Due to the complexity and chronicity of heart failure, engaging yet simple patient self-management tools are needed.This study aimed to assess the feasibility and patient engagement with a smartphone app designed for heart failure.Patients with heart failure were randomized to intervention  or control  groups. All intervention group patients were interviewed and monitored closely for app feasibility while receiving standard of care heart failure management by cardiologists. The Atlanta Heart Failure Knowledge Test, a quality of life survey (Kansas City Cardiomyopathy Questionnaire), and weight were assessed at baseline and final visits.P=.04) and quality of life . The correlation between app use and weight change was ρ=–0.40 (P=.19). Only 1 out of 11 patients in the control group retained education material by the follow-up visit.Patients  with heart failure  were enrolled, and 82% of patients  completed both baseline and final visits (median follow up 60 days). In the intervention group, 2 out of the 12 patients who completed the study did not use the app after study onboarding due to illnesses and hospitalizations. Of the remaining 10 patients who used the app, 5 patients logged ≥1 interaction with the app per day on average, and 2 patients logged an interaction with the app every other day on average. The intervention group averaged 403 screen views (per patient) in 56 distinct sessions, 5-minute session durations, and 22 weight entries per patient. There was a direct correlation between duration of app use and improvement in heart failure knowledge (Atlanta Heart Failure Knowledge Test score; ρ=0.59, The Habits Heart App with a Bluetooth-linked scale is a feasible way to engage patients in heart failure management, and barriers to app engagement were identified. A larger multicenter study may be warranted to evaluate the effectiveness of the app.ClinicalTrials.gov NCT03238729; http://clinicaltrials.gov/ct2/show/NCT03238729 The COVID-19 pandemic has disrupted chronic disease management -4. SmartIn this context, we employed a heart failure–specific smartphone-based app intervention with content designed by clinicians with the following features: daily to-do lists for heart failure–related activities including patient education, symptom monitoring, wireless weight tracking, sodium intake tracking, exercise logs, and action prompts based on weight and symptom severity. There was also a messaging platform for patients to reach out to study staff and physicians. Our goal was to test feasibility and usability in this pilot randomized proof-of-concept study.All study procedures were approved by the Partners Healthcare Institutional Review Board and carried out in accordance with the Declaration of Helsinki, and the study was registered (NCT03238729). The Habits Heart App was developed in conjunction with cardiologists who care for patients with heart failure at Massachusetts General Hospital and software engineers at Jana Care. Cardiologists designed the main concepts  and content in writing and worked with Jana Care to develop the app. While the main goal of Track was to monitor weight changes, tracking sodium content (in diet) and exercising logging were also included, in an exploratory fashion, using a publicly available database of exercises and a database from University of Minnesota of the sodium content in food items. The Habits Heart App was downloaded onto a study Android smartphone.Heart failure with reduced ejection fraction was defined as symptomatic heart failure with a left ventricular ejection fraction ≤40%, and heart failure with preserved ejection fraction was defined as symptomatic heart failure with left ventricular ejection fraction ≥50% by any imaging modality. A randomized list with the order of intervention or control group allocations corresponding to the order of enrollment was generated at the start of the study. After informed consent was obtained, patients were then allocated to either the intervention group or the control group , the Atlanta Heart Failure Knowledge Test (AHFKT-V2)  and a quSpearman correlations were used to determine the relationships between the average session duration of app use and the AHFKT-V2 score, the KCCQ-12 score, and weight loss.A total of 28 patients were enrolled in the study . Of the patients enrolled, 54% (15/28) had heart failure with reduced ejection fraction, 71% (20/28) were male, and the median age was 63 years. The median follow-up was 60 days (range 50-66 days); 80% of patients (12/15) in the intervention and 85% of patients (11/13) in the control group completed both baseline and final visits . In the All patients in the intervention group  were able to demonstrate their competency using the app by performing actions in 5 key areas at the end of the 30-minute onboarding session; however, 2 out of the 12 patients (17%) who completed both study visits were unable to use the app after study onboarding due to intermittent illnesses and hospitalizations. Of the 10 patients who were able to use the app, 7 patients (70%) logged ≥1 interaction with the app every other day on average throughout the study duration, and 5 patients (50%) logged ≥1 interaction with the app per day on average throughout the study duration. At the final visit, 9 out of 12 patients (75%) in the intervention group subjectively reported regularly interacting with the app .There was an average of 403 screen views per person over 56 distinct sessions . The patients who reported using the app regularly (n=9) averaged 527 screen views per person over 73 distinct sessions .As of the 6-week follow-up visit, 0 out of 12 patients had problems opening and watching education sessions on Learn. Only 1 out of 12 patients (8%) reported synching issues between the scale and the app, 1 out of 12 patients (8%) reported not being able to send additional messages on the app messaging platform for several minutes after sending the first message, and 2 out of 12 patients (17%) had problems with the smartphone intermittently disconnecting from the internet. There were 28 app crashes among the entire group. Only 1 out of 11 patients (9%) in the control group retained reading materials by the follow-up visit.Patients rated the app (average 3.8 out of 5) and described enjoying the app’s educational lessons and videos most, followed by the weight recording feature, and the messaging platform. Patients reported that sometimes they could not find the exact food that they had consumed in the diet database, so they input the next-best option. Throughout the study, 48 symptoms were logged by app users, of which 25 triggered a prompt to contact the study team, and 4 required that patients call their doctors or call 911. In total, 11 messages were sent by app users to the study coordinator through the messaging platform regarding symptoms and trouble-shooting of the app.P=.04) and KCCQ-12  scores from baseline to follow-up (P=.19).The more a patient interacted with the app (higher the average session duration), the greater the improvement in AHFKT-V2  bodyweight during the study while patients in the app arm lost 3.8 lbs (1.7 kg) bodyweight.In this pilot proof-of-concept study in a sample of patients with heart failure, we found that the Habits Heart App with a Bluetooth-linked scale was feasible and easily implemented. All of the patients in the intervention group were able to demonstrate that they could use the 5 key areas of the app on their own after a 30-minute onboarding session, and almost all of the patients were able to use the app without major stability, performance, or standards adherence issues. The Habits Heart App with scale is unique in that it was designed by clinicians who care for patients with heart failure to address a patient’s complex self-management needs for patient education, tracking, and coaching without requiring resource-intensive telemonitoring. While intensive monitoring may help to decrease adverse events in patients with heart failure, telemonitoring can require a lot of resources and be financially taxing . ResourcCompared with a previously published study , our conWhile there are several patient-centered heart failure apps available, few have been designed by clinicians who care for patients with heart failure or have been evaluated with a scientific assessment or publication in a medical journal to date ,9,15-17.There are several areas identified in the feasibility assessment to address for the future iteration of the app including Bluetooth connectivity with the app, messenger delays, and sodium and exercise logging issues. Enabling app download onto patients’ own smartphones, whether iPhone or Android, will solve the internet connectivity issue and decrease the inconvenience of carrying 2 smartphones. An ability to sync summary data with electronic health records may further reduce administrative burden. Addition of mobile laboratory capabilities to check B-type natriuretic peptides, N-terminal pro–B-type natriuretic peptides, and basic metabolic panel may expand the utility of the app.Despite randomization, there were differences in patient characteristics between the app and control groups, likely due to the small sample size of this feasibility study. The control group appeared to have more advanced heart failure , while the intervention group had a higher burden of comorbidities such as atrial fibrillation, coronary artery disease, chronic obstructive pulmonary disease, and chronic kidney disease. The differences between the two groups may potentially result in more improvement in the app group compared with that in the control group; patients with less advanced heart failure may score better. However, in order to minimize the potential impact of baseline differences in the two groups, we looked at the relative change in heart failure knowledge, quality of life, and weight loss rather than the final or absolute values. Nonetheless, any definitive conclusions about the app’s effects on quality of life or heart failure knowledge should be evaluated with a large multicenter trial.The Habits Heart App is a feasible way to dynamically engage patients in heart failure management and equip patients with effective self-management tools to potentially improve heart failure knowledge, quality of life, and adherence to medical recommendations. A larger multicenter trial is needed to further test the use of the app and clinical outcomes.


