Cryoballoon ablation (CBA) has been proven to be very effective for pulmonary vein (PV) isolation (PVI) if complete occlusion is achieved and conventionally assessed by angiographic injection of contrast within PV lumen. The aim of our study was to assess the usefulness of saline contrast intracardiac echocardiography in guiding CBA with respect to PV angiography.Thirty consecutive patients with paroxysmal atrial fibrillation were randomly assigned fluoroscopy plus color-flow Doppler  or contrast intracardiac echocardiography plus color-flow Doppler  for guidance of CBA.We evaluated 338 occlusions of 107 PVs. The intracardiac echocontrastography-guided assessment of occlusion, defined as loss of echocontrastographic back-flow to the left atrium after saline injection regardless of the visualization of PV antrum, showed a high level of agreement with the angiographic diagnosis of occlusion. PVI rate was similar in both groups and effectively guided by intracardiac echocontrastography . Group 2 patients had significantly shorter procedure  and fluoroscopy times  and used a lower iodinated contrast .PV occlusion and PVI during cryoablation can be effectively predicted by intracardiac saline echocontrastography. This technique reduces procedural time, radiological exposure and iodinated contrast use. Pulmonary veins (PVs) electrical isolation (PVI) is the cornerstone of the ablative treatment of atrial fibrillation (AF) . RadiofrNevertheless angiography-guided CBA of PVs usually requires exposure to radiation doses and use of a contrast medium with the potential risks of inducing cancerous processes, allergic reactions and contrast nephropathy .To reduce these risks, transesophageal echocardiography has been proposed [10], although general anesthesia is required in order to avoid patient discomfort.More recently, intracardiac echocardiography -13 has bA bolus of saline solution can be injected through the internal lumen of cryoballoon, thereby inducing the rapid appearance and diffusion of microbubbles within the left atrium. When the cryoballoon is positioned at the PV ostium, the absence of this contrastographic effect suggests that occlusion has been achieved.The aim of our study was to prospectively assess the usefulness of saline contrast intracardiac echocontrastography in comparison with angiography  in diagnosing PVs occlusion and in guiding CBA procedure.Thirty consecutive patients with symptomatic drug-refractory paroxysmal AF, undergoing CBA, were enrolled in this study from October 2009 to December 2010. They provided written informed consent. Oral anticoagulation was administered for at least 4 weeks prior to the procedure, interrupted and replaced with low molecular weight heparin 5 days before hospital admission. A transesophageal echocardiography was performed within 24 hours of the procedure in order to exclude left atrial thrombus. Contrast-enhanced cardiac magnetic resonance imaging (MRI)  was perfThe procedure was performed under conscious sedation and analgesia with fentanyl and midazolam. An 8/10-F ultrasound imaging catheter  was inserted through a 9/11-F sheath, positioned in the right atrium and connected to an ultrasound platform .Using intracardiac echo-guidance, two trans-septal punctures  were perPV angiography was performed in two oblique projections  to facilitate the following positioning of guide-wire and ablation catheter. PV angiographic ostium was defined as an abrupt angular change between the left atrium and the tubular portion of the PVs.A single 28-mm 'big-balloon' strategy was adopted in accordance with suggestions from Chung et al. . BrieflyThe internal lumen of the cryoballoon allows either iodinated contrast medium or saline solution to be injected in order to evaluate occlusion, using different imaging techniques according to the study protocol: fluoro-based and intracardiac echocardiography-based (echocontrastography and color-flow Doppler echocardiography). Multiple saline solution injections were used to cautiously advance the cryoballoon at the PV ostium to the most proximal wedged position, until the echocontrastographic effect disappeared and a brisk increase in resistance to manual injection was felt. Flushing of sheaths was temporarily stopped during saline injection and assessment of PV occlusion. Direct coaxial, hockey-stick and big-loop occlusive approaches were used, as already described .In the case of failure of occlusion before freezing using the above-mentioned techniques, the PD maneuver was performed, aiming at occlusion during freezing ,10,11,13Angiographic occlusion of PVs was defined as absence of contrast medium back-flow jet in the left atrium in multiple projections, contrast retention with dense delineation of the adjacent proximal portion of plugged PV and antegrade flow of contrast medium toward the PV tree periphery  8]..8].Intracardiac echocontrastography occlusion of PVs was defined as the absence of the back-flow of microbubbles into the left atrium after rapid injection of 1-5 cc saline solution into the targeted PV within a timeframe of a few seconds.Color-flow Doppler occlusion of PVs was defined if anatomic contact at the PV ostium-balloon interface was evident and no venous jets reflowing from residual gaps were observed -13. In cA high-velocity turbulent reflow jet was carefully distinguished from double-peaked accelerated compensatory flow originating from the adjacent PV by means of pulsed wave Doppler. PW Doppler peak flow velocities of all PVs were determined before and after ablation. Intra-ostial recordings from all PVs were made by a 14-pole circular catheter . After CBA, bidirectional electrical PVI was assessed in sinus rhythm and re-checked 30 minutes after the last application [15], using differential pacing when the left PV voltage amplitude was > 0.15 mV -18. In tStudy patients were randomly assigned to the following imaging support groups:In Group 1 (n=15 patients) contrast medium injection was used to assess complete occlusion of targeted PV before freezing. Occlusion was assessed by angiography and intracardiac echocontrastography imaging. While the first electrophysiologist, evaluating the angiographic readings and handling the cryo-ablation catheter, was unable to see the echocontrastography readings, the second electrophysiologist, who was experienced in performing echocardiography, was unabled to see the angiographic readings. This was due to the necessary orientation and positioning of the ultrasound platform in respect to the fluoroscopy monitors in the operating room. CBA was carried out if angiography showed occlusion, irrespective of echocontrastography readings.In Group 2 (n=15 patients) PV occlusion shown solely by real-time echocontrastography was required before freezing. However, in order to gather a larger number of data on the correlation between the imaging techniques, an angiographic bolus of contrast medium was also injected after echocontrastography confirmation of occlusion; this was done just at the onset of freezing, thereby not interfering in the decision-making process of cryoenergy delivery.For PD-type cryofreezes, color flow-Doppler imaging alone was used to document the achievement of occlusion after freezing following the withdrawal of the cryoballoon, and to guide CBA in both groups, due to inadequacy of angiography and intracardiac echocontrastography readings.Diagnostic agreement between angiography, which is regarded as the gold standard, and intracardiac echocontrastography in documenting occlusion before freezing was assessed in the overall population, in group 1 by means of single contrast medium injections and in group 2 by means of separate injections of saline solution and contrast medium. The efficiency of angiographic and echographic diagnoses of occlusion in guiding CBA was compared in the two groups by evaluating the achievement of PVI with ≤ 2 double cryofreezes for each PV. Finally, procedural time, radiological exposure and use of contrast medium were assessed in the 2 groups.Follow-up visits performed at 2, 4, 6, 9, 12, 15, 18 and 21 months after ablation included clinical examination, periodic interviews, 12-lead surface ECG and 24-h ECG Holter monitoring. All AF episodes lasting >30 seconds that were documented after a 2-month blanking period were regarded as recurrences.Mean ± standard deviation was used to describe continuous variables with normal distribution. Student's t-test for paired samples was applied for comparison. Categorical data were shown as absolute and relative frequencies and they were compared by means of Chi-squared or Fisher's exact test, as appropriate. The diagnostic agreement between angiography and intracardiac echocontrastography on the overall population, and in the A and B subgroups has been measured using the Kappa statistics. A p value <0.05 was considered statistically significant. All the analyses were performed using the statistical software SPSS 12.0 .Thirty-four patients were evaluated for inclusion in the study. Four patients were excluded from CBA owing to left atrium appendage thrombus (1), long-standing persistent AF (1), previous surgical repair of an atrial septal defect (1) or decompensated heart failure (1). The study therefore included 30 patients with symptomatic paroxysmal AF episodes. Patients' characteristics are detailed in Preprocedural MRI revealed a left common trunk in 13 patients (6 in group 1) and an accessory right pulmonary vein in 1 patient (group 1). In the 30 patients, all 108 PVs were analyzed by means of intracardiac echocardiography. In 78 PVs, the PV antrum was adequately explored by means of color flow-Doppler analysis; in the remaining 30 PVs, exploration was deemed to be insufficient. Ostial diameters of all PVs, as assessed by pre-procedural MRI, were not statistically different from those assessed by intracardiac echocardiography, as illustrated in In both groups, a total of 107 out of 108 PVs were actually treated; one right inferior PV was not treated due to tachycardia-induced AF coming from the left upper PV . In the Indeed, 285 occlusions (84%) were achieved before freezing; the remaining 53 occurred during PD-type maneuvers, in which occlusion was achieved after freezing . Out of In 206 out of 283 cases (73%) occlusion was adequately evaluated also by color flow-Doppler and was confirmed in all cases; in the remaining cases (27%) color flow-doppler visualization was deemed insufficient or inadequate for the diagnosis of complete occlusion.In 252 cases, angiography injections did not show occlusion before the beginning of freezing and were not followed by cryoenergy delivery. In 250 out of the 252 cases (99%), echocontrastography confirmed the angiographic absence of occlusion (clear contrastographic effect in the left atrium).In all 107 of the PVs treated, PVI was achieved by means of CBA without the need for additional focal touch-up applications. Angiography in non-PD cryofreezes in group 1 and echocontrastography in non-PD cryofreezes in group 2 (both plus color flow-Doppler in PD-cryofreezes) proved equally efficient in guiding CBA, enabling PVI to be achieved by means of 1-2 double cryoenergy deliveries in 89% (48 of 54) and 92% (49 of 53) of PVs, respectively .In the remaining cases, another 3-4 applications of cryoenergy enabled PVI to be achieved in all the treated PVs.In group 1 in which the diagnosis was guided by angiography, procedural time and radiological exposure time were longer and more iodinated contrast medium was used than in group 2 in which the diagnosis of pre-ablation occlusion was guided by saline contrast echocardiography .In 53 cases without initial demonstration of occlusion, PV was