Mean diffusivity, fractional anisotropy, axial diffusivity, radial diffusivity were quantified in the left and right optic nerves and surrounding sheaths at supine baseline and after 4.5 h head-down tilt for each condition. In the optic nerve sheath, mean diffusivity was increased with all head-down tilt conditions by (Best Linear Unbiased Predictors) 0.147 (SE: 0.04) × 10−3 mm2/s (P < 0.001), axial diffusivity by 0.188 (SE: 0.064) × 10−3 mm2/s (P < 0.001), and radial diffusivity by 0.126 (SE: 0.04) × 10−3 mm2/s (P = 0.0019). Within the optic nerve itself, fractional anisotropy was increased by 0.133 (SE: 0.047) (P = 0.0051) and axial diffusivity increased by 0.135 (SE: 0.08) × 10−3 mm2/s (P = 0.014) during head-down tilt, whilst mean diffusivity and radial diffusivity were unaffected (P > 0.3). These findings could be due to increased perioptic cerebral spinal fluid hydrodynamics during head-down tilt, as well as increased cerebral spinal fluid volume and movement within the optic nerve sheath.More than half of astronauts present with significant neuro-ophthalmic findings during 6-month missions onboard the International Space Station. Although the underlying cause of this Microgravity Ocular Syndrome is currently unknown, alterations in cerebrospinal fluid dynamics within the optic nerve sheath may play a role. In the presented study, diffusion tensor imaging was used to assess changes in diffusivity of the optic nerve and its surrounding sheath during head-down tilt, a ground-based model of microgravity. Nine healthy male subjects (mean age ± SD: 25 ± 2.4 years; mean body mass index ± SD: 24.1 ± 2.4 kg/m Changes to the optic nerve and surrounding sheath during microgravity could explain why space flight is harmful to an astronaut’s vision. Darius Gerlach from the German Aerospace Center in Cologne and colleagues studied the tissue architecture of the optic nerve and its surrounding sheath in nine healthy men who experienced head-down tilt, a commonly used ground-based model of weightlessness. Using a neuroimaging technique called diffusion tensor imaging, the researchers documented fluid dynamic changes wrought by the microgravity-like conditions that could be due to alterations in the volume and movement of cerebrospinal fluid within and around the optic nerve. The findings may help explain why many astronauts experience poorer vision after long-duration space flights, although more work is needed to explore the effects of true microgravity on the visual system. The hyperopic shift can be transient or result in permanent degradation of visual acuity or visual field loss.6 In addition, animal studies have found that a sizable amount of CSF could also be extruded via cranial nerves into the lymphatic system.10 In mice, Furukawa et al. demonstrated lymphatic pathways in the dura matter of the ON by immunohistochemistry and electron microscopy.11 The link between intracranial, spinal, and ON sheath CSF is likely not a simple circuit, but rather a complex open system interacting with arterial and venous blood as well as lymphatics. Accordingly, CSF fluid dynamics are largely unknown, especially under microgravity conditions.3On a time-scale of minutes to hours, CSF pressure changes can result from alterations in its formation or resorption, the latter of which occurs primarily through the arachnoid granulations into the dural venous sinuses in a pressure-dependent way.14 By measuring the signal loss due to proton spin random translational motion, DTI can noninvasively examine microstructural changes and the integrity of white matter. Molecular diffusion captured in three dimensions provides information on both the orientation and magnitude including the fractional anisotropy , the mean diffusivity , the axial diffusivity  and the radial diffusivity .Diffusion tensor imaging (DTI) is a valuable tool to assess the ON and its surrounding sheath at early pathological stages of various diseases before severe symptoms occur.15 Additionally, AD and RD increased in the patient group.13 Patients with optic neuritis have been found to have increased RD, MD, and decreased FA,16 with similar findings reported in patients with multiple sclerosis.17 In retinitis pigmentosa, higher MD, AD, and RD have been found compared to healthy controls, as well as lower FA.14 Moreover, CSF dynamics have been assessed with computer tomography cisternography in patients with glaucoma,18 as well as papilledema,19 and CSF compartmentation was reported as the cause for these alterations.Several studies have found strong correlations between the severity of glaucoma and a reduction in FA, as well as an increase in MD in the ON.20 Moreover, we aimed to differentiate between the ON and the perioptic CSF-filled ON sheath within the DTI acquisition. We hypothesized that HDT-induced cephalad fluid shifting would increase MD in the ON sheath due to altered CSF hydrodynamics, as potentially caused by pulsatile or cohesive CSF movement and thus causing signal loss from the phase shift.21 Finally, we were interested in whether lower-body negative pressure (LBNP), a technique known to reduce centrally available venous blood volume, and inspiratory carbon dioxide, a potent arterial vasodilator, in combination with HDT would have additional effects.Thus, whilst DTI has provided valuable insight into disease-related alterations in the ON, it has not been used to study the physiological effects of sustained variations in intracranial pressure. Therefore, we aimed to determine the short-term effects of increased intracranial pressure in healthy subjects on ON diffusivity using the head-down tilt (HDT) model.22 Briefly, nine healthy male subjects  participated in five experimental HDT conditions: −6°, −12°, −18°, −12° plus 1% CO2 atmosphere, and −12° plus LBNP. These five conditions were done in random order and on different experimental days. The LBNP was maintained at −20 mmHg during the entire 5 h HDT protocol. Magnetic Resonance Imaging (MRI) scans were performed at supine baseline after lying supine for at least 1 h and again after 4–5 h of intervention. Subjects were not allowed to sleep and remained in the HDT position during all experimental conditions. Custom-built MRI-compatible wedges in the three HDT positions, breathing masks and an LBNP chamber were used for the interventions. The HDT wedges ended at the neck of the subject, which coincided with the beginning of the head coil.This study was performed in compliance with the Declaration of Helsinki and was approved by the local ethics commission (Ärztekammer Nordrhein). Written and informed consent was obtained from all subjects prior to study inclusion. The study design has been previously reported.The total duration of the MRI examination was ~1:05 h, with DTI performed at about ~30 min into the scan. MRI acquisitions were obtained on a 3 T scanner with a maximum gradient amplitude of 45 mT/m and maximum slew rate of 200 T/m/s (mMR Biograph—Positron Emission Tomography-Magnetic Resonance Imaging (PET-MRI) scanner based on the Verio system—Siemens, Erlangen, Germany) with an appendant 16 channel head and neck coil.b value = 1000 s/mm−2 and one without diffusion weighted acquisition (b = 0 s/mm−2) were acquired. Parallel imaging with an acceleration factor of two was enabled . A bandwidth of 1086 Hz/Px. with 1.03 ms echo spacing was applied. The DTI scan time was 6:04 min. A twice-focused single-shot spin-echo EPI was utilized to reduce geometric distortions.23 The phase encoding direction was A to P. To avoid foldover artefacts, a saturator of 55 mm was used in the posterior brain region. Scanner stability was assessed by spherical water phantom acquisition.24The orbital DTI parameters were: TR 5600 ms, TE 100 ms, FA 90°, Matrix 128 × 128, Filed of View 160 mm, 25 slices, NEX 2. The nominal resolution was 1.25 mm× 1.25 mm × 2.0 mm and the interpolated resolution was 0.625 mm × 0.625 mm × 2.0 mm, with an additional inter slice spacing of 2 mm. Thirty non-collinear diffusion directions with 2 turbo spin echo (TSE) scans of left and right ON were used for the positioning of the orbital DTI. Oblique transversal sections through both ONs were planned in addition to a coronal magnetization-prepared rapid acquisition gradient-echo reconstruction. The positioning was further adjusted and controlled in the left and right sagittal TSE ON central sections. This procedure allowed for an exact adjustment of the DTI slices through both ONs . Eddy current corrected diffusion weighted images were used for voxel-by-voxel based tensor calculation. The following parameter maps were calculated from the Tensor data: FA, MD /3), AD  and RD /2)26:DTI data was post-processed with FSL software library (http://www.itksnap.org). 2D image planes of the middle ON section were utilized for segmentation. Depending on the shape of the ON, the segmented length was ~0.8 to 1.5 cm starting from the eye globe. The B0 non-diffusion weighted images were used as a reference for segmentation. FA, MD, AD, and RD values were calculated as mean values of the left and right ON and ON sheath region of interests.The ON and its surrounding sheath were segmented manually with itk-SNAP  were used as fixed effects and subject as a random effect. Interactions of the main fixed effects were also included in the model, however, the three-way interaction term was removed in all instances during model simplification, as well as the term for side and interaction terms with it. Notably, the main effect of time then reflects effect of HDT, and the time × condition interaction reflects diversity of the different conditions over time. Significant overall ANOVA effects were inspected with a-priori defined treatment contrasts with −12° HDT as reference. Significance was set to p < 0.05. Means of the diffusion parameters (± standard deviation) for the ON sheath and ON MD, FA, AD, and RD are given for all conditions in Table Statistical analysis was carried out with R statistical software version 3.2.0  using ANOVA and linear mixed effects models to analyze the ON and ON sheath MD, FA, AD, and RD. Linear mixed effect models take care of inter-subject sources of variation and are thus suitable for repeated measurements. Time (baseline vs. 4.5 h HDT), side (left vs. right) and condition  generated during and analyzed during the current study are not publicly available due to containing personal details of subjects, but are available from the corresponding author on reasonable request.With the exception of two scans , all data were acquired and analyzed as foreseen by the study protocol.P < 0.001), suggesting that MD increased by 0.147 (SE: 0.04 × 10−3 mm2/s). There was also a significant effect of condition (P = 0.003), which however, did not reflect in any significant a-priori contrasts . Notably, there was no significant time × condition interaction (P = 0.37). Taken together, the results suggest that MD in the ON sheath was increased after exposure to HDT, with all HDT conditions having similar effects as the −12° condition. FA depicted a significant main effect of time (P = 0.036) and a trend for the time × condition interaction (P = 0.054). AD and RD, respectively, in the ON sheath revealed significant main effects of time (both P < 0.001), suggesting increases by 0.188 (SE 0.042) × 10−3 mm²/s for AD and 0.126 (SE 0.04) × 10−3 mm²/s for RD. Significant main effects of condition for AD and RD  were inconclusive during inspection with a-priori contrasts , and there were no time × condition interaction effects found for AD and RD , indicating that AD increased after exposure to HDT by 0.135 (SE 0.08) × 10−3 mm²/s. However, no effect of condition was found (P = 0.96), nor was there a time × condition interaction (P = 0.96), suggesting that time-related changes in the various HDT conditions were all comparable to the −12° condition. No significant