With rapid expansion of offshore renewables, a broader perspective on their ecological implications is timely to predict marine predator responses to environmental change. Strong currents interacting with man-made structures can generate complex three-dimensional wakes that can make prey more accessible. Whether localised wakes from man-made structures can generate predictable foraging hotspots for top predators is unknown. Here we address this question by quantifying the relative use of an anthropogenically-generated wake by surface foraging seabirds, verified using drone transects and hydroacoustics. We show that the wake of a tidal energy structure promotes a localised and persistent foraging hotspot, with seabird numbers greatly exceeding those at adjacent natural wake features. The wake mixes material throughout the water column, potentially acting like a prey conveyer belt. Our findings highlight the importance of identifying the physical scales and mechanisms underlying predator hotspot formation when assessing the ecological consequences of installing or removing anthropogenic structures. Lilian Lieber et al. examined seabird foraging around natural and man-made wakes, finding that wake from a turbine structure generates a more intense and predictable foraging hotspot for seabirds. This shows the importance of changes in physical forcing to top predators when installing or removing offshore structures. Predators rely on a multitude of physical processes, which dynamically influence foraging behaviour3 and success4. In the open ocean, predator foraging has been associated with mesoscale (10−100 km) physical features, such as fronts and eddies7. However, even fine-  or local-  scale physical features may create small-scale predator hotspots10. The importance of these fine and local-scale physical processes is heightened in seabirds restricted to shallow plunge diving techniques, such as gulls and terns, where prey availability near the sea surface governs foraging site selection13. Consequently, tern species (Sternidae) tend to focus their foraging activity in areas of bathymetry-generated turbulence or shallow upwellings that consistently make prey available near the surface15. Such physically enhanced prey availability and its predictability seem to determine seabird foraging habitat rather than prey density alone20. Therefore, the identification of local flow processes interacting with bathymetric features  can improve our understanding of the physical mechanisms promoting foraging hotspot formation and persistence in dynamic coastal systems21.In an era of intense marine urbanisation22—are characteristic of strongly tidal seas. The introduction of anthropogenic structures into such dynamic environments adds further complexity to local flow processes, potentially triggering ecological implications23. Man-made structures modify local hydrodynamics24, including flow velocities25 and wake effects28. Further, a von Kármán vortex street29, characterised by distinct and repeatable eddy trajectories, may occur in the wake of embedded structures when placed in strong, near-laminar flows30. While fish may exploit the lee of a structure as a flow refuge31 or use small-scale vortices  to Kármán gait32, an extreme downstream wake with eddy vortices of sufficient size and vorticity33 can vertically displace or overturn fish in fast, unsteady flows36, potentially making them accessible to surface-foraging predators.The periodic emergence of tidally driven bathymetry-induced turbulence, shallow upwellings or more ephemeral turbulent structures such as boils—circular regions of local upwellingSternidae), comparable to those at adjacent natural wake features. SeaGen, the world’s first grid-connected tidal energy turbine, currently being decommissioned, produces a wake with vortex shedding approaching a von Kármán vortex street30. The device consisted of a monopile structure (3 m diameter) attached to a quadropod foundation fixed on the seabed (water depth about 25 m) with a 27 m long crossbeam supporting the original rotors on either side of the tower 15 m above the seabed. During this study, the rotors had already been removed, however, the monopile itself contributes considerably to the vortex shedding in the downstream wake as shown through large eddy simulations30. SeaGen is situated in a dynamic tidal channel (‘‘the Narrows’’) in Strangford Lough, Northern Ireland, in proximity to colonies of summer-breeding tern species . The channel also provides diverse foraging opportunities with natural wake features commonly used by terns, therefore presenting a suitable study system. Two neighbouring extreme natural wake features, an island  and a whirlpool structure (Routen Wheel), within the channel were selected to compare the terns’ use of the natural wakes with the man-made wake  anthropogenically generated wake can present a reliable foraging location for surface-feeding seabirds ( km anthr37) or if aggregations were coupled to the ebb-flood tidal cycle.The number of terns foraging at each wake feature was assessed using vantage point surveys (July–August 2018) with observations covering different tidal states , recording variations in tern abundance across hydrodynamic conditions. The occurrence of conspicuous topographic and anthropogenic landmarks allowed the construction of plots with approximately the same area, with calculations based on bearings and distances from the vantage point. For SeaGen, observations were spatially divided into North (area of flood tide wake) and South (area of ebb tide wake) of the foundation, respectively. While the physical structure of SeaGen’s wake does not differ between the flood and ebb tide, the spatial separation was needed to ensure equal spatial extent per site. Further, it helped to assess whether terns were solely attracted to the environmental cue of turbulence , triggering a fine-scale investigation of its wake dynamics. Unmanned aerial vehicle (UAV) transects above SeaGen over several tidal cycles visualised the dynamic vortex shedding of the wake and the exact spatial extent of tern foraging, thereby overcoming the oblique angle of the vantage point observer. Consistent with the vantage point surveys, these transects recorded that terns focused their foraging activity almost exclusively over the flood wake  transects were run either side of the SeaGen foundation throughout a flood-ebb tidal cycle. The upstream near-laminar flow exceeding 5 msGen Fig. . The corake Fig.  is most 2, Fig. Applying machine learning algorithms to distinguish terns from other moving targets , flight trajectories recorded over the wake region Fig.  showed aFinally, the persistent use of the SeaGen (North) wake by the terns limited to the flood tidal cycle was explored using a hydrodynamic model coupled to an ecological module. Passive particles as a proxy for small prey organisms were released from the Irish Sea, outside the entrance of the Lough at the beginning of a flood tide Fig. . The flu45, distinct mechanisms may be in place to explain such associations. Namely, (1) natural reefing can increase fish biomass46, (2) fish can seek flow refuge in the immediate lee of a structure34 and (3) downstream wake effects can make incoming prey available near the surface through displacement47. The latter mechanism is currently the least explored in a natural setting despite its importance in high-flow environments, highlighting the relevance of our findings. While natural bathymetric features and associated patterns of shear lines and wake effects have been shown to attract top predators8, the man-made wake in this study promoted the most persistent and intense foraging aggregations of terns among all wake features investigated. While we did not assess prey vertical distribution, turbulent vertical velocity fluctuations within the wake were greater than 0.5 ms−1  and may have the potential to displace prey. Therefore, our future studies will focus on assessing prey distribution and availability within both the inflows and wakes under different tidal states.To our knowledge, this is the first study to link indirect physical interactions (a downstream wake) of a renewable energy structure with top predators, highlighting the hitherto overlooked ecological implications of localised physical forcing around man-made structures. While top predator use of anthropogenic structures has been observed elsewhere Namely,  natural 23 and requires the identification of such localised physical processes underlying top predator hotspot formation. For seabirds, there is concern that the introduction of renewable energy devices could lead to avoidance, thereby negatively impacting on energy expenditure50. Likewise, it has been suggested that hydrodynamic forces around hard structures could modify prey availability, thereby increasing a seabird’s rate of energy acquisition51. While our findings suggest that terns exploit the flood wake of a device, an overall ecological (population-level) benefit through increased individual energy acquisition can only be determined when accounting for parameters relating to, e.g., foraging success, prey profitability, and breeding performance52.With the intensification of man-made structures in coastal seas, new synergies between these and marine predators are likely. Our findings demonstrate that wake features, predictable in time and space, persistently attract top predators at highly localised scales. We also provide the first empirical evidence that localised hydrodynamic forcing attributable to an anthropogenic structure can present a mechanism to promote a foraging hotspot, where predator aggregations exceed those at adjacent natural wake features. A broader perspective on the ecological implications of offshore installations is critical53), monopile foundations similar to the SeaGen design present the most common substructure (66%53) and lead to comparable wake vortices54. However, even submerged tidal turbines, and more so arrays, placed in unsteady flows will change the local hydrodynamic regime, including wake effects55 and more empirical data are required to predict changes in hydrodynamics and foraging habitat.In the expanding renewable energy sector  the Routen Wheel , turbulent whirlpool structures that are generated from a shallow pinnacle (5 m depth) surrounded by 20 m deep waters. Here, the asymmetrical bathymetry of the channel promotes a more intense turbulence field at the surface during the ebb tide. While all three wake features differ in composition, they all predictably create local zones of extreme turbulent flow structures and tern feeding flocks had been observed at all three features prior to the study. With various tern  colonies located across Strangford Lough, Swan island presents the nearest colony to any of these wake features , SeaGen (n = 13) and Routen Wheel (n = 11) with a total observation time of 23.38 h, 25.26 h and 22.14 h, respectively. A general-additive mixed effect model (GAMM) was performed to quantify variances in the probability of encountering terns and the number of terns if encountered among tidal states and locations. A binomial model was used for the probability of encountering terns, and a negative binomial was used for the number of terns if encountered. Location was used as a categorical explanatory variable. Tidal state (hours after high water) was used as a continuous and non-linear explanatory variable. The number of knots was constrained to six to avoid over-fitting. Tidal state was also modelled as an interaction with location to account for differences in patterns among locations. An AR1 structure was used to account for temporal autocorrelation in model residuals within locations. Model parameters were used to predict variations in the probability of encountering terns and the number of terns if encountered across different locations and tidal states. Differences in probabilities and numbers across locations and tidal states were tested for significance (p < 0.05) using F-tests. Models were performed in the mgcv packages in R Statistics65.A vantage point study was designed to collect count data of terns over the wake features between 18th July 2018 and 12th August 2018. Vantage points were located on the shore with a 200 m–1 km distance from each feature and covered an area of ~0.05 km² for each site to assess bird numbers associating with each localised wake feature. Observations covered all tidal states over a spring and neap tidal cycle. Using binoculars (Opticron Verano BGA HD and Nikon Monarch 10 × 42), counts of hovering