subsequently occluded after the onset of freezing, during a PD maneuver . NeitherDuring 53 PD maneuvers, 40 color flow-Doppler reflow jets were documented; the maximum velocity of the turbulent flow was ≥ 1.2 m/sec. Post-occlusion flow peak velocity of adjacent PVs transiently increased by 0.1-0.2 m/s, its maximum value being < 1.1 m/sec.After a 2-month blanking period and a mean follow-up of 14.5±3 months, 20 (66%) patients were free from AF recurrences without antiarrhythmic therapy, and 3 patients (10%) were free from AF with antiarrhythmic therapy. No differences were observed between the 2 groups of patients .In one group 1 patient, the creatinine level transiently rose from a basal value of 1.8 to a peak value of 2.8. Pericardial effusion requiring drainage occurred in one patient of group 1. No acute PV stenosis occurred.PVI remains the cornerstone for successful percutaneous AF ablation . CBA hasThe trans-esophageal echocardiography has been adopted as a cost saving technique for supporting CBA. However, it requires general anesthesia due to the discomfort that it causes. In a study by Siklody et al., the disThe use of intracardiac echocardiography during CBA  in 22 paThe present study compared the usefulness of saline solution echocontrastography in guiding CBA. We have observed that saline intracardiac echocontrastography can effectively guide CBA with a high diagnostic agreement in respect to angiography in assessing PV occlusion regardless of venous anatomy and echographic visibility. Moreover, procedural time, fluoroscopy and contrast use were reduced, showing a high predictive value with regard to PVI and allowing a low number of cryofreezes in 90% of PVs.The diagnostic capability of the echocontrastographic method in documenting pre-ablation occlusion proved to be very high in both groups of patients. Saline solution echocontrastography proved feasible even in cases of moderate or poor color flow-Doppler visualization.Indeed, the echocontrastographic occlusion is an "all or nothing" type of finding, based on the absence of microbubbles into the left atrium immediately after saline injection. As such, it does not depend on the availability of adequate visualization of the PVs, though admittedly the technique is not practical after the freezing of the internal lumen of the cryoballoon, as occurs in PD-type applications .Nevertheless, in 2 of the 285 occlusions, a diagnostic disagreement was observed. Despite the echocardiography diagnosis of complete occlusion, an intermittent reflux jet of contrast medium was seen after angiographic injection; this was due to the greater injection pressure in the venous chamber proximal to the occlusion, which enabled the contrast medium to seep through the seal between the cryoballoon and the PV ostium in an intermittent manner. However, echocardiographic reassessment by means of both color flow-Doppler and echocontrastography with saline injection documented complete occlusion. Indeed, unlike with angiography, a high pressure injection is not necessary in order to obtain an adequate contrastographic effect with the saline solution, and the injection can be repeated, even at different pressures, without fear of increasing the use of contrast medium. Although we regarded these two cases as false positive diagnoses of occlusion on echocontrastography, they could also be regarded as angiographic false negatives.PVI, when supported by contrastographic intracardiac echocardiography integrated with color flow-Doppler, was achieved in a very high proportion of group 2 patients. This success rate has been practically identical to that recorded in group 1 patients, in whom the procedure was supported by color flow-Doppler-integrated angiography (98%), despite greater left atrial dimensions assessed by MRI were being observed in group 2 patients. Moreover, as demonstrated by the achievement of PVI in the 13 cases of left common trunk and the 1 case of right accessory PV, the venous anatomy made no difference to the therapeutic efficacy of intracardiac echocontrastography-supported CBA. In group 2 patients, in whom echocontrastography alone was used to support CBA in pre-ablation occlusive applications, radiological exposure and the overall use of iodinated contrast medium were reduced in comparison to group 1 patients with fluoroscopy guidance.Our results led to the conclusion that the combined contribution of echocontrastography and color flow-Doppler may be used, though the spheres of application of each remain distinct. The ability of both techniques to diagnose occlusion and to predict PVI may be optimized within an integrated algorithm of imaging diagnostics during CBA, in which intracardiac echocontrastography would be used as an essential guide to initially occlusive applications  while color flow-Doppler would fundamentally be dedicated to PD maneuvers.Finally, intracardiac echocontrastography could be particularly useful in patients with iodinated contrast allergy.This paper reports a single-center experience on a limited number of patients. A larger multi-center experience could attenuate the influence of inter-operator variability. The feasibility of performing CBA with almost exclusive echocardiographic support should be evaluated prospectively on a larger case series over longer periods of follow-up. Pre-procedural contrast MRI, routinely performed in our patients to achieve a 3D anatomical reconstruction of the left atrium and PVs, is not so strictly necessary in balloon-based ablation procedures.Higher injection pressures were required for iodinated contrast due to its viscosity, despite this no differences in the contrastographic effects were observed using either iodinated or saline contrast. Intracardiac echocontrastography involves loss of anatomical information about distal PV anatomy and side-branches, provided by the iodinated contrast/fluoroscopic technique. However, in our experience the echocontrastographic effect is able to mitigate the diagnostic yield of this information.Intracardiac echocontrastography is feasible in documenting complete occlusion of the PV before freezing begins and it can therefore support CBA procedure.This technique showed a high diagnostic agreement with angiography, the actual gold standard in documenting PV occlusion, and is not limited by either the type of echographic visualization or the venous anatomy.Moreover, intracardiac echocontrastography markedly reduced procedural time, radiological exposure and use of iodinated contrast medium during the procedure. The best diagnostic yield could be obtained through a diagnostic algorithm that integrates echocontrastography with color flow-Doppler imaging, the latter being preferentially dedicated to pull-down approaches.


Self-monitoring is a key behavior change mechanism associated with sustained health behavior change. Although Web-based interventions can offer user-friendly approaches for self-monitoring, engagement with these tools is suboptimal. Increased use could encourage, promote, and sustain behavior change.To determine whether email prompts or email plus telephone prompts increase self-monitoring of behaviors on a website created for a multiple cancer risk reduction program.We recruited and enrolled participants (N = 100) in a Web-based intervention during a primary care well visit at an urban primary care health center. The frequency of daily self-monitoring was tracked on the study website. Participants who tracked at least one behavior 3 or more times during week 1 were classified as meeting the tracking threshold and were assigned to the observation-only group . This group was followed but did not receive prompts. Participants who did not meet the threshold during week 1 were randomly assigned to one of 2 prompting conditions: automated assistance  or automated assistance + calls . During prompting periods (weeks 2–3), participants in the AA and AAC conditions received daily automated emails that encouraged tracking and two tailored self-monitoring reports  that provided feedback on tracking frequency. Individuals in the AAC condition also received two technical assistance calls from trained study staff. Frequency of self-monitoring was tracked from week 2 through week 17.P < .001). Self-monitoring rates were greater in the OO group than in either the AA or AAC condition (P < .001).Self-monitoring rates increased in both intervention conditions during prompting and declined when prompting ceased. Over the 16 weeks of observation, there was a significant between-group difference in the percentage who met the self-monitoring threshold each week, with better maintenance in the AAC than in the AA condition ClinicalTrials.gov NCT01415492; http://clinicaltrials.gov/ct2/show/NCT01415492 (Archived by WebCite at  Self-monitoring of behaviors and health measures such as diet, smoking, physical activity, and weight is a key behavior change mechanism. Adherence to self-monitoring regimens is associated with greater behavior change -6, weighParticipants in Web-based interventions who self-monitored their weight or physical activity, or both, have been shown to have greater success than those who do not ,13,14. HDespite its importance, there is limited information about the frequency of self-monitoring in Web-based interventions. This may be because process data are seldom reported ,21. NoneThe prompting study (hereafter referred to as substudy) described in this paper was a substudy of Healthy Directions 2, a randomized controlled trial of a multiple risk factor cancer prevention intervention conducted in two urban primary care health centers located in metropolitan Boston, USA. In Healthy Directions 2, patients from 33 participating providers were recruited and randomly assigned to study arms at the provider level. Enrollment eligibility included being a health center patient, being 18+ years of age, having a scheduled well visit or chronic disease management appointment, and being able to read English. Patients were ineligible if they had undergone cancer treatment in the previous year or had a diagnosis of dementia, blindness, neurodegenerative disease, or psychiatric illness .The Healthy Directions 2 intervention targeted multiple cancer risk factors and was designed to (1) promote physical activity, (2) reduce red meat intake, (3) increase fruit and vegetable consumption, (4) promote daily multivitamin use, and (5) promote smoking cessation, as applicable. Intervention components included an endorsement of behavior change by the participant’s health care provider; materials delivered through the study website or in print, based on participant preference; intervention materials for participants’ friends and family members; and links to community-based resources. The materials emphasized the importance of consistent and continued tracking of health behaviors. The website included a user-friendly section where patients could self-monitor all targeted behaviors at once. Although daily self-monitoring was encouraged, the website allowed participants to enter data for the day they logged into the website and for the 2 days prior. After entering data, participants received immediate feedback in the form of graphs and descriptive text. Participants could also view their data over time, to assess overall progress. Intervention materials were available via the Web or as a print packet. When joining the study, each participant randomly assigned to the intervention received a bottle of multivitamins, a pedometer, intervention materials or logon information for the study website, and a US $5 gift card. The Healthy Directions 2 study was approved by the institutional review board at Harvard