main or interaction effects were found for MD or RD in the ON . For FA, there was no main effect of condition (P = 0.51), and a trend for time (P = 0.055). Moreover, FA depicted a time × condition interaction  after −6° HDT compared to −12°HDT and by 0.207 (SE 0.068) after −12° plus LBNP.  was utilized in this study due to the multiple hour exposure. LBNP has been shown to have sizable effects on intraocular pressure at −40 mmHg, whilst LBNP at −20 mmHg does not.2 was hypothesized to further increase intracranial volume and thus pressure during HDT and elevate diffusivity further. However, exposure to increased ambient CO2 during HDT was not found to have any additional effects on diffusivity parameters of the ON or ON sheath.On the contrary, as an arterial vasodilator, increased ambient CO15 However, all diffusion values, especially AD, are slightly lower than previously reported values,14 although there is great variation in the reported literature. Heterogeneity in reported diffusivity values in the literature may be the result of different age groups of healthy control subjects. In the presented study, young male subjects (25 ± 2.4 years) were enrolled and although not reported for healthy subjects, ON FA is known to decrease with age in patients with glaucoma.15 Furthermore, Wang et al. (2013) and Zhang et al. (2016) report diffusivity values for controls across a wide age range and mixed genders.14 Interestingly, ocular changes in astronauts predominantly affects middle-aged men 4 and thus, age could should be taken into consideration as a potential factor in MOS.The FA and MD baseline values of the ON found in this study are in line with previously reported values in healthy subjects.29 Choroidal folds, cotton wool spots, ON sheath distension, globe flattening, elevated CSF opening pressure or papilledema have been identified in 42% of 36 astronauts post-flight, with 19 examinations still pending.4Optical Coherence Tomography, cycloplegic refraction, fundus examinations, visual acuity, visual field thresholds are standard measures for astronauts ocular health pre and post flight according to the clinical practice guidelines of long-duration missions.30 gynecological or robotic prostate surgery.31 Intraocular pressure assessed in patients with head-down (Trendelenburg) surgical positioning has been found to increase, however, retinal nerve fiber layer thickness measured with optical coherence tomography does not appear to be altered in patients with vision impairments after surgery.33 Similarly, no clinically relevant findings were found during 14 day exposure to −6° HDT bed rest,34 whereas 70 day −6° HDT bed rest could provoke an increase in the superior, nasal, and inferior peripapillary retinal thickness.35In the presented study, subjects did not suffer from significant visual impairment during or after the HDT. The time frame used in the presented study may be beneficial to understand perioperative visual loss due to ischemic optic neuropathy, seen in patients undergoing prolonged surgery requiring head-down positioning such as spinal,37 First, successful acquisition depends greatly on subject compliance as eye movement can result in blurred scans, resulting in an indefinable strong partial volume effect in DTI parameters. Furthermore, a long TA signal quality is limited by motion artifacts. This issue can be overcome by an eye triggered DTI examination, however, the problem persists with echo train lengths of 7–10 s. To prevent this, we chose to use high in-plane resolution with a lower slice thickness resolution. Prior to the DTI scans, we performed three high resolution anatomical scans of the orbits . This allowed the operator to adjust the DTI slices exactly through both ONs in the oblique transverse direction. This procedure, however, has two disadvantages: First, it is operator-dependent, and second, it is sensitive to subject movement which can cause partial volume artifacts. Therefore, we conclude that a scan with an isotropic resolution benefits from operator independence and insensitivity from subject movement between scans and no high resolution 3D cuts are needed for slice positioning. While a volumetric scan would be sufficient for slice positioning, sophisticated segmentation techniques would also be necessary. In addition, considering potential tortuosity of the ON, a DTI acquisition perpendicular  to the long axis is considered more robust to partial volume effects.38There are several limitations inherent to the presented study that need to be taken into consideration when interpreting results. Notably, the study included a small number of subjects, a detriment that may affect statistical analysis, as well as interpretation. In addition, DTI of the ON has several limitations.40 Even though the signal from the ON and CSF voxels is suppressed, partial volume effects cannot be avoided with a low resolution.Notably, the measurement technique may also be the cause of variation in reported DTI values compared to previously reported values in the literature. This may be due to motion artifacts, partial volume effects in the ON sheath or different acquisition approaches with fat and water suppression. The majority of related publications fail to describe how eye movement was avoided. Some studies have also used water and fat suppression techniques for ON scans to avoid signal acquisition from surrounding CSF spaces.42 Advanced sequences such as diffusion-sensitized multishot rapid acquisition with relaxation enhancement could also be promising with higher magnetic fields resolution.43 The examination of deeper sections of the optic path (ON closer to the chiasm) would also benefit from such a non-distorted technique.Another obstacle was the strong susceptibility for artifacts in the orbital region caused by tissue susceptibility differences between fatty tissue, bony structure, and the nasal cavities. A promising approach to avoid distortions by single shot echo planar imaging, especially at higher magnetic fields , is by utilizing spin echo-based sequences. These sequences are not affected by magnetic field inhomogeneities.Finally, we would like to remind the reader that the implemented statistical approach tested for differences between any condition and the −12° condition. It is therefore possible that differences e.g. between −6° and −18° went unnoticed in the analyses, which would have provided further evidence to our understanding of HDT effects on perioptical hydrodynamics. In any case, tilt-angle dependent effects on ON DTI should be explored in more detail in future studies.36Overall, the presented data constitute evidence to suggest facilitated diffusivity along the ON with HDT. This may indicate increased perioptic CSF hydrodynamics with increased CSF volume and/or movement within the ON sheath during HDT exposure. The sensitivity of DTI to detect altered physiology with HDT demonstrates its capability to be used to further investigate ocular changes in astronauts. This work can also be extended to the entire visual system including the optic chiasm, the optic tract, and radiations.


Aspergillus penicillioides, Eurotium halophilicum, Xerochrysium xerophilum (formerly Chrysosporium xerophilum) and Xeromyces bisporus, were produced by cultures growing on media supplemented with glycerol (and contained up to 189 mg glycerol g dry spores−1). The ability of these spores to germinate, and the kinetics of germination, were then determined on a range of media designed to recreate stresses experienced in microbial habitats or anthropogenic systems (with water‐activities from 0.765 to 0.575). For A. penicillioides, Eurotium amstelodami, E. halophilicum, X. xerophilum and X. bisporus, germination occurred at lower water‐activities than previously recorded . In addition, the kinetics of germination at low water‐activities were substantially faster than those reported previously. Extrapolations indicated theoretical water‐activity minima below these values; as low as 0.570 for A. penicillioides and X. bisporus. Glycerol is present at high concentrations (up to molar levels) in many types of microbial habitat. We discuss the likely role of glycerol in expanding the water‐activity limit for microbial cell function in relation to temporal constraints and location of the microbial cell or habitat. The findings reported here have also critical implications for understanding the extremes of Earth's biosphere; for understanding the potency of disease‐causing microorganisms; and in biotechnologies that operate at the limits of microbial function.For the most‐extreme fungal xerophiles, metabolic activity and cell division typically halts between 0.700 and 0.640 water activity (approximately 70.0–64.0% relative humidity). Here, we investigate whether glycerol can enhance xerophile germination under acute water‐activity regimes, using an experimental system which represents the biophysical limit of Earth's biosphere. Spores from a variety of species, including  Aspergillus penicillioides and Xeromyces bisporus which dominate league tables for ability to grow in high‐solute environments and/or at low water‐availability . For instance, cells of fungi and algae can contain 7–8 M glycerol (see below); high intracellular glycerol is a determinant for vigour ; though the germination process was severely inhibited: germ‐tube formation only occurred after a 2‐year incubation period .Glycerol, which can be present in the extracellular environment or within the cytosol at high concentrations, is a recurring motif in the physiology of extremophilic microbes. It is fungal xerophiles such as od Snow, . Snow , Eurotium amstelodami, E. echinulatum, Eurotium halophilicum and Eurotium repens  and Xerochrysum xerophilum (formerly Chrysosporium xerophilum). We hypothesized that (i) composition of biophysical stressors within a fungal substrate determines ability to germinate at low water‐activity; and glycerol can both (ii) speed up the kinetics of germination and (iii) enable germination at hitherto unprecedented water activities.This study was carried out, taking inspiration from the natural ecology of extremophiles, to investigate whether glycerol can determine biotic activity at the water‐activity limit for life. A dual approach was used: extreme xerophilic fungi were encouraged to produce spores which accumulated glycerol, following which these propagules were assayed for ability to germinate on high‐glycerol substrates. These germination assays were carried out in the range 0.765–0.575 water activity, i.e. at biologically hostile water activities, for seven fungal species of the order Eurotiales. They were: et al., et al., et al., et al., et al., in press). In relation to germination processes, spores containing 2.8–8.5% w/v glycerol are known to germinate more vigorously, at lower water‐activity and (for pathogenic fungi) exhibit higher levels of virulence , and D‐shaped ascospores for X. bisporus strains .