or diving birds deemed foraging were completed every 2nd/3rd min for 15 min with a 5 min rest period to avoid observer fatigue . Number of surveys varied minimally per site, with Walter’s Rock  over the flood wake of SeaGen to capture seabird flight tracks over time. Surveys reported here were conducted on 11 July 2018 during a flood tidal cycle (07:30 h – 08:30 h GMT) with a total flight time of 41 min. All missions were completed in accordance with local regulations and flown by the same qualified (UK Civil Aviation Authority) pilot. The UAV camera was calibrated in the lab and video sequences post-processed using MATLAB . Georeferenced composite panoramic images captured the distribution of terns up-and downstream of SeaGen. Machine learning approaches were used to identify, count and track terns over SeaGen’s flood wake. Briefly, moving objects were detected using frame-to-frame differencing, segmentation and then filtered by size to remove sun-glint speckles and large foam patches. Images of potential targets were then passed through a trained “Bag of Features” classifier before using Kalman filters to compile tracks of those targets identified as terns only. The classifier was trained using 806 manually identified images each of foam and terns, with an average accuracy of 93% when applied to a validation set of 3764 images.To record fine-scale foraging behaviour in relation to the wakes, UAV surveys were performed from the nearest accessible shore location to each feature using a DJI Mavic Pro quadcopter recording 4 K video at 25 fps. The UAV was flown manually using the DJI Go v4.0 application. In order to comply with best practices39, volume-backscattering strength  was calculated across a maximum of 40 bins from the ADCP’s recorded raw echo intensity data using a working version of the sonar equation as originally described in Deines67 and updated by Mullison68. The backscatter equation accounts for two-way transmission loss, time-varying gain, water absorption, and uses an instrument- and beam-specific RSSI scaling factor to convert counts to decibels. This makes it a more robust measure of scattering compared to raw echo intensity, which can be more readily extracted from the ADCP. Sv was calculated for each bin along each of the four beams of the ADCP. For each range bin, the maximum of the four beams (Svmax) was taken to create depth profiles of the maximum level of scattering across the water column. In high-flow environments, high values of acoustic scattering are dominated by enhanced surface bubble entrainment and sediment re-suspension69.Vessel-mounted ADCP transects were performed on 13 Aug 2018 using a pole-mounted (1.15 m depth) RDI Workhorse Monitor broadband ADCP (600 kHz) in bottom-tracking mode with a vertical bin size of 1 m. All data was acquired using VMDas software  and post-processed in WinADCP . True current velocities were computed by subtraction of the bottom-tracked boat velocity. To quantify the acoustic scattering in the water column as a metric for macro-turbulencewww.dhisoftware.com)70 was used to simulate particle movement in the Narrows. In short, the model uses a finite volume method by solving a depth averaged shallow water approximation. Full details of the model setup can be found in Kregting and Elsäßer70. The Strangford Lough model was coupled to a particle tracking module that incorporates advection and dispersion resolved using the Langevin equation. For horizonal movement, in the absence of any dispersion  information, the scaled eddy viscosity was used with the software recommended constant value of 1.0. For the vertical dispersion, a constant dispersion value of 0.01 m2 per second was used. Changes in flow velocity throughout the water column were calculated based on the bed friction velocity, a parameter calculated directly in the hydrodynamic model. Passive particles as proxy for microscopic or small organisms were released from the Irish Sea at a depth of 10 m, approximately half the water column height (Fig. The Strangford Lough hydrodynamic model developed using MIKE21 modelling software (DHI Water and Environment software package: Further information on experimental design is available in the Reporting SummarySupplementary Information


Plaque being protective, as it immobilizes cytotoxic amyloid-β, rather than AD's causative agent. The presence of natural antibodies against cytotoxic amyloid-β implies the existence of a protective anti-AD immunity. Hence, for vaccines to induce a similar immunoresponse that prevents and/or delays the onset of AD, they must have adjuvants that stimulate a sole anti-inflammatory Th2 immunity, plus immunogens that induce a protective immunoresponse against diverse cytotoxic amyloid-β conformers. Indeed, amyloid-β pleomorphism may explain the lack of long-term protection by monoclonal antibodies that neutralize single conformers, like aducanumab. A situation that would allow new cytotoxic conformers to escape neutralization by previously effective monoclonal antibodies. Stimulation of a vaccine's effective immunoresponse would require the concurrent delivery of immunogen to dendritic cells and their priming, to induce a polarized Th2 immunity. An immunoresponse that would produce besides neutralizing antibodies against neurotoxic amyloid-β oligomers, anti-inflammatory cytokines; preventing inflammation that aggravates AD. Because of age-linked immune decline, vaccines would be significantly more effective in preventing, rather than treating AD. Considering the amyloid-β's role in tau's pathological hyperphosphorylation and their synergism in AD, the development of preventive vaccines against both amyloid-β and tau should be considered. Due to convenience and cost, vaccines may be the only option available to many countries to forestall the impending AD epidemic.The apparently near-term effects of the monoclonal antibody BAN2401 in slowing the progression of prodromal Alzheimer's disease (AD) has created cautious optimism about the therapeutic use of antibodies that neutralize cytotoxic soluble amyloid- Epidemiological data shows that the onset of AD in Latin Americans occurs on the average seven years earlier than in white Americans and that they also have a higher incidence of diabetes, which is an AD risk factor , 114. Ye


The aim of this study was to determine the effect of vitamin E andpolyphenols on the antioxidant potential and meat quality of broiler chickensfed diets supplemented with low-quality oil. The experimental materialscomprised 120 male Ross 308 broilers . Dietary supplementation with vitamin E and/orpolyphenols was applied in the following experimental design: group I(negative control) – without supplementation without low-quality oil; groupII (positive control) – without supplementation  Low-quality oil added tobroiler chicken diets decreases productivity , increases mortality  and decreases thequality of animal products . Diets rich inpolyunsaturated fatty acids (PUFAs) increase susceptibility to lipidperoxidation and decrease the antioxidant capacity of animals. Rancid lipidsthat undergo auto-oxidation processes contain free-radical-generatingsubstances that exert adverse effects on the health status of birds.Oxidation reactions produce harmful peroxides that are converted intohydrocarbons, ketones, alcohols, organic acids and aldehydes  including malondialdehyde (MDA) with mutagenic and carcinogenicproperties. Oxidation reactions also decrease the content of vitamins A andE and carotenoids . Increasedproduction of reactive oxygen species (ROS) disrupts redox balance andcontributes to oxidative stress with harmful implications for health .Supplementation with antioxidants is an effective method of minimising theadverse consequences of low-quality oils in animal diets. Recent years havewitnessed growing levels of awareness among consumers and food producersregarding the origin of feed additives in livestock nutrition. The demandfor products of natural origin continues to increase.Polyphenols are substances of plant origin which possess antioxidantproperties and may reduce the negative effects of oxidative stress . Their antioxidant potential iscomparable with that of the major biological antioxidants: The results of in vitro and in vivo studies demonstrated that polyphenolsdeliver positive effects in animal nutrition , including in animals whose diets contain oxidisedoils or oils rich in PUFAs .The research hypothesis postulated that polyphenols from onions and grapeseeds can partially replace vitamin E in terms of antioxidant activity inbroiler chickens fed oxidised oil. The aim of this study was to determinethe effect of polyphenols and increased inclusion levels of vitamin E on theantioxidant potential and meat quality of broilers fed diets supplementedwith low-quality oil.22.1The experiment was approved by the Local Ethics Committee for AnimalExperimentation in Olsztyn, Poland. The experimental materials comprised 120male Ross 308 broiler chickens . The chickens were kept in cages without litter (two birds percage). The experiment lasted 35 d. The body weights of birds weredetermined at weekly intervals. Feed intake and mortality rates were alsomonitored, and the data were used to calculate the feed conversion ratio(FCR).The birds were fed ad libitum starter (1–14 d of age) and grower (15–35 dof age) diets in mash form, which fully met the nutrient requirementsof broiler chickens , and they hadfree access to water.The Proviox Nucleus preparation  used in the study containspolyphenols obtained from onions (quercetin and flavonols) and grape seeds. Broiler chickendiets were supplemented with 50 % vitamin E in the form of dl-2.2Feed and breast meat samples were assayed for dry matter (DM), crude ash,crude protein (CP), ether extract (EE) and crude fibre (CF) by standardmethods . Vitamin E concentrations were measured bychromatography (HPLC – high-performance liquid chromatography) , according to the method described byPolish Standard PN-EN ISO 6867 (2002). The amount of metabolisable energy andthe content of amino acids and minerals in broiler chicken diets werecalculated according to the Nutrient Requirements of Poultry (2005). Thecomposition and energy value of diets without supplemental vitamin E arepresented in Table 2.Blood samples were collected from broiler chickens on day 35. The activity ofsuperoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) was determinedin heparinised whole blood. Total antioxidant status (TAS) and theconcentrations of vitamins A and E were determined in the blood serum. Bloodsamples were stored at a temperature of Total antioxidant status, SOD and GSH-Px activity were determined with theuse of the Randox kit according to the manufacturer's recommendations. Theactivity of SOD was measured as the percent inhibition of the rate offormazan dye formation (505 nm wavelength). The activity of GSH-Px inheparinised whole blood was determined with the use of cumene hydroperoxide.In the presence of glutathione reductase and NADPH (nicotinamide adenine dinucleotide phosphate), oxidised glutathione isconverted to a reduced form, and NADPH is oxidised to NADPSerum samples (1 mL) were deproteinised with anhydrous ethanol (1 mL),extracted with 5 mL of n-hexane (Vortex 5') and centrifuged  was calculated based on the activity oftocopherols relative to At 35 d of age, 10 birds per group were slaughtered and carcass qualitywas evaluated. Samples of fresh breast muscles and liver were assayed forthe concentrations of lipophilic vitamins  asdescribed by Rettenmaier and Schüep (1992), hydrophilic vitamins(vitamin C) as described by Omaye et al. (1979), and thiobarbituricacid-reactive substance (TBARS) values according to Sørensen andJørgensen (1996).The percentage content of breast muscles in the carcass and abdominal fat,heart and liver in live body weight was determined. Samples of fresh breastmuscles were assayed for proximate chemical composition  and meatquality parameters including acidity , colour in the CIE peroxidisability index (PI) – (% monoenoic atherogenicity index (AI) – (C12thrombogenic index (TI) –  by chromatography,including saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs),n-3 and n-6 polyunsaturated fatty acids (PUFAs), hypercholesterolemic fattyacids (OFAs) and hypocholesterolemic fatty acids (DFAs), in accordance withthe criteria proposed by Barowicz et al. (2000) and Leskanich andNoble (1997). The fatty acid percentages were calculated with the MicrosoftExcel (Microsoft Excel 2013) software and the mean value of each fatty acidwas used to calculate the following