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TITLE: Hyperbaric treatment for children with autism: a multicenter, randomized, double-blind, controlled trial ABSTRACT.BACKGROUND: Several uncontrolled studies of hyperbaric treatment in children with autism have reported clinical improvements; however, this treatment has not been evaluated to date with a controlled study. We performed a multicenter, randomized, double-blind, controlled trial to assess the efficacy of hyperbaric treatment in children with autism. ABSTRACT.METHODS: 62 children with autism recruited from 6 centers, ages 2–7 years (mean 4.92 ± 1.21), were randomly assigned to 40 hourly treatments of either hyperbaric treatment at 1.3 atmosphere (atm) and 24% oxygen ("treatment group", n = 33) or slightly pressurized room air at 1.03 atm and 21% oxygen ("control group", n = 29). Outcome measures included Clinical Global Impression (CGI) scale, Aberrant Behavior Checklist (ABC), and Autism Treatment Evaluation Checklist (ATEC). ABSTRACT.RESULTS: After 40 sessions, mean physician CGI scores significantly improved in the treatment group compared to controls in overall functioning (p = 0.0008), receptive language (p < 0.0001), social interaction (p = 0.0473), and eye contact (p = 0.0102); 9/30 children (30%) in the treatment group were rated as "very much improved" or "much improved" compared to 2/26 (8%) of controls (p = 0.0471); 24/30 (80%) in the treatment group improved compared to 10/26 (38%) of controls (p = 0.0024). Mean parental CGI scores significantly improved in the treatment group compared to controls in overall functioning (p = 0.0336), receptive language (p = 0.0168), and eye contact (p = 0.0322). On the ABC, significant improvements were observed in the treatment group in total score, irritability, stereotypy, hyperactivity, and speech (p < 0.03 for each), but not in the control group. In the treatment group compared to the control group, mean changes on the ABC total score and subscales were similar except a greater number of children improved in irritability (p = 0.0311). On the ATEC, sensory/cognitive awareness significantly improved (p = 0.0367) in the treatment group compared to the control group. Post-hoc analysis indicated that children over age 5 and children with lower initial autism severity had the most robust improvements. Hyperbaric treatment was safe and well-tolerated. ABSTRACT.CONCLUSION: Children with autism who received hyperbaric treatment at 1.3 atm and 24% oxygen for 40 hourly sessions had significant improvements in overall functioning, receptive language, social interaction, eye contact, and sensory/cognitive awareness compared to children who received slightly pressurized room air. ABSTRACT.TRIAL REGISTRATION: clinicaltrials.gov NCT00335790 BODY.BACKGROUND: Autistic Disorder (autism), along with Asperger syndrome and pervasive developmental disorder–not otherwise specified (PDD-NOS), comprise a spectrum of neurodevelopmental disorders (collectively termed autism spectrum disorders or ASD) that are characterized by restrictive and repetitive behaviors along with impairments in communication and social interaction [1]. The number of children diagnosed with ASD has increased over the last decade [2-4] and ASD currently affects as many as 1 out of 150 individuals in the United States (U.S.) [5]. ASD is generally considered a "static" neurological disorder [6] without any known cure. The use of hyperbaric treatment in children with ASD has increased in recent years [7] and traditionally involves inhaling up to 100% oxygen at a pressure greater than one atmosphere (atm) in a pressurized chamber [8]. Most typical indications for hyperbaric treatment involve the use of hyperbaric pressures above 2.0 atm. Higher atmospheric pressures are generally required to treat conditions such as carbon monoxide poisoning and to improve wound healing [8,9]. However, improvements have been observed via treatments with 95–100% oxygen and hyperbaric pressures of 1.5–2.0 atm for some chronic neurological conditions, including autism [7], fetal alcohol syndrome [10], cerebral palsy [11,12], and chronic or traumatic brain injury [13-16]. Furthermore, improvements in some of these conditions, including autism [7,17] and cerebral palsy [12], have been observed with the use of hyperbaric pressures of 1.3 atm and oxygen levels of 21–24%. In one study, significant improvements were observed in children with autism with the use of hyperbaric treatment at both 1.5 atm/100% oxygen and 1.3 atm/24% oxygen; neither hyperbaric protocol worsened markers of oxidative stress and both reduced C-reactive protein (a marker of inflammation) [7]. Rationales for the use of hyperbaric treatment in autism include decreasing inflammation [18-20], improving cerebral hypoperfusion [21,22], and modulating immune dysregulation [23-25], all reported as problems in some individuals with autism [26-34]. Several case reports [21,22] and three uncontrolled studies enrolling between 6 and 18 children with autism [7,17,35] have reported clinical improvements with hyperbaric treatment at 1.3 atm. However, to our knowledge, the efficacy of hyperbaric treatment in children with autism has not been evaluated to date with a controlled study. Given this background, we decided to study the effects of hyperbaric treatment in children with autism using 1.3 atm and 24% oxygen compared to near-placebo hyperbaric conditions (slightly pressurized room air at 1.03 atm and 21% oxygen). Hyperbaric treatment for children is generally regarded as safe, even at pressures of 2.0 atm and 100% oxygen for two hours per day [36]. In descending order, the most common side effects observed during hyperbaric treatment are barotrauma (2% incidence), sinus squeeze, serous otitis, claustrophobia, reversible myopia, and new onset seizure (which occurs in 1–3 per 10,000 treatments) [8]. In children with autism, the use of hyperbaric treatment using pressures up to 1.5 atm and 100% oxygen has been shown to be safe and well-tolerated [7,17]. BODY.METHODS.STUDY DESIGN: This was a prospective, randomized, double-blind, controlled trial involving treatment in parallel groups for 4 weeks. Active treatment was hyperbaric treatment at 1.3 atm and 24% oxygen for 40 sessions lasting 1 hour each at pressure ("treatment group"), whereas the control treatment consisted of slightly pressurized room air at 1.03 atm and 21% oxygen for 40 sessions lasting 1 hour each at pressure ("control group"). Comparison of the clinical effects of parallel treatments for 4 weeks was the primary objective of this study. The number of treatments (40 sessions) and the overall treatment period (4 weeks) were chosen because these were previously shown to be safe in two other studies of hyperbaric treatment in children with autism [7,17]. BODY.METHODS.PARTICIPANTS: ELIGIBILITY CRITERIA AND RECRUITMENT: This study was approved by the Liberty Institutional Review Board and enrolled children, 2 to 7 years of age, who had a diagnosis of Autistic Disorder and had not previously received any type of hyperbaric treatment. All children met the DSM-IV criteria for Autistic Disorder [1] and this diagnosis was also corroborated by psychologists using the Autism Diagnostic Interview–Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS). Children with PDD-NOS, Asperger syndrome, seizure disorder, current ear infection, uncontrolled asthma, inability to equalize ear pressure, fragile X syndrome, and ongoing treatment with chelation medication were excluded from participation in this study. Written informed consent was obtained from the parents and, when possible, the child. Sixty-six children were evaluated for inclusion in the study from six clinics throughout the U.S. Four children were excluded from participation because the diagnosis of Autistic Disorder could not be corroborated by ADI-R and ADOS. Therefore, the recruitment process yielded 62 eligible participants, who were randomized as depicted in Figure 1. Figure 1Consolidated Standards of Reporting Trials (CONSORT) flow diagram. BODY.METHODS.INTERVENTIONS: The active treatment was hyperbaric treatment at 1.3 atm and 24% oxygen in a monoplace hyperbaric chamber for 60 minutes at this pressure per session (this length of time did not include approximately 10–15 minutes for pressurization and depressurization). Oxygen flowing at 10 liters per minute from an oxygen concentrator was mixed with room air and pumped into the chamber following a protocol previously described [7]. This resulted in a final chamber oxygen concentration of approximately 24% as measured by an oxygen monitor. This treatment was given twice a day separated by a minimum of 4 hours, 5 days per week, for 4 consecutive weeks, for a total of 40 treatments per child. Control treatment consisted of slightly pressurized room air (1.03 atm and 21% oxygen) in a monoplace hyperbaric chamber for 60 minutes at this pressure per session (this length of time did not include approximately 10–15 minutes for pressurization and depressurization). This treatment was given twice a day separated by a minimum of 4 hours, 5 days per week, for 4 consecutive weeks, for a total of 40 treatments per child. For blinding purposes, participants underwent a brief compression to 1.1 atm at the beginning of each treatment. The chamber was then slowly decompressed from 1.1 to 1.03 atm where the pressure stayed for the remainder of the treatment. No oxygen was added to the chamber and thus the chamber was pressurized with room air (approximately 21% oxygen). The children in the control group remained in the chamber for the same length of time as children in the treatment group. At the end of each treatment, the pressure was slowly increased to 1.1 atm over about 5 minutes and then the chamber was depressurized. Procedures were developed and applied to mimic, for the control group, the experience of hyperbaric treatment at 1.3 atm, and thereby to keep participants and parents unaware of the nature of the intervention. These procedures included covering control switches, inflating and deflating the chambers to simulate pressure changes, and masking the sounds from the chambers. To further mask the group assignments, the equipment (including chambers) used for the control group was indistinguishable from the equipment used for the treatment group. Moreover, the same type of equipment was used at each study site. A pressure of 1.03 atm (with increases to 1.1 atm for several minutes at the beginning and at the end of the treatment) was chosen for the control group because this pressure represented the lowest that could be applied and still effectively simulate hyperbaric treatment at 1.3 atm. To verify its effectiveness, prior to beginning the study, this pressure protocol was tested in six adult individuals who were randomly and repeatedly exposed to both the treatment group pressure (1.3 atm) and the control group pressure (1.03 atm with short increases to 1.1 atm) and none of these individuals were able to reliably distinguish between the two pressures. At each study site, the primary investigator (DAR) visited and trained each hyperbaric technician to ensure that the same protocol was followed to minimize variances between study sites. An analysis performed after the study was finished demonstrated no significant differences (p = ns) between the six study sites for age, initial autism severity, and initial and final scores on all of the scales used in this study (this analysis can be found in the results section). Initial screening for this study included medical history taking and a physical examination by one of the study physicians. This included examination of the ears and tympanic membranes. Throughout each treatment, children were closely monitored by a hyperbaric technician for any signs of ear pain or other problems, and parents were instructed on how to recognize ear pain in their child. In both groups, in order to facilitate treatments, a parent or primary caretaker accompanied the child into the chamber as it was deemed that the children were too young to enter and remain in the chamber alone for the duration of each treatment. Children finishing more than one full session were included in the final analysis in an intention-to-treat manner. Daily treatment logbooks for each child were maintained by the hyperbaric technician and any side effects during treatment were recorded. At the end of the study, all children assigned to the control group were offered 40 hyperbaric treatments at the treatment pressure (1.3 atm and 24% oxygen) if the parents desired (all parents chose this option; treatments were provided without charge). During the study period, children in both groups were not allowed to begin any new therapies or stop any current therapies, including medications and nutritional supplementation. At the onset of the study, the use of nutritional supplements, medications, and applied behavioral analysis (ABA) therapy was similar in both the treatment and control groups (p = ns), see Table 1. Table 1 Initial characteristics of children in the treatment and control groups Treatment Control Comparison between groups (p-value) Age 4.97 ± 1.29 4.86 ± 1.13 0.7288* Male 30/33 22/29 0.1672** Use of nutritional supplements 23/33 20/29 0.9999** Use of medications 16/33 10/29 0.3915** Applied Behavioral Analysis Therapy 15/33 11/29 0.733** ABC Total Score 55.2 ± 28.7 53.3 ± 24.0 0.7843* ABC Irritability 13.2 ± 9.5 12.2 ± 7.9 0.6714* ABC Social Withdrawal 10.5 ± 6.9 11.2 ± 6.9 0.7048* ABC Stereotypy 7.5 ± 4.9 6.2 ± 4.7 0.3205* ABC Hyperactivity 20.7 ± 9.9 20.1 ± 8.2 0.8279* ABC Speech 3.4 ± 3.1 3.6 ± 3.6 0.8567* ATEC Total Score 75.3 ± 19.5 75.6 ± 21.0 0.9592* ATEC Speech/Language/Communication 16.3 ± 5.0 15.9 ± 6.1 0.7958* ATEC Sociability 17.4 ± 6.6 17.8 ± 6.2 0.849* ATEC Sensory/Cognitive Awareness 18.1 ± 5.2 19.6 ± 5.6 0.3676* ATEC Health/Physical/Behavior 23.5 ± 11.5 22.4 ± 8.3 0.72* ** Chi-square test with Yates' correction or Fisher's exact test. * Student's t test BODY.METHODS.RANDOMIZATION AND ALLOCATION: From the 62 children who were enrolled in the study, 33 were randomly allocated to the treatment group and 29 were randomly allocated to the control group. To achieve this allocation, a random allocation sequence (1:1) was generated and stratified on both the participant's group (treatment or control) and center, and this sequence was equilibrated every eight patients. The allocation sequence remained concealed to all investigators, study participants, parents, nursing staff, and all other clinic staff. The only person at each center who was aware of the group assignment for each particular child was the hyperbaric technician, who had no input in the evaluation (outcome measures) of the child. The hyperbaric technician was specifically instructed not to discuss the treatment nature or group assignments with anyone else in the clinic, including participants, parents, psychologists, and physicians. It was not possible to blind the hyperbaric technician due to the nature of the study (the technician had to know the group assignment in order to adjust the chamber to the correct pressure for treatment). However, all individuals involved in evaluating the child (parents, physicians, and psychologists) remained blinded to the group assignment for each child throughout the entire study period. In hyperbaric treatment studies, the study is considered double-blinded if the study participants and the evaluators of outcome measures are both blinded to group assignment (as they were in this study), even though the hyperbaric technician is aware of the assignment [37,38]. After the study was completed, parents in both groups were surveyed to determine the effectiveness of the blinding process, and there was no significant difference between groups in their ability to determine which group their child had been assigned (p = ns). BODY.METHODS.OUTCOME MEASURES: The primary outcome measures were changes compared to baseline observed after 4 weeks (40 sessions) of treatment, in parallel groups, on (1) Aberrant Behavior Checklist–Community (ABC) total score and subscale scores, based on the parent's or primary caretaker's rating, (2) Autism Treatment Evaluation Checklist (ATEC) total score and subscale scores, based on the parent's or primary caretaker's rating, and (3) Clinical Global Impression–Improvement (CGI) scale for changes in overall functioning and subscales, as rated by the parent or primary caretaker and also as rated separately by the treating physician (without knowledge of the parental ratings). Previous studies of hyperbaric treatment in children with autism have utilized the ABC and/or ATEC [7,17]. The ABC is a 58-item questionnaire that assesses communication, reciprocal social interaction, play, and stereotypical behaviors [39]. It is used to evaluate the effectiveness of medications and other therapeutic interventions and is scored from 0 ("not at all a problem") to 3 ("problem is severe in degree"). For this study, a total score was calculated as well as scores in 5 subscales: irritability, social withdrawal (also termed lethargy), stereotypy, hyperactivity, and inappropriate speech. The ABC was administered immediately prior to beginning the study (to determine baseline scores) and immediately after finishing 40 sessions. Lower scores on the ABC indicate lower autism severity. The ATEC is a questionnaire developed by the Autism Research Institute to evaluate treatment efficacy in individuals with autism. It consists of four subscales: Speech/Language/Communication, Sociability, Sensory/Cognitive Awareness, and Health/Physical/Behavior. The scores are weighted according to the response and the corresponding subscale. The higher the subscale and total scores, the more impaired the subject. A split-half reliability analysis on 1,358 checklists indicated high internal consistency among the questions within each subscale [40]. The ATEC is designed to allow evaluators to assess outcomes of certain treatments commonly used in individuals with autism. In this study, scores were calculated for the total score and the four separate subscales. The ATEC was administered immediately prior to beginning the study (to determine baseline scores) and immediately after finishing 40 sessions. Due to an administration error, the baseline ATEC was not performed at one of the study centers, and thus data was available for analysis for 23 children in the treatment group and 21 children in the control group. Scores for the CGI scale were obtained immediately after 40 sessions. The CGI scale gives an impression of global changes in certain areas for each child compared to baseline [41]. A total score for change in overall functioning was rated by a parent or primary caregiver and separately by the treating physician (the same physician who initially evaluated the child) using the following ratings: 1 ("very much improved"), 2 ("much improved"), 3 ("minimally improved"), 4 ("no change"), 5 ("minimally worse"), 6 ("much worse"), and 7 ("very much worse"). Children who received a score of "very much improved" or "much improved" on the physician CGI overall functioning score were considered to be "good responders" to treatment. Data was also collected from parents and physicians as to whether or not there were improvements in the following CGI subscales: receptive language, expressive language, sleep pattern, attention span, activity level, bowel movement pattern, self-stimulatory behavior, social awareness/alertness, social interaction, play skills, self-injurious behavior, eye contact, mood, anxiety level, aggression, general health, gross motor skills, and fine motor skills. BODY.METHODS.ANALYSIS: All data were prospectively collected and analyzed using StatsDirect statistical software (version 2.7.2) and are presented as mean ± SEM (standard error of the mean). Data analysis was based on an intention-to-treat approach on all participants who finished more than one treatment or control session. BODY.METHODS.ANALYSIS.POWER CALCULATIONS: Because this was the first controlled study of its kind, power calculations were based on the closest comparable study that had outcome data available at the time of this study design [17]. Analysis of this data demonstrated a medium to large effect size, depending on the scale examined (Cohen's d = 0.44 to 0.77) [42]. Using the most conservative effect size (d = 0.44), a power calculation using G*Power 3 [43] indicated that a total sample of 43 children would achieve a power of 80% with alpha set at 0.05 (two-tailed). BODY.METHODS.ANALYSIS.PLANNED COMPARISONS: Planned group comparisons were performed on the primary outcome measures. The normal distribution of data was checked using the Kolmogorov-Smirnov test. In parameters with normal distribution, comparisons were analyzed using the Student's t test. When normality was not present and equal variance could not be assumed, nonparametric tests (Mann-Whitney and Wilcoxon signed rank tests) were used. The Pearson's chi-square (χ2) test with Yates' correction or the Fisher's exact test (when subgroups contained less than 10 children) was applied to assess differences in the percentage of children responding to treatment in each group. In all analyses, a p-value less than 0.05 (two-tailed) was considered significant. BODY.RESULTS.STUDY SAMPLE: The flow of participants throughout the study is depicted in Figure 1. This study consisted of 52 boys and 10 girls, which is consistent with the male/female ratio observed in children with autism [44]. The mean age of all children was 4.92 ± 1.21 years and was similar (p = ns) in the treatment group (4.97 ± 1.29 years) and the control group (4.86 ± 1.13 years), see Table 1. There were more girls in the control group compared to the treatment group, but this difference was not significant (p = ns). Initial ABC and ATEC scores were similar in both groups (p = ns). At the onset of this study, the use of nutritional supplements, medications, and applied behavioral analysis (ABA) therapy was similar in both groups (p = ns), see Table 1. One-way analysis of variance (ANOVA) demonstrated no significant differences (p = ns) between the six centers that participated in this study for: age, initial autism severity, initial ABC total scores, final ABC total scores, initial ATEC total scores, final ATEC total scores, physician CGI scores, or parental CGI scores. Attrition rates during the study were low (see Figure 1). In the treatment group, two children dropped out of the study prior to beginning any treatments due to an illness (one with otitis media, the other with bronchitis). Another child dropped out before finishing one full treatment due to anxiety in both the child and the parent. Finally, one child was removed from the study after nine sessions because asthma symptoms worsened (neither the parents nor the treating physician felt that the hyperbaric treatments contributed to the increased asthma symptoms, but the child was removed from the study as a precaution); this child's scores performed at time of drop-out showed mild improvements in behavior (as separately ranked by both the physician and the parents) and these scores were included in the intention-to-treat analysis. The inclusion or exclusion of this child's scores had no significant effect on the statistical analysis. The remaining 29 children completed all 40 hyperbaric treatment sessions at 1.3 atm and 24% oxygen. Therefore, data from 30 children were analyzed in the treatment group. In the control group, two children dropped out of the study prior to beginning any treatments (one because of a death in the family, the other because of the time commitment). One child dropped out prior to finishing one full treatment due to parental claustrophobia. The remaining 26 children finished all 40 sessions at 1.03 atm and 21% oxygen. BODY.RESULTS.OUTCOME MEASURES.PHYSICIAN CLINICAL GLOBAL IMPRESSION (CGI) SCALE: After 40 sessions, the mean physician CGI score for change in overall functioning compared to baseline significantly improved (p = 0.0008) by 1.13 points in the treatment group (2.87 ± 0.78, score of 4 = "no change") compared to 0.38 points in the control group (3.62 ± 0.75), see Figure 2. Furthermore, 9/30 (30%) children in the treatment group had a "very much improved" or "much improved" rating compared to 2/26 (7.7%) in the control group (p = 0.0471). An improvement on the CGI scale (score of 1, 2, or 3) was noted in 24/30 (80%) children in the treatment group compared to 10/26 (38%) in the control group (p = 0.0024). Conversely, 16/26 (62%) children in the control group had a "no change" or "minimally worse" score (CGI score of 4 or 5) compared to 6/30 (20%, all 6 had a score of 4) in the treatment group (p = 0.0024). In the control group, two children received a score of 5 ("minimally worse"), whereas none received this score in the treatment group (p = 0.211). No child received a score worse than 5 in either group. Examination of the physician CGI subscales demonstrated that more children improved in the treatment group compared to the control group in receptive language (p < 0.0001), social interaction (p = 0.0473), and eye contact (p = 0.0102); a trend towards improvement was also observed in activity level (p = 0.0545). Figure 2Absolute change compared to baseline on the mean CGI overall functioning score in the treatment and control groups as rated separately by physicians and parents. * p < 0.001; ** p < 0.05. BODY.RESULTS.OUTCOME MEASURES.PARENTAL CLINICAL GLOBAL IMPRESSION (CGI) SCALE: The mean parental CGI score for change in overall functioning compared to baseline significantly improved (p = 0.0336) by 1.30 points in the treatment group (2.70 ± 0.81, score of 4 = "no change") compared to 0.83 points in the control group (3.17 ± 0.73), see Figure 2. A "very much improved" or "much improved" rating was observed in 9/30 (30%) children in the treatment group compared to 4/26 (15%) in the control group (p = 0.2238). Furthermore, 27/30 (90%) children had an improvement on the CGI scale (score of 1, 2, or 3) in the treatment group compared to 19/26 (73%) in the control group (p = 0.1616). A score of "no change" or "minimally worse" (CGI score of 4 or 5) was reported in 3/30 (10%, all scored 4) in the treatment group versus 7/26 (27%) in the control group (p = 0.1616). One child received a score of 5 ("minimally worse") in the control group compared to none in the treatment group (p = 0.4643). No child received a score worse than 5 in either group. Examination of the parental CGI subscales demonstrated that more children improved in the treatment group compared to the control group in receptive language (p = 0.0168) and eye contact (p = 0.0322). Examination of the mean CGI score for change in overall functioning in the treatment group as rated by the physicians compared to the parental ratings demonstrated that there was no significant difference (p = 0.4716). A significant correlation existed between the physician and parental CGI scales for the treatment group (r = 0.60, p = 0.0005). However, parents of children in the control group were significantly more likely to rate an improvement on the CGI score for change in overall functioning than were physicians (p = 0.0245) and therefore the correlation between the physician and parental CGI scales was not significant (r = 0.27, p = 0.1819). BODY.RESULTS.OUTCOME MEASURES.ABERRANT BEHAVIOR CHECKLIST (ABC) SCORES: In the treatment group, the ABC total score significantly improved after 40 sessions (p = 0.0118), see Additional file 1. Improvements in ABC subscales were also observed in the treatment group for irritability (p = 0.0147), stereotypy (p = 0.0124), hyperactivity (p = 0.0211), and speech (p = 0.0155). No significant improvements were observed in the control group on the ABC total score or any of the ABC subscales (p = ns). Analysis of changes in the ABC total score and subscale scores between the treatment and control groups demonstrated no significant changes (p = ns), although there was a trend towards improvement in the treatment group for irritability (p = 0.0976, see Figure 3) and 20/30 (67%) children in the treatment group had an improvement in irritability compared to 9/26 (35%) in the control group (p = 0.0311). Figure 3Changes compared to baseline on the ABC total score and subscales (percentage change) in the treatment and control groups. *** p < 0.1. BODY.RESULTS.OUTCOME MEASURES.AUTISM TREATMENT EVALUATION CHECKLIST (ATEC) SCALE: In the treatment group, significant improvements were observed on the ATEC scale in total score (p = 0.002), sociability (p = 0.0009), sensory/cognitive awareness (p = 0.0017), and health/physical/behavior (p = 0.0446), see Additional file 2. In the control group, ATEC improvements were found in total score (p = 0.0385) and sociability (p = 0.0134). Analysis of changes in ATEC total score and subscale scores between the treatment and control groups showed a significant improvement in sensory/cognitive awareness in the treatment group (p = 0.0367), see Figure 4. Non-significant improvements in the treatment group compared to the control group were observed in the other ATEC subscales (p = ns). Figure 4Changes compared to baseline on the ATEC total score and subscales (percentage change) in the treatment and control groups. ** p < 0.05. BODY.RESULTS.OUTCOME MEASURES.ANALYSIS BY AGE AND AUTISM SEVERITY: Because we had previously observed that both younger children and children who had higher initial autism severity improved more robustly with hyperbaric treatment at 1.3 atm [7,17], two separate sub-analyses of the effects of age and initial autism severity on the outcome scales used in this study were performed to determine if a subgroup could be identified that had a better response to hyperbaric treatment. BODY.RESULTS.OUTCOME MEASURES.ANALYSIS BY AGE AND AUTISM SEVERITY.AGE: Post-hoc analysis of children in the treatment group demonstrated a better improvement on the ABC total score in children who were over age 5 compared to those age 5 and under (p = 0.0482). Comparison of children who were over age 5 in the treatment and control groups demonstrated that children in the treatment group had significantly better improvements on the ABC in irritability (p = 0.0149), social withdrawal (p = 0.0086), and stereotypy (p = 0.0434). There was no significant difference in ABC scores between the treatment and control groups for children age 5 and younger (p = ns). When examining the ATEC scale, comparison of children who were over age 5 between the treatment and control groups demonstrated that children in the treatment group had significantly better improvements in sociability (p = 0.0095) and sensory/cognitive awareness (p = 0.0384). No significant difference between the two groups was observed for children age 5 and younger on the ATEC scale (p = ns). No significant age effect (p = ns) was observed between the treatment and control groups on the parental or physician CGI scales. BODY.RESULTS.OUTCOME MEASURES.ANALYSIS BY AGE AND AUTISM SEVERITY.AUTISM SEVERITY: Post-hoc analysis of children in the treatment group demonstrated that those who had an initial ADOS score below the 50th percentile for all children (less initial autism severity) had similar improvements in ABC total score and subscales compared to children with an initial ADOS score above the 50th percentile (p = ns). However, comparison of children in the treatment group and the control group who had an initial ADOS score below the 50th percentile (less autism severity) demonstrated that the children in the treatment group had significantly better improvements in ABC irritability (p = 0.0348) and ABC stereotypy (p = 0.0359). There was no significant difference in ABC scores between the treatment and control groups for children with an initial ADOS score above the 50th percentile (p = ns). When examining the ATEC scale, comparison of children in the treatment group and the control group with an initial ADOS score below the 50th percentile demonstrated a significantly better improvement in the treatment group in sociability (p = 0.0333). No significant difference between groups was observed for children with an initial ADOS score above the 50th percentile (p = ns). No significant effect (p = ns) was observed for autism severity between the treatment and control groups on the parental or physician CGI scales. BODY.RESULTS.ADVERSE EVENTS AND TOLERANCE: Hyperbaric treatment in this study was safe and well-tolerated. In the treatment group, one child developed both urinary frequency (urinalysis was normal) and a skin rash that the treating physician thought was yeast-related. As previously described, one child had worsening of asthma symptoms after nine treatment sessions and was removed from the study, and another child had anxiety and dropped out of the study before finishing one full treatment. None of the children in the treatment group received a score worse than 4 ("no change") on the physician or parental CGI for change in overall functioning. In the control group, one child developed abdominal distension and diarrhea during the study, but was able to complete the study. Another child in the control group had worsening of eczema during the study. No other adverse events including barotrauma or seizures were observed in either group. BODY.DISCUSSION: To our knowledge, this trial represents the first controlled study of hyperbaric treatment in children with autism. Previous studies examining this treatment in autism have described improvements that could have been due, in part, to a participation (placebo) effect. The results of uncontrolled studies in autism should be interpreted with caution, especially since some randomized, double-blind, placebo-controlled studies in individuals with autism have reported relatively high improvement rates in the placebo group. For example, one prospective study comparing a single dose of IV secretin to a placebo found that 30% of the children receiving the placebo had a significant improvement immediately after the infusion [45]. Another prospective study comparing daily treatment with amantadine to a placebo over a 4-week period found a mean placebo response rate of 37% [46]. In the current study, 80% of children in the hyperbaric treatment group had an improvement on the CGI scale for change in overall functioning as rated by blinded physicians; however, 38% of children in the control group were also rated as improved. This 38% improvement rate in the control group may have occurred because these children received a very low level of hyperbaric pressure (1.03 atm with short increases to 1.1 atm), and therefore, strictly speaking, this pressure did not represent a true placebo-control group. Hyperbaric pressure as low as 20 mmHg (approximately 1.03 atm) has been shown to decrease in vitro pro-inflammatory cytokine release (including IL-1beta) from human monocytes and macrophages [47]. Some children with ASD have elevations in certain pro-inflammatory cytokines, including IL-1beta [32,48]. Therefore, some of the improvements observed in the control group could have been due to the slight hyperbaric pressure received. Because the control group experienced pressure conditions closer to those of the treatment group than a true placebo (e.g., 1.00 atm and 21% oxygen) would have provided, the difference in clinical outcomes between the treatment and control groups may have been less significant than what would have been observed with a placebo. However, a true placebo could not have been used with this study design because some degree of hyperbaric pressure was needed to mimic hyperbaric treatment at 1.3 atm, otherwise blinding of the group assignment would have not been possible. A pressure of 1.03 atm (with short increases to 1.1 atm) was chosen for the control group because testing performed prior to the study indicated that this pressure was the lowest that could be given and still effectively simulate, from the perspective of the blinded parents and children, hyperbaric treatment at 1.3 atm. The blinding procedure in this study appeared to be adequate because there was no significant difference between the two groups in the ability of parents to correctly guess the group assignment of their child. Furthermore, 73% of parents of children in the control group rated their child as improved on the CGI scale which also suggests that the blinding procedure was adequate, because if parents thought that their child was in the control group, they probably would have been less likely to rate an improvement after treatment. In the hyperbaric treatment group, parental CGI scores significantly correlated with physician CGI scores (r = 0.60, p = 0.0005) which strengthens the CGI results in this group. In the control group, the parents were significantly more likely to rate their child as improved on the CGI scale compared to the physicians (p = 0.0245) and therefore the parental and physician CGI scales did not significantly correlate (r = 0.27, p = 0.1819). This finding further suggests that the blinding procedure was adequate in this study and also demonstrates evidence of a participation effect in the control group. In this trial, the use of hyperbaric treatment at 1.3 atm was well-tolerated and confirmed previous reports of safety. This study also demonstrated clinical improvements that were similar to previous uncontrolled studies of hyperbaric treatment in children with autism [7,17,21,22,35]. The