−1 . For glycerol‐only media, the glycerol concentration varied between 7.0 and 7.7 M; all other media types were supplemented with 5.5 M glycerol plus additional solute(s), over a range of concentrations for the latter (Supporting Information Table S2). The 0.765–0.605 water‐activity range represents the tip of the biotic windows for growth or germination of the most extremely xerophilic strains . These media were supplemented with glycerol + NaCl; glycerol + sucrose; glycerol + glucose +fructose; glycerol only; glycerol+ NaCl + sucrose or glycerol + NaCl + sucrose + KCl; representing various natural habitats and/or anthropogenic systems in which xerophiles are found . Please note that both hyphal growth and germination of the xerophiles used in the current study are typically optimal in the range 0.930–0.830 , glycerol + sucrose at ≤0.619 water activity , glycerol + glucose + fructose at ≤0.611 water activity (22.74 kJ g−1 chaotropic activity), glycerol only at ≤0.654 water activity (21.58 kJ g−1 chaotropic activity), glycerol + NaCl + sucrose at ≤0.651 water activity , and glycerol + NaCl + sucrose + KCl at ≤0.639 water activity  , and glycerol only at ≤0.707 water activity (18.43 kJ g−1 chaotropic activity); for other media its profile was the same as that of strain FRR 0025 , if also present in natural substrates, would render the latter effectively sterile and uninhabitable. Generally, germination of most strains occurred on media in the range 0.765–0.674 water activity; approximately half of strains were able to germinate from 0.668 to 0.664; and only occasional strains were able to germinate on selected media in the range 0.654–0.637 Fig. . Whereas−1) appeared hostile to fungal germination, this was attributable to their low water‐activity (i.e. <0.637) rather than kosmotropicity per se  prevented mycelial growth for a range of xerophile strains, including a number of those used in this study; a finding also consistent with the limit for Aspergillus wentii strain IMI 017295ii on high‐glycerol media , A. penicillioides strains JH06GBM and JH06THJ, and E. halophilicum FRR 2471 . These strains were first isolated from fruits, wooden surfaces (both JH06GBM and JH06THJ) and plant seeds respectively (Supporting Information Table S1). The germination of X. bisporus FRR 0025 was most rapid on glycerol + sucrose and glycerol + glucose + fructose media in the water‐activity range 0.734–0.637, according to rates of germination and germ‐tube extension . The progress of germination and germ‐tube extension for strain JH06GBM showed a close relation to water activity, regardless of medium composition, suggesting that this strain would make a useful model system to study water relations of xerophilic fungi , as evidenced by the advanced germination performance observed on glycerol + NaCl, glycerol + sucrose as well as glycerol + NaCl + sucrose media  may arise from the lack of a Na+‐exporting ATPase, Ena, according to studies of its genome  by stressors such as glycerol, sorbitol, glucose, ethanol, NaCl, KCl, MgCl2, NH4NO3 and urea  at ∼0.700 water activity, regardless of strain or species  .Studies of microbial ecology have demonstrated that chaotropicity can determine the outcomes of competitive interactions, both qualitatively and quantitatively  . The germination rates in the water‐activity range 0.765–0.637 were between 37.5% and 0.02% of the optimum rates , depending on the strain and culture‐medium . Similarly, pH and temperature curves for these three strains  confirm that the pH of media used in this study  were within the optimal pH range , and that the 30°C incubation‐temperature used in this study was optimal regardless of strain.The water‐activity range assayed in this study was considered in the context of the entire windows for germination of A. penicillioides, E. halophilicum and X. bisporus in the high‐glycerol system occurred 16–73 days earlier in the water‐activity range 0.700–0.640, depending on fungal species , germination took place at lower water activities in the current study, regardless of species  so it may be that part of the differences observed in Fig. For each species of xerophile assayed, the kinetics of germination reported in the current study were more rapid than those reported previously. Below 0.700 water activity, germination had been observed only after a lag period of 38–80 days prior to this study, regardless of species  than low‐glycerol spores; i.e. ≥0.989 , for non‐xerophilic fungi , 0.640 for A. penicillioides strain JH06THJ on glycerol + NaCl‐supplemented media, and 0.651 for E. halophilicum strain FRR 2471 and A. penicillioides strains JH06GBM and JH06THJ on glycerol + NaCl + sucrose‐supplemented media . In the current study, theoretically determined water‐activity minima were derived by extrapolation of trend lines for strains which germinated at ≤0.674 water activity for three or more water‐activity values on the same type of medium, or two data points with the lower water‐activity value at <0.665 , whereas X. bisporus FRR 2347 was germinating on only three types of media by Day 10, and all germination was complete by Day 35 ; and diverse fungal xerophiles such as X. bisporus and A. penicillioides . Based on manipulations of these interacting parameters, recent studies have revealed that extremely halophilic bacteria and Archaea are not constrained to water activities of ≥0.755 , but retain activity down to values close to 0.600 water activity (see above); and the lower limit for mycelial growth of extremely xerophilic fungi has been revised from 0.656 ; spores and hyphae of fungi such as in situ , little is known about the biophysics of glycerol in relation to ecosystem function. For instance, glycerol is hygroscopic in nature and so may draw external water into microbial biomass that is located in water‐constrained habitats; applications of glycerol can correct water‐repellency in non‐wetting sandy soils . The high‐glycerol system used in this study can be viewed as an anthropogenic intervention in the usual biology of the fungal system. However, both artificial and natural fungal substrates which have stressfully low water‐activity values yield spores with high amounts of the low‐Mr polyols glycerol and, to a lesser extent, erythritol . It is, therefore, plausible if not, indeed, likely that glycerol facilitates the activity of diverse types of microbial cell/community under low water‐activity conditions. In this way, glycerol may determine the extent of, and failure points for, the functional biosphere on Earth  are those of halophilic Archaea growing in bittern brine , will facilitate their greater exploitation in biotechnology (in a similar way to the use of thermophiles and their heat‐tolerant biomolecules). Furthermore, our work here suggests that careful supplementation of fermentation media with glycerol (possibly in combination with other stressors) can enable the rational manipulation of microbial metabolism and/or cell division targeted towards specific industrial applications.Understanding the biophysical and ecophysiological factors which interact to enable and constrain life has important implications. The majority of microbiological studies, even for extremophilic taxa, focus on organisms growing under relatively benign conditions. Characterizing life under hostile conditions may be critical to understand and manipulate nutrient cycling in the biosphere; saprotrophic activity in arid soils for instance. Furthermore, understanding what is (and is not) possible on Earth will inform our search for potential habitats on other planets. Some infectious organisms can inhabit low water‐activity environments (either in the human body or in spaces cohabited with humans) and so knowledge of these limits may facilitate novel antimicrobial treatments or sterilisation techniques based on reducing the water availability below these limits. Many microbe‐driven industrial processes, including food‐, drinks‐ and biofuel fermentations take place in low water‐activity milieu . Please note that E. echinulatum, E. halophilicum and E. repens have recently been renamed as Aspergillus brunneus, Aspergillus halophilicus and Aspergillus pseudoglaucus respectively  supplemented with 5.5 M glycerol (0.821 water activity) at 30°C.llsworth  and are in vitro or, for microbial habitats, in situ ; A. penicillioides, Eurotium spp., X. bisporus (e.g. strain CBS 328.83) and X. xerophilum are found within saprotroph communities on surfaces such as dried leaves, straw, wood and paper .Only 13 microbial species/communities have been observed to grow and/or germinate in the water‐activity range 0.690–0.605 according to empirical data obtained from experiments carried out hristian  to have A. penicillioides, E. amstelodami, E. echinulatum and E. repens; and for 21–28 days for the slower‐growing/later‐sporulating E. halophilicum, X. bisporus and X. xerophilum at 30°C. Media were inoculated using 2‐mm‐diameter plugs of agar taken from the periphery of an exponential‐phase culture growing on medium of the same composition, and plates of each medium were placed in a sealed bag of low‐density polyethylene to maintain a constant relative humidity (thus maintaining water activity), while allowing gaseous exchange . Spores were harvested into a sterile solution of NaCl (5.3 M) and the resulting suspensions were filtered through glass wool to remove hyphal fragments. Spore samples were then freeze‐dried and extractions were carried out with 5‐mg samples of spores that were sonicated in AnalaR water  and then immersed in a boiling water bath as described previously  for 10–14 days for For the 12 xerophile strains, ability to germinate and germination kinetics at biologically hostile water activities (0.765–0.570) were assessed using a range of 36 media (Supporting Information Table S2), designed to emulate physicochemical stresses experienced by microbes in both natural habitats and anthropogenic systems; these media include MYPiA supplemented with glycerol + NaCl; glycerol + sucrose; glycerol + glucose + fructose; glycerol only; glycerol + NaCl + sucrose and glycerol + NaCl + KCl + sucrose (see above). All media used in this study were incubated in polyethylene bags with identical media types at 30°C (except for those used in temperature assays described above), and were sterilized by autoclaving at 121°C (1 atm) except for those containing glucose + fructose.Data from germination assays were used to plot percentage germination and germ‐tube length versus time Figs , 3 and 5A. penicillioides, E. amstelodami, E. echinulatum and E. repens; and 21–28 days for strains of X. bisporus, X. xerophilum and E. halophilicum; X. bisporus, X. xerophilum and E. halophilicum. Spores were harvested from colonies growing on MYPiA + glycerol (5.5 M) media by covering Petri plates with sterile solutions of 5.5 M glycerol (15 ml); aerial spores were then dislodged by gently brushing with a sterile glass rod. The resulting suspension was then passed through sterile glass wool twice to remove hyphal fragments as described in earlier studies  onto the medium; the suspension was then distributed across the agar surface using a sterilize glass spreader.Spores were obtained from cultures incubated on MYPiA + glycerol (5.5 M) for 10–14 days for strains of Germination was assessed by removing a 4‐mm agar disc, and immediately quantifying percentage germination, spore diameter and germ‐tube length using light microscope. Plates were immediately resealed and placed back in the incubator after removal of agar discs. Percentage germination was determined via counts of 200 spores, and 50 individual germinated spores were measured for germ‐tube length; spores with germ‐tubes longer than their diameter were considered to have germinated  were presented as percentage spore germination over time  were plotted against those for spores harvested from a basal medium containing 2% w/v glucose  were plotted against the highest rates available for A. penicillioides strain FRR 3722, which had been pre‐cultured on a basal medium with no added solutes ; Gock et al. ; Snow (E. echinulatum); Andrews and Pitt  and Wheeler and Hocking (E. amstelodami) for use in Fig. Germination kinetics for the three model strains, which had been cultured on high‐glycerol media, were compared with those from extant datasets Fig. . For X. , et al. , and theet al., et al. . Water‐activity measurements were taken at the same temperature at which cultures and germination assays were to be incubated, and several precautions were employed to ensure accuracy of readings, as described previously  at 30°C. Data are means of three replicates, and grey bars indicate standard errors.Supplementary figure legends Supporting referencesClick here for additional data file.