Vanguard Medical Associates.After completing recruitment for the parent study, we recruited an additional 100 participants for the substudy. This substudy was separate from the parent study. Eligibility requirements were the same as those for the parent study, plus the following additional criteria: (1) having an email address, (2) having the ability to access the Internet daily, and (3) being willing to receive the Healthy Directions 2 intervention via the Web only.Recruitment for both the parent study and the substudy was the same. Eligible patients were sent an introductory letter that outlined the study and let them know that they may be approached and invited to join the study at their upcoming appointment. At check-in, study staff met the patient and verbally introduced the study, and interested individuals provided written informed consent and completed a self-administered baseline survey.During recruitment for the substudy, eligible participants were made aware that they may receive an additional intervention that would include emails and possibly two technical assistance calls. After completing the survey, each substudy participant received a bottle of multivitamins, a pedometer, login information for the study website, and a US $5 gift card for completing the survey. Recruitment for the substudy was limited to one site (8 providers) and took place in March 2010. As We set a minimum threshold of self-monitoring at least one behavior 3 or more times per week . ParticiParticipants assigned to the AA condition (n = 36) received 2 weeks of daily emails during the prompting period (weeks 2 and 3) that encouraged them to track their behaviors via the study website. Email messages changed daily and included a brief message about the benefits of self-monitoring and a hyperlink to the study website. Participants could choose to respond directly to the email with their tracking information instead of logging into the website; study staff uploaded emailed self-monitoring data to the study website. Participants also received two tailored self-monitoring reports: the first at the end of week 2 and the second at the end of week 3. Reports provided feedback to the individual about his or her frequency of tracking for each of the behaviors during the previous week. The reports mirrored the self-monitoring graphs available on the website; we hoped that seeing the graphs would encourage individuals who had not self-monitored to visit the website and track their behaviors. Reports were viewed as part of the prompting intervention. If participants did not self-monitor, their reports reiterated the information on the benefits of self-monitoring that was included on the daily emails and encouraged self-monitoring via the study website.Participants randomly assigned to the AAC condition (n = 50) were sent the emails and tailored self-monitoring reports, detailed above, and received two technical assistance calls. The first call was made at the end of the first week of prompting (week 2) and the second call took place at the end of the second week of prompting (week 3). The calls, conducted by a trained health coach, were designed to be brief (less than 5 minutes) and focused on troubleshooting technical questions .We created two categories of tracking measures. The first, self-monitoring measures, focused on the frequency of monitoring. We created the second, threshold measures, to examine the minimum weekly therapeutic threshold of self-monitoring 3 or more times per week. We used multiple measures to attempt to fully capture participants’ interaction with monitoring.We determined the total number of self-monitoring events (range 0–112). We then determined the total number of weeks during which participants self-monitored at least once (range 0–16), the greatest number of continuous weeks in which participants self-monitored (range 0–16), and the frequency with which participants self-monitored each week (range 0–7).Using the frequency of self-monitoring each week, we determined whether participants met the weekly threshold each week (yes or no), total number of weeks during which participants met the threshold (range 0–16), and the greatest number of continuous weeks in which participants met the threshold (range 0–16).The baseline survey included items to assess race, Latino ethnicity, marital status, frequency of Internet use, comfort level in using computers, and participants’ financial situation by asking participants to rate the “money situation” in their household . Age, sex, and primary care provider was determined by data obtained from participants’ electronic medical records.All behaviors were assessed using validated measures -28. The We obtained descriptive statistics for key variables . We used chi-square statistics and analysis of variance to assess differences in demographics and in meeting the behavioral recommendations between those who met and those who did not meet the weekly self-monitoring threshold of tracking at least one behavior 3 or more times during week 1 (OO vs AA and AAC). We used analyses of variance to assess differences between the three groups  and the self-monitoring variables and the threshold variables.To examine the impact of the prompting conditions from baseline through the prompting period, we tested a series of 2 (group: prompting conditions) × 3 (time: weeks 1–3) repeated-measures models. Separate models were used for each dependent variable . We used binomial repeated-measures models for the dichotomous outcomes and general liner models for the continuous outcomes. The models were specified with a within-group factor of time and a between-group factor of prompting condition. We conducted similar analyses to examine the impact of the prompting conditions over the 16 observational weeks. We then included the OO group in the analyses and conducted repeated-measures models with 3  × 16 (time: weeks 2–17) and post hoc tests to explore any differences between the groups.The sample (N = 100) was 53% (53/100) male, with a mean age of 45.6 years, and was racially and ethnically diverse, with 37% (37/100) of the sample being black and 8% (8/100) being Latino or Latina. Most participants were college graduates  and reported frequent Internet use see . DemograOverall, 99.92% of participants who self-monitored, regardless of prompting condition and frequency of self-monitoring, tracked all four (nonsmokers) or five (smokers) of the targeted behaviors each time that they self-monitored (1219 of 1220 times tracked). In addition, during 76.8% (205/267) of the weeks when participants self-monitored at least once, they met the weekly threshold for self-monitoring during that week (tracking 1 or more behaviors 3 or more times). In all conditions, self-monitoring rates decreased over time.The OO group consisted of study participants who met the weekly self-monitoring threshold (tracking 1 or more behaviors 3 or more times) during week 1 and therefore did not receive prompts. Within this group, some self-monitoring occurred in 100% of the observational weeks.P < .001), on whether the threshold was met each week (P = .006), and on frequency of self-monitoring each week (P < .001). The between-group differences and group-by-time interactions were not significant.Time had a within-group effect on whether self-monitoring occurred each week (P < .001) but not a between-group effect. The analyses examining whether the threshold was met each week revealed a within-group effect (P < .001) and a between-group effect (P = .009). The AA condition was less likely than the AAC condition to meet the threshold each week . The group-by-time interaction was not significant. The analyses examining frequency of self-monitoring each week determined that there was a within-group effect (P < .001) but not a between-group effect.The repeated-measures models examining whether self-monitoring occurred each week revealed a within-group effect for time (P = .04). There were, however, no significant differences between the two prompting conditions in the self-monitoring variables  or in the greatest number of continuous weeks when the participants met the threshold.In the AAC condition, there were more weeks when people self-monitored their behaviors at least once than in the AA condition , with results favoring the OO group.The results of the repeated-measures models and post hoc analyses examining the three self-monitoring outcomes determined that the OO group was significantly different from both of the prompting conditions on all outcomes  replied to the tracking reminder emails with their tracking information, but of those who responded, 67% (6/9) replied multiple times.Consistent and continued self-monitoring is an instrumental strategy for initiating and maintaining behavior change -5,7-12, The purpose of this study was to examine the effect of two limited prompting interventions on increasing participants’ use of self-monitoring tools available on the study website. In this study, participants who did not meet the self-monitoring threshold of tracking at least one behavior 3 or more times during the first week of the study were enrolled in one of two prompting conditions. Both prompting conditions yielded a modest but significant increase in self-monitoring each week (yes vs no), meeting the weekly threshold (yes vs no), and frequency of self-monitoring/week during the prompting period. After prompting ceased, self-monitoring rates decreased in both prompting conditions. Although the significant within-group effect remained throughout the observation period, the increased self-monitoring during the prompting period coupled with the subsequent decline suggest that reminder emails can increase prompting, but that additional subsequent email reminders may be useful to help sustain this increase. Future research is needed to examine the effectiveness for different prompting intervals .Over the course of the study, a greater percentage of participants in AAC, the group that received technical assistance calls, met the weekly self-monitoring threshold, suggesting that brief contact, even in the context of technical assistance calls, may be beneficial for promoting and sustaining tracking. This limited contact with study staff may have increased motivation to track by removing technical barriers, or simply served as a gentle push to self-monitor. In total, 80% of participants in the AAC condition completed call 1, and 70% completed call 2; higher completion rates may have resulted in the AAC prompting intervention having a greater impact on self-monitoring. Future research is needed to explore the nature of interactions with staff that prompt higher levels of engagement , and whether a more explicit focus on enhancing motivation to track may yield greater self-monitoring.Over the course of the study, the OO group had the highest rates of self-monitoring each week. However, even with this group’s strong start, their self-monitoring during weeks 3–5 declined precipitously, suggesting that this may be a critical time to intervene. Beginning with week 4, when prompting ended, self-monitoring rates decreased in both the AA and AAC groups. This general across-the-board attrition is similar to those seen in other studies ,32 and iIt is noteworthy that participants who self-monitored chose to track multiple behaviors simultaneously 99.9% of the time. This is encouraging and suggests that tracking multiple behaviors is not burdensome. The willingness to track multiple behaviors in this study may have been due, in part, to the beta testing of the website that was conducted to inform a user-friendly design. The website was specifically structured so that