indexes:The AV and POV of rapeseed oil were determined according to Polish Standards PN-88/C-04288/06 and PN-88/C-04288/10, and MDA concentration was determined asdescribed by Esterbauer et al. (1991).2.3The results were processed statistically by one-way analysis of variance andDuncan's test. The arithmetic mean (33.1The applied antioxidants did not improve the growth performance of broilerchickens fed diets containing oxidised rapeseed oil (Table 3). Broilermortality was not recorded in groups receiving diets supplemented with 100 mg kg3.2At 35 d of age, no differences in TAS or SOD activity were found betweengroups (Table 4). The addition of polyphenols to diets (groups V and VI)increased GSH-Px activity in the blood of broiler chickens (The addition of polyphenols to diets (group VI) led to an increase .The lowest retinol content .However, dressing percentage tended to increase in broilers fed dietswithout rancid oil (group I) and diets containing rancid oil andsupplemented with polyphenols (group VI).No differences in the percentage content of the heart, liver and abdominalfat were found between groups (Table 5). The analysed antioxidants had nosignificant effect on the weight and pH of the crop and proventriculus inall groups. Higher gizzard weight  and dietssupplemented with 100 mg kgThe breast muscles of broilers fed diets supplemented with polyphenols werelighter . The analysed meatsamples did not differ in the content of dry matter or protein (Table 6).Crude fat content was nearly two-fold lower in the breast muscles ofbroilers fed diets without oxidised oil (group I), compared with birds feddiets with oxidised oil and without antioxidants (group II) and diets withthe highest inclusion level of polyphenols (group VI) . Broilers whose diets did not contain oxidised oil(group I) were characterised by lower concentrations of total MUFAs andhigher content of PUFAs, relative to the remaining groups.The content of n-3 PUFAs tended to decrease in broilers whose diets weresupplemented with polyphenols only relative to the negative control group(group I). The breast muscles of birds given polyphenols only (group VI) andbroilers fed low-quality oil without antioxidants were characterised by alower content of n-6 PUFAs, DFAs, a lower4Vegetable oils are added to poultry diets to increase the energy value offeed rations. Oils rich in PUFAs are more susceptible to peroxidation.Oxidised products are characterised by high ROS levels and a reduced contentof fat-soluble vitamins . Research shows thatdietary supplementation with oxidised oil compromises production results. In the present study, oxidised rapeseed oil as dietarystressor did not affect the growth performance of broilers, but birds whosediets were supplemented with antioxidants had higher final body weights,although the differences noted were not statistically significant. Theevaluated antioxidants did not improve performance parameters. Similarresults were reported by Açıkgöz et al. (2011), in whose studymoderately oxidised oil did significantly affect the body weights of malebroilers, feed intake and feed conversion, and a vitamin E dose of200 mg kgDietary stressors like oxidised feeds are a source of free radicals, whichinduce oxidative stress in birds . The results of numerousstudies have confirmed that oxidised dietary oils decrease theconcentrations of antioxidants  and Tavarez et al. (2011) did not observe differences in carcass dressing percentage, carcassweight or breast muscle weight of broilers whose diets contained rancid oiland were supplemented with vitamin E or antioxidants . Zduńczyk et al. (2002) did not report differences in thecarcass dressing percentage or abdominal fat content of turkeys fed oxidisedoil. In the work of Anjum et al. (2004) and Racanicci et al. (2008),chickens fed oxidised or fresh oil did not differ in carcass dressingpercentage or gizzard weight. Liver weight increased in birds givenlow-quality oil . In the literature, there is a generalscarcity of studies investigating the effect of polyphenols on carcassdressing percentage and the structure of the gastrointestinal tract inbroilers fed low-quality dietary oils.Peroxidation induces changes in the fatty acid profile of oils and fats.Low-quality oils with high AV and POV are sources of free radicals that exertadverse effects on unsaturated fatty acids and meat quality. Oxidative damagecompromises the quality of poultry meat, including its colour, structure andwater-holding capacity, and increases drip loss . In the current study, low-quality rapeseedoil increased pHIn the present study, the content of PUFAs was higher and the AI was lowerin chickens that did not receive oxidised oil and in broilers whose dietswere supplemented with vitamin E alone or vitamin E and polyphenols relativeto birds that were fed oxidised oil without antioxidants. Similarobservations were made by Sheehy et al. (1994) in whose study, PUFAconcentrations were higher in the lung tissue of chicks receiving fresh oiland in birds given oxidised oil with vitamin E, and they were lowest inthe group of birds fed oxidised oil without antioxidants. Racanicci et al. (2008)also reported a decrease in the concentration of C5It can be concluded that the applied antioxidants had no effect on thegrowth performance of chickens fed oxidised oil. Increased dietary inclusionlevels of vitamin E and/or polyphenols improved the antioxidant status inthe blood and increased the content of non-enzymatic antioxidants in theliver and breast muscles of broilers fed low-quality oil. The testedantioxidants had no influence on carcass quality parameters in chickens fedoxidised oil. However, birds fed diets with the addition of vitamin E werecharacterised by higher gizzard weight and higher pH of gizzard digesta.Dietary supplementation with vitamin E and polyphenols or polyphenols alonecontributed to a lighter colour and lower pH of breast muscles and anincrease in the content of fat and ash in the breast muscles of broilers fedoxidised oil. The breast muscles of birds receiving supplemental vitamin Ewere characterised by higher concentrations of n-6 PUFAs and DFAs, a moredesirable


Anaplastic thyroid carcinoma (ATC) is a deadly disease with very limited therapeutic options. There is an urgent need for new and efficacious drugs. Unfortunately accrual in clinical trials is problematic because of the rarity of the disease and often poor performance status at diagnosis. Recently some data have emerged suggesting a role for immunotherapy in the treatment of ATC. We describe the case of a 75-year-old patient with poor performance status and compromised airway and oesophagus at diagnosis, showing a rapid and dramatic response to first line single agent pembrolizumab. Disease progression in the brain occurred 16 months after initial diagnosis. At that time there was ongoing extracranial disease control. Anaplastic thyroid carcinoma (ATC) is a rare and aggressive disease representing 1-2% of all thyroid cancers but accounting for 50% of annual thyroid cancer related mortality. Approximately 50% of patients present with metastatic disease and treatment options are limited except for a subset of patients with BRAF-mutated tumors. Recent data suggest a potential role for immunotherapy in this disease desperate for new therapies. We describe a patient with poor performance status at presentation showing a rapid and dramatic response to first line single agent pembrolizumab.A 75-year-old female never smoker presented to the ear, nose and throat specialist with hoarseness, dry cough, difficulty swallowing and a 3 kg weight loss since 1 month.Ic well-differentiated serous papillary ovarian carcinoma treated with surgery and adjuvant chemotherapy.5 years earlier she was treated with radioactive iodine for a toxic multinodular goiter. She also had a past medical history of a stage On clinical examination, a multinodular and enlarged thyroid gland was palpated. A computed tomography (CT) scan of the neck and chest revealed a large mass (8 × 9 cm) in the anterior mediastinum involving the right thyroid lobe and causing deviation of both trachea and oesophagus. There were multiple pathologically enlarged cervical, supraclavicular and mediastinal lymph nodes as well as multiple bilateral lung metastases . FurtherThe case was discussed at the multidisciplinary tumor board and based on clinical presentation, imaging and pathology a diagnosis of primary anaplastic thyroid cancer was made. Next generation sequencing  was negative for molecular drivers. PDL1-staining was positive with a high expression (60% of tumor cells). At that point the clinical condition of the patient was rapidly deteriorating due to dysphagia with impaired food intake. The possibility of palliative stenting of the oesophagus was discussed with the gastroenterologist but was considered technically impossible. The patient was unfit for a clinical trial. Based on occasional case reports describing exceptional response to immunotherapy in patients with anaplastic thyroid cancer, the patient was offered the possibility of treatment with pembrolizumab. After only 2 cycles, the patient noticed a marked improvement of dysphagia and after 3 cycles, a near complete response was seen on CT . ThyroidAfter 8 cycles however, the patient developed a severe grade 4 colitis  requiring aggressive fluid resuscitation and treatment with systemic steroids. Due to further clinical deterioration infliximab was administered, after which gradual improvement occurred. 3 months after the last administration of pembrolizumab, there were no signs of tumor progression. As most guidelines recommend permanent discontinuation of immunotherapy after grade 4 gastro-intestinal toxicity, a watchful waiting approach was proposed with follow-up CT of the chest and neck every 3 months.6 months later (16 months after diagnosis), the patient presented to the emergency department with gait difficulties, falling tendency to the left, left-sided facial palsy and hypoesthesia of the left upper arm. Magnetic resonance imaging (MRI) of the brain revealed a 19 × 18 × 19 mm lesion in the right thalamus. Stereotactic biopsy of this lesion was compatible with metastasis of known anaplastic large cell carcinoma. There were no signs of extra-cranial tumor progression . StereotAnaplastic thyroid carcinoma (ATC) is a rare and aggressive disease with a median overall survival of less than 6 months and a 1-year survival of only 20%. In metastatic disease particularly, treatment options are limited [Lately promising results were reported with targeted therapies. In a single-arm, open-label study 17 ATC-patients were treated with the multikinase-inhibitor lenvatinib. The objective response rate was 24%, the median progression-free survival (PFS) was 7.4 months and the median overall survival (OS) 10.6 months . More imNCT02688608; nivolumab plus ipilimumab in NCT03246958).The role of immunotherapy in ATC remains to be elucidated. Kolllipara et al. described an exceptional response with immunotherapy (nivolumab) in a patient with BRAF positive ATC who showed a mixed response to the BRAF-inhibitor vemurafenib . In a smIn most ATC-trials however, accrual is hampered by the rarity of the disease and the often poor performance status at initial diagnosis. In a review article on diagnosis and management of ATC, Chintakuntlawar et al. state that it might therefore be reasonable to consider PD-1 inhibitors in these patients [We hereby describe a case of metastatic ATC treated with single agent pembrolizumab as first line therapy. Our case illustrates that a rapid and clinically meaningful response can be obtained even in a very sick patient with compromised airway and oesophagus. Clinical benefit occurred after only 2 cycles of pembrolizumab and near complete response was obtained after 3 cycles. Unfortunately, Pembrolizumab had to be discontinued because of grade 4 colitis and relapse occurred in the brain 14 months after initial diagnosis. At that moment there was ongoing extra-cranial disease control. As the initial impressive response to immunotherapy in our patient is intriguing we retrospectively assessed tumor infiltrating lymphocytes (TILs) on H&E slides from a metastatic lymph node. Stromal TILs (sTILs) could not be scored as no stroma was present in the biopsies. The percentage of intratumoral infiltrating lymphocytes (iTILS) was 30%. Whether PD-L1 or H&E based assessment of TILs (and at which cut-off) are predictive biomarkers in anaplastic thyroid cancer is a question that hopefully can be answered by ongoing or future clinical trials.