We hypothesized that we would see an improved accuracy at withholding a response to affectively congruent stimuli (i.e. increased accuracy at withholding a response to fearful ‘no-go’ distractors) under threat of shock. Induced anxiety slowed reaction time, and at the second event quicker responses were made to fearful stimuli. However, we did not observe improved inhibition overall during induced anxiety, and there was no evidence to suggest an interaction between induced anxiety and stimulus valence on response accuracy. Indeed Bayesian analysis provided decisive evidence against this hypothesis. We suggest that the presence of emotional stimuli might make the safe condition more anxiogenic, reducing the differential between conditions and knocking out any threat-potentiated improvement.Studying the effects of experimentally induced anxiety in healthy volunteers may increase our understanding of the mechanisms underpinning anxiety disorders. Experimentally induced stress (via threat of unpredictable shock) improves accuracy at withholding a response on the sustained attention to response task (SART), and in separate studies improves accuracy to classify fearful faces, creating an affective bias. Integrating these findings, participants at two public science engagement events ( A state of anxiety, thought to be related to pathological anxiety  can be iThreat of shock can also instantiate a negative affective bias: enhancing the processing of affectively congruent stimuli ,14. WhenThreat of shock can therefore selectively enhance processing of affectively congruent stimuli and promote inhibitory control. In this study, conducted over two public scientific engagement events and a follow-up laboratory sample, we used an adapted version of the SART to investigate whether inhibitory effects interact with affective bias. In this new task, participants were instructed to respond to happy faces and withhold responses to fearful faces (and vice versa) whilst they were alternately at risk and safe from unpredictable shock. We predicted that the threat of shock manipulation would induce a state of transient anxiety in participants, and we would see an improvement in accuracy to withhold a response to affectively congruent stimuli (i.e. increased accuracy at withholding a response to fearful ‘no-go’ distractors) under threat of shock. The nature of the scientific engagement events also enabled us to investigate effects within a more naturalistic setting than in highly controlled laboratory conditions.2.1 and a second at the Wellcome Collection, entitled ‘Feeling Emotional’2. We also ran a follow-up study at UCL. At the engagement events, data were collected in a large room, with two computers set up, but event attendees were free to wander around the room and watch the testing. The overall sound levels and potential for distraction were considerably greater than a controlled testing environment but can be thought of as a naturalistic representation of an informal social event. All participants provided written informed consent (UCL ethics reference: 1764/001) and completed a screening form that verified they had no history of neurological, psychiatric or cardiovascular conditions.This study was completed as part of two public engagement events: the first at the Royal Institution, entitled ‘Questioning Reality’2.1.Two electrodes were attached to the back of the participants' wrists (on their non-dominant hand) to induce stress via threat of unpredictable electric shock. They were instructed to make their responses using the opposite hand. For each participant, the shock level was set to a level where it was ‘unpleasant but not painful’ . Shocks 2.2.Immediately after completing the task participants were asked to report how anxious, afraid and stressed they had felt in the safe and threat conditions. They made a response between 1  and 10 (very much so).2.3.a priori power analysis was run in G*Power [An  G*Power . The pow G*Power  that gav2.4.Participants completed a new task adapted from our previously used SART  under tw2.5.2.5.1.M = 30.48, s.d. = 9.38) were included in the analysis.Forty-seven subjects completed the study. One subject was removed due to inattention (mean target accuracy = 0.02). In total, 46 participants  aged between 19 and 65 (2.5.2.a). These durations are the same as in the original design [There were a total of four blocks (two threat and two safe blocks), with each block lasting 52 s. Participants received either one shock  or two shocks , during the shock condition. They were informed that the shocks would be delivered at random and were uncontrollable (i.e. they were not punished as a result of an incorrect key press). Forty-seven ‘go’ stimuli and five ‘no-go’ stimuli were randomly presented in each block. Face stimuli were presented for 250 ms, followed by an interstimulus interval of 750 ms, before presentation of the next stimulus a. These l design , but the2.5.3.Thirty-two participants also completed self-report measures of Trait Anxiety . Blood a2.6.2.6.1.M = 30.80, s.d. = 9.33) completed the study. One participant was removed due to an absence of responses throughout the task. In total, 55 participants  aged between 18 and 60  were included in the analysis.Fifty-six participants  aged between 18 and 60 (2.6.2.b). The stimulus presentations were longer in this task to ensure that the duration was sufficient to allow for the processing of the emotion. As in Study 1, the number of blocks was fewer than in the original design [This was the same as above, with the exception that each block lasted 60 s. There were also fewer stimuli in each block: 36 ‘go’ stimuli (35 in third block) and four ‘no-go’ stimuli (five in third block) were randomly presented. As above, participants received either one shock  or two shocks . In contrast to Study 1, face stimuli were presented for 750 ms, while the interstimulus interval remained the same at 750 ms b. The stl design  in order2.7.We collected data from a further 18 participants at UCL. The task design remains exactly the same as described under ‘Study 2’ with the exception of the background colour. During safe conditions, the background was red, and during threat conditions the background was blue. These extra data were collected to check whether effects might be driven by colour rather than condition.2.8.p = 0.05, was considered significant. Bayesian statistics were also run for these frequentist analyses .Reaction time (RT) and accuracy was analysed using two repeated-measures general linear models in SPSS version 22 . For all analyses, Performance accuracy for each condition (threat/safe) and trial type (‘go’/‘no-go’) was calculated by dividing the number of correct trials by the total number of trials.10 factors for models of interest relative to a null model (main effect of subject). The ‘winning’ model BF10 was defined as the highest BF10 relative to the null. The following labels were assigned to BF10: anecdotal (1–3), substantial (3–10), strong (10–30) decisive (more than 100) to interpret the magnitude differences between models [Accuracy analysis was performed on ‘no-go’ trials only . Response accuracy on ‘no-go’ trials was analysed using a two-way ANOVA with factors valence (happy/fearful) and condition (threat/safe). RT analysis was performed on ‘go’ stimuli only . Prior to analysis, a log transform was applied to RT data as they were not normally distributed. RT to ‘go’ stimuli was analysed using a two-way ANOVA with factors valence (happy/fear) and condition (threat/safe). Bayesian ANOVAs were used to generate BFn models .r correlations between symptom measures  and RT/accuracy data were run. Only n = 28 participants could be included in the secondary analysis of STAI anxiety scores in Study 1 due to missing data. All participants' scores (n = 55) were included in the secondary analysis of STAI anxiety scores in Study 2.Post hoc analyses were run to investigate the effect of STAI trait anxiety scores and blood alcohol concentration on performance. Pearson's 3.3.1.3.1.1.t42 = 5.31, p < 0.001, d = 0.73; safe M = 3.37, s.d. = 2.21, threat M = 5.19, s.d. = 2.75). Bayesian analyses confirmed a model with main effect of condition was the winning model, with a decisive magnitude difference relative to the null (BF10 = 4816).Subjective ratings of anxiety levels were significantly higher during the threat condition relative to the safe condition  worse than the null enabling us to reject our hypothesis of a threat × valence interaction.Bayesian analysis confirmed that no models had a BF3.1.3.F1,45 = 18.25, p < 0.001, M = 306.84 ms, s.d. = 48.01 ms, threat M = 311.51 ms, s.d. = 45.24 ms; F1,45 = 1.395, p = 0.24, F1,45 = 0.02, p = 0.89, We found a significant effect of condition . Bayesian analyses confirmed a model with main effect of condition was the winning model, with a decisive magnitude difference (BF10 = 120 160).Subjective ratings of anxiety levels were significantly higher during the threat condition relative to the safe condition  worse than the null, providing evidence against our hypothesis of a threat × valence interaction.Bayesian analysis confirmed that no models had a BF3.2.3.F1,54 = 8.78, p = 0.005, M = 387.30 ms, s.d. = 66.69 ms; safe M = 370.51 ms, s.d. = 55.84 ms; F1,54 = 8.81, p = 0.004, M = 371.12 ms, s.d. = 53.40 ms; happy M = 386.70 ms, s.d. = 67.12 ms; p = 0.61; see There was a main effect of condition  better than the null.3.2.4.r = 0.256, p = 0.188, r = 0.233, p = 0.087).Correlations were run to look at response accuracy and response time differences  across threat and safe conditions against STAI trait scores in both studies, and blood alcohol measures in Study 1. The correlation between trait anxiety and threat minus safe RT to fearful targets had the highest correlation in both studies 1 and 2 . Crucially, however, there was no significant interaction between colour and condition . Indeed, a model containing this interaction term was 41.36 times worse than a model including a main effect of condition, enabling us to reject the hypothesis that background colour is driving the effect of condition.Bayesian analysis confirmed a non-significant interaction between condition and background colour .Across studies, we replicated a previous finding that threat of shock slows down reaction times to target ‘go’ stimuli ,13,24. WIn the first study, there were no main effects of valence or improved inhibition under threat overall . Bayesian analysis confirmed that a null model was in fact 90.91 times better than a model including this interaction term. Thus, although threat of shock had an influence on affective state, demonstrated by significantly different subjective ratings of anxiety between sessions, and slower reaction times, it had no effect on response inhibition (i.e. improved accuracy at inhibiting a response to fearful faces under threat of shock). One explanation for this could be the stimulus presentation times. Here, the stimuli were presented for 250 ms, while in a previous study (which demonstrated an affective bias) the faces were presented for 1000 ms. These different presentation times across studies correspond with different average reaction times . As it is thought that conscious processing of faces occurs above chance for presentations of 330 ms or more [In Study 2, our results did not support our hypothesis that there would be an improvement in accuracy at withholding a response to fearful distractors under threat