findings of this study are significantly strengthened because of the presence of a control group which previous hyperbaric treatment studies in autism lacked, and also because of the use of six separate centers which should have minimized potential bias, especially since there were no significant differences between study sites in age, initial autism severity, and initial and final scores on all of the scales used in this study. In this current trial, significant improvements were observed in several domains with the use of hyperbaric treatment at 1.3 atm and 24% oxygen compared to slightly pressurized room air, including overall functioning, receptive language, social interaction, eye contact, and sensory/cognitive awareness. The reason for these different areas of improvement is not clear. The mechanism of action of hyperbaric treatment in autism is not entirely known, although it may act by diminishing gastrointestinal and cerebral inflammation and by improving immune dysregulation and cerebral hypoperfusion [24]. Multiple studies have reported that these problems are relatively common in children with autism [26-34]. Cerebral hypoperfusion, especially of the temporal lobes, is a very common finding in children with autism compared to typically-developing children, affecting up to 75% [28,49]. This hypoperfusion is an indirect measure of diminished brain activity [28] because cerebral blood flow is normally tightly coupled to brain metabolic rate and function [50,51]. Several studies have reported that the anatomical location of cerebral hypoperfusion significantly correlates with certain autistic behaviors [24]. For example, in a study of 30 individuals with autism compared to 14 non-autistic individuals, hypoperfusion of the thalamus as measured by Single Photon Emission Computed Tomography (SPECT) was observed in the autism group and significantly correlated (r = 0.42, p < 0.01) with repetitive behaviors and unusual sensory interests [52]. In another SPECT study of 23 children with autism compared to 26 non-autistic children, hypoperfusion of the right medial temporal lobes was found in the autism group and was correlated with obsessive desire for sameness (p < 0.001), and hypoperfusion of the medial prefrontal cortex and anterior cingulate gyrus was associated with impairments in social interaction and communication (p < 0.001) [27]. Furthermore, two SPECT studies in individuals with autism have reported that cerebral hypoperfusion significantly worsens with increasing age [53,54]. In one of these studies, hypoperfusion of brain areas that controlled speech (left temporal lobe and frontal areas) significantly worsened with increasing age (p < 0.001) and was associated with deficits in language formation and "subsequently prevent [ed] development of true verbal fluency and development in the temporal and frontal areas associated with speech and communication" [54]. Furthermore, in another study of 45 children with autism, children with the highest degree of left temporal lobe hypoperfusion, as measured by Positron Emission Tomography (PET), also had the most severe autistic behavior [55]. The cause of cerebral hypoperfusion in children with autism is not known. Several studies have described apparent vascular-associated cerebral inflammation in children with autism compared to controls including perivascular macrophage and microglia accumulation in post-mortem autistic brain samples [33] as well as the presence of serum IgM and IgG autoantibodies that bind to small blood vessels in the brain in about 30% of children [26,56]. These findings could be consistent with a cerebral vasculitis [24]. Elevated urinary levels of 8-isoprostane-F2α have also been reported in some children with autism [57]. In some studies, this isoprostane elevation has been shown to cause in vivo vasoconstriction and increase the aggregation of platelets [58]. Furthermore, elevations in 2,3-dinor-thromboxane B2 (associated with increased platelet activation) and 6-keto-prostaglandin F1α (a marker of endothelium activation) have been described in some children with autism [59]. These inflammatory-related findings could contribute to the cerebral hypoperfusion described in autism [24]. Cerebral hypoperfusion is associated with hypoxia [24] and several studies in children with ASD have reported evidence of cerebral hypoxia, as measured by a reduction in brain Bcl-2 and an increase in brain p53 [60-63]. Elevated p53 is induced by hypoxia [64] and a decrease in Bcl-2 is associated with increased apoptosis provoked by hypoxia [65]. Hypoxia leads to higher brain concentrations of hypoxia-inducible factor 1α (HIF-1α) [66]. An increase in HIF-1α causes an increase in inflammation, including redness and swelling of tissues, and the attraction of lymphocytes [66]. HIF-1α is essential for inflammation mediated by myeloid cells [67]. In fact, in one study, rats that were null for HIF-1α demonstrated almost complete inhibition of the inflammatory response [68]. HIF-1α is responsible for angiogenesis that is secondary to hypoxia [68,69] and also induces Vascular Endothelial Growth Factor (VEGF), which increases the permeability of blood vessels [66] and causes tissue edema. Evidence of cerebral edema in 19 children with autism compared to 20 typically-developing children was suggested by one recent T2-magnetic resonance imaging (MRI) study [70]. This edema can lead to increased interstitial space between cells [71] and cause an increase in the distance that oxygen must diffuse from blood vessels to reach brain cells and can thus lead to cellular hypoxia [72]. Inflammation is also associated with blood-brain barrier disturbances which can further increase cerebral edema [24]. Chronic inflammation is commonly associated with the infiltration of polymorphonuclear neutrophils and other immune cells, along with the cytokines that are released by these cells. This causes an increase in local oxygen usage due to the elevated oxygen requirements created by these newly infiltrated cells. Yet, at the same time, inflammation causes reduced oxygen extraction by normal cells [73]. For instance, in one study, elevated markers of inflammation (including IL-6, tumor necrosis factor receptors 1 and 2, and high-sensitivity C-reactive protein) were significantly correlated with decreased maximum oxygen uptake at peak exercise (VO2max) in patients with known or suspected coronary artery disease [74]. Therefore, inflammation prevents maximal uptake of oxygen by cells. Inflammation also increases oxidative stress and can cause neutrophils to become more adherent and attach to vessel walls [75]. This infiltration and increased adherence of inflammatory cells can contribute to brain injury by decreasing microvascular blood flow, causing thrombosis, and increasing the production of free radicals [76]. Hyperbaric treatment can overcome the effects of cerebral hypoperfusion and hypoxia by: increasing the plasma oxygen tension which transfers more oxygen into tissue, including the brain [77,78], decreasing cerebral edema [79], inhibiting the expression of HIF-1α and its target genes [80], and by causing angiogenesis over time [18]. Several case reports in children with autism have described improved cerebral perfusion after hyperbaric treatment at 1.3 atm, as measured by post-hyperbaric treatment SPECT scans compared to pre-hyperbaric SPECT scans [21,22]. If the hypoperfusion in children with autism is related to cerebral inflammation, then hyperbaric treatment could potentially improve cerebral perfusion by decreasing this inflammation [24]. Hyperbaric treatment possesses strong anti-inflammatory properties [18-20] and has been shown to significantly decrease neuroinflammation [81] as well as cerebral edema and blood-brain barrier damage in animal models [79]. At 1.3 atm, hyperbaric treatment decreased a marker of inflammation (C-reactive protein) in one study of children with autism [7]. It is unknown if any of the improvements observed in this study were mediated through an improvement in cerebral hypoperfusion and/or a decrease in cerebral inflammation as this study was not designed to examine these possibilities. However, since cerebral hypoperfusion is relatively common [28,49] and can be diffuse in location in children with autism [82,83], and the anatomical location of hypoperfusion significantly correlates with certain autistic behaviors [27,52,54], then improving hypoperfused brain areas with hyperbaric treatment could account for the different areas of improvement observed in this study. Our previous studies suggested children who were younger and those who had higher initial autism severity responded more robustly to hyperbaric treatment [7,17]. However, these studies were small and uncontrolled, and thus we analyzed these two parameters (age and autism severity) in this study with a post-hoc analysis. An interesting finding from this current study was that children who were over age 5 had significantly better improvements on the ABC total score with hyperbaric treatment at 1.3 atm compared to younger children (p = 0.0482). Given the fact that older children with autism generally have a higher degree of cerebral hypoperfusion compared to younger children [53,54] and that hyperbaric treatment can improve cerebral hypoperfusion [21,22], these factors could have accounted for the age findings observed in this study. Additional studies examining the use of hyperbaric treatment in children with autism that also incorporate SPECT or PET scans to measure changes in cerebral blood flow might be helpful in further delineating these possibilities. Moreover, children who had lower initial autism severity also had the most improvements with hyperbaric treatment in this study. The reason for this finding is not known, but may be due to greater levels of oxidative stress and other metabolic problems recently described in children with higher autism severity compared to those with lower severity [84]. Because this study was not designed to measure the long-term outcomes of hyperbaric treatment in children with autism, additional studies are needed to determine if the significant improvements observed in this study last beyond the study period. It is possible that ongoing treatments would be necessary to maintain the improvements observed, but this study was not designed to examine that possibility. Our clinical observations in children with autism suggest that additional hyperbaric treatments beyond 40 total sessions can lead to additional improvements; however, further studies are needed to formally validate these observations. Recently, several companies have started producing and marketing portable hyperbaric chambers that are approved by the U.S. Food and Drug Administration (FDA) for home use and are able to supply the hyperbaric treatment parameters used in this study. Therefore, the widespread and long-term use of this potential treatment is feasible and not necessarily costly (on a per treatment basis). Finally, this study was not designed to determine if higher hyperbaric treatment parameters (higher atmospheric pressure and oxygen levels, which can only be provided in a clinic setting) would lead to better or more long-lasting results. Additional studies are needed to investigate that possibility. BODY.CONCLUSION: Given the positive findings of this study, and the shortage of proven treatments for individuals with autism, parents who pursue hyperbaric treatment for their child with autism can be assured that it is a safe treatment modality at the pressure used in this study (1.3 atm), and that it may improve certain autistic behaviors. Further studies are needed by other investigators to confirm these findings; we are aware of several other planned or ongoing studies of hyperbaric treatment in children with autism. However, in light of the positive results of this study and those of several previous studies [7,17,21,22,35], the use of hyperbaric treatment appears to be a promising treatment for children with autism. BODY.ABBREVIATIONS: ABA: Applied Behavioral Analysis; ABC: Aberrant Behavior Checklist; ADI-R: Autism Diagnostic Interview-Revised; ADOS: Autism Diagnostic Observation Schedule; ASD: Autism Spectrum Disorder; ATEC: Autism Treatment Evaluation Checklist; atm: atmosphere; CGI: Clinical Global Impression; FDA: Food and Drug Administration; HIF-1α: Hypoxia-Inducible Factor 1α; IHA: International Hyperbarics Association; MRI: Magnetic Resonance Imaging; ns: not significant; PDD-NOS: pervasive developmental disorder-not otherwise specified; PET: Positron Emission Tomography; SPECT: Single Photon Emission Computed Tomography; U.S.: United States; VEGF: Vascular Endothelial Growth Factor. BODY.COMPETING INTERESTS: DAR, LWR, SS, CS, AU, JN, EMM, and EAM treat individuals with hyperbaric treatment in their clinical practices and derive revenue from hyperbaric treatment. DAR, LWR, and EAM had previously received research funding from the International Hyperbarics Association for an earlier study of hyperbaric treatment in autism [7]. EAM has also received hyperbaric chambers and financial support (unrelated to this study) from OxyHealth LLC for remodeling the Rimland Center, a center for mentoring clinicians interested in learning how to care for children with autism spectrum disorders. The remaining authors (SL, GH, and BG) declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: DAR, LWR, and EAM conceived of the study and the study design. DAR, SS, CS, AU, JN, EMM, and EAM performed initial physical exams on the children and were responsible for completing the physician CGI scales. SL and GH performed psychological testing on the children including ADOS and ADI-R. DAR and BG performed the statistical analysis. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: BODY.SUPPLEMENTARY MATERIAL: Additional File 1Table 2. Changes on the ABC scale and subscales in the treatment and control groups (lower scores denote improvement). # Wilcoxon signed rank tests. ## Mann Whitney test.Click here for file Additional File 2Table 3. Changes on the ATEC scale and subscales in the treatment and control groups (lower scores denote improvement). # Wilcoxon signed rank tests. ## Mann Whitney test.Click here for file

TITLE: Ketamine perturbs perception of the flow of time in healthy volunteers ABSTRACT.RATIONALE: Disturbances in the subjective experience of time have been observed both in schizophrenia and following acute administration of ketamine. However, effects of ketamine on more objective timing tasks have not yet been measured in humans, nor has it been established that timing effects are not merely secondary to a more general dysfunction in working memory (WM). ABSTRACT.OBJECTIVE AND METHODS: In a double-blind placebo-controlled crossover study, we characterised the effects of ketamine (100 ng/ml blood plasma level) on performance of perceptual timing and colour discrimination tasks, which were matched for WM and attentional demands. To test the ubiquity of ketamine's effects on timing, we also examined two distinct measures of temporal predictability. ABSTRACT.RESULTS: Ketamine significantly distorted the subjective experience of time as measured by the Clinician-Administered Dissociative States Scales. Critically, ketamine also impaired accuracy on the perceptual timing task while having no effect on performance of the colour perception task. Although ketamine did not impair the ability to use prelearned temporal (or spatial) cues to predict target onset (or location), it did slow reaction times at long delays following non-informative neutral cues, suggesting an impaired ability to use the unidirectional flow of time itself to make temporal predictions. ABSTRACT.CONCLUSIONS: Ketamine induced selective impairments in timing, which could not be explained by more fundamental effects on the ability to hold information in WM. Rather our collected findings suggest that ketamine may disturb timing by selectively impairing the way in which information is temporally manipulated within WM. BODY.INTRODUCTION: The glutamatergic N-methyl-d-aspartate (NMDA) receptor antagonist ketamine is a useful pharmacological model of psychosis (Krystal et al. 1994; Corlett et al. 2007a). In healthy participants, it induces perceptual changes (Krystal et al. 1994; Duncan et al. 2001; Stone and Pilowsky 2006), thought disorder (Krystal et al. 2005; Malhotra et al. 1996; Duncan et al. 2001) and negative symptoms (Krystal et al. 2005; Duncan et al. 2001), as well as delusion-like thinking (Pomarol-Clotet et al. 2006). Moreover, ketamine induces selective impairments in cognitive function (Morgan and Curran 2006) that are comparable to those observed in schizophrenia (Fletcher and Honey 2006). Notably, both ketamine (Honey et al. 2003, 2004) and schizophrenia (Perry et al. 2001; Kim et al. 2004) are associated with difficulties in manipulating, rather than maintaining, information in working memory (WM). While ketamine does not reproduce the signs and symptoms of established schizophrenia, it has been argued that the perceptual and cognitive changes it induces resemble those found during prodromal stages of the illness (Pomarol-Clotet et al. 2006; Corlett et al. 2006, 2007a, 2007b, 2011). One feature of schizophrenia that has not been well-explored under ketamine is that of the experience of time. Numerous studies have demonstrated that patients with schizophrenia show reduced accuracy in estimating periods of time in the minutes-to-hours range (Rabin 1957; Johnson and Petzel 1971) and in timing of stimulus durations in the range of several hundreds of milliseconds to a few seconds (Lhamon and Goldstone 1956; Tysk 1983; Elvevåg et al. 2003; Davalos et al. 2003; Carroll et al. 2008, 2009). However, the effects of ketamine on timing are uncertain: in rats, ketamine had no direct effect on timing (Cheng et al. 2006) but instead 'unlocked' the clock-speeding effects of cocaine that had otherwise been masked by extensive training (Cheng et al. 2007a). Yet, subjective reports in healthy volunteers indicate that ketamine can distort the experience of time (Pomarol-Clotet et al. 2006; Stone and Pilowsky 2006), a phenomenon also observed in schizophrenia (Freedman 1974; Seeman 1976; Vogeley and Kupke 2007). Although the effects of ketamine on timing have not yet been explored experimentally in humans, Rammsayer (2006) noted that memantine (a lower-affinity NMDA antagonist) impaired timing of auditory stimuli in the seconds (~1 s.) but not tens of milliseconds (~50 ms) range. Timing in the range of seconds, but not milliseconds, requires support from WM and sustained attention processes (Gibbon et al. 1984; Zakay and Block 1996): a seconds-range stimulus must be continually attended from stimulus onset to offset (as opposed to estimates of, e.g. pitch or colour, which can be estimated almost instantaneously), and the contents of WM must be continuously updated as stimulus presentation unfolds. Given the pattern of memantine's effects, Rammsayer (2006) concluded that its selective effects on seconds-range timing were secondary to its effects on WM. Given differences in their pharmacological specificity, we cannot presume that ketamine's effects would be similar to those of memantine nor, from a more cognitive point of view, that such effects necessarily reflect a more fundamental effect on WM. Timing of longer, seconds-range durations depends not only upon WM but also, of course, upon an index of elapsed time itself, for example accumulation of temporal pulses (Gibbon et al. 1984; Treisman 1963) or temporal integration of steadily climbing neuronal activity (Reutimann et al. 2004). Critically, psychophysical evidence suggests that this timing mechanism may be quite distinct for millisecond- and seconds-range timing (Gibbon et al. 1997; Buonomano et al. 2009). Thus, it is possible that a deficit in the seconds but not milliseconds, range reflects a specific temporal, rather than WM, deficit. We therefore sought to both establish whether ketamine, like memantine, is associated with a disturbance in an objective measure of time perception in the seconds time range and, crucially, to dissociate such an effect from concurrent mnemonic disturbance by directly comparing the effects of ketamine on performance of timing and colour discrimination tasks that were matched for WM demands (Coull et al. 2004, 2008). A ketamine-induced impairment of the timing, but not colour, task would strongly suggest a selective timing impairment over and above any effects on WM. However, 'timing' is an imprecise term (Michon 1985; Pöppel 1997; Coull and Nobre 2008) that can refer equally well to how long a current event lasts (duration estimation) or when a future event is likely to occur (temporal prediction). Since ketamine has previously been shown to modulate neural indices of prediction error (e.g. Corlett et al. 2007a; 2011), we examine performance not only on time perception but also on measures of temporal prediction. Specifically, we tested the impact of ketamine on two distinct indices of temporal prediction derived from the temporal-orienting paradigm (Coull and Nobre 1998; Nobre 2001), a temporal analogue of the classic Posner covert orienting of spatial attention task (Posner et al. 1980). In the task, visual cues provide valid, invalid or neutral information concerning the likely delay before an imminent target is presented. The behavioural benefit of temporally informative cues is indexed by faster reaction times during valid compared to invalid trials (Coull and Nobre 1998; Griffin et al. 2001). Yet, even when the onset of the target cannot be predicted by temporal cues (i.e. in the neutral condition), the unidirectional flow of time itself, or 'time's arrow' (Eddington 1928), provides a degree of predictive power: since time flows inexorably forward an event that we expect to occur, but has not yet occurred, must do so at some time in the future. The 'hazard function' measures the increasing conditional probability (and, hence, increasing sense of temporal expectation) over time that an event will occur, given that it has not already occurred (Elithorn and Lawrence 1955; Luce 1986). Increasing temporal certainty of target presentation over time allows the response plan to be updated dynamically, as the trial unfolds, which ultimately translates into faster RTs at longer delays (Niemi and Näätänen 1981). The orienting paradigm can therefore index temporally predictive information that is conveyed either by valid, prelearned cues (temporal condition) or by the very passage of time itself (neutral condition). In order to control for possible, though unlikely (Morgan and Curran 2006), effects of ketamine on non-specific attentional processes, we also examined effects of ketamine on the more classic, spatial, version of the task. BODY.METHODS.PARTICIPANTS: We collected data from 13 (6 male and 7 female; mean age 23.1 years) right-handed, healthy volunteers. Although three further volunteers had been recruited, their data could not be included in the final analysis due either to technical difficulties or subject dropout. Research was approved by Addenbrookes NHS Trust Research Ethics Committee. Participants spoke fluent English, had normal or corrected-to-normal vision with no colour blindness, and reported no history of clinical drug or alcohol abuse or of psychiatric illness. BODY.METHODS.EXPERIMENTAL DESIGN: Each participant was tested on two occasions, with a minimum of 1 week between sessions. A within-subject, double-blind crossover design was used such that participants received ketamine in one session and placebo in the other. Participants also completed two learning tasks during the testing session, and performed all tasks in the same order from one session to the next. Here, we report data for the two timing tasks (data for other tasks will be reported elsewhere). BODY.METHODS.KETAMINE INFUSION PROTOCOL: Racemic ketamine (2 mg/nl solution) or saline solution was administered intravenously by a computerised target-controlled infusion system, which calculated infusion rates necessary to achieve the 'target' blood concentration: 100 ng/ml plasma for 75 min immediately followed by a further 40 min at 200 ng/ml plasma. The timing tasks reported here were performed during the 100 ng/ml infusion period only. The infusion pump (Graseby 3500, Smiths Medical, Ashford, UK) was controlled by a personal laptop computer running Stanpump software, programmed to use a two-compartmental pharmacokinetic model for ketamine (Absalom et al. 2007) to calculate infusion rates. The order of infusion was counterbalanced across participants. BODY.METHODS.COGNITIVE TASKS: Both tasks were performed at 100 ng/ml blood plasma level of ketamine. Twelve participants completed the discrimination tasks and 11 completed the orienting tasks. BODY.METHODS.COGNITIVE TASKS.TEMPORAL AND COLOUR DISCRIMINATION: Task The trial structure and stimuli used in the temporal and colour tasks were identical (Fig. 1), with the only difference being whether participants were asked to make time or colour judgements. In the time condition, participants estimated whether the duration of the second (probe) stimulus was shorter (S), equal to (=), or longer (L) than the previous (sample) stimulus. In the colour condition, participants estimated whether the probe was redder (R), equal to (=), or bluer (B) than the sample. The response screen presented three alphanumeric characters, indicating each of the three possible responses for each condition. Their spatial position could vary on a trial-by-trial basis. Participants pressed one of the three corresponding response buttons (left, middle or right) to indicate their estimate of relative duration or colour. The response screen appeared for 1,500 ms, during which time the subject gave their response, and any responses slower than 1,500 ms were not recorded. Inter-trial intervals varied pseudo-randomly from 1 to 2 s. Participants performed 36 trials per condition, which comprised equal numbers of trials in which the probe was shorter than/longer than/equal to the sample in the time condition, or bluer than/redder than/equal to the sample in the colour task. The time and colour conditions were presented twice each in a blocked manner, with each block comprising 18 trials of either the time condition or the colour condition. Fig. 1Temporal and colour discrimination task. Participants estimated either the duration or colour of two consecutive stimuli. The first (sample) and second (probe) stimuli were presented for one of three durations (540, 1,080, 1,620 ms) and had an overall percept of one of three shades of purple (maroon, violet or indigo). Stimuli were not of a uniform colour but instead comprised rapidly flickering (90 ms) presentations of three of five different shades of purple (see inset) to give the overall colour percept. Participants indicated whether the probe was shorter (S), longer (L) or the same (=) duration as the sample ('time' condition) or redder (R), bluer (B) or the same (=) shade of purple as the sample ('colour' condition) using a three-choice button press at the onset of the response screen Stimuli The sample and probe stimuli were presented consecutively, separated by a pseudo-random inter-stimulus interval of 1–2 s. Each stimulus was presented for one of three durations (540, 1,080 or 1,620 ms), and had an overall percept of one of three shades of purple (maroon, violet or indigo) ranging from a reddish to a bluish hue. Duration and colour stimulus attributes were counterbalanced such that any of the three stimulus durations could be paired with any of the three colours. The stimuli to be estimated were not a uniform colour for the entire duration of stimulus presentation. Rather, rapidly alternating presentations (90 ms) of five different shades of purple across the entire stimulus duration gave an overall percept of maroon, violet or indigo. During the colour task, the participant estimated the average shade of purple by amalgamating all shades presented during the flickering percept. This rather unusual colour task was designed to ensure that subjects maintained attention, and continually updated their colour estimate, throughout the entire stimulus presentation. This manipulation is crucial for equating sustained attention and working memory demands across the time and colour tasks since (1) time perception necessitates attention being sustained for the entire duration of the stimulus (~1 s.), whereas perception of a static colour would occur within the first hundred milliseconds or so, and (2) time perception requires stimulus onset to be held in working memory and then continually updated as time elapses, whereas perception of a static colour would require no such updating of working memory. BODY.METHODS.COGNITIVE TASKS.TEMPORAL AND SPATIAL ORIENTING OF ATTENTION: Task The task manipulated participants' expectations of where or when target stimuli would appear within an experimental display. A central cue (inset Fig. 2) predicted either the location (spatial condition) or onset (temporal condition) of an imminent target or else it provided neither temporal nor spatial information (neutral condition). In the spatial and temporal conditions, cues could either correctly (valid trials) or incorrectly (invalid trials) predict where or when the target would appear, with an overall ratio of 80:20 valid/invalid trials. In the neutral conditions, the participants could predict neither where nor when the target would appear. The participants' task in all three conditions was simply to detect the target as rapidly as possible, while avoiding mistakes. Participants indicated covert detection of the target by pressing a response button with their right index finger. Inter-trial intervals varied between 600 and 1,000 ms. The computer recorded reaction times to target stimuli. Subjects were thoroughly familiarized with the cues prior to performing 120 experimental trials of each of the three conditions (360 in total). For the temporal and spatial conditions, this corresponded to 96 valid trials and 24 invalid trials. Fig. 2Temporal and spatial-orienting task. Participants detected a target appearing at one of two peripheral locations (left/right) after one of two cue-target intervals (short/long) as quickly as possible. In the spatial or temporal condition, the visual cue predicted where or when (respectively) the target was likely to appear, with 80% validity. In the neutral condition, the visual cue carried neither spatial nor temporal information Stimuli The basic visual display consisted of a central cueing stimulus (1° eccentricity) and two peripheral boxes (7° eccentricity), inside which the target ('x' or '+') appeared (Fig. 2). The central cue was a compound stimulus consisting of a diamond and two concentric circles. One part of the cue was briefly (100 ms) highlighted to inform the subject whether to attend to the location or time of onset of the target. During the spatial condition, the left (or right) side of the diamond brightened to inform the subject that the target was likely to appear in the left (or right) peripheral box. During the temporal condition, brightening of the inner or outer circle indicated that the target would appear after a short (750 ms) or long (1,500 ms) delay, respectively. During the neutral-cue condition, the entire cue brightened, providing no spatial or temporal information. Targets appeared for 100 ms in one of the two peripheral boxes and after one of two time intervals, according to the nature of the cue: in the spatial condition, targets appeared randomly at the short or long time delay, but their spatial location was determined by the nature of the preceding cue (left/right; valid/invalid); in the temporal condition, targets appeared randomly in the left or right box but their temporal onset was determined by the nature of the preceding cue (short/long; valid/invalid). Spatial locations and temporal onsets were counterbalanced across trials for all three experimental conditions. BODY.METHODS.SUBJECTIVE RATING SCALES: Subjective experience induced by the drug was recorded using a series of clinician-administered questionnaires: the Clinician-Administered Dissociative States Scales (CADSS, Bremner et al. 1998), Brief Psychiatric Rating Scale, Startup and Startup and a Rating Scale for Psychiatric Symptoms. The time perception subscale of the CADSS (Krystal et al. 1994; 1998) indexed the subjective experience of time more specifically (see Appendix). BODY.DATA ANALYSIS.TEMPORAL AND COLOUR DISCRIMINATION: Mean accuracy (percentage of trials correct), omissions (percentage of missed responses) and reaction times (RTs) were analysed in 2 × 2 × 2 repeated measures ANOVAs, with task (time/colour) and treatment (ketamine/placebo) as within-subjects factors, and treatment order (ketamine–placebo/placebo–ketamine) as a between-subjects factor. In addition, proportion of under or overestimates in the time condition were analysed in a 2 × 3 × 2 × 2 repeated measures ANOVA, with error type (under/overestimate), trial type (probe shorter/equal to/longer than sample) and treatment as within-subjects factors, and treatment order as a between-subjects factor. BODY.DATA ANALYSIS.TEMPORAL AND SPATIAL ORIENTING OF ATTENTION: Mean RTs for temporal and spatial orienting conditions were analysed in 2 × 2 × 2 × 2 repeated measures ANOVAs with treatment, cue validity (valid/invalid) and either target onset (short/long) or target location (left/right) (for the temporal and spatial conditions, respectively) as within-subject factors and treatment order as a between-subject factor. Mean RTs for the neutral condition were analysed in a 2 × 2 × 2 × 2 repeated measures ANOVA with target onset, target location and treatment as within-subject factors and treatment order as a between-subject factor. BODY.DATA ANALYSIS.SUBJECTIVE RATING SCALES: Overall CADSS and CADSS time perception subscale scores were analysed in separate 2 × 2 repeated measures ANOVAs with treatment as a within-subject factor and treatment order as a between-subjects factor. Spearman's rank correlations further explored the relationship between these subjective CADSS scores and objective discrimination task performance. Specifically, the association between task performance and (1) global dissociative experience and (2) subjective experience of time was tested by correlating drug-induced change (ketamine–placebo) in mean timing or colour discrimination accuracy with drug-induced change in (1) overall CADSS and (2) CADSS time perception subscale scores, respectively. BODY.RESULTS.TEMPORAL AND COLOUR DISCRIMINATION: A significant main effect of task [F(1, 10) = 11.80, p < 0.01] revealed that the colour task was performed less accurately than the timing task. A significant main effect of treatment [F(1, 10) = 7.34, p < 0.05] was qualified by a significant interaction between treatment and task [F(1, 10) = 8.73, p < 0.05]. Post-hoc analysis with paired t tests indicated that ketamine, as compared to placebo, significantly reduced accuracy in the timing task (t (11) = 3.35, p < 0.01) but had no effect on performance of the colour task (t(11) = −0.44, ns) (Fig. 3a). Ketamine had no significant effect on reaction time or number of omissions. There were no significant main effects of, or interactions with, treatment order. Fig. 3Ketamine impairs perceptual timing. a Ketamine selectively impaired accuracy (% correct) of time discrimination, while having no effect on accuracy of colour discrimination. b During the temporal discrimination task, ketamine significantly increased the likelihood (% trials) that the probe stimulus would be overestimated when the probe was shorter than (<) the sample. Error bars represent the standard error of the mean Given the selective effect of ketamine on timing, we explored these timing data further to examine whether the drug-induced impairments in accuracy were caused by a consistent bias to under or overestimate probe duration. First, each trial was classified according to whether the second stimulus (probe) was longer, shorter or of equal duration to the first stimulus (sample). Then, for each of these three trial types, we calculated the proportion of trials in which a subject under or overestimated the probe compared to the sample. If the probe and sample were of the same duration but the subject judged the probe to be shorter, then this was classed as an underestimate, whereas if the probe was judged to be longer, then this was classed as an overestimate. If the probe was longer than the sample but the participant judged it to be shorter or of equal length, then this was classed as an underestimate. Conversely, if the probe was shorter than the sample but the subject judged it to be longer or of equal length, then this was classed as an overestimate. A main effect of treatment showed that ketamine significantly increased the proportion of both over- and underestimates [F(1, 10) = 11.50, p < 0.01], but did not differentially induce one type of error more than the other (treatment × error-type interaction [F(1, 10) = 0.09, ns]). However, when data were broken down into trial type (probe shorter/equal to/longer than sample), a significant interaction between treatment, error type and trial type [F(2, 20) = 7.34, p < 0.005] indicated that ketamine, as compared to placebo, induced an increased incidence of overestimates, particularly when the probe was shorter than