Similar procedures were conducted with the using of CRC HCT116 and HT29 cell lines. We found that the detailed configuration of CSC-like markers significantly influenced the maturation and activation of DCs after stimulation with cancer cells lysates or culture supernatants. This basic stimulatory effect was enhanced by LPS that is normally present in CRC CSCs niche. The increased number of CD29+ and CD44+ CSCs presented the opposite impact on treated DCs as showed by many significant correlations. The CD133+ CSCs seemed to impair the functions of DCs. The more CD133+ CSCs in tumor sample the lower number of activated DCs evidenced after stimulation. Moreover, our results showed superiority of the spherical culture model over the adherent one since spherical HCT116 and HT29 cells presented similar influence on DCs properties as CRC patients cancer cells. We concluded that the DCs features may depend directly on the properties of CSCs affected by progression status of tumor.The therapy of colorectal cancer (CRC) patients is often unsuccessful because of the presence of cancer stem cells (CSCs) resistant to conventional approaches. Dendritic cells (DC)-based protocols are believed to effectively supplement CRC therapy. Our study was aimed to assess how the number and properties of CSCs isolated from tumor tissue of CRC patients will affect the biological characteristics of  Despite increasing knowledge concerning pathogenesis, genetic and epigenetic alterations associated with the CRC development, effectiveness of the therapy remains unsatisfactory because of cancer recurrence and metastases. Cancer stem cells (CSCs) were showed to be responsible for metastasis, recurrence, relapse and resistance to conventional chemotherapy4 which can destroy only proliferating and mature cancer cells while quiescent CSCs survive. Therefore, elucidation of the mechanisms of CSCs maintenance is important for the understanding of cancer cell persistence and relapses. Additionally, that may enable specific CSCs targeting as a potential therapeutic strategy to definitively eradicate cancer7. The CSC-specific immune responses in breast cancer and glioblastoma10 were proved; despite the immune evasion of the CSCs. Vaccination of dendritic cells (DCs) with irradiated glioma tumorspheres was demonstrated to increase the survival rate in a mice cancer model9.Colorectal cancer (CRC) is one of the most frequent malignancies and the fourth most common cause of cancer-related deaths in the world with 1.2 million new cases being diagnosed every year. The 5-year survival rate of patients with stage IV CRC is less than 10%+ and effector CD8+ T lymphocytes12. Although many tumor-associated antigens (TAA) have been already found in CRC cells, such as CEA (carcinoembryonic antigen)14, WT1 (Wilms’ tumor gene 1)16, MUC1 (mucin 1)13, MAGE (melanoma-associated antigen gene)19, p5320, the heterogeneity and patient-specificity are severe obstacles to use the anti-TAA therapies. A very promising strategy to prime cancer-specific T cell responses is dendritic cell-based immunotherapy. Autologous cancer cells lysates could provide a wide range of ‘personalized’ cancer epitopes including neoantigens which result from cancer–specific DNA mutations21. Cancer immunotherapy approaches based on the vaccination with the use of TAA, whole cancer cells or viral vectors, have been tested to treat CRC patients. However, despite the relative effectiveness of these treatments side-effects are still observed in the large proportion of patients and the number of recurrences is still high25.The main goal of current efforts worldwide is to incorporate recent discoveries into novel treatment algorithms. One of the potential methods is immunotherapy which is hoped to induce CRC-specific cytotoxic reactions mediated by antigen presenting cells (APCs) (including DCs), helper CD428. The main role of DCs is to mediate innate immune responses and induce adaptive responses acting as powerful APCs29. DCs represent a widely distributed heterogeneous population of professional APCs that originate from bone marrow precursors known as MDPs (monocyte and DC progenitors)30. The critical issues underlying DC-immunotherapy is limited number of DCs available from each patient and, additionally, those DCs can represent variable activities: antigen presenting, cytotoxic32 or suppressive35.Dendritic cells qualitatively and quantitatively coordinate the function of the immune system cells such as various populations of T lymphocytes, also naïve and memory B cells, natural killer (NK) cells and NKT cells through the secretion of cytokines  or the presence in their cell membranes various proteins such as CD1, CD54, CD80, CD83, CD86, CCR742. These aspects of cancer cells biology primarily depend on the access to oxygen, nutrients and growth factors. Studies conducted on tumorospheres derived from various types of cancers, including breast45, colon46, lung48 and prostate49 cancer as well as glioma8 and melanoma50 showed that sphere-based assays could be a reliable platform for development of immunotherapy targeting CSCs. It is also believed that spherical cultures can provide short-term patient-derived CSCs for the evaluation of DC-based therapies what defined the main goals of our study.It was reported that spheroid (3-D) cultures of cancer cell lines better than adherent (2-D) cell cultures resemble original cancer in such areas as gene expression profiles, cellular heterogeneity, morphology and distribution of cancer cells3, since they were found to present lower levels of TAA and MHC class I proteins3. Additionally, it was showed that spherical cell cultures can secrete IL-4 that inhibited functions of cytotoxic T lymphocytes and NK cells53.Cancer cells expanded in the spheroid form were showed to induce different immune response in comparison to cells cultured in adherent form55. These cells are also responsible for the main clinical complications of anti-cancer therapies including appearance of relapses, sometimes years after chemo- or radiotherapy56. Therefore, elucidating the mechanisms of CSC maintenance is important for our understanding of tumor cells persistence and relapses and may enable specific targeting of CSCs, a promising therapeutic strategy to eradicate cancer58. The identification and classification of CSCs remains controversial, as none of the known markers are universal and reliable for the identification of CSCs in all types of tumors59. The most commonly used marker of CSCs (including CRC) is CD133 protein (prominin-1)59. According to some analyses only CD133+ cells are able to reproduce a CRC tumor in a mouse xenotransplantation model60. However, some other groups showed independently that CD133− cells also possess high proliferative and differentiating potential, comparable to those of CD133+ CRC-CSCs61. Proteins which are also considered as reliable CSCs markers are CD24, CD29, CD44, CD166, Aldh1 and other63.The discovery of cancer stem cells enables the understanding of some of the most lethal features of cancer. The CSC model states that cells within the tumor are hierarchically organized with the subpopulation of cancer stem cells with high tumorigenicity on the top. CSCs were showed to be able to self-renew and to generate differentiated cells within the tumor mass often described as non-CSCs53. Although CSCs could inhibit differentiation of CD8+ naïve T cells, they could not inhibit the cytotoxicity of previously differentiated and activated CTLs53. Vaccination of CRC patients with autologous, cancer lysate-loaded DCs has been showed recently to be a well-tolerated therapeutic approach65 but the role of CSCs has not yet been analyzed.The analyses of the expression profiles of co-stimulatory molecules and cytokines suggest that CSCs might contribute to the formation of an immune‐suppressive cancer microenvironment including immune escape from cytotoxic T lymphocytes (CTLs)in vitro experiments. We hoped to provide the valuable information which could contribute to the clinical applications of such therapies.The aim of our study was to evaluate how the CSC-like properties may influence the effectiveness of DC stimulation and modulate their phenotype, endocytotic activity and ability to induce the proliferation of lymphocytes. According to our best knowledge no other study has addressed the evaluation of specific DCs pulsed with CSC-antigens-rich lysates and tumor conditioned medium (TCM) activities in such an ample repertoire of ex vivo or lost their sphere-forming capacity after first passages. At the same time we wanted to highlight the fact that our functional tests were conducted with cells, TCM or lysates obtained from third passage, although the phenotypic analyses showed that CSCs along the expansion were stable what is presented in supplementary figures . The cancer tissue samples which gave the most stable spherical cultures were collected from four patients in stage III and one patient in stage IV of CRC (according to the current TNM staging). Moreover, the presence of lymph node metastasis correlated with the proportion of CD133+CD44−CD29+ CSCs during expansion .We identified significant correlation between TNM status and the life span in culture of CSCs derived from CRC patients  and IL12-p70  in the media from DCs culture. The substantially higher concentrations of IL-12p70 and granzymes in the supernatants from DCs cultures were found in samples which originated from CRC patients with TNM I-II stages in comparison to patients with more advanced stage (TNM III-IV). The same relationships were observed in all applied options of the DCs stimulation  increased equally.The lysates and TCM of CSCs from CRC patients increased the proportion of CD11cres Fig.  and even+CD83+ DCs proportion in the most significant manner and it is even more meaningful as lysates alone lowered the MFI of CD80+CD83+ cells below the LPS-stimulated DCs level. At the same time, TCM exerted much weaker influence since we observed the proportion of CD80+CD83+ DCs at the level of iDCs and significantly lower than LPS-stimulated DCs  would affect the number of FasL+ DCs. We found that factors derived from adherent cultures of HCT116 and HT29 cells significantly decreased the proportion of FasL+ DCs in comparison to iDCs +HLA-DR+and CD80+CD83+ DCs and the proportions of CSCs with specific phenotype. The negative correlations between CD11c+HLA-DR+ DCs and the presence of CD44+CD29− CSCs in both CD133+ and CD133− fractions indicated the negative impact of CD44+ CSCs on the in vitro modified DCs. In parallel, the same configuration of CSCs markers positively influenced the activation status of DCs since the positive correlations were found between CSCs lacking CD29 marker and CD80+CD83+ DCs proportion. Moreover, the CD133 marker was found to exert the most significant impact on the CD80+CD83+ DCs number , we decided to conduct the co-culture of DCs with autologous effector cells for 24 hours following the