of shock—i.e. a condition by valence interaction. These hypotheses would be expected based on previous research demonstrating an increased response to aversive stimuli ,14 and iWe also highlight that the effect of threat of shock on RT remained when pooling results from the second and third studies, and that background colour did not interact with condition. This indicates that the effect of shock we see is not driven by the background colour and is consistent with prior research that has also demonstrated no influence of background colour on the effects of threat of shock ,32.It should be noted that our paradigm, which uses a threat of shock manipulation, attempts to exploit anxiety and not fear responses. Fear can be thought of as a short lasting response to a predictable, threatening stimulus, whereas anxiety can be thought of as a response to unpredictable and prolonged threats. These different responses are behaviourally, neurally and pharmacologically discrete –35. Howep > 0.05) it should be noted that the breathalyser measures may have been unreliable. Since alcohol reduces anxiety and the key to investigating this affective bias in our task is comparison of performance across safe and threat (stress-inducing) conditions, the presence of unaccounted for alcohol-driven variance might explain our null finding on the first event. Nonetheless, unlike traditional controlled laboratory settings, these results can be considered a naturalistic representation of performance during a social event and demonstrate collecting data during public engagement events is possible. Indeed, we were especially keen to publish this data as part of this continuing engagement process such that participants will eventually be able to track their involvement all the way to this paper.It should be noted that the STAI measure we used to determine dispositional anxiety may not be the best measure to assess anxiety related to social stimuli such as faces. Future work may seek to determine individual differences in social anxiety. We also highlight the somewhat unique data collection environments which may have impacted on the data. Data were collected in very busy rooms with event attendees free to wander around and watch the testing. After optimization of the task in Study 2, we observed a main effect of valence, which suggests that participants were concentrating on the task. However, attention on the task might plausibly be less than it would have been in a controlled laboratory setting. This could be particularly relevant as individual differences in attentional control mechanisms interact with performance across threat and safe conditions  and promTo summarize, across two studies we replicated a previously observed effect, namely that threat of shock reliably slows down reaction times for ‘go’ responses ,24,26, a


P. vivax and P. falciparum malaria in patients from the Brazilian Amazon.A proliferation‐inducing ligand (APRIL) and B cell activation factor (BAFF) are known to play a significant role in the pathogenesis of several diseases, including BAFF in malaria. The aim of this study was to investigate whether APRIL and BAFF plasma concentrations could be part of inflammatory responses associated with P. vivax and P. falciparum malaria patients (n = 52) resident in Porto Velho before and 15 days after the beginning of treatment and from uninfected individuals (n = 12). We investigated APRIL and BAFF circulating levels and their association with parasitaemia, WBC counts, and cytokine/chemokine plasma levels. The expression levels of transmembrane activator and calcium‐modulating cyclophilin ligand interactor (TACI) on PBMC from a subset of 5 P. vivax‐infected patients were analyzed by flow cytometry.Blood samples were obtained from P. vivax and P. falciparum infections whereas BAFF levels were only increased during acute P. falciparum malaria. Although P. vivax and P. falciparum malaria patients have similar cytokine profiles during infection, in P. vivax acute phase malaria, APRIL but not BAFF levels correlated positively with IL‐1, IL‐2, IL‐4, IL‐6, and IL‐13 levels. We did not find any association between P. vivax parasitaemia and APRIL levels, while an inverse correlation was found between P. falciparum parasitaemia and APRIL levels. The percentage of TACI positive CD4+ and CD8+ T cells were increased in the acute phase P. vivax malaria.APRIL plasma levels were transiently increased during acute These findings suggest that the APRIL and BAFF inductions reflect different host strategies for controlling infection with each malaria species. Plasmodium species that infect humans, P. falciparum and P. vivax are responsible for 95% of malaria cases around the world. Although P. falciparum accounts for the vast majority of morbidity and mortality, P. vivax has a wider geographic distribution and causes significant symptomatic disease Plasmodium spp. Malaria is one of the most important human parasitic diseases. Nearly half the world's population is at risk of contracting malaria, with an estimate global annual incidence of about 212 million clinical cases and almost 429,000 deaths The mechanisms regulating BAFF and APRIL‐system molecule expression are poorly known. However, it is recognized that cytokines such as interferon (IFN)‐γ, IFN‐α, TNF, and Interleukin (IL)‐10 as well as granulocyte colony‐stimulating factor (G‐CSF), CD40 ligand (CD40‐L), lipopolysaccharides, and peptidoglycans can upregulate BAFF or APRIL expression in different cells P. falciparum malaria, BAFF levels are increased in plasma samples from infected children in Kenya P. falciparum antigens and hemozoin enhanced BAFF surface expression as well as secretion by human monocytes and increased B cell proliferation and IgG secretion P. falciparum malaria, as before mentioned, the role of APRIL in malaria is still unknown.In the recent years, several studies have been shown that BAFF, APRIL, and their receptors play a significant role in the pathogenesis of various noninfectious and infectious diseases P. vivax accounts for more than 85% of all malaria cases P. falciparum and P. vivax malaria in some adults from the Brazilian Amazon who were followed up during the acute and convalescent phases of infection.Brazil has a peculiar malaria epidemiological situation, in which P. vivax (n = 33) or P. falciparum (n = 19) malaria who resided in the communities of Porto Velho, the capital of the Rondônia state , in the Brazilian Amazon malaria endemic region . All malaria patients who were enrolled in this study complied with the following criteria: 1) they presented symptoms; 2) they had thin‐thick blood smears positive for malaria parasites; 3) they were not indigenous; 4) they were not prisoners; 5) they were all over 12 years old; and 6) no females were pregnant or breast‐feeding.P. vivax and/or P. falciparum were subsequently treated according to Brazilian Ministry of Health standards for malaria therapy: for uncomplicated malaria is recommended chloroquine combined to primaquine to P. vivax infection and artemether‐lumefantrine (Coartem®) to P. falciparum infection. Asexual blood forms of P. vivax and P. falciparum were cleared from the peripheral blood of all patients included in the study following therapy and no parasite reappearance was observed during follow‐up. The control group (n = 12) was composed of apparently healthy individuals who lived in the same geographic area, but were negative for malaria parasites as assessed by thick blood films and had not reported any malaria episodes for at least 5 years.The patients sought health care at the government outpatient clinic in Porto Velho after the onset of malaria symptoms. Blood samples were collected on the day of diagnosis before treatment (in the acute phase = D0) and 15 days later . After giving written informed consent which was reviewed and approved by the Ethical Committee of the Oswaldo Cruz Foundation (354/06), questionnaires were administered inquiring about demographic data, time of residence in the endemic area, personal and family malaria histories, presence of malaria symptoms, and use of malaria prophylaxis for each survey participant. After blood collection, donors positive for Plasmodium sp.) and species  were performed in all samples as previously described Venipuncture drew venous blood samples (5 mL) in EDTA‐containing tubes. Thin and thick blood smears of all donors were examined for malaria parasites. Parasitological evaluations were done by examination of 200 fields at 1,000‐fold magnification under oil‐immersion. The number of parasites was counted against 200 white blood cells (WBC). The parasite density per microliter of blood was calculated by multiplying the number of parasites counted by number of WBC divided by 200 7cells/mL in the freezing medium consisting of 90% heat‐inactivated fetal bovine serum  and 10% dimethyl sulfoxide  and placed into Nalgene cryogenic freezing container . The freezing container was then stored at −70°C overnight, and frozen cell samples were transferred to liquid nitrogen container (−196°C).WBC count was performed in the acute and during the convalescence phase using an automatic hematology analyzer (Pentra ABX). The cell counters provided data on leukocyte, lymphocyte, eosinophil, neutrophil, band cell, monocyte, and basophil counts. Qualified pathologists examined the smears. Of the subjects who agreed, venous peripheral blood (10 mL) was collected in heparin‐containnig tubes for flow cytometry analysis (five patients and four controls). Peripheral blood mononuclear cells (PBMC) were separated from whole blood by density gradient centrifugation . Cell viability was determined using the trypan blue dye exclusion assay. Cells were resuspended to a concentration of 2 × 10Cytokine and chemokine concentrations in plasma samples were determined by Luminex technology . Fourteen cytokines IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, IL‐12 p70, IL‐13, IL‐17, IFN‐γ, TNF‐α, G‐CSF, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and three chemokines  were analyzed using a BioPlex‐Kit assay . The assay was performed according to the manufacturer's instructions using a BioPlex‐kit in combination with the Luminex system. Briefly, 50 μL of standard or test sample along with 50 μL of mixed beads were added into the wells of a pre‐wetted 96‐well microtitre plate. After 1 h of incubation and washing, 25 μL of detection antibody mixture was added and the samples were incubated for 30 min and then washed. Finally, 50 μL of streptavidin‐PE was added and after 10 min of incubation and washing, the beads were resuspended in 125 μL assay buffer and analyzed using a BioPlex suspension array system (Bio‐Rad Laboratories) and the Bio‐Plex manager software (v.3.0). A minimum of 100 beads per region were analyzed. A curve fit was applied to each standard curve according to the manufacturer's manual and sample concentrations were interpolated from the standard curves. The limit of cytokine detection using this method was 2 pg/mL for all cytokines and chemokines. The median cytokine and chemokine levels in 12 healthy controls were 2 pg/mL for IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, IL‐12 p70, IL‐13, IL‐17, and GM‐CSF, 5.85 pg/mL for IL‐1β, 17.64 pg/mL for IFN‐γ, 23.23 pg/mL for TNF‐α, 4.40 pg/mL for G‐CSF, 379.13 pg/mL for MCP‐1, and 994.8 pg/mL for MIP‐1β.The plasma concentrations of APRIL and BAFF were measured with Human APRIL platinum ELISA Kit  and Human BAFF Instant ELISA kit  according to the manufacturer's instructions. Assays were performed in duplicate on 1/2 diluted samples. Plasma APRIL and BAFF levels were quantified using the in‐Kit standard curves for the respective cytokines. The minimal detectable doses of APRIL and BAFF in ELISA assay were 1.43 and 3.03 ng/mL, respectively. The median APRIL levels in 12 healthy controls were 5.02 ng/mL and the median BAFF were 6.72 ng/mL.Cryopreserved PBMC were thawed at 37°C, and Benzonase‐supplemented complete medium was slowly added  T test to calculate significance levels between patients and control group. Spearman‐rank correlation analysis was used to calculate correlations. Reported P values are two‐tailed, and statistical differences were considered significant when P values were less or equal to 0.05.The survey data were recorded and entered into a database created with EPI Info 2007 . Statistical analysis was carried out with GraphPad Prism 5.0  and R software. We previously checked whether the data was normally distributed by performing the D'Agostino's and Pearson omunibus normality test. As the data was not normally distributed we used Wilcoxon matched‐paired test to calculate significance levels between patients (D0 and D15) and Mann–Whitney unpaired P. vivax and P. falciparum malaria patient groups. Although the median of parasitemia appeared to be higher in P. vivax than in P. falciparum infected patients, this difference was not statistically significant. All patients were symptomatic at the time of enrollment independent of Plasmodium species and the most common symptoms reported by them were fever (91%), headache (89%), myalgia (77%), chills (77%), and vomiting (64%). However, no correlation was found between parasitaemia and axillary temperature (P < 0.05). There was no significant difference among the general features of the patient groups and control group, except for the absence of circulating parasites and the lowest number of previous malaria in the control group. All patients were parasitemia negative by day 15 of follow‐up after receiving effective drug treatment.General features of the volunteers are displayed in Table P. vivax (n = 33) or P. falciparum (n = 19). To investigate changes in APRIL and BAFF levels during the infection, we compared levels in the plasma from the same patient during the acute (D0) and convalescent (D15) phases.We analyzed APRIL and BAFF plasma levels from 12 healthy controls and 52 patients infected with P. vivax  and P. falciparum  than those of the controls . To investigate changes in APRIL levels during the infection, we compared APRIL levels in the plasma from the same patient during the acute (D0) and convalescent (D15) phases. During the convalescent phase P. vivax  and P. falciparum  patients had significantly lower levels of APRIL than during the acute phase. The levels of APRIL were similar between P. vivax and P. falciparum patients during both acute and convalescent phases .As shown in Figure P. falciparum patients  during the acute phase and returned to the levels similar to those of the controls . During the convalescent phase, P. falciparum  patients had significantly lower levels of BAFF than during the acute phase. In P. vivax patients, although the median BAFF levels were increased during the acute  and convalescent  phases, these levels were not statistically different from those of the controls . The levels of BAFF were similar between P. vivax and P. falciparum patients during both acute and convalescent phases . Furthermore, no correlation was found between APRIL and BAFF plasma levels during both acute and convalescent phases of P. vivax  and P. falciparum  malaria. The correlation between APRIL plasma levels and parasitaemia was only observed in P. falciparum malaria . There was no correlation between BAFF plasma levels and parasitaemia in P. falciparum  or P. vivax malaria .As shown in Figure P. vivax and P. falciparum‐infected patients  and convalescent (D15) phases. P. vivax and P. falciparum patients presented higher levels of IFN‐γ, TNF‐α, IL‐6, IL‐8, IL‐17, and MIP1β in the convalescent phase than in the acute phase. Although P. vivax and P. falciparum malaria patients have similar cytokine profiles during infection, P. falciparum patients presented higher levels of IFN‐γ and TNF‐α, in acute phase and of IFN‐γ, IL‐6, IL‐8, IL‐17, and G‐CSF in convalescent phase than P. vivax patients. Moreover, the levels of IL‐2, IL‐4, and IL‐12 were increased in the convalescent phase of P. falciparum infection. On the contrary, IL‐10 levels decreased in the P. vivax and P. falciparum malaria convalescent phase and returned to healthy normal levels. The cytokines IL‐5, IL‐7, and GM‐CSF were not detectable in most plasma samples. No differences were observed in MCP‐ 1 levels compared with controls. The lL‐10 and G‐CSF levels were positively correlated with parasitaemia in the P. vivax  and P. falciparum  malaria acute phase. The same was true for IFN‐γ , IL‐2  and IL‐4  in P. falciparum malaria patients.Table P. vivax acute phase, APRIL plasma levels were positively correlated with IL‐ 1 , IL‐2 , IL‐4 , IL‐6 , and IL‐13  while BAFF plasma levels were not statistically correlated with any of the cytokines/chemokines assayed. No correlation was observed between APRIL, BAFF, and cytokine plasma levels during the P. vivax convalescent phase. No relationship was found between any of the cytokines/chemokines assayed and the APRIL or BAFF plasma levels in P. falciparum acute or convalescent phases and control group .As shown in Table P. vivax malaria patients during the acute (D0) and convalescent (D15) phases and four healthy controls was performed  compared to the controls   .Transmembrane Activator and CAML Interactor (TACI) is a receptor of the APRIL/BAFF system and is expressed on B cells and activated T cells. To determine whether malaria induces increased expression of TACI, phenotypic analysis of PBMC of five ed Figs. A and 3B.Plasmodium infection Plasmodium immune‐modulatory molecules P. falciparum malaria, nothing is known about the role of the follows: 1) APRIL in any malaria or 2) BAFF/APRIL system in P. vivax malaria. In this regard, the main objective of this study was to investigate whether BAFF and APRIL plasma concentrations could be part of inflammatory response associated with uncomplicated P. falciparum and P. vivax malaria in some adults from the Brazilian Amazon that were followed up during the acute and convalescent phases of infection.Malaria is characterized by many pathophysiological changes including alterations in the immune system involving myeloid‐cells, T‐cells and B‐cells. Regarding B‐cells, a number of observations clearly demonstrate that B‐cell homeostasis is affected by P. vivax and P. falciparum infections whereas BAFF levels were only increased during acute P. falciparum malaria. These findings raise a possibility that the regulation of BAFF secretion may differ between the two strain infections. Although a range of myeloid cells produces BAFF and APRIL, the mechanisms regulating production and expression are poorly understood and likely differ from cell type to cell type. Plasmodium spp. infections elicit significant changes including immunological alterations involving myeloid cells and cytokine levels. In line with previous studies P. vivax and P. falciparum malaria  in malaria‐naive adults P. falciparum antigens (sPfAg) are capable of inducing BAFF release from monocytes and B‐cell co‐cultures P. falciparum and P. vivax are genetically distant malaria parasites P. vivax acute phase malaria, APRIL but not BAFF levels correlated positively with IL‐1, IL‐2, IL‐4, IL‐6, and IL‐13 levels. In addition, we did not find any relationship between P. vivax parasitaemia and APRIL levels, while an inverse correlation was found between P. falciparum parasitaemia and APRIL plasma levels. Although APRIL and BAFF share several biological characteristics and receptor‐specificity, they have distinct functions. While BAFF is fundamental for B‐cell maturation and survival, APRIL is essential for antibody class‐switching and plasma‐cell survival and is involved in the late stage of B‐cell differentiation First, we observed that the APRIL plasma levels were increased during acute ia Table . In bothConsistent with our previous findings P. vivax and P. falciparum malaria P. vivax and P. falciparum malaria.The APRIL and/or BAFF plasma levels are upregulated in many noninfectious P. vivax and P. falciparum acute phase malaria. However, lymphopenia was a transient finding and 15 days after the beginning of treatment, the lymphocyte counts were similar to those of control subjects, indicating that this period was sufficient for the patients to achieve lymphocyte homeostasis, according to our previous findings Consistent with previous findings P. falciparum malaria in Kenyan children. We have access to PBMC of five P. vivax malaria patients in acute and convalescence phases. Our results showed a transient and significant increase in the percentage of TACI‐positive CD4+ and CD8+ T cells during the acute phase, as well as a transient but not significant increase in the percentage of TACI‐positive CD19+ B cells in P. vivax infected donors. Aware that our sample size is too small to make inferences, we also can not rule out the possibility that the discrepancies observed between this study and ours with respect to TACI‐positive B cells, could be associated with the fact that patients involved in our study were adults with uncomplicated P. vivax malaria living in different epidemiological scenarios. In relation to T cells, TACI expression presented a transient kinetics similar to IL‐10 and APRIL levels. Thus, the APRIL and IL‐10 plasma levels as well as the proportion of CD4+ and CD8+ T cells expressing TACI might reflect the acute phase of P. vivax malaria, and may contribute to calcium‐dependent constitutive activation of nuclear factor of activated T cells (NF‐AT) TACI is a receptor of the APRIL/BAFF system and is predominantly expressed on B cells and in some subsets of activated T cells P. vivax infection. Moreover, it seems that the APRIL and BAFF inductions reflect different host strategies for controlling infection with different malaria species, P. vivax and P. falciparum. Further larger cohort study is ongoing in the Brazilian Amazon to provide better knowledge of the APRIL/BAFF system as well as the TACI B‐ and T‐cell expression in the immunomodulation of malaria.Taken together, we demonstrate that APRIL plasma levels as well as the percentage of TACI positive CD4+ and CD8+ T cells were increased in the acute phase of uncomplicated RAP performed experiments, statistical analysis and wrote the paper. ACS performed experiments and manuscript review. DSPS performed statistical analysis and manuscript review. RNRS performed experiments. LAS performed experiments. MRA performed statistical analysis. FS did fieldwork support. JCLJ performed collection of blood and epidemiological data and manuscript review. JOF performed collection of blood and epidemiological data. DMVV designed the study and performed manuscript review. PMDL performed the experiments, statistical analysis and manuscript review. CECP designed the study and performed manuscript review. DMB conceived, designed and coordinated the study, performed collection of blood and epidemiological data and wrote the paper. All the authors read and approved the manuscript for publication.None.


