients are less sedated and low tidal volume ventilation is more common than before and since cardiac arrhythmias are not uncommon. A recent prospective study reported an incidence of 17% of instances where the reliability of PPV and SVV could be used without limitation .In the operating room setting, PPV and SVV monitoring (invasively or non-invasively obtained) retain their predictive value since the conditions of their applicability are generally fulfilled. The limitations of PPV and SVV must always be kept in mind by the intensivists or anaesthesiologists, since ignoring them could lead to serious misinterpretations. However, a recent survey demonstrated that a large proportion of intensivists did not have full knowledge of all factors confounding PPV and SVV interpretation .The principle behind these indices is also based on heart–lung interactions, but they do not operate in the same way as PPV. The changes in intrathoracic pressure induced by mechanical ventilation may induce some variations in the venae cavae in close proximity to the heart when the central blood volume is low. The variation of the inferior vena cava diameter measured by transthoracic echocardiography has been reported to detect preload responsiveness with reasonable accuracy. The “collapsibility” of the superior vena cava has also been shown to reflect fluid responsiveness; however, it requires transesophageal echocardiography . It has Compared to PPV and SVV, the vena caval indices have been less well studied and the diagnostic thresholds that were reported have varied from 12%  to 40%   has been used for decades by rescuers as a first-line measure in patients with dizziness and syncope. Its interest in critical care has emerged after a study demonstrating that it induces significant changes in right and left cardiac preload . A few yIt has been recently shown that the infusion of blood induced by the postural change during a PLR is sufficient to induce a significant increase in the mean systemic pressure . The resSince 2006, many studies have confirmed the reliability of the PLR test with a remarkable consistency. Two meta-analyses of these studies have been recently published , 59. In A number of practical aspects regarding the technique of performing the PLR test are important to consider, as recently reviewed by Monnet and Teboul . The mosNevertheless, this does not imply that the PLR test necessarily requires invasive monitoring. Many studies have used non-invasive or minimally invasive techniques to estimate the PLR-induced changes in cardiac output , 59. Bot2) [2 are only related to changes in cardiac output. A recent study showed that the changes in end-tidal CO2 were able to detect the changes in cardiac output during PLR, but not during a mini-fluid challenge with 100 mL of saline [An original and totally non-invasive method is to measure the PLR-induced increase in end-tidal carbon dioxide (CO2) –71. Thisf saline .It has been suggested that intra-abdominal hypertension invalidates the PLR test . HoweverIn patients undergoing mechanical ventilation, each insufflation decreases cardiac preload and tends to impede venous return. Interrupting mechanical ventilation for a few seconds stops this cyclic impediment in venous return. Cardiac preload transiently increases. If cardiac output increases in response to this end-expiratory occlusion (EEO) test, this indicates preload responsiveness of both ventricles Fig. . Of note2O as well as of 15 cmH2O [The test has the advantage of being very easy to perform. It simply requires “stopping” the ventilator, as when measuring the intrinsic positive end-expiratory pressure, and measuring the changes in cardiac output. The EEO technique does not have the technical constraints of PLR. The EEO test is valid in patients with ARDS, a condition where PPV and SVV are not sensitive enough  Fig. 2)2). In a 15 cmH2O . The maiIn published studies, the EEO test’s effects on cardiac output were assessed by pulse contour analysis , 75, 76.2O. The effects were measured through CO2 elimination, which was used as a surrogate of cardiac output in these patients who were well sedated [The idea of using heart–lung interactions to challenge preload responsiveness has led to other novel tests, less studied than the ones detailed above. In a recent study in patients undergoing cardiac surgery, fluid responsiveness was predicted by the haemodynamic effects of a sudden increase in positive end-expiratory pressure from 5 to 10 cmH sedated . The res sedated . This te sedated .Infusing fluid is obviously the most direct way to challenge fluid responsiveness . NeverthThe second major drawback of a fluid challenge is that it is not a test but a treatment in its own right. In patients where multiple fluid challenges must be repeated in a short time, this inevitably leads to administering a volume of fluid that is far from negligible. For instance, in a patient with haemodynamic instability, where four or five episodes of hypotension occur in one day, performing fluid challenges will lead to infusing 2000 to 2500 mL of fluid that, by constitution, do not increase cardiac output. This obviously contributes to fluid overload and haemodilution with inherent risks of decrease in oxygen delivery to the tissues.2 [The idea has emerged to perform a fluid challenge with a volume of fluid much smaller than the “conventional” challenge. In a study where a “mini-fluid challenge” was performed with 100 mL of colloid, the changes in the velocity time integral of the left ventricular outflow tract measured with echocardiography predicted preload responsiveness . The sta2 . Thus, aRecently, some authors sought to determine the smallest volume of fluid required to perform an effective fluid challenge by investigating the effects of different doses of intravenous fluids on changes in cardiac output  and mean circulating filling pressure. In this study, a bolus of 4 mL/kg over 5 min was the smallest volume that could reliably increase the mean circulating filling pressure and make fluid challenge interpretable in every circumstance .Some important points must be kept in mind at the bedside. First, there are some instances where the tests we described in detail above are pointless because fluid