stimulation of DCs with lysates or TCM. Next, we analyzed the changes in the MFI of T regulatory-like CD4+CD25+ lymphocytes on the same level as iDCs after co-culture. At the same time these values were lower than MFI measured for LPS-stimulated DCs  was followed by the colorimetric proliferative assay to find out if DCs could change the proliferative potential of the effector cells  prevailsThe most effective endocytosis was observed for DCs stimulated with adherent HCT116-derived agents, significantly higher than in iDCs and LPS-stimulated DCs. The cancer cells derived from adherent and spherical cultures triggered different effects on the analyzed phenomenon and these differences were cell line-specific Fig. . HCT116 −CD44+CD29− cells within whole cancer cells population correlated negatively with the endocytotic activity of DCs stimulated with TCM . In parallel, the CD133−CD44−CD29+ cells exerted the opposite effects since the correlation rank was positive . These data suggest that the presence of CD44 protein on a surface of CSCs seemed to lower the endocytotic activity of DCs when used for DC stimulation. For the population of CD133+ CSCs we could observe similar tendencies.When we analyzed the correlations between phenotype of CRC lines-derived CSCs and endocytotic activity of stimulated DCs, we found no relationships between those parameters when adherent lines were taken for DC stimulation. On the contrary, when we used spherical CRC cells to induce DCs maturation, the numerous correlations were identified. Surprisingly, lysates and TCM exerted opposite effects. Lysate/LPS seemed to enhance the endocytotic activity of DCs regardless of CSCs phenotype whereas DCs treated with TCM/LPS demonstrated diverse effectiveness of particulate material uptake. The proportion of CD133+HLA-DR+ and CD80+CD83+ DCs after specific stimulation with TCM, the higher endocytotic activity of these DCs  found. Nonetheless, lysate/LPS appeared to decrease the antigen uptake simultaneously with increased proportion of activated DCs .Additionally, our results demonstrated that the higher the proportion of CD11cIn order to evaluate the direct cytotoxic potential of DCs obtained during our procedures, we conducted co-cultures of healthy donors blood monocyte-derived DCs with HCT116 and HT29 cells in both adherent and spherical forms.+7AAD+ cancer cells after incubation with DCs treated with adherent cancer cell-derived agents was similar to the effect triggered by iDCs. The exceptions were DCs stimulated with HT29-derived TCM and HCT116-derived lysate/LPS . Similarly, the spherical culture-derived TCM in the cooperation with LPS exerted positive effect on the abilities of DCs to increase the proportion of NKT cells . Surprisingly, the adherent CRC cell lines seemed not to influence DCs cytotoxic potential.Surprisingly, after the analysis of the correlations between DCs phenotype and their cytotoxic properties, we could notice the positive relationship only within DC populations stimulated with spherical culture-derived agents, especially with TCM/LPS  presented the highest secretory efficiency. Moreover, DCs in our study were the most potent to secrete IL-12p70 from all measured soluble proteins Fig. . The higThe highest concentrations of granzymes were found in supernatants from the cultures of HT29-dervived lysates and TCM-stimulated DCs, especially when lysates and TCM were administrated with LPS, the values were 230 times higher than these obtained for iDCs Fig. . When weThe lowest values of secreted proteins were obtained for soluble form of FasL since in many cases we even found its concentration to decrease below the level acquired by iDCs Fig. . Only adIFNγ was the most effectively secreted by DCs stimulated with lysate/LPS or TCM/LPS originated from both adherent CRC cell lines Fig.  whereas We found the highest concentrations of IL-10, IL-12p70, granzymes and IFNγ in cultures of DCs stimulated with adherent HT29-derived lysate/LPS and TCM/LPS. The most dominating seemed to be the secretion of proinflammatory IL-12p70 (up to 667-fold increased concentration than iDCs), but also granzymes (up to 230x) were found outstanding.69, it became crucial to find an alternative source of fully effective DCs for clinical use72. Another problem is a limited access to monocytic precursors from individuals with advanced cancer. The characterization of the optimal environment supporting development of effective DCs subpopulations requires relatively high number of these cells. We found out that leukocytes from healthy volunteers can provide monocytic precursors which64 can be in relatively simply way differentiated toward DCs with the use of GM-CSF and IL-4. The isolation and in vitro differentiation of circulating monocytic precursors enabled us to establish the appropriate methodology for specific evaluation of anti-cancer properties of in vitro modified DCs. The clinical application of DC-based procedures for the treatment of cancers with low immunogenicity including colorectal73, prostate and renal74 cancers and hematological neoplasms75 has been previously reported. In vitro modified DCs applied as monotherapy for cancer patients were found to be safe and rarely induced toxic side effects in contrast to treatments that used cytokines and other biologically active molecules77.In order to evaluate the DCs suitability for anti-cancer regiments, the first requirement is to get a system in which they could be modified and propagated. Since it was found in cancer patients that DCs mature defectively or they could even be kidnapped to support the cancer progressionin vivo environment for CSCs survival37. Such diversification of the cellular forms allowed us to directly compare the usefulness of both forms of CRC cell lines for the in vitro investigations. According to our best knowledge no other study has addressed the evaluation of specific DCs activities in such an ample repertoire of in vitro experiments.Since we conducted most of the laboratory tests simultaneously on CSCs isolated from cancer tissue of CRC patients and on two commercially available CRC cell lines (HCT116 and HT29), it was possible to compare corresponding panels of DCs activities. Moreover, we expanded CRC lines in two distinct forms: adherent  and spherical, which is thought to resemble 36, the adherent forms of the studied HCT116 and HT29 CRC cell lines are characterized by 100%-enrichment in CD133+CD44+CD29+ CSCs, however, when the culture conditions were modified into sphere-forming environment, a decrease of CD133+ cells number by 39% and 59% in HCT116 and HT29 cell cultures, respectively, was found. CRC patients-derived spherical cultures appeared to contain varying proportions of CD133+ cells (ranging from 2 to 96%). Importantly, that seemed to be related to the cells survival time in culture since we found significant correlation between the number of passages conducted on particular population and the TNM status of CRC patient who was a donor of these cells.As we reported earlierin vitro experiments on CRC patients-derived DCs, we used monocyte-derived DCs of healthy volunteers that were stimulated with TCM or lysates of HCT116 and HT29 CRC cell lines. The diverse results observed for DCs stimulated with CRC lines-derived lysates and TCM may be associated with different origin of these lines and epigenetic and genetic differences between them78. HCT116 cells represent non-differentiated and highly aggressive cell line that corresponds to the TNM III stage and HT29 line is known as more differentiated and less invasive (TNM II)78. The interpretation of our findings in respect of line dependency (and their origin dependency) may explain many differences between results obtained on both CRC cell lines. The obtained results were generally consistent with those conducted with DCs stimulated with agents derived from CSCs of CRC patients. For instance, stimulatory factors for DCs derived from spherical cultures of HT29 cells allowed us to obtain the higher proportions of CD11c+HLA-DR+ and slightly higher  proportions of FasL+ dendritic cells, less CD4+CD25+ and more CD3+CD56+ lymphocytes after co-culture with DCs, more pronounced proliferation of lymphocytes, slightly enhanced endocytosis and cytotoxicity, lower IFNγ and IL-10, higher IL-12p70 and sFasL concentrations in comparison to DCs stimulated with spherical HCT116-derived lysates and TCM. This summarized comparison suggests that the progression status of cancer significantly influenced the DCs features, probably due to more developed evasion mechanism typical for less differentiated and more aggressive cancers (such as HCT116 cell line). The CD80+CD83+ DCs number appeared to depend on stimulatory agent used during procedure. The lysates or TCM when used alone let us gain lower number of activated DCs when HT29 cells were a source of stimulatory factors in comparison to HCT116-derived agents, but the adding of LPS inverted these relationships. Moreover, we found that adherent cultures of CRC cell lines are not an appropriate model for analyzing any in vitro modifications and any therapeutic options for future anti-cancer protocols. Since the adherent model of cancer cells expansion is unnatural  it should be avoided in in vitro cancer studies if only the cultures of cancer cells in spherical form can be obtained and processed.Primarily, our results showed superiority of the spherical culture model over the adherent one since spherical HCT116 and HT29 cells presented similar impact on DCs properties as the cancer cells collected from tumors of CRC patients. Since we could perform only a limited number of + and CD44+ CSCs presented the opposite impact on the potential of in vitro modified DCs since the correlations (number of CSCs vs number of DCs) presented opposite correlation ranks. The presence of CD133+ cells seemed to suppress DCs activities. The higher CD133+ CSCs proportion in patients tumor samples the lower number of activated DCs was detected after stimulation. The concomitant experiments on CRC cell lines supported this conclusion since their CD133+ cells analogically suppressed the features of DCs after stimulation with lysates or TCM. Moreover, the lower concentrations of granzymes and IL-12p70 in cultures of DCs derived from CRC patients with TNM III-IV stage in comparison to patients with less advanced disease (TNM I-II) deserves a special attention. Thus, our observations suggest that the progression status of colorectal cancer may play a key role for the effectiveness of in vitro modified DCs. Our observations may be associated with the tumor evasion phenomenon reported to complicate therapy of many different types of malignancies, including CRC79.The analysis of the phenotypes of CSCs and autologous DCs demonstrated that the detailed configuration of CSC-like markers may have a crucial impact on functions of stimulated DCs. The increased number of CD2926. For ex vivo modification strategies, the most serious