This randomized clinical trial evaluates the effectiveness of spinal manipulation and spinal mobilization compared with a placebo at reducing pain and disability among young adults with mild to moderate chronic low back pain. What is the comparative effectiveness of spinal manipulation compared with spinal mobilization relative to a placebo control in reducing pain and disability in chronic low back pain?In this randomized clinical trial that included 162 young adults, there was no difference in reduction of pain and disability when comparing spinal manipulation to spinal mobilization relative to the placebo control treatment.Neither spinal manipulation nor mobilization appeared to be an effective intervention for young adults with mild to moderate chronic low back pain. Low back pain (LBP) is one of the most common reasons for seeking medical care. Manual therapy is a common treatment of LBP, yet few studies have directly compared the effectiveness of thrust  vs nonthrust  techniques.To evaluate the comparative effectiveness of spinal manipulation and spinal mobilization at reducing pain and disability compared with a placebo control group (sham cold laser) in a cohort of young adults with chronic LBP.This single-blinded (investigator-blinded), placebo-controlled randomized clinical trial with 3 treatment groups was conducted at the Ohio Musculoskeletal and Neurological Institute at Ohio University from June 1, 2013, to August 31, 2017. Of 4903 adult patients assessed for eligibility, 4741 did not meet inclusion criteria, and 162 patients with chronic LBP qualified for randomization to 1 of 3 treatment groups. Recruitment began on June 1, 2013, and the primary completion date was August 31, 2017. Data were analyzed from September 1, 2017, to January 20, 2020.Participants received 6 treatment sessions of (1) spinal manipulation, (2) spinal mobilization, or (3) sham cold laser therapy (placebo) during a 3-week period.Coprimary outcome measures were the change from baseline in Numerical Pain Rating Scale (NPRS) score over the last 7 days and the change in disability assessed with the Roland-Morris Disability Questionnaire  48 to 72 hours after completion of the 6 treatments.P = .45), spinal manipulation and placebo , or spinal mobilization and placebo . There was no significant difference in change in self-reported disability scores between spinal manipulation and spinal mobilization , spinal manipulation and placebo  or spinal mobilization and placebo .A total of 162 participants  with chronic LBP  were randomized. Fifty-four participants were randomized to the spinal manipulation group, 54 to the spinal mobilization group, and 54 to the placebo group. There were no significant group differences for sex, age, body mass index, duration of LBP symptoms, depression, fear avoidance, current pain, average pain over the last 7 days, and self-reported disability. At the primary end point, there was no significant difference in change in pain scores between spinal manipulation and spinal mobilization (0.24 ; In this randomized clinical trial, neither spinal manipulation nor spinal mobilization appeared to be effective treatments for mild to moderate chronic LBP.NCT01854892ClinicalTrials.gov Identifier:  Manipulative therapies are a common conservative treatment approach for low back pain, with more than 18 million American adults receiving manipulative therapies at a total annual out-of-pocket cost of $3.9 billion.4Low back pain is one of the most common reasons for seeking care from a physician, with total costs exceeding $100 billion annually in the US alone.5 In contrast, mobilization-based techniques use a low-velocity, low-force approach that generally does not produce audible joint sounds. Although the clinical effectiveness of manipulative therapy techniques for acute and subacute low back pain has been reported,8 the evidence on the effectiveness for reducing pain in patients with chronic low back pain is mixed.11 Reviews have generally found that manipulation is more effective compared with standard of care,6 but few studies have compared the clinical effectiveness of these 2 manipulative therapies,17 and existing studies have yielded discrepant results. Some studies indicate that manipulation-based techniques are more effective for low back pain compared with nonthrust techniques16; however, other studies report no significant difference in clinical outcomes between spinal manipulation and mobilization.17 Finally, most studies on manipulative therapies have used standard of care or waiting list controls as the comparison group.6 The current trial sought to use stronger control given the prevalence of both techniques in clinical practice and that understanding their relative effectiveness is an important and unresolved issue.Spinal manipulative treatments can be broadly classified as manipulation-based or mobilization-based techniques. Manipulation-based techniques apply a high-velocity, low-amplitude force to the spine and are often accompanied by an audible sound from 1 or more joints.Accordingly, the RELIEF (Researching the Effectiveness of Lumbar Interventions for Enhancing Function) Study was conducted as a phase II randomized clinical trial to compare the effectiveness of spinal manipulation, spinal mobilization, and placebo (sham laser) in the treatment of pain and disability among young adults with chronic low back pain.18 and registered on ClinicalTrials.gov (NCT01854892). This study was conducted according to the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline. Recruitment began on June 1, 2013, and the primary completion date was August 31, 2017. Participants were randomized in a 1:1:1 ratio into 1 of 3 groups , placebo-controlled, 3-arm, parallel-groups trial with repeated measurements. The Ohio University institutional review board approved this study, and written informed consent was obtained from all participants. The detailed study protocol  is publi3 groups .Group 1 received spinal manipulation treatment 2 times per week for 3 weeks . Group 2 received spinal mobilization treatment 2 times per week for 3 weeks. The placebo group received sham cold laser treatment 2 times per week for 3 weeks. Randomization was stratified by sex, using a permuted block design, for the purpose of maintaining balance across treatment groups. This-computer generated randomization table was maintained by the study coordinator. All outcome measures were obtained by study staff blinded to the assignment of treatment group.19 In the original clinical prediction rule, an individual had to meet 4 of 5 characteristics .19 As this trial sought to examine spinal manipulative therapies in a cohort with chronic back pain, none of the participants had symptom duration of less than 16 days. Interventions allowed during the trial were over-the-counter pain relief medications  and application of heat or ice packs. However, narcotics, muscle relaxants, manipulation, mobilization, acupuncture, or massage treatments were prohibited.Patients with chronic low back pain (n = 162) were randomly assigned to the following treatment groups (54 patients per group): (1) spinal manipulation, (2) spinal mobilization, or (3) placebo. The Box. Between 18 and 45 years of ageAnswer yes to the following questions:Have you had low back pain constantly or on most days for the last 3 months?Has your back pain caused you to seek care or consultation from a health care provider?Average pain intensity assessed using the Numerical Pain Rating Scale over the past week ≥2 on a 0-10 numerical pain scaleRoland-Morris Disability Questionnaire score ≥4Exhibit at least 3 of the following findings reported in the clinical prediction rules for spinal manipulation:No symptoms distal to the kneeFear Avoidance Beliefs Questionnaire work subscale score <19At least 1 hypomobile lumbar spinal segmentAt least 1 hip with >35 degrees internal rotation range of motionHave a personal history of the following neurological disorders: Alzheimer disease, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson disease, or strokeHave a history of the congestive heart failure or heart attack in past 24 months2Have a body mass index greater than 35 kg/mHave used narcotics or muscle relaxants within 30 days prior to study enrollmentHave clinical depression Report having pending litigation related to an episode of low back pain or are receiving any type of disability services related to low back painHave active cancerAre blindReport being pregnant, lactating, or that they anticipate becoming pregnant in the next 3 monthsCurrent drug or alcohol use or dependence that, in the opinion of the primary investigators, would interfere with adherence to study requirements.We used print and media advertisements, including mass mailing campaigns, to recruit a broad set of individuals with low back pain. This recruitment resulted in 4903 people completing a brief online survey to determine initial eligibility. In general, the vast majority of these potential participants were excluded owing to (1) insufficient pain levels, (2) insufficient duration of symptoms, (3) receiving prohibited interventions, or (4) age outside of the criterion. Individuals who met initial study criteria (n = 182) were then scheduled for an in-person physical screening prior to assignment to a treatment group. Our inclusion and exclusion criteria were designed to optimize the potential for observing an effect of manipulative intervention. Accordingly, a relatively homogenous group of individuals with chronic low back pain who were young and without obesity was enrolled . We postulate that these criteria minimized the confounding effects of aging and obesity and likely the comorbidities associated with these factors. Careful investigator bias control measures included the following: (1) study personnel conducting the outcome assessments were blind to group assignment throughout the length of the study, (2) clinicians were not aware that the cold laser was a sham intervention, and (3) the statistician and principal investigators remained blind to group assignment until data collection and analyses were completed. While participants could not be blinded to the assigned treatment group, they were unaware that the cold laser was inert .20 The credibility and expectancy instrument consists of 7 questions with a 9-point Likert scale to assess how confident the participant is that the treatment they have received has the potential to reduce their symptoms. It is often used to assess the strength of the placebo treatment group in randomized trials. Three (±1) days after the final treatment, participants reported back to the laboratory for a follow-up assessment of all clinical measures . Participants returned approximately 4 weeks after their last treatment for a follow-up assessment of the clinical outcome measures.Eligible participants were enrolled into the study and randomized to an intervention group, baseline clinical measures were obtained, a treatment intervention was performed, and then the clinical measures were repeated . Baseline measures to characterize the sample are provided in the The participant was placed in a side-lying position facing the clinician with the more painful side facing upward. The clinician passively flexed the participant’s hips and knees to induce lumbar spine flexion until they felt the spinous process of the affected lumbar vertebrae begin to move. Next, the clinician passively rotated the participant’s torso opposite to the side they were lying on until they felt rotation in the vertebra above the suspected lesion. The clinician applied a rapid thrust to the shoulder (anterior to posterior force) and pelvis (posterior to anterior force) resulting in a rotation force couple on the hypomobile segment. If a cavitation  occurred, the treatment was considered complete. If no cavitation was produced, the participant was repositioned and the manipulation was attempted again. A maximum of 2 attempts per side was permitted. If no cavitation was produced after the 4 attempts , the treatment was considered complete.The participant was positioned using the same technique as described above. The clinician then placed 1 hand on the anterior shoulder and 1 hand on the posterior pelvis. The participant was asked to try to gently push the shoulder and pelvis into the clinician’s hands. Thus, they performed an isometric rotation of the torso resulting in a rotation force couple on the hypomobile segment. The clinician had the participant hold the contraction for a minimum of 7 seconds and a maximum of 60 seconds if the patient could comfortably tolerate the stretch. If a cavitation occurred or the clinician assessed a release in tissue tension, the treatment was considered complete. Otherwise, the clinician repositioned the patient as needed and repeated the treatment for up to 10 minutes.The laser manufacturer  provided a MedX 1100 system that did not deliver any significant amount of light energy or heat but otherwise appeared operational to participants and clinicians. The sham laser was delivered over the painful region with the study participant positioned as described for spinal manipulation and spinal mobilization treatments.Licensed clinicians , with at least 3 years of clinical experience using manipulative therapies provided all treatments. A yearly workshop organized by the study team harmonized treatment interventions to ensure fidelity to the interventions described above. Successful adherence was defined as a study participant receiving at least 5 of the 6 treatments (83%). At each visit, all negative changes in health status were recorded as adverse events with attribution and grading determined based on the National Cancer Institute’s Common Terminology Criteria for Adverse Events.18 In brief, based on extant studies of spinal manipulation, we used a Cohen d effect size of 0.5 for simulation studies that indicated 54 subjects per treatment group would be sufficient to achieve power of 0.8 to detect a clinically meaningful treatment effect for coprimary outcomes  at our primary end point. Sample size calculations also assumed a 10% attrition rate.Sample size calculations for this study have been previously described.21 and a 3-point reduction in Roland-Morris Disability Questionnaire score22 were considered clinically meaningful important differences. P < .05 was used to indicate statistical significance.The primary hypotheses were that (1) the spinal manipulation and the spinal mobilization groups would display a significantly larger reduction in NPRS level from baseline than the placebo at the primary end point and (2) the spinal manipulation and the spinal mobilization groups would display a significantly larger reduction in Roland-Morris Disability Questionnaire score than the placebo at the primary end point. One-tailed tests were used to assess these directional hypotheses. A 2-point reduction in NPRS scoreData were examined to check for the following key assumptions: (1) normal distribution of random intercepts and slopes as well as residuals and (2) homogeneity of residual variances across the 3 treatment groups. Linear mixed-effects regression analyses served as the main analytic framework. This regression technique incorporates miscellaneous factors  as random effects so that the variances due to those factors can be partitioned. Furthermore, the maximum-likelihood estimation allows participants with missing data to be included in the model if the observations were missing completely at random or missing at random.All regression models incorporated random intercept terms to accommodate the within-subject variance associated with longitudinal experimental designs and fixed-factor parameters for the main effects of group (sham-laser control as reference), main effect for time (baseline as reference), and the simple interaction terms of group by time (relative to control and baseline). Biological sex was included in each of the models. Additional a priori contrasts comparing changes from baseline between the 2 intervention groups  were also performed, with Holm alpha adjustments to preserve the familywise error rates to 0.05.All randomized study participants with at least a baseline assessment were included in the intention-to-treat (ITT) analysis. This trial enrolled and randomized participants following an in-person physical examination. Baseline measures were then completed in a follow-up visit within 7 days of the physical screening. Four of the randomized participants dropped out before baseline measures. Accordingly, our primary analysis does not meet the strict definition for ITT analysis. Thus, we used a near ITT analysis as the primary method for assessing outcomes, and maximum-likelihood estimation was used to estimate any missing observations.For the per-protocol analysis (PPA), we excluded study participants who (1) attended fewer than 5 treatment sessions and (2) received prohibited concomitant interventions or (3) developed an exclusionary medical condition while on the study protocol. The outcomes from the ITT analysis and PPA were nearly identical . Accordingly, only the results of the ITT analysis are presented.A total of 162 participants (92 women [57%] and 70 men [43%]) with a mean (SD) age of 25.0 (6.2) years  were enrolled in this trial. At baseline, this cohort reported a mean (SD) pain score over the last 7 days of 4.3 (2.5) on the NPRS , a mean (SD) score of 9.9 (4.5) on the Roland-Morris Disability Questionnaire , and mean (SD) length of chronic pain of 6.2 (5.1) years, indicating mild to moderate back pain of long duration. Fifty-four participants were randomized to the spinal manipulation group (31 women [57%] and 23 men [43%]), 54 participants to the spinal mobilization group (31 women [57%] and 23 men [43%]), and 54 participants to the placebo group (30 women [56%] and 24 men [44%]). Four participants were withdrawn from the study after the randomization process owing to scheduling issues  and never completed baseline measures or any part of the treatments and follow-up assessments. An additional 5 individuals did not complete the allocated intervention and follow-up visits. Participant characteristics as a function of treatment group are reported in P = .45), spinal manipulation and placebo , or spinal mobilization and placebo  (P = .14), spinal manipulation and placebo , or spinal mobilization and placebo   A. There P = .17) B. There F2,151 = 1.70, P = .19), indicating that, on average, participants in each group had similar expectations regarding the likely benefit of their assigned treatment. Next, we examined the relationship between these individual treatment expectancy ratings , indicating that those who had higher expectations of treatment success reported larger decreases in pain with treatment. When these expectancy effects were examined within each treatment group, significant inverse correlations were observed for placebo  and spinal manipulation  but not spinal mobilization . The changes in Roland-Morris Disability Questionnaire ratings  were not significantly related to treatment expectancy.A comparison of treatment credibility and expectancy ratings across groups was not statistically significant , this hypothesis was not supported by the data.8; however, the evidence on the effectiveness for reducing pain in chronic low back pain sufferers is mixed.11 We developed a modified version of a clinical prediction rule to identify individuals with chronic low back pain most likely to respond to a manipulative therapy intervention. Specifically, we modified the clinical prediction rule by Childs and colleagues19 by removing 1 of the 5 criteria  for this study. It should be noted that there were no data to indicate that acuteness of symptom onset was more heavily weighted than the 4 other criteria . Even with a modified clinical prediction rule to identify a cohort of individuals with chronic back pain most likely to respond to manipulative therapies, our data indicate that spinal manipulation and spinal mobilization were not very effective in reducing pain and disability in this cohort. This finding is consistent with those of Dougherty and colleagues,23 who also found that a modified version of this rule was not effective in discriminating which patients with chronic low back pain are most likely to respond to spinal manipulation. It is important to note that findings were reported by Dougherty and colleagues more than a year after subject recruitment began for the RELIEF Study.The clinical effectiveness of manipulative therapy techniques for acute and subacute low back pain has been reported6 whereas the results are more mixed in chronic low back pain.11 Although the young age cohort in our study, which likely arose from our primary recruitment strategy being direct emails and online advertisements, is a limitation with respect to generalizability, a recent paper by Manogharan and colleagues24 suggests there are relatively small differences in baseline characteristics and prognosis among young, middle-aged, and older cohorts of patients with low back pain. In sum, these findings provide additional evidence about the limits of manipulative therapies in chronic low back pain.There is also the possibility that manipulative therapy techniques are not as effective for treating mild to moderate pain conditions. Across treatment groups, participants in this study reported a mean pain score of 4.2  when asked to rate their pain over the last 7 days. While our data show that manipulative therapy techniques were no more effective than an effective placebo treatment for mild to moderate chronic low back pain, it is possible that these techniques would be more effective for individuals with more severe pain. Alternatively, the finding that manipulative therapies were not very effective in a young cohort of patients with mild to moderate low back pain with few confounding factors may provide additional evidence that this approach is not ideal for chronic back pain. That possibility would be consistent with the literature that indicates manipulation is a reasonable, effective intervention for those with acute low back pain25 A review of low back pain trials by Machado and colleagues25 reported that 20% of trials had a placebo that was a potentially genuine treatment, 13% maintained adequacy of blinding, and most that assessed treatment expectations found that participants had lower expectations of placebo as compared to the active treatment groups.25 Another review reported that the quality of placebos typically used in studies on manipulative therapy is inadequate and that few studies controlled for or assessed treatment expectations.26 We used a validated treatment expectancy measure,20 and, consistent with Puhl et al,26 our data clearly showed that expectancy of treatment success was related to reductions in pain but not disability. Although there was no effect of treatment group on participant treatment expectancy ratings, the sham cold laser treatment group showed the strongest relationship between expectancy and pain reduction. Taken in total, these data suggest that sham cold laser is an effective placebo for spinal manipulation studies and that the power of participant expectations can be quite large. These findings reinforce the conclusions from Puhl et al26 that future trials should carefully select the control treatment, and the effectiveness of the control must be evaluated to provide confidence for assessing the effectiveness of manipulative therapy.Another consideration, not only in the RELIEF trial but in all studies on manipulative therapy, is the effectiveness of the placebo to create a true control condition. Whereas an effective placebo is critical to maintain blinding and reduce the risk of bias, poor placebos are quite common in low back pain studies.This study has limitations that should be noted. First, our participants were relatively young adults with only mild to moderate chronic low back pain with low levels of self-reported disability. Although this age cohort may not seek medical care for low back pain as often as middle-aged and older adults, it is worth noting the average duration of symptoms for this cohort was greater than 6 years. Thus, this is potentially an understudied cohort, and data on treatment effectiveness of manual therapies is needed. Second, the trial used a single-blinded design. However, this design is consistent with all studies of spinal manual therapies, as it is impossible to have a true double-blind design because the clinician will obviously know what treatment they are providing. We attempted to minimize any bias by having the data collection team assessing clinical outcomes blinded to treatment assignment. Another potential limitation is that the interventions were restricted to the isolated application of manipulation, mobilization, or placebo  using a classic randomized trial design. This approach was designed to reduce potential confounds by the application of concomitant interventions, such as stretching and strengthening exercises. Thus, a future study that uses a pragmatic clinical trial design approach could provide further understanding of the potential additive benefits of these manual therapies.Our findings indicated that spinal manipulation and spinal mobilization were no more effective than a well-chosen placebo in reducing pain and disability in patients with chronic low back pain. We conclude that these manipulative therapy techniques do not appear to be effective for chronic low back pain, at least among relatively young individuals with mild to moderate back pain.


Compared to Ly6Chi monocytes, less is known about Ly6Clow monocyte recruitment and mechanisms involved in the recruitment of this subset. Furthermore, the role of Ly6Clow monocytes during infections is largely unknown. Here, using intravital microscopy, we demonstrate that Ly6Clow monocytes are predominantly recruited to the brain vasculature following intravenous infection with Cryptococcus neoformans, a fungal pathogen causing meningoencephalitis. The recruitment depends primarily on the interaction of VCAM1 expressed on the brain endothelium with VLA4 expressed on Ly6Clow monocytes. Furthermore, TNFR signaling is essential for the recruitment through enhancing VLA4 expression on Ly6Clow monocytes. Interestingly, the recruited Ly6Clow monocytes internalized C. neoformans and carried the organism while crawling on and adhering to the luminal wall of brain vasculature and migrating to the brain parenchyma. Our study reveals a substantial recruitment of Ly6Clow monocytes to the brain and highlights important properties of this subset during infection.Monocytes exist in two major populations, termed Ly6C hi and Ly6Clow monocytes. In contrast to the recruitment of Ly6Chi monocytes shown in other infection models, we observed the predominant recruitment of Ly6Clow monocytes to the brain post-capillary venules during intravenous infection with C. neoformans, a fungal pathogen causing brain infection. The recruitment is mainly mediated by the interaction of VCAM1 and VLA4, which are expressed on the brain endothelium and monocytes, respectively. We further demonstrate that TNFR signaling plays an essential role during Ly6Clow monocyte recruitment through enhancing VLA4 expression on monocytes. We also observed that Ly6Clow monocytes internalize C. neoformans and, together with the ingested organism, crawl along the luminal wall of brain vasculatures and migrate to the brain parenchyma. Thus, VCAM1/VLA4 interaction mediates Ly6Clow monocyte recruitment to the brain in a TNFR signaling dependent manner during fungal infection.Monocytes are white blood cells, circulating in the bloodstream and playing important roles during infections. There are two subsets of monocytes in mice: Ly6C Noverr, Ph.D.Associate EditorPLOS PathogensAaron MitchellSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XGrant McFaddenEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-2556-3526***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: This intravital microscopy study, combined with analysis of CNS immune response responses focuses on the of Ly6Clow monocytes during the early stages of CNS disseminating fungal infection caused by C. neoformans (C.n). Study demonstrates rapid/early recruitment to the brain vasculature (within 12-24h) of monocytes, chielfly LY6Clow subset, following intravenous inoculation with a high-dose of C.n. H99. The recruitment depends primarily on the interaction of VCAM1 expressed on the brain endothelium with VLA4 expressed on Ly6Clow monocytes. TNFR signaling is essential for the recruitment through enhancing VLA4 expression on Ly6Clow monocytes. Some of these Ly6Clow monocytes contain internalized C. neoformans and carried the organism while crawling on and adhering to the luminal wall of brain vasculature and migrating to the brain parenchyma. This study highlights important involvement of this subset during infection. While focus on the Ly6Clow monocytes in the context of disseminated C.n., followed by CNS infection is novel and interesting and could potentially be an important advance, there are some significant concerns:First, the presented results are difficult to reconcile with previous studies showing that CCR2/CCL2 axis is involved in human patients with CNS C.n. infection  and CD11b+Ly6C+ myeloid cells are recruited in high quantities into the brain of mice with CNS C.n. infection . These studies have not been cited or discussed by the authors. However, these published data STRONGLY argue against proposed here "unique link" of Ly6Clow monocytes with cryptococcal brain infection, as opposed to other organs with C.n-infection or the CNS infections with other microbes. The more likely reason for predominance of Ly6Clow monocytes in this model is that the relatively early time points (1-3 days) are used prior to CCR2+ monocyte BM mobilization, and the known delay in recruitment of Ly6Chi/CCR2+ monocytes, typically past day 7 and day 14 in the C.n-infected lungs or CNS, respectively.Second, it is not clear what fraction of these intravascular monocytes actually crosses to the brain . Thus, at this point, it is hard to understand relative importance of these monocyte subsets in host defenses/pathogenesis of the C.n-induced CNS disease without any quantitative data in this respect.Reviewer #2: Overall, the studies were well executed and the findings/interpretations are supported by the data presented. Additionally, the article is well-written. The strengths of the study is that the authors findings were supported using multiple animal models and good visuals. Nonetheless, a weakness of the study is a concern that the infectious dose combined with the route of inoculation used in the study (20 x 10^6 i.v.) is so over-bearing on the immune response that the resulting observations are not relatable clinically. The overall consensus is that infection begins via intranasal inhalation and that the organisms may eventually disseminate to the CNS . This allows for the development of a much different type of immune response and possibly more time for monocytes to differentiate into various other cell types such as DCs. Thus, the findings presented in the manuscript may only be a reflection of the model system. This may need to be addressed to put the observations in context or some additional studies done using a much smaller infectious dose and/or route of infection.Reviewer #3: This is a study by Sun et al that defines the kinetics of accumulation of Ly6c low monocytes in the brain of Cryptococcus-infected mice and examines molecular factors that are associated with these cells' recruitment into the tissue. The authors utilize intravital microscopy to visualize some of these events and show that VCAM1/VLA4 and TNF receptor signaling is important for Ly6c low accumulation in the context of cryptococcal meningitis. This is a potentially interesting topic, with description of a cell type that has not been examined thoroughly during infections, and particularly during fungal infections. The authors carefully perform experiments to outline the molecules that may be critical for cell accumulation in the fungal-infected brain. The paper is well-written. However, the manuscript lacks 2-3 very important pieces of information to provide it with the significance in advancing the field as outlined below.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: The key experiment to address the first concern would be to monitor the vasculature of other organs (e.g. kidney and liver) in this C.n high-dose IV bolus disseminated infection model, to determine if similar microvasculature recruitment of Ly6Clow versus Ly6Chigh monocytes will be observed outside of the CNS in other organs during fungemia.It would be also worthwhile to induce an inhalation C.n infection 1-2 week prior to the IV-bolus infection to activate bone marrow to produce both subsets of monocytes first, and then follow with the IV infection/microscopy studies. Such model would be more consistent with natural history of cryptococcal infection, when the infection initially develops in the lungs, triggers some level of host response, but then disseminates into the CNS. This approach would allow to verify the relative contribution of each monocyte subsets in the CNS in a more "natural history disease setting" and allow for better comparisons with other studies .The second major concern regarding true monocyte recruitment to the brain could be addressed using short-term injection with fluorescent antibody or other monocyte tracer IV infusion (such as pkH20) at the onset of monocyte vascular accumulation and compare with similar approach at day 3 after infection and flow cytometry study to determine what fractions of Ly6C low monocles remained in the vasculature (tracer positive) and what fraction crossed BBB (tracer negative) over time. This study would help to clarify the claim of monocyte recruitment to the brain (as stated in the title of the manuscript).If authors cannot produce the requested data the conclusions should be significantly toned down, limitations of the study acknowledged and alternative interpretations of their data should be provided.Reviewer #2: 1. The authors should perform studies using a much lower infectious dose via the i.v. to show the necessity of VCAM1/VLA4 interactions and TNFR signaling on the recruitment of Ly6C lo monocytes to the brain. Doing these studies via the pulmonary route of infection may not be feasible using this strain of C. neoformans (H99) b/c mice typically succumb to infection via asphyxiation.Reviewer #3: 1. Although the authors describe accumulation of Ly6c low monocytes in the fungal-infected brain and define some molecules important for this process, there is absolutely no information with regard to whether Ly6c low monocytes are protective or detrimental during infection or whether their accumulation plays no role. Therefore, the authors should perform two sets of experiments to enlighten the readers on this very important topic:1a- Is Cryptococcus fungal load greater in any of the models that the ly6c low monocyte accumulation is low? That is in mice with impaired VCAM1 or VLA4 or CD11a or ICAM1?1b- Is Cryptococcus fungal burden increased in Nr4a1-deficient mice that have been reported to specifically lack Ly6c low monocytes? .These 2 sets of experiments are in this reviewer's opinion critical to establish a functional role of Ly6c low accumulation in this model. Without that, it is unclear what the significance of modest decreases of Ly6c low monocytes in this model mean.2. In Figure 5C, are the authors sure that the CD31+ cells are indeed endothelial cells and that the staining is not an artifact? Have they used CD102 to also stain? FACS sorted the cells to confirm by pPCR or WB that indeed these are endothelial cells? Brain preparations don't typically contain many endothelial cells unless specific protocols are followed, and such protocols are not specified in the methods by the authors. To extend this, the data on TNFR expression by these CD31+ cells in Figure 5E is quite unimpressive. The authors should use a positive control Ab that is known to be expressed in endothelial cells to prove that these are indeed valid data.3. In Figure 5G the authors "normalize" numbers of Ly6c low monocytes relative to microglia. This is not the correct way to do this analysis. Absolute numbers of Ly6c low monocytes is important to show. In addition, these chimera data are suggestive but not proving of the point. Competitive repopulation studies of Ly6clow monocytes from 50% WT and 50% TNFR KO mice  is the way to show that there is differential recruitment/accumulation. Therefore, the authors should not "normalize" numbers of monocytes to microglia and they should perform a competitive repopulation study to conclusively examine their hypothesis.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: Ln 91-92: the 600,000 numbers come from the older reports from the peak years of infections. Present estimate is around 180,000 deaths per year. please use more current reference such as the recent colloquium report of Academy of ASMLn 108-9 "following brain infection": to be precise the study is not restricted to brain infection but follows the acute fungemia, a model of acutely disseminated C.n. infection.Ln: 117 the paragraph title is somewhat misleading since it is not clear from the study if the monocytes were recruited to the brain, brain vasculature or the vasculature of all other tissuesLN 138 In the absence of additional data it is not clear which subset of monocytes predominantly was recruited/crossed to the brainOther titles Ln 167, 189, 206 should be probably changed from "to the brain" to "to the brain vasculature", as most images seem to show monocytes in the vasculature.Since microglia express CX3CR1, the author should how they gate out microglia in their flow cytometry analysis such as Fig 2C. Does the CXCR3+Ly6Clow gate also contain microglia if you initially gated on CD45+ cells?It is somewhat arbitrary what is "massive" cell recruitment to the brain  but total cell numbers reported here are not that overwhelming in numbers. Perhaps this could be toned down to a "substantial".Reviewer #2: 1. It is not clear by the monocyte population is compared to microglia .2. Do the authors suspect a difference in morbidity/mortality will be observed by blocking VCAM1/VLA4 interactions and/or TNFR signaling (perhaps due to less inflammatory cells trafficking to the brain)?3. Adhesion misspelled in Y axis of Figure 4C.Reviewer #3: 1. Why did the authors use VLA4 and VCAM1 Abs and not genetic deletion of these molecules? Are these mice not available?2. Please show CD11b in Figure 2C. Are all CX3CR1+ cells (Ly6c hi and low) all CD11b+?3. Please label Figure 6 appropriately (is #7 now)**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article : 21 Feb 2020Dear Dr. Shi,We are delighted to inform you that your manuscript, "VCAM1/VLA4 interaction mediates Ly6Clow monocyte recruitment to the brain in a TNFR signaling dependent manner during fungal infection," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


Remote earthquake triggering is a well-established phenomenon. Triggering is commonly identified from statistically significant increases in earthquake rate coincident with the passage of seismic energy. In establishing rate changes, short duration earthquake catalogs are commonly used, and triggered sequences are not typically analyzed within the context of background seismic activity. Using 500 mainshocks and four western USA 33-yearlong earthquake catalogs, we compare the ability of three different statistical methods to identify remote earthquake triggering. Counter to many prior studies, we find remote dynamic triggering is rare . For the mainshocks associated with remote rate increases, the spatial and temporal signatures of triggering differ. We find that a rate increase coincident in time with mainshock energy alone is insufficient to conclude that dynamic triggering occurred. To classify dynamically triggered sequences, we suggest moving away from strict statistical measurements and instead use a compatibility assessment that includes multiple factors, like spatial and temporal indicators. For these three examples, the rate increases were not subtle, but represented significant increases in rates of local earthquakes coincident in time with the passage of the surface waves from the triggering mainshocks. There are many other cases of documented remote dynamic triggering (see reviews and references therein10). In many of these examples, the seismicity rate change is subtle and the spatial extent very localized. Causality is assumed primarily because local events are either coincident with the passage of the surface waves, as determined by the presence of local earthquakes in the wavefield15 or the triggered events initiate within several hours after the passage of the mainshock seismic waves, leading to the concept of delayed dynamic triggering19. The prevalence of identified mainshocks generating remote, small magnitude earthquakes has led to two hypotheses: (1) remote dynamic triggering is ubiquitous13 and (2) dynamically triggered events are small, often below the magnitude of completeness (Mc) and to identify these events requires catalog enhancement24.Clear examples of remote dynamic triggering include increases in local earthquake rates over large areal extents as observed following the Mw 7.3 1992 Landers, California, earthquake25. This movement argues that we should abandon relying on a given significance value, like a p-value of 0.05 or 95% confidence interval (which were arbitrarily chosen to begin with), and instead move toward a compatibility assessment. In our analysis, we examine statistical significance in context of the spatial characteristics of the triggered events and the temporal behavior of the rate changes .The difference between the obvious remote triggering following the Landers, Denali Fault, and Izmit earthquakes, and the subtler instances of triggering and instances of delayed dynamic triggering leads to the question: what are the indicators for dynamic triggering and do different indicators imply differences in the physical mechanisms generating the triggered earthquakes? Here, we step back and first ask whether statistically significant changes in rate are sufficient to be the sole indicator for dynamic triggering. Currently, within the statistics community there is a debate over removing “statistical significance” from the lexicon of scientific study6. For these examples, it appears there is something fundamental in the passing seismic energy, amplitude or frequency, and/or that large areas of the crust are critically stressed and primed for earthquakes27. This has led many to look for a triggering threshold29 or other characteristics of the dynamic wave31 that could be used to anticipate remote dynamic triggering. However, in many subsequent studies the change in earthquake rate is subtler and the spatial extent more localized. For these cases, it seems plausible that the driving force is spatially confined and perhaps related to the interaction between the incoming waves and the orientation of local faults35 or the activation of fluids36.In the initial cases of dynamic triggering, there was an increase in the rate of earthquakes over a large spatial extent37 to ± several days39. To identify rate changes, many statistical methods compare the number of events in the window prior to the mainshock energy to the number of events in the window post the mainshock arrival.One of the main challenges of remote dynamic triggering studies is how to determine the background seismicity rate and subsequently how to determine significant rate changes. Previously, researchers have used many different statistical approaches. Commonly, these statistics compare windows prior to and following the arrival of the transient mainshock energy. The windows range from as small as ± one to ± three hours41. This statistic was originally developed to identify periods of earthquake quiescence. However, since the statistic measures differences in seismic rate between the number of earthquakes in a pre-window and the number predicted for a post-window normalized by the variance in the rate calculated using the pre-window, it can also be used to find periods of seismicity increases. Beta is defined as:Ta and Tb are the window lengths for counting the number of earthquakes after (Npost) and before (Npre) the time of interest, respectively, and pre * The most common statistic cited in dynamic earthquake triggering studies is the beta-statistic40 cautioned that significant anomalies found for short windows be treated “suspiciously.” They also suggested that when evaluating the significance of the beta-statistic, one look at the distribution of beta over an interval. Prejean and Hill39 investigated beta-statistic thresholds using Monte-Carlo resampling of data from Alaskan Volcanoes and found beta thresholds ranged from 2.7 to 16. Importantly, their minimum 2.7 value is larger than a beta threshold of 2, which is often used when assuming a Gaussian distribution42.In originally deriving the beta-statistic, it was assumed that: (1) the dataset was stationary  and (2) the pre-event window used to determine the rate contain at least one earthquake. The second assumption sets limits on how small the windows can be, and Matthews and Reasenberg45. The Z-statistic, like the beta-statistic, examines the rate of events in two different time windows using a stationary catalog.Npre, Npost, Tb and Ta are as defined above. Here, the difference in the mean number of events between the two-time windows is normalized by the sum of the variance, and the 95% and 99% significance thresholds are 1.96 and 2.57, respectively44. Note that when calculating the beta-statistic, if Tb and Ta are combined to predict the number of events in Ta (pre + Npost)* 39, then the calculated value more closely approximates a Z-statistic46.Alternative statistics to measure rate changes include the Z-statistic24. In this statistic, since a Poisson distribution assumes a constant rate, the number of events in the Tb window is taken to be the mean and the standard deviation is the square root of this value. For data sets with large number of samples generating the mean, 95% and 99% significance is equal to 1.96 and 2.58 times the standard deviation (σ), respectively, and significant rate changes are found when Npost > Npre + [1.96 or 2.58] * (σ), for 95% and 99%, respectively.A third statistic assumes a Poisson distribution and calculates the difference from the mean (DFM)The three statistics described above depend on the ability to reliably predict the rate of earthquakes based on a chosen time window. For comparison, we propose a fourth empirically derived statistic where we calculate the background rate over three-year windows using the distribution of the number of events in a shorter window (here 5-hours). However, we first construct a uniform catalog by correcting for Mc (see full explanation in the Methods section). Using the distribution of number of events in the 5-hour windows, we use percentiles to identify threshold rates that occur less than 5% and 1% of the time.pre is zero. Given this, it is not possible to correctly calculate the beta-statistic and we are careful to heed the warning regarding assigning significance when using small windows40. For the DFM method, if Npre is zero, we assign a value of one to preclude the case when < 2 events can be called triggering. Using the three chosen statistics, we identify 38 and 24 rate increases coincident with a mainshock at the 95% and 99% significance levels, respectively , this method is inherently plagued by the statistics of small numbers. In addition, there is no correction for variance in these data. The results from the proposed empirical method and the Z-statistic flag fewer triggering mainshocks than the DFM method.Applying the DFM method using the short, 5-hour, pre-event time window identifies the most instances of rate increases coincident with the arrival of the seismic waves, 38 and 24, at the 95% and 99% confidence levels, respectively. However, because of the short 5-hour duration of the observation window  and that the widespread seismicity tended to form clusterspost window: widespread . Of note, temporal class one sequences tend to exceed the statistical thresholds by the largest factors and the triggered events tend to have the largest spatial footprints. Temporal classes two and three sequences tend to activate single source zones.For each mainshock deemed capable of remote triggering, we categorize the time history into four classes Fig. . The sigead Fig. , single ead Fig. , a signaead Fig. , and tooL 2.7 quake followed by four smaller shocks. For the 2012 event, there were 3 larger and several smaller sequences within the two-month time period. Again, the seismicity is quite diffuse  with a few localized clusters. One of these clusters began ~3 hr after the origin of the mainshock with an ML 2.0 quake followed by three smaller shocks. These examples demonstrate the required additional scrutiny needed to truly assess if triggering occurred.When looking at the increases identified by multiple statistical measurements Figs.  and 3, w44 that can generate larger surface waves does not appreciably change these results  mainshocks or 1%-2% are coincident with significant increases in earthquake rates at the 99% and 95% levels, respectively, and may represent remote dynamic triggering. Of these, only 8 are consistent with a temporal step change coincident with the arrival of the mainshock energy. Notably, the rate change for these eight events is significant at both the 95 and 99% levels. Eleven and three  of the sequences are bursty and as discussed not easily associated with dynamic triggering. For the remaining 11 sequences, the rate increase is within an ongoing sequence. While difficult to conclusively associate with the passage of dynamic stresses, it is notable that like the step increase case all 11 events are significant at both the 95 and 99% levels. Overall, we find that Yellowstone and Utah are more susceptible to remote triggering than Anza and Montana. Yellowstone and Utah exhibit all three temporal classes, while rate changes in Anza exhibit step increases and rate changes in Montana exhibit enhancements within on-going sequences. Limiting our mainshocks to only shallow events (≤100 km)There is precedent in the seismological community that analysis using only changes in local earthquake rates can be used as an indicator for remote dynamic triggering. Here, we explore whether an increase in earthquake rate is always indicative of dynamic triggering. For example, should temporally bursty sequences be treated the same as temporal step increases? Can deep earthquakes trigger remote crustal earthquakes, as suggested by the Anza rate increase following the ~600 km deep 2013 Mw 8.3 Sea of Okhotsk earthquake? And, should increases in on-going sequences constitute remote dynamic triggering?48. When applying the Z-statistic and DFM to catalogs not corrected for Mc, we find 30–50% additional events  are flagged using the Z-statistic and 100–200%, using DFM . However, declustering may be counterproductive because the goal of dynamic triggering studies is to identify changes in what could be termed remote aftershocks, so these statistics should be used with caution, especially when using short window lengths. We propose using an empirical approach to describe the rates. A potential disadvantage to the empirical approach is the need for a longer duration catalog. However, because a longer catalog is used the distribution of earthquake rates can be established using a statistically significant pool—thousands of windows each with a rate measurement. The longer catalog can also be used to establish Mc. Not correcting for Mc can significantly change the triggering results47. We suggest following the lead of the statisticians who have recently called into question the usefulness and validity of statistical significance. In doing so, we contend that the seismological community should also transition from using hard-set rules of statistical significance and instead transition to the concept of compatibility25. However, if following this path, then we will also need to define indicators of dynamic triggering beyond rate. These could include the spatial and temporal classes identified in this and other papers21. Our findings suggest down-weighting the link to dynamic triggering for bursty sequences and increasing the weight for widespread spatial triggering. Other factors to consider include peak ground velocity and strain changes50 and the orientation of incoming seismic waves with respect to the local faults35.A third issue raised here is what constitutes a significant increase. If significance is set at 95% more sequences are identified as potentially triggered. However, many are bursty or within on-going sequences. Whereas if significance is set at 99% some triggered sequences may be missed, including the recently established triggering in Anza following the 2010 El Mayor Cucapah earthquakeMoving forward, future research on remote earthquake triggering should de-emphasize use of the beta-statistic and DFM and move beyond exploring only narrow time window rate changes. Additionally, a change in rate should be one of many indicators used to identify remote earthquake triggering. Here, we place the most confidence in sequences exhibiting either a strong step increase in rate and/or that the triggered seismicity has a distinct spatial footprint.51, and to fully understand triggering in the MIS region, one would first need a means to account for variations in rate that are the result of production efforts. In the four regional catalogs, the reported magnitudes are typically local (ML) or duration (Md). In rare instances, for some of the larger earthquakes, moment (Mw) or body (Mb) magnitudes are also reported. For less than 1% of the data the regional catalogs contain events that do not have assigned magnitudes, for these events we assume the magnitudes are below the Mc.The mainshock earthquake catalog includes global and regional seismic data from 01 January 1985 through 31 December 2017 (33 years). These data include 11 earthquakes M ≥ 6.7 from within the western US and Canada  and 489 global earthquakes M ≥ 7 Fig. . For theL and Md events for each of the four target catalogs. We are careful, however, to check if the catalog for a given region has different ML and Md magnitude distributions. We take this cautionary step because previous work found that in some catalogs, especially for small magnitude events (<1.5), Md is consistently larger than ML53. If this is the case in our data, then when the two different distributions are combined they would produce a double peak in the magnitude histogram. To help negate these limitations, when there are multiple peaks, we choose the larger value as the completeness level, recognizing that this approach will underestimate the completeness level. This method of calculating Mc approximates the Maximum Curvature Technique54.We divide the 33-year regional catalogs into 3-year sub-catalogs, netting a total of 44 sub-catalogs for the four study regions. For each 3-year sub-catalog, we create histograms of the earthquake magnitudes and determine Mc for each sub-catalog from the mode of the histogram  to a timestamp at the start of the window  when the cumulative sums equals or exceeds 95% and 99%  to the number of events in the 5-hour post-window (Npost). If Npre > Npost, we rule out triggering. Importantly, for triggering cases not only do we require Nthresh < Npost, but we also require that Npre < Nthresh. Given this secondary restriction, our 95% level results will not necessarily be a subset of the 99% level results.To determine time periods coincident with the arrival of the mainshocks, we establish when the first teleseismic waves arrive at the centroid of each study region using the travel time toolkit TauPSupplementary Information.Supplementary Data 1.Supplementary Data 2.Supplementary Data 3.


Neuroprotective treatments in ischemic stroke are focused to reduce the pernicious effect of excitotoxicity, oxidative stress and inflammation. However, those cellular and molecular mechanisms may also have beneficial effects, especially during the late stages of the ischemic stroke. The objective of this study was to investigate the relationship between the clinical improvement of ischemic stroke patients and the time-dependent excitotoxicity and inflammation. We included 4295 ischemic stroke patients in a retrospective study. The main outcomes were intra and extra-hospital improvement. High glutamate and IL-6 levels at 24 hours were associated with a worse intra-hospital improvement . High glutamate and IL-6 levels at 24 hours were associated with better extra-hospital improvement . Effective reperfusion after recanalization showed the best clinical outcome. However, the long term recovery is less marked in patients with an effective reperfusion. The variations of glutamate and IL6 levels in the first 24 hours clearly showed a relationship between the molecular components of the ischemic cascade and the clinical outcome of patients. Our findings suggest that the rapid reperfusion after recanalization treatment blocks the molecular response to ischemia that is associated with restorative processes. Neuroprotective treatments in acute IS are mainly focused to reduce the pernicious effect of excitotoxicity, oxidative-nitrosative stress, and inflammation8, however, these cellular and molecular mechanisms could also have reparative and beneficial effects for the patients. The reduction of glutamate concentration in the synaptic cleft has been the target of several clinical trials using antagonists of glutamate N-methyl-D-aspartate receptors to alleviate excitotoxicity, however those trials have failed to show clinical efficacy9. Targeting glutamate receptors can also block the synaptic transmission, and even though it could protect from glutamate excitotoxicity, it could also hinder the promotion of neuronal survival and cerebral plasticity in the late phase of IS9. Inflammation can also amplify the ischemic lesion and a considerable number of clinical trials were designed to target the inflammatory response10. Nonetheless, it is now clear that inflammation also promotes critical events necessary for tissue repair during the late stages of the IS11.The ischemic cascade is a complex molecular and cellular process that is triggered immediately after an ischemic stroke (IS). During this process, several mechanisms are developed sequentially as a consequence of the deprivation of blood and oxygen supply to the ischemic core15. Preclinical studies have shown that reperfusion could also modify the molecular profile of the ischemic cascade18, although the clinical implications of these findings are yet to be proven. In addition, reperfusion is associated with detrimental side effects. For instance, the potential toxic effect of tPA has been demonstrated in patients who did not reperfuse after the recanalization treatment20. Reperfusion is also associated with secondary damage due to oxidative stress21, although its repercussion on the clinical evolution of patients has not been demonstrated.Recanalization treatments aimed to achieve reperfusion, either with the tissue Plasminogen Activator (tPA) or by mechanical thrombectomy, are the only treatments that have shown clinical benefit in IS, with a better clinical outcome observed in patients treated with recanalization therapies compared with untreated patients, both at short and long termThe clinical benefits of reperfusion are limited by several factors, including its narrow therapeutic window, the topography of the vascular occlusion and the type of thrombus, as well as by other factors related to the patients, such as age and the hemodynamic situations. The risks associated with reperfusion therapies require the best possible selection of patients amenable to recanalization therapies. Since recanalization treatment is known to modify the patient’s outcome, in this work we studied the improvement of patients in relation with the success of the recanalization treatment, both at short term or intra-hospital improvement (patient improvement between admission and discharge) and at long term or extra-hospital improvement (patient improvement between discharge and 3 months afterwards). We also evaluated the relation between patient improvement with the time-dependent excitotoxic and/or inflammatory mechanisms. The aim of the present study was to evaluate whether the levels of glutamate, IL6 and the reperfusion have a direct influence in the intra-hospital and extra-hospital improvement, in patients with IS.The IS patients included in the analysis were classified into 3 groups: patients who did not receive recanalization treatment (PNR); patients with recanalization treatment and effective reperfusion (PER) and patients with recanalization treatment and uneffective reperfusion (PWER). The results are summarized in Table The selection criteria of patients for recanalization partially determined the statistical differences between the groups. In this regard, PER showed the highest clinical improvement, and the benefit persists during the first three months after the treatment .The NIHSS at hospital discharge was significantly higher for PWER compared to PNR and PER . Regarding the intra-hospital improvement, it was much higher for PER compared with PNR and PWER . However, this trend was different for the extra-hospital improvement, with the highest values observed in PNR, followed by PWER and PER .3 mL vs 9.7 ± 3.1×103 mL, p < 0.0001), fibrinogen , C-reactive protein  and sedimentation rate .Intra-hospital improvement was defined as a positive value of the NIHSS scale on admission minus the value of the NIHSS scale at discharge. Among the total sample (4295 patients), 1176 patients (27.4%) did not show any improvement during hospitalization, including 398 deaths, while 3119 patients (72.6%) showed an improvement of at least 1 point in the NIHSS between admission and discharge. It can be seen . Also, extra-hospital improvement was higher in patients with more serious infarcts . The type of reperfusion treatment did not influence the extra-hospital improvement, however, it was less frequent in PER (2.7%) compared with PNR (79.7%) and with PWER (17.6%).Extra-hospital improvement was defined as a positive value of the NIHSS scale at discharge minus the value of the NIHSS scale at three months. Extra-hospital improvement was analyzed in 3853 patients ; however, this relationship was not demonstrated for basal IL-6 levels . After 24 hours, both glutamate and IL6 levels correlated with a worse intra-hospital evolution  Table . These rSurprisingly, and opposed to the trend observed for intra-hospital improvement, the extra-hospital improvement Table  was assoThe association of glutamate and IL6 levels with intra and extra-hospital improvement was further confirmed using a multiple linear regression analysis Table . GlutamaTo the best of our knowledge, this is the first study aimed to analyse the early  and late  evolutionary outcome of IS patients in relation to the recanalization treatment.15. In our study, we also observed the best clinical outcome in patients with effective reperfusion as a response to the most appropriate recanalizing treatment, both at the time of hospital discharge and after 3 months. Indeed, the multivariate analysis demonstrates that early improvement is 38 times more frequent in patients with effective reperfusion compared with patients without recanalization treatment. Patients who received an uneffective recanalization treatment were the ones with the worst clinical situation. These results are in agreement with other clinical studies, as it is now recognized that effective reperfusion is an accurate indicator for clinical outcome in IS patients25.Recanalization treatments have demonstrated high survival rates and good outcome in clinical trialsThe main factors independently associated with a negative intra-hospital evolution are hyperthermia, infarct volume and especially the uneffective reperfusion. However, effective reperfusion is related to worst extra-hospital improvement. Logically, the extra-hospital evolution is greater in patients who present a higher NIHSS at discharge, and therefore, the margin for improvement is lower for PER Fig. . However27, although the clinical significance of these findings is rather low taking into account the nature of these variables and its units. Surprisingly, we observed an association between high glutamate levels at 24 hours and better extra-hospital evolution, and we determined that high glutamate levels at 24 hours are independently associated with an 11% better extra-hospital improvement, with both statistical and clinical significance. These observations suggested that the intense reduction of glutamate in the first 24 hours may be a factor that leads to a lower late recovery after an effective reperfusion  for the purposes of this analysis. The use of a clinical criterion to define effective reperfusion could lead to a bias in the classification in therapeutic response groups. The Doppler and MR angiography study after recanalization treatment was limited to a small group of patients. The narrow therapeutic margin available for patients with good therapeutic response could reduce the benefits in this group of patients; however, to avoid this effect we used the percentage deviation, not absolute, between the score of the NIHSS at discharge and at 3 months. The 3-month follow-up time is probably too short to evaluate the long-term effects. We have made an analysis in a subgroup of 287 patients that we have followed for a year, and the results were similar (data not shown).Effective reperfusion after recanalization treatments provided the best clinical outcome for IS patients. However, this clinical improvement is usually achieved at an early stage, and long term recovery is less marked in PER. It is likely that the rapid reperfusion blocks the molecular response to ischemia, which is associated with restorative processes and cerebral plasticity, and some degree of excitatory and inflammatory mechanisms may be associated with the late recovery of patients.22. The administration of anti-inflammatory drugs in large therapeutic windows could have negative effects. In this sense, the use of inflammatory markers like IL6 could be used not only for diagnosis but also as neuroprotectors in the management of IS32.The results of this work may involve the following practical suggestions: treatments targeting excitotoxicity may be optimal if administered in very short therapeutic windows and the inhibition of excitotoxicity after the first hours could block molecular responses for brain healing. The use of peripheral glutamate grabbers, which do not affect the mechanisms of the excitatory response in the brain, could be therefore an interesting therapeutic alternativeThis is a retrospective analysis conducted on a prospective registry of patients with acute cerebrovascular disease consecutively admitted to the Stroke Unit of the University Clinical Hospital of Santiago de Compostela (Spain) from September 2007 to March 2018. We prospectively included all IS patients admitted to the Stroke Unit in the BICHUS registry. The study was carried out in accordance with the Declaration of Helsinki of the World Medical Association and approved by the Research Ethics Committee of Galicia. Written informed consent was obtained on admission from each patient or from their relatives after full explanation of the procedures.The clinical questions addressed in this study expressed in PICO  format were:Among IS patients, do high levels of glutamate, IL6 and effective reperfusion compared with low levels of the mentioned makers and with uneffective reperfusion and non recanalization treatments improve the functional outcome at short and long term after hospitalization?All patients were handled by the same stroke team according to the Stroke Unit protocol. The variables of our registry included the date, onset-inclusion and wake-up strokes. Moreover, demographic data, previous modified Rankin scale score (mRS) and vascular risk factors were included. Data on previous carotid disease and processes of carotid revascularization were also collected, and in patients with a previous transient ischemic attack (TIA), the time between the TIA and stroke, as well as the coincidence or not with the topography. Medical history recording potential vascular risk factors, blood and coagulation test, 12-lead ECG and chest x-ray were collected. Carotid and transcranial Doppler were performed on admission; the transcranial Doppler study was repeated as a control of the recanalization treatment in 18% of the treated patients. Axillary temperature was obtained on admission and every 4 hours during the first 48 hours. The outcome clinical variables recorded were: National Institute of Health Stroke Scale (NIHSS), an internationally validated scale used to quantify the neurological impairment caused by a stroke, with values ranging from 0 (no stroke symptoms) to 42 (severe stroke), measured on admission, 24 hours, 48 hours, at discharge and at 3 ± 1 months; and the modified Ranking Scale (mRS), which is commonly used for measuring the degree of disability or dependence in the activities of stroke patients, ranging from 0 (no symptoms) to 6 (dead), measured at discharge and at 3 ± 1 months. Good outcome was considered for patients with a mRS value of 0,1 and 2. Both scales were performed by accredited neurologist.On the other hand, IS stroke subtypes were classified according to the TOAST criteria and hemorrhagic strokes were classified as hypertensive, amyloid or related to antiplatelet/anticoagulant. The type of reperfusion treatment, its characteristics and complications were also recorded.At discharge, 28.7% of the patients were institutionalized and the rest were reintegrated into their family environment. During hospitalization, candidate patients for rehabilitation were evaluated by the Rehabilitation Service according to a common protocol. The outpatient rehabilitation was coordinated by the primary care physician, but it was not protocolized in an even way.Biochemistry, haematology and coagulation test were assessed in the central laboratory of the hospital. For the molecular determinations, venous blood samples were collected in Vacutainer tubes  on admission, and 24 ± 12 hours and/or 48 ± 12 hours from stroke onset. After clotting for 60 minutes, blood samples were centrifuged at 3000 × g for 10 minutes, and the serum was immediately aliquoted, frozen and stored at −80 °C until analysis.33. Serum levels of interleukin 6 (IL6) were also measured on admission and after 24 hours using an immunodiagnostic IMMULITE 1000 System .Glucose levels, glycosylated hemoglobin, leukocytes, red blood cells, platelets, fibrinogen, C-reactive protein, total and fractionated cholesterol, triglycerides, proBNP, vitamin D and cholecalciferol were measured on admission. Serum levels of glutamate were determined on admission and after 24 hours by high performance liquid chromatography (HPLC) analysis  using the AccQ‐Tag derivatization method for amino acid analysis following the manufacturer´s technical specifications (Waters)34 in DWI-MRI on admission and betwen the 4th and 7th day in IS patients. In 2012, recanalization by endovascular treatment was protocolized in our hospital. Since 2012, a MRI angiography is performed in all patients candidate for reperfusion treatments. Neuroradiologists blinded to clinical and analytical data performed all imaging studies.All patients underwent cerebral computed tomography (CT) on admission and between days 4 and 7 after IS. Multimodal magnetic resonance imaging (MRI) was also performed on admission in IS patients candidates to recanalization treatments. Lesion volumes were measured using ABC/2 methodWe included 6565 stroke patients. Excluded patients were: ICH (1011), transient ischemic attack patients (512), patients transferred to other hospital during the acute phase (44), dead by other causes (40), no final stroke diagnosis (85), admitted outside the acute phase (97) and lacked follow-up at 3 months (480). Finally, 4295 patients were valid for the analysis.35) was used as a clinical marker of effective reperfusion.Early neurological improvement /42 × 100], and the extra-hospital improvement between the NIHSS at discharge and at 3 ± 1 months, using the formula [(NIHSS at discharge-NIHSS at 3 months)/42 × 100] .We categorized the intra-hospital and extra-hospital improvement as a decrease ≥1 in the NIHSS between admission and discharge and between discharge and at 3 months, respectively. The deceased patients were given the highest NIHSS value.Firstly, an analysis was performed to describe the sample. Frequencies and percentages were calculated for categorical variables while mean and standard deviation [SD] or median and interquartile range were used for continuous variables depending on their adjustment to normality. Kolmogorov-Smirnov test with Lilliefors was applied to assess it. ANOVA and squared test were performed to determine differences in patients according with the reperfusion group . The main objective of the study was to evaluate the intra and extra hospital improvement. In order to achieve it, multivariable models were built using a lineal regression analysis. The dependent variables were adjusted by clinical significant variables calculated in the bivariate analysis. Also, logistic regression analyses were carried out for patients with the same dependent variables but categorized. These results were shown as odds ratios (ORs) with 95% confidence intervals (CI 95%). P-value <0.05 was considered to be statistically significant. The statistical analyses were conducted in SPSS 21.0 .Supplemental Information.