the sample (t(11) = 4.14, p < 0.005) (Fig. 3b). Although there was a corresponding increase in underestimates when the probe was longer than the sample (Fig. 3b), this trend merely approached statistical significance (t(11) = 1.84, p = 0.09). BODY.RESULTS.TEMPORAL AND SPATIAL ORIENTING OF ATTENTION: Any RTs faster than 100 ms were considered anticipatory and removed from the analysis. All remaining RTs were log transformed to normalize distributions (resulting RTs non-significantly different from a normal distribution as assessed by the Shapiro–Wilks test). BODY.RESULTS.TEMPORAL AND SPATIAL ORIENTING OF ATTENTION.TEMPORAL ORIENTING OF ATTENTION: A main effect of cue validity [F(1, 9) = 11.54, p < 0.01] indicated that targets appearing when expected were detected more quickly than targets appearing at an unexpected moment (Table 1). But, as predicted by the results of previous studies using the temporal orienting paradigm (e.g. Coull and Nobre 1998; Coull et al. 2000; Correa et al. 2006), this main effect was qualified by a significant validity × onset interaction [F(1, 9) = 18.24, p < 0.005]. Specifically, the benefit of temporally valid cues was significant at the short (t(10) = −4.71, p = 0.001) but not long (t(10) = −0.48, ns) interval (Table 1): when a target does not appear after the short interval as expected it must, by process of elimination, appear at the longer one. Such temporal predictability effectively removes the cost of targets appearing unexpectedly late. Crucially however, ketamine did not interact with either cue validity [F(1, 9) = 0.25, ns], temporal onset [F(1, 9) = 0.06, ns], nor the interaction between the two [F(1, 9) = 0.05, ns]. Nor did ketamine have any overall effect on performance (main effect of drug [F(1, 9) = 0.43, ns]). Ketamine, therefore, had no effect on the use of temporal cues to predict stimulus onset. Table 1Mean (±standard error) reaction times (RT) to detect targets in a cued simple RT taskTemporal orientingSpatial orientingShortLongLeftRightValidInvalidValidInvalidValidInvalidValidInvalidPlacebo310.06 (±14.37)341.84 (±17.57)290.88 (±15.87)295.50 (±13.78)311.31 (±19.58)339.03 (±18.21)308.30 (±15.78)346.53 (±22.06)Ketamine316.68 (±11.00)346.75 (±10.68)301.53 (±14.45)301.82 (±14.81)321.00 (±14.93)361.01 (±19.41)314.79 (±17.54)354.18 (±24.58)In separate blocks, the cue predicted either the onset (temporal-orienting task) or location (spatial-orienting task) of the upcoming target. RTs are shown for targets appearing after either a short (750 ms) or long (1,500 ms) delay in the temporal-orienting task or on the left or right-hand side in the spatial-orienting task. The predictive information conveyed by the cue could be either valid (80% of trials) or invalid (20% of trials) BODY.RESULTS.TEMPORAL AND SPATIAL ORIENTING OF ATTENTION.SPATIAL ORIENTING OF ATTENTION: As predicted by prior studies (Posner et al. 1980), a main effect of cue validity (F(1, 9) = 29.24, p < 0.0001) confirmed that targets were detected more quickly when they appeared in expected versus unexpected spatial locations (Table 1). Cue validity did not interact with any other factor, including treatment [F(1, 9) = 0.05, ns]. Ketamine, therefore, had no effect on the use of spatial cues to predict stimulus location. BODY.RESULTS.TEMPORAL AND SPATIAL ORIENTING OF ATTENTION.HAZARD FUNCTION: As predicted by numerous previous studies (see Niemi and Näätänen 1981 for a review), a main effect of onset [F(1, 9) = 30.81, p < 0.0001] confirmed that RTs in the neutral cue condition were significantly faster at long versus short intervals. This speeding effect is due to the increasing conditional probability of target occurrence as a function of the passage of time, i.e. the hazard function, which allows for improved response preparation over time, and thus faster RTs. However, a significant treatment by onset interaction [F(1, 9) = 5.83, p < 0.05] revealed that, as compared to placebo, the relative speeding of RTs at the long interval was attenuated by administration of ketamine (Fig. 4). Therefore, although ketamine did not significantly slow performance in a global manner (main effect of drug [F(1, 9) = 1.71, ns]), it selectively compromised the beneficial effects of the hazard function on performance. Fig. 4Ketamine attenuates the beneficial effect of the hazard function. The neutral cue condition of the orienting task produces faster RTs for targets appearing after long rather than short intervals due to the "hazard function". Ketamine slowed RTs selectively at the long, not short, intervals, thus significantly attenuating the benefit that is normally afforded by long delays. Error bars represent the standard error of the mean BODY.RESULTS.SUBJECTIVE RATING SCALES: Consistent with numerous previous reports (e.g. Krystal et al. 1994, 1998; Fu et al. 2005) a main effect of treatment on the overall CADSS score [F(1, 10) = 10.95, p < 0.01] confirmed that ketamine induced a general dissociative state in healthy volunteers, though this effect tended [F(1, 10) = 4.61, p < 0.06] to be more pronounced in subjects receiving ketamine in the first session and placebo in the second (Table 2). More specifically, ketamine significantly [F(1, 10) = 7.22, p < 0.05] increased scores on the CADSS time perception subscale (see also Krystal et al. 1998), indicating a drug-induced distortion in the subjective experience of time. Again, this effect was greater [F(1, 10) = 5.09, p < 0.05] in subjects receiving ketamine in the first session and placebo in the second (Table 2). Table 2Mean (±standard error) scores on the CADSS index of dissociative experienceCADSS overallCADSS time perceptionK/PP/KK/PP/KPlacebo0.04 (±0.04)0.10 (±0.06)00.28 (±0.18)Ketamine1.06 (±0.39)0.32 (±0.13)1.28 (±0.40)0.39 (±0.18)Higher scores represent a greater sense of dissociation and disturbed perception. Overall CADSS score represents the average score of all 27 questions, whereas the time perception subscale is calculated as the mean of the three questions relating specifically to the subjective experience of time. Scores are shown as a function of whether the participant received ketamine in the first session and placebo in the second (K/P) or vice versa (P/K) We used Spearman's rank correlations to search for a link between these subjective measures of perceptual experience and more objective measures of visual perception. Neither the overall CADSS score, nor the time perception subscale, correlated with overall accuracy (% correct) of the time (Spearman's rho = −0.02; Spearman's rho = 0.29, respectively, ns) or colour (Spearman's rho = −0.30; Spearman's rho = −0.06 respectively, ns) discrimination task. However, the overall time accuracy score incorporates errors of both over- and underestimates of probe duration. Prior reports of biases to either over- (Johnson and Petzel 1971; Tysk 1983) or under- (Rabin 1957; Elvevåg et al. 2003) estimation of duration in patients with schizophrenia prompted us to explore our data further for potential links between the subjective experience of time and the direction of objective temporal error (i.e. proportion of over- or underestimates). We found a significant correlation between scores on the CADSS time perception subscale and the proportion of overestimates (Spearman's rho = −0.60, p < 0.05), though not underestimates (Spearman's rho = 0.25, ns) in the time discrimination task. BODY.DISCUSSION: Administration of an acute dose of ketamine (100 ng/ml blood plasma level) selectively impaired perception of temporal, but not colour, attributes of visual stimuli in healthy volunteers. As compared to placebo, ketamine reduced accuracy of perceptual timing while having no effect on accuracy of a colour perception task that was matched to the timing task for accessory cognitive processes, such as visual processing, sustained attention and working memory (WM). This is the first time that selective effects of ketamine on a perceptual timing task have been shown. Despite our relatively small sample size (n = 12), the selectivity of the results, demonstrated by a significant task by drug interaction, is striking. Although the colour task was more difficult than the timing task, the lack of drug effect on the colour task is unlikely to merely reflect a floor effect since participants' performance in both sessions (~70% accuracy) was well above chance (33%), allowing sufficient margin for further deterioration in performance. Selective impairment on performance of the timing task was reflected in participants' subjective assessment of time. Ketamine significantly altered the inner experience of time and drug-induced changes in subjective ratings correlated with objective measures of duration overestimation in the perceptual timing task. Further evidence of timing disturbances were noted during performance of a cued reaction time (RT) task with variable cue-target intervals. In this task, RTs are generally faster for targets appearing at long, rather than short, intervals due to the increasing conditional probability of target appearance over time (Niemi and Näätänen 1981). However, ketamine slowed RTs selectively at the long interval, thereby attenuating the increase in speed that is normally seen at long delays. By contrast, ketamine had no significant effect on RT when temporal pre-cues could be used to predict in advance when the target would appear. Similarly, ketamine had no effect on the ability to use spatial cues to predict target location, consistent with previous results in schizophrenic patients (Carter et al. 1992). BODY.DISCUSSION.ARE EFFECTS OF KETAMINE ON TIMING DUE TO WM DEFICITS?: Rammsayer (2006) concluded that effects of the NMDA receptor antagonist memantine on timing in the seconds (~1 s.) but not tens of milliseconds (~50 ms) range were due to a primary effect on the additional WM processes that are needed to process seconds-range durations. We aimed to disentangle drug effects on timing from effects on WM by examining the effects of ketamine on two tasks that were matched for WM demands, in terms of both manipulation and maintenance in WM. During the 500–1,500 ms stimulus presentation time in both tasks, subjects had to maintain attention and integrate (i.e. manipulate) either duration or colour information in WM: for the timing task, duration estimates had to be updated incrementally throughout stimulus presentation; for the colour task, colour estimates had to be averaged across stimulus presentation. Both tasks also required that a sample stimulus be stored (i.e. maintained) in WM for a delay of 2–4 s for comparison with a later probe, and for the resulting decision to be maintained in WM until appearance of the response screen. Since mnemonic (and attentional) load were thereby matched across tasks, selective effects of ketamine on timing, but not colour, discrimination, suggests that ketamine-induced impairments do not simply reflect a functional side effect of the drug on the WM processes that are necessary for processing seconds range rather than millisecond timing. However, one difference between the timing and colour task was the specific way in which information was manipulated in WM. For the timing task, information was incrementally accumulated, whereas for the colour task, it was averaged. Accumulation implies a unidirectionality, a fundamental feature of the flow of time itself ('time's arrow', Eddington 1928). Averaging does not imply this unidirectionality. It is possible therefore, that ketamine influences timing behaviour by selectively impairing the ability to manipulate information in WM in a particular direction or order. A related possibility is that subjects were accumulating not time, but a numerical count of the flickering stimulus. However, this is unlikely given that (1) each flicker was presented for a duration of only 80 ms necessitating an abnormally fast rate of counting and (2) the vast majority of our trials contained at least one stimulus whose duration was below the critical 1,200 ms value at which counting begins to become a useful strategy (Grondin et al. 1999). BODY.DISCUSSION.TEMPORAL EXPECTATIONS AND THE FLOW OF TIME: As expected, both spatial (Posner et al. 1980) and temporal (Coull and Nobre 1998) cues speeded RTs when targets appeared at expected, rather than unexpected, locations or delays, respectively. These RT effects were not modulated by ketamine, in either the spatial or temporal domain. Although the glutamatergic system has previously been implicated in signaling mismatch between expected and actual outcome (also known as the prediction error) (Corlett et al. 2007a; 2011), we found no evidence in this study for a selective effect of ketamine on RTs to unexpected target locations or onsets. This null effect could represent a lack of effect of ketamine on the processing of unexpected events. Alternatively, it may represent the insensitivity of our behavioural measures: the behavioural consequences of prediction error are rather elusive, and it has previously proven easier to demonstrate the underlying neural responses to prediction error, and their modulation by ketamine, using, for example electrophysiological recordings (e.g. Schultz 1998; Umbricht et al. 2002) or fMRI (e.g.McClure et al. 2003; O'Doherty et al. 2003; Corlett et al. 2006). Future studies examining the effects of ketamine on neural responses to unexpected locations or onsets could help resolve this issue. A final possibility is that these null effects simply reflect a lack of power in the data. However, the predicted behavioural advantages of valid versus invalid cues were clearly present in both the temporal and spatial conditions, itself suggestive of sufficient power. Moreover, although ketamine had no effect on speed of responding in the temporal or spatial conditions, it selectively slowed RTs to long delay targets in the neutral condition. Together, these patterns of data suggest a selectivity of effect rather than insufficient power. In the orienting task, temporal cues allow subjects to predict event onset, thus generating temporal expectations that help speed performance (Coull and Nobre 1998; Griffin et al. 2001). Yet, even when the onset of the imminent event cannot be predicted by arbitrary, pre-learned cues, the unidirectional flow of time itself (Eddington 1928) provides a degree of predictive power via the hazard function. In our study, the neutral cue condition of the orienting task, in which participants did not know in advance, whether the target would appear at the short or long delay, provides an index of the hazard function. We observed the expected speeding of RTs for targets appearing at long versus short delays following administration of placebo. Ketamine, however, significantly attenuated this increase in response speed (Fig. 4), slowing RTs selectively at the longer delay. Previous studies using a fixed foreperiod paradigm have also reported ketamine-induced slowing selectively at long delays (Guillermain et al. 2001; Micallef et al. 2004). This was interpreted as reflecting an effect of ketamine on motor preparation processes due to particularly impaired temporal resolution at long delays. However, this is an unlikely explanation for our own results since ketamine did not slow RTs at long intervals when participants could use arbitrary temporal cues to predict in advance when the target would appear (i.e. the temporal cue condition of the orienting task), suggesting they were perfectly able to estimate and prepare for the long interval as long as it had been cued beforehand. Ketamine did not therefore affect the ability to use prelearned cues to predict in advance when the target would occur but, instead, interfered with the ability to use the very passage of time itself to predict target occurrence when no other sources of temporal information were available. Ketamine effects have previously been interpreted within a Bayesian framework (Corlett et al. 2009; 2011), in which information processing is contingent upon prior expectations. In Bayesian terms, our data show that ketamine does not disrupt the ability to make use of prior expectations to interpret sensory events, but does interfere with the ability to generate expectations online as a function of sensory evidence accumulated over time. BODY.DISCUSSION.RELEVANCE TO SCHIZOPHRENIA: Temporal predictability in the temporal cue condition of the orienting task is driven by external, visual cues. Conversely, temporal predictability in the neutral cue condition, where visual cues are uninformative, depends upon an internal sense of the passage of time. After administration of ketamine, participants made good use of external visual cues to make temporal predictions, but were less able to use internal estimates of time to make predictions. This psychopharmacological dissociation is reminiscent of findings in patients with schizophrenia who rely more upon external sensory stimuli to interpret the consequences of their actions, rather than internal sensorimotor processes (Synofzik et al. 2010; Voss et al. 2010). Moreover, while the temporal cue condition requires straightforward maintenance of a fixed a priori temporal prediction in WM, the neutral cue condition requires updating (i.e. manipulation) of the prediction as a function of elapsing time. Notably, both ketamine and schizophrenia have previously been associated with deficits in manipulation, rather than maintenance in WM (Fletcher and Honey 2006). Our orienting data, taken together with the results on the temporal discrimination task discussed above, support the proposal that ketamine interferes with the way in which the unidirectional flow of time is integrated within WM. Intriguingly, disturbances in the temporal flow (past–present–future) of causal experience have previously been suggested to underpin certain symptoms of schizophrenia (Fuchs 2007). The timing deficits observed in schizophrenia may therefore be more accurately characterised as impairments in temporal flow, such that the beginning, middle and end of the event to be timed are not necessarily processed (or retained in WM) in the correct order, thus compromising duration estimates. Further experiments are clearly needed to test this hypothesis more directly. Patients with schizophrenia have previously been reported to both over- (Johnson and Petzel 1971; Tysk 1983) and underestimate (Rabin 1957; Elvevåg et al. 2003) stimulus duration. We therefore examined whether ketamine produced a consistent temporal error in one direction or the other by measuring the incidence of over- or under-estimation of probe duration. Overall, ketamine induced both types of error, with no significant bias in one direction or the other (see also Cheng et al. 2006). However, a particularly high incidence of drug-induced overestimation was evident whenever the probe stimulus was shorter than the sample. This result is unlikely to reflect a ketamine-induced increase in speed of an internal clock (Gibbon et al. 1984), because any change in clock speed would apply equally to both sample and probe stimuli. Instead, ketamine appears to have either selectively elongated perception of the current (probe) event, or contracted the memorized representation of the sample. This issue could be addressed by future experiments in which the sample duration is encoded either before or after administration of ketamine (see Malapani et al. (2002) for an example of this approach in patients with Parkinson's disease). Finally, patients with schizophrenia (Carroll et al. 2008) or individuals at high risk for schizophrenia (Penney et al. 2005) have previously been reported to show larger than normal differences in the relative accuracy of auditory versus visual timing, potentially due to a preferential impairment of timing in the auditory modality (Carroll et al. 2008). If ketamine can be considered a useful model of schizophrenia, then we may expect to replicate this 'modality effect' with ketamine and, furthermore, to predict that its effects on timing would be even more evident when tested in the auditory domain. BODY.DISCUSSION.POSSIBLE NEURAL SUBSTRATES: We have shown that ketamine impairs accuracy on a temporal discrimination task that has previously been shown to engage Supplementary Motor Area, right prefrontal cortex and basal ganglia (Coull et al. 2004; 2008). Although ketamine had no effect on the ability to use visual cues to predict stimulus onset in the temporal orienting task, a process previously shown to engage left inferior parietal cortex and intraparietal sulcus (Coull and Nobre 1998; Coull et al. 2011), it did impair the ability to use the flow of time to make temporal predictions (the hazard function), a process that has consistently been shown to depend upon right prefrontal cortex (Vallesi et al. 2007a, 2007b, 2009; Bueti et al. 2010). Taken together with data from the temporal discrimination task, we suggest that ketamine may impair timing by modulating right prefrontal cortex function. This hypothesis is supported by functional imaging data showing that ketamine modulates activity in prefrontal cortex, among other areas, during tasks of WM and executive function (Fu et al. 2005; Honey et al. 2008) or prediction error (Corlett et al. 2006). These data might indicate therefore that timing depends upon glutamatergic function in prefrontal cortex. Alternatively, ketamine is known to increase prefrontal dopamine in rats (Bowers and Morton 1994; Verma and Moghaddam 1996), suggesting that its effect on timing may instead be a result of drug-induced modulation of prefrontal dopamine. Therefore, just as dysregulation of dopaminergic innervation of prefrontal cortex has been suggested as the common underlying mechanism for both schizophrenia and ketamine-induced impairments in WM (Fletcher and Honey 2006), it may also explain the timing deficits associated with these two states. Notably, the dopaminergic system has repeatedly been implicated in timing (Meck 1996), with converging evidence from the animal and clinical literatures (Coull et al. 2011). Yet dopaminergic manipulations provoke consistent changes in clock speed, with enhanced dopaminergic activity leading to overestimates of stimulus duration (Maricq and Church 1983; Meck 1983; Matell et al. 2004, 2006) and reduced activity inducing underestimates (Maricq and Church 1983; Meck 1983, 1986; MacDonald and Meck 2005) and results of the present study, as well as prior work in rats (Cheng et al. 2006, 2007a), suggest ketamine has no effect on clock speed. Instead, Cheng et al. (2007a, 2007b) suggest ketamine 'unlocks' the clock-speeding effects of dopaminergic agonists that are otherwise attenuated by extensive training. Specifically, although administration of dopaminergic agonists is known to speed the internal clock, extensive training on the task abolishes this effect (Cheng et al. 2007a, 2007b). The hypothesis is that as performance on the timing task becomes more and more automated through training, the dopaminergic system becomes less critical for timing, and instead, the glutamatergic system takes over to mediate a more habitual form of timing behaviour. If a glutamatergic antagonist, such as ketamine, is administered, then control reverts to the dopaminergic system, meaning dopaminergic agents will once more produce their clock-speeding effects. So why, in contrast to Cheng et al. (2006, 2007a), does ketamine modulate timing in our relatively naive participants? The answer may lie, once more, with the hazard function. Keeping track of the unidirectional flow of time in WM in order to time current events, as well as to predict the onset of future events, is an overlearned, automated process that, as suggested above, appears to be subject to disruption by ketamine. Therefore, the results of this study appear not to reflect a dopamine-dependent clock-speed effect, but may instead indicate a glutamate-dependent modulation of the unidirectional nature of 'time's arrow'. Alternatively, in line with ketamine's effects on the manipulation of information in WM (Honey et al. 2003, 2004), our results may reflect dopamine-dependent modulation of the way in which temporal information is integrated within WM. Future studies combining functional neuroimaging and psychopharmacological methodologies could ascertain more directly the role of prefrontal dopamine or glutamate in the effects of ketamine on timing or, more specifically, on the ability to integrate the unidirectional flow of time in WM.

TITLE: Experience of unpleasant sensations in the mouth after injection of saline from prefilled syringes ABSTRACT.BACKGROUND: Nurses at The Norwegian Radium Hospital have reported that some patients notice an unpleasant smell or taste in accordance with flushing of intravenous lines with commercially available prefilled syringes. We have conducted a study in healthy volunteers to investigate the occurrence, consistency and intensity of this phenomenon. ABSTRACT.METHODS: A randomised, blinded, crossover study comparing commercial available prefilled saline 9 mg/ml syringes to saline 9 mg/ml for injection in polyethylene package was performed in 10 healthy volunteers. The volunteers were given intravenous injections of varying volume and speed. Data were analysed using descriptive statistics, and also Wilcoxon Signed Rank Test to compare groups. ABSTRACT.RESULTS: After intravenous injection, 2 of 15 recordings demonstrated any sensation of smell or taste after injection of saline from polyethylene package, while 14 of 15 recordings noted a sensation after injection of saline from prefilled syringes. The intensity of the unpleasant sensation was rated significantly higher after injection of saline from prefilled syringes compared to saline from polyethylene (p = 0.001). ABSTRACT.CONCLUSIONS: Injection of saline from prefilled syringes in healthy volunteers resulted in an experience of bad taste or smell. It is important that nurses and health workers are aware of the phenomenon as described in this article in order to choose the preferred product for a given patient. BODY.BACKGROUND: In order to maintain the patency of vascular access devices like intravenous cannulaes and central venous lines, these devices are regularly flushed with saline [1]. Commercially available prefilled syringes are in some institutions used to ease the work of the nursing staff. In addition, there have been arguments in favour of these prefilled saline syringes regarding decreased risk of infection and medication errors [2]. In addition, there are organizations and regulatory agencies in some countries that recommend the use of single dose containers for all medications including catheter flushing and locking solutions. However, nurses report that some patients notice an unpleasant smell or taste in accordance with the flushing procedure. This is also mentioned in the Instruction for Use enclosed in the package, where it is noted "Some patients may experience a transitory taste or odour during flushing. This minor effect ceases shortly after the procedure." We have conducted a study in healthy volunteers to investigate the occurrence of this phenomenon and the consistency and intensity of the experience. BODY.METHODS: A literature search regarding prefilled saline syringes and experience of unpleasant taste, flavour, or odour was carried out. Becton Dickinson Pharmaceutical Systems was contacted for possible background information. A High performance liquid chromatography (HPLC) analysis of saline from BD Saline XS (Becton Dickinson, 10 ml pre-filled syringe) and from Mini-Plasco® connect (B. Braun, 20 ml polyethylene bottle) was conducted. Fifty microliters of sample was injected on an Agilent 1100 system and separated through a 0.46 × 10 cm C18 Prodigy ODS column using a linear gradient from 10% to 90% acetonitrile in water over 30 minutes. Drift and background was corrected using 18.2 MΩ/cm Milli-Q water). Then, a randomised, blinded, crossover study comparing prefilled syringes (BD Saline XS, Becton Dickinson [BDs]), to normal saline (from B. Braun, in polyethylene packaging [rSaline]) was performed. 10 healthy volunteers participated, 2 male and 9 female, age 27 to 58. Written informed consent was obtained from all participants. Randomisation list was constructed from Research Randomizer http://www.randomizer.org/form.htm. The volunteers were asked to describe taste and smell after an injection of the two different types of saline (BDs and rSaline). Since this was done in a cross-over fashion (5 volunteers were given BDs - rSaline - BDs, and 5 volunteers rSaline - BDs - rSaline), 15 registrations were recorded for each test. For each experiment we registered taste or smell with Yes or No. We also used a Numeric Rating Scale where 0 represents no bad taste or smell what so ever and 10 represents the worst taste or smell conceivable. The volunteers were given the following categories of smell and taste to describe their experience: Bitter, sweet, sour, salt, metallic, stale, nauseous. The volunteers were also given the opportunity to describe taste/smell with their own words. The experiment was conducted in the following manner: An intravenous cannula was inserted into the arms of the volunteers. The volunteers were blindfolded. They were given a 10 ml intravenous injection of BDs or rSaline, in the course of 5 seconds. The volunteers gave feedback on the extent of the smell and/or taste they experienced after receiving the injection. The same procedure for the second and third injection was used. All three injections (of BDs and rSaline) were given in a random order. After 15 minutes one injection of 10 ml BDs was given to all volunteers within a 30 second period. Finally, (after 15 minutes) one injection of 3 ml of BDs was given to all volunteers within a 3 second period. The purpose with the different injections was to register if the degree of any smell/taste was influenced by low/high injection speed or low/high volume (ml). The volunteers flushed their mouths with tap water after each test. The healthy volunteers received 30 € to participate in the project. The Regional Committees for Medical Research Ethics in Norway and the Norwegian Social Science Data Services approved the study. BODY.METHODS.STATISTICS: SPSS software, version 16.0 (SPSS, Chicago, IL), was used for statistical analyses. Descriptive data and Wilcoxon Signed Rank Test (2-tailed) were applied since normal distribution was not guaranteed and we tested a small number of volunteers. A P value < 0.05 was regarded as statistically significant. BODY.RESULTS: A literature search in PubMed, EMBASE and SveMed using the search terms, Becton Dickinson, prefilled syringes, saline, odour, and taste (single terms and in combination), revealed no published articles addressing this phenomenon. Chromatograms from HPLC-analysis conducted in our laboratory revealed that both products tested contained a number of unidentified substances generating chromatographic peaks (fig. 1). Figure 1Chromatograms of saline from BD Saline XS (-) and from Mini-Plasco® connect (-). Detection by UV-absorbance at 240 nm. Acetonitrile gradient from 10% to 90% as indicated by solid line. Drift and background was corrected using 18.2 MΩ/cm Milli-Q water. Information from Becton Dickinson showed that the presence of volatile substances in the plastic material of the syringes was discovered in 2001 and that these substances were linked to the experience of minor reactions like bad taste or smell. Becton Dickinson concluded that these substances represented no toxic or pharmacological risk to patients' health. The identification and saline-solution concentrations provided by Becton Dickinson were: 2-methyl-2-propanol: 8.5 ppm; 2-methyl-2-butanol: 0.7 ppm; ethyl-buthyl-ether: 0.4 ppm. Two of 15 recordings demonstrated any sensation (smell/taste) after injection of rSaline, while 14 of 15 recordings noted a sensation after injection of BDs. Stale, metallic and sweet were the standard descriptors most used. In addition 4 persons used the word plastic. The intensity of an unpleasant sensation after injection of BDs 10 ml over 5 seconds was rated significantly higher compared to rSaline, 10 ml over 5 seconds (p = 0.001), see figure 2. BDs 10 ml over 30 seconds and BDs 3 ml over 3 seconds are demonstrated in figure 3. Although the sensation when injected at slower speed, or less volume was lower than with 10 ml over 5 seconds, these differences were not statistical significant compared to an injection of 10 ml BDs over 5 seconds. Figure 2Box plot of volunteers' experience of intensity of unpleasant taste or odour after injection of 10 ml saline (during 5 seconds) from prefilled syringes from Becton Dickinson (BDs) and regular saline from polyethylene bottles (rSaline). Y-axis: Numeric rating scale where 0 = no smell or taste and 10 = worst taste or smell conceivable. Figure 3Box plot of volunteers' experience of intensity of unpleasant taste or odour after injection of 10 ml saline (during 30 seconds) and injection of 3 ml (during 3 seconds) from prefilled syringes from Becton Dickinson (BDs). Y-axis: Numeric rating scale where 0 = no smell or taste and 10 = worst taste or smell conceivable. BODY.DISCUSSION: Injection of saline from BD-syringes resulted in a sensation of bad taste or odour in most volunteers. The phenomenon of bad taste or smell is explained by the mechanism of volatile substances, released from saline when injected into the bloodstream and then eliminated by the respiratory system and thus detected by the patients in the air expired by the olfactory system. A sensation of taste, mediated by the taste buds, is usually combined with stimulation of touch and smell. Thus it is not surprising that the volunteers confused taste, flavour, and odour. We cannot find any documentation regarding the frequency or the intensity of this unpleasant experience after flushing of saline with these prefilled syringes. The phenomenon is probably underreported as few patients will find it logical to associate an unpleasant odour with flushing of intravascular devices. The chromatographic peaks demonstrated in the HPLC-analysis (fig. 1) identify substances in the saline (from BD-syringes) that are not found in regular saline. Whether these peaks represent the volatile substances are likely, but not established. According to Becton Dickinson, these volatiles substances (2-methyl-2-propanol: 8.5 ppm, 2-methyl-2-butanol: 0.7 ppm, ethyl-buthyl-ether: 0.4 ppm) present no toxic or pharmacological risk to the patient's health even when administered in dosages 1000 times that observed with normal flushing. However, these effects can be unpleasant for the patients, and might be even worse in nauseated patients whether from anaesthesia or chemotherapy. Probably, the experience is especially disturbing for cancer patients under lengthy chemotherapy who often are more sensitive to taste perceptions [3]. It is claimed from Becton Dickinson that the effect usually can be avoided by using a flushing procedure with a slower flow rate or smaller volume (Becton Dickinson - personal communication). This was not the case in our study. A limitation is the small number of experiences with low injection rate and small volume. The benefit of prefilled syringes could improve clinician work flow and reduced time required to prepare flush syringes. Furthermore, labeling as part of the manufacturing process increases patient safety. Potential reduction in microbial contamination when used for vascular access devices is also an attractive argument, although documentation from good clinical controlled studies are lacking. However, infectious outbreaks have been associated with improper use of multiple dose vials of saline and heparin. As mentioned previously, different organizations and regulatory agencies in some countries (particularly in the United States) recommend the use of single dose containers for medications including flushing solutions. This means that prefilled syringes are the only options in many places. When weighing the overall risk versus benefits of prefilled syringes, the issue of altered taste could be argued to represent a minor complaint compared to the potential benefits or reduced risk of infection and medication errors. BODY.CONCLUSION: Injection of BD-saline in healthy volunteers resulted in an experience of bad taste or smell. When considering the pros and cons of prefilled saline syringes, this phenomenon should be taken into consideration. The manufacturers of these prefilled saline syringes should find ways to reduce the phenomenon. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: UEK participated in all aspects of the project and secured funding. AA helped design the study, performed the HPLC analysis, commented on the results and revised the manuscript. MØ participated in the practical implementation of the project, acquisition of data and commented on the results. I-AYO helped design the study and participated in the practical implementation of the project, acquisition of data and helped draft the manuscript. LIB initiated the project, helped design the study, and commented on and interpreted the results. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1472-6955/9/1/prepub

TITLE: Randomized, Double-Blinded, Phase 2 Trial of WR 279,396 (Paromomycin and Gentamicin) for Cutaneous Leishmaniasis in Panama ABSTRACT: In this randomized, double-blinded Phase 2 trial, 30 patients with Leishmania panamensis cutaneous leishmaniasis were randomly allocated (1:1) to receive once daily topical treatment with WR 279,396 (15% paromomycin + 0.5% gentamicin) or Paromomycin Alone (15% paromomycin) for 20 days. The index lesion cure rate after 6 months follow-up was 13 of 15 (87%) for WR 279,396 and 9 of 15 (60%) for Paromomycin Alone (P = 0.099). When all treated lesions were included, the final cure rate for WR 279,398-treated patients was again 87%, but the final cure rate for Paromomycin Alone-treated patients was 8 of 15 (53.3%; P = 0.046). Both creams were well tolerated with mild application site reactions being the most frequent adverse event. The increased final cure rate in the WR 279,396 group in this small Phase 2 study suggests that the combination product may provide greater clinical benefit than paromomycin monotherapy against L. panamensis cutaneous leishmaniasis. BODY.INTRODUCTION: Leishmania infection is endemic in 98 countries or territories, with more than 350 million people at risk. Published figures indicate an estimated incidence of 2 million new cases per year (0.5 million visceral leishmaniasis [VL] and 1.5 million of cutaneous leishmaniasis [CL]).1 In the United States (U.S.), CL has been reported in southern Texas along the Mexican border2 and in travelers returning from endemic areas.3 In the U.S. Military over 2,878 cases of CL has been parasitologically confirmed since April 2003 at the Leishmania Diagnostics Laboratory at the Walter Reed Army Institute for Research. Although CL ultimately self-cures, the infection can create substantial morbidity caused by the continued presence of a skin ulcer and the psychological impact of disfigurement4; there is no U.S. Food and Drug Administration (FDA) approved drug for CL in the United States, no available vaccines, and no chemoprophylaxis. Several antileishmanial chemotherapeutic agents have been developed and evaluated for the treatment of VL,5 a life-threatening disease where the need for systemic therapy is compelling.6 An inherent difficulty with using anti-VL drugs for CL is that benefit may easily outweigh the toxicity of systemic agents for a fatal disease such as VL, but such toxicity may be harder to justify for a non-fatal disease such as CL.6 As a result, treatment of CL is unsatisfactory and is currently the major unmet medical need for the leishmaniases. A new approach for CL treatment favored by the World Health Organization (WHO) and other experts is the use of a local treatment followed by parenteral treatment only if the local treatment fails or cannot be performed.7,8 This step-wise approach is intended to limit the risk of severe adverse events, increase compliance, facilitate CL treatment, and reduce cost while preserving efficacy. One prominent non-systemic treatment is the topical application of paromomycin-containing creams. To create a paromomycin cream that would be simultaneously effective, well tolerated, and improve healing, we developed a cream (WR 279,396) containing paromomycin sulfate 15% plus gentamicin sulfate 0.5% in a complex base to aid drug penetration. Gentamicin was included in the formulation because gentamicin augmented the effect of paromomycin in mouse models of CL, particularly against the New World species, Leishmania panamensis and Leishmania amazonensis.9 In a Phase 2 clinical trial in Tunisia,10 WR 279,396 was significantly more effective than vehicle control versus Leishmania major at the same study site at which a WHO topical formulation (paromomycin without gentamicin) was ineffective.11 In the course of developing WR 279,396, two studies of the pharmacokinetics (PKs) of this combination formulation and of Paromomycin Alone in the same complex base were performed in CL patients in Panama and Peru. The combined PK data from both sites is being reported in a separate publication (accepted for publication). Efficacy and safety data were secondary endpoints in these studies. Here, we report the efficacy and safety of WR 279,396 compared with Paromomycin Alone against CL in the Panama study. BODY.METHODS.ETHICS.: The study was sponsored by the Office of the Surgeon General, Department of the Army, USA, and it is registered with ClinicalTrials.gov identifier NCT 01083576. The protocol was approved by the Panamanian National Committee of Bioethics for Research, Panama City, Panama, and by the Human Research Protections Office, U.S. Army Medical Research and Materiel Command, Ft. Detrick, Maryland. The study complied with all applicable laws, rules, and regulations of the United States and Panama, and it was conducted in accordance with the International Conference on Harmonization Good Clinical Practice Guidelines, the Belmont Principles, and the ethical principles that have their origins in the Declaration of Helsinki. The investigators adhered to the policies for protection of human subjects as prescribed in U.S. Army Regulation 70-25. Informed consent was obtained from all study participants and/or guardians before enrollment. Minors also provided assent. BODY.METHODS.STUDY PATIENTS.: Eligible patients were males or non-pregnant/non-lactating females; ≥ 5 years of age; with ≤ 10 lesions; and with one of these lesions (the index lesion) having the following characteristics: ulcerative, at least 1 cm, and < 5 cm in greatest diameter of lesion, including induration, and confirmed to contain Leishmania by culture or microscopic examination of lesion material. The reason to designate one lesion as the index lesion is that the response of at least that lesion would reflect the efficacy of treatment on an ulcer known to be caused by Leishmania. Subjects could not have signs of disseminated disease, against which a topical treatment would not be expected to be effective or recent treatment (within 8 weeks of starting study treatments) with a recognized antileishmanial, and were generally healthy otherwise. BODY.METHODS.SAMPLE SIZE.: As the study was primarily a PK evaluation that was not designed to compare the efficacy of the two topical creams, the selection of the number of subjects (15 in each arm) was based on the following objectives: 1) to obtain PK data which, when combined with PK data from a similarly designed Phase 2 study in Peru, would provide a collective body of data to determine the extent of systemic drug exposure; and, 2) to obtain sufficient data to have a preliminary estimation of the initial clinical cure rate as a basis for calculating sample sizes for a possible larger trial. BODY.METHODS.STUDY DESIGN.: This was a randomized, double-blinded, parallel-group study in which the PKs, efficacy, and safety of WR 279,396 and Paromomycin Alone creams were compared for the treatment of L. panamensis CL in Panama. After establishing eligibility for the study, a total of 30 patients were randomly assigned in 1:1 allocation to the two treatment groups in a blinded manner. To balance treatment assignments by age group, a permuted block randomization method was used to generate the treatment randomization within age groups. Subjects were stratified by age group: 5–11 years, 12–7 years, and ≥ 18 years of age. No more than 18 subjects could be randomized in any age range, so that there would be at least six subjects in each age stratum evaluable for the PK analysis. Subjects were treated once daily for 20 days with the topical creams. Blood samples for PKs were collected during the first 20 days, local application site toxicity was assessed daily, and lesion sizes were measured five times during the treatment period. Subjects were followed at weekly intervals after completing treatment of safety and efficacy up to Day 63, and then had final follow-up visits at Days 100 and 168. BODY.METHODS.STUDY AREA.: This study was conducted between 16 February 2010 and 28 March 2011 on patients recruited from the area surrounding Panama City, Panama, where L. panamensis is prevalent. BODY.METHODS.PARASITOLOGY.: Lesion samples were obtained from ulcerative lesions by scraping and aspiration at baseline and 1 day after the end of therapy on Day 21. Specimens were smeared onto microscope slides, placed in culture medium, and analyzed at the Parasitology Laboratory of the Instituto Conmemorativo Gorgas de Estudios de la Salud (ICGES), Panamá City, Panamá. At the ICGES, proof of infection caused by leishmaniasis was documented through 1) the microscopic identification of Leishmania amastigotes by Giemsa staining of specimen slides; and/or 2) by the demonstration of motile Leishmania promastigotes in aspirate cultures. Whenever possible, Leishmania species identification (speciation) was also performed using polymerase chain reaction (PCR) analysis or isoenzyme analysis. For PCR, a sample of lesion scrapings from each subject was sent to the ICGES Parasitology Laboratory for analysis12,13; for isoenzyme analysis, only samples of cultures that were positive at the ICGES were sent for isoenzyme analysis14 to the Leishmania Diagnostics Laboratory, Walter Reed Army Institute of Research, Silver Spring, MD. BODY.METHODS.DRUG ADMINISTRATION.: WR 279,396 and Paromomycin Alone creams were manufactured by Teva Pharmaceuticals USA, Sellersville, PA, in accordance with Good Manufacturing Practice. For each patient, all lesions (i.e., the index lesion plus any non-index lesions) were treated topically once daily for 20 days. Before the first application, the lesions were cleaned with soap and water, debrided, and then dried. At each subsequent application of the investigational creams, the previous day's application and dressing were removed, the lesion sites were cleaned with soap, water, and sterile 0.9% saline, and then dried using sterile cotton gauze sponges, and redressed. A generous amount of cream was applied to each lesion by rubbing the cream into the lesion for about one minute before applying the dressing. BODY.METHODS.STUDY PROCEDURES.: Medical history, physical exam, vital signs, parasitology, and baseline clinical laboratory measurements (alanine aminotransferase, aspartate aminotransferase, glucose, sodium, potassium, creatinine, white blood cells, hemoglobin, and platelet count) were performed to establish patient eligibility for inclusion in the study. Females of childbearing potential had a pregnancy test. Serum creatinine was repeated at the end of treatment (Day 20), and parasitology was also repeated one day after completing treatment (Day 21). Adverse events were assessed at every visit during the study. Lesion measurements and vital signs were taken before treatment, five times during treatment, and at all follow-up visits. Lesions were photographed before treatment and at all follow-up visits. BODY.METHODS.FOLLOW-UP AND TOXICITY EVALUATION.: Efficacy was assessed by measuring the CL lesion area (calculated according to the formula for an ellipse) at baseline, at the end of therapy on Day 20, and at follow-up on Days 28, 35, 42, 49, 56, 63, 100, and 168 (end of study). Application sites (CL lesions and surrounding area) were inspected daily during treatment of erythema and edema/swelling and any other clinical signs, and the patient was asked about pain using the Wong-Baker FACES pain rating scale.15 General questions about adverse events were also asked at each visit. Medications to treat side effects were recorded. Serum creatinine was measured at Day 20 to assess potential aminoglycoside-related renal toxicity. BODY.METHODS.CLINICAL ENDPOINTS CRITERIA.: Final clinical cure was defined as (A and C) or (B and C), where A = patient had initial clinical cure (100% re-epithelialization of index lesion by nominal Day 63); B = patient had initial clinical improvement (> 50% re-epithelialization of index lesion by nominal Day 63 followed by 100% re-epithelialization of the index lesion on or before nominal Day 100; and C = patient had no relapse of index lesion. Relapse was defined as an index lesion meeting the criteria for initial clinical cure or initial clinical improvement that had any new ulceration (> 0 × 0 mm measurement) by nominal Day 168. The protocol-specified primary efficacy endpoint was the number of subjects with an index lesion that exhibited final clinical cure. If the subject was withdrawn early from the study, this subject was then considered a treatment failure. The occurrence of new lesions was not considered a treatment failure, as the investigational drugs were administered directly to the lesion and no systemic effects were expected. Secondary endpoints included number of subjects where all baseline lesions that received treatment met the definition for final clinical cure and all lesions treated independently of the subject. The safety endpoints were adverse events in general, application site reactions, and aminoglycoside renal toxicity determined by serum creatinine measurements at the end of therapy on Day 20. BODY.METHODS.STATISTICAL ANALYSIS.: Statistical analyses were performed using SAS software, version 9.2 (SAS Institute, Inc., Cary, NC). The modified intention-to-treat (mITT) population, which included all randomized subjects who received at least one application of a study drug, was used for efficacy and safety analyses. There were no formal hypotheses tested with respect to efficacy endpoints as the study was primarily a PK evaluation. It was not designed to compare the efficacy of the two topical creams, but rather to collect data to support the design of a possible Phase 3 trial. However, exploratory analyses were performed on efficacy endpoints to determine if the observed differences in the two study groups were statistically significant. The null hypothesis was that there was no difference in the primary and secondary efficacy endpoints between the 2 study groups. Final clinical cure rates were compared between the two study groups by two-sided uncorrected χ2 analysis.16 Although the sample size in this study was small (30 total subjects), as this study was "One Margin Fixed Design," the χ2 analysis was considered more appropriate than the more conservative Fisher's exact test to explore the clinical endpoints.17 Baseline variables were explored statistically to identify differences that might have an impact on outcomes to be considered in possible future study designs. BODY.RESULTS: Of the 54 patients who were screened, 30 were eligible for the study and were randomized equally between the two treatment groups (Figure 1). Of the 24 subjects not eligible to participate in the study, 10 had lesions that were parasitologically negative, 5 had a single lesion that was < 1 cm, 2 had a single lesion > 5 cm, 2 had > 10 lesions, 2 had started antimonial therapy, 1 did not have an ulcerated lesion, and 2 started screening but recruitment ended. Six subjects in the Paromomycin Alone group and one subject in the WR 279,396 group were withdrawn from the study by the investigator before the final visit at Day 168 because of treatment failure. All of these subjects were included in the mITT analysis. Figure 1.Subject disposition. A total of 54 subjects were consented and screened of which 30 were randomized. All but one of the 30 subjects received 20 days application of study drug. One subject missed one day. Subjects who were withdrawn early from the study were withdrawn by the investigator as judged to be treatment failures: one subject in the WR 279,396 group and six subjects in the Paromomycin Alone group. Most patients (80%) were male (Table 1). There were 17 adults, 7 patients aged 12–17 years, and 6 patients aged 5–11 years. There were a total of 64 lesions on the 30 patients, with most patients having two lesions; the index lesion and one other. Because index lesions were ulcerative by protocol, the majority of lesions were ulcerative. The mean lesion area was ∼175 mm2, although lesions ranged in area between 2 and 839 mm2. Forty-seven percent of parasites in index lesions were positive by culture and were speciated; all were L. panamensis. All but one subject received 20 days of daily drug application. One subject in the WR 279,396 group missed the Day 3 visit, but received the rest of the scheduled treatments. BODY.RESULTS.EFFICACY.: For the patients in the WR 279,396 group, the index lesion final clinical cure rate was 13 of 15 (87%) (Table 2). The all-lesions-per-patient cure rate was also 13 of 15 (87%). Because there were a total of 34 presenting lesions of which 32 cured, the per-lesion cure rate was 94%. For the two patients in the WR 279,396 group whose index lesions did not meet the protocol criteria for cure, one index lesion failed because it never re-epithelialized and one index lesion failed because of relapse. All non-index lesions on these patients also cured. The typical response of a lesion to treatment is shown in Figure 2. Figure 2.Example of response to treatment with WR 279,396. The subject presented with a deep ulcerous lesion surrounded by a large area of induration on the foot. At 1 week after completing treatment (Day 28), the induration had resolved and the ulcer was nearly completely cured. The ulcer completely cured by Day 42 and remained cured for the duration of follow-up. For the patients in the Paromomycin Alone group, the index lesion final clinical cure rate was 9 of 15 (60%, P = 0.099 versus the WR 279,396 group) (Table 2). The all-lesions-per-subject cure rate was 8 of 15 (53.3%, P = 0.046 versus the WR 279,396 group). Because there were a total of 30 presenting lesions of which 20 cured, the per-lesion cure rate was 66.7% (P = 0.005 versus the WR 279,396 group). For the six patients in the Paromomycin Alone group whose index lesions did not show a final clinical cure, one patient was removed by the investigator on Day 35 because the lesion had doubled in size (Table 2), one failed to show at least 50% re-epithelialization on Day 63, and two had not 100% re-epithelialized on Day 100. For the remaining two who did not cure, the index lesion re-epithelialized on Day 63, but had evidence of infiltration at that time, presumably caused by continued parasitic infection. One patient also had a non-index lesion that did not meet the protocol defined criteria for cure. In addition, although this did not contribute to the failure rate, a new cutaneous lesion arose on two subjects (one subject in each of the two study groups). These new lesions were treated for 20 days with the same treatment to which each subject had been randomized. Both new lesions cured. When repeat parasitological testing was performed on Study Day 21 (1 day after treatment completion), 15 of 15 (100%) of index lesions in the WR 279,396 group were negative for parasites by both smear and culture examination. In the Paromomycin Alone group, 13 of 14 (92.9%) were negative. For index lesions that cured, median time to cure was almost identical in the two treatment groups: 49 days for the WR 279,396 group and 48 days for the Paromomycin Alone group (Figure 3). Figure 3.Mean percentage cure rate over time for the index lesion. Index lesions start to completely re-epithelialize at Day 28 and continue to heal until about Day 100 after which time, if subjects had a lesion that did not cure; the subject was taken off study and offered other treatment. At Day 49 the curves for the two treatment groups start to separate with lesions treated with WR 279,396 ultimately reaching a higher final cure rate than that for the Paromomycin Alone group. Two adult subjects, one in each of the two treatment groups, developed erythema secondary to an erosive lesion in the nostril and turbinate during the trial; both were grade 1 (mild). In the first subject, an erosive lesion in the left nostril was observed at Study Day 62 and a second lesion was observed at Day 132. In the second subject, an erosive lesion was observed in the right inferior turbinate at Day 63. These lesions were biopsied for pathology, culture, and PCR. Although no lesion showed evidence of Leishmania parasites by microscopy or culture, lesions from both patients were positive by PCR. Both subjects were removed from the study (therefore they were considered treatment failures) and were treated with meglumine antimoniate off-study. BODY.RESULTS.SAFETY.: For both treatment groups, essentially all patients had mild adverse events and less than half of the patients had moderate adverse events (Table 3). The one severe adverse event was a migraine headache in the WR 279,396 group that was not considered to be drug related. The most frequently observed treatment-related adverse events were application site reactions, with edema being reported in 13.3% of subjects in the WR 279,396 group and 20% in the Paromomycin Alone group. Erythema was reported in 20.0% of subjects in the WR 279,396 group and 13.3% in the Paromomycin Alone group. Application site pain was noted more frequently in the Paromomycin Alone group (33.3%) than in the WR 279,396 group (6.7%). Contact dermatitis reactions (caused by the gauze and tape of the dressing) occurred in roughly the same number of subjects in both groups, 6 (40.0%) for the WR 279,396 group and 8 (53.3%) for the Paromomycin Alone group. BODY.DISCUSSION: Two topical antileishmanial formulations, WR 279,396 (paromomycin 15% plus gentamicin 0.5%) in a complex cream base, and Paromomycin Alone (15% paromomycin in the same complex cream base, were each administered to 15 subjects with uncomplicated CL in Panama. The index lesion (the lesion confirmed to be caused by Leishmania) and all other lesions were treated once daily for 20 days. Although the primary purpose of this trial was PKs, the need to provide effective, well-tolerated treatment of New World CL makes the efficacy and safety data also of interest. The protocol-defined primary efficacy endpoint was final clinical cure of the index lesion (at least 50% reduction in the index lesion area by Day 63, continued or complete re-epithelialization by Day 100, and no relapse by the end of the trial on Day 168). This compound definition is in accord with clinical practice. Treatment of New World CL is viewed as failing if the lesion is not appreciably smaller at early time points and not completely healed at 6 months7; the cure rates for all of the clinical endpoints were greater in the WR 279,396 group than the Paromomycin Alone group. As expected from a topical formulation, there were no systemic toxicities, and drug-related adverse events were limited to application site reactions. Hypersensitivity reactions were also observed. These were attributed to the tape that had been used in the dressings overlying treatment application sites. Topical treatment with WR 279,396 can be compared with topical treatment with Paromomycin Alone in this study, to topical treatment with paromomycin 15% plus methylbenzethonium chloride (MBCL) 12% in the literature, and to treatment with vehicle/no treatment in the literature. In this study, WR 279,396 trended toward superiority versus Paromomycin Alone on the basis of the index lesion cure rate, and was statistically superior to Paromomycin Alone on the basis of per patient and per-lesion cure rate. Although the gentamicin component that differentiates WR 279,396 from Paromomycin Alone is not known as an antileishmanial agent, Daneshvar and others18–20 succeeded in producing attenuated Leishmania by culturing promastigotes under pressure of gentamicin. Promastigotes of the attenuated lines could enter but not survive in macrophages derived from murine bone marrow, and mice inoculated subcutaneously with attenuated parasites did not develop CL skin lesions. In lesions treated with WR 279,396, it may be that parasites' constant exposure to the gentamicin component of WR 279,396 leads to their attenuation, thereby increasing their vulnerability to both paromomycin treatment, and host immune mechanisms. Paromomycin 15% plus MBCL 12% has not been evaluated alone against L. panamensis. However, the combination of this topical formulation with short courses of parenteral antimony was tested in an L. panamensis-endemic area of Colombia, and it did not augment the cure rate compared with short courses of antimony alone.21 In contrast, Paromomycin 15% plus MBCL 12% was tested in Guatemala (infecting species L. braziliensis and L. mexicana), where it was very effective (86% of patients were cured) compared with vehicle control (39% of patients were cured).22 The mITT and per protocol cure rates for this formal study were identical, and can be compared with the per protocol cure rate for L. panamensis treated with placebo/no-treatment in investigator-initiated studies. The WR 279,396 cure rate in this study was considerably higher than published placebo/no-treatment cure rates of 33–36% reported for L. panamensis in Colombia.23,24 The index lesion cure rate of 87% for WR 279,396 also compares favorably with cure rates after treatment with the antimonial Glucantime for L. panamensis in Colombia of 36–81% in three different studies after the same 6-month follow-up period.24–26 However, the cure rate of WR279,396 in L. panamensis observed in this study will be confirmed in a Phase 3 study (N = 300), planned to start in Panama in Spring 2013. Topical treatments are being developed for uncomplicated CL where the less tolerable systemic antileishmanial therapies may not have an acceptable benefit-risk profile. Patients with uncomplicated CL present with diagnostically confirmed CL, but lesion location and infecting Leishmania species are such that the parasite has little or no potential to disseminate to the mucosal tissue or elsewhere. Entry criteria for patients in our studies of WR 279,396 include patients with < 10 lesions and no evidence of systemic dissemination to mucosal tissue by qualified physician examination. Compared with systemic treatments, important advantages of all topical or local treatments for CL are 1) their failure to generate clinically meaningful systemic drug exposure, and 2) their relatively low risk for systemic side effects. Topical or local interventions may, however, generate considerable application site reactions, depending on the formulation. For example, topical paromomycin 15% formulated with MBCL 12% causes unacceptable stinging27; intralesional injections of antimony are painful28 and particularly problematic for children and patients with facial lesions; the ThermoMed device requires anesthetics to dull the blistering pain that results from intense heating of the skin29; and cryotherapy creates edema and bullae, which necessitate 1–2 weeks of saline compresses and antibiotic creams.29 In contrast, both WR 279,396 and Paromomycin Alone were formulated to promote drug penetration into the dermis while avoiding substantial local toxicity. With WR 279,396, any application site reactions are typically mild and have yet to cause a patient to withdraw from treatment. In conclusion, new topical therapies containing paromomycin with or without gentamicin may offer an effective low toxicity alternative to the current standard of care pentavalent antimonials for the treatment of uncomplicated CL in Panama.

TITLE: Hormone-Balancing Effect of Pre-Gelatinized Organic Maca (Lepidium peruvianum Chacon): (II) Physiological and Symptomatic Responses of Early-Postmenopausal Women to Standardized doses of Maca in Double Blind, Randomized, Placebo-Controlled, Multi-Centre Clinical Study ABSTRACT: This was a double-blind, randomized, placebo-corrected, outpatient, multi-centre (five sites) clinical study, in which a total of 168 Caucasian early-postmenopausal women volunteers (age>49 years) participated after fulfilling the criteria: follicle stimulating hormone (FSH) >30 IU/ml and estrogen (E2) <40 pg/ml levels at admission. They were randomly allocated to Placebo and Pre-Gelatinized Organic Maca (Maca-GO) treatment, according to different monthly treatment sequences scheduled for each site. Two 500 mg vegetable hard gel capsules with Maca-GO or Placebo powder were self-administered twice daily with meals (total 2 g/day) during three (Trial I; n=102) or four (Trial II; n=66) months study periods. At the baseline and follow- up monthly intervals, blood levels of FSH, E2, progesterone (PRG) and lutinizing hormone (LH), as well as serum cholesterol (CHOL), triglycerides (TRG), high- and low density lipoproteins (HDL and LDL) were measured. Menopausal symptoms were assessed according to Greene's Score (GMS) and Kupperman's Index (KMI). Data were analyzed using multivariate technique on blocs of monthly results in one model and Maca versus Placebo contrast in another model. A total of 124 women concluded the study. Maca-GO significantly stimulated production of E2 (P<0.001) with a simultaneous suppression (P<0.05) of blood FSH, increase (P<0.05) in HDL. Maca-GO significantly reduced both frequency and severity of individual menopausal symptoms (hot flushes and night sweating in particular) resulting in significant (P<0.001) alleviation of KMI (from 22 to 10), thus, offering an attractive non-hormonal addition to the choices available to early-postmenopausal women in the form of a natural plant alternative to Hormone Replacement Therapy (HRT) – hence, reducing dependence on hormone therapy programs. BODY.INTRODUCTION: Amongst a variety of traditional applications, Maca has been used for years by the native people of Peru as an energizing vegetable for maintenance of health at high altitudes in the Andes and as a dietary supplement. It has been used as a traditional remedy to treat common health problems in both men and women (1-3) as well as being used successfully to treat various, specific female-related disorders such as hormonal imbalances, menstrual irregularities, infertility, and menopausal symptoms, including hot flashes, vaginal dryness, loss of energy and libido, and depression (4, 5). There are indications (3-5), that Maca can be helpful in reducing discomfort caused by menopausal symptoms. Limited case studies on pre- (6) and postmenopausal women (7) followed by an in-depth model study on laboratory animals (8, 9), conducted in this laboratory, may support those indications. Results of laboratory studies conducted by Chacon in the late 1950's (10) suggested that the action of Maca relies on plant sterols, which act as biochemical triggers to help the body itself to increase or reduce production of individual hormones, balancing their levels appropriate to age and gender, with this trend confirmed in studies on animal models (8, 9). Since then, numerous technical reports have been published, describing the benefits of inclusion of Maca in reducing dependence of women on hormone replacement therapy (HRT) programs (3-5). Maca was introduced to the medical profession in the USA (4) as a non-hormonal plant preparation with potential use in anti-aging treatments, adding to a list of estrogenic herbal preparations including Soy-based products, Red Clover, Black Cohosh, Wild Yam etc. Maca preparation was classified as non-toxic for use by humans (8) at the LD50>7.5 g/kg body weight (BW), which was well above the 2 g/kg BW limit determined by the OECD (11) for dietary supplements. Previous paper (Part I) from this series (9) reported on the biochemical and pharmacodynamic effects of pre-Gelatinized Organic Maca (Maca-GO) preparation used in a model laboratory study on ovariectomised rats. Four weeks administration of Maca-GO (250 mg/kg BW twice daily) to ovariectomised rats significantly reduced both Estrogen (E2) and Progesterone (PRG), while in non-ovariectomised, sexually-experienced female rats, the level of E2 was reduced only, with a simultaneous increase in PRG level. This trend has not confirmed earlier pilot observations on postmenopausal women (7) where levels of both E2 and PRG significantly increased with simultaneous decrease in Follicle Stimulating Hormone (FSH) level. Maca-GO showed a distinctive, anti-depressant-like and sedative effect, without disturbing cognitive functions or anxiolytic activity. This was associated with lowering of serum Cortisol (CT) and Adrenocorticotropic hormone (ACTH) levels, which was identical to the anti-depressive response observed in animal models. Maca-GO balanced the thyroid hormones (TSH and T3) to within physiologically-normal range, which was significantly lower for TSH and elevated for T3 in ovariectomised rats, but did not affect serum Iron level. This did not confirm earlier observations (8) nor the results reported in the literature (12) demonstrating the stimulating effect of Maca on absorption of Iron from the digestive tract. On the basis of results from the pilot study on early-postmenopausal women conducted so far (7) and other in-depth biochemical and physiological observations made in laboratory models on animals (8, 9), it was reasonable to conclude that non-hormonal Maca-GO preparation exhibits hormone-balancing effects on the female organism and thus may reduce the discomfort experienced by women in the early-postmenopausal stage. This fact warranted in-depth clinical confirmation and clarification in view of some discrepancies between results obtained in the pilot study on women and observations made in laboratory models on mice and rats. Therefore, in this double blind, randomized, coordinated multi-centre, outpatient, full-scale clinical study, an attempt has been made to determine the effect of Maca-GO treatment in early-postmenopausal women, administered intermittently with placebo, during various, month-long intervals, on changes in levels of sex hormones and serum lipids. Simultaneously, symptoms of menopausal discomfort in participants were subjectively assessed using menopausal tests according to Greene and Kupperman. BODY.MATERIAL AND METHODS.MACA: The plant species, its cultivation and proprietary processing have been described in detail in previous papers from this series (7, 8) and etno-botanical description with traditional and current applications was described in details by Chacon (1) and Obregon (2). Maca roots, used in production of Maca-GO preparation, represented typical distribution of three main root ecotypes: black, yellow and purple/red, observed in this cultivation area – averaging to approximately 16%, 48% and 9% respectively and 27% other colors. Dried Maca roots were processed at the National Institute of Agricultural Research (NIAR), National Agricultural University La Molina in Lima (Peru), after previous verification of their origin, organic status and scientific authenticity as cultivated organic Maca - Lepidium peruvianum Chacon. Gelatinization index for starch component in the final product (99.8%) was confirmed analytically (according to the BRI Laboratory Assay, Sydney, Australia). Chemical characteristics of Maca-GO used in this study were identical to the one described previously (8, 9). BODY.MATERIAL AND METHODS.TOXICITY (LD50): Toxicity of the batch of Maca-GO received for use in the study was determined on male and female rats according to the harmonized OECD procedure (11) reported in details in Part I of this work (9). BODY.MATERIAL AND METHODS.DEMOGRAPHICS: The study was conducted on groups of early-postmenopausal women selected from patients regularly visiting gynecologic clinics in two geographically-distant locations (some 150km apart), representing two different social matrices of population. Four private clinics (CP-1; CP-2, CP-3 and CP-4) were located in urban areas belonging to the municipality of Poznan (northern-west region of Poland) and one clinic (CG-1) in the municipality of Glogow (mid-west region of Poland). The first location (CP) may be broadly described as a light to medium industry-based city, with rich traditions and recorded history dating back to the ninth century, with numerous academic, research and cultural centers and for 50 years a host to various international expositions and Fairs (Poznan - 800,000 inhabitants). The second location (CG) represents a town which has been 100% rebuilt after World War II, with a mixed population (75,000), who settled in this town as a result of work-related migration based on economic needs. The population is entirely dependent on the local copper mining industry which was established in this region in early-1950s (Glogow – the largest copper mining and processing region in Europe). BODY.MATERIAL AND METHODS.SUBJECTS: All subjects considered for inclusion in the study were early-postmenopausal women in good overall health and fulfilling menopausal criteria set at the levels: for FSH of 30 mIU/mL level or more and E2 of 40 pg/mL levels or less. Subjects had experienced absence of menses for at least 6 months, were not on HRT, or had discontinued HRT at least 12 months before admission to the study. Subjects were excluded if they had a history of breast cancer, hyperplasia, endometrial carcinoma, or cervical neoplasia; undiagnosed abnormal vaginal bleeding; a bilateral hysterectomy; history of cardiovascular disease; liver disease; history of chronic alcoholism, medication hypersensitivity, or allergy to dietary supplement ingredients; uncontrolled addiction or severe depression; acute systemic infection or abnormal laboratory values. As a result of screening procedures in five clinics, a total of 168 participants representing Caucasian, early-postmenopausal women volunteers aged 49 to 58 years were selected. They represented healthy subjects willing to participate in the study, of three (Trial I) and four month duration (Trial II), each addressing separate issues as per aims of the study: TRIAL I: A follow-up, three-month Trial, to confirm outcome of pilot study (7) and to establish residual effects of Maca-GO treatment. This double blind, randomized, coordinated multi-centre, outpatient clinical study was established in five clinics (four in Poznan=CP and one in Glogow=CG with two different admission periods #a and #b). A total 102 women volunteers were enrolled (12 subjects in CP-1; 10 in each CP2; CP-3 and CP-4 and 24 in CG-1a and 36 in CG-1b) with five MD specialist Gynecologists being responsible for the implementation of unified experimental protocol according to coded treatment sequence, allocated at random to individual clinics, the schedule of which was known to the study coordinator only. In one treatment, a group of women (n=62 in five clinics) was exposed to experimental protocols adopted in the pilot study (7) in which, after admission point (A), one month Placebo treatment (P) was introduced prior to the two months Maca-GO (M) treatment (A-P-M-M). In order to observe "residual effect" of Maca treatment after Maca-GO was withdrawn from participants for one month, the second group of women (n=40 in two clinics) was given Maca-GO capsules for the two months period from the Admission point, followed by one month Placebo capsules (A-M-M-P). All voluntarily participating women, after being assessed by a Gynecologist and confirmed on the basis of adopted analytical blood criteria as early-postmenopausal, were enrolled in this 3 month long Trial. All participants agreed to four, monthly "in-clinic" visits (at admission and then after each month of the treatment) to be physically examined by the Gynecologist, with blood sampling for hormone analysis and a simultaneous interview with the Gynecologist to assess their menopausal symptoms according to questionnaire by Greene (GMS) and Kupperman (KMI). In the clinic CG-1, admission series CG-1a, total of 24 participating women (12 women in each treatment sequence), all four sex hormones (FSH, E2, PRG and Lutinizing Hormone – LH), as well as GMS and KMI were determined, while in admission series CG-1b (18 women in each treatment sequence), in addition to GMS and KMI, only FSH and E2 were analyzed and four blood lipid indices (Triglycerides - TRGL, Total Cholesterol - CHOL, High- and Low Density Lipoproteins - HDL and LDH) were determined. TRIAL II: Assessing Placebo effect when introduced in different sequence and length intervals with Maca-GO (four months multi-centre Trial). In this double blind, randomized, coordinated multi-centre, outpatient study on 66 early-postmenopausal women, an attempt was made to determine the effect of Placebo administered intermittently with Maca-GO (Placebo as a resting period from Maca-GO treatment), on the degree of womens' responses to Maca-GO administered in various monthly intervals during the four month treatment program. In five clinics, six individual treatment sequences were used (A-P-M-M-P; A-P-P-M-M; A-M-M-P-P; A-M-M-P-M; A-P-P-P-M; A-M-M-M-P). Eleven women were assigned to each treatment sequence. Blood levels of FSH, E2, PGR and LH were measured and a degree of menopausal discomfort was determined according to Greene and Kupperman. BODY.MATERIAL AND METHODS.PROCEDURE: The protocol and amendments were approved by the Bioethics Committee of Medical Review Board in Poznan (No. 11/2004). Written informed consent was obtained from each enrolled subject regarding voluntary participation in the trial conducted under the supervision of the specialist Gynecologist, who had explained the purpose, benefits and possible risks of the study, its requirements and procedures. In all Trials, one of the responsible investigators enrolled all patients in each clinic and randomly allocated subjects to a predetermined sequence of treatment after previous clinical confirmation of blood FSH and E2 eligibility criteria. The patients as well as research team were kept blind throughout the study. At the admission and during further consultation visits to the Gynecologist, each eligible subject was given a complete physical and pelvic examination, blood was sampled for clinical tests and an assessment of specific symptoms describing menopausal status was made using questionnaires according to GMS and KMI. Subjects were instructed to return any unused portion of monthly volume of capsules so as to determine compliance. BODY.MATERIAL AND METHODS.EXPERIMENTAL PROTOCOL: The study was carried out by specialist Gynecologists and researchers from the Research Institute of Medicinal Plants (RIMP) and Medical University in Poznan, under the strict supervision of the Study Coordinator between January 2004 and June 2005 according to experimental design previously approved by the Bioethics Committee. Encapsulation and coding of monthly doses of the appropriate treatments was conducted at the RIMP. Maca-GO or Placebo powder (sorbitol & cellulose) were encapsulated in identical-looking hard gelatin vegetable capsules (500 mg net per capsule). Capsules used in this study were kept in numbered containers for delivery to appropriate centre, according to the pre-determined experimental design known to the study coordinator only. During a monthly interview with a Gynecologist who was one of the investigators, all participants received a numbered container with a monthly allocation of capsules to be taken as a dose 4 × 500 mg capsules (2,000 mg) daily, either Maca-GO, or Placebo. The appropriate monthly capsule doses were self-administered according to the following schedule: 2 capsules some 30 minutes before the morning meal and 2 capsules before the evening meal for the period of three (Trial I) or four months (Trial II) where four or five visits to the clinic were required respectively. The 2 g/day dose of Maca-GO was identical to the one applied in a pilot study (7) and was adopted from practical clinical experience by Muller (4, 5) who recommended such a dose for alleviation of menopausal discomfort in women involved in various therapeutic programs in clinics in the USA. BODY.MATERIAL AND METHODS.ASSAYS: Blood serum level of hormones was measured on monthly basis for: 17β-estradiol (E2), FSH, LH and PRG and lipid profiles (CHOL, TRGL, HDL, LDL). Indices of menopausal discomfort were determined in personal interviews by a doctor using the Menopausal Score and Index questionnaire according to Greene (7) and Kupperman (6). Blood analyses were conducted by two clinical laboratories (for clinics in Poznan by "Labo-Med" Medical Diagnostic Centre, Pod Lipami, Poznan, Poland, and for Glogow by "Prodok" Medical Laboratory, Przylep, Poland), using officially accepted standard clinical procedures on Immulite – DPC equipment. Precision of analytical techniques in both Laboratories is monitored by National Center of Quality of Diagnostic Medical Laboratories in Poland and both Laboratories are participants in the International Quality Control RIQAS maintained by Randox Company. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: Data within each Trial were expressed as means with a linear mixed model fitted to data, allowing the comparison between groups of individuals within each month and taking into account the effects of individuals on each response variable (combined fixed and random effect). The errors were assumed to follow a Gaussian (normal) distribution. All parameters were estimated using the Restricted Maximum Likelihood (REML) estimation (13). The comparisons between groups within months were made using least significant difference (LSD) test with the differences considered significant at P<0.05 and highly significant at P<0.01 and P<0.001 levels. Greene's and Kupperman's scores of postmenopausal symptoms: Total scores defined by both GMS and KMI were analyzed using the conventional analysis of variance to compare the individual groups (Menopausal status by Maca-GO treatment combinations). Weighted total scores of 21 in GMS and indexed 11 symptoms based on KMI formula were analyzed in a similar manner to the hormone data. Significant differences were tested using the least significant difference (LSD) at 5% level. A multivariate analysis via the canonical variate technique (14) was used to relate 21 (GMS) or 11 (KMI) response variates to groups of menopausal women treated in various sequence combinations of Maca-GO & placebo treatment. Monthly data were analyzed separately. All analyses were performed using SAS statistical software (15). BODY.RESULTS.TOXICITY (LD50) OF MACA-GO PREPARATION: As reported in Part I of the paper (9), all animals survived the LD50 toxicity test within the range of administration 0.75 g/kg BW and 15 g/kg BW without any adverse effects noted on the basis of abnormal behavior and histopathology of internal organs (liver, spleen, pancreas and testis or ovaries). BODY.RESULTS.SUBJECTS’ PARTICIPATION: The study began with total 168 subjects registered at the admission point: 102 in Trial I and 66 in Trial II. In Trial I, total 14 subjects failed to present themselves to consulting doctors for their monthly checkup and to obtain next portion of monthly supply of capsules, with total 88 participants completing the three month study (55 in APMM group and 33 in AMMP group). Seven subjects resigned while on APMM treatment prior to conclusion of the first month on Placebo treatment, with reason given that they were discouraged after feeling no expected effect of the treatment, and seven subjects on the AMMP after the first two months on Maca treatment, admitting that they were not taking capsules while on holidays or were inconsistent with daily capsule administration routine. In Trial II, out of 66 subjects allocated into six groups of 11 each at admission point, only 40 women satisfactorily concluded the four month Trial. In the two sequence groups: APPPM and AMMMP located in two clinics in Poznan (CP-4 and CP-5), out of the total 22 admitted participants, only two women concluded the four month Trial. Women resigned during the first and the second month participation due to admitted irregular intake of capsules; hence, both these groups were excluded from statistical analysis. Four women from APMMP and two from AMMPM sequence group failed to present themselves for monthly examination after the first or second month of study (admitting that they were not taking capsules regularly or after not feeling anticipated reduction in menopausal discomfort). Therefore, records from only 38 subjects in four treatment groups were submitted to statistical analysis in this Trial (sequence: APMMP, APPM, AMMPP and AMMPM). BODY.RESULTS.TRIAL I: Results from hormone assays (Table 1) showed that, in comparison to placebo, out of four hormones studied, a significant (P<0.05) decrease in FSH and a highly significant (P<0.001) increase in E2 were observed in sequence group APMM only, without significant changes (P>0.05) recorded in PRG and LH levels within the same sequence treatment. There were no significant changes recorded in any of the hormones within a sequence group AMMP (P>0.05), nor between Placebo and Maca-GO treatment in subjects who received Placebo after previous two months Maca-GO treatment (P>0.05). There were no statistically-significant differences (P>0.05) between the two sequence groups in levels of four hormone profiles recorded on monthly basis in any of the four blood sampling points. Table 1 TRIAL I. Average Admission (A) and monthly values for hormones FSH, E2, PRG and LH levels at four sampling times (Month Model) a and Placebo (P) versus Maca-GO (M) contrast (Treatment Model) for two application sequences: APMM (n=55) and AMMP (n=33) during three months long study Hormone Treatment Admission (A) End of Month 1 End of Month 2 End of Month 3 SED b Placebo c Maca SED P d FSH (IU/ml) APMM 60.86 61.70 56.11 54.66 4.117 61.72 55.43 3.19 <0.05 AMMP 67.14 61.09 50.51 56.4 4.537 55.68 55.1 3.96 ns SED 6.36 6.48 6.65 6.25 5.77 P ns ns ns ns ns E2 (pg/ml) APMM 22.39 17.15 30.41 32.04 5.183 17.16 31.21 4.23 <0.001 AMMP 17.22 22.97 30.8 24.8 5.727 24.84 27.36 4.99 ns SED 5.94 5.96 6.2 5.83 4.66 P ns ns ns ns ns PRG (ng/ml) APMM 0.425 0.494 0.392 0.496 0.201 0.510 0.472 0.156 ns AMMP 0.469 0.541 0.606 0.331 0.225 0.331 0.574 0.195 ns SED 0.27 0.296 0.221 0.233 0.2416 P ns ns ns ns ns LH (mU/ml) APMM 26.7 29.13 28.24 29.71 2.25 29.12 29.12 1.74 ns AMMP 31.55 31.67 28.29 26.18 2.477 26.18 29.98 2.18 ns SED 3.86 4.12 3.99 3.89 3.64 P ns ns ns ns ns a A linear mixed model was fitted to data, allowing the comparisons between treatment groups: treatment by month differences in one model and Maca versus Placebo contrast in another model. The random effects included both individual variation and residuals. Two models may be written as follows: Month Model, Fixed (Treatment + Month + Interaction) + Random (Individuals + error); Treatment Model, Fixed (Treatment + Maca + Interaction) + Random (Individuals + error). The errors are assumed to follow a Gaussian (normal) distribution. All parameters were estimated using the Restricted Maximum Likelihood (REML) estimation. The differences between treatments within each month and between Maca and placebo were tested using least significant difference (LSD) test at 5% and 1% significance levels; b SED , Standard error of differences; c Capital letters attached to the means indicate significant difference between values in columns. Small letters indicate significant differences between values in rows. d P , Probabilities of significance. ns, not significant at P >0.05; P <0.05, significance at 5% probability level; P <0.01, significance at 1% probability level; P <0.001, significance at 0.1% probability level. In comparison to the admission point, after two months of Maca-GO treatment, there was statistically-significant (P<0.05) reduction in blood TRGL and LDL concentrations, at a simultaneous significant (P<0.05) increased in HDL levels (Table 2). Comparing to Placebo, one month Maca-GO intake was not long enough for the treatment to produce statistically-significant changes in lipid indices, but after two month a significant (P<0.05) increase in HDL concentration was observed. Table 2 TRIAL I. Average Admission (A) and monthly values for Lipids: Cholesterol (CHOL), Triglycerides (TRGL), High Density Lipoproteins (HDL) and Low Density Lipoproteins (LDL) levels at four sampling times (Month Model)1 and Placebo (P) versus Maca-GO (M) contrast (Treatment Model) for two application sequences: APMM (n=18) and AMMP (n=18) during three months long study a Measurement Treatment Admission (A) End of Month 1 End of Month 2 End of Month 3 SED b Placebo Maca SED P c CHOL(mg/100ml) APMM 255.2 234.5 224.1 218.6 10.53 234.5 222.3 8.1 ns AMMP 236.9 236.0 226.9 238.6 7.87 238.6 231.6 6.9 ns SED 15.13 15.17 16.3 14.43 14.53 P ns ns ns ns ns TRGL (mg/100ml) APMM 130.0 125.2A 107.2AB 83.8B 19.7 * 125.2 98.8 15.5 ns AMMP 137.4 130.5 123.7 129.7 14.5 129.7 127.0 12.3 ns SED 26.4 26.63 28.4 24.7 25.3 P ns ns ns ns <0.05 HDL (mg/100ml) APMM 67.07 59.54B 62.63AB 73.02A 5.39 * 59.54 65.68 3.91 ns AMMP 61.54 66.38AB 69.83A 62.65B 3.13 * 62.65B 68.07A 2.7 <0.05 SED 5.42 5.73 6.62 5.07 5.42 P ns ns ns LDL (mg/100ml) APMM 187.7 171.7 165.0 149.5 13.8 171.7 157.2 9.8 ns AMMP 169.2 166.8 155.2 170.6 8.0 170.6 160.7 7.1 ns SED 15.1 16.1 18.6 14.4 15.4 P ns ns ns ns ns a For explanation see Table 1 ; b Standard error of differences; Asterisk (*) indicates existence of significant differences between monthly measurements within the same treatment sequence group. Values marked with unlike capital letters are considered statistically significant at P <0.05; c Significance probabilities: ns = not significant at P >0.05; P <0.05 = significance at 5% probability level; P <0.01 = significance at 1% probability level; P <0.001 = significance at 0.1% probability level. BODY.RESULTS.TRIAL II: Placebo introduced at the start of the Trial has slightly (P>0.05) lowered the FSH in group APMMP in relation to Admission point, however taking Placebo for one month again after two months Maca-GO treatment, resulted in a distinctive and highly significant (P<0.01) increase in concentration of this hormone in relation to both Admission point and both monthly measurements on Maca-GO (Table 3). Two month Placebo at the beginning of the Trial or during the last two months following two months of Maca-GO treatment had no significant (P>0.05) effect on FSH results – although Maca-GO had a distinctive tendency to lower level of FSH. Women in sequence Group AMMPM showed highest degree of reduction in FSH after two months of Maca-GO intake (from 60 to 28.8 IU/ml) with one month Placebo treatment significantly (P<0.05) increasing blood FSH and after returning to Maca-GO treatment for another month giving a distinctive, although not statistically significant (P>0.05) reduction in this hormone. In all sequence groups, two months of Maca-GO treatment was more pronounced as compared to one month intake of capsules only. Table 3 TRIAL II. Average Admission (A) and monthly values for FSH, E2, PRG and E2 levels at five sampling points (Month Model) a and Placebo (P) versus Maca-GO (M) contrast (Treatment Model) for five application sequences (n=42) during the four months long study: APMMP (n=7), APPMM (n=11), AMMPP (n=11) and AMMPM ( 9 ) Hormone Treatment b Admission (A) End of Month 1 End of Month 2 End of Month 3 End of month 4 SED c Placebo Maca SED P d FSH (IU/ml) APMMP A 85.73 P e 78.48 M 70.39 M 64.94 P 97.88 10.10 88.18 68.12 6.95 <0.01 APPMM A 64.62 P 70.54 P 71.28 M 65.60 M 60.66 8.08 70.91 63.56 5.6 ns AMMPP A 69.03 M 62.01 M 57.27 P 53.63 P 59.22 7.46 56.42 59.64 5.29 ns AMMPM A 60.06 M 49.60 M 28.77 P 53.67 M 40.35 11.46 53.67 40.47 6.81 ns SED 17.301 17.348 17.371 19.208 16.565 15.618 P ns <0.05 ns <0.05 <0.05 ns E2 (pg/ml) APMMP 30.42 35.73 37.26 31.20 35.93 28.34 35.87 34.23 18.84 ns APPMM 19.11 8.33 15.14 45.72 58.43 22.19 11.73 50.95 15.36 <0.05 AMMPP 8.62 12.08 25.3 25.61 17.14 21.60 21.13 18.69 15.05 ns AMMPM 30.19 39.94 77.25 54.27 87.97 21.5 54.27 65.69 16.47 ns SED 31.661 28.375 29.016 35.96 18.992 19.124 P ns <0.05 ns ns <0.05 <0.05 PRG (ng/ml) APMMP 0.426 0.659 0.574 0.484 0.351 0.259 0.505 0.529 0.181 ns APPMM 0.201 0.233 0.295 0.209 0.286 0.250 0.264 0.241 0.174 ns AMMPP 0.225 0.233 0.373 0.358 0.302 0.233 0.330 0.303 0.163 ns AMMPM 0.561 0.432 0.4309 0.557 0.370 0.242 0.557 0.431 0.187 ns SED 0.241 0.237 0.240 0.270 0.188 0.165 P ns ns ns ns ns ns LH (mU/ml) APMMP 21.62 30.69 26.81 28.78 28.01 4.18 29.35 27.74 2.9 ns APPMM 43.79 38.92 38.91 34.05 32.41 3.985 38.91 33.38 2.74 <0.05 AMMPP 36.66 32.69 32.58 30.03 32.5 3.678 31.27 32.64 2.59 ns AMMPM 26.43 30.65 24 22.35 28.3 3.77 22.35 27.58 2.91 ns SED 8.6 8.566 8.622 8.805 6.203 6.035 P ns ns ns ns <0.05 ns a For explanation see Table 1 ; b In this Table (Trial II), participants in the two sequence groups: APPPM and AMMMP have been excluded from analysis of data due to the two women only, out of the total 22 admitted women concluded the four months Trial located in two separate clinics in Poznan (CP-4 and CP-5); c SED , Standard error of differences; d P , Probabilities of significance. ns, not significant at P>0.05 ; P<0.05 , significance at 5% probability level; P<0.01 , significance at 1% probability level; P<0.001 , significance at 0.1% probability level; e Letters preceding the values in columns for consecutive blood sampling points within the individual hormone, indicate hormone level as recorded after one month of treatment with either Placebo (P) or Maca-GO (M) within the relevant four treatment sequences analyzed in this Trial. Letters’ pattern for the E2, PRG and LH are identical to those as marked in the FSH data block. After exposure to Maca-GO intermittently with the Placebo, levels of E2 showed opposite trends to the one observed in FSH blood contents, which resulted in Maca-GO significantly (P<0.05) increasing E2 values in sequence group APPMM and nearly-significantly in group AMMPM (P>0.05). Two months Maca-GO treatment magnified this trend in comparison to one month of treatment. There were no significant (P>0.05) differences recorded in PRG concentrations when Maca-GO was intermittently introduced to women in various sequence patterns and for different length of time. Maca-GO treatment significantly (P<0.05) lowered LH in a sequence group where two months Placebo treatment was followed by Maca-GO application. There were significant differences (P<0.05) detected between sequence groups within both Placebo (FSH, E2, and LH) and Maca-GO (E2 only) blocks of data, with sequence groups APMMP and AMMPM representing clinics located in Poznan and the other two in Glogow which shows differences existing in dynamics of responses of participating women to Maca-GO in two geographically-distant locations. BODY.RESULTS.KUPPERMAN’S MENOPAUSAL INDEX (KMI) AND GREENE’S MENOPAUSAL SCORE (GMS): Responses of participants to two questionnaires collected by Gynecologists were computed to derive menopausal index according to Kupperman (KMI) and menopausal score according to Greene (GMS), allowing for an evaluation of menopausal discomfort expressed as sums of recorded points derived from scores of individually-assessed symptoms. In Trial I, as compared to the Admission point, after one month Placebo treatment, before introduction of Maca-GO (APMM), there was already statistically-high significant reductions (P<0.001) in total KMI and GMS values(Table 4). Similar (P<0.001) reductions in feelings of discomfort, typically observed in early- postmenopausal stage, were recorded in women receiving Maca-GO treatment immediately after the admission point (AMMP). After the second month of Maca-GO treatment, in both sequence groups, there was further significant (P<0.001) lowering in both KMI and GMS total values. On the other hand when Placebo was introduced after two months of Maca-GO treatment, there was an overall statistically-high increase (P<0.001) in total KMI and GMS values, these being a reflection of a significant increase (between P<0.009 and P<0.001 - depending on the symptom) in the severity of individual menopausal symptoms as demonstrated in Tables 5 and 6. Table 4 TRIAL I. Dynamics of monthly changes in total values from symptoms determined according to Kupperman’s Menopausal Index (KMI) and Greene’s Menopausal Score (GMS) recorded between Admission point (A), at three monthly sampling points (Month Model) a and Placebo (P) versus Maca-GO (M) contrast (Treatment Model) according to two treatment sequences applied in Trial I (for APMM n=55 and for AMMP n=33) Treatment Admission Month 1 Month 2 Month 3 SED Placebo Maca SED b P c Kupperman’s Menopausal Index (KMI)   APMM 26.61 22.04 12.83 7.52 1.13 22.04 10.17 1.16 <0.001   AMMP 27.53 14.09 8.50 19.14 1.25 19.14 11.3 1.28 <0.001    SED 1.68 1.68 1.68 1.7 1.7    P <0.01 <0.01 <0.001 ns ns Greene’s Menopausal Score (GMS)   APMM 21.42 17.74 12.26 8.33 0.92 17.74 10.29 0.89 <0.001   AMMP 25.92 14.18 9.71 14.82 0.98 14.82 11.95 0.94 <0.01    SED 1.68 1.68 1.68 1.73 1.56    P <0.05 ns <0.001 ns ns a For explanation see Table 1 ; b SED , Standard error of differences; c P, Probabilities of significance. ns, not significant at P>0.05 ; P<0.05 , significance at 5% probability level; P<0.01 , significance at 1% probability level; P<0.001 , significance at 0.1% probability level. Table 5 TRIAL I. Monthly changes in values of individual symptoms determined according to Kupperman’s Menopausal Index (KMI) recorded between Admission (A), and at three monthly sampling points (Month Model) a with one month Placebo (P) followed by two months Maca-GO (M) contrast according to the sequence APMM (at n = 55) applied in Trial I Code# Symptom in KMI Admission A After 1m P After 1m M After 2m M ± SED P Placebo Maca-GO ± SED b P c K-1   Hot flushes 3.94 3.17 2.16 1.64 0.580 0.002 1.761 0.651 0.152 <0.001 K-2   Excessive sweating 2.41 1.24 1.11 0.85 0.276 <0.001 1.391 0.673 0.162 <0.001 K-3   Interrupted sleep 1.98 1.15 0.75 0.78 0.267 <0.001 1.196 0.605 0.144 <0.001 K-4   Nervousness 2.63 1.63 1.18 0.61 0.237 <0.001 1.630 0.802 0.145 <0.001 K-5   Depression 1.24 0.61 0.45 0.46 0.197 <0.001 0.826 0.619 0.160 0.198 K-6   Losing body balance 0.87 0.39 0.32 0.32 0.186 0.006 0.696 0.395 0.125 0.017 K-7   General weakness 1.24 0.72 0.68 0.56 0.201 0.011 1.239 0.874 0.161 0.025 K-8   Joint pain 1.02 0.65 0.70 0.69 0.208 0.001 1.000 0.719 0.158 0.079 K-9   Headaches 1.20 0.61 0.45 0.34 0.188 0.009 0.717 0.452 0.132 0.047 K-10   Heart palpitations 1.02 0.46 0.32 0.22 0.185 <0.001 0.543 0.336 0.131 0.116 K-11   Numbness hands & legs 0.57 0.39 0.20 0.20 0.190 <0.001 0.543 0.336 0.135 0.126   Total value for  KMI symptoms 26.61 22.04 12.83 7.52 1.13 <0.001 22.04 10.170 1.408 <0.001 a For explanation see Table 1 ; b SED , Standard error of differences; c P, Probabilities of significance. ns, not significant at P>0.05 ; P<0.05 , significance at 5% probability level; P<0.01 , significance at 1% probability level; P<0.001 , significance at 0.1% probability level. Scoring index: 0, symptom not experienced; 1, occasionally; 2, often; 3, very often. Kupperman’s Indexing Factors: K-1, ×4; K-2 to K-5, ×2; the remaining, ×1. Table 6 TRIAL I. Monthly changes in values of individual symptoms determined according to Greene’s Menopausal Score (GMS) for APMM sequence treatment (n=55) in early-postmenopausal women recorded between Admission (A), and three monthly sampling points (Month Model) a with one month Placebo (P) followed by two months Maca-GO (M) contrast (Treatment Model)1 according to the sequence applied in Trial I Individual Menopausal Symptoms’ Scores according to Greene Symptom Admission A After Month 1 (P) After Month 2 (M) After Month 3 (M) ± SED b P c Placebo Maca ± SED b P c Abnormally-fast heart rate 0.66 0.45 0.35 0.28 0.149 0.094 0.455 0.315 0.118 0.240 Nervousness 1.51 1.68 1.00 0.76 0.200 <0.001 1.682 0.879 0.169 <0.001 Difficulty falling asleep 1.15 1.12 0.77 0.52 0.212 0.009 1.121 0.646 0.169 0.006 Excessive alertness 1.33 1.33 1.00 0.55 0.175 <0.001 1.333 0.776 0.147 <0.001 Sudden feeling of anxiety 0.82 0.70 0.45 0.45 0.193 0.145 0.697 0.450 0.169 0.147 Difficulty concentrating 0.97 1.09 0.94 0.41 0.182 0.002 1.091 0.675 0.153 0.008 Feeling of tiredness/lack of energy 1.39 1.54 1.32 0.72 0.205 <0.001 1.545 1.023 0.173 0.003 Lack of interest 0.36 0.33 0.26 0.21 0.129 0.612 0.333 0.233 0.111 0.037 Unhappy/depressed 1.00 0.94 0.74 0.45 0.201 0.030 0.939 0.595 0.174 0.050 Excessive crying 0.45 0.27 0.23 0.24 0.129 0.263 0.273 0.234 0.109 0.721 Irritability 1.27 1.15 0.84 0.65 0.189 0.005 1.152 0.747 0.165 0.016 Loss of consciousness 0.61 0.88 0.45 0.41 0.170 0.030 0.879 0.433 0.147 0.003 Nervous tension 0.72 0.59 0.52 0.41 0.156 0.238 0.591 0.465 0.133 0.345 Numbness/“pins & needles” 0.67 0.61 0.45 0.31 0.183 0.207 0.606 0.381 0.153 0.144 Headaches 0.97 0.85 0.58 0.34 0.206 0.014 0.848 0.463 0.167 0.023 Muscle and joint aches and pains 1.24 1.18 0.97 0.62 0.224 0.027 1.182 0.794 0.193 0.047 Loss of feeling in feet & hands 0.45 0.36 0.19 0.24 0.148 0.284 0.364 0.217 0.123 0.239 Difficulty breathing 0.27 0.12 0.16 0.10 0.097 0.303 0.121 0.132 0.073 0.879 Hot flushes 1.73 1.73 1.10 0.52 0.257 <0.001 1.727 0.807 0.210 <0.001 Excessive night sweating 1.45 1.39 1.06 0.48 0.253 <0.001 1.394 0.774 0.203 0.003 Loss of interest in sex life 1.06 1.12 0.84 0.60 0.200 0.036 1.121 0.712 0.173 0.020 TOTAL GMS 20.09 19.45 13.36 8.15 1.869 <0.001 19.45 10.76 0.16 <0.001 a For explanation see Table 1 ; b SED , Standard error of differences; c P , Probabilities of significance. ns, not significant at P>0.05 ; P<0.05 , significance at 5% probability level; P<0.01 , significance at 1% probability level; P<0.001 , significance at 0.1% probability level. Taking APMM sequence group as an example, after Placebo administration for a one month period immediately after the Admission point (prior to Maca-GO treatment), a significant (P<0.001) reduction in severity of most of the 11 KMI symptoms was observed, except for the two: hot flushes and numbness in hands & legs (Table 5). With introduction of Maca-GO to participants after previous Placebo treatment, out of 11 symptoms, there was a distinctive, almost significant (P>0.05) lowering effect recorded in hot flushes values only, with other symptoms only slightly, not significantly (P>0.05) reduced. However two months of Maca-GO treatment in APMM sequence group provided the most pronounced, highly significant (P<0.001) reduction of hot flushes and nervousness as compared to both Admission point and Placebo treatment, while index values in other symptoms, although noticeably lowered, were not statistically confirmed (P>0.05). Analyzing results for the same sequence group according to the questionnaire by Greene, which comprises 21 questions (Table 6), as compared to Placebo after one month of Maca-GO treatment, the most distinctive and highly significant (P<0.001) reduction in absolute GMS values was observed in hot flushes, nervousness, difficulties in falling asleep, irritability and excessive alertness. Extending Maca-GO application for another month resulted in further highly significant (P<0.001) lowering in the GMS values for the following symptoms: lack of energy, tiredness, excessive night sweating, hot flushes and excessive alertness. It appears that of all 21 GMS symptoms, the most sensitive in both the degree of reduction in absolute GMS value for individual symptom and the level of statistical significance (P<0.001), were the following: hot flushes, excessive sweating, nervousness, excessive alertness, lack of energy/feeling of tiredness and to a lesser degree (P<0.01) irritability and headaches. In Trial II, as already observed in Trial I, the second month of Maca-GO treatment, magnified positive effect on alleviation of menopausal symptoms as measured by either KMI and/or GMS. Differences between Placebo and Maca-GO treatments were more pronounced in the KMI as compared to GMS (Table 7). The GMS values showed significant (P<0.01) effect of Placebo in one sequence group only (APPMM) while recorded KMI values showed highly significant (P<0.001) differences between Placebo and Maca-GO treatment in all sequence groups. Table 7 TRIAL II. Dynamics of overall monthly changes in total values from individual symptoms determined according to Kupperman’s Menopausal Index (KMI) and Greene’s Menopausal Score (GMS) recorded between Admission point (A), at four monthly sampling points (Month Model) a and Placebo (P) versus Maca-GO (M) contrast (Treatment Model) according to four intermittent sequence of Placebo application during four month study as per Trial II & III (n=42) Treatment Admission A After Month 1 P After Month 2 P After Month 3 M After Month 4 M SED b Placebo Maca SED P c Kupperman’s Menopausal Index (KMI)   OPMMP 21.56 14.44 8.44 7.67 16.33 1.62 15.39 8.06 1.38 <0.001   OPPMM 30.40 15.20 24.00 10.80 6.99 1.69 19.60 9.65 1.31 <0.001   OMMPP 29.64 13.09 9.45 14.88 19.58 1.52 17.21 11.27 1.30 <0.001   OMMPM 25.27 14.59 9.64 17.77 11.32 1.47 17.77 11.85 1.25 <0.001    SED 3.52 3.52 3.53 3.59 3.47 3.47 Greene’s Menopausal Score (GMS)   OPMMP 25.00 19.11 12.67 10.00 8.00 1.64 13.56 11.33 1.52 ns   OPPMM 25.80 10.90 8.60 8.90 3.30 1.55 9.75 6.10 1.44 <0.01   OMMPP 30.91 12.45 10.91 13.00 7.18 1.48 10.09 11.68 1.37 ns   OMMPM 26.91 18.64 15.91 16.36 12.73 1.48 16.36 15.76 1.58 ns    SED 4.28 4.28 4.28 4.28 4.35 4.04 a For explanation see Table 1 ; b SED , Standard error of differences; c P , Probabilities of significance. ns, not significant at P>0.05 ; P<0.05 , significance at 5% probability level; P<0.01 , significance at 1% probability level; P<0.001 , significance at 0.1% probability. BODY.DISCUSSION.THE EFFECT OF MACA-GO ON HORMONAL BALANCE IN EARLY-POSTMENOPAUSAL WOMEN: According to clinical definition established by WHO, post-menopause is characterized by increased levels of gonadotropins and reduction in estrogens, with low level or absence of LH (16). Lowering of estrogen is assumed to trigger depression observed in women entering menopause, therefore, hormone therapy - through influencing an increase in blood and brain serotonin levels, particularly when combined with antidepressant medication - may alleviate stages of depression and symptoms of mood swing which are commonly observed in menopausal women (17). However, in view of the side-effects observed in women using HRT which include high blood pressure, heart complications, obesity etc. it is apparent that women are opting for non-hormonal programs, or using phyto-hormone sources such as phytoestrogens to help reduce discomfort of menopause and achieve daily comfort at this stage of life (18). The E2 levels of 30 pg/ml and above are considered as adequate for woman (with average 60-75 pg/ml) to avoid symptoms of discomfort characteristic to menopause (19). In this study levels of E2 were below 30 pg/ml – typically indicating postmenopausal stage with impaired ovarian function (16). It is reasonable to assume that Maca-GO, although it contains no plant hormones (10), stimulated and/or contributed to the regulatory mechanism responsible for optimizing ovarian function and secretion of the quantity of estrogen – in some cases– well above the 30pg/ml considered as desired minimum (19). This may contribute to reduction in menopausal discomfort in women taking Maca-GO, avoiding problems linked to mental, physiological and physical symptoms indicated by results as measured in GMS and KMI and expressed by overall values of obtained scores (Tables 4, and 7). Overall positive responses of participants in this study may be supported by previous pilot observations on early-postmenopausal women (7) and in-depth biochemical and physiological observations made in bioassays using sexually-experienced (8) and ovariectomised laboratory animals (9), confirming an existence of positive relationship between Maca-GO treatment and its hormone balancing function in early-postmenopausal women. BODY.DISCUSSION.PLACEBO EFFECT IN MACA-GO TREATMENT: When Placebo was introduced for one month prior to daily dose of 2 g Maca-GO self-administered by women for two consecutive months (group APMM -55 women), there was a high degree of responses recorded by individual participants in the following key menopausal indicators, expressed either in relation to the Admission point (%A) or as Placebo-corrected values (%P) for: FSH (89%A and 78%P), E2 (80%A and 72%P), GMS (100%A and 76%P) and KMI (96%A and 85%P). As judged by absolute results (Tables 1, 3, 4 and 7) and lower %P values in relation to %A, there was a distinctive Placebo effect observed in this study, as shown by responses of early-postmenopausal women to one month Placebo treatment, which was similar, but reduced in magnitude, to the effect observed after administration of Maca-GO. Extending Placebo treatment for another month reduced or eliminated the earlier (one month) Placebo effect on women, while the effect of Maca-GO was magnified in both absolute and relative terms. When results from the second sequence group (AMMP – 33 women) were expressed in a similar manner to the first treatment group, generally lower responses in calculated values were obtained: for FSH (93%A and 63%P), E2 (87%A and 66%P), GMS (90%A and 84%P) and KMI (96%A and 78%P). The lower degree of responses of women to the Placebo administered after Maca-GO treatment, compared to Placebo introduced prior to the two months of Maca-GO use, may confirm an existence of a residual effect of Maca-GO treatment during the first month after its withdrawal when Placebo was used instead - the trend clearly demonstrated in Figure 1 on an example of four hormones: FSH, E2, PRG and LH. Figure 1TRIAL I: Placebo-corrected relative values (P= 100%) for four hormones (FSH, E2, PRG and LH) in two treatment sequences (APMM and AMMP) showing different effect of Placebo, depending on the time in the sequence during the three months Trial, when Placebo was introduced prior to, or after two months Maca-GO administration to early-postmenopausal women volunteers (n=55 & n=33 respectively). P<0.05 = statistical significance at P<0.05 level. When in Trial II results were expressed as a percentage of Placebo introduced in different sequence patterns concurrently with Maca-GO treatment (Figure 2), it was apparent that: there was different magnitude of relative responses of women to Maca-GO depending on whether Placebo was introduced prior to or after previous Maca-GO treatment; irrespective of the sequence in which Placebo was introduced during the four month trial, two months of Maca-GO application magnified the therapeutic effects of the treatment. Similar trends were observed in total GMS and KMI values (Table 4), as well as on the example of individual menopausal symptoms summarized in Figure 3. Figure 2TRIAL II: Placebo-corrected values (P=100%) for four hormones: FSH (IU/ml), E2 (pg/ml), PRG (ng/ml) and LH (mU/ml) in four treatment sequences (AMMPM, APMMP, AMMPP and APPMM) showing different effect of Placebo on the outcome of Maca-GO treatment (M), depending on the time in the sequence during the four months Trial when Placebo (P) was administered to early-postmenopausal women volunteers (n= 40). Figure 3TRIAL I: Comparison of Placebo Corrected (%P) results in relative values for individual symptoms comprising Total Kupperman's Menopausal Index (KMI) in response of early- postmenopausal women (n=55) to two consecutive monthly periods of Maca-GO application (M-1 = for one month and M-2 = for 2 months) administered after one month Placebo (P) treatment in a sequence treatment APMM (results for M-1 and M-2 are expressed as a percentage of values recorded after one month P administration). It seems appropriate to express results attributed to full Maca-GO effect on women after two months treatment and calculate Placebo corrected values against results recorded after two month of Placebo treatment, when Placebo effect based on emotional factors is expected to be greatly reduced or eliminated in most of the measurements. BODY.DISCUSSION.TOXICITY AND LD50 FOR MACA-GO: Toxicity results obtained in laboratory model using rats (8) showed the value LD50>7.5 g of Maca-GO per kg body weight (BW). However, depending on the test laboratory animal being used, applied doses were between 0.75 g/kg BW through to 7.5 g/kg BW (rat's model) to 15g/kg BW (mice model). Within the above range up to 15 g Maca-GO per kg BW (the highest dose possible to be applied in the study), no detectable negative physiological, clinical, histo-pathological, nor toxic effects existed which could be attributed to the used doses of Maca-GO and administered to rats and/or mice during model laboratory bioassays (9). Therefore, the LD50>15 g/kg BW was considered as the value for Maca-GO preparation. Such a value is considerably higher than the 2 g/kg BW limit adopted by the OECD (11) as non toxic and safe for use as dietary supplement, and therefore, Maca-GO fully conforms to such a classification as a "non toxic" with a "safe for human consumption" status. BODY.DISCUSSION.DISTINCTION BETWEEN MACA-GO AND OTHER COMMONLY USED HERBAL THERAPIES: Several herbal alternatives have already gained acceptance by medical, health practitioners and phyto-pharmaceutical industries for potential substitution for synthetic hormones used in hormone therapies. Positive effect of soy and red clover extracts is attributed to action of flavonoids with biochemical structure resembling steroids in human hormones (20) and may induce some side effects similar to those observed after synthetic hormones are used. It has been reported that Maca preparations tend to be more effective for menopausal patients as compared to treatment with phyto-estrogenic herbs (4). Maca contains no hormones or their precursors and its action is linked to a group of compounds resembling sterols which are known to stimulate hormonal activity in ovaries, thyroid and pancreas via pituitary gland and hypothalamus. Also, alkaloids, non-steroidal compounds present in Maca-GO, which, although acting much more slowly than soy and clover extracts, are much better tolerated by women remaining on Maca program (5) and positively affect physiological actions along the hypothalamus - pituitary gland – gonadal axis. Maca was successfully used to wean women off hormone replacement therapy (some of them had been on HRT for many years), and for those women who still had some symptoms, a combination protocol involving Maca extract and a minute amount of natural estrogen together with natural progesterone was suggested, which, unlike progestin, is not considered to be carcinogenic (5). BODY.DISCUSSION.THE EFFECT OF MACA-GO ON INDIVIDUAL MENOPAUSAL SYMPTOMS: Significant increase in E2 level observed in this study after two months of Maca-GO treatment was associated with alleviation of a number of individual menopausal symptoms, most distinctive being reductions in frequency and severity of hot flushes, excessive sweating, interrupted sleep pattern, nervousness depression, headaches and loss of libido/interest in sex (Table 5 and 6). Studies reported by other authors (2, 21) also indicate that Maca can be helpful in reducing discomfort caused by menopausal symptoms. There was a distinctive effect of the length of Maca-GO treatment on the degree of reduction in severity of individual symptoms, with two months of treatment magnifying the positive results already observed after one month of treatment. (Figure 3). Demonstrated in this study, the positive effect of Maca-GO on lowering E2, and alleviation of the menopausal symptoms not restricted to hot flushes and night sweating only, shows Maca-GO as a natural non-hormonal treatment, superior to HRT in terms of all the additional benefits such as simultaneous reduction in FSH and an increased HDL, not delivered by HRT programs or phyto-estrogenic preparations. BODY.DISCUSSION.COMPARISON OF SENSITIVITY OF THE GMS AND KMI TESTS: Comparing degree of statistical significance in influences of Maca-GO on KMI and GMI in terms of detected reduction in frequency and severity of menopausal symptoms, it appears that KMI is more sensitive in displaying the effect of both Placebo and Maca-GO treatments on the recorded scores of menopausal tests as compared to GMS (Table 7). BODY.DISCUSSION.RESPONSES OF WOMEN TO MACA-GO TREATMENT IN CLINICS LOCATED IN TWO DISTANT REGIONS: In early- postmenopausal stage, women experience noticeable and unpredictable waves of discomfort with differing intensity of complex physical and psychological manifestations. These are expressed differently by individual patients-subjects and/or groups of women of different lifestyles and/or ethnic backgrounds etc. Results observed in this multi-centre study in women volunteers attending clinics in two geographically distant locations, which differed greatly in both socio-cultural and economic terms, may support such an assumption. Apparent differences were reported (22) in responses of women to HRT due to geographical location (Europe and USA) – the fact, behind a decision made in this study to observe clinical, physiological and vasomotor responses of postmenopausal women to non-hormonal phyto-preparation, as an alternative to HRT, in two geographically-distant locations representing different socio-economic and cultural matrix of participants. There were significant differences in dynamics of E2 responses observed in individual sequence groups to Maca-GO and Placebo treatment. In addition to the effect of Placebo or Maca-GO treatment, responses recorded in individual groups may be associated with participants attending different clinics, located in two geographically-distant regions representing the different cultural and socio-economic population matrixes characterizing Poznan and Glogow. Although it could not be statistically tested in this study, it may be assumed that different responses to Maca-GO treatment may be confounded by differences in dietary habits amongst populations linked to two regions and groups of women participating in the study (preference for potato-based dishes with fewer leafy vegetables in Poznan and pasta or rice with more leafy vegetables and fruits in Glogow). It has to be emphasized that there were no instructions given to participants regarding use of a specific diet or exercise program nor were records of dietary intake kept by participants in this study. BODY.DISCUSSION.THE EFFECT OF MACA-GO ON LIPID METABOLISM INDICES: Lipid metabolism data, observed in Trial I showed a significant effect of Maca-GO on increasing HDL only without significantly (P>0.05) affecting CHOL, TRGL and LDL when relating results to Placebo treatment. There was a distinctive reduction in LDL/HDL ratio from 2.7 to 2.3. In previous study on ovariectomised rats (9), serum HDL also increased together with CHOL and LDL with TRGL noticeably – but not significantly reduced. Reported in the literature (1) positive influence of Maca on overall lipid metabolism has not been confirmed in this study, with one exception of an elevated HDL level. BODY.DISCUSSION.FURTHER CONSIDERATIONS: In addition to the positive effects of Maca-GO on hormonal balance, HDL and alleviation of menopausal symptoms, results presented in this part of the clinical study on early-postmenopausal women, also demonstrated an existence of Placebo effect, residual Maca-GO effect and a necessity for two month treatment period for Maca-GO to display its full therapeutic functionality. In order to confirm the above findings, in the next, concluding part (III) of this clinical study, using a crossover configuration experimental design, levels of gonadal, pituitary, thyroid and adrenal hormones, lipids and key minerals, were measured in relation to changes in severity of menopausal symptoms contributing to overall degree of discomfort felt by early-postmenopausal women after two months of Maca-GO administration. BODY.CONCLUSIONS: Two months administration of Maca-GO to early-postmenopausal women volunteers, resulted in significant reduction of Placebo-corrected results for FSH (P<0.05) with a simultaneous increase in E2 (P<0.001) and HDL (P<0.05). Maca-GO treatment resulted in a significant (P<0.01) alleviation of menopausal symptoms as expressed by reduction in frequency and severity of hot flushes and night sweating – in particular, followed by reductions in nervousness, mood swings, interrupted sleep pattern, fatigue, stress, headaches depression, and decreased libido. Maca-GO treatment significantly reduced menopausal discomfort in early-postmenopausal women as measured by total KMI and GMS values (from 22 to 10 and from 18 to 11 respectively). Toxicity of Maca-GO assessed in a laboratory model, showed its safety for use as dietary supplement for humans at the level determined as LD50>7.5 g/kg BW (on rats) and LD50>15 g/kg BW (on mice) – much higher than the minimum OECD limit LD50>2 g/kg BW. For Maca-GO to exhibit its significant hormone-balancing and therapeutic effect, it was essential for women to use it continuously during two consecutive months. Results indicated an existence of "residual effect" of Maca-GO in most measurements taken, after the treatment was withdrawn and replaced by Placebo for one month. There were significantly different responses of women in E2 to Maca-GO treatment in clinics located in two distant regions which may be attributed to socio-cultural and economic differences.

TITLE: A randomized prospective controlled trial comparing the laryngeal tube suction disposable and the supreme laryngeal mask airway: the influence of head and neck position on oropharyngeal seal pressure ABSTRACT.BACKGROUND: The Laryngeal Tube Suction Disposable (LTS-D) and the Supreme Laryngeal Mask Airway (SLMA) are second generation supraglottic airway devices (SADs) with an added channel to allow gastric drainage. We studied the efficacy of these devices when using pressure controlled mechanical ventilation during general anesthesia for short and medium duration surgical procedures and compared the oropharyngeal seal pressure in different head and-neck positions. ABSTRACT.METHODS: Eighty patients in each group had either LTS-D or SLMA for airway management. The patients were recruited in two different institutions. Primary outcome variables were the oropharyngeal seal pressures in neutral, flexion, extension, right and left head-neck position. Secondary outcome variables were time to achieve an effective airway, ease of insertion, number of attempts, maneuvers necessary during insertion, ventilatory parameters, success of gastric tube insertion and incidence of complications. ABSTRACT.RESULTS: The oropharyngeal seal pressure achieved with the LTS-D was higher than the SLMA in, (extension (p=0.0150) and right position (p=0.0268 at 60 cm H2O intracuff pressures and nearly significant in neutral position (p = 0.0571). The oropharyngeal seal pressure was significantly higher with the LTS-D during neck extension as compared to SLMA (p= 0.015). Similar oropharyngeal seal pressures were detected in all other positions with each device. The secondary outcomes were comparable between both groups. Patients ventilated with LTS-D had higher incidence of sore throat (p = 0.527). No major complications occurred. ABSTRACT.CONCLUSIONS: Better oropharyngeal seal pressure was achieved with the LTS-D in head-neck right and extension positions , although it did not appear to have significance in alteration of management using pressure control mechanical ventilation in neutral position. The fiberoptic view was better with the SLMA. The post-operative sore throat incidence was higher in the LTS-D. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT02856672, Unique Protocol ID:BnaiZionMC-16-LG-001, Registered: August 2016. BODY.BACKGROUND: Laryngeal Tube Suction Disposable, LTS-D (VBM Medizintechnik GmbH, Sulz, Germany) [1, 2] and Supreme Laryngeal Mask Airway, SLMA (Intavent Orthofix, Maidenhead, UK) [3, 4] are second generation, single-use, supraglottic airway devices (SADs), with added gastric access, for use in spontaneously and mechanically ventilated patients undergoing general anesthesia (Fig. 1) Supreme Laryngeal Mask Airway and (Fig. 2) Laryngeal Tube Suction Disposable. Fig.1 Supreme Laryngeal Mask Airway Fig. 2Laryngeal Tube Suction Disposable The effectiveness of the LTS-D and the SLMA has been well established [5–11]; however, the oropharyngeal seal pressure of both devices in different head/neck positions and the performance of these devices using positive pressure ventilation have not been evaluated. Changing the head/neck position can alter the sealing capabilities of the SAD. To our best knowledge there are no studies comparing the oropharyngeal seal pressure in different head and neck position when using these devices. Our study compared the LTS-D with the SLMA, hypothesizing that the two devices would provide adequate oropharyngeal seal pressure in different head and neck positions and perform similarly during pressure controlled ventilation in neutral position, despite differences in their structural design. BODY.METHODS: One hundred sixty patients, ASA physical status I and II weighing between 50 and 100 kg, with normal airways, undergoing general anesthesia for minor elective surgical procedures in supine position were randomly assigned to have either a LTS-D or a SLMA for airway management. Two hospitals in two different countries (Bnai Zion Medical Center, Haifa, Israel and Italiano Hospital in Buenos Aires, Argentina) participated in the study. IRB approval was obtained separately for each institution and written informed consent was obtained from each of the study subjects. The patients were blinded to their device type assignment. Each study center conducted eighty cases with even randomization for the type of airway device. Randomization was performed using a computer generated list and by opening a sealed envelope immediately before induction. The surgery types were from a wide range of elective minor general surgery, orthopedic, urologic, gynecologic, and plastic surgery of short and moderate duration. Exclusion criteria were: age <18 years, weight <50 kg, or >100 kg, body mass index >35 kg/m2, cervical spine disease limiting neck movement, a known difficult airway, intrinsic lung disease and patients having active gastro esophageal reflux disease. The following demographic data were collected for each patient: sex, age, height, weight, body mass index, type of operation, and total time of anesthesia. Two attending anesthesiologists in each medical center participated in the trial. Each had previously performed more than 50 insertions of the LTS-D and SLMA. Midazolam 0.05 mg/kg IV and IV fentanyl 2 mcg/kg were administered during a 3 min period of time during preoxygenation. General anesthesia was induced with IV propofol 2 mg/kg and neuromuscular blockade was achieved with IV rocuronium bromide 0.6 mg/kg. After induction, sevoflurane end-tidal concentration up to 1.5 % in 33 % oxygen and 66 % nitrous oxide was started and ventilation was controlled by facemask was controlled by facemask for three min, and then the SAD was inserted. A size 4 or 5 LTS –D and size 4 or 5 SLMA was used according to manufacturer's recommendations. In accordance with these recommendations the cuffs were inflated to 60 cm H2O immediately after insertion, using a cuff pressure gauge (VBM Medizintechnik GmbH, Sulz, Germany). The airway was judged to be effective and adequate if an expiratory volume of at least 6 mL/kg was obtained during gentle manual ventilation, at a peak airway pressure of at least 15 cm of H2O, no oral leak as judged by stethoscope auscultation over the neck, and a normal square-wave capnograph trace. The expiratory volume data and the square wave capnograph trace were obtained using the integrate spirometer and capnograph of the S/5TM Anesthesia Delivery Unit (EDU) (Datex-Ohmeda, Helsinki, Finland). The total time to achieve an effective airway was measured as the time after the anesthesiologist removed the facemask until the square-wave capnograph tracing was observed after insertion. Two attempts at insertion were permitted. If unsuccessful (as judged by an inability to insert the device or by a total lack of ventilation), we planned to secure the airway using an endotracheal tube. If after the insertion of the device the airway was judged to be inadequate by the above criteria, but some ventilation was obtained, further maneuvers were allowed to properly position the device. These adjustments included minor interventions: (adjusting head or neck position, applying jaw lift or changing depth of insertion) or major interventions (reinsertion of the device, or changing the size). Insertion of the devices was classified as 1 = easy, 2 = moderate, 3 = difficult and 4 = impossible, as evaluated by the anesthesiologist. Once an effective airway was achieved, oropharyngeal cuff seal pressures were obtained by closing the expiratory valve of the anesthesia circuit with a fixed gas flow rate of 3 L/min and noting the airway pressure at which equilibrium was reached. The maximum allowed airway pressure during this evaluation period was 40 cm H2O [12]. The oropharyngeal cuff seal pressure was obtained in neutral position (occiput on standard firm pillow 7 cm in height), maximal flexion (chin touching the thorax) maximal extension and maximal lateral left and right head/neck rotation. Before checking the oropharyngeal seal pressure in each different head and neck position the intra cuff pressure was re-checked and adjusted to 60 cm H2O if necessary, in order to standardize the measurement conditions. This way, the oropharyngeal seal pressure was only influenced by the change of the anatomical position. During the maintenance of the anesthesia the patients' lungs were initially ventilated with 17 cm H2O pressure control ventilation, a respiratory rate of 12/min, and adjustments made to maintain the ETCO2 to 35–40 mmHg. Respiratory variables were measured using a Datex AS-5 monitor. The fiberscope score was determined in all cases in neutral position by passing a pediatric flexible bronchoscope (Storz, Germany, 3.1 mm) through the airway tube of the devices to a level of the one of two ventilation apertures of the LTS-D or to the unique ventilation aperture of the SLMA. In this position, the operator could redirect the tip of the fiberoptic bronchoscope in order to obtain best possible glottic view. We used a previously described scoring system scoring the fiberoptic view [13]. The SLMA has the drain tube on the central part of the airway channel. Therefore, the fiberoptic scope can pass on either side of the drain tube until reaching the ventilation aperture situated between the epiglottis fins. The LTS-D has a separate drain channel, so that the fiberoptic scope is passed in the middle of the airway channel and the view could be obtained through the two frontal apertures of the device. The fiberoptic score was ranged from grade 4 (full view of arytenoids and glottis), 3 (arytenoids and glottis partly visible), 2(view of arytenoids, glottis or epiglottis), and 1 (no part of larynx identifiable). A single attempt was made to pass a lubricated (K-Y gel, Johnson and Johnson, USA) 18 gauge - French gastric tube through the LTS-D and a 16-French gauge gastric tube through the drain tube of the SLMA. Placement of the gastric tube in the stomach was confirmed by aspiration of gastric contents or synchronous injection of air and epigastric auscultation. After successful insertion, the gastric tube was removed. The following data were recorded every 5 min commencing after checking the oropharyngeal seal pressure until the administration of the reversal of the muscular relaxant: oxygen saturation, inspired and expired tidal volumes, respiratory rate, fraction of inspired oxygen, ETCO2, peak inspiratory pressure, plateau pressure, and PEEP. The integrity of the flow volume loops to monitor for leak was controlled during all the procedure. At the end of the surgery sevoflurane and nitrous oxide were discontinued and the patients were allowed to breathe 100 % O2. Reversal of the neuromuscular blockade was obtained with neostigmine and atropine. The airway device was removed when the patients could open their mouths to command and after a train-of four count of four was obtained. Each device was examined for the presence of blood and the mouth was inspected for dental or mucosal trauma. Patients were interviewed to determine the incidence and severity of postoperative airway related adverse events. Non-leading questions were asked by a blinded research assistant after release of the patient from the Post Anesthetic Care Unit (PACU) and 24 h postoperatively. Perioperative adverse events were graded: mild = coughing or gagging on insertion, hiccups, gastric insufflations; moderate = bronchospasm, airway obstruction, blood staining of the device, oral or tongue pain, sore throat, hoarseness, difficulty in swallowing, sore neck, mandibular pain, dysphasia and dysphonia; severe = hypoxia, regurgitation, aspiration, dental trauma, soft tissue trauma, gross blood-staining of the device. BODY.METHODS.STATISTICAL ANALYSIS: For the demographic continuous variables (age, weight, height and BMI) means and standard deviations were calculated. The results of the demographic continuous variable between the two study groups (Supreme vs LTS) were analyzed by the 2 sample T-test for differences of mean. For the categorical variables (gender, ASA, device insertion categorical parameters and postoperative complications), numbers and percentages were calculated. The distributions for the categorical variables between the two study groups were compared and analyzed by the Chi square test (a parametric test) or by Fisher-Irwin exact test (a non-parametric test for small numbers). For the continuous of seal pressure variables means, standard deviations and ranges were calculated. The results of the leak pressure continuous variable between the two study groups (SLMA vs. LTS-D) were analyzed by the two sample T-test for differences of mean. The results of the leak pressure continuous variable comparing neutral to each of the other head/neck positions in each of the two study groups were analyzed by the T-test paired. For the respiratory continuous variables of inspirium, expirium, saturation, ETCO2, peak inspiratory pressure, PEEP the average of the 12 repeated measurements (every 5 min in first the hour) for each patient was calculated and then means and standard deviations were computed. The results of the respiratory continuous variables between the two study groups (Supreme vs. LTS) were analyzed by the two sample T-test for differences of mean. All statistical tests were analyzed to a significance level of 0.05. Based on previous studies [14, 15], the sample size was calculated to find a difference of 5 cm of H2O in the oropharyngeal seal pressure between the two devices with standard deviation of 8 cm H2O. for a type I error of 0.05 and power of 0.88. This calculation allowed us to establish sample size of 40 patients in each group being (80 per each medical center with a total of 160 patients). BODY.RESULTS: There was no difference in the demographic and surgical data between the groups, Table 1. LTS-D and the SLMA were inserted successfully in all patients requiring one attempt in 87.5 and 86.2 % of patients for SLMA and LTS-D respectively and a second attempt in 12.5 and 13.8 % of patients for SLMA and LTS-D respectively (p = 0.8150). The insertion time was 31.6 ± 12 and 29.4 ± 12.8 seconds for the SLMA and LTD-S respectively (p = 0.2680).Table 1Demographic and surgical dataSLMA (n = 80)LTS-D (n= 80) p valueAge (years)55.1 ± 11.355.5 ± 13.3 †0.8080Weight (kg)68.5 ± 8.369.3 ± 10.7 †0.6136Height (cm)167 ± 9167 ± 8 †0.9548BMI (kg/m2)24.7 ± 2.425.0 ± 3.4 †0.5067Gender (M/F)29/5136/64 %32/4840/60 % ├0.6250ASA(I/II) I17 (21)14 (18) ├0.5480 II63 (79)66 (82)Duration of anesthesia (min)45.2 ± 20.948.3 ± 22.7 †0.3725Data are mean ± SD or numbers (%) P value by †2- sample T-test for differences of mean or ├chi square test; p >0.05 (NS) Significantly less minor airway interventions were needed with the SLMA (p = 0.005). Data describing the ease of insertion and airway interventions is described in Table 2.Table 2Insertion dataSLMA (n = 80)LTS-D (n = 80) p valueEase level 164 (80.0)58 (72.5) ┴0.5900 211 (13.8)15 (18.8) 35 (6.2)7 (8.7)Minor intervention No intervention35 (43.8)19 (23.8) ├0.0050** Adjusting head/neck position16 (20.0)33 (41.2) Jaw lift or changing depth of insertion29 (36.2)28 (35.0)Data are mean ± SD or numbers (%) P value by ├chi square test; or ┴Fisher exact test; **p ≤0.01 (Sig) or p >0.05 (NS) The oropharyngeal seal pressure of the LTS-D was higher that the SLMA in extension (p=0.0150) and right position (p=0.0268 at 60 cm H2O intracuff pressure. This probably reflects the different mechanism of seal of the two devices. The cuff of the SLMA surrounds the laryngeal inlet in longitudinal position and forms a seal with the periglottic tissues, whereas the LTS-D forms a seal by exerting pressure against all the circumference of the pharyngeal mucosa. No significant differences have been detected between devices in all other positions, Table 3.Table 3Oropharyngeal seal pressureLeak pressureSLMA (n = 80)LTS-D (n= 80) p valueNeutral (cmH2O)33 ± 6 (19–40)35 ± 6 (22–40) †0.0571Extension (cmH2O)31 ± 5 (17–40)33 ± 5 (20–40) †0.0150*Flexion (cmH2O)35 ± 6 (19–40)35 ± 5 (21–40) †0.4711Right (cmH2O)32 ± 5 (19–40)34 ± 5 (19–40) †0.0268*Left (cmH2O)32 ± 5 (18–40)34 ± 5 (18–40) †0.1140Balloon pressure (cmH2O)60 ± 0.2 (58–60)60.0 ± 0 (60–60) †0.3188Data are mean ± SD (range) P value by †2- sample T-test for differences of mean *p ≤0.05 (Sig) or p >0.05 (NS) When compared with neutral position the oropharyngeal seal with SLMA was significantly higher with head/neck flexion (p = 0.0007) and significantly lower with head/neck extension (0.0002) and right rotation. (0.0480). When compared with neutral position the oropharyngeal seal pressure for the LTS-D was significantly lower with head/neck extension. (0.0038). The mean peak inspiratory pressure and plateau pressure in patients with SLMA was significantly lower than in patients with LTS-D (p = 0.006) and (p = 0.008). Compared with the neutral position, the leak pressure for the SLMA was significantly higher from extension (0.0002) and from right position (0.0480), and significantly lower from flexion (0.0007). Compared with the neutral position, the leak pressure for the LTS-D was significantly higher from extension (0.0038). No significant differences have been detected between devices for all other respiratory parameters (Table 4).Table 4Respiratory data SLMA (n = 80) LTS-D (n = 80) p valueVentilation inspirium (ml)501 ± 125524 ± 138 †0.2787Ventilation expirium (ml)444 ± 110475 ± 123 †0.0965Ventilation delta (ml)57 ± 3548 ± 26 †0.0792O2 Saturation (%)99 ± 0.899 ± 0.8 †0.8632ETCO2 (mmHg)33 ± 334 ± 4 †0.4356Peak (cmH2O)16 ± 318 ± 4 †0.0060**Peep (cmH2O)2 ± 0.72 ± 0.6 †0.2631P plat (cmH2O)11 ± 212 ± 2 †0.0080**Data are mean ± SD P value by †2- sample T-test for differences of mean; **p ≤0.01; p >0.05 (NS) There was a significant difference in fiberoptic view score favoring the SLMA. Fiberoptic position (4/3/2/1/) was 25/34/15/6 for the SLMA and 15/25/11/29 for the LTS-D (p = 0.0001). Upper airway trauma, as evaluated by the presence of blood staining of the devices after their removal was not significant statistically between the two devices. Gastric tube insertion was successful in all patients in both groups. In PACU, there was no significant difference between the groups regarding the post-operative complications. (Sore throat: 12 % in the SLMA group (95 % C.I: ±7.8) and 14 % in the LTS-D group, (95 % C.I.: ±8.3) (p = 0.668), dysphagia: 0 % in the SLMA group and in the LTS-D 2 % group (p = 0.497), (95 % C.I.: ±3.4) dysphonia: 0 % in the SLMA group and 3 % in the LTS-D group (95 % C.I.: ±4.2) (p = ˫0.245). A significantly higher incidence of post-operative sore throat was detected in patients with LTS-D (26.3 %) (95 % C.I.: ±9.7) as compared to patients with SLMA (6.3 %) (95 % C.I.: ±5.3) after 24 h (p = 0.001). The percentage of patients with at least one complication was 13 % in the SLMA group and 18 % in the LTS-D group (p = 0.317). Gastric tube insertion was successful in all patients in both groups. BODY.DISCUSSION: The main finding of this study was that the oropharyngeal seal pressure for the LTS-D is higher that the SLMA in right and head- neck extended position, at 60 cm H2O intracuff pressures. Even though there was statistically significant difference favoring the LTS – D, this finding has no clinical significance as both devices proved to have an excellent seal pressure. Our results are consistent with previous studies reporting high seal pressure with both devices [6, 10, 16–18]. Genzwuever et al [10] found a oropharyngeal seal pressure of 33.1 cm H2O using LTS II, a device similar to LTS –D. However, there are also reports of a very low oropharyngeal seal pressure for both devices [15, 19]. Kikuchi et al. [15] found an oropharyngeal seal pressure as low as 16 cm H2O with the LTS II, which the author attributes to the Asian ethnicity of the participants. Tham et al. [19], in a crossover study also reports a low oropharyngeal seal pressure for the SLMA (19.6 cm H2O). The value of the fiberoptic anatomical position assessment of a SAD in predicting a functional oropharyngeal seal is controversial [20, 21, 22]. The better fiberoptic score we obtained with the SLMA may have a potential benefit when considering an endotracheal tube placement thought the device. The vocal cords could not be visualized in 36.3 % of the patients with LTS-D, however in all these patients ventilation was adequate, confirming findings in a previous report that there is no correlation between the adequacy of ventilation and a low fiberoptic score [6]. We found an increased oropharyngeal seal pressure in the SLMA and in the LTS-D during neck flexion. Our results are in accordance with the Brimacombe et al. [23] reports on the influence of the head and neck flexion and extension on the oropharyngeal seal pressure. A reduction or increase of approximately 25 % in pharyngeal volume during head and neck extension or flexion may increase or decrease the oropharyngeal seal pressure [23]. Early reports with the Classic LMA described an increased oropharyngeal seal pressure during neck flexion and decreased during extension [24, 25]. Neck flexion causes a reduction in the antero posterior pharyngeal diameter and with the better seal provided by the SLMA the oropharyngeal seal pressure is increased. Neck extension increases the pharyngeal antero-posterior diameter by raising the laryngeal inlet, leading to a reduced contact of the SLMA cuff with the mucosa and therefore a drop in the oropharyngeal seal pressure. The oropharyngeal seal pressure decreased for both devices in right and left head and neck rotation suggesting that the contact with the pharyngeal mucosa could be affected by the change in the antero-posterior and lateral diameter. Similar to Park et al. [26], our study showed that the LTS-D can maintain an acceptable oropharyngeal seal pressure after extension, flexion and rotation of the head and neck. Even though we found statistically significant decreases of the oropharyngeal seal pressure in right head/neck and extension position, the seal pressures remained high enough to potentially allow effective mechanical ventilation. We cannot specifically comment of the ventilation adequacy in this positions as we did not address this question in our study. Surgeries in different head/neck positions using SAD's to secure the airway are frequently performed and additional studies need to be performed to check the effectiveness of the devices in these positions. In our study, we found both the LTS-D and the SLMA easy and quick to insert, with an equally high success on the first attempt. However the data concerning the insertion of both devices is different in comparison with other studies. The insertion time for the LTS-D in our study was longer than that reported by Russo et al. [16] with the LTS-D, (14 s) and from Mihai et al. [6] with the LTS II, (15 s), and faster than the one of Schalk et al. [27] performed by paramedics and emergency physicians using LTS-D, (45 s) and Kikuchi T et al. [15] using the LTS II, (40 s). The insertion time for the SLMA was longer in comparison with other studies. Verghese et al. [4] reported an insertion time of 15 s and Cook et al [28] 18 s, however it was faster than the insertion time for SLMA was described by Zhang et al. 38 s [29]. We believe that the differences in the insertion time could be related to the investigators experience and different definition to define an effective airway. Despite the high percentage of the maneuvers necessary to optimize the position of the device, the success rate after two insertion attempts was 100 % reflecting the excellent clinical effectives of both devices. Cook et al. [28] reported 30 maneuvers necessary to optimize the airway patency in 24 patients. Most studies report no failures during inserting the LTS-D [6, 10] and SLMA [18, 28, 30]; however, success rate of as low as 70 % with the LTS-D has also been reported [16]. Both devices provided optimal oxygenation and ventilation during pressure control ventilation. We found a higher peak pressure inspiratory pressure with the LTS-D. Previous studies with the first version of the Laryngeal Tube Suction (LTS) (with only two ventilation orifices) reported a high inspiratory pressure attributed to the narrow size of the ventilations holes or to the obstruction by the soft tissue causing increased resistance to gas flow [11, 31]. The LTS-D used in the present study has four additional ventilation orifices to improve ventilation. Our results confirming those of Russo et al. [16] proving that the resistance to gas flow is still high and probably additional improvement of the ventilation outlet is still needed. We had a high success rate in passing a gastric tube trough the gastric port of both devices. The LTS-D allows passage of the gastric drain tube number 18F whereas the largest size tube that can be used for a SLMA is 16F. We did not find a correlation between the successful insertion of a gastric tube and the fiberoptic score, even though that was previsously reported for the Proseal Laryngeal Mask Airway and has been associated with a good fiberoptic score [32, 33]. The reported sore throat related to LTS-D use varies largely from 7 to 71 % [6, 10, 16]. Probably depending on the level of the providers experience with the device. We found 14 % of patients in the LTS-D group complaining of sore throat when leaving PACU. Our incidence of sore throat with the SLMA is higher than the results reported by others studies [18, 31, 33] (8–15 %) but similar with the that reported by Bermann et al. [34]. However, the causes of postoperative adverse events such as sore throat after general anesthesia using supraglottic devices are multifactorial, with the overall incidence influenced by the depth of anesthesia at the time of insertion, the method of insertion [35], the number of insertion attempts [36] the duration of the anesthesia [36], the mode of ventilation used [37], and the type of postoperative analgesia provided [38]. Similar to Cook et al [28] and Mihai et al [6] we found symptoms of upper airway trauma after 24 h, 6. 3 % in the SLMA group and 26.3 % of patients in the LTS group complained for sore throat. The incidence of sore throat after 24 h was higher than it the PACU, presumably because of the masking effect of analgesics administered in the immediate post-operative period. Twenty four hours after the discharge of PACU, 8.8 % of SLMA patients group and 31.3 % of LTS-D patients group had at least one minor perioperative complication event suggesting that the SLMA is less traumatic that the LTS-D. Our study has several limitations. Although the investigators were experienced with both devices, the large prior experience with all types of the LMAs may have given the SLMA a possible advantage. In addition, the operators were obviously unblinded to the type of the SAD so that bias could not be excluded. The devices were inserted three minutes after the administration of the neuromuscular blocking agent however, we did not use neuromuscular blockade monitoring to confirm to ensure similar SAD placement conditions. Our results may not be applicable to patients with spontaneous ventilations. We didn't ventilate the patients in the different head and neck positions, so we cannot know the performance of the devices during mechanical ventilation despite the good seal pressure measured. Cuffs constructed from PVC are less susceptible to nitrous oxide diffusion [39]. However, as we did not check the intracuff pressures throughout the procedure, we cannot exclude possible nitrous oxide diffusion that could affect the seal pressure of the devices. BODY.CONCLUSIONS: Better oropharyngeal seal pressure was achieved with the LTS-D in head-neck right and extension positions, although it did not appear to have significance in alteration of management using pressure control mechanical ventilation in neutral position.

TITLE: Letrozole in the neoadjuvant setting: the P024 trial ABSTRACT: Neoadjuvant chemotherapy trials have consistently reported lower response rates in hormone receptor-positive (HR+) breast cancer when compared with HR− cases. Preoperative endocrine therapy has therefore become a logical alternative and has gained considerable momentum from the finding that aromatase inhibitors (AIs) are more effective than tamoxifen for HR+ breast cancer in both the neoadjuvant and adjuvant settings. The most convincing neoadjuvant trial to demonstrate the superiority of an AI versus tamoxifen was the P024 study, a large multinational double-blind trial in postmenopausal women with HR+ breast cancer ineligible for breast-conserving surgery. The overall response rate (ORR) was 55% for letrozole and 36% for tamoxifen (P < 0.001). Significantly more letrozole-treated patients underwent breast-conserving surgery (45 vs. 35%, respectively; P = 0.022). In addition, ORR was significantly higher with letrozole than tamoxifen in the human epidermal growth factor receptor HER1/HER2+ subgroup (P = 0.0004). The clinical efficacy of letrozole in HER2+ breast cancer was confirmed by fluorescent in situ hybridization analysis and was found to be comparable to that of HER2− cases (ORR 71% in both subsets). Biomarker studies confirmed the superiority of letrozole in centrally assessed estrogen receptor-positive (ER+) tumors and found a strong relationship with the degree of ER positivity for both agents. Interestingly, letrozole was effective even in marginally ER+ tumors and, unlike tamoxifen, consistently reduced the expression from estrogen-regulated genes (progesterone receptor and trefoil factor 1). Furthermore, when analyzed by Ki67 immunohistochemistry, letrozole was significantly more effective than tamoxifen in reducing tumor proliferation (P = 0.0009). Thus, neoadjuvant letrozole is safe and superior to tamoxifen in the treatment of postmenopausal women with HR+ locally advanced breast cancer. BODY.INTRODUCTION AND RATIONALE: Early-stage breast cancer is traditionally treated with an initial surgery such as lumpectomy or mastectomy followed by subsequent adjuvant therapy, including radiotherapy, chemotherapy, and hormone therapy [1, 2]. Numerous studies have investigated the use of preoperative (neoadjuvant) hormonal therapy or chemotherapy prior to surgical intervention, with the goals to improve surgical outcome and obtain long-term disease-free survival (DFS) (see Table 1). The National Surgical Adjuvant Breast and Bowel Project B-18 trial demonstrated that doxorubicin and cyclophosphamide administered as neoadjuvant or adjuvant therapy had equivalent outcomes in terms of both DFS and overall survival (OS) [3]. Adding a taxane in the B-27 trial significantly reduced the local recurrence rate but did not significantly increase DFS or OS [4]. Importantly, neoadjuvant chemotherapy has been shown to increase the rate of breast-conserving surgery (BCS) without adversely affecting DFS or OS [3, 5–7]. Therefore, neoadjuvant chemotherapy has become the standard treatment approach for locally advanced breast cancer and an accepted option for patients with primary operable disease [8]. Table 1Aims of neoadjuvant therapy in different breast cancer populationsPopulationAimsTreatment optionLocally advanced breast cancerPrimary: to improve surgical optionsFit and healthy patients: chemotherapySecondary: to obtain freedom from disease, to gain information on tumor responseUnfit patients with hormone-sensitive disease: endocrine treatmentOperable breast cancer and candidates for adjuvant chemotherapyPrimary: to obtain freedom from diseaseChemotherapy ± OFS and/or AIsSecondary: to improve surgical options, to gain information on tumor responseSequence versus combinationLonger versus shorterOperable breast cancer and candidates for adjuvant endocrine treatment alonePrimary: to improve surgical optionsEndocrine treatment longer versus shorterSecondary: to gain information on tumor responseTamoxifen versus AIsReprinted from [8] with permission from the American Society of Clinical OncologyAIs aromatase inhibitors, NST neoadjuvant systemic therapy, OFS ovarian function suppression Significant tumor reduction from neoadjuvant chemotherapy occurs in only subsets of breast cancer, however. For example, patients with estrogen receptor-negative (ER−), high-grade, and highly proliferative tumors appear to benefit the most from neoadjuvant chemotherapy [9–11]. In contrast, significantly lower response rates have been reported in patients with ER+ tumors in neoadjuvant chemotherapy trials [12–15]. The German Breast Group demonstrated that the pathologic complete response (pCR) rates were 6.2 versus 22.8% for ER+ and ER− tumors, respectively (odds ratio 3.23, 95% confidence intervals [CIs] 1.91, 5.46; P = 0.0001) [12]. Recent studies indicate that the sensitivity to chemotherapy is likely determined by the underlying gene expression pattern and the molecular subtype of the tumor [16, 17]. The luminal subtype categorized by gene expression profiling, which encompasses most of the ER+ tumors, is less responsive to preoperative chemotherapy than the basal-like and human epidermal growth factor receptor 2-positive (HER2+) cancer. In addition, neoadjuvant chemotherapy is associated with substantial toxicity that may limit its clinical benefits and acceptability, especially in the elderly patient population [13, 18–20]. Thus, there is great need for effective alternatives to cytotoxic chemotherapy in hormone-responsive locally advanced breast cancer. Endocrine treatment is an attractive alternative to chemotherapy as neoadjuvant or primary systemic therapy for women with hormone-responsive primary breast tumors [21–23]. A study comparing neoadjuvant endocrine therapy with exemestane versus chemotherapy in 152 patients with ER+/progesterone receptor-positive (PgR+) breast cancer found that while efficacy outcomes were comparable in the two groups, chemotherapy treatment was significantly more toxic, confirming that endocrine therapy could be used as an alternative to chemotherapy in older women [24]. Early studies of primary tamoxifen as an alternative to surgery were conducted in older women with hormone-responsive breast cancer who were unfit for chemotherapy [23, 25–27]. While these studies demonstrated a reduction in tumor size with primary tamoxifen, long-term local disease control was found to be poor. The omission of primary surgery resulted in an increased rate of progression, therapeutic intervention, and mortality [28]. Thus, primary tamoxifen treatment is indicated for only the most frail, medically ill, or noncompliant patients [29]. Although primary therapy with tamoxifen was found to be well-tolerated, adverse effects were reported including hot flushes, skin rash, vaginal discharge, breast pain, sleepiness, headache, vertigo, itching, hair loss, cystitis, acute thrombophlebitis, nausea, and indigestion [29]. Furthermore, more serious adverse effects, such as an increased risk of endometrial cancer and thromboembolic events, have been reported in large trials of adjuvant tamoxifen [30]. In view of the limitations of tamoxifen as a single modality treatment, attention has switched to the neoadjuvant use of endocrine therapy to increase the rate of breast conservation [21]. Data from preclinical models [31] and clinical studies in advanced breast cancer [32] predict that aromatase inhibitors (AIs) may be more effective than tamoxifen in the neoadjuvant setting. Furthermore, preclinical and clinical evidence suggests that letrozole may be the most effective AI in this setting [33–35]. In postmenopausal women with endocrine-responsive locally advanced or metastatic breast cancer, first-line treatment with letrozole was shown to be significantly more effective than tamoxifen in terms of response rate (overall response rate [ORR], 30 vs. 20%, P = 0.0006) and time to disease progression (41 vs. 26 weeks) [32]. A phase 1–2 pilot study showed a clinical response rate of 88% (21/24 patients) in postmenopausal women with ER+ locally advanced breast cancer treated with letrozole for 3 months prior to surgery [36]. All patients in the study were eligible for breast conserving surgery following neoadjuvant letrozole. A large multinational trial, P024, was designed to assess the efficacy of neoadjuvant letrozole compared with tamoxifen in postmenopausal women with hormone receptor-positive (HR+) breast cancer who were ineligible for breast-conserving surgery [21]. The clinical objectives of the trial were to compare response rates and surgical outcomes between the two treatment arms. In addition, the trial provided the opportunity to conduct prospective biomarker studies to explore the biologic basis for response to neoadjuvant endocrine therapy [37]. This review describes the results of the P024 study and the follow-up biomarker studies, focusing on the treatment implications in the neoadjuvant setting. BODY.TRIAL DESIGN AND PATIENTS: PO24 was a multinational, randomized, double-blind controlled trial comparing letrozole and tamoxifen in postmenopausal women with hormone-responsive primary invasive breast cancer who were not eligible for breast-conserving surgery [21]. The trial was conducted in 55 centers in 16 countries between March 1998 and August 1999. Local ethics review boards approved the protocol and all patients gave written informed consent before study enrollment. BODY.TRIAL DESIGN AND PATIENTS.RANDOMIZED CLINICAL TRIAL DESIGN: Patients were randomly assigned to receive letrozole 2.5 mg or tamoxifen 20 mg administered orally once daily for 4 months prior to scheduled surgery [21]. Patients were considered to have completed the study when they had received 4 months of treatment and had been assessed for surgery. Following surgery, patients were treated at the investigator's discretion and were followed for 5 years for local recurrence, distant metastasis, and survival. BODY.TRIAL DESIGN AND PATIENTS.PATIENT POPULATION: A total of 337 postmenopausal women were enrolled into the trial, and 324 were included in the intent-to-treat population (see Fig. 1). Eligible patients were postmenopausal women with untreated, primary HR+ (≥10% nuclear staining for ER or PgR) invasive breast tumors (stages T2–4a–c, N0–2, M0). The minimum tumor size was ≥3 cm, and all patients were considered inoperable or ineligible for breast-conserving surgery, mostly because of a non-favorable ratio tumor size/breast size. Exclusion criteria included previous exposure to AIs, uncontrolled endocrine or cardiac disease, bilateral or inflammatory breast cancer, distant metastasis, and other malignant disease. In addition, administration of other cancer treatment or hormone replacement therapy was not allowed during study participation. Fig. 1Patient disposition [21] BODY.TRIAL DESIGN AND PATIENTS.TRIAL END POINTS: The primary end point was ORR, defined as the percentage of patients in each treatment arm with a complete response (CR) or a partial response (PR) as determined by breast palpation [21]. Response categories were CR, PR, no change, progressive disease, or not assessable/not evaluable. The secondary end points were the percentage of patients who underwent breast-conserving surgery and the response rate (CR + PR) determined at 4 months by mammography and by ultrasound [21]. Safety was assessed and adverse events graded according to the National Cancer Institute Common Toxicity Criteria. BODY.TRIAL DESIGN AND PATIENTS.CORRELATIVE SCIENCE STUDIES: In parallel, studies were conducted in which tumor biopsies and blood samples were taken from all patients prior to the initiation of therapy and at the end of treatment to generate a database from which changes in molecular markers could be assessed [37–41]. These studies were prospectively designed to assess the biological basis for letrozole efficacy. The specific objectives of the biomarker sub-studies were as follows:To confirm ER and PgR status in a central laboratory. ER and PgR were determined using immunohistochemistry (IHC) performed on pretreatment biopsies; the criterion for minimal ER positivity was 10% positive cells [37]. The Allred histopathological score was applied to further assess the level of ER and PgR expression in both the baseline and surgical specimens [42].To explore relationships between ER and PgR expression levels and response to treatment [37].To examine the relationship between the expression of HER1 and HER2 and the likelihood of primary response [37]. Previous studies have shown that HER2 overexpression may be a predictor of tamoxifen resistance [43, 44], and HER1 has also been linked with endocrine therapy resistance [45]. HER2 IHC was initially scored as 0/+ (negative) or ++/+++ (positive or overexpressed) [46]. Since fluorescent in situ hybridization (FISH) testing has replaced IHC as the gold standard for HER2 assessment, the P024 tumor bank was reassessed using HER1 and HER2 FISH probes, and these data on HER2 gene amplification status were supplemented with 106 tumor samples [40]. Pre- and post-treatment tumor biopsy samples were also analyzed for ER and several indices of ER function, including PgR and trefoil factor 1 (TFF1), HER1 and HER2, and the proliferation marker Ki67 [38].To assess gene expression profiling as a means to further investigate the transcriptional programs that underlie resistance and sensitivity to estrogen deprivation [39]. Gene expression (mRNA) profiles were collected from the tumor biopsies collected prior to surgery, at 1 month of treatment, and at surgery. The analysis was done using an Affymetrix U 133 subA Gene Chip.To measure aromatase expression in tumors before and after 4 months of letrozole or tamoxifen treatment. Aromatase is the key enzyme responsible for estrogen biosynthesis and is present in about 70% of tumors. IHC was performed with a monoclonal aromatase antibody (677) on trial samples prior to and following letrozole or tamoxifen treatment (n = 185); scoring was measured as a proportion of immuno-positive cells and their intensity of reactivity in malignant epithelial, stromal, adipose, and normal compartments [41]. BODY.EFFICACY: In the intent-to-treat population, 154 patients received letrozole and 170 received tamoxifen. Similar proportions of patients in the letrozole and tamoxifen groups had inoperable tumors (13 and 14%, respectively). Other baseline characteristics were also well-balanced for age, race, HR status, and tumor/nodal stage of disease. More patients in the tamoxifen group (n = 41) discontinued treatment than in the letrozole arm (n = 23). The main reason for premature discontinuation was disease progression. BODY.EFFICACY.CLINICAL RESPONSE AND BREAST-CONSERVING SURGERY: Letrozole was consistently superior to tamoxifen for primary and secondary efficacy end points [21]. The ORR was 55% for letrozole versus 36% for tamoxifen (P < 0.001). Median time to response was 66 days in the letrozole group and 70 days in the tamoxifen group. The odds ratio for achieving CR + PR was more than doubled with letrozole (2.23, 95% CI 1.43, 3.50; P = 0.0005). In terms of clinical progression, 12% of patients on letrozole and 17% on tamoxifen progressed, while 24% of letrozole- and 35% of tamoxifen-treated patients had stable disease. Letrozole was also shown to be significantly more effective than tamoxifen when response rates were assessed by mammography (34 vs. 16%, respectively; P < 0.001) and ultrasound (35 vs. 25%, respectively; P = 0.042). The proportion of patients able to undergo breast-conserving surgery was significantly higher in the letrozole group than in the tamoxifen group (45 vs. 35%, respectively; P = 0.022). Of note, the odds ratio for breast-conserving surgery was 4.56 (P = 0.0001) for patients presenting with T2 tumors compared with all other T stages. The only other factor that increased the odds of undergoing breast-conserving surgery was treatment with letrozole (odds ratio 1.71, P = 0.03). BODY.EFFICACY.ER AND PGR: A supportive analysis of clinical efficacy data conducted in patients with centrally confirmed ER+ or PgR+ tumors also showed that outcomes were significantly improved with letrozole (n = 124) compared with tamoxifen (n = 126) [37]. In this subgroup, the ORR was 60% for letrozole versus 41% for tamoxifen (P = 0.004), and breast-conserving surgery was performed in 48 versus 36%, respectively (P = 0.036). There was a linear relationship between ER Allred expression scores and response rates to both letrozole and tamoxifen (see Fig. 2). Tumors with low ER expression were still responsive to letrozole but not to tamoxifen [37]. Of note, letrozole response rates were numerically superior to tamoxifen response rates in every ER Allred category from three to eight, indicating that letrozole is more effective than tamoxifen regardless of the level of ER expression [37]. This finding is important, because of all the AIs, only letrozole has demonstrated clear ORR superiority over tamoxifen in ER-poor tumors. When the effects of letrozole and anastrozole on tumors with low ER values (Allred scores 2–5) were compared in the neoadjuvant setting, only letrozole achieved a significant reduction in cell proliferation in ER-poor tumors [35]. Fig. 2Clinical response rate versus estrogen receptor (ER) Allred score for letrozole and tamoxifen. The P value for a linear logistic model was 0.0013 for letrozole and 0.0061 for tamoxifen according the Wald test. In this analysis, ER−, PgR+ cases (determined by conventional cut points) were excluded. Reprinted from [37] with permission from the American Society of Clinical Oncology Letrozole, but not tamoxifen, significantly reduced the expression of estrogen-regulated proteins PgR and TFF1 [38]. Average tumor PgR expression decreased dramatically on letrozole treatment (P = 0.0001), and only 4.4% of surgical specimens exhibited an Allred score of between six and eight for PgR expression. The significant decrease in PgR expression with letrozole remained significant in the ER+, HER2+ subpopulation. In contrast, changes in PgR expression with tamoxifen therapy were not consistent, with both increases and decreases in expression frequently observed in the overall and ER+, HER2+ subpopulations. Analysis of changes in TFF1 produced similar conclusions, with letrozole markedly reducing expression (P = 0.0001) and tamoxifen producing no overall trend in either direction. BODY.EFFICACY.HER1/HER2 AND RESPONSE: Approximately 15% of tumors were ER+ and overexpressed both HER1 and HER2. The response rate in this patient subgroup was significantly higher with letrozole than with tamoxifen (88 vs. 21%) [37]. The odds ratio for response to letrozole versus tamoxifen was 28 (95% CI 4.5, 177; P = 0.0004). Letrozole was equally effective for HER1/HER2+ and HER1/HER2− tumors, whereas tamoxifen was significantly less effective in HER2+ compared with HER2− tumors (P = 0.045). These data suggest that although HER1 and HER2 status might not be the only explanation for the superiority of letrozole over tamoxifen, overcoming resistance pathways associated with HER1 and HER2 expression is a significant component of the improvement in outcomes associated with letrozole treatment observed in this clinical trial. FISH analysis of tumor samples confirmed the clinical efficacy of letrozole in breast cancers with or without HER2 amplification (ORR 71% in both subsets; P = 0.98). In contrast, tamoxifen-treated tumors with HER2 gene amplification had lower clinical response rates than tamoxifen-treated HER2− tumors (33 vs. 49%, P = 0.49) (see Table 2) [40]. Table 2Analysis of clinical, ultrasound, and mammogram response data according to HER2 FISH status in letrozole-treated patients and tamoxifen-treated patientsResponse CategoryNo. responsesTotal No. (%)No. responsesTotal No. (%)HER2 FISH-positive Tamoxifen-treated patientsHER2 FISH-negative Tamoxifen-treated patientsP-valueaClinical39 (33)4490 (49)0.49Ultrasound 39 (33)2674 (35)0.99Mammography 19 (11)2290 (24)0.68HER2 FISH-positive Letrozole-treated patientsHER2 FISH-negative Letrozole-treated patientsP-valuebClinical1217 (71)131185 (71)0.98Ultrasound817 (47)91170 (54)0.61Mammography716 (44)84178 (47)0.79Reprinted from [40] with permission from the American Society of Clinical OncologyHER2 human epidermal growth factor receptor 2, FISH fluorescence in situ hybridizationaFisher test P-valuebMantel–Haenzel P-value BODY.EFFICACY.BIOMARKERS OF TUMOR PROLIFERATION: Letrozole inhibited tumor proliferation, measured by the biomarker Ki67, to a greater extent than tamoxifen (reduction in geometric mean Ki67 level 87 vs. 75%, respectively; P = 0.0009). The differences in Ki67 reduction were also observed in ER+, HER1 and/or HER2 overexpressing tumors (88% for letrozole vs. 45% for tamoxifen, respectively; P = 0.0018) [38]. Changes in the percentage of Ki67-positive cells in HER1/2+ tumors treated with letrozole or tamoxifen are shown in Fig. 3. Fig. 3A box plot of before and after treatment Ki67 values in the estrogen-receptor-positive, human epidermal growth factor receptor (HER) 1/2+ subset. With letrozole (n = 15), 11 showed a decrease, one exhibited no change, and three showed an increase, of which only one was >2-fold (0.1–0.3%). With tamoxifen (n = 17), ten showed a decrease and seven an increase, of which three were relatively dramatic (9.5–22.7, 20.9–40.7, and 0.1–17.3%). Reprinted from [38] with permission from the American Association for Cancer Research More recently, it was found that HER2 FISH-positive tumors showed higher histologic grade (P = 0.009), higher pretreatment Ki67 (P = 0.005), and less Ki67 suppression after letrozole when compared with HER2 FISH-negative tumors (P = 0.0001) [40]. Letrozole significantly decreased the geometric mean Ki67 level in HER2 FISH-negative tumors (from 6.25 [95% CI 5.16, 7.58%] to 0.68% [95% CI 0.53, 0.87%]; P = 0.0001), but the decrease in HER2 FISH-positive tumors was blunted (from 14.73 [95% CI 9.67, 22.44%] to 8.1% [95% CI 4.16, 15.75%]; not significant). A similar observation for Ki67 was made in a smaller cohort of tamoxifen-treated tumors [40]. The relationship between cell cycle CR, defined as ≤1% of post-treatment Ki67 staining in the infiltrating component of the tumor, and HER2 status of tumors treated with letrozole was also analyzed [40]. Significantly more HER2 FISH-negative than FISH-positive tumors met the definition of a cell cycle CR at the time of surgery (60 vs. 12%; P = 0.0001). There was a high level of correlation between lack of cell cycle CR and the presence of a positive HER2 FISH test (P = 0.0001) for letrozole- and tamoxifen-treated tumors, which is consistent with the conclusion that HER2 gene amplification generates resistance at the level of cell cycle progression regardless of which endocrine therapy is used [40]. These biomarker findings are discordant with clinical observations that tumor regression is unaffected by HER2 amplification status in patients treated with neoadjuvant letrozole and may imply therapeutic resistance that could manifest later in the clinical course of the disease. Consistent with this finding, Miller and colleagues also reported that neoadjuvant letrozole produces rapid and profound decreases in expression of Ki67 and PgR that do not always correlate with clinical and pathological responses [47]. BODY.EFFICACY.GENE EXPRESSION PROFILING: Preliminary gene expression profiling analysis of biopsies taken pretreatment and 1 month post letrozole treatment showed down-regulation of genes involved in DNA replication and synthesis, cell cycle progression, apoptosis suppression, and tissue invasion [39]. These results illustrate the molecular basis for estrogen-deprivation letrozole therapy which may be useful in the development of predictive models of ER+ breast cancer. BODY.EFFICACY.AROMATASE: Sufficient pre- and post-treatment tumor material was available from 171 cases (81 on letrozole and 90 on tamoxifen) from the P024 trial for immunohistochemistry analysis of aromatase protein expression [41]. Aromatase was detected in all tumor compartments, with the strongest staining observed in malignant epithelial cells. Median aromatase values did not change significantly with letrozole or tamoxifen treatment; however, changes in score did occur in individual cases, with more noticeable effects observed in letrozole-treated patients [41]. A positive correlation existed between baseline ER and aromatase staining in cancer cells, while a negative correlation was observed between baseline Ki67 and aromatase expression in cancer plus stroma. Baseline aromatase expression did not predict response to letrozole or tamoxifen, or changes in Ki67 induced by treatment. However, negative staining in both stroma and cancer after treatment was strongly associated with fewer cell cycle CR and smaller Ki67 declines with letrozole (but not tamoxifen) treatment [48]. BODY.SAFETY AND DURATION OF THERAPY: P024 demonstrated that letrozole is well-tolerated in the neoadjuvant setting [21]. There were no major tolerability differences between letrozole and tamoxifen, and adverse effects of a similar nature were seen in 57% of patients in each arm [21]. The most common treatment-related adverse event was hot flushes, occurring in 20% of patients in the letrozole group and 24% of patients in the tamoxifen group. The excellent tolerability, predictable pharmacokinetics, and minimal drug–drug interactions [49, 50] make letrozole a particularly suitable option for older women unable to tolerate or unwilling to accept neoadjuvant chemotherapy and in whom the presence of comorbidities and use of concomitant therapies complicate treatment selection [20]. The median age of patients in P024 treated with letrozole was 68 years, and 46% of patients were at least 70 years old. The feasibility and safety of letrozole was also reported recently from another trial using letrozole as primary systemic therapy in elderly patients (median age 79 years) with breast cancer [51]. In addition, letrozole has been successfully administered to elderly patients (median age of the elderly subgroup 75 years; range 70–96 years) in the advanced breast cancer setting [52]. The favorable safety profile of letrozole also allows for the extension of the neoadjuvant treatment beyond the 4 months used in the P024 trial. In a recent study, 33 postmenopausal women with HR+ breast cancer ineligible for breast-conserving surgery were treated with letrozole for 4 months. Continued administration of letrozole for a further 4 months in responders and patients with stable disease resulted in a statistically significant improvement in tumor size reduction (P = 0.039); ORR was 90% in patients receiving preoperative treatment for longer than 4 months compared with 57% in patients receiving treatment up to 4 months [53]. In another study, 42 patients who were unsuitable for breast-conserving surgery or had refused surgery after responding to initial neoadjuvant therapy with letrozole for 3 months benefited from continuing tumor volume reduction during further letrozole treatment administered for up to 12 months [54]. The median reductions in tumor volume were 52% (95% CI 37, 62) from 0 to 3 months, 57% (95% CI 26, 100) from 3 to 6 months, and 66% (95% CI 22, 100) from 6 to 12 months. Extending the duration of letrozole also improved the CR rate, which increased from 4/42 patients (9.5%) at 3 months to 12/42 (29%) by 6 months and 8/22 (36%) by 12 months. BODY.CONCLUSIONS: Neoadjuvant therapy with AIs is a safe and effective treatment option for postmenopausal women who are unwilling or unable to undergo surgery or preoperative chemotherapy [21, 22, 55–57]. Letrozole is significantly more effective than tamoxifen in the neoadjuvant setting in terms of ORR and increased rate of breast-conserving surgery [21, 55]. One of the advantages of using a neoadjuvant strategy is the opportunity to gain information on tumor response early in the course of treatment. Detailed studies correlating clinical response to neoadjuvant therapy with changes in tumor biomarkers and gene expression may ultimately prove useful to tailor therapy for individual patients and to gain a better understanding of the biology of HR+ breast cancer. Correlative studies conducted using tumor samples from P024 have revealed important information about how breast tumors respond to letrozole [37–40]. Letrozole was shown to be significantly more effective than tamoxifen in the inhibition of ER+ tumor proliferation, since letrozole produced a greater reduction in levels of the proliferation biomarker Ki67 [38]. It has been suggested that a greater suppression in proliferation could lead to greater long-term survival in the adjuvant setting. Preliminary data from the Immediate Preoperative Anastrozole Tamoxifen or Combined with Tamoxifen trial have indicated that short-term changes in Ki67 levels, after 2 and 12 weeks, may be a useful predictive marker for relapse-free survival in patients treated with neoadjuvant AI therapy [58]. It has also been suggested that changes in proliferation and concurrent changes in apoptosis may be expected to be more predictive of adjuvant benefit from endocrine therapy than clinical response [59]. Correlative studies have also highlighted the complexity of breast cancer biology and revealed discordance between clinical and biomarker responses [40]. Amplification of HER2 was shown to be associated with a more aggressive breast cancer phenotype and greater resistance to tamoxifen [37, 40]. Clinical response data from the P024 trial have shown that letrozole is equally effective in HER2+ and HER2− tumors, whereas tamoxifen is less effective in HER2+ tumors [37]. These data suggest that letrozole could be a superior option to tamoxifen for postmenopausal women with HER2+, HR+ tumors [37]. However, analysis of proliferation markers has provided evidence of estrogen-independent proliferation of ER+, HER2+ breast cancer despite neoadjuvant letrozole [40]. It appears that cell-cycle regulation is partially or completely estrogen-independent in the majority of primary tumors showing HER2 gene amplification, and patients with such tumors may eventually develop resistance to adjuvant AI therapy. Novel strategies to delay or overcome hormone resistance are described elsewhere in this supplement, in the article, "Femara and the future." Gene expression profiling has demonstrated that letrozole targets genes responsible for DNA replication and synthesis, cell cycle progression, apoptosis, and tissue invasion [39]. Research into genetic profiling is continuing, with the aim of developing clinically relevant predictive models that can accurately classify ER+ disease according to likely response to specific neoadjuvant therapies. Predictive models will improve treatment individualization and help to avoid unnecessary treatment-related toxicity in patients unlikely to benefit from systemic therapies [17, 60]. The P024 trial has clearly demonstrated the therapeutic superiority of letrozole over tamoxifen for the neoadjuvant management of primary breast cancer. The trial has also provided the oncology community with a validated research setting within which to gain valuable insights into the molecular features of ER+ breast cancer and its treatment that will help shape new therapies in the years to come.