pitfall is an improper DCs maturation which fails to induce antigen-specific responses and may in fact provide the immunoregulatory signals to DCs that could induce an immunosuppressive response. This limiting factor contributes to the unsatisfactory success rate of the DC vaccines83. This may be caused by many reasons but the salient one seems to be the insufficient knowledge about regulatory mechanisms of DC maturation and mechanisms of cancer immune evasion which may completely invert the status of injected DCs67. Although, our results presented diverse stances in relation to that issue, we suggest that the activation pathway of DCs triggered in our culture condition could be beneficial for the purpose of cancer immunotherapy. The stimulatory agents which were used for DC modification generally did not elevate the proportion of T regulatory-like CD4+CD25+ cells. The only exceptions were DCs stimulated with adherent HCT116-derived lysates and TCM. Additionally, the analysis of IL-10 secretion by DCs showed its low concentration in supernatants from DCs cultures in all experimental options. Unfortunately, at the same time the colorimetric assay showed the inhibition of lymphocytes proliferation during their co-culture with DCs, however, DCs stimulated by cancer cells lysates increased the number of lymphocytes in all experimental variations. The observed decreased division rates of effector lymphocytes evidenced the development of immune evasion mechanism mediated by cancer cells but DCs treated with TAA-rich lysates could avoid that according to our results and other research groups85. However, the CRC patients-derived CSCs presented the significant negative impact on DCs abilities to induce proliferation of lymphocytes during co-culture. That effect was the most prominent after DCs were stimulated with lysate/LPS or TCM/LPS. Our observation seems to confirm that CSCs are responsible for cancer evasion phenomenon since the decrease of lymphocytes division rate is one of the cancer evasion elements. The differences observed in this part of our study may be associated with the origin of lymphocytes taken for co-cultures since DCs and lymphocytes used for experiments with CRC cell lines were obtained from healthy volunteers. Lymphocytes separated from the blood of cancer patients probably were encumbered by cancer-related immunosuppresion what could have markedly implicated our final results.The activation and maturation of resting (immature) DCs is supposed to be associated with the series of complicated, antigen-acquisition-dependent, tightly controlled functional and phenotypic changes. DCs are aimed to acquire all essential tools for migration to lymphoid tissue and optimal activation of target-specific immune responsein vivo or in vitro86, we decided to generate in vitro CSC-antigens-specific DCs by using either tumor conditioning medium or lysates of CSCs. Both, lysates and TCM, are known to be rich sources of a wide variety of cancer antigens necessary for DCs activation. The analysis of DC phenotype after their incubation with TCM or lysates from CSCs revealed that the proportions of CD11c+HLA-DR+ and CD80+CD83+ activated effector DCs depended on the stimulatory agent used. The basic stimulatory effect with TAA-rich lysates or TCM provided some kind of leading signal for the whole maturation but LPS seemed to intensify these effects. The usage of LPS in our experimental procedures seemed to be appropriate for colon-derived cells bacause the presence of LPS-rich bacteria is the common physiological phenomenon for this particular niche. For that reason our results suggest that LPS should be introduced into analyses of anticancer activities of DCs in in vitro studies.Since it was earlier showed that multiple tumor-associated antigens specifically expressed by cancer cells are very efficient and produce effective anti-cancerous response ex vivo generated DCs83. Other researchers applied γ-irradiated dying or staurosporin-treated HT29 cells which after being engulfed during phagocytosis were more potent in DC activation than cell lysates87. In our study lysates alone induced weaker effects on DCs functions than lysates with LPS because lysate/LPS provided significantly enhanced stimulation especially in regard to DC phenotype, their ability to kill cancer cells during co-culturing and to secrete cytokines. Our results showed that apart from applying lysates for DC stimulation, the additional support is necessary to achieve the more prominent effect of treatment.The cancer cell-derived lysates were confirmed to be a strong immune stimulus for therapeutic use of 88 study conducted on samples of CRC patients tumors, showed that TCM added to DC culture did not affect cytokine secretion or DC phenotype when compared to iDCs indicating constant hypo-responsiveness of CRC patients DCs. That is partially in accordance with our study which demonstrated that TCM inhibited patients-derived DCs secretory activity. However, these DCs presented significantly higher proportion of mature CD11c+HLA-DR+ DCs in comparison to iDCs. At the same time the numbers of CD80+CD83+ DCs and FasL+ DCs maintained at the same level as within iDCs. Additionally, experiments conducted with the use of spherical CRC lines indicated some differences in comparison to these made with CRC patients cells. We could observe elevated proportions of matured and activated DCs, but TCM only in the combination with LPS exerted statistically significant effect. In contrast, lysates  of HCT116 and HT29 cells significantly stimulated DCs activation, even to a larger extent than LPS alone.O’Toole’s+CD56+) was significantly enhanced after incubation with DCs stimulated with TCM/LPS from spherical cultures of both CRC cell lines. These results were found positive in regard to the goals of our study since the increased proportion of CD3+CD56+ NKT cells was demonstrated earlier as one of the most desirable effects of DC-based therapy89. The Lindenberg group90 published similar observations after they stimulated skin DCs with supernatants from the 3-D culture of CRC cells from cancer patients.However, in the functional tests assessing endocytotic and cytotoxic activities of DCs, the results appeared to be more heterogeneous and were CRC line-dependent. The proportion of NKT cells  which displayed similar tendencies in cultures of DCs treated with both CRC patients-derived and spherical CRC lines-derived stimulatory factors. At the same time, the secretion of IFNγ and IL12-p70 appeared to be more intense after the stimulation with lysate/LPS and TCM/LPS derived from spherical CRC lines in comparison to stimulated DCs of CRC-patient origin. The more effective secretion of IL12-p70 after DCs stimulation with spherical HT29 cells in comparison to stimulation with HCT116 cells is consistent with the results concerning DCs of cancer patients. Individuals with less advanced CRC (TNM I – II) exhibited higher secretion of this proinflammatory protein in comparison to patients with TNM III-IV stage whereas HT29 cells are referred to as TNM II while HCT116 – TNM III.in vitro DC stimulation, on how effectively DCs mature and conduct endocytosis.Moreover, we identified numerous correlations between various cellular parameters and these relationships were significant when TCM and lysates from spherical cultures were used for DC stimulation. We feel that the negative correlations between the number of HCT116 and HT29 spherical cancer cells with specific phenotype and DCs endocytotic activity deserve a special attention. These relationships confirmed the negative impact of cancer cells bearing the CSC-like markers on the effect of + DCs was not observed during our study and the concentration of soluble FasL was extremely low, we could conclude that FasL was not engaged in the cytotoxicity against FasR+ lymphocytes and CRC cancer cells under the conditions of our experiments. Moreover, the equal or even lower number of FasL+ DCs in comparison to iDCs confirmed the cancer evasion capabilities of CRC cells.Because the elevated proportion of FasLWe showed that tumorsphere assay-based drug evaluation of anti-cancer agents will provide more translatable results compared to those obtained from the traditional 2-D monolayer culture system. Our observations, a first of that type, contribute to the expansion of knowledge about the use of DCs in the studies on cancer biology, with special focus on cancer stem cells. It is hoped that the obtained results can assist in efforts to successful clinical application of some novel anti-cancer DC-based therapies.in vitro modifications. For the CRC niche the usage of LPS seems to be justified and beneficial. In view of the strong immunosuppression, which accompanies neoplastic diseases, unmodified DCs are not sufficient to effectively stimulate a cellular response. The use of in vitro modified DCs in cancer therapy is certainly an interesting and promising direction of research. However, the protocols of generation and stimulation of DCs with tumor antigens should be worked out before vaccination of patients is introduced into standard therapy.However, our results show that in addition to lysates and TCM, the additional support is necessary to achieve the desired effect of DC Freshly isolated colorectal cancer tissues were used to isolate and expand CSCs. CSCs were also isolated and expanded from two CRC cell lines, HT116 and HT29, that were cultured both in adherent and spherical forms. Peripheral blood of CRC patients and healthy volunteers was used to isolate and expand dendritic cells from their monocytic precursors. Autologous lymphocytes were also used for co-cultures with dendritic cells. At the end of expansion of DCs they were modified with lysates or tumor conditioned media or LPS as well as the combinations of these stimulants. Furthermore, the biological properties of DCs subjected to these procedures were determined.All human tissues samples  used in the study were donated freely and written informed consent was obtained from all patients. Ethical approval was obtained from The Independent Bioethics Commission for Research of the Medical University of Gdansk and all research was performed in accordance with relevant guidelines. The general clinicopathological characteristics of CRC patients were presented in the Table Freshly resected colorectal cancer tissues from 27 CRC patients treated in the Department of General, Endocrine and Transplant Surgery, Medical University of Gdansk, Poland, were collected and immediately processed in culture. All experimental chemicals were purchased from SigmaAldrich, Poznan, Poland, except for growth factors, which were purchased from R&D Systems, Biokom, Warszawa, Poland. Tissues were washed several times in serum-free Dulbecco’s modified Eagle medium (DMEM) - F12 supplemented with antibiotic-antimycotic agents. The specimens were minced into 1–2 mm3 pieces followed by incubation in collagenase (20 ng/ml) and hyaluronidase (20 ng/ml) for 1.5 h at 37 °C. Single cell suspension was obtained by mixing every 15 minutes and by filtration through a 70 μm cell strainer.36. Briefly, SCs were maintained on low adherent culture flasks and plates in serum-free stem cell medium containing DMEM-F12 supplemented with ITS Liquid Media Complement (1x), BSA (4 mg/ml), glucose (3 ml/ml), Hepes (5 mM), L-glutamine (2 mM), progesterone (20 nM), putrescine (9.6 µg/ml), Heparin (4 μg/ml), EGF (20 ng/ml), bFGF (20 ng/ml), and antibiotic-antimycotic solution (1x). For the need of current study, we used early passaged (from third to fifth passage) SCs for analysis. During each passage the medium was collected and stored in −80 °C for DC stimulation and was referred to as tumor conditioned medium (TCM).Primary colon spheroid cultures (SC) were treated as we described previouslyFor passage spheroid cultures were collected into the tube, centrifuged and resuspended in fresh SC medium following rigorous pippeting aimed to dissociate the spheres into cells suspension.2. The cells were serially treated with trypsin when they achieved 80% confluence and the medium was renewed 2–3 times per week.The HT29 and HCT116 human colon adenocarcinoma cell lines , Manassas, VA, USA) were used in this study. The cells were cultured routinely as a monolayer in McCoy’s medium supplemented with fetal bovine serum (10%), penicillin-streptomycin (1%) and L-Glutamine (2 mM) and incubated at 37 °C under a humidified atmosphere of 5% COFor the spheroid culture of HCT116 and HT29 cell lines, they were grown in SC medium characterized in previous paragraph. Cancer cultures were maintained under these conditions for 5–6 passages before being used for experiments. During each passage the TCM was collected and stored in −80 °C for DCs stimulation.n = 36) obtained from volunteers recruited during the routine medical consultation in the Regional Blood Bank in Gdansk, Poland, and only the healthy individuals were included into this study. Peripheral blood mononuclear cells were separated by Histopaque®−1077 gradient centrifugation at 1200 g, 30 min, RT. After isolation and erythrocytes lyses, cells were washed and prepared to further isolation steps. To separate monocytes, PBMCs were cultured for 24 h on adhesive Petri dish in RPMI1640 supplemented with FBS (10%), L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml), at 37 °C, 5% CO2, 95% humidity. After incubation, medium containing non-adherent cells was gently removed and the plate with adherent cells was put on ice for 30 minutes. Afterwards, monocyte layer was harvested using a scraper.We used leukocyte-platelet coats (Non-adherent cells were placed in a separated plate and cultured in RPMI1640 with FBS (10%), L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml) and IL-2 (5 ng/ml) and used as effector cells for co-culture with DCs.6 adherent cells (comprising mostly monocytes)/1 ml were placed on 24-well plates in medium supplemented with GM-CSF (50 ng/ml) and IL-4 (100 ng/ml) for 7 days. On day 3 half of the medium was replaced with fresh medium containing cytokines. On day 6, cells were subjected to maturation for 24 hours in the presence of LPS (50 µg/ml), cancer cell lysates, TCM or mixtures of these. Lysates and TCM were obtained from the culture of HCT116 and HT29 cell lines .A total of 1 × 10Cell differentiation and maturation were monitored and documented with the use of Olympus CKX53 inverted microscope coupled with digital camera Olympus SC50 . The analysis and measurements of cells sizes were performed with the use of Olympus cellSens SoftwareSome cancer cells were counted and afterwards directed to the lysate preparation. Lysates were prepared by 4 repeating freeze-thaw cycles  followed by filtration through 0.2 µm strainer. DCs were stimulated with lysates and the proportion between the number of cancer cells taken for lysates preparation and DCs was 1:1.+ monocytes were positively selected using anti-CD14 antibody conjugated with magnetic microbeads  according to the manufacturer’s instruction. Briefly, cells were incubated with anti-CD14 tetrameric antibody complexes and dextran-coated magnetic particles. Labeled cells were separated using an EasySep™ magnet. Stained cells remained in tube while CD14− cells were poured off to another tubes and used as effector cells during co-culture with DCs.3–6 ml of peripheral blood was collected from CRC patients before surgical resection of CRC. CD145 monocytes/200 µl were placed on 96-well plates and expanded according to the same protocol as we described in previous paragraph for peripheral blood monocyte–derived DCs. The lysates and TCM used for DC stimulation were obtained from the autologous cancer cells isolated from tumor fragments of CRC patients.A total of 2 × 10CRC lines, cells separated from human CRC tissue fragments, DCs and effector leukocytes (mentioned in previous paragraphs) were stained with the following cocktail of monoclonal antibodies purchased from BD Biosciences, USA: anti-CD29-APC , anti-CD44-FITC , anti-CD95-PE , anti-FasL Biotin  coupled with Streptavidin-APC, anti-CD3-PE , anti-CD4-FITC , anti-CD11c-APC ,anti-CD14-PerCP , anti-CD25-PE , anti-CD56 , anti-CD80-PE , anti-CD83-APC , anti-HLA-DR-PerCP . Anti-CD133/2-PE  monoclonal antibodies were purchased from MiltenyiBiotec. After 30 min incubation in the dark, samples were fixed with 1% PFA or PBS + 1 mM EDTA for adherent or spherical cells, respectively, and prepared for further analysis. Flow cytometric analysis was performed using FACS Calibur flow cytometer  with BD CellQuest Pro Software.5 cells/ 100 µl/ well DCs was assessed by adding FITC-dextran  to the culture medium for 2 h at 37 °C (control on ice). Thereafter, cells were extensively washed, and the uptake of FITC-dextran was analyzed by flow cytometry .Endocytotic activity of 1 × 10in vitro modified DCs derived from both peripheral blood monocytes and CRC patient blood – monocytes using BD Cytometric Bead Array Flex Set System kits  according to the manufacturer’s instruction. Briefly, Capture Beads were transferred into all tubes and samples were incubated for 1 h at RT. Afterwards, PE Detection Reagents were added to each tube, which than were incubated next 2 h at RT. Captured beads, detection reagents (reporter antibodies) and samples were incubated together to form sandwich complexes. After double washing the samples were resuspended in washing buffer and analyzed in FACS Calibur flow cytometer . Fluorescence intensity was proportional to the amount of a given cytokine in a vial and estimated according to the standard curves acquired after analysis of standard dilutions. Data from cytometric analysis were transformed into graphical and tabular formats using FCAP Array Software. Finally, results were presented as pg/ml.The levels of soluble IL-10, IL-12p70, IFNγ, FasL and granzymes were analyzed in supernatants from cultures of Modified peripheral blood monocyte–derived DCs were co-cultured with HCT116 and HT29 CRC cell lines  for 24 h. Unstimulated immature DCs (iDC) served as a negative control. Following the incubation cells were harvested and used for viability assays. The experiments were performed in triplicate.The 7-aminoactinomycin D (7-AAD) dye  was used for death cell evaluation. After adding 10 µm of 7AAD and anti-CD133 antibodies our samples were incubated for 30 minutes, washed and resuspended in PBS prior to cytometric analysis FACS Calibur flow cytometer . Anti-CD133 antibodies were used to confirm that the appropriate population of CSCs was analyzed.− cells saved after magnetic isolation of CRC patients blood monocytes were co-cultured with in vitro modified DCs for 24 h. iDCs served as a negative control. Following the incubation cells were harvested and the changes of T regulatory-like lymphocytes (CD4+CD25+) and NKT cells (CD3+CD56+) proportions were evaluated with the using of FACS Calibur flow cytometer . The experiments were performed in triplicate.Nonadherent PBMCs from peripheral blood leukocyte-platelets coats and CD14in vitro modified DCs and specific effector cells (mentioned above) were put in triplicate on 96-well plate in different proportions, ranging from 0.0625 to 2 (DC/effector cell number) and incubated for 24 h. After that time, cell proliferation was assessed by colorimetric CellTilter 96® Aqueous One Solution Cell Proliferation Assay (Promega Corporation), according to the manufacturer’s instructions. Briefly, 20 μl of CellTiter 96®AqueousOne Solution Reagent were added into each well of the 96-well assay plate containing the samples in 200 μl of culture medium. After 4 h of incubation the absorbance at 490 nm was recorded using a microplate reader .The p < 0.05 were considered as statistically significant.All data obtained during the study were analyzed with the use of GraphPad Prism ver. 6.05  and the software Statistica 12  according to some non-parametric tests: U Mann-Whitney, Kruskal-Wallis ANOVA, Spearman’s rank correlation coefficient. Values of Supplementary data


ECG, breathing rate, and EEG were recorded. CWL participants: The Eating Restraint scores (Three Factor Eating Questionnaire) of the CWL participants correlated negatively with their heart rates recorded during the Food Stroop task . There was no such relationship in CTL participants. The P200 latencies in CWL participants evoked by the Stroop color-word cues at the C3 electrode were positively correlated to the log high frequency power in their cardiac spectrograms during the Food Stroop . There were no such relationships in the Office Stroop task nor in CTL participants. Combined Groups: Participants' heart rates were significantly lower (p < 0.05) and their RMSSD values and the log Total Power in their cardiac spectrograms were significantly greater during the Food Stroop vs. Office Stroop . In conclusion Eating Restraint scores in CWL participants correlated with their Stroop heart rates, while the P200 latencies evoked by the Stroop cues correlated with the log high frequency power in their cardiac spectrograms  during the Food Stroop task. This provides evidence that even 12 months after successful weight loss maintenance the cardiac ANS reactivity to food cues while completing a cognitive performance test was still different to that in individuals of normal weight who never weight cycled. Across all participants the cardiac ANS reactivity evoked by performing the Stroop task was lowered by food cues suggesting that the dampening effect of food cues on cardiac ANS reactivity may be one of the drivers of ‘stress induced' eating.Successful weight loss maintainers are more vulnerable to stress induced eating. The aim of our study was to determine what effect an attention-demanding cognitive performance task had on brain-heart reactivity to visual food cues in women who maintained clinically relevant weight loss vs. women who had never weight cycled. A clinical weight loss group (CWL,  The increasing rates of obesity worldwide