responsiveness is obvious. In cases of haemorrhagic shock, clear-cut hypovolemic shock and the early phase of septic shock when fluid has not been administered, cardiac output will undoubtedly increase with fluid infusion. In such circumstances, delaying fluid administration is likely harmful, and therefore, tests of fluid responsiveness should not be used. Second, testing fluid responsiveness makes sense only in cases of circulatory failure Fig. . The queTesting for fluid responsiveness may help one to decide to administer fluid. However, equally important, testing for fluid responsiveness may help in the decision to stop fluid administration or not to administer fluids at all. The decision to stop fluid administration should be made on the disappearance of signs of circulatory failure, the appearance of signs of fluid overload and when the tests of preload responsiveness become negative . TestingBecause of the risk of fluid overload and the inconstant efficacy of volume expansion, the decision to administer fluid cannot be taken lightly. Fluids are drugs whose dose must be carefully titrated to the needs of the patient. Several methods and tests are currently available to identify preload responsiveness. All these techniques have some limitations Table , but the


Electrophysiological methods are the gold standard in neuroscience because they reveal the activity of individual neurons at high temporal resolution and in arbitrary brain locations. Microelectrode arrays based on complementary metal-oxide semiconductor (CMOS) technology, such as Neuropixels probes, look set to transform these methods. Neuropixels probes provide ∼1000 recording sites on an extremely narrow shank, with on-board amplification, digitization, and multiplexing. They deliver low-noise recordings from hundreds of neurons, providing a step change in the type of data available to neuroscientists. Here we discuss the opportunities afforded by these probes for large-scale electrophysiology, the challenges associated with data processing and anatomical localization, and avenues for further improvements of the technology. Current Opinion in Neurobiology 2018, 50:92–100NeurotechnologiesThis review comes from a themed issue on Polina Anikeeva and Liqun LuoEdited by Issue and the EditorialFor a complete overview see the Available online 13th February 2018https://doi.org/10.1016/j.conb.2018.01.009http://creativecommons.org/licenses/by/4.0/).0959-4388/© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license . The daDespite these algorithmic advances, no spike sorting algorithms are yet truly automatic, requiring manual supervision to improve results. One reason for this is the problem of electrode drift, the movement of the brain relative to the probe. Algorithmic approaches can be taken to join groups of spikes whose shapes have shifted over time , 46, butHowever, the biggest impediment to developing fully-automatic spike sorting algorithms is the difficulty of ground truth validation. No algorithm can be trusted blindly without quantitative evidence that it has low error rates, and the development of metrics to assess these error rates is an area of active research , 47. UltThe best form of ground truth, then, is actual simultaneous recording of spikes with another method such as a juxtacellular electrode , 50. ThiGiven this context, careful manual curation of the results of spike sorting algorithms is still critical, and public, open-source software packages have been developed to improve the efficiency of this process . Even afTargeting a probe to a desired brain structure and subsequently localizing the recording sites is a challenge for any electrophysiology experiment. With Neuropixels probes, targeting is easier: it is difficult to miss the desired brain location along the insertion vector, given that all sites over a 4 mm span along that vector will be recorded when using the densest site configuration. Localization, however, is a challenge. Neuropixels probes cannot produce electrolytic lesions, and their relatively small cross-section may not leave visible tracks through DAPI- or Nissl-stained tissue b. FortunTo determine the brain regions in which neurons were recorded, one could then proceed as usual by manually identifying each structure along the recording track. However, this problem may be solved another way using recently developed 3D atlases of the mouse brain . After Neuropixels probes may also offer the opportunity to systematically take advantage of the electrophysiological signatures of brain regions and layers that have long been known ‘by ear’ to electrophysiologists c. With aNeuropixels probes are manufactured with CMOS technologies, so they are inexpensive to produce in large volumes and open to significant further improvement. As circuit fabrication technologies improve, the device size will shrink. For instance, a version of the probe with a significantly smaller base area may be possible, and would increase the utility of Neuropixels probes for chronic implantations in small animals such as mice.When light is delivered extrinsically, Neuropixels probes are compatible with optogenetic experiments involving activation of local circuitry or opto-tagging, i.e. the identification of neurons on the basis of their expression of genetically-encoded light-activatable channels ••. HowevCertain use cases are not ideally served by Neuropixels probes. First, chronic implants in primates or other large animals require probes that have either flexible shafts or that ‘float’ on the brain to minimize tissue-probe movement, which can damage either or both. Research into materials and designs for such applications is ongoing , 63 but To record from greater numbers of sites simultaneously, other projects have taken advantage of the capabilities of CMOS electronics to develop probes that rapidly switch between recording sites , 69. ThoThus electrophysiology enters an exciting new era, as datasets explode and our capability to measure global brain dynamics at fine spatial and temporal scales reaches new heights. Making sense of the resulting data explosion will be a major challenge, but that is a good problem to have.Nothing declared.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as: