Despite hundreds of studies, there is continuing debate about the extent to which violent video games increase aggression. Believers argue that playing violent video games increases aggression, but this stance is disputed by skeptics. The present study addressed believers' and skeptics' responses to summaries of scientific studies that do or do not present evidence for increased aggression after violent video game play.N = 662) indicated whether they believed that violent video games increase aggression. Afterwards, they evaluated two opposing summaries of fictitious studies on the effects of violent video play. They also reported whether their initial belief had changed after reading the two summaries and indicated again whether they believed that violent video games increase aggression. Results showed that believers evaluated the study showing an effect more favorably than a study showing no effect, whereas the opposite was observed for skeptics. Moreover, both believers and skeptics reported to become more convinced of their initial view. In contrast, for actual attitude change, a depolarization effect was found.Participants (These results suggest that biased assimilation of new information leads believers and skeptics to become more rather than less certain of their views. Hence, even when confronted with mixed and inconclusive evidence, the perceived gap between both sides of the argument increases. The question of whether playing violent video games causes aggression has received considerable attention in scientific and public debate. This is not surprising insofar as video game play has become an integral part of the lives of many people. In the United States, 91% of children between the age of two and seventeen play video games Numerous studies have shown that playing violent video games is associated with increased aggression. Correlational work revealed positive correlations between violent video game play and aggression in real world contexts However, there are also studies that failed to find that violent video games cause aggression Likewise, many lay people are concerned that playing violent video games increases aggression, whereas this stance is disputed by others. For instance, a recent representative sample of 2,278 American adults showed that 58% somewhat agreed or strongly agreed that there is a link between playing violent video games and teenagers showing violent behavior, whereas 32% somewhat disagreed or strongly disagreed. The remaining 10% were unsure The present research examined believers' and skeptics' responses to scientific studies examining the link between violent video game play and aggression. Abundant research has shown that people are more willing to search for congruent information and abstain from seeking out incongruent information biased assimilation). As a consequence, both believers and skeptics should feel validated and report to become more convinced of their initial view (attitude polarization). Hence, even when learning about mixed evidence, the perceived gap between believers' and skeptics' views should increase.But what happens if one is confronted with inconclusive evidence? Many news media reports operate according to a fairness doctrine, in that both sides of a debate are presented even when the overall evidence clearly supports one view over the other Biased assimilation is the tendency to evaluate belief-consistent information more positively than belief-inconsistent information. In a seminal study, participants were first asked whether the death penalty is an effective deterrent against murder After evaluating the two opposing studies, Lord and colleagues asked their participants about the perceived changes in their attitudes toward capital punishment. Results showed that both proponents and opponents became more polarized in their opinions (attitude polarization): When they were asked to compare their current attitude toward capital punishment with the attitude they had at the beginning of the experiment, proponents reported that they were even more in favor of capital punishment, whereas opponents reported that they were even more against capital punishment. Note that Lord and colleagues did not ask their participants to indicate their actual attitude toward capital punishment after reading the essays, so actual attitude change was not assessed. This is important insofar as subsequent research has replicated attitude polarization for reported attitude change The present research employed a similar design as these previous investigations. Participants were asked to what extent they believe that playing violent video games causes aggression . Afterwards, they were given summaries of two fictitious studies about the effects of violent video games. Whereas one study apparently found support for increased aggression after violent video game play (positive effect study), the other study found no significant association (no-effect study). After evaluating both studies, participants were asked to indicate their perceived attitude change as well as their belief toward the effect of violent video games on aggression after learning about these two studies . It was predicted that believers would evaluate the positive effect study more favorably than the no-effect study, whereas the opposite pattern should be observed for skeptics. Moreover, this biased assimilation should lead to differences in reported attitude change, in that believers report to become even more convinced that violent video games increase aggression, whereas skeptics report to become even less convinced. In terms of actual attitude change, no attitude polarization was expected.As a further aim, the present study examined whether regular players of violent video games would be less likely to believe that violent video games increase aggression than non-regular players. Most, if not all, people are motivated to perceive themselves in a positive light and to make their beliefs consistent with their behaviors. Because behaviors are often more difficult to change than beliefs, people tend to alter their beliefs to make them consistent with discrepant behavior. There is wide consensus that aggression is an unwanted behavior. Thus, if players of violent video games learn that their behavior makes aggressive, their self is threatened. This reasoning holds that regular, more than non-regular, players tend to deny the existence of the negative consequences of violent video game play. In fact, people scrutinize information that is not compatible with a positive self-view, whereas they tend to accept information that does not threaten the self at face value Finally, previous research suggests In Austria, it is not necessary to get explicit ethical approval if the study conforms to the guidelines of the German Psychological Society. Because this is the case for the current study, I got tacit approval from our ethics review board. That is, approval was waived. At the beginning of the study, participants read detailed instructions regarding ethical guidelines . This instruction page serves as documentation of participants' informed consent. Our ethics committees/IRBs approve this consent procedure.SD = 5.8).Participants were 666 students who were recruited via a university mailing list. Because psychology students may have learned about the relation between violent video games and aggression in class, only non-psychology students received the invitation to participate. Attentive participation was verified with an item manipulation check At the onset, participants learned that the study was about the effects of violent video game play. About half of the participants were then asked to name their three favorite video games, to estimate the number of hours per week spent playing each video game, and to rate how violent the content of each video game was. For each video game, the amount of playing time was multiplied by violent content. These three violent video game exposure scores were averaged to provide an overall index of violent video game exposure. This approach has been successfully employed in previous video game research strongly decrease aggression), 0 (no effect), +5 (strongly increase aggression). There were 397 participants who thought that violent video games would increase aggression, 142 participants thought violent video games had no effect on aggression, and 123 participants who thought that violent video games would decrease aggression. For the following analyses, the latter two categories were combined, so that participants who thought that violent video games would increase aggression (believers) were compared with participants who thought that violent video games would not increase aggression (skeptics). Because no study had to be evaluated that allegedly shows that violent video games decrease aggression, the pieces of new information employed in the present study  were inconsistent and consistent with the initial beliefs for both participants who thought that violent video games had no effect on aggression and participants who thought that violent video games would decrease aggression.Then, participants indicated their initial belief toward whether violent video games increase aggression. To this end, participants responded to a single Likert-type item. The anchors were: −5  to 7 (very much). The positive effect summary read:Afterwards, participants were presented with two opposing summaries of fictitious studies on the effects of violent video play. These two studies allegedly appeared in scientific journals in 2012. After each summary, participants indicated to what extent the study was convincing and replicable in future studies, respectively. The scale for both items was from 1 , 0 (unchanged), +5 (more convinced). Participants also indicated their final belief toward whether violent video games increase aggression, using the same anchors as before. Finally, participants were thanked and debriefed.Participants were randomly assigned to read the positive effect study either first or second. However, study order did not qualify any of the main findings and is thus not considered further. Afterwards, reported attitude change was assessed by asking participants whether their belief toward the increasing effect of violent video game play on aggression had changed after reading the two summaries. The anchors were: −5  = 272.03, p<.001, η2 = .29  more favorably than the no-effect study , F = 289.76, p<.001, η2 = .42, whereas skeptics evaluated the no-effect study  more favorably than the positive effect study , F = 54.39, p<.001, η2 = .17. Thus, there was strong evidence for biased assimilation.Ratings of how convincing and replicable the study is were highly correlated and were, thus, averaged into a general perceived quality index . This general perceived quality index was analyzed in a 2 ×2 (study: positive effect vs. no effect) analysis of variance (ANOVA), with study as a within-groups factor. Results revealed the predicted significant interaction between initial belief and study, η2 = .29 . BelieveF = 104.29, p<.001. Most importantly, initial belief significantly predicted the difference score, β = .50, t(658) = 13.44, p<.001. Moreover, participant sex received a significant regression weight, β = −.07, t(658) = 2.01, p = .045, whereas violent video game play marginally significantly predicted the difference score, β = −.07, t(658) = 1.93, p = .054. In sum, the regression analysis replicated the main finding that initial belief had an effect on biased assimilation, while controlling for participant sex and the extent to which participants reported to play violent video games. Participant sex and violent video game play effects will be addressed more thoroughly below.Splitting the initial belief variable into believers and skeptics is the standard procedure in the literature. It also tellingly illustrates the point that people with opposing beliefs differ in the way how they evaluate novel, conflicting information. Nevertheless, the use of a continuous measure rather than dichotomizing the beliefs increases statistical power. In fact, the increase in power should enable participant sex and video-game play to be included in the analyses. As in past research M = +0.12, SD = 0.92), whereas skeptics reported becoming less convinced , F = 14.26, p<.001, η2 = .02. That is, attitude assimilation was observed for reported attitude change.Attitude assimilation was observed for reported attitude change: believers reported becoming more convinced that violent video games increase aggression  = 9.50, p<.001. Initial belief significantly predicted reported attitude change, β = .15, t(658) = 3.40, p = .001, whereas participant sex, β = −.03, t(658) = 0.78, p = .435, and violent video game play, β = −.06, t(658) = 1.40, p = .162, received non-significant regression weights. Participant's differential evaluations of the studies (biased assimilation) were significantly correlated with reported attitude change, r(662) = .19, p<.001.This finding was replicated in a multiple regression where reported attitude change was regressed on the continuous measure of initial beliefs, participant sex, and the extent to which participants reported to play violent video games. The overall regression was significant, F = 79.70, p<.001, η2 = .11  than before , F = 62.26, p<.001, η2 = .14, whereas skeptics became less convinced that violent video games do not increase aggression after reading the summaries  than before , F = 25.61, p<.001, η2 = .09.In the following, it was examined whether there was attitude polarization for actual beliefs. That is, believers should become more convinced that violent video games increase aggression after reading the summaries, whereas skeptics should become more convinced that violent video games do not increase aggression. To test this hypothesis, a 2 ×2  ANOVA, with belief as a within-groups factor, was performed on the data. Results revealed a significant interaction between initial belief and belief, η2 = .11 . HoweverF = 48.54, p<.001. Initial belief significantly predicted actual attitude change, β = −.48, t(658) = 11.87, p<.001. Whereas participant sex was not associated with actual attitude change, β = −.03, t(658) = 0.78, p = .438, violent video game play did receive a significant regression weight, β = −.17, t(658) = 4.18, p<.001. Biased assimilation was not significantly correlated with actual attitude change, r(662) = .02, p = .658. Moreover, reported attitude change was not related to actual attitude change, r(662) = .02, p = .624. Thus, biased assimilation may underlie the effect of initial attitude on reported attitude change, but could not mediate the effect on actual attitude change.For the multiple regression analysis, initial beliefs were subtracted from final beliefs. The overall regression was significant, p<.05, 95% confidence interval ), indicating that biased assimilation accounted for the finding that believers and skeptics reported to become more convinced of their initial attitude.The mediation analysis examined whether participant's differential evaluations of the studies (biased assimilation) would mediate the effect of initial belief on reported attitude change. In fact, a bootstrapping analysis based on 1,000 bootstraps confirmed that the indirect effect was significantly different from 0 (r(662) = −.46, p<.001, rating of the positive effect study, r(662) = −.25, p<.001, rating of the negative effect study, r(662) = .27, p<.001, and reported attitude change, r(662) = −.15, p<.001. That is, the more participants reported to play violent video games, the less they were convinced that violent video games increase aggression, the more negatively the positive effect study was evaluated, the more favorably the no-effect study was evaluated, and the less they reported becoming convinced that violent video games increase aggression. In contrast, violent video game play was not related to actual attitude change, r(662) = .04, p = .324.Violent video game play was related to initial belief, M = 9.60, SD = 9.20) reported to play more violent video games than female participants , F = 123.54, p<.001, η2 = .16. As predicted, female participants  were more likely to be convinced that violent video games increase aggression than male participants , F = 99.63, p<.001, η2 = .13. They  evaluated the positive effect study more favorably, F = 31.92, p<.001, η2 = .05, and the no-effect study less favorably , F = 34.22, p<.001, η2 = .05, than did the male participants . Female participants  also reported becoming more convinced that violent video games increase aggression, whereas male participants reported becoming less convinced , F = 8.46, p = .004, η2 = .01. In terms of actual attitude change, however, female participants  became less convinced that violent video games would increase aggression after reading the studies than before, whereas male's actual attitudes were almost unchanged , F = 3.84, p = .050, η2 = .01.Male participants  = 33.40, p<.001, η2 = .05, evaluation of the positive effect study, F = 11.45, p = .001, η2 = .02, and evaluation of the negative effect study, F = 11.56, p = .001, η2 = .02, remained significant, but were considerably reduced. In all analyses, the effect of violent video game exposure was significant, all Fs>27.29, all ps<.001, all ηs2>.04. That is, differences in the amount of violent video game play accounts for some of the sex differences in terms of initial beliefs and evaluation of the studies. Moreover, for both reported attitude change and actual attitude change, the sex difference was no longer significant when controlling for violent video game exposure, both Fs<2.91, both ps>.089, both ηs2<.01.When controlling for violent video game exposure, the sex differences in terms of initial belief, The notion that violent video games cause aggression is a topic of hot debate. The present research also shows that there is considerable disagreement on this issue. Out of 662 participants, 397 thought that violent video games would increase aggression, whereas the remaining 265 participants did not believe so. In line with previous research into biased assimilation because they are convinced that playing violent video games is harmless. Future experimental work may test these competing accounts.It is noteworthy that females much more than males believe that violent video games increase aggression. Whereas 297 out of 390 female participants (76%) indicated that they thought violent video games would increase aggression, only 100 out of 272 male participants (37%) did so. Part of this substantial sex difference was accounted for by the fact that male participants reported to be more likely to play violent video games than female participants. In general, participants who regularly play violent video games were less likely to believe that doing so makes the players aggressive than participants who do not regularly play violent video games. Because this finding is based on a correlational design, one does not know whether players of violent video games are motivated to defend their own behavior or whether people play violent video games The present study addressed participant's responses to new information about possible negative effects of violent video game play . But video game play may also positively affect social outcomes Some limitations of the present study have to be acknowledged. Biased assimilation was assessed by two items, whereas only one measure was employed to assess initial and final beliefs. Moreover, participants only responded to two conflicting pieces of evidence. Note that this is the standard procedure in the biased assimilation literature Future research may address some behavioral implications of increased perceived attitude change after learning mixed video game evidence. For instance, people's future amount of violent video game play may be affected by their perceived attitude change. Interestingly, the increased gap was perceived but not real. In fact, the actual final beliefs of believers and skeptics were more similar than the initial beliefs —a pattern of findings that is consistent with a regression toward the mean interpretation.Because of the differences in terms of how evidence on the relation between violent video games and aggression is interpreted, it may not suffice when both sides of the argument know the most important pertinent facts. As shown in many studies, people are very able to maintain their existing beliefs by counterarguing information that does not fit their preexisting opinions Of course, it is possible that people's beliefs can shift in response to learning about new facts. If new evidence clearly contradicts one's beliefs, people may abandon belief maintenance. For instance, following Japan's Fukushima Daiichi nuclear disaster, Chancellor Angela Merkel's coalition announced that Germany's nuclear power stations will be shut down by 2022. This constituted a reversal of their own policy, because just one year ago the government had overturned the phase-out plan of the previous government and agreed on a 12-year delay of the schedule. Likewise, if substantial evidence accumulates on whether violent video games increase aggression, some people may consider changing their beliefs.But because most partisans will find a way how incongruent new evidence can be discredited, it is likely that the threshold is very high before this really happens. So debiasing techniques may be needed to decrease assimilation of new information. Previous research has shown that increasing the availability of counterexplanations


A pilot study suggests that playing video games may enhance a range of spatial vision functions in adults with amblyopia. n = 10), non-action videogame group (n = 3), and crossover control group (n = 7). Our experiments show that playing video games (both action and non-action games) for a short period of time  using the amblyopic eye results in a substantial improvement in a wide range of fundamental visual functions, from low-level to high-level, including visual acuity (33%), positional acuity (16%), spatial attention (37%), and stereopsis (54%). Using a cross-over experimental design , we can conclude that the improvement cannot be explained simply by eye patching alone. We quantified the limits and the time course of visual plasticity induced by video-game experience. The recovery in visual acuity that we observed is at least 5-fold faster than would be expected from occlusion therapy in childhood amblyopia. We used positional noise and modelling to reveal the neural mechanisms underlying the visual improvements in terms of decreased spatial distortion (7%) and increased processing efficiency (33%). Our study had several limitations: small sample size, lack of randomization, and differences in numbers between groups. A large-scale randomized clinical study is needed to confirm the therapeutic value of video-game treatment in clinical situations. Nonetheless, taken as a pilot study, this work suggests that video-game play may provide important principles for treating amblyopia, and perhaps other cortical dysfunctions.Abnormal visual experience during a sensitive period of development disrupts neuronal circuitry in the visual cortex and results in abnormal spatial vision or amblyopia. Here we examined whether playing video games can induce plasticity in the visual system of adults with amblyopia. Specifically 20 adults with amblyopia  were recruited and allocated into three intervention groups: action videogame group , positional acuity , spatial attention , and stereoacuity (stereo-vision / 3-D depth perception) in a small group of teenagers and adults. We found that they tended to recover vision much faster than we would have expected from the results of conventional occlusion therapy in childhood amblyopia. Additional experiments and modelling suggest that the improvements are a result of decreasing spatial distortion and increasing information processing efficiency in the amblyopic brain. Thus, video games may include essential elements for active vision training to boost visual performance. Most importantly, our findings suggest that video-game play may provide important principles for treating amblyopia, a suggestion that we are pursuing with larger scale clinical trials. The most frequent cause of permanent visual loss in childhood is amblyopia (“lazy eye”) Playing video games results in enhancement of a broad range of visual tasks in adults with normal vision, including light sensitivity In the present study, we aimed to assess with a small pilot group whether playing video games with an amblyopic eye can induce cortical plasticity and improve spatial vision in adults with amblyopia, well beyond the “sensitive period” of brain development. We hypothesized that the intense sensory-motor interactions while immersed in video-game play might push brain functions to the limit, enabling the amblyopic visual system to learn, on the fly, to recalibrate and adjust, providing the basis for functional plasticity. Moreover, game playing requires the allocation of spatial attention, detection, and localization of low contrast, fast moving targets, and aiming (in first-person shooter games). Thus, we speculated that video games may include several essential elements for active vision training to boost visual performance, and thus could potentially be useful in improving amblyopic vision.We tested a range of visual functions to examine the neural alternations, if any, following video-game play in a small group of adults . These vWhile action video games are reported to be useful in enhancing visual function in normal humans, non-action video games are not effective Our participants played video games for 40 h with their fellow eye patched. One might argue that the visual improvements, if any, might have resulted from the eye patching alone. To address this point, we used a cross-over treatment design in which a group of amblyopes first underwent occlusion therapy, i.e. patching the fellow sound eye, for a period of time before the video-game phase. With this experimental design, we can compare the efficacy of the two treatment approaches (passive patching and video-game playing).Our study had several limitations: small sample size, lack of randomization, and differences in numbers between groups. A large-scale randomized clinical study is needed to confirm the therapeutic value of video-game treatment in clinical situations. Nonetheless, taken as a pilot study, this work suggests that video-game play may help guide future treatment of amblyopia.To evaluate how video-game play alters amblyopic vision, we monitored the changes, if any, in visual acuity in 10 adults with amblyopia while they played a first-person shooter game—Medal of Honor: Pacific Assault (MOH)—using their amblyopic eye, with the fellow sound eye patched with a black eye patch. Visual acuity (VA) is a standard clinical procedure to quantify spatial vision by determining the smallest letter on a chart that can be identified at a given viewing distance. In amblyopia, vision is often substantially poorer when the target letter is presented with surrounding letters than when it is presented alone, a phenomenon known as crowding t = 10.154, p<0.0001) and 27.2%±3.2%  improvements in minimum angle of resolution (MAR—bottom panels). Two mild amblyopes (SA2 & SA4) completely “normalized” according to a criterion of 20/20 . It might be argued that the improvements could be due to learning the letter charts. Therefore, instead of taking measurements every 10 h, we tested observer SS1's acuity only before and after the video-game intervention, and similar to what we observed in other observers, his acuity improved substantially (≈2.5 letter-lines or ≈44% for both measurements).Surprisingly, playing video games rapidly reversed their amblyopia. After 40 h of video-game play (2 h/d), acuity improved, on average, by 1.6 and 1.4 lines on a LogMAR letter chart for crowded letters and single letters, respectively , and one, a mild amblyope (SA6) normalized to ≈20/16. On average, this group was able to read 1.5 more letter-lines (28.4% improvement) for crowded-letter acuity and 0.8 more lines (15.1% improvement) for single-letter acuity. These findings suggest that non-action games share useful properties for enhancing amblyopic vision. To determine the limits of plasticity, the three players who participated in the SimCity experiments were then asked to play MOH for another 40 h (phase 2: 40th to 80th h). Additional improvements of about one letter-line  were observed. Note that SA6's amblyopia was completely normalized at the end of phase I and no further significant improvement was observed.Interestingly, we found that similar to the action game group, all three non-action game players showed enhanced vision (th h); the dashed line in the bottom panels shows the mean data . In contrast, for the same amount of time, the video-game group (n = 9) showed a marked improvement in acuity of ≈20% . Five of the seven participants who completed the patching experiment continued to the video-game phase for another 40 h. In this phase of the experiment, we used both action  and non-action (SC1) games. Although none of the five showed any significant change in acuity in the patching phase, all improved substantially in the video-game phase . From this small-scale “cross-over” experimental design, we can conclude that it is the video-game experience, and not simply the patching, that enhances amblyopic vision.Since our participants played video games with the fellow eye patched, the vision enhancement we observed could have been the result of wearing an eye patch alone. Thus, in a control experiment, another group (OT) of seven amblyopic adults wore a patch, but instead of playing video games they were required to engage in other visually demanding activities, such as watching television, reading books, knitting, and surfing the Internet, using the amblyopic eye. After 20 h, however, no significant change in acuity was observed  at the 20th h were 3.03 and 1.33 for crowded acuity and isolated acuity, respectively. The recovery of crowded acuity was slightly faster than uncrowded acuity. An exponential fit y = oy+a(1−ebx−) to the data revealed time constants (b) of 0.064 and 0.054 h−1 for crowded acuity and uncrowded acuity, respectively. It is worthwhile noting that the recovery rate we observed here in adults is ≈5-fold faster when compared with the conventional occlusion therapy in children. It would take >200 h to obtain comparable treatment effects in children (≈0.1 logMAR unit/120 h) There was no significant correlation between the amount of acuity improvement and the baseline acuity .While visual acuity represents one important limit to spatial vision, positional acuity, which represents the ability to localize visual objects, is another important aspect of spatial vision. While positional acuity is remarkably acute in normal vision (often referred to as hyperacuity), it is often severely impaired in amblyopia. We found that positional acuity . The mean counting threshold (the number of dots that can be reliably counted) increased significantly by 37%, from 3.3±0.3 to 4.4±0.4 dots  and the mean response latency decreased by 16.5% (N = 1–10), though not significantly . In short, video-game play increases the number of items the amblyopic brain can direct attention to simultaneously, reduces undercounting deficits, and increases the processing speed of visual counting.Video-game play also appears to increase visual attention in amblyopia. We used a visual counting task to determine how many visual locations the brain can direct attention to in a very brief time period, 200 ms . Previoun = 5 [MOH40: 3+SIM40: 2]). Mean improvement in stereoacuity was 53.6%±8.4% , noting that SA6 failed the stereo test and had no recordable stereopsis in the baseline session. Three participants  fully regained normal stereoacuity (20 arc sec) as measured by this test, and were basically “cured” in this aspect of vision.Amblyopia is associated with abnormal binocular vision and reduced or absent stereopsis (binocular depth perception or 3-D). With improved monocular vision following video-game play, for some amblyopes binocular vision also recovered to a substantial extent. Five of the six anisometropic amblyopes (with straight eyes) were tested for stereopsis following the training. All five showed improved stereopsis  and high-level  visual processing. Importantly, we provide preliminary characterization of the time course, limits, and underlying mechanisms of video-game experience-dependent cortical plasticity. The findings of our “cross-over control” experiment show that the treatment effects cannot be simply explained by eye patching, suggesting that it is indeed the video-game experience which improves amblyopic vision.The visual plasticity stimulated through video-game training has been well documented in the “normal” visual system, however the neural mechanisms are not yet clear. Using positional noise, we are able to reveal the underlying mechanisms. As we previously reported, repeatedly practicing a Vernier task in positional noise, with response feedback, can improve sampling efficiency and re-calibrate the distorted retinal topographical mappings of the amblyopic visual field Perhaps most importantly, we show that playing video games can indeed improve visual acuity and sharpen amblyopic vision. Note that visual acuity is the gold standard for examining spatial vision in clinical situations. To our knowledge, our work is the first to report that uncrowded visual acuity can be improved through video-game training. Green and Bavelier Playing a non-action game for 30 h has been found to be ineffective in enhancing attentional performance in participants with normal vision Perceptual learning has shown to be useful in improving amblyopic vision Treatment of adult amblyopia has recently received considerable attention ever since the introduction of perceptual learning techniques in the past few years Before a video-game-based approach is used to treat amblyopia clinically, there are still many questions to be addressed . The current study serves as a “pilot” trial and, as such, has several design limitations: lack of randomization, small study size, and differences in numbers between arms. The lack of randomization and differences in numbers between arms may have resulted in potentially imbalanced makeup of the study arms on baseline characteristics. For example, the action game group was much more likely to be male and younger than the other groups. In addition, the small number of participants (four) in the non-action game group makes it difficult to draw strong conclusions. A much larger sample size is necessary for future studies to investigate which type, action or non-action, is more effective in treating amblyopia. Specifically, a large-scale randomized double-blind clinical trial  is needed to eliminate differences between people, placebo effects, and measurement differences. Despite these limitations, the present pilot study provides new insights into how video-game play sharpens visual functions in adult amblyopia and, most importantly, reveals that video-game play may provide important principles for improving treatment in amblyopia, and perhaps other clinical abnormalities.The experimental procedures were approved by the University Committee for the Protection of Human Subjects, and the research was conducted according to the principles expressed in the Declaration of Helsinki. Informed consent was obtained from each participant. There was no known risk involved in the experimental procedures.Altogether 20 adults with amblyopia participated in three video-game experiments . They were recruited through advertisements in newspapers and through the Internet websites. Thorough eye examination was carried out by an experienced optometrist . Our participant inclusion criteria included: (1) age >15 years; (2) all forms of amblyopia, e.g. strabismic, anisometropic, refractive, deprivative, and meridional amblyopia; and (3) interocular visual acuity difference of at least 0.1 LogMAR. Exclusion criteria included any ocular pathological conditions  and nystagmus. All of our participants had a difference in crowded visual acuity of two lines or more between the two eyes, and had normal vision in the sound eye (∼20/12–20/16). The maculae of all participants were assessed as normal, and they all had clear ocular media . Their clinical data are summarized in n = 4; SIM: n = 1, SC1) in phase 2. The two video games used were Medal of Honor Pacific Assault and SimCity Societies . Since there has been no previous clinical evidence indicating that video games can modify vision in adult amblyopia in any way, in this pilot trial we decided to recruit participants for the video game treatment groups in the beginning, in order to evaluate the feasibility of this treatment approach. It is important to note that the participant allocation was not based on the clinical characteristics of participants.Participants were allocated into three intervention groups—two video-game treatment groups and one conventional occlusion therapy cross-over control group . The firn = 20; positional acuity, n = 16; visual counting, n = 14; stereoacuity, n = 5). Those participants in the control experiment (OT group) were given a log sheet to keep track of the patching hours and the visual tasks performed during patching.In the main experiments, participants were required to play the assigned video games in our research laboratory for 40 or 80 h (2 h/d) using the amblyopic eye, with the fellow eye occluded with a black eye patch. They were given full optical correction for the viewing distance. A battery of vision function tests listed below was used to examine the effects of video-game experience on amblyopic vision . All visTwo Bailey-Lovie logMAR letter charts , National Vision Research Institute of Australia 1978, were used in measuring visual acuity . The cal2, and the contrast of each Gabor patch was 99%. Positional noise to the Gabor patches was introduced by varying their vertical positions according to a Gaussian distribution function. The average offset of each jittered Gabor patch grouping was constrained to be 0 by uniformly shifting the eight patches. An offset cue was produced by randomly shifting the right Gabor patch grouping up or down. The stimulus size and spatial frequency were scaled in rough proportion to their visual acuity in the amblyopic eye by varying viewing distance from 0.5 m to 4 m . Note that both the amblyopic eye and the fellow sound eye were tested at the same viewing distance.The stimuli and methods are essentially identical to those used in our previous studies of perceptual learning in amblyopia d′ = 1.1) were obtained on a Weibull function .A modified interleaved staircase method was adopted to control the offset magnitude between the two Gabor patch groupings and track the individual thresholds. Trial-by-trial feedback was provided. Positional threshold was defined as the offset at which 66% correct responses (eσ) on the threshold (thσ):iσ is the internal spatial distortion and k is the number of samples extracted. Sampling efficiency (E) was defined as:iσ and k can be estimated with a least-square algorithm.A positional averaging model was used to quantify the effects of external noise (N) of black circular dots (0.5 cd/m2) was then presented for 200 ms against a gray background (42 cd/m2) , with Weber contrast of 99%. N ranged from 1–10 dots; the dots were randomly positioned in 10×10 square cells. The target stimulus was followed by a checkerboard pattern for another 100 ms, which was used to mask any after images of the dot stimuli. Each dot subtended 3 arcmin in diameter and was centered in its corresponding cell (6 arcmin×6 arcmin); the entire dot stimulus subtended 1° by 1° at a testing distance of 4 m. The distance between dots was at least two cells . The dot size was scaled with visual acuity (AE) by varying viewing distance , and therefore the dots displayed on the screen were very visible. The amblyopic eye and the fellow eye were both tested at the same viewing distance.Visual counting is used to examine spatial selective attention capacity of the brain to shift the focus of attention to individuate and attend to a number of objects at different locations in the visual field. A schematic diagram of the visual stimulus is illustrated in N. The threshold reported for each observer was based on four blocks of measurement, i.e. a total of 400 trials. Response latency was measured using the time it took the observer to say the number into a microphone. Data acquisition of observers' voice responses was performed by an analog-to-digital converter .Observers were asked to enumerate the number of dots as quickly and accurately as they could. No feedback was given with respect to observers' responses. Counting threshold was taken as the midway between the upper and the lower free floating asymptotes (free floating) of a Weibull function as illustrated in Randot Stereotest, Stereo Optical Co., Inc., was used to measure stereoacuity with wearing polarizing viewer .


Endothelium removal or L-NAME (10−4 M) incubation increased the response to phenylephrine, but the L-NAME effect was greater in the aortic rings from the post-weaning protein malnutrition rats. The protein expression of the endothelial nitric oxide isoform increased in the aortic rings from the post-weaning protein malnutrition rats. Incubation with apocynin (0.3 mM) reduced the response to phenylephrine in both groups, but this effect was higher in the post-weaning protein malnutrition rats, suggesting an increase of superoxide anion release. In the tail artery of the post-weaning protein malnutrition rats, the vascular reactivity to phenylephrine (0.001–300 µg) and the relaxation to acetylcholine (10−10–10−3 M) were increased. Post-weaning protein malnutrition increases blood pressure and induces vascular dysfunction. Although the vascular reactivity in the aortic rings did not change, an increase in superoxide anion and nitric oxide was observed in the post-weaning protein malnutrition rats. However, in the resistance arteries, the increased vascular reactivity may be a potential mechanism underlying the increased blood pressure observed in this model.Malnutrition during critical periods in early life may increase the subsequent risk of hypertension and metabolic diseases in adulthood, but the underlying mechanisms are still unclear. We aimed to evaluate the effects of post-weaning protein malnutrition on blood pressure and vascular reactivity in aortic rings (conductance artery) and isolated-perfused tail arteries (resistance artery) from control (fed with Labina®) and post-weaning protein malnutrition rats (offspring that received a diet with low protein content for three months). Systolic and diastolic blood pressure and heart rate increased in the post-weaning protein malnutrition rats. In the aortic rings, reactivity to phenylephrine (10 Proper nutrition in terms of quality and quantity is essential for the growth and development of organisms, including humans. Furthermore, nutritional deficiencies due to a decrease in or absence of the consumption of macro and micronutrients in food causes malnutrition in which the degree depends on the type of diet, age and the length of decrease consumption. Malnutrition is the result of an interaction of socioeconomic, political, cultural and environmental factors that more strongly affect children who live in extreme poverty Previous reports have shown that not only intrauterine malnutrition but also its occurrence during childhood might be a risk factor for the development of hypertension In northeast Brazil, nutritional studies on the food consumption of the population allowed for the development of an experimental multideficient diet model, the regional basic diet (RBD) However, the factors that promote the development of hypertension associated with malnutrition are still unclear. Therefore, we evaluated the impact of the post-weaning malnutrition in rats on blood pressure, vascular reactivity of the rat tail vascular bed (resistance vessels) and aortic rings (conductance vessels).ad libitum.The studies were performed on male Wistar rats. All experiments were conducted in compliance with the guidelines for biomedical research as stated by the Brazilian Societies of Experimental Biology and approved by the Ethics Committee on Animal Experimentation of the Biological Sciences Center of the Federal University of Pernambuco (Process Number 23076.008507/2007-16). All rats had free access to water and were fed rat chow Animals were divided into two experimental groups: control (CT) and post-weaning protein malnutrition over 3 months. In the control group, the mothers were fed a chow diet during the pre-mating, mating, pregnancy and lactation. After weaning (21 days), the offspring had the same diet as their mothers (Labina®). In the post-weaning protein malnutrition group, mothers were fed a Labina® diet during the pre-mating, mating, pregnancy and lactation phases. The offspring received the RBD ration post weaning for 3 months.Phaseolus vulgaris), sweet potatoes (Ipomea batatas), jerked beef and manioc flour (Manihot esculenta). These components were macerated, molded into “pellets” and heated to 50°C. The RBD pellets providing a total of (g/g%): proteins 9, carbohydrates 78, lipids 1.1, fiber 7, minerals 4, sodium chloride 0.17 and Kcalorie 356. No vitamin supplement was added. Moreover, part of the diet was supplemented with 0.2% (g/g) sodium chloride. The control diet was a commercial diet (Labina®). The standard diet contains the following content (g/g%): protein 23, carbohydrates 41, lipids 2.5, fibers 9, minerals 8, sodium chloride 0.37 and Kcalorie 278 Two types of diets were used for this study. The diet used to induced malnutrition was the RBD  as descrAt the end of treatment of malnutrition (3 months), the rats were anesthetized with urethane , and a polyethylene catheter (PE50) filled with heparinized saline (50 U/mL) was introduced into the carotid artery to record arterial systolic (SBP) and diastolic blood pressure (DBP). Recordings were performed over 30 min with a pressure transducer (TSD 104A- Biopac) and with an interface and software for data collection . The heart rate (HR) was determined from the intra-beat intervals.After the hemodynamic measurements, rats were killed by exsanguination. The thoracic aortas were carefully dissected and separated from the connective tissue. For the reactivity experiments, the aortas were divided into cylindrical segments 4 mm in length. To analyze the isoforms of endothelial and inducible nitric oxide synthase protein expression levels, the arteries were rapidly frozen in liquid nitrogen and kept at −70°C until the day of analysis.3; 2.5 mMCaCl2; 1.2 mM KH2PO4; 1.2 mM MgSO4; 11 mM glucose and 0.01 mM EDTA, gassed with 95% O2 and 5% CO2, and maintained at a resting tension of 1 g at 37°C as a previously described Segments of thoracic aorta were mounted in an isolated tissue chamber containing Krebs–Henseleit solution: 118 mM NaCl; 4.7 mM KCl; 23 mM NaHCOAfter a 45 min equilibration period, the aortic rings were initially exposed twice to 75 mM KCl once to check their functional integrity and a second time to assess the maximal tension. Afterwards, the endothelial integrity was tested by adding acetylcholine (10 µM) to segments previously contracted with phenylephrine (1 µM). A relaxation equal to or greater than 90% was considered to be demonstrative of functional integrity of the endothelium. After a 45-min washout, concentration-response curves to phenylephrine were determined. Single curves were performed in each segment. The effects of the following drugs were evaluated: (1) a nonspecific NOS inhibitor N-nitro-L-arginine methyl ester , (2) a non-selective cyclooxygenase (COX) inhibitor , (3) an NADPH oxidase inhibitor . These drugs were added to the bath 30 min before performing the phenylephrine curve.The influence of the endothelium on the response to phenylephrine in the post-weaning protein malnutrition and control groups were investigated after performing a mechanical removal by rubbing the lumen with a needle. The absence of endothelium was confirmed by the inability of 10 µM acetylcholine to produce relaxation.Proteins from homogenized arteries (80 µg) were separated by 10% SDS-PAGE. The proteins were transferred to nitrocellulose membranes that were incubated with mouse monoclonal antibodies for endothelial nitric oxide synthase . After washing, the membranes were incubated with anti-mouse  immunoglobulin antibody conjugated to horseradish peroxidase. After thorough washing, the immunocomplexes were detected using an enhanced horseradish peroxidase/luminal chemiluminescence system  and film . Signals on the immunoblot were quantified with the National Institutes of Health Image V1.56 computer program. The same membrane was used to determine α-actin expression using a mouse monoclonal antibody .i.p.). Ten minutes after the administration of heparin, 1 cm of the tail artery was dissected free and cannulated with an intracath  near the base of the tail. The vascular bed was flushed with Krebs-Henseleit buffer (KHB in mM)  and bubbled with 5% CO2 −95% O2 at 36±0.5°C. The tail artery was then severed from the body and placed in a tissue bath and perfused with KHB at a constant flow of 2.5 mL/min with a peristaltic pump .Isolated rat tail arteries were used in this study as previously reported After a 45-min equilibration period, the experimental protocol was initiated. The mean perfusion pressure (MPP) was measured by using a pressure transducer , and the data were recorded using an interface and software for data acquisition . Because a constant flow was used, changes in the perfusion pressure represented changes in vascular resistance.−10–10−3 M) was evaluated in arteries previously contracted with potassium chloride (65 mM) added to the perfusion fluid. Finally, after a 30-min stabilization period, the dilatory responses to sodium nitroprusside (10−10–10−3 M) were determined in arteries previously contracted with potassium chloride (65 mM) added to the perfusion fluid.The following protocols were used to investigate the effects of post-leaning malnutrition on vascular reactivity to phenylephrine, acetylcholine and sodium nitroprusside. After a 45-min stabilization period, increasing doses of phenylephrine (0.001–300 µg as bolus injections of 100 µL) were administered into the perfusion medium in preparations from the controls and post-weaning protein malnutrition groups. The arteries were then maintained for a 30-min stabilization period. Then, the vasodilatation induced by acetylcholine  was expressed as the percentage of relaxation of the maximal contractile response to phenylephrine (1 µM).Results regarding the perfusion pressure measurements for the tail artery experiments are presented as the changes in the mean perfusion pressure after subtraction of the peak pressure from the baseline pressure. The concentration-responses curves to acetylcholine or sodium nitroprusside are expressed as the percentages of relaxation of the maximum contractile response to KCl 65 mM.max) and the concentration of agonist that produced 50% of the maximal response (log EC50) were calculated using non-linear regression analysis . The sensitivity of the agonists is expressed as pD2 (−log EC50). To compare the effects of endothelium denudation or drug incubation on contractile responses to phenylephrine, some results were expressed as the differences of the area under the concentration–response curves (dAUC) in both experimental groups. These values indicate whether the magnitude of the effect of endothelial denudation or drug incubation were different in the control and post-weaning protein malnutrition groups.For each concentration-response curve or dose-response curve, the maximum effect . Salts and reagents used were of analytical grade from Sigma  and Merck .No difference in body weight between the groups was observed either at birth or 21 days after birth . HoweverIn order to verify if the loss in weight gain induced by hypoproteic diet produces arterial pressures alterations, blood pressure and heart rate were measured. At the end of treatment of malnutrition (90 days), significant increases in systolic and diastolic arterial blood pressure and heart rate were observed in the post-weaning protein malnutrition group compared to the controls . TherefoBlood pressure changes could occur by an increase of cardiac output or/and by increased vascular resistance. Therefore, to further investigate the underlying mechanisms that produced the increase in arterial pressure, the effect of post-weaning malnutrition on vascular reactivity was investigated in isolated aortic rings (conductance arteries) and in the tail vascular bed (resistance arteries). In aortic rings, malnutrition did not affect the maximal response to 75 mM KCl in segments with endothelium  and after endothelium removal . These results demonstrated that the maximal tension that developed in aortic rings of post-weaning protein malnutrition group compared to the controls were not different. After that, vasodilatation and vasoconstriction mechanisms were investigated in aortic rings from post-weaning malnutrition and controls rats.The endothelium-dependent relaxation induced by acetylcholine in phenylephrine-contracted arteries was similar in aortic rings isolated from controls and post-weaning protein malnutrition , suggesting that the oxide nitric release induced by acetylcholine was not modified. The contractile response induced by phenylephrine did not change in aortic rings from post-weaning protein malnutrition rats compared to the control group . The infTo investigate whether the nitric oxide release in response to phenylephrine in aortic rings from post-weaning protein malnutrition was altered, rings were incubated with L-NAME. L-NAME (100 µM) induces a hyper contraction and left-shifted the concentration-response curve to phenylephrine in the aortic segments from both groups . HoweverHypertension might be associated with increased oxidative stress. To investigate the role of superoxide anion generated by NADPH oxidase activity on vascular reactivity to phenylephrine in post-weaning protein malnutrition rats, the NADPH oxidase inhibitor apocynin (0.3 mM) was used. Incubation with apocynin reduced reactivity to phenylephrine in all groups , but theIndomethacin (10 µM), a cyclooxygenase inhibitor, was used to investigate the putative role of prostanoids on vascular reactivity to phenylephrine in post-weaning protein malnutrition rats. Indomethacin did not alter the phenylephrine responses in aortic segments from both groups (results not shown).Because vascular reactivity in the conductance arteries was almost unaffected, we also investigated the effect of post-weaning malnutrition on the vascular reactivity of the resistance arteries (tail artery). This protocol was used to obtain information regarding the effects of post-weaning protein malnutrition on the resistance vasculature.In the endothelium-intact tail vascular bed preparations from the post-weaning protein malnutrition group, the mean perfusion pressure was similar in both groups, as observed from the initial dose of phenylephrine response curve, which are similar . HoweverThis study demonstrated that post-weaning protein malnutrition increased blood pressure, which was accompanied by increased vascular reactivity in resistance arteries (tail artery). However, in conductance arteries (aortic rings), malnutrition did not change the vascular reactivity due to increased basal release of NO, probably resulting from eNOS overexpression, although there was an increased release of free radicals derived from NADPH oxidase. Therefore, this balance prevented apparent changes in the phenylephrine concentration-response curves.In this study, we investigated the effects of post-weaning protein malnutrition on blood pressure and vascular reactivity of resistance and conductance arteries in rats to elucidate some mechanisms that are correlated with two relevant issues in public health: malnutrition and hypertension. Post-weaning protein malnutrition was induced by a diet that reproduces a regional basic diet (RBD) Animals submitted to post-weaning protein malnutrition showed a marked reduction in weight gain. These results highlight the importance of nutritional status in the growth phase of the animal. They show a greater impact of the post-weaning protein malnutrition period including the stage of growth and development into adulthood. Similar results were previously observed An alteration in body weight is an indicator of the nutritional status of the individual and might be associated with alterations in the cardiovascular system In accordance to these studies, Sawaya et al. Malnutrition did not change the vascular reactivity in the isolated aortic rings. However, in the presence of L-NAME, a non-specific inhibitor of NOS, there was a greater vasoconstrictor response to phenylephrine in post-weaning protein malnutrition group, suggesting increased NO production. However, the endothelium-dependent relaxation induced by acetylcholine was similar in aortic rings isolated from controls and post-weaning protein malnutrition, suggesting that the oxide nitric release induced by acetylcholine was not modified.As mentioned above, we observed that the malnutrition triggers hypertension. Hypertension might be associated with reduced nitric oxide bioavailability, although there are studies that have shown an increase in this vasodilator as a compensatory mechanism Franco et al. These results might be a compensatory mechanism induced by increased blood pressure or oxidative stress. The increase in superoxide anion, the most important free radical generated by NADPH oxidase activity, could decrease nitric oxide bioavailability Therefore, in order to investigate if the post-weaning malnutrition per se or the increment of blood pressure that was observed after the treatment with RBD could increase the release of free radicals, apocynin was incubated in isolated aortic rings from both groups. Based on the effects of apocynin on reducing phenylephrine reactivity, our findings point towards this possibility. Therefore, we believe that post-weaning protein malnutrition increases oxidative stress that is initially compensated by increased nitric oxide production. However, for a long period, this compensatory response can be lost, and the endothelial dysfunction might increase or maintain hypertension.Because the vascular reactivity to phenylephrine in conductance arteries was not modified after the post-weaning malnutrition, other endothelium-derived vasoconstrictors are probably released. Therefore, we investigated the prostanoids derived from the cyclooxygenase pathway. In the presence of indomethacin, a COX inhibitor, there was no difference in the vasoconstrictor response to phenylephrine in the post-weaning protein malnutrition and control groups, suggesting that vasoactive prostanoids did not seem to be altered in this artery after malnutrition.Considering that findings obtained from the aortic rings did not provide a clear explanation for the increased blood pressure, we investigated the effects of malnutrition on a resistance vessel. In fact, we demonstrated that in the tail artery of the post-weaning protein malnutrition group, the vascular reactivity to phenylephrine increased. Therefore, this increase in vascular reactivity might increase vascular resistance, which increases diastolic blood pressure. However, in the same artery, we observed that the relaxation response to acetylcholine increased. This effect could be a compensatory mechanism to the increased vascular reactivity or blood pressure.in vitro by an increase in vascular reactivity in the resistance arteries. However, in the conductance arteries, malnutrition did not modify the vascular reactivity probably due to the increased release of nitric oxide, which balances free radicals and prevents changes in the phenylephrine concentration-response curves. We suggest that the increase in free radicals and nitric oxide in the aortic rings of post-weaning protein malnutrition rats might be a compensatory mechanism to the increase in blood pressure.Therefore, our results used a qualitative malnutrition model with low-protein to show an increase in blood pressure in the post-weaning protein malnutrition group, which was accompanied


PEI) were successfully engineered for widespread deposition in the lung and prolonged release of intact dec-ODN in vitro. LPPPEI caused a prolonged inhibition of IL-8 and MUC2 expression in CF human bronchial epithelial cells and human epithelial pulmonary NCI-H292 cells, respectively, as compared to naked dec-ODN. Nonetheless, as compared to previously developed LPP, the presence of PEI was essential to construct a dec-ODN delivery system able to act in mucoepidermoid lung epithelial cells. In perspective, engineering LPP with PEI may become a key factor for tuning carrier properties, controlling lung inflammation and mucin production which, in turn, can foster in vivo translation of dec-ODN therapy.A specific and promising approach to limit inflammation and mucin iperproduction in chronic lung diseases relies on specific inhibition of nuclear Factor-κB (NF-κB) by a decoy oligonucleotide (dec-ODN). To fulfill the requirements dictated by translation of dec-ODN therapy in humans, inhalable dry powders were designed on a rational basis to provide drug protection, sustained release and to optimize pharmacological response. To this end, large porous particles (LPP) for dec-ODN delivery made of a sustained release biomaterial (poly(lactic-co-glycolic) acid, PLGA) and an “adjuvant” hydrophilic polymer  were developed and their effects on LPS-stimulated human airway epithelial cells evaluated. The composite PLGA/PEI particles containing dec-ODN (i.e., LPP In fact, the failure of nucleic acid delivery to the airway epithelia is largely attributed to extracellular and cellular barriers Taking a look at the ON therapeutic pipelines for chronic lung diseases, it is apparent that the most widely employed dosage forms for ON delivery are solutions for injections or inhalation P. aeruginosa, both in vitroin vivoa priori can be beneficial to promote ON transport toward epithelium. Thus, once biocompatibility issues are sufficiently addressed, pulmonary delivery of PEI along with decoy ONs against NF-κB may become an attractive therapeutic strategy for the treatment of complex lung pathologies  requiring a multidrug approach aimed at controlling chronic inflammation, infection and iperproduction of a viscous mucus An approach recently proposed to overcome airway barriers and transfect differentiated respiratory epithelial cells with nucleic acids, relies on the use of cationic polymers As a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA), which is already approved for human use by parenteral injection, can be of great help in developing novel inhalable formulations for biotech drugs, such as decoy ONs PEI) on the expression of IL-8 in LPS-stimulated CF human bronchial epithelial IB3-1 cells was assessed. Afterwards, taking into account that mucin gene expression in lung disease is transcriptionally and post-transcriptionally regulated by inflammatory mediators P. aeruginosa LPS induces the expression of MUC2 gene via NF-κB PEI on MUC2 gene expression in LPS-stimulated mucoepidermoid carcinoma cells (NCI-H292).Taking for granted the beneficial properties of LPP developed in our previous studies, here we try to add “adjuvant” functionality by engineering LPP containing dec-ODN with PEI. We investigate how PEI addition in LPP may affect those carrier properties considered crucial to the development of therapeutically-relevant delivery systems. In analogy to our previous findings, the effect of dec-ODN released from PEI/PLGA composite LPP , LPPPEI displayed a volume mean diameter significantly higher than the corresponding LPPDPPC (≈31.5 µm) t was estimated for LPPPEI as compared to LPPDPPC (≈6 µm). Anyway, MSLI tests demonstrated that the developed LPPPEI had very good aerosolization properties, with more than 95% of the capsule content being emitted during aerosolization, very high FPF and an experimental MMAD<4 µm. Concerning LPP entrapment efficiency, about 70% and 40% of the total amount of dec-ODN and PEI respectively added to the formulation were effectively entrapped within LPPPEI.The overall properties of LPPin vitro release studies are reported in Results of DPPC displayed a triphasic release profile of dec-ODN, characterized by low burst (8±2% of dec-ODN released after 6 h), followed by a slower release phase lasting about 15 days and a final rapid release of dec-ODN. Finally, the release of dec-ODN from LPPPEI was a two-stage process where a burst as low as 5±1% was followed by a continuous release of dec-ODN approximating a zero-order kinetics. We have also tried to assess LPP release kinetics from simulated CF mucus in simulated interstitial lung fluids. Nevertheless, to avoid the interference of CF mucus components on the quantitation of dec-ODN, it was necessary to modify LPP formulation by increasing dec-ODN actual loading. Noteworthy, no significant difference in dec-ODN release from modified LPP was observed in simulated lung fluids as compared to results achieved in phosphate buffer, suggesting that CF mucus components did not affect dec-ODN release from the developed LPP (data not shown).On the contrary, LPPPEI and naked dec-ODN in NCI-H292 cell culture medium collected at 24, 48 and 72 h was assessed by gel electrophoresis. As shown in PEI at 24 h exhibited a marked band which intensity was reduced at 48 and 72 h, indicating a slow release of dec-ODN from LPPPEI. In contrast, the band of naked dec-ODN was evident at 24 h and smeared at 48 and 72 h, suggestive of its partial degradation. Furthermore, the band of dec-ODN extracted from LPPPEI pellet .Since an important concern in using PEI relates to its toxicity profile, we investigated the effects of PEI alone, naked dec-ODN/PEI or both entrapped in LPPPEI on IL-8 mRNA expression in ΔF508 CFTR-mutated human bronchial epithelial IB3-1 cells stimulated with LPS from P. aeruginosa. LPS challenge of cells for 24 and 72 h induced a significant increase in the IL-8 mRNA levels, as compared to unstimulated cells. Treatment of cells with LPPPEI at 0.125 and 0.250 µM did not cause any effect, whereas 0.5 µM LPPPEI significantly prevented IL-8 mRNA expression . Notably, when used at the same concentration, naked dec-ODN prevented IL-8 mRNA expression only at 24 h   . A compa3)  induced a significant increase of DNA/protein complexes, as compared to untreated cells. Incubation of cells with LPPDPPC did not cause any effect on NF-κB/DNA binding activity and MUC2 mRNA levels even at concentrations 4 folds higher than those used in IB3-1 cells (LPPDPPC 0.5 µM) (data not shown). Contrariwise, incubation of cells with LPPPEI (2.0 µM) resulted in a significant reduction of NF-κB/DNA binding activity at 24 and 72 h  (n = 3). Scramble dec-ODN released from LPPPEI (Scramble LPPPEI) as well as blank LPP did not exhibit any effect  . Naked dy effect .The composition of the NF-κB complex was determined by competition and supershift experiments in nuclear extracts from LPS-stimulated NCI-H292 cells . The spePEI or naked dec-ODN on NF-κB/DNA binding activity was confirmed by evaluating nuclear levels of p50 and p65 by Western blot (The effect of dec-ODN released from LPPern blot .PEI (2.0 µM) caused, at 24 (n = 3) and p65 . Naked dec-ODN, at the same concentration, reduced p50 and p65 nuclear translocation only at 24 h . LPPPEI containing scramble dec-ODN (scramble LPPPEI) as well as blank LPPPEI did not exhibit any effect . Contrariwise, incubation of cells with LPPPEI (2.0 µM) significantly reduced MUC2 protein expression at 24 and 72 h . Naked dec-ODN, at the same concentration, exhibited this effect only at 24 h . Scramble dec-ODN released from LPPPEI (scramble LPPPEI) as well as blank LPPPEI did not exhibit any effect (The expression of MUC2 protein was significantly augmented in cytoplasmic extracts from NCI-H292 cells stimulated with LPS (10 µg) at 24 and 72 h, as compared to untreated cells. As observed for NF-κB transcriptional activity, incubation of cells with LPPy effect .PEI (2.0 µM) inhibited in a significant manner MUC2 mRNA levels . Naked dec-ODN, at the same concentration, reduced MUC2 expression only at 24 h . Scramble dec-ODN released from LPPPEI as well as blank LPPPEI did not exhibit any effect.To assess whether the decrease in MUC2 protein expression was related to a reduced MUC2 gene expression, we analyzed MUC2 mRNA levels by RT-PCR . Stimulain vitro/in vivo degradation; ii) promote ON transport through lung mucus barriers; iii) facilitate ON uptake by lung epithelial cells, where its target is located. In this scenario, we are developing large porous particles (LPP) for pulmonary delivery, focusing on a decoy oligodeoxynucleotide against NF-κB (dec-ODN) DPPC) may cause a prolonged inhibitory effect of dec-ODN on NF-κB/DNA binding activity and related IL-6 and IL-8 expression in LPS-stimulated CF cells DPPC in inhibiting IL-6 and chemoattractant IL-8 secretion from ΔF508 CFTR-mutated human bronchial epithelial cells stimulated with LPS from P. aeruginosa, as shown in DPPC), PLGA/PEI composite LPP have been developed and their effects on human airway epithelial cells stimulated with LPS were evaluated. In fact, PEI could represent an interesting candidate for lung delivery of ONs in the treatment of chronic lung disease, such as CF, owing to its proved antibacterial properties toward Gram (-) bacteria and potential mucoactive effects.The translation of oligonucleotide (ON)-based respiratory therapies in humans runs into the need of specifically tailored inhalable formulations able to: i) protect the entrapped ON from PEI are conceived for local and prolonged dec-ODN release in the lung, some crucial technological aspects of the particles were taken into account: primary bulk properties , encapsulation efficiency and in vitro dec-ODN release profile. In term of size and density, large but “light” particles  may represent a useful mean to reach more distal airways and avoid natural macrophage-mediated lung clearance mechanisms ensuring that encapsulated dec-ODN is effectively delivered to lung epithelium. We have found that processing of PLGA/PEI mixtures in the presence of ammonium bicarbonate may allow the production of gas-foamed LPPPEI displaying large size and a highly porous structure. Pore formation, due to gas bubbles evolution during LPPPEI preparation, was well controlled in analogy to what found for previously developed LPPDPPC. LPPPEI showed a higher geometric diameter as compared to LPPDPPC, likely ascribable to PEI partitioning in the internal water phase of the double emulsion. The osmotically active ingredient may generate an osmotic pressure gradient, allowing the transport of water across the organic phase. As previously observed, the occurrence of this process during particle hardening results not only in a higher particle porosity but also in a potential increase of particle dimensions t suggests good aerodynamic properties for LPPPEI with values lower than 10 µm, 4.5-fold lower than the corresponding geometric mean diameter. Nonetheless, if particle porosity is essential to achieve respirable particles, this can be detrimental for ON entrapment efficiency and release profile. Furthermore, particles that are “too porous” may easily imbibe extracellular fluids, being reach of nucleases, and the encapsulated ON is expected to be more exposed in such loose polymer matrices.Since LPPPEI provide not only an effective encapsulation of dec-ODN within LPP, but also its sustained release in vitro for more than 30 days. Notably, the release profile of dec-ODN depended upon the adopted helper excipient and was approximately of zero-order in case of LPPPEI. During the diffusion-erosion phase, dec-ODN molecules located inside LPP macropores will naturally diffuse towards one of the endpoints of the pore and, where pores are interconnected, reach release medium PEI the contribution of the electrostatic interactions occurring between dec-ODN and PEI on release rate plays an important role. Actually, dec-ODN is negatively-charged and likely able to interact with the polycation at the interfaces, where PEI is thought to be preferentially located Release rate is a critical factor to exert a temporal control over the administered ON dose. Notably, the approach of using PEI to optimize powder properties is further strengthened by the results obtained in this work. Unmodified LPP prepared without helper excipients show a poorly modulated biphasic release of the encapsulated ON, which is typical for porous PLGA particles PEI on the expression on IL8 and MUC2 expression in human airway epithelial cells stimulated with LPS. Several studies showed that PEI may itself stimulate the immune system and cause various degrees of pro-inflammatory cytokine secretion PEI does not induce cytotoxic effects in all the cell lines tested whereas free PEI or PEI/dec-ODN significantly reduce cell viability. These results likely suggest that the use of PEI in PLGA formulations able to exert a control over PEI dose administered to the cells in time may result in a lower toxicity as compared to its bolus administration. Actually, recent in vitro toxicological data suggest that when PEI is used within a therapeutically relevant concentration range either as polyplex or free, no cytotoxicity and only minor immunomodulatory responses are elicited on murine alveolar epithelial–like type II cells and murine alveolar macrophages On the basis of the encouraging technological premises, we evaluated the effects of LPPDPPC, LPPPEI cause a prolonged inhibition of IL-8 expression in LPS-stimulated IB3-1 cells up to 72 h, whereas naked dec-ODN exhibits these effects only at 24 h. The fact that LPPDPPC and LPPPEI work by a similar extent likely suggest that endocytosis mediated by caveolin, already reported to be abundantly expressed in lung tissue including IB3-1 cells In analogy to previously developed LPPPEI in human lung mucoepidermoid carcinoma cells (NCI-H292), a well-established in vitro model for investigating MUC gene expression in airway diseases P. aeruginosa) and several cytokines/chemokines relevant to CF have been reported to transcriptionally upregulate MUC2 gene expression P. aeruginosa caused a significant increase of NF-κB/DNA binding activity as well as MUC2 protein and mRNA expression. Noteworthy, cell exposure to LPPDPPC did not result in any inhibitory effect, even at concentrations 4-folds higher than those tested on CF cells. By contrast, due to the presence of PEI and dec-ODN slow release, LPPPEI showed a persistent inhibition of NF-κB related MUC2 gene expression at concentrations much lower as compared to naked dec-ODN. These results suggest that different mechanisms control internalization of dec-ODN in mucoepidermoid cells. Thus, engineering LPP with PEI may play a crucial role in controlling mucin gene expression and, in turn, become a key factor for in vivo translation of dec-ODN therapy.Very interesting results were obtained on LPPPEI features could all contribute to better control local chronic inflammation and prompt us to go in depth into their in vivo safety and potential. To date, the safety and effectiveness of decoy ONs against NF-κB to prevent restenosis has been assessed in an open-label phase I/IIa clinical trial and no significant systemic adverse effect occurred in any of the patients along six months of observation Taken all together, our findings suggest that the developed LPP are respirable upon delivery from a breath-actuated DPI, exert a temporal control over dec-ODN released amounts, while preserving its integrity, and cause a persistent inhibition of IL-8 and MUC2 expression as compared to naked dec-ODN. LPPPEI in CF treatment, with particular regard to further roles of PEI as multipurpose agent.Dry powders for dec-ODN delivery in the lung were successfully engineered to provide a new therapeutic strategy for inhibiting NF-κB transcriptional activity and its related gene expression in chronic lung inflammation of CF patients. Formulation of dec-ODN in respirable particles based on combinations of PLGA and PEI allowed its protection in biologically relevant conditions and sustained release. Our findings demonstrate a persistent inhibition of IL-8 and MUC2 expression in LPS-stimulated airway epithelial cells and highlight the role of PEI in enhancing carrier properties while suppressing its toxicity. These promising indications prompt us to go in depth into the potential of respirable LPPsn-glycero-3-phosphocholine (DPPC), and ammonium hydrogen carbonate were obtained from Sigma-Aldrich . Analytical grade sodium chloride, potassium chloride, sodium phosphate dibasic anhydrous, sodium bicarbonate, methylene chloride, were supplied by Carlo Erba .Phosphorothioate oligodeoxynucleotide synthesis was performed by Tib Molbiol . Poly (50∶50) (PLGA)  was purchased from Boehringer Ingelheim (Germany). Polysorbate 80, polyvinylalcohol , sodium azide, 1,2-dipalmitoyl-scramble dec-ODN were prepared by annealing sense and antisense phosphorothioate ODNs in vitro in filtered water solution. In order to denature any secondary structure and to pair sense and antisense nucleotide sequences by hydrogen bonds, the mixture was heated at 80°C for 5 min and allowed to cool slowly at room temperature over 18 h. For each experiment, dec-ODN and scramble dec-ODN were annealed before use.A plain double-stranded decoy oligodeoxynucleotide to NF-κB (dec-ODN) and its corresponding The wild-type NF-κB consensus sequence used (dec-ODN) was:G GAC TTT CCC TAG C-3′5′-GAT CGA GGC CTG AAA GGG ATC G-5′3′-CTA GCT CCscramble dec-ODN) was used:Mutant NF-κB consensus sequence with a mutation of the bases written in bold (GGAC to AAGC) of the wild-type NF-κB consensus sequence (A AGC TTT CCC TAG C-3′5′-GAT CGA GGT TCG AAA GGG ATC G-5′3′-CTA GCT CC4)HCO3 at 10% (w/v) and 0.6 mg of dec-ODN were poured into 2.5 ml of methylene chloride containing 375 mg of PLGA. DPPC at 0.1% (w/v) or PEI (PEI/PLGA 1∶100 w/w) were added to the PLGA-containing organic phase, so as to achieve LPPDPPC and LPPPEI dec-ODN formulations, respectively. Emulsification was achieved using a high-speed homogenizer  operating at 755 g (tool 6G) for 3 minutes. The primary w/o emulsion was rapidly added to 25 ml of a 0.5% (w/v) PVA (Mowiol® 40–88) aqueous solution and homogenised at 676×g (tool 10F) for 2 minutes to produce the multiple emulsion. Solvent evaporation and subsequent particle hardening was achieved under magnetic stirring  at room temperature. After 3 h, particles were collected, washed three times with distilled water by centrifugation  at 4°C and frozen in liquid nitrogen. Samples were then dried for 36 h by a Modulyo freeze-drier  operating at 0.01 atm and −60°C. Each batch was prepared in triplicate.PLGA-based large porous particles containing dec-ODN were prepared by a modified double emulsion-solvent evaporation technique assisted by gas-foaming DPPC and blank LPPPEI) and LPP containing scramble dec-ODN (scramble LPPDPPC and scramble LPPPEI) were prepared as controls.Unmodified LPP without helper excipient (unmodified LPP), blank LPP without dec-ODN , labeled LPP (FITC-LPP) were prepared with a fluorescent PLGA (5% w/w) covalently coupled with fluorescein isothiocyanate (FITC) at carboxylic end-group (PLGA-FITC).PEI was investigated by CLSM analysis carried out on a LSM 510 Zeiss confocal inverted microscope equipped with a Zeiss 63/1.25 oil objective lens . An argon laser  was used.Particle shape and external morphology were analysed by Scanning Electron Microscopy (SEM)  as previously described The mean geometric diameter and size distribution of the particles were determined by laser light scattering  on a dispersion of freeze-dried microspheres in 0.2% w/v aqueous PVA. Particle size is expressed as volume mean diameter ± S.E.M. of values collected from three different batches.t) was estimated on the basis of the definition:0 is a reference density of 1 g/ml and X is the dynamic shape factor, which is 1 for a sphere. In the case of porous particles of approximately spherical shape an approximate bulk measure of ρ is provided by tapped density Powder density was evaluated by a tapped density tester according to European Pharmacopoeia 7 ed. (Ph.Eur.), whereas theoretical mass mean aerodynamic diameter  operating at 265 nm. The linearity of the response was verified over the concentration range 0.020–1.0 nmol/ml (r2>0.99). Blank LPP were used as control to verify that particle components did not interfere with dec-ODN quantification. The amount of PEI in the solution was contemporary quantified by UV spectrophotometric analysis at 285 nm after complexation with cupper(II) The amounts of dec-ODN and PEI encapsulated within LPPPEI were tested in vitro using an Astra Type Multi-Stage Liquid Impinger (MSLI), Type ALI 1000  after delivery from Turbospin®, a breath-activated, reusable DPI that works with single unit capsule . For each test, a hard gelatin capsule  was filled with about 20 mg of the powder and placed in the Turbospin®. Briefly, 20 ml of methylene chloride were poured into each of the four stages of the impinger to wet the collection surfaces. The capsule was then pierced and the liberated powder drawn through the impactor operating at 60 l/min for 4 s using an electronic digital flow meter (DFM mode). This allowed the aspiration of 4 L of air through the apparatus as recommended by Ph.Eur. The powder deposited on the four MSLI stages, in the induction port and on the final collection site (i.e. filter) was recovered by washing with methylene chloride. In each case, after removal of the solvent by rotary evaporation  the resulting precipitate was dissolved in 1.2 ml of methylene chloride and dec-ODN was extracted into 1.2 ml of water. The suspension was centrifuged  and the supernatant analysed for dec-ODN content in the aqueous phase by spectrophotometric analysis as described above.The aerosolization properties of LPPThe emitted dose (ED) was calculated by accurately weighing the capsule before and after Turbospin® actuation. Results are expressed as percentage of powder actually delivered ± S.E.M. of values collected from three different batches.exp) and the geometric standard deviation (GSD) were calculated according to Ph.Eur. deriving a plot of the cumulative mass of powder detained at each stage  versus the cut-off diameter at that stage.The fine particle fraction (FPF), that is the fraction of particles with an aerodynamic diameter lower than 5 µm, the experimental mass median aerodynamic diameter . A known amount of particles (8 mg) was suspended in 0.35 ml of PBS and placed in a dialysis membrane bag . The sample was dropped into 4.0 ml of PBS (sink condition), and kept at 37°C. At scheduled time intervals, 1.5 ml of external medium was withdrawn and replaced by the same amount of fresh PBS. The withdrawn medium was analyzed for dec-ODN content by spectrophotometric analysis as described above. Experiments were run in triplicate for each time point of release kinetics.The Human epithelial bronchial IB3-1 cells (with ΔF508 CFTR mutation) as well as human epithelial pulmonary NCI-H292 cancer cells (over-expressing MUC2 gene)  were purchased by LGC Standards .2/95% air in LHC-8 medium with 5% fetal bovine serum. Petri dishes as well as multiwells were pre-coated with albumin 1 mg/ml, collagen 3 mg/ml and fibronectin 1 mg/ml. The cells were plated in 48 culture wells at a density of 125×104 cells/ml per well or in 10-cm-diameter culture dishes at a density of 5×106 cells/ml per dish and allowed to adhere for 2 h.IB3-1 cells were cultured at 37°C in humidified 5%CO5 cells/ml/well or 10 cm diameter culture dishes at a density of 3×106 cells/ml/dish and allowed to adhere for 2 h.NCI-H292 cells were cultured at 37°C in humidified 5% CO2/95% air in RPMI containing 10% fetal bovine serum, 2 mM glutamine, 100 UI/ml penicillin, 100 µg/ml streptomycin, 25 mM Hepes, 1 mM glucose, 0.5 mM sodium bicarbonate and 5 mM sodium pyruvate. The cells were plated in 24 culture wells at a density of 2.5×10P. aeruginosa (10 µg/ml) for 24 and 72 h in the absence or presence of naked dec-ODN (2.0 µM), LPPPEI containing scramble dec-ODN (2.0 µM), LPPPEI containing dec-ODN (2.0 µM) or the same amount of blank LPPPEI.After adhesion, the medium was replaced with fresh medium and cells (IB3-1 or NCI-H292 cells) were stimulated with LPS from PEI was determined by using 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion assay. Briefly, NCI-H292 cells were incubated as described above. Thereafter the medium was replaced with fresh medium and cells were stimulated with LPS from P. aeruginosa (10 µg/ml) for 24 h in the presence of either naked dec-ODN (2.0 µM), PEI alone , dec-ODN (2.0 µM)/PEI (0.1 mg), LPP prepared without PEI , LPPPEI  or blank LPPPEI (10 mg/ml). The optical density (OD) of each well was measured with a microplate spectrophotometer (Titertek Multiskan MCCC/340) equipped with a 620 nm filter. The cell viability in response to the treatment with test compounds was calculated as % dead cells = 100 − (OD treated/OD control) × 100.The cytotoxicity of LPPPEI containing dec-ODN (2.0 µM) in NCI-H292 cells. At 24, 48 and 72 h, cell culture medium was collected and centrifuged . dec-ODN was extracted from the pellet by a solvent extraction method employing methylene chloride and water PEI were loaded into 1% agarose gel in TBE buffer . Freshly annealed naked dec-ODN was used as internal control. In each case, dec-ODN was visualized by ethidium bromide staining. Images were captured by ImageQuant 400 .The stability of dec-ODN was evaluated by gel electrophoresis on naked dec-ODN or LPP6) were washed two times with ice-cold PBS and centrifuged at 180 g for 10 min at 4°C. The cell pellet was resuspended in 100 µl of ice-cold hypotonic lysis buffer  and incubated on ice for 15 min. The cells were lysed by rapid passage through a syringe needle for 5 or 6 times and the cytoplasmic fraction was then obtained by centrifugation for 1 min at 13,000 g. The supernatant containing the cytosolic fraction was removed and stored at –80°C. The nuclear pellet was resuspended in 60 µl of high salt extraction buffer  and incubated with shaking at 4°C for 30 min. The nuclear extract was then centrifuged for 15 min at 13,000 g and supernatant was aliquoted and stored at −80°C. Protein concentration was determined by Bio-Rad  protein assay kit.Cytosolic and nuclear extracts of NCI-H292 cells were prepared as previously described with some modifications 5′-CAACGGCAGGGGAATCTCCCTCTCCTT-3′) were end-labeled with 32P-γ-ATP. Nuclear extracts containing 5 µg protein were incubated for 15 min with radiolabeled ONs (2.5–5.0×104 cpm) in 20 µl reaction buffer containing 2 µg poly dI-dC, 10 mM Tris-Hcl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol. The specificity of the DNA/protein binding was determined by competition reaction in which a 50-fold molar excess of unlabeled wild-type, mutant or Sp-1 ON was added to the binding reaction 15 min before radiolabeled probe. In supershift assay, antibodies reactive to p50 or p65 or a mixture of both were added to the reaction 15 min before the addition of radiolabeled probe. Nuclear protein-ON complexes were resolved by electrophoresis on a 6% non-denaturing polyacrylamide gel in 1× TBE buffer at 150V for 2 h at 4°C. The gel was dried and autoradiographed with intensifying screen at −80°C for 20 h. Subsequently, the relative bands were quantified by densitometric scanning of the X-ray films with a GS-700 Imaging Densitometer (Bio-Rad) and a computer program .Double stranded ONs containing the NF-κB recognition sequences  of each sample were electrophoresed in a 6–12% discontinuous polyacrylamide minigel. The proteins were transferred onto nitro-cellulose membranes, according to the manufacturer’s instructions . The membranes were saturated by incubation at room temperature for 2 h with 10% non-fat dry milk in PBS and then incubated with (1∶1000) anti-MUC2, anti-p50 or anti-p65 at 4°C overnight. The membranes were washed three times with 0.1% Tween 20 in PBS and then incubated with anti-rabbit or anti-mouse immunoglobulins coupled to peroxidase (1∶1000) . The immunocomplexes were visualised by the ECL chemiluminescence method . The membranes were stripped and re-probed with β-actin (for cytosolic protein extracts) or GAPDH (for nuclear protein extracts) antibodies to verify equal loading of proteins. Subsequently, the relative expression of MUC2, p50 or p65 proteins in cytosolic and nuclear fraction was quantified by densitometric scanning of the X-ray films with a GS 700 Imaging Densitometer and a computer programme .Cytosolic and nuclear fraction proteins were mixed with gel loading buffer  in a ratio of 1∶1, boiled for 3 min and centrifuged at 10,000 CCA TGC GTG CCT CTC TGC AA (forward) and 5′-GTG GGT TGG GTG ACA CAC TC-3′ reverse; for IL-8, 5′-TGC CAA GGA GTG CTA AAG-3′ (forward) e 5′-TCT CAG CCC TCT TCA AAA-3′ (reverse); for GAPDH, 5′-CGC TGA GTA CGT GGA G-3′ (forward) and 5′-GAG GAG TGG GTC TCG CTG TT-3′(reverse). The PCR products were run on a 1% agarose gel and visualised by Sybr Safe staining.Total RNA extraction from NCI-H292 cells, by using TRIzol, was performed as described elsewhere Results were expressed as the mean ± S.E.M. of n experiments. Statistical significance was calculated by one way analysis of variance (ANOVA) and Bonferroni-corrected p value for multiple comparison test. The level of statistically significant difference was defined as p<0.05.Figure S1vs. unstimulated cells; **p<0.01; ***p<0.001 vs. LPS.Representative ELISA shows IL-6 and IL-8 protein levels induced by LPS in IB3-1 cells at 24 and 72 h. Data are expressed as mean ± S.E.M. of three experiments. °°°p<0.001 (TIF)Click here for additional data file.Figure S2DPPC or naked dec-ODN collected from cell culture medium at 24, 48 and 72 h. (A) dec-ODN released from LPPDPPC or naked dec-ODN from supernatant; (B) dec-ODN extracted from LPPDPPC pellet and annealed naked dec-ODN . Data are from a single experiment and are representative of three separate experiments.Agarose gel electrophoresis of LPP(TIF)Click here for additional data file.Supporting Information S1DPPC containing dec-ODN on IL-6/IL-8 secretion from CF cells - Integrity of dec-ODN in cultured CF cells.Effects of LPP(DOC)Click here for additional data file.