CI) 1.50‐2.04; p < .001]. The relative risks in individuals with and those without diabetes were 2.10  and 1.50 , respectively. In all individuals, the relative risk of hyperglycaemia requiring initiation of hypoglycaemic therapy, comparing glucocorticoid treatment with placebo, was 1.73 . In conclusion, glucocorticoid therapy increases the risk of hyperglycaemia in all individuals with underlying respiratory disease but not when diabetic status is analysed separately.The relative risk of glucocorticoid‐induced hyperglycaemia is poorly quantified. We undertook a meta‐analysis to estimate the association between glucocorticoid treatment and hyperglycaemia, overall and separately in individuals with and without diabetes and underlying respiratory disease. We searched electronic databases for clinical trials of adults randomized to either glucocorticoid treatment or placebo. Eight articles comprising 2121 participants were identified. We performed a random effects meta‐analysis to determine relative risks for the associations between glucocorticoid use and both hyperglycaemia and starting hypoglycaemic therapy. In all individuals, the relative risk of hyperglycaemia comparing glucocorticoid treatment with placebo was 1.72 [95% confidence interval ( In the UK, the prevalence of glucocorticoid use at any one time is ~1%.Current data regarding glucocorticoid‐induced hyperglycaemia are based on data from retrospective prescription databasesUsing meta‐analytical techniques, the present study estimates the relative risk of glucocorticoid treatment and hyperglycaemia in individuals with and without diabetes and underlying respiratory disease. It also estimates the association between glucocorticoid use and initiation of hypoglycaemic therapy.22.1ClinicalTrials.gov. We searched MEDLINE using standard medical subject heading (MeSH) terms  and free text  terms. We searched EMBASE using “map term to subject heading” in Advanced Ovid Search. We scanned reference lists of relevant articles to supplement our literature search. We did not restrict the date of study publication.We searched the electronic databases MEDLINE, EMBASE, Cochrane and 2.2ClinicalTrials.gov (n = 112), plus 48 additional citations obtained through scanning reference lists. We included studies with patients aged ≥18 years not treated at baseline with systemic  glucocorticoids for underlying COPD, pneumonia or interstitial lung disease. We included trials if participants received orally or intravenously administered glucocorticoids  or placebo and/or additional therapy for underlying pulmonary disease for at least 72 hours. We included studies that documented glycaemia throughout, including diabetic status at baseline, and that documented when therapy for hyperglycaemia started. Our primary outcome was hyperglycaemia, as defined by each study‐specific protocol. Our secondary outcome was hyperglycaemia requiring therapy. Eight studies, comprising 2121 participants, met our inclusion criteria .We identified citations in MEDLINE (n = 262), EMBASE (n = 1475), Cochrane Database (n = 138) and 2.3We collected the following data from each study: study design; year of publication; number of participants; age; sex; underlying respiratory disease; diabetes status at study entry; type and dose of glucocorticoid; method and frequency of quantifying blood glucose; new hyperglycaemia; new hypoglycaemic therapy in response to hyperglycaemia; type of hypoglycaemic agent; and length of follow‐up. Where published data were not available, we communicated with investigators of included studies to obtain additional data about the diabetic status of participants.2.42‐statistic. To investigate small study effects, we used Begg's funnel plot.We calculated a pooled estimate of the association between glucocorticoid treatment and hyperglycaemia by combining the study‐specific relative risks and absolute risk difference using random effects meta‐analysis. We assessed heterogeneity between studies using the I33.1Supplementary Table S1 displays the details of the trial characteristics, all of which were placebo‐controlled.3.22 = 0%  1.50‐2.04; p < .001; I3.32 = 0% ,A Cochrane review systematically assessed the risk of hyperglycaemia in six studies that administered glucocorticoids to treat acute exacerbations of COPD; however, diabetic status was not ascertained.Observational studies have estimated odds ratios ranging between 1.36 and 2.31 for the association between glucocorticoid therapy and new‐onset diabetes mellitus; however, the primary endpoints for these studies were new prescriptions filled for glucocorticoidsGlucocorticoids increase hepatic gluconeogenesis, reduce peripheral glucose uptake, inhibit insulin secretion and cause postprandial hyperglycaemia. Seven studies included in the present analysis did not define the timing of blood glucose measurements. One study, in which patients were randomized to once‐daily intravenous dexamethasone or placebo, sampled blood daily at 08:00 hours, which may not be sensitive to hyperglycaemiaIn summary, the data suggest that glucocorticoids increase the risk of hyperglycaemia in patients with respiratory disease. In subjects analysed based on diabetic status alone, if an association between treatment with glucocorticoids and hyperglycaemia exists, then this study did not find it. Our estimates may reflect publication bias. We recommend that future trials of glucocorticoids determine glycaemia at baseline, during follow‐up, and at time intervals that reflect the pharmacokinetic actions of glucocorticoids.S. J. S. is supported by the Medical Research Council (Grant: MC_UU_12015/1).B.G.C and A.A designed the study. S.B and B.G.C collected the data. S.S performed statistical analysis. B.G.C and A.A wrote the manuscript.FigureS1. Study flow diagram illustrating selection process for published studies included in the final meta‐analysis.Click here for additional data file.FigureS2. Funnel plot to assess for small studies effect in the analysis of the relative risk of hyperglycaemia comparing glucocorticoid treatment with placebo in all individuals. Studies above the dark contour have p < .01, studies between the light and dark contour have .01 < p < .05, studies below the light contour have p > .05.Click here for additional data file.FigureS3. Funnel plot to assess for small studies effect in the analysis of relative risk of hyperglycaemia requiring initiation of hypoglycaemic therapy comparing glucocorticoid treatment with placebo in all individuals. Studies above the dark contour have p < .01, studies between the light and dark contour have .01 < p <.05, studies below the light contour have p > .05.Click here for additional data file.TableS1. Baseline characteristics of participants in the seven studies included in the meta‐analysis. N/A, not available.Click here for additional data file.