has been attributed to the increased abundance of processed foods at more affordable pricing , furtherThese anticipatory increases in heart rate and BP observed during food cue exposure and during completion of the Stroop task, is thought to be modulated by the “central autonomic network” (CAN). Given the impact that this network, that incorporates the insular cortex, anterior cingulate cortex and amygdala , 12, hasTwo further drivers of obesity in our modern fast paced lifestyles are low physical activity levels  and highOur highly obesogenic environments then necessitates more extreme weight control strategies in successful weight loss maintainers compared to normal weight individuals . Indeed In addition, the sensory brain areas in successful weight loss maintainers have been found to be more reactive to food cues as compared to normal weight and obese individuals . This suFurther research linking peripheral ANS responses and the brain are provided by Geisler and Polich . These aGiven that both Stroop task performance  and foodFurthermore, the reward/effort processes underpinning the above cognitive performances/ingestive behavioral prompts has been found to be modulated by both dopamine and noradrenaline neurotransmission . Both ofmaintained attentional processing of visual food cues  in an effort to mute the incentive value of external food cues  for at least 12 months and BMI matched normal weight individuals who had never weight cycled (CTL). Given that successful weight loss maintainers have (1) alterations in brain processing of food cues  and (2) Our hypotheses were set out as follows:Thepresence of calorie dense food images vs. neutral images during a Stroop task would have significant modulating effects on:ttention processing speeds, specifically that there will be a relationship between P300 ERP latency periods and heart rates and/or HRV measures and (1b) that the P300 ERP latency vs. heart rate modulation will be different in CWL vs. CTL participants;(1a) aarly attention processing speeds, specifically that there will be a relationship between P200 ERP latency periods and heart rate and/or HRV measures and 2b) that the P200 ERP latency vs. heart rate modulation will be different in CWL vs. CTL participants;(2a) eheart rate and HRV 3b) and it would have a significantly greater modulating effect in CWL vs. CTL participants;(3a) participants' * women was recruited via local primary school newsletters, through social media, and notice board announcements at a commercial wellness center. Participants were excluded if they reported a history of a known metabolic disease, pregnancy or lactation in the last 3 months. The women were allocated to a clinical weight loss (CWL) group, or to a body mass index (BMI)-matched control (CTL) group with no reported history of clinically meaningful weight reduction. The experimental protocol was approved by the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee (HREC reference: 214/2012), and all participants provided informed consent prior to assessment. Participants were tested at our EEG laboratory situated at Valkenberg Hospital, Observatory, Cape Town.A convenience sample of apparently healthy*Here the term “apparently healthy” refers to an absence of diagnosed chronic disease  which may confound our results. These individuals were identified via a standardized inclusion/exclusion screening questionnaire.Weight , height , waist circumference, and hip circumference measurements were collected. A demographic questionnaire verified socio-cultural and economic similarity between groups, and a health questionnaire was implemented to exclude applicants with known metabolic disease, eating pathology, positive HIV status, and chronic medication usage. A reproductive survey was administered to exclude women presenting with menstrual dysfunction, hysterectomy or menopause.The validated 51-item Three Factor Eating Questionnaire (TFEQ) was employed to measure dietary restraint, disinhibition, and trait-related hunger. Higher scores being indicative of greater degrees of dietary restraint, disinhibition and hunger. The reason we chose the TFEQ was to compare our data to existing data (the TFEQ has been used widely in the South African literature pertaining to obesity) and also for us to draw parallels with work published on the National Weight Control Registry (NWCR) in the U.S. Furthermore the TFEQ provides a means to ensure homogeneity between our participants and to allow for correlation-based analyses.ECG activity was recorded from 3 electrodes  placed in positions representing Eindhoven's triangle namely, subclavicular bilaterally and over the left anterior superior iliac crest. The skin surface was cleaned and gently abraded with an alcohol swab before electrodes were attached. Electrode cables were taped down to prevent movement artifact. The 3 electrodes were connected to a Biopac MP150 system  ECG amplifier set to band-pass filter between 0.5 and 35 Hz and a sampling frequency of 500Hz. ECG recordings were analyzed with AcqKnowledge for Windows (version 4.1). The filtered ECG recording tachograms were then visually inspected to determine the correct recognition of QRS complexes and T waves. Missed and ectopic beats were corrected by either adding or spacing beats .Only after each tachogram showed no spurious beats were the data analyzed using HRV analysis software (Kubios v 2.1) from the Biomedical Signal Analysis Group . Data were transformed using autoregressive (AR) analysis, with an AR model order of 15, into low frequency (LF) (0.04–0.15 Hz) and high frequency (HF) (0.15–0.4 Hz) components . We compThe breathing rate per minute was measured via a Biopac force transducer fixed to a belt placed around the chest wall. Subjects were asked to expel the air from their lungs when the transducer belt was first fitted and then instructed to breathe normally. The chest transducer was connected to a Biopac MP150 RSP100C amplifier with a low-pass 10 Hz filter. Breathing rates were manually counted.1, Fp2, F3, F4, F7, F8, C3, C4, P3, P4) with EEG cap (http://electro-cap.com/) arranged via the international 10/20 montage system using 10 EEG Biopac amplifiers connected to the MP150 system and ear-linked reference electrodes . Preceding extraction, the raw EEG data were corrected for EOG artifact (automated ICA) within Acqknowledge 4.1. ERP waveforms were extracted using an automated Matlab-designed program . The data were band pass filtered (FIR) with a Hamming window of 0.1–30 Hz. ERP Epochs were set at 200 ms prior to and 600 ms post cue presentation to capture an 800 ms window. Extraction was set to reject ERPs ± 100 μV where each subject must have presented viable ERPs within rejection limits for ≥75% of image trials  of food and office image trials to be included in the analyses which followed. ERPs were baseline corrected, and grand average waveforms were generated to identify robust ERP components. Latency (ms) values were obtained for each ERP wave component. A detailed description of the components extracted (and their respective windows) are outlined in an earlier work ) and P300-like ) components. Geisler and Polich  to display the color word conflicts and embedded images between the color words. As described previously  three StThe experimental design has been published  that rept-tests were performed to assess for differences in parametric data and Pearson pairwise correlations were calculated between the parametric HRV measures and ERP latencies. Mann-Whitney U tests were performed to examine non-parametric data. The assumptions of statistical tests regarding the homogeneity of variances were tested with Bartlett's test for parametric data and Levene's test for non-parametric data. Parametric data in Table and text are described as means ± standard deviation (SD). Alpha was set at <0.05.Stata 12 software package  was used for statistical analyses. Shapiro-Wilk W tests were conducted to examine data normality. Student's Forty five women with no known illness or disorder, similar socioeconomic status, BMI and age participated in the study. Five of the 45 women were excluded from subsequent data analyses, 3 due to BMIs > 2 SDs more the mean, 1 due to being hypertensive (148/84 mmHG and >2 SDs higher than mean) and the Stroop reaction times of 1 participant was >2 SDs slower than the mean. Of the remaining 40 women 17 retained a clinically meaningful weight loss (≥5% of body weight) for a period of at least 12 months by non-surgical means, while 23 reported no history of clinically relevant weight reduction. No differences were found between groups for BMI, weight (kg), height (cm), BP or hip circumference (cm) . These relationships were not found in the CTL group  was significantly higher in CWL participants prior to the performance of Stroop Tasks with record of EEG, ECG and breathing rates  were found Table .4) electrode upon food image presentation and their heart rates during the Food Stroop Task  electrode upon Stroop image presentation and the logHFpower in their cardiac spectrograms during the Food Stroop Task  were significantly different during the Food vs. Office Stroop Tasks , had a significant dampening effect on the attendant cardiac ANS reactivity  electrode evoked by food cue exposure and their heart rates, agrees with Geisler & Polich's findings  for such a correlations in the combined two groups during the Office Stroop task, which suggests that the food cues had an additional modulating effect on attention processing in all participants completing the Food Stroop task.Our third finding, a negative correlation between the latencies of participants' P300 wave at the right parietal  between baseline Eating Restraint scores and heart rates during the Stroop tasks and (2) between P200 latencies evoked by Stroop cues and the high frequency power in the participants' cardiac spectrograms during the Food Stroop task. This provides evidence that even 12 months after successful weight loss maintenance the cardiac ANS reactivity to food cues during a cognitive performance test was still altered in CTL participants. Finally, across all participants the cardiac ANS reactivity evoked by completing the Stroop color-word conflict task was lowered by food cues suggesting that this dampening effect that food cues has on cardiac ANS reactivity may form part of the physiological pathway underpinning “stress induced” eating.HR co-developed research, wrote part of protocol, was responsible for HRV analysis/design and wrote the first draft. DH co-developed, co-wrote protocol, executed research, and was responsible for data collection/analysis/design, EEG write up and editorial input. FH was responsible for EEG analysis/design and EEG write up. JK co-developed research, and contributed editorial input. EL conceptualized the idea for the research, co-wrote protocol and provided research funding and editorial input. All authors have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.