all tracking could be completed on one page, thus making it easy to track all behaviors at the same time. In addition, it is striking that in 76.8% 205/267) of the weeks when participants self-monitored they met the threshold for that week, suggesting that specific strategies could be helpful for individuals who are willing to self-monitor but do not reach a weekly therapeutic threshold. The response rate to the tracking emails was low. It is possible that participants who were motivated to track simply logged in to the website and recorded their self-monitoring data themselves without assistance. It also could be that participants found it difficult to respond to the emails because of the timing of the sent messages . One Web-based study found that participants who actively responded to emails had a greater increase in fruit and vegetable intake than did participants who did not reply to emails [5/267 of Most often tracking has been limited to pencil and paper. Burke and colleagues reported that participants found self-monitoring via a personal digital assistant to be more socially acceptable than monitoring in paper logs . The HeaStudy limitations include a modest sample size; a largely well-educated sample, which may limit generalizability; and the lack of a nonprompting control group that included only participants who did not self-monitor during the first week of the study. Study strengths include having a diverse racial and ethnic sample and a design that allowed comparison with a group already motivated to record their behavior.It would be helpful if interventions that include self-monitoring provided information about these rates or used consistent definitions of self-monitoring. But, to date, the literature has not provided either, making it difficult to compare adherence across studies . In addiThe 2010 US Affordable Care Act emphasizes prevention and engaging patients as active participants in their health care ,35. Enco


Besides stimulants and hallucinogens, whose psychotropic effects are shared by many structurally related molecules exhibiting different efficacies and potencies in humans, the phenylisopropylamine MDMA  is the prototypical representative of a separate class of psychotropic substance, able to elicit the so-called entactogenic syndrome in healthy humans. This reversible altered state of consciousness, usually described as an “open mind state”, may have relevant therapeutic applications, both in psychotherapy and as a pharmacological support in many neuropsychiatric disorders with a high rate of treatment failure. Nevertheless, a comprehensive and systematic exploration of the structure-activity relationships associated with entactogenic activity has remained incomplete and controversial, highlighting the possibility that MDMA might represent a pharmacological rarity in the field of psychotropics. As the latter is still an open question, the pharmacological characterization of MDMA analogues remains the logical strategy to attempt the elucidation of the structural requirements needed to elicit typical MDMA-like effects. Intriguingly, almost no experimental evidence supports the existence of actual MDMA analogues that truly resemble the whole pharmacological profile of MDMA, probably due to its complex  mechanism of action that includes a disruption of monoaminergic neurotransmission. The present review presents a brief summary of the pharmacology of MDMA, followed by the evidence accumulated over the years regarding the characterization of classical structurally related MDMA analogues in different models and how this state of the art highlights the need to develop new and better MDMA analogues. Indeed, whereas an impressive amount of experimental data obtained using animal models and drug consumers has accumulated showing that chronic exposure to MDMA may elicit anxiogenic effects . It is difficult to predict it in humans, because susceptible users are unusually sensitive to small variations in dose. Nevertheless, due to its status of “most popular street drug” and also because it remains placed on Schedule I in the U.S.A. since 1985 (followed by similar decisions enacted in many other countries) as a drug deemed to have no medical uses and a high potential for abuse, research has been focused during the last decades in constructing a detailed pharmacological profile of MDMA based on its behavioral and toxic effects 5-HT and [3H]dopamine in whole brain synaptosomal preparations were measured. The results obtained indicated that two of them, 2-methyl-MDA and 5-methyl-MDA, exhibited high potency and selectivity as serotonin-releasing agents, although they cannot be classified as “pure” entactogens in vivo [3H]5-HT and [3H]dopamine release from rat hippocampal slices, whereas IDA (isopropylidenedioxyamphetamine) was considerably less potent [125I]DOI displacement. In contrast, MDE , which is believed to be less neurotoxic than MDMA in animal models [2) may possess a non-amphetamine-like profile, even more distinct than that of MDMA itself [S)(+)-MDMA, (±)-MDE, (S)(+)-N-ethylamphetamine and (±)-N-hydroxyamphetamine generalized to amphetamine, indicating that entactogenic activity is probably stereo-selective [N-methyl derivative 5,6-methylenedioxy-2-methylaminoindan produced similar results. The authors propose that this demonstration of entactogen-like discriminative stimulus properties, for drugs devoid of neuronal degenerative toxicity potential, may serve as reliable evidence of the independent mechanisms for these effects in rats [Various MDMA and MDA analogues have been tested in different experimental models to determine if they share some of the pharmacological properties of both structural templates. In an interesting old study, the exploration of the behavioral properties of a series of methoxylated phenylisopropylamines in order to determine the effect of other substitution patterns and the relative importance of individual methoxy groups was attempted using a drug discrimination task and DOM -1-aminoethane) were studied in rats. It was demonstrated that, in contrast to reasonable interpretations of the reports available regarding this drug, 2C-T7 seems to be best classified as a DOM-like hallucinogen, whereas 4-MTA (1-(4-methylthiophenyl)-2-aminopropane), PMA and PMMA  may be considered as MDMA-like molecules, as confirmed by recreational consumers and rat behavioral studies. Indeed, PMA is a weak central stimulant compared to amphetamine, whereas methamphetamine is as potent as the latter drug [S)(+)-PMMA was able to completely generalize to (±)-PMMA, suggesting that the PMMA stimulus may be stereoespecific [S)(+)-MBDB, (R)(-)-MBDB, (S)(+)-3,4-DMA, (R)(-)-3,4 DMA, (S)(+)-MDMA and (±)-MDMA in rats. In addition, it was suggested also that MBDB and 3,4-DMA are probably closer to PMMA [R)(-)-PMA, like MDMA, seems also to share an entactogenic-like profile [So far, among the MDMA and MDA derivatives tested, only MDE and MBDB have been reported to exert entactogenic-like effects  and haveter drug -127. Theter drug . In a sespecific , as in tspecific . Additio to PMMA . Interes profile . 1A receptor activation mediated by the massive 5-HT release in the hypothalamus induced by MDMA at acute doses. The latter causes the release of the neuropeptide oxcytocin that, in turn, should be the direct effector of the pro-social behaviors induced by MDMA [2A receptor activation), probably induced by 5-HT depletion that may arise after repetitive drug exposure [As described earlier, MDMA induces in humans an altered state of consciousness characterized by increased empathy to others . Consequ by MDMA , 142-144 by MDMA , 147. Mo by MDMA , 146. Inexposure . To the exposure , no repo2).Interestingly, despite of the detailed but ambiguous descriptions of the subjective effects in humans of at least 40 synthetic psychotropic phenylalkylamines which remain underinvestigated , a carefN-methyl by an α-(MBDB) or N-ethyl group (MDE) or by including it in a 2-aminoindan ring structure (MMAI) [et al. [3). Bioisosteric replacement of oxygen by sulfur has been shown to increase potency in a number of cases that bear some structural analogy to the molecules , and it seems reasonable to extend this concept based on the MDMA structure. As an extension of the latter, the study of isoxazoles and their dihydro derivatives  might be possible suitable templates for the development of novel MDMA-like molecules.Variations on the MDMA structure for pharmacological purposes have been limited to the modification of the aminoalkyl side chain by replacing the α- and the e (MMAI) . In addie (MMAI) . Parker  [et al.  found thFig. 3B, ). A singLophophora willliamsii . At the potency . For thi potency -154 or tcategory . Moreovecategory , so theycategory .para to the ethylamine chain, either with a sulfur atom, a longer alkyl chain, or both, lead to compounds with potent central nervous system activity. The 4-n-propoxy and 4-n-butoxy homologues and their corresponding 4-thio analogues were also synthesized and pharmacologically evaluated. The propyl homologues retained high potency, but a butyl group (either with or without a sulfur atom) leads to a decrease in activity. The m-ethyl or m-thio analogues retained some effect but the diethoxy and especially the triethoxy homologues seem to be inactive as psychotomimetic drugs [The monothio analogues of the mono-, di-, and triethoxy homologues of mescaline have been evaluated in man. Modifications at the ring position ic drugs . MDMA seems to possess peculiar pharmacological properties that cannot be resembled easily by standard medicinal chemistry approaches. Moreover, MDMA-like molecules were not actually found, highlighting the need to search for more and effective experimental approaches in order to answer the central question about MDMA as a “unique” psychotropic drug. One possible explanation for the lack of success looking for true MDMA analogues might reside in the fact that most of the key events associated with the pharmacological effects of MDMA remain still not fully understood. In particular, the complex links between serotonin, dopamine and norepinephrine taking place at both the presynaptic and postsynaptic level seem to be critical to reach full MDMA-like activity. As in the case of behavioral approaches, exhaustive designed physiological profiles of these interactions are required to further comparison with possible, rationally designed MDMA analogues.in vitro, probably because the presence of a methyl group may allow the molecule to adopt a favorable conformation to interact with SERT. Similar modifications at specific positions on the aromatic ring  do not favor hallucinogenic or entactogenic-like activity [in vitro approaches evaluating monoamine- releasing effects must be attempted and compared with those of MDMA.Besides the latter, the development of new analogues and bioisosteres starting from the basic phenylalkylamine moiety is based on the fact that rather subtle structural modifications of the basic structure of the phenylethylamine template may induce dramatic changes in the ability of the molecule to evoke stimulant, hallucinogenic and/or entactogenic effects: for instance, although an additional methyl group or bromine atom may not necessarily generate major electronic disruptions inside the molecule, they may enhance its lipophilicity compared to MDMA and consequently should enhance brain penetrability. Such modifications have been related to the ability of these drugs to act as 5-HT releasers activity . One mayet al. [Additionally, the comparison of the behavioral profiles in animals referred to prototypical amphetamines representing entactogens (MDMA), and structurally related stimulants and/or hallucinogens through an appropriate choice of a series of animal behavioral paradigms instead of the evaluation of single behaviors might be a reliable strategy to find MDMA-like molecules  as an exet al.  showed fet al. . 2A receptor antagonist [The unique locomotor activity pattern induced by MDMA in rodents seems to be strongly dependent on the differential activation of central dopaminergic D1, D2 and D3 receptors , 163. Intagonist , 165. Outagonist . In additagonist .3 and/or R5 with bromine  may lead to compounds sharing at least some of the in vivo effects elicited by MDMA. The latter justifies the pharmacological characterization of those types of compounds, as well as a selected group of mescaline-like brominated analogues . In this regard, preliminary data for 12 brominated MDMA analogues and a selection of mescalinoids indicate that these molecules exhibit restricted orientations in the binding site at rSERT and hSERT, retaining a similar affinity compared to MDMA [Another almost unexplored possibility to find new entactogenic-like drugs is the search for new possible MDMA-like structural templates. One intriguing possibility is a systematic exploration of mescalinoids. Here, the encouraging subjective reports in humans for 2,4-DMA and 2,4,6-trimethoxyphenylisopropylamine (TMA-6) are consistent with MDMA-like effects . Consequ to MDMA -172. Cor to MDMA , includi to MDMA .in vivo long-term potentiation (LTP) assay. Preliminary results show that MDMA almost duplicate prefrontal cortex LTP, whereas spatial memory is disrupted [in vivo LTP at key brain locations  may offer some hints about the actual central effects associated with operative cognition after MDMA intake. As already mentioned, the available data regarding possible disruptive effects on fear acquisition processes are controversial and incomplete. In particular, the operative relationship between prefrontal cortex and amygdala activities should be addressed comparing the effects of MDMA at both locations with those elicited by the compounds proposed. Considering that MDMA is believed to be non-anxiogenic but it enhances acquisition  and proin vivo, in vitro and in silico methodologies, it seems that the particularly peculiar pharmacological nature of MDMA makes the use of such a multidisciplinary strategy much more critical, in order to answer the complex question about the biological basis of the “altered state of consciousness” elicited (apparently only) by MDMA. However, the limitations of the animal models currently available to address the complex pharmacology of MDMA to predict the occurrence of entactogenic effects in humans must be kept in mind, as human MDMA-like effects are expected to be always dependent upon individual on set  and setting  influences besides purely structural  considerations of the drug interacting with a single and/or several molecular targets.Regardless of the obvious assumption that the pharmacological characterization of psychotropic drugs requires an integrated approach combining


Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasite's ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum.The  Plasmodium has proven to possess powerful anti-Plasmodium defense capabilities. As the major regulators of these immune responses, signaling pathways, particularly the Imd pathway which seems especially capable of eliminating malaria parasites, have become attractive candidates targets for malaria-control interventions. Although the general anti-parasitic activity of the Imd pathway has been established, the particular components of the pathway involved and the physiological conditions under which the pathway is capable of limiting infection are mostly unknown. Awareness of these major players and conditions is crucial for adapting the Imd pathway into an intervention strategy. We report that while several members of the Imd pathway are critical for such a response, others are dispensable. We also show that timing of the response with regard to infection and intensity of infection exposure both influence the effectiveness of an Imd-derived anti-Plasmodium response while the status of the gut flora does not. Taken together, this data lays the essential groundwork for effective intervention based on manipulation of this pathway that can severely limit mosquito infection with human malaria parasites.The immune response of the mosquito vector of  Plasmodium. In fact, such an immune response is responsible for one of the most dramatic reductions in the parasite population during the Plasmodium life-cycle: a log-fold loss in parasite numbers as ookinetes cross the mosquito's midgut cells and develop into oocysts. This drop in parasite number is due, at least in part, to an effective cellular and systemic anti-Plasmodium immune assault. Boosting the mosquito's immune response to a level that would fully eliminate this already vulnerable parasite stage could effectively terminate the malaria transmission cycle.Malaria remains one of the world's most devastating infectious diseases, and its successful control will require a multifaceted approach involving a combination of multiple strategies P. berghei, P. falciparum, and/or P.vivaxP. falciparum in laboratory reared A. gambiae, A. stephensi, and A. albimanus; others have observed a requirement for PGRP-LC, one of the receptors activating the Imd pathway, in the response of wild-caught A. gambiae to field isolates of P. falciparum, and still others have reported a role for REL2, in the response of the recently colonized Ngousso A. gambiae strain to NF54 P. falciparumP. falciparum, and this influence is translatable from laboratory to field, most likely across multiple Anopheles species and Plasmodium strains.Immune signaling pathways are key regulators of insect immune defenses and are therefore attractive candidates for genetic modification to create a mosquito with an immune response that overwhelms the parasite. Although the Toll and Jak-Stat pathways control immune attacks that limit the development of P. falciparumcaspar silencing), indicated that thioester-containing protein 1 (TEP1), fibrinogen immunolectin 9 (FBN9), and a leucine-rich repeat family member (LRRD7/APL2) are three of the major players in the Plasmodium killing that occurs in caspar-silenced mosquitoes of the A. gambiae Keele strain Plasmodium at the physiological level.We have shown that an early activation of the REL2 transcription factor in the midgut tissue, at 6–14 hours after ingestion of an infected blood meal is affective at targeting P. falciparum infection in the A. gambiae Keele strain, with the goal of facilitating the identification of optimal strategies for Imd pathway manipulation.In this study, we addressed some of the remaining key questions regarding the impact of the Imd pathway on Plasmodium defense. Existing data have implicated the negative regulator Caspar, the transcription factor REL2, and the pattern recognition receptor PGRP-LC in the defense against malaria so we concentrated our efforts on the Imd pathway components known in Drosophila to interact with or between these components and that have a clear 1-to-1 ortholog in Anopheles  and the artificially enhanced (infection plus caspar silencing) immune response against P. falciparum.We first addressed the contribution of specific Imd pathway components in the anti-nopheles , though Plasmodium completes its fertilization to form a zygote, transitions to the motile ookinete form, penetrates the midgut epithelium, and transforms into the oocyst stage. Since most assessments of immunity enumerate parasites at the oocyst stage, it is often unknown which stage(s) are targeted by anti-Plasmodium mechanisms, yet this information could drastically affect how an anti-Plasmodium strategy is designed.We then determined which parasite life-cycle stages are vulnerable to an Imd pathway-derived attack. After it enters the midgut lumen, Plasmodium development Plasmodium densities in mosquitoes P. falciparum because gut-specific over-expression of REL2 in A. stephensi has a significant effect on P. falciparum, highlighting the immune contribution of tissues harboring bacteria caspar silencing in limiting P. falciparum.It has long been known that mosquitoes' endogenous bacteria can affect P. falciparum infection have utilized an exceptionally virulent strain of P. falciparum that is capable of producing high intensities of infection. Recent work from Mendes et al.Plasmodium response can vary with the infection intensity. To assess the potential role the Imd pathway in this differential response, we also assessed the outcome of infection following three different levels of parasite exposure in mosquitoes in which several Imd pathway members and downstream effectors had been silenced.To date, all analyses of the effect of the Imd pathway and its downstream components on Plasmodium defense, we used an RNA interference approach to silence individual genes in order to determine whether each was necessary for P. falciparum infection. A double-silencing assay was also performed to determine whether those components with an effect on P. falciparum could reverse the decreased infection that was observed when caspar was silenced, thereby identifying any factors that are essential for the caspar silencing-mediated immune defense in addition to the routine defense against P. falciparum.To assess the contributions of Imd pathway components to the anti-imd, fadd, caspL1 (dredd), and rel2 resulted in median P. falciparum oocyst infection intensities that were at least two-fold greater than those of the control group treated with dsRNA against GFP  with caspar completely reversed the typical resistance to infection observed when only caspar was silenced; i.e., median infection intensities were not significantly different from those of the single-silenced groups and did not exhibit the absence of infection typically observed following caspar silencing   (Single-silencing of  levels) . Similar levels) .P. falciparum infection; median oocyst levels in silenced mosquitoes reached 22 oocysts per mosquito, nearly identical to the median of 22.5 oocysts per mosquito in GFP dsRNA-treated controls .Depletion of the TAK1 protein had no significant effect on controls . Accordisilenced . TAK1 isPlasmodium parasites are susceptible to mosquito immune responses at multiple stages of their development, previous analyses of caspar silencing have measured only the changes in oocyst numbers, an approach that cannot distinguish between activity against a specific stage and that against multiple stages. To discover which stage(s) of development is (are) halted by caspar silencing, we developed an assay in which the time of dsRNA injection was varied. In this assay, peak silencing occurred only after most parasites had matured to a specific stage. Time course analysis of caspar silencing revealed that efficient depletion of caspar mRNA throughout the mosquito body could be achieved within a day of injection, and the effect would persist for at least 6 days .Administered 3 days post-infection, of 77.3% . For comcaspar-silenced and control mosquitoes, although, surprisingly, an appreciable degree of infection inhibition was still observed. In these experiments, the median number of surviving oocysts in the caspar-silenced mosquitoes was 36, as compared to 56 in the GFP dsRNA-treated mosquitoes, representing a decrease of 45.7% while prevalence was not impacted  were far less effective in limiting the number of parasites per midgut in impacted .Plasmodium infection Drosophila, the Imd pathway is a primary regulator of the response against intestinal bacteria caspar silencing, or whether the Imd pathway activation provided by the bacteria under normal conditions is simply supplanted by the artificial Imd pathway activation mediated by caspar silencing. To answer this question, we administered an antibiotic cocktail to mosquitoes before treating them with dsRNA against caspar or GFP and subjecting them to P. falciparum infection. We have previously shown that antibiotics can be used to eliminate the majority of the bacterial population from the mosquito midgut Plasmodium infection if they had been pre-treated with antibiotics; aseptic mosquitoes harbored almost twice as many oocysts as their GFP dsRNA-treated septic counterparts  from the levels in GFP dsRNA- treated control mosquitoes (apl1 significantly increased the infection levels (when compared to the GFP dsRNA-treated group) only at low (a 2-fold increase) and medium (a 2.6 -fold increase) infection intensities. Specific paralog silencing suggested that APL1C was most effective in influencing low-level infections (a 2-fold increase), while APL1B was the most influential paralog at medium infection intensities (a 2.3-fold increase). In our experiments, APL1A depletion had no effect on P. falciparum infection under any condition.Earlier studies have featured a model system composed of a highly virulent parasite strain and highly susceptible mosquito strain. While we have previously shown that the Imd pathway is effective against squitoes  and thatsquitoes . For exaapl1, knockdown of another member of the leucine-rich repeat-containing family, lrrd7, was effective in low- and medium-, but not high-intensity infections. Importantly, we noted that both the prevalence and intensity of infection were significantly increased by silencing lrrd7 prior to low levels of exposure to P. falciparum, but only infection intensity was significantly altered by lrrd7 silencing prior to medium-level exposure. Thus, the contribution of a gene to the anti-Plasmodium response is not only dependent on the level of exposure but also on the method of data collection used   .caspar silencing was effective in limiting infection, regardless of the exposure dose; in essence, caspar silencing was effective in limiting low-, medium- and high-intensity P. falciparum infections. The only other tested effector to display this consistent effect was TEP1 , the level closest to that at which pathway analysis is typically performed . Neverthwas TEP1 .As a control, we checked the efficiency of the gene silencing of the Imd pathway components and downstream effectors genes by measuring the expression of their gene-specific RNAs and observed a reduction of 48–92% in their regular expression patterns .Despite the fact that the negative regulator of the Imd pathway, Caspar, controls a branch of the mosquito immune system that determines the mosquito's resistance to human malaria parasites, until now little has been known about the genes/proteins controlled by this regulator, when they exert their effects on the parasite, and whether this activity is dependent on a tripartite relationship with endogenous bacteria or is influenced by infection intensity. The answers that we have obtained to these essential questions about the biology of Caspar should facilitate the use of the Imd pathway for the development of malaria control strategies.P. falciparum (single-silenced and infected) and the Caspar-controlled response against P. falciparum in relation to other Imd pathway factors (double-silenced and infected). These experiments revealed three different phenotypes for Imd pathway members: 1) full participation in both anti-P. falciparum responses and caspar-mediated resistance; 2) weaker participation in anti-P. falciparum responses, with unclear contribution to the response directed by caspar; and 3) no participation in anti-P. falciparum immunity.In the present study, we first addressed the contribution of selected components of the Imd pathway to the normal mosquito response against caspar silencing. Importantly, co-silencing Imd, FADD, CaspL1, and REL2 with caspar gave median infection intensities that were almost identical to those of the single-silenced groups. This result suggests a complete dependence of the caspar-silenced infection phenotype on these pathway members. This finding is not surprising, since Drosophila Caspar has been suggested to regulate the Dredd-dependent cleavage of REL2, and Imd and FADD are co-activators of Dredd For the first group of components, silencing genes encoding four of the known components of the Imd pathway  resulted in a significant increase in the intensity of the midgut infection when compared to GFP dsRNA-treated controls and an ability to reverse the resistance caused by caspar phenotype. Reduced levels of IKK-gamma led to an appreciable increase in oocyst load when compared to control silencing treatments; the co-silencing of caspar and IKK-gamma showed a mild reversion of the caspar phenotype, but to levels that were not significantly elevated over those for the GFP dsRNA-treated control (as was consistently observed for the four genes in the first group). This result could suggest either a lesser involvement in caspar-dependent immune responses than for the other components, a dual role for IKK-gamma in caspar-dependent and caspar-independent responses (the independent form interfering with a complete reversion of the dependent), or a caspar-independent role in antagonizing P. falciparum response that overwhelms the agonistic output of Caspar.In contrast, the second group of components produced reasonable increases in infection level in response to single silencing, yet only a partial reversion of the Drosophila model system have strongly suggested that Imd pathway activation results in the cleavage of the ankyrin repeat region of the C-terminus of Relish, likely by Dredd, freeing the active form of Relish for translocation to the nucleus Anopheles, both a full form of the Relish ortholog (REL2-L) and a short form lacking ankyrin repeats (REL2-S) are independently transcribed rel2-L has no effect on the prevalence of P. falciparum infection, although infection intensities were not reported in their study. We observed in the present study that silencing rel2-L had a noticeable effect on oocyst burden in single-silencing assays but did not result in the same potent immune response against P. falciparum as did silencing all forms of rel2. In addition, while co-silencing of caspar and rel2-L did not result in complete refractoriness, double-silencing was unable to reverse the phenotype, even to the level of infection intensity observed for the GFP dsRNA-treated control group, much less to the single-silencing levels observed when all forms of rel2 were targeted  during other infections in Anopheles.The third group of components showed no effect on infection when either single-silenced or double-silenced and had no effect on the outcome of P. falciparum and that the Imd pathway is uniquely potent against this parasite. Based on these data, we propose a model of the A. gambiae Imd pathway  in mosquitoes. Nevertheless, the strong infection-intensity phenotypes obtained by silencing multiple Imd pathway members suggest that this module within the pathway is a major player in the anti-P. falciparum defense employed by A. gambiae.Taken together, these data suggest that the proteins hypothesized to act most intimately with Caspar  are the most effective players in the mosquito defense against Plasmodiumcaspar occurs quickly, but mRNA levels recover within 5 to 6 days , any control strategy based on the Imd pathway will be affected by the mosquito's individual flora. Detectable infection levels in mosquitoes lacking endogenous bacteria suggest that the lack of negative regulation in the Imd pathway is sufficient to confer resistance to P. falciparum. Standard antibiotic treatments eliminate 99% of the endogenous bacteria Plasmodium. However, the observation that the required basal level of elicitation can occur in mosquitoes with <1% of the normal flora suggests that the differences in bacterial loads or flora composition would have only minimal effect on P. falciparum resistance directed by the Imd pathway.Several studies have reported a significant contribution of the endogenous flora to the generation of a mosquito's anti-parasitic responses. In addition to the direct interaction between bacteria and parasites, the exponential increase in midgut bacterial loads during blood-feeding elicits immune gene expression and activates immune mechanisms at the same time that P. falciparum gametocytes available during blood feeding, we have been able to control the average infection intensity in a given group of mosquitoes. These experiments showed that the requirement of a given pathway component or effector, assessed by RNAi, is often dependent on that average infection intensity   . EffectoPlasmodium effectors are produced and therefore available for combating parasites. This type of effector would not have an effect on infections at high intensities in control mosquitoes, and therefore silencing the gene(s) that encode them would show no effect. Inefficiency of some effectors at higher Plasmodium doses may also be governed by infection-intensity gene regulation if high levels of infection cause those genes to be down-regulated, as in, for example, a feedback loop. Such genes would theoretically be produced at sufficient levels in control mosquitoes during low- or medium-level infections but would be down-regulated, and therefore less effective, at higher doses.Effectors known to be required at lower but not higher doses could be influenced by a saturation effect, in which Imd pathway effectors are being produced in quantities that are sufficient to kill lower numbers of parasites but insufficient to deal with infections above a certain level. This would be the case if only a small, finite number of anti-Plasmodium defenses under laboratory conditions, since measurable effects can be seen for most genes, and statistical analyses can be made (in contrast to very low-infection assays). However, our data also indicate that looking only at medium-level intensities can lead to unfounded assumptions about the physiological relevance of the examined genes during infection under natural field conditions, and complexities of pathway regulation can be masked.Our data suggest that the medium- intensity infection levels are optimal for studying anti-Plasmodium defenses that have not been addressed in previous studies using only standard infection intensities:Our study also offers a number of interesting and novel insights into other aspects of anti-P. falciparum, our data are not in agreement with regard to the specific paralogs involved. Most strikingly, we did not find a significant role for the APL1A paralog in the anti-P. falciparum immune response  . The fact that LRIM1 has been linked to the TEP1/APL1 anti-Plasmodium mechanism Plasmodium activity, but it is possible that it may not be required (or is redundant) at very low infection intensities, or that RNAi-mediated depletion is not be sufficient to reduce it to a level that would have any impact on a low-intensity infection.Second, the results of infection exposure have revealed a novel role for LRIM1 during tensity) . We belicaspar silencing had an effect at very high infection intensity whereas Imd silencing did not may suggest that other branches of the Imd pathway act upstream of Caspar and in parallel with the Imd receptor. The existence of multiple downstream signal circuits would also explain the robustness of the caspar silencing phenotype and the variety of downstream effectors it regulates. However, the identities of such circuits are unknown.Third, the fact that P. falciparum-laden blood meal, silencing caspar still greatly reduced the resulting oocyst infection and, conversely, silencing tep1 significantly increased the infection. These results suggest that these two components are essential factors in the anti-Plasmodium defense for which no substitutes are allowed, whereas the other factors may be to some degree redundant.Fourth, we found that no matter how concentrated or dilute a caspar and transgenic over-expression of REL2) has no observable impact on mosquito fitness, yet is potently anti-parasitic DrosophilaTaken together, our finding suggest that members of the Imd pathway are reasonable first targets for vector-based malaria control interventions, and analyses of such interventions should necessarily include facets of mosquito-parasite infection biology such as the level of the infection and the parasite stage(s) affected. Clearly, some pathway members would be more effective than others: Specifically, Imd, CaspL1, or REL2 would be good positive regulators to manipulate, along with Caspar as the negative regulator, and we have confirmed this conclusion for REL2 using transgenic methodology Plasmodium have emerged as favorable candidates for targeted malaria-control interventions. However, the immense technical effort required to build such interventions is not trivial, and therefore the preliminary refinement of candidate selection must be thorough. Here we present data that not only identify the biological mediators of the Imd pathway-driven immune response that can render mosquitoes almost completely refractory to P. falciparum but also answer major questions about when and how that response is generated. We show that while several members of the Imd pathway are critical for such a response, others are indispensable, and the anti-Plasmodium response decreases in potency as the parasite matures, with the pre-oocyst stages being most vulnerable and the mature oocyst stage the least. We also show that caspar-mediated refractoriness can occur without regard to microbial flora or the intensity of the Plasmodium exposure, revealing the delicate yet robust control this negative regulator exerts over the Imd pathway and indicating the pathway's potential for broad applicability.Components of the immune response of the mosquito vector of This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee of the Johns Hopkins University (Permit Number: M006H300). Commercial anonymous human blood was obtained from Interstate Bloodbank and used for parasite cultures and mosquito feeding and informed consent was therefore not applicable. The Johns Hopkins School of Public Health Ethics Committee has approved this protocol.A. gambiae mosquito larvae were raised in 30×34 cm trays (∼250 larvae per tray) with cat food pellets added daily and ground fish food supplemented upon water change. Adults were reared in a 20×20×20 cm3 cage and sustained using a 10% sugar solution at 27°C and 80% humidity with a 12-h light/dark cycle according to standard procedures Keele strain Anopheles Imd pathway response and genes encoding 1-to-1 orthologs of Imd components known in Drosphila were targeted by synthesizing sense and antisense RNAs from ∼300- to 600-bp PCR-amplified gene fragments using the T7 Megascript kit (Ambion) and primers indicated in Assays were performed according to a standard protocol Mosquitoes were fed on NF54 gametocytes in human blood through a membrane feeder at 37°C at various time points, depending on the stage being examined: 3–4 days after dsRNA injection for assessment of ookinetes , and 6 dcaspar or GFP (as a control) 2 days after emergence and incubated under normal conditions. Each day at the same time, ∼10 mosquitoes from each group were collected and homogenized for total RNA extraction. Assays were then performed according to a standard protocol Mosquitoes were silenced for A. gambiae mosquitoes were sequestered in pint-capacity paper cups and maintained on either 10% sucrose or 10% sucrose supplemented with 10 µg/mL penicillin-streptomycin and 15 µg/mL gentamicin Plasmodium infection, RNA extraction, PCR amplification, and other methods described above. Primer sequences are given in Groups of 80–100 recently emerged (>1-day-old) female Table S1Imd pathway phenotypic dissection.(DOCX)Click here for additional data file.Table S2(A) Early oocysts. (B) Late oocysts.(DOCX)Click here for additional data file.Table S3Caspar silencing with and without antibiotics.(DOCX)Click here for additional data file.Table S4A) Effect of silencing of IMD pathway members and effectors on low exposure infections. (B) Effect of silencing of IMD pathway members and effectors on medium exposure infections. (C) Effect of silencing of IMD pathway members and effectors on high exposure infections.((DOCX)Click here for additional data file.Table S5Primers used for dsRNA synthesis and for verification of knockdown of target genes.(DOCX)Click here for additional data file.


Crassostrea gigas is considered to be tolerant to relative low salinity. The genes that regulate C. gigas responses to osmotic stress were monitored using the next-generation sequencing of whole transcriptome with samples taken from gills. By RNAseq technology, transcript catalogs of up- and down-regulated genes were generated from the oysters exposed to low and optimal salinity seawater.Low salinity is one of the main factors limiting the distribution and survival of marine species. As a euryhaline species, the Pacific oyster Through Illumina sequencing, we reported 1665 up-regulated transcripts and 1815 down-regulated transcripts. A total of 45771 protein-coding contigs were identified from two groups based on sequence similarities with known proteins. As determined by GO annotation and KEGG pathway mapping, functional annotation of the genes recovered diverse biological functions and processes. The genes that changed expression significantly were highly represented in cellular process and regulation of biological process, intracellular and cell, binding and protein binding according to GO annotation. The results highlighted genes related to osmoregulation, signaling and interactions of osmotic stress response, anti-apoptotic reactions as well as immune response, cell adhesion and communication, cytoskeleton and cell cycle.C. gigas transcriptome will not only provide a better understanding of the molecular mechanisms about the response to osmotic stress of the oysters, but also facilitate research into biological processes to find underlying physiological adaptations to hypoosmotic shock for marine invertebrates.Through more than 1.5 million sequence reads and the expression data of the two libraries, the study provided some useful insights into signal transduction pathways in oysters and offered a number of candidate genes as potential markers of tolerance to hypoosmotic stress for oysters. In addition, the characterization of  The marine environment is changing rapidly around the world due to global warming Crassostrea gigas is a dominant species in many intertidal locations as well as an important aquacultured bivalve species. Summer mortality of the Pacific oyster attracted extensive attention C. gigas grows in optimal salinity ranged from 20‰ to 25‰, whereas they can occur at salinities below 10‰ and will survive salinities in excess of 35‰C. gigas and contribute to the hypoosmotic adaption The Pacific oyster Recently, the next-generation sequencing technologies make large-scale sequencing possible by high-throughput and cost-efficiency In the present study, we examined the whole transcriptome responses to low salinity stress of the Pacific oyster for the first time using the Illumina's sequencing technology. Considering individual monitoring of the oyster responses to salinity stress, two libraries were established from the gills of oysters that exposed to optimal and low salinity seawater, respectively. The study aimed to compare the expression patterns of the two libraries to better understand the transcriptomic regulation in oyster to low salinity stress and identify genes involved in osmoregulation of the Pacific oyster. The results of this study are an important resource for future researches on mechanism of tolerance to hypoosmotic stress for marine invertebrates.To have access to the transcriptome of the oyster and obtain the quantitative and qualitative gene expression database for hypoosmotic stress, two libraries were established from the control group (PC library) and the treated group (PT library). Characteristics of the two libraries are summarized in Aplysia californica (GenBank Accession: AASC00000000) . We acquired 45,771 putative proteins that can be annotated matching biological function. In addition, sequences without annotations may represent poorly conserved region  in C. gigas.The contigs were annotated taking into consideration the identity of the translation frame, and 281,800 contigs were predicted to mapping to protein-coding sequences by GetORF of EMBOSS firstly. Then, the putative protein-coding sequences used Blastp searching on the Swiss-Prot database, the NCBI non-redundant (Nr) protein database and the genome of Secondly, Gene Ontology (GO) analysis was carried out, which conducted on the putative proteins blasting the Swiss-Prot and its supplement TrEMBL database. Of 45,771 putative proteins, 16,291 unique sequences were assigned with one or more GO terms, and 113,677 GO assignments were finally obtained. All GO assignments fell into broad categories for all three major GO functional domains  as presented in versus PT library, a list of transcripts has been established that included different expressions to responses to salinity stress (Table S1 and Table S2). Osmoregulation is implicated by the up-regulation of the potassium channel tetramerisation domain containing 1 (KCTD1), NFX1-type zinc finger-containing protein 1 and Cysteine-rich secretory protein Mr30, and the down-regulation of the ion and amino acid transporter genes, such as transient receptor potential cation channel subfamily M member 8 (LTrpC-8), neutral and basic amino acid transport protein rBAT and Na/Pi-cotransporter. In addition, signaling and interaction molecules such as cAMP-responsive element-binding protein-like 2, LRIG3 were evidenced as well as recognition molecules such as lectins. Besides, stress proteins such as heat shock proteins were found. Immune response is characterized by genes related to signaling pathways notably involved in innate immunity, which includes the up-regulated genes such as C-type lectin, thioester-containing protein and C1q domain containing proteins. In addition, the genes related to antimicrobial immunity are down-regulated including 2′–5′ oligoadenylate synthetase 1 and zinc proteinase. Apoptosis is noticed with not only regulating anti-apoptotic genes such as the up-regulation of Baculoviral IAP repeat-containing protein, but also apoptosis triggers such as the down-regulation of caspases, programmed cell death 1 ligand 1 (PD-L1). Cell adhesion and communication is mainly in down-regulated genes such as collagen and integrins that related to cell membrane molecules. Cytoskeleton is organized by the up-regulated expression of actin, tubulin and ankyrins. In addition, genes coding for receptors associated to cytoskeleton network are also up-regulated. Cell cycle is dominated by up-regulated genes related to promote cellular proliferation like protein fosB and MYC homolog. Besides, MEGF 10 is a negative regulator of cell cycle and down-regulated. Interestingly, 2+-binding proteinsCa were highlighted, owing to their significantly down-regulated expression. Ca2+-binding proteins included protocadherin, calmodulin, copine family proteins and calcium-binding EGF domain protein that combine with Ca2+ for the control of signaling processes, defense responses and other biological processes.We focused on biological functions that may characterize the responses to hypoosmotic stress. Comparing their occurrences in PC C. gigas. It is noteworthy that C. gigas, as a marine shelled mollusc, keeps up the osmotic disbalance with the environment by means of temporary inhibition of the water-salt exchange to seal mantle cavity In this study, we compared the Pacific oyster transcriptomes of two groups, one in optimal salinity seawater (25‰), and the other in low salinity seawater (8‰). Our data show significant changes of up- or down-regulated genes between two groups, which indicate the putative molecular mechanism of adaption to hypoosmotic shock in Functional annotation of genes differentially represented between PT and PC libraries has revealed biological processes that may characterize the responses to salinity stress. From the data, we found enrichment for genes related to signal transduction, cell adhesion and communication, ion channels and so on, implicated in the regulation of hypoosmotic stress response. Moreover, cellular processes related to immune response, cell cycle and differentiation, cytoskeleton rearrangements, as well as anti-apoptotic processes, were all highly represented.C. gigas, regulates the intracellular concentration of solutes to adapt to the surrounding conditions + (Kv) channels are homologous. With four Kv channel subunits aggregating to create a transmembrane ion conduction pathway, KCTDs had been anticipated to bind to and regulate Kv channels C. gigas, which mediates the permeation for cations such as sodium, potassium, calcium Similar to most marine molluscs C. gigasC. gigas. In our results, C-type lectin, thioester-containing protein and C1q domain containing proteins were all up-regulated, demonstrating their involvement in innate immunity. Lectins are a family of carbohydrate-recognition proteins that play crucial self- and non-self-recognition roles in innate immunity and can be found in soluble or membrane-associated forms Litopenaeus vannamei, mRNA of zinc proteinase is more expression in WSSV resistant individuals, which proved it played a role in immune responses Besides genes participating in ion and amino acid channels, genes related to immune responses were also found. Stress-induced immune changes have been elucidated in many marine invertebrates 2+-binding proteins that were all down-regulated significantly such as protocadherin, calmodulin, copine family proteins and calcium-binding EGF domain protein. These calcium-binding genes are involved in the maintenance of calcium homeostasis 2+ levels and they take part in the calcium-binding signal transduction 2+ levels have been taken into account as immune-related parameters 2+, which acts as a second messenger of signal transduction 2+ concentration is higher with exposure to live bacteria 2+ dropped with the decrease in Ca2+ concentration in the external environment in C. gigas2+ and no artificial live bacteria stimulus, Ca2+-binding signaling pathways and proteins tended to be down-regulated.Our study highlighted CaC. gigas.Apoptosis, programmed cell death, also called cell suicide, was also evidenced in our data with significant difference. Among them, Baculoviral IAP repeat-containing protein, an apoptosis suppressor, inhibits the caspase activity directly C. gigas.The data presented here reveal that genes involved in cell adhesion and communication, signal receptors, and cytoskeleton may play an important role in salinity shock. Cell adhesion is intimately related to the processes of cell motility and cell migration Using the next-generation sequencing technologies, the present study demonstrated the value of whole transcriptome analysis generated by RNAseq for accurate quantification of gene expression. Through more than 1.5 million sequence reads obtained, we established transcriptome data set for oysters subjected to hypoosmotic stress for the first time, and the data we generated could enrich on genomic resources of this non-model organism. Moreover, we provided a catalog of genes and cellular functions that are potential targets for mechanisms of adaption to low salinity by quantifying gene expression. The results provide not only a better understanding of the molecular mechanisms that control or contribute to the responses to osmotic stress of the oysters, but also an initial look at the cascade of gene expression patterns of marine organisms that occur in response to osmotic stress.C. gigas were purchased from an oyster farm in Weihai, Shandong Province, China, in 2010, and acclimatized over a week in 25‰ filtered seawater at 20°C. The Pacific oyster is not the endangered or protected species, and no specific permits were required for our studies. Twenty-four oysters were individually tagged and divided into two groups in separate tanks. One group was exposed in the optimal salinity filtered seawater (25‰), as the control group (PC); another group was exposed in the simulated conditions of increased fresh water input (8‰), as the treated group (PT). A part of the shell edge (about 10 mm long and 5 mm wide) of each specimen was chipped away in order to ensure the free exchange of seawater between the inside and outside of the shell. Each group was distributed in two tanks, and disposed for 8 hours simultaneously. After that, six oysters of each group were chosen randomly to be sacrificed, and their gill tissues were dissected and saved in RNA store (Dongsheng Biotech.).Adult individuals of The gills from 6 specimens saved in RNA store of each group were balanced mixed. Each sample was lysed in 1 ml of TRIzol reagent (Invitrogen) for total RNA extraction according to the manufacturer's instructions. Total RNA concentration was measured on a NanoDrop (Thermo Fisher Scientific) and RNA integrity cDNA samples were prepared following the protocol described in The synthetic cDNA was mode to 300–500 bp by a UTR200 sonication device (Hielscher Ultrasonics GmbH), and was selected using AMpure beads (Agencourt). This was followed by amplifying with TruSeq PE cluster kit v3-cBot-HS (Illumina) and constructing libraries with TruSeq™ DNA sample prep kit-set A (Illumina) according to the manufacturer's instructions. In the end, the cDNA library was sequenced on the Illumina sequencing platform (HiSeq 2000) from both ends of each fragment.Raw data generated from Illumina sequencing were preprocessed to remove nonsense sequences and were assembled as contigs by Velvet http://www.genome.jp/kegg/kaas/) using bi-directional best-hit method To annotate the assembled sequences with GO terms, which have three main categories , the Swiss-Prot and its supplement TrEMBL BLAST results were imported into GoPipe Firstly, we counted the number of reads from two samples that can be mapped to the assembly contigs respectively and transformed into RPKM (Reads Per Kilo bases per Million reads) Table S1List of functional groups and related gill up-regulated genes from oysters in response to hypoosmotic stress versus in optimal salinity conditions.(XLSX)Click here for additional data file.Table S2List of functional groups and related gill down-regulated genes from oysters in response to hypoosmotic stress versus in optimal salinity conditions.(XLSX)Click here for additional data file.


Recent advances in the field of neuromyelitis optica (NMO) research provided convincing evidence that anti-AQP4 antibody (AQP4-Ab) not only serves as a highly specific disease marker, but also plays an essential role in the pathogenesis of the disease. Although it is now widely recognized that AQP4-Ab induces astrocytic necrosis in a complement-dependent manner, additional triggers are also suspected as a prerequisite for the development of the disease. Unraveling these unresolved aspects of the disease will provide substantial insight into still controversial issues in the pathogenesis of NMO. During the past several years there has been a huge progress in our understanding of the pathogenesis of neuromyelitis optica (NMO). The discovery of a disease-specific autoantibody, anti-aquaporin-4 antibody (AQP4-Ab), in the sera of patients with NMO has attracted enormous attention of researchers within the field , 2. Amonin vitro and in vivo studies. It is reported from several groups that AQP4-Ab-positive sera induce necrosis of astrocytes in a complement-dependent manner in vitro [in vitro and in vivo and plays a pivotal role in the pathogenesis of NMO.Since the identification of a highly disease-specific autoantibody, AQP4-Ab, in the sera of patients with NMO, several clinical observations suggested the pathogenicity of AQP4-Ab , 2. It hin vitro –16. Anotin vitro . Most imin vitro –5. The ain vitro –5. The sin vitro , 4. Thesin vivo [in vivo. When recipient rats were pretreated with CVF, the loss of GFAP-positive cells in the spinal cords was much less observed. In vitro studies also showed that AQP4-Ab-positive sera are capable of inducing astrocytic death only in the presence of complement [Apart from the remarkable loss of astrocytes in NMO, the active lesions are also characterized by the deposition of activated complement and immunoglobulins . The majin vivo . Similarmplement , 15. Furmplement . When ramplement . These omplement .In vivo studies also suggest that pathological changes are not reproducible even after transferring patients' immunoglobulins to juvenile naïve rats where the integrity of BBB is still fragile. Therefore, the access of AQP4-Ab beyond the BBB is presumably not sufficient to induce the astrocytic cytotoxicity [Although the passive transfer models of NMO confirmed the pathogenicity of AQP4-Ab on astrocytes, it still remains unclear whether AQP4-Ab is a disease-modifying factor or a primary initiator of the disease . Since Atoxicity . This ditoxicity , 27. Sintoxicity . Taken tIf the leakage of BBB is not sufficient for AQP4-Ab to become fully pathogenic, then what other factors are necessary? We have recently shown that pretreatment of recipient rats with complete Freund's adjuvant (CFA) alone is sufficient for the AQP4-Ab transferred in the periphery to induce astrocytic cytotoxicity in the spinal cords . CFA is Although we have learned through our animal model that nonspecific inflammation is sufficient to induce the pathogenic effect of AQP4-Ab, it is noteworthy that the lesions observed in CFA model were much less extensive compared to those of recipient rats preimmunized with MBP (EAE model) , 20. OnlThe prototypical presentation of NMO is characterized by preferential involvement of central gray matter in the spinal cord and brain stems, where AQP4 is known to be expressed at high level . Recent In vitro studies did not confirm the pathogenic effect of activated granulocytes on astrocytes even after they showed degranulation [in vivo should elucidate whether granulocytes are actively involved in the formation of pathology in NMO.In spite of the huge progress in the mechanism of AQP4-Ab-related pathology, much less is investigated as to how granulocytes recruited in the active lesions of NMO are related to the disease . The granulation . More deex vivo spinal cord slice model [Recent progress of animal studies has provided substantial insight into our knowledge of NMO pathogenesis. Nonpathogenic recombinant monoclonal anti-AQP4 Ab, called “aquaporumab,” was recently shown to dramatically reduce NMO-like lesions in an ce model . Althougce model . The corce model , 48. We