Neutrophils from overweight/obese group had a significantly higher free radical production compared to the normo-weight group. In neutrophils, the lowest CPE concentration significantly reduced free radical production in overweight/obese group only, and higher CPE concentrations were effective in both groups. In monocytes, the CPE concentration that was significantly effective in reducing free radical production was lower in overweight/obese subjects than in normo-weight subjects. Chocolate polyphenol extracts inhibit oxidative burst in human neutrophils and monocytes with a higher efficiency in subjects characterized by an unphysiological oxidative/inflammatory stress, such as overweight and obese. Results of this study provide further evidence about a differential role of dietary antioxidant strictly related to the “stress” condition of the subjects.Oxidative and inflammatory stress represents a major risk factor for cardiovascular disease (CVD) in overweight and obese subjects. Between the different plant foods, chocolate has been shown to decrease CVD risk due to its antioxidant and anti-inflammatory properties. However, as we recently showed in epidemiological studies, meta-analyses, and human trials, dietary antioxidants resulted more effective in subjects characterized by an ongoing oxidative stress, than in healthy people. Aim of this work was to investigate the effect of different concentrations of chocolate phenolic extract (CPE) on  Oxidative stress, imbalance between reactive oxygen species (ROS) production and the neutralizing capacity of antioxidant mechanism, represents a prepathological status involved in the development of majority of degenerative diseases . A raisiThe tight link between oxidative and inflammatory stress, in the mechanisms of body’s defenses against stress, is highlighted in the oxidative burst of leukocytes, the innate immune response involving the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase yielding to a massive production of ROS and reactive nitrogen species . PolymorCocoa and, among its principal products, dark chocolate are functional foods that have been shown to display an antioxidant role in humans . The antin vitro neutrophils and monocytes, isolated from normo-weight and overweight/obese subjects, from oxidative damage and to identify the correlation between body mass indices and oxidative stress.The aim of the present study was to investigate the ability of polyphenol extracts from dark chocolate rich in flavonoids to protect Different types of dark chocolate bars rich in polyphenols and having a cocoa percentage between 60 and 70%, identified in previous studies, were analyzed in order to choose the sample with higher antioxidant  content and antioxidant activity.g for 10 min, and the hexane was decanted. Hexane was evaporated at room temperature overnight. The defatted sample was extracted with a solvent mixture . After the addition of solvent, the tube was vortexed for 30 s and eventually subjected to sonication at 37°C for 10 min. At the end of extraction, the tube was centrifuged at 2,000 × g for 15 min. The supernatant, representing the chocolate phenolic extract (CPE), was collected and used for further analyses.Sample extraction was carried out according to the procedure described by Adamson et al. . Briefly−1 with a linear gradient from A (dichloromethane) to B (methanol) and a constant 4% level of C  according to Counet and Collin (Flavanols and proanthocyanidins determination was carried out by high-performance liquid chromatography (HPLC) analysis according to Ioannone et al. . The samd Collin . Gradiend Collin  and subm−1 by a syringe pump; all the analytes were detected in negative ionization with a capillary voltage of −4,500 V, nebulizer gas (air) at 0.21 N mm−2, curtain gas (nitrogen) at 0.21 N mm−2. The declustering potential was set at −22 V for m/z < 1,000 amu; −80 V for m/z > 1,000 amu. For the MS/MS experiments, the collision gas was set at 3  and the collision energy was −20 eV. The spectra were acquired using the AB-Sciex Analyst Software 1.5.The MS analysis of the HPLC fractions (P1–P10) has been carried out by means of a triple quadrupole mass spectrometer API 2000 from AB-Sciex  equipped with a TurboIon-Spray source. The spectra were acquired by injecting each solution at a flow of 10 μL minThe quantification of single proanthocyanidins was carried out by HPLC analysis using diode array detection. Since proanthocyanidins show a similar absorption coefficient , a calib3+-TPTZ) complex to the ferrous form at low pH. Briefly, 160 µL of FRAP reagent, prepared daily, was mixed with 30 µL of water and 10 µL of diluted CPE; the absorbance at 595 nm was recorded after a 30 min incubation at 37°C with the Sunrise absorbance plate reader. FRAP values were calculated using a calibration curve obtained with increasing concentrations of Fe2+ and expressed as micromoles of Fe2+ per micromoles per gram of sample.The total phenolics index (TPI) was determined according to Singleton and Rossi . A total−2] and seven overweight and obese subjects  were recruited for the study. Criteria for inclusion were based on physical examination and BMI. Exclusion criteria are diabetes mellitus, CVD, gastrointestinal tract disease, pulmonary disease, psychiatric disorder, alcohol and drug dependence, history of organ transplant, surgery within 12 months, positive test results for immunodeficiency virus, evidence for hepatitis B virus or hepatitis virus C infection, chronic liver disease or nephropathies, cancer, organ failure, taking lipids-lowering, anti-inflammatory or other medications, taking vitamins, minerals, polyphenols or other supplements, regular consumer of fruits and vegetables of more than four servings per day, following caloric restriction diet, and unbalanced intake of macro and micro nutrients. Waist circumference (WC) was measured in subjects who were categorized as normal or overweight on the BMI scale. All subjects gave written informed consent in accordance with the Declaration of Helsinki.Eight normo-weight [four men and four women aged 46 ± 9 years, body mass index (BMI) = 20.54 ± 0.94 kg mVenous peripheral bloods from health volunteers were collected in vacutainer tubes containing ethylene diamine tetraacetic acid (EDTA). All the tubes were stored at room temperature and were immediately used as a source of polymorphonuclear leukocytes (PMNs) for oxidative burst generation assay. PMNs were isolated after bulk erythrocyte lysing. After 10 min of incubation with lysis buffer , the cells were washed twice with phosphate buffer saline (PBS). The method adopted for the measurement of oxidative burst in neutrophils use dihydrorhodamine 123 (DHR123) as probe and phorbol 12-myristate 13-acetate (PMA) for activation . DHR123 in vitro ability of dark chocolate to protect neutrophils and monocytes, leukocytes were split into different aliquots containing PMA and different concentration of CPE (previously dried under nitrogen flow and then suspended in dimethyl sulfoxide): 5, 10, 20, 50, and 100 µg mL−1. To test whether polyphenols in chocolate extract preparation were responsible for the inhibitory effect of oxidative burst, the effect of EC, one of the major polyphenols in cocoa, was studied at a concentration of 100 µM. Flow cytometric analyses were carried out using a FACScan flow cytometer . Lymphocytes, monocytes and neutrophils were sorted out using the cytometer by segregating them in gates using forward angle light scatter and side scatter. ROS production was quantified by mean channel fluorescence (MCF) of DHR123 green fluorescence histogram (FL1). Free radicals production (stimulation index) was obtained by dividing the MCF of PMA-stimulated granulocytes by the MCF of unstimulated granulocytes (To investigate the ulocytes .t-test for independent (unpaired) samples. Statistical analyses were performed using the Microsoft Excel software.All determinations were carried out at least in triplicate. Experimental data are presented as mean values ± SD. Differences between means were compared using the Student’s The antioxidant  content, the total phenolic index, and antioxidant activity of six commercial dark chocolate samples with 60–70% cocoa were determined. Table ex vivo study aimed to investigate the effect of chocolate phenolic extract (CPE) addition on PMA-induced oxidative burst of leukocytes isolated by normo-weight and overweight/obese subjects.Sample 1, showing the highest content of total polyphenols as well as the highest antioxidant capacity, was characterized for its phenolic profile Table  and was In order to evaluate relationship between BMI and oxidative stress, the response to PMA for all subjects in both neutrophils and monocytes was first compared. Eventually, the effect of chocolate polyphenols was evaluated by comparing the monocytes and neutrophils activation between the two groups of subjects after adding different concentrations of chocolate extract.P < 0.001) positive correlation  between the considered morphometric indices and the response of neutrophils after PMA stimulation. No correlation was found between the same morphometric indices and the response of monocytes after PMA stimulation (data not shown).In Figure P < 0.05) intracellular ROS generation when compared with those from healthy normo-weight subjects since the mean of fluorescence in normal individuals was 1,954 ± 61 and in overweight/obese ones was 2,353 ± 78  significantly reduced fluorescent intensity (P < 0.01 vs. PMA) in overweight/obese subjects only, with an inhibition of radical production of 27% respect to PMA. A 10 µg mL−1 chocolate concentration fraction showed a significant reduction (P < 0.01 vs. PMA) of fluorescent intensity for overweight/obese subjects and a less significant reduction (P < 0.05 vs. PMA) in normo-weight subjects . Higher chocolate polyphenol concentration (from 20 to 100 µg mL−1) showed a more significant reduction (P < 0.001 vs. PMA) in fluorescence intensity than low concentration in both groups . EC, used as positive control, exerted a significant (P < 0.001 vs. PMA) antioxidant effect in normo-weight and overweight/obese subjects, at a concentration of 100 µM.In neutrophils, as shown in Figure −1 of chocolate polyphenol extract significantly reduced (P < 0.01) fluorescence intensity (MCF = 37 ± 12) in overweight/obese subjects group respect to PMA fluorescence (MCF = 72 ± 25), while in normal weight group, the reduction of fluorescence intensity (MCF = 35 ± 11) with respect to PMA (78 ± 14) was present but less significant (P < 0.05). The concentration of 50 µg mL−1 resulted the second most effective polyphenol concentration in reducing fluorescence both for normo-weight and overweight/obese subject groups (P < 0.05 vs. PMA). At 20 µg mL−1 polyphenol concentration, a significant reduction was observed only for overweight/obese subjects (P < 0.05 vs. PMA). The lowest chocolate polyphenol concentration (10 and 5 µg mL−1) did not show any significant activity in monocytes. EC of 100 µM determined a significant fluorescence reduction for normal weight (P < 0.05 vs. PMA) and overweight/obese subjects (P < 0.01). A significant correlation was observed between percentage of inhibition of ROS in neutrophils and monocytes and dark chocolate concentrations of phenolics in the normo-weight  was effective in reducing significantly ROS production and a 50 µg mL−1 (69 µM) chocolate extract determined a protection against ROS formation not significantly different from the 100 µg mL−1 (147 µM) catechin positive control. Since the chocolate phenolic extract contains catechin, EC, and polymeric proanthocyanidins, it is possible to affirm that polymeric proanthocyanidins, that are present in a certain amount in chocolate, could show an inhibitory effect of ROS production even higher than catechin.The first evidence of an inhibitory effect of flavonoids on the respiratory burst of neutrophils was by Pagonis et al. , who shoAt low dosages, the CPE was more effective in inhibiting the oxidative burst of neutrophils than that of monocytes. Most of the studies on oxidative burst concern analysis of neutrophils and not monocytes. In fact, these two types of cells differ in structure, time of activation, and behavior during the inflammation response. In a typical acute inflammatory response, there is a well-defined sequence: neutrophils accumulate first and usher monocytes into sites of inflammation where the latter accumulates second . ProbablPostprandial stress, arising from the consumption of unbalanced high caloric meals, has been associated with an increased risk for atherosclerosis and obesity . Indeed,Dark chocolate polyphenols showed an inhibitory effect on PMA activated oxidative burst in white blood cells, in a concentration-dependent manner. This inhibitory effect is significantly more evident in subjects characterized by an unphysiological oxidative/inflammatory stress, such as overweight and obese subjects. Although antioxidant capacity of dark chocolate in human is deeply related to the low bioavailability of the flavonoids contained in the foodstuff, our results suggest that chocolate polyphenols might efficiently display a higher antioxidant/anti-inflammatory effect in overweight and obese subjects, characterized by an ongoing oxidative and inflammatory stress, than in normo-weight subjects. Results of this study provide further evidence about a differential role of dietary antioxidant related to the “stress” condition of the subjects. In future human trials, one criteria of selection of the subjects should be the presence of oxidative stress condition in order to maximize the chance of success of the antioxidant intervention. Specifically, more evidences in human trials are needed in order to unravel the role of cocoa and polyphenols in reducing oxidative and inflammatory stress in overweight and obese subjects.Due to the nature of the study, simple collection of blood specimens, the involvement of ethical committee was not necessary. All subjects remain anonymous and gave written informed consent in accordance with the Declaration of Helsinki.MS designed the experiment; FI and GS conducted the experiments; MS and GS contributed to data analysis and interpretation; FI, GS, and MS contributed to the manuscript drafting.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.