TITLE: Changes in Weight, Waist Circumference and Compensatory Responses with Different Doses of Exercise among Sedentary, Overweight Postmenopausal Women ABSTRACT.BACKGROUND: It has been suggested that exercise training results in compensatory mechanisms that attenuate weight loss. However, this has only been examined with large doses of exercise. The goal of this analysis was to examine actual weight loss compared to predicted weight loss (compensation) across different doses of exercise in a controlled trial of sedentary, overweight or obese postmenopausal women (n = 411). ABSTRACT.METHODOLOGY/PRINCIPAL FINDINGS: Participants were randomized to a non-exercise control (n = 94) or 1 of 3 exercise groups; exercise energy expenditure of 4 (n = 139), 8 (n = 85), or 12 (n = 93) kcal/kg/week (KKW). Training intensity was set at the heart rate associated with 50% of each woman's peak VO2 and the intervention period was 6 months. All exercise was supervised. The main outcomes were actual weight loss, predicted weight loss (exercise energy expenditure/ 7700 kcal per kg), compensation (actual minus predicted weight loss) and waist circumference. The study sample had a mean (SD) age 57.2 (6.3) years, BMI of 31.7 (3.8) kg/m2, and was 63.5% Caucasian. The adherence to the intervention was >99% in all exercise groups. The mean (95% CI) weight loss in the 4, 8 and 12 KKW groups was −1.4 (−2.0, −0.8), −2.1 (−2.9, −1.4) and −1.5 (−2.2, −0.8) kg, respectively. In the 4 and 8 KKW groups the actual weight loss closely matched the predicted weight loss of −1.0 and −2.0 kg, respectively, resulting in no significant compensation. In the 12 KKW group the actual weight loss was less than the predicted weight loss (−2.7 kg) resulting in 1.2 (0.5, 1.9) kg of compensation (P<0.05 compared to 4 and 8 KKW groups). All exercise groups had a significant reduction in waist circumference which was independent of changes in weight. ABSTRACT.CONCLUSION: In this study of previously sedentary, overweight or obese, postmenopausal women we observed no difference in the actual and predicted weight loss with 4 and 8 KKW of exercise (72 and 136 minutes respectively), while the 12 KKW (194 minutes) produced only about half of the predicted weight loss. However, all exercise groups had a significant reduction in waist circumference which was independent of changes in weight. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT 00011193 BODY.INTRODUCTION: The increasing prevalence of overweight, obesity and the associated comorbidities of excess weight are well documented and represent a major challenge to public health and the health care systems. [1]–[4] Regular exercise is considered a central component of weight loss and there are many organizations that recommend an hour or more per day of exercise to prevent weight gain, promote weight loss and/or prevent weight regain. [1], [5]–[8] Given the potential for exercise to create a negative energy balance, the relative importance of exercise in promoting weight loss is far more complicated than expected as exercise-based weight loss studies produce considerably less weight loss than predicted. For example, in a comprehensive review of clinical exercise and weight loss trials, Ross et al noted that in studies lasting more than 25 weeks in duration the average weight loss was only ∼30% of predicted. [9] The difference between actual weight loss and predicted weight loss has been termed "compensation" and participants whose exercise induced weight loss is less than predicted based on caloric expenditure have been identified as "compensators". [10] In all previous exercise studies exploring weight compensation, only one dose of exercise was examined and these were typically large doses of exercise prescribed specifically for weight loss. [9]–[11] This is important because the exercise recommendation for general health is 30 minutes per day on most days of the week, which is considerably less than the weight loss recommendation of 60 minutes per day most days of the week. [1], [5]–[8], [12], [13] To our knowledge there are no studies that have examined weight compensation across different doses of exercise. The Dose-Response to Exercise in postmenopausal Women (DREW) study was designed to examine the health benefits of 50%, 100%, and 150% of the NIH Consensus Panel physical activity recommendation in sedentary, overweight or obese, postmenopausal women with elevated blood pressure. [14], [15] The results from the primary outcomes, cardiorespiratory fitness and blood pressure, have been reported but due to the large undertaking and costs associated with conducting the DREW trial a number of important secondary outcomes were included a priori in the study design including changes in weight. [15] In DREW, participant retention was excellent (92%), non-exercise activity was monitored and the exercise training was supervised with outstanding adherence (∼97% for completers). [15] Thus the DREW study represents a unique opportunity to examine actual compared to predicted weight loss (compensation) across different doses of exercise. BODY.METHODS: A complete description of the DREW design, methods and primary outcomes has been published elsewhere.[14], [15] In brief, the study was a randomized, dose-response exercise trial with a no-exercise control group and 3 exercise groups with incrementally higher doses of energy expenditure. The study was originally reviewed annually by The Cooper Institute and subsequently approved by the Pennington Biomedical Research Centers Institutional Review Board (IRB) for continued analysis. Prior to participation, all participants signed a written informed consent document outlining the procedures involved in the DREW study. The protocol for this trial and supporting CONSORT checklist ate available as supporting information; see Checklist S1 and Protocol S1. An abbreviated CONSORT diagram is provided in Figure 1 . The full CONSORT diagram for the DREW study has been previously published. [15] We conducted a total of 4545 telephone screens between April 2001 and June 2005. After baseline testing, a two week run-in period, and giving informed consent, 464 postmenopausal women within the age range of 45 to 75 years, who were sedentary (not exercising more than 20 minutes on 3 or more day a week, and <8000 steps per day assessed over the course of one week), overweight or obese (BMI 25.0 to 43.0 kg/m2), and had a systolic blood pressure of 120.0 to 159.9 mm Hg were randomized to 1 of 3 exercise groups or a non-exercise control. Exclusion criteria included history of stroke, heart attack, or any serious medical condition that prevented participants from adhering to the protocol or exercising safely. Participants were recruited using a wide variety of techniques including newspaper, radio, television, mailers, community events, and email distributions. [14], [15] 10.1371/journal.pone.0004515.g001Figure 1Participant flow diagram. The non-exercise control group was asked to maintain their baseline levels of activity and dietary habits during the six-month study period. All participants were asked to record their daily steps (see below) and fill out monthly medical symptoms questionnaire forms. We calculated the prescribed exercise energy expenditure for women in the DREW study to meet the consensus public health recommendation from the NIH and other organizations.[13], [16] We used data from sedentary women recruited for previous exercise trials conducted by our group, and also from our large cohort study.[17]–[20] Specifically, we estimated that the typical sedentary postmenopausal woman who started an activity program and followed the consensus public health recommendation would expend 8 kcal/kg/week (KKW) in the exercise program. Details of these calculations are presented in the DREW design and methods report.[14] A major objective of DREW was to evaluate exercise levels 50% below and 50% above the current public health recommendations to test whether the lower dose provides any benefit and whether the higher dose provides proportionally more benefit than the standard 8-KKW exercise level. Thus, women were assigned to either a non-exercise control group or to groups that expended 4, 8, or 12 KKW. Exercising women participated in 3 or 4 training sessions each week for 6 months with training intensity at the heart rate associated with 50% of VO2. During the first week, each group expended 4 KKW. Those assigned to the 4 KKW group remained at this dose for the duration of the study while those assigned to the 8 and 12 KKW groups continued to increase their energy expenditure 1 KKW until their assigned exercise level was reached. All exercise sessions were performed under supervision in an exercise laboratory, and strict monitoring of the amount of exercise completed during each session was performed. Participants were weighed each week, and their weight was multiplied by their exercise dosage to determine the number of calories to be expended for the week. Women in the exercise groups alternated training sessions on semi-recumbent cycle ergometers and treadmills. Adherence to exercise training over the entire 6-month period was calculated for each individual by dividing the caloric expenditure during the exercise training by the caloric expenditure prescribed for the training period ×100% Fitness testing was conducted using a Lode Excalibur Sport cycle ergometer (Groningen, Netherlands), an electronic, rate-independent ergometer. Participants cycled at 30 Watts (W) for 2 min, 50 W for 4 min, followed by increases of 20 W every 2 min until they could no longer maintain a pedal cadence of 50 rpm. Respiratory gases were measured using a Parvomedics True Max 2400 Metabolic Measurement Cart. Breath-by-breath respiratory gases were measured using a Parvomedics True Max 2400 Metabolic Measurement Cart. Volume and gas calibrations were conducted before each test. Gas-exchange variables (VO2, CO2 production, ventilation, and respiratory exchange ratio [RER]) were averaged every 15 s. Heart rate was measured directly from the ECG monitoring system. Ratings of perceived exertion (RPE) were obtained using the 20-point Borg scale. Two fitness tests were performed on separate days at baseline and follow-up, and the average value from these two tests were used for baseline and follow-up values for analyses. There were two sources of weight data. The primary analysis was performed using the weights measured at baseline and follow-up in the clinical assessment laboratory. These visits occurred in the morning under fasting conditions. Weight was measured on an electronic scale (Siemens Medical Solutions, Malvern, PA) and height was measured using a standard stadiometer. This scale was calibrated weekly. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. For the individuals in the exercise groups there was a second source of weight data available for analysis. These were the weights taken during the first exercise session of each week in the exercise training room. We used the same type of electronic scale for these measurements and each scale was calibrated weekly. These weights were used to determine the exercise prescription for the week and were taken in the non-fasted state with participants wearing their workout clothes but no shoes. Waist and hip circumference were measured using the recommendations of the Airlie Conference.[21] Body fat was estimated from the sum of four skinfolds; triceps, abdomen, iliac crest and thigh. [22] To calculate predicted weight change we assumed that 1 Kg of weight represented 7700 kcal in energy. Caloric expenditure from supervised exercise (total and weekly) was divided by 7700 kcal to predict weight change. To assess potential changes in nonsupervised physical activity, all randomized participants wore a step counter (Accusplit Eagle, Japan) over the 6-month experimental trial to record their daily steps. Exercisers removed the step counter during supervised exercise sessions. Smoking history and medication use were assessed by detailed questionnaire. Diet was assessed by the Food Intake and Analysis System semi-quantitative food frequency questionnaire. [23] Participants were repeatedly informed that the study was not a weight loss trial and were asked not to change their dietary or physical activity habits. The primary purpose of our current analysis was to examine weight compensation across different doses of exercise. Accordingly, we limited our analysis to participants with baseline and follow-up weight values and in participants randomized to an exercise group we excluded individuals with a supervised exercise energy expenditure adherence of 85% or less. Descriptive baseline characteristics of groups were tabulated as means and SDs or as percentages, but not tested for differences. We calculated average step data per month for each randomization group. Compensation was defined as the difference between predicted and actual weight loss. Participants whose weight compensation was greater than zero were defined as compensators. For each group we also calculated the mean percent of predicted weight loss achieved (actual/predicted ×100%). Weight compensation cannot occur in the control group; therefore, their data were not eligible for analysis. Differences in outcomes among the randomization groups were tested by ANOVA with adjustment as appropriate. For statistically significant ANOVAs (p<.05), all pairwise comparisons among the randomization groups were tested using Tukey studentized range adjustment. Results are presented as adjusted least-squares means with confidence intervals. For each exercise group, we examined mean predicted weight loss, actual weight loss and compensation for weeks 1 through 23. Using ANOVAs (p<.05) with adjustment for multiple comparisons we examined if the mean compensation for each group was statistically different from zero for each week of the intervention. This analysis was limited to individuals with at least 19 weeks with recorded body weight data. Body weight values from the previous week were carried forward when body weight data was missing. All reported P values are two-sided. All analyses were performed using SAS version 9.0 (Cary, NC). BODY.RESULTS: Of the 464 randomized participants, 427 returned for follow-up testing of which 3 individuals had missing weight data and 13 of the participants from exercise groups had an adherence value ≤85%, resulting in 411 (88.6%) participants with complete data. Table 1 summarizes the participant descriptive characteristics by group. The study sample had a mean age of 57.2 (6.3), BMI of 31.7 (3.8) and 63.5% of the sample was Caucasian. The mean adherence to the exercise prescription was 99.5, 99.3 and 99.2% across the 4, 8 and 12 KKW groups, respectively. To assess potential changes in non-exercise activity daily step counts were examined for individuals with complete step counter data. The mean steps per day at baseline for all participants were 4858 (1715) with no significant differences between groups. The mean steps per day during the first month of intervention were 5399 (1873), 5288 (1870) and 5264 (1933) for the 4, 8 and 12 KKW group respectively. There were no significant differences in mean weekly steps during the first month among any of the exercise intervention groups, but the non-exercise control group (6098 (2065)) had significantly (P<0.05 for each) more steps compared to all of the exercise groups. However, by the 6th month of intervention there was no statistically significant difference in mean steps between any of the groups. During month 6 the daily steps for the control group was 5830 (2046) and 5416 (2054), 5444 (1866) and 5473 (1749 for the 4, 8 and 12 KKW groups. respectively. This finding is virtually identical to what we observed with the full cohort. [15] Also similar to previous reports from the full study cohort there was a dose response in change in fitness (P-trend<0.001) across the study groups with the 4, 8 and 12 KKW groups having a 3.7 (95% CI:1.9, 5.4), 6.9 (4.7, 9.2), and 8.0 (5.9, 10.2) percent change in fitness, respectively, each of which was statistically significant (p<0.001) compared to the control group (−2.7 (−4.9, −0.6)). [15] 10.1371/journal.pone.0004515.t001 Table 1 Participant Characteristics * . Characteristics Randomization Groups All Control 4 kcal/kg/wk 8 kcal/kg/wk 12 kcal/kg/wk (n = 411) (n = 94) (n = 139) (n = 85) (n = 93) Demographics Age, y 57.2 (6.3) 57.2 (5.9) 57.9 (6.5) 56.7 (6.4) 56.4 (6.3) Ethnicity, No. (%)  Caucasian 261 (63.5) 62 (65.9) 83 (59.7) 49 (57.7) 67 (72.0)  African American 123 (29.9) 23 (24.5) 47 (33.8) 29 (34.1) 24 (25.8)  Hispanic/other 27 (6.6) 9 (9.6) 9 (6.5) 7 (8.2) 2 (2.2) Weight, kg 84.2 (11.9) 85.6 (12.4) 83.4 (11.6) 85.0 (12.8) 83.1 (11.1) Body mass index, kg/m 2 † 31.7 (3.8) 32.2 (3.9) 31.4 (3.7) 32.2 (4.1) 31.1 (3.6) Body fat, % 28.9 (4.9) 30.8 (5.5) 27.6 (4.1) 29.3 (4.8) 28.7 (4.8) Waist circumference, cm 100.9 (11.8) 102.8 (12.0) 100.0 (11.3) 101.9 (12.0) 99.2 (11.9) Peak absolute VO 2 , L/min 1.30 (0.24) 1.33 (0.27) 1.28 (0.24) 1.28 (0.22) 1.32 (0.23) Peak relative VO 2 , ml/kg/min 15.6 (2.8) 15.7 (2.9) 15.5 (2.9) 15.1 (2.2) 16.1 (2.8) Data From Exercise Training Laboratory Total Exercise Energy Expenditure Prescribed, Kcal N/A N/A 7975 (1188) 15305 (2235) 20730 (2753) Total Exercise Energy Expenditure Achieved, Kcal N/A N/A 7932 (1193) 15197 (2249) 20560 (2734) 6-Month Adherence, % N/A N/A 99.5 (2.1) 99.3 (1.8) 99.2 (2.7) Time, min/week N/A N/A 72.2 (12.4) 136.3 (19.4) 193.7 (31.0) Abbreviations: VO 2 , volume of oxygen consumed, N/A, not applicable. * Continuous variables presented as means (SD) and dichotomous variables presented as count (percentage). † Calculated as weight in kilograms divided by height in meters squared. Figure 2 depicts the heterogeneity in the individual amount of weight change in each of the groups. As depicted in Figure 3 , all groups had a significant reduction in weight compared to weight at baseline (P<0.05 for each) but there were no significant differences between groups in weight change (P>0.05 for all between group comparisons). Change in weight was adjusted for age, baseline weight and ethnicity. Within the exercise groups the predicted weight change in the 4 and 8 KKW groups closely matched the actual weight loss while in the 12 KKW group the actual weight loss (−1.5 kg) was considerably less than the predicted weight loss (−2.7 kg). Analyses repeated without adjustment produced similar results. 10.1371/journal.pone.0004515.g002Figure 2Distribution of weight loss for each study group. 10.1371/journal.pone.0004515.g003Figure 3Actual weight loss (white bars) and predicted weight loss (black bars) for each study group.Actual weight loss was adjusted for age, baseline weight and ethnicity. Predicted weight loss represents caloric expenditure from supervised exercise divided by 7700 kcal/kg. Error bars represent 95 percent confidence intervals. The three panels of Figure 4 more closely depict compensation with all values adjusted for age and ethnicity. In the top panel, the amount of compensation in each exercise group is plotted. The 12 KKW (1.2 kg) had significantly greater compensation compared to the 4 and 8 KKW groups (−0.3 and −0.2 kg, respectively). The middle panel depicts the prevalence of compensation in the exercise groups. In the 4 and 8 KKW groups, 54.3% (p = 0.009) and 52.8% (p = 0.01) of participants, respectively, were compensators both of which were lower compared to 72.6% in the 12 KKW group. The lower panel summarizes the percent of predicted weight loss achieved in each of the exercise groups. The 4 and 8 KKW achieved over 100% of the predicted weight loss while the 12 KKW group only achieved 53.5% of the predicted weight loss. 10.1371/journal.pone.0004515.g004Figure 4The top panel depicts weight compensation (actual weight loss minus predicted weight loss) across exercise groups.The middle panel depicts the prevalence of compensation across exercise groups and the lower panel presents the percent of predicted weight loss for each exercise group. All values adjusted for age and ethnicity. Error bars in the top panel represent 95 percent confidence intervals. Figure 5 graphically depicts body weight data obtained in the exercise training lab for determination of exercise prescription. It should also be reiterated that this analysis was limited to individuals with at least 19 weeks with recorded weights resulting in 132, 84 and 87 participants used in the analysis for the 4, 8 and 12 KKW groups, respectively. The left column of the graphs compares the actual weight loss to predicted weight loss for each exercise group during weeks 1 through 23 of the intervention period. The right column of graphs plots the compensation during the exercise intervention for each of the groups. For the 4 KKW group, not only was there no compensation but the weight loss was more than expected at weeks 2, 3, 8, 16 and 18 through 23. For the 8 KKW group the weight loss was more than expected at weeks 2 through 6; however during weeks 7 through 23 actual and predicted weight loss were closely matched. For the 12 KKW group there was no evidence of compensation until week 10, at which point compensation started to trend upward and reached significance at week 19 and remained significant for the remainder of the intervention weeks. It should be noted that exercise during weeks 1–4 for the 8 KKW group and weeks 1–8 for the 12 KKW group were not performed at the full dose, as these weeks were part of the exercise dose ramping phase. 10.1371/journal.pone.0004515.g005Figure 5Predicted weight loss and actual weight loss are presented in the left column while compensation is presented in the right column for each exercise group during weeks 1 through 23 of the intervention period.This analysis was limited to individuals with at least 19 weeks with recorded weights and for missing values the weight from the previous week was carried forward. This resulted in 132, 84 and 87 participants used in the analysis for the 4, 8 and 12 KKW groups, respectively. It should be noted that exercise during week's 1–4 for the 8 KKW group and weeks 1–8 for the 12 KKW group were not performed at the full dose as these weeks were part of the exercise dose ramping phase. For compensation, statistical deviation from zero for each week was tested using ANOVAs with adjustment for multiple comparisons. Table 2 summarizes the energy intake data from baseline and follow-up across groups. Both at baseline and follow-up there were no between group differences in mean energy intake. However, for all groups, the reported mean energy intake was lower at follow-up compared to baseline. There were no between groups difference in baseline or follow-up in dietary protein, fat and carbohydrate intake (data not included). To explore mechanisms of weight loss in the control group, we categorized individuals in the control group into those that lost weight (n = 51) and those that maintained or increased weight (n = 37), and we examined mean follow-up energy intake adjusted for baseline energy intake. The individuals in the control group that lost weight had a lower energy intake at follow-up compared to individuals in the control group that did not lose weight (1766 [1618, 1927] versus 2035 [1820, 2239] kcal, P = 0.04). In the exercise groups we observed no difference in mean follow-up energy intake adjusted for baseline energy intake between the compensators and non-compensators. Given the considerable limitations of our self-report food frequency questionnaire, all the dietary results need to be interpreted with caution. 10.1371/journal.pone.0004515.t002 Table 2 Energy Intake at Baseline and Follow-up. Randomization Groups Control 4 KKW 8 KKW 12 KKW Between Groups P-Value (n = 88) (n = 120) (n = 77) (n = 80) Energy Intake, kcal/day * Baseline 2138 (1995, 2344) 2000 (1866, 2138) 2228 (2040, 2399) 2188 (2040, 2399) .16 Follow-Up 1862 (1698, 1995) † 1730 (1585, 1819) † 1831 (1656, 1995) † 1870 (1698, 2040) † .42 Abbreviations: KKW, kcal/kg/week. * Values are expressed as fitted mean (95% CI) of normalized data. † Within Group Differences Between Baseline and Follow-Up P<0.001. Because changes in weight do not provide any insight into where the weight has changed or whether the weight loss is primarily fat, muscle or both we examined changes in body fat and waist circumference. The change in body fat was not statistically different across the control, 4,8 and 12 KKW groups: 1.0 (−0.1, 2.1), −0.7 (−1.6, 0.2), −0.5 (−1.7, 0.6) and −0.1 (−1.2, 1.0), respectively (p>0.05 for each). In contrast, as depicted in Figure 6 all exercise groups had a reduction in waist circumference compared with the control group and these decreases were largely unchanged by adjustment for changes in weight. Interestingly the correlation between change in weight and change in waist circumference was only 0.34 (p<0.001) ), indicating that weight change accounted for only 11.6% of the variance in waist circumference change. To further explore the exercise-weight loss and waist circumference relations, we examined changes in waist circumference within stratifications of weight compensation and weight loss with the all data from the exercise groups combined. As depicted in Figure 7, with both the weight compensators and non-compensators there were significant decreases in waist circumference but reduction in waist circumference in the non-compensators was approximately twice as much as observed in the compensators. Similar results were found when the combined exercise data was stratified by weight loss (yes/no). These data suggest that waist circumference is decreased in response to exercise training even in the absence of weight loss but that when weight loss is present the decrease in waist circumference is significantly greater. 10.1371/journal.pone.0004515.g006Figure 6Change in waist circumference for each group with (diagonal striped) and without (open) adjustment for change in weight.The change in waist circumference was significantly different from control for each exercise groups in both analyses. All values adjusted for age and ethnicity. Error bars in the top panel represent 95 percent confidence intervals. 10.1371/journal.pone.0004515.g007Figure 7Changes in waist circumference within stratifications of weight compensation and weight loss with the all data from the exercise groups combined.The set of bars to the left represent change in waist circumference with categorization by weight compensation (yes/no) and set of bars to the right represent categorization by weight loss (yes/no). All values adjusted for age and ethnicity. Error bars in the top panel represent 95 percent confidence intervals. BODY.DISCUSSION: The primary finding from this large exercise intervention trial in postmenopausal women is that the difference between actual weight loss and predicted weight loss (compensation) increases with exercise dose. We confirmed the findings of previous studies that a relatively high dose of exercise (12 KKW or 194 minutes per week) results in compensatory mechanisms that attenuate weight loss in previously sedentary women. However, a dose of exercise consistent with the exercise prescription for general health (8 KKW or 136 minutes per week) did not result in compensation as the actual weight loss closely matched the predicted weight loss. An exercise dose of 4 KKW (72 minutes per week) also resulted in weight loss that slightly exceeded the predicted weight loss. Our findings are important because most exercise guidelines for weight loss recommend 200–300 minutes per week and we provide evidence that this amount of exercise induces compensation that results in significantly less weight loss than predicted. Our findings of different doses of exercise resulting in different amounts of compensation may in part explain the discrepant results from previous exercise and weight loss studies. If different doses of exercise result in different amounts of compensation it is not surprising that when exercise studies of varying doses and duration are examined collectively there is no relation between exercise dose and weight loss. Our findings provide an excellent example of the complex nature of the energy expenditure and energy intake relation, and the importance of considering both sides of the equation in creating weight loss programs. Numerous well conducted clinical exercise trials of long duration have reported less weight loss than expected. [9] For example, Donnelly et al observed that after 18 months of exercise training and achieving 2000 kcal per week of exercise, college-aged women had no weight loss, although women in the control group actually gained weight. [11] In a follow-up report Donnelly et al suggested that the observed "compensation" was due to increased energy intake, not changes in metabolic rate or non-exercise activity. [24] A recent report by King et al provides support that increased energy intake may be a major source of energy compensation as they noted that after 12 weeks of 2500 kcal per week in exercise training energy expenditure, in those individuals that did not lose the predicted amount of weight (compensators) there was a increase in caloric intake while in those that lost the predicted amount of weight (non-compensators) there was no change in caloric intake. [10] Unlikely sources of the observed compensation in the DREW study include changes in resting metabolic rate and the thermogenic effect of food as previous studies have found these variables not to be influenced by exercise training. [24] Our step counter data suggests that there was no change in outside activity in the exercise groups, nor were there any differences in average daily steps among the exercise groups. [15] Though doubly-labeled water or accelerometer data would have presumably been more accurate than our step data, it is still unlikely that substantial changes in non-exercise activity explain our findings. In the previously published DREW primary outcomes paper we reported that the mean follow-up waist circumference was lower in all exercise groups compared to the control group and there was no difference in mean follow-up weight across groups. [15] The current analysis allowed us to further explore the change in weight data including examining individual level data as well as weight compensation. Further we were able to examine if exercise induced changes in waist circumference are independent of weight loss. Our observation that adjusting for changes in weight did not meaningfully affect the change in waist circumference in any of the exercise groups as well as the observation that even in the exercise sub-group that did not lose weight there was a reduction in waist circumference is in agreement to previous published reports. [25] It is worth noting that the exercisers that did lose weight had approximately two times the loss of waist circumference compared to the exercisers that did not lose weight which is also in agreement with previous reports. [25] Excess abdominal obesity is associated with increased risk of mortality, CVD, diabetes, insulin resistance and metabolic syndrome. [26]–[29] These findings reinforce the positive health benefits of engaging in physical activity even in the absence of substantial weight loss. The DREW study was not designed to examine differences in actual and predicted weight loss nor the mechanism responsible for these observations. As such our measures of food intake, a food frequency questionnaire, is not optimal to measure small changes in energy intake. While we cannot conclude that an increase in energy intake in response to increased energy expenditure is the source of the observed compensation, based on the work of others we conclude it is the most likely cause. Our findings should not be interpreted as suggesting that lower doses of exercise are more effective in producing weight loss than higher doses. We emphasize that DREW was not a weight loss study and it was not designed to examine the nuances of exercise induced weight loss. As such we hope our findings stimulate more research into the variability and mechanisms of exercise induced compensation. In the 12 KKW group approximately 27% of individuals did not have significant compensation in response to exercise training suggesting there are individuals that can achieve higher doses of exercise without compensation. Though primarily focused on weight loss maintenance, not weight loss, data from the Weight Control Registry (WCR) demonstrates that individuals who have lost a substantial amount of weight and have maintained weight loss typically achieve 45 or more minutes a day of moderate intensity activity. [30] These data from the WCR provides further data that there are individuals that either are not prone to compensation or who have developed strategies to combat compensation. Developing methods to identify individuals likely to compensate, and developing/testing strategies to combat this compensation are areas deserving of future work. Our findings also offer important insight into the exercise dose required for the prevention of weight gain. Based largely on data from cross-sectional doubled labeled water studies, the 2005 USDA Dietary Guidelines and Institute of Medicine recommend obtaining at least 60 minutes of moderate intensity activity per day to prevent weight gain. [6], [8] Both the 4 and 8 KKW group in DREW not only did not gain weight but actually lost weight during the 6 month study period. These two groups performed 72 and 136 minutes per week of moderate intensity physical activity, which is considerably less than the recommended 60 minutes per day to prevent weight gain. Our findings highlight the need for more research, in particular large randomized controlled clinical trials, exploring the dose of physical activity required to prevent weight gain as 60 minutes a day may present itself as a daunting undertaking for most individuals but even more so for a habitually sedentary individuals. In addition, this dose may well lead to compensation by more individuals, and to less success in weight loss. Strengths of the DREW study include that it is an efficacy study, using a tightly controlled exercise doses, with all exercise completed in the laboratory and extensive monitoring of exercise energy expenditure. We obtained excellent exercise adherence and had a low dropout rate. Monitoring of steps per day throughout the 6-month period indicates that outside physical activity remained constant throughout the trial for all exercise groups, thus ensuring that observed group differences were due to the prescribed exercise dose. Further, the exercise doses, including intensity, are easily obtainable and are well tolerated by sedentary women; and this has important public health implications for refining future physical activity recommendations. The study has limitations because the sample is limited to sedentary, overweight or obese, postmenopausal women with elevated blood pressure. Thus, we do not know if the results will apply to other women or men. However, the study sample is a group that is likely to benefit from exercise training and represents a sizeable proportion, probably a majority, of U.S. women in the age range of 45 to 75 years. Further the study could have benefitted from a more rigorous and sensitive measure of energy intake. The food frequency questionnaire utilized in DREW prevented us from being able to definitively demonstrate that the observed compensation is the result of an increase in energy intake. However, it should be reiterated that examining compensation was not the a priori goal of DREW. The exercise training intensity was moderate and while this makes for good public health and clinical applicability, it is possible that higher levels of training intensity might produce different results in regard to compensation and changes in waist circumference. In this study of previously sedentary, overweight or obese, postmenopausal women we observed no difference in the actual and predicted weight loss with 4 and 8 KKW of exercise (72 and 136 minutes respectively), while the 12 KKW (194 minutes) produced only about half of the predicted weight loss. We need to gain a better understanding of the mechanisms responsible for this exercise dose dependent phenomenon and develop strategies to identify and treat potential compensators. BODY.SUPPORTING INFORMATION: Checklist S1CONSORT Checklist(0.13 MB PDF)Click here for additional data file. Protocol S1Trial Protocol(0.47 MB PDF)Click here for additional data file.

TITLE: Patient Empowerment Improved Perioperative Quality of Care in Cancer Patients Aged ≥ 65 Years – A Randomized Controlled Trial ABSTRACT.PURPOSE: This randomized controlled, clinical prospective interventional trial was aimed at exploring the effect of patient empowerment on short- and long-term outcomes after major oncologic surgery in elderly cancer patients. ABSTRACT.METHODS: This trial was performed from February 2011 to January 2014 at two tertiary medical centers in Germany. The study included patients aged 65 years and older undergoing elective surgery for gastro-intestinal, genitourinary, and thoracic cancer. The patients were randomly assigned to the intervention group, i.e. patient empowerment through information booklet and diary keeping, or to the control group, which received standard care. Randomization was done by block randomization in blocks of four in order of enrollment. The primary outcome were 1,postoperative length of hospital stay (LOS) and 2. long-term global health-related quality of life (HRQoL) one year postoperatively. HRQoL was assessed using the EORTC QLQ C30 questionnaire. Secondary outcomes encompassed postoperative stress and complications. Further objectives were the identification of predictors of LOS, and HRQoL at 12 months. ABSTRACT.RESULTS: Overall 652 patients were included. The mean age was 72 ± 4.9 years, and the majority of patients were male (68.6%, n = 447). The ^median of postoperative length of stay was 9 days (IQR 7–14 day). There were no significant differences between the intervention and the control groups in postoperative LOS (p = 0.99) or global HRQoL after one year (women: p = 0.54, men: p = 0.94). While overall complications and major complications occurred in 74% and 24% of the cases, respectively, frequency and severity of complications did not differ significantly between the groups. Patients in the intervention group reported significantly less postoperative pain (p = 0.03) than the control group. Independent predictors for LOS were identified as severity of surgery, length of anesthesia, major postoperative complications, nutritional state, and pre-operative physical functional capacity measured by the Timed Up and Go-test by multiple robust regressions. ABSTRACT.CONCLUSION: Patient empowerment through information booklet and diary keeping did not shorten the postoperative LOS in elderly onco-surgical patients, but improved quality of care regarding postoperative pain. Postoperative length of stay is influenced by pre-operative nutritional state, pre-operative functional impairment, severity of surgery, and length of anesthesia. ABSTRACT.TRIAL REGISTRATION: Clinicaltrials.gov. Identifier NCT01278537 BODY.INTRODUCTION: Cancer is among the leading cause of morbidity and death worldwide]. The incidence of (solid) cancer increases substantially with age [1]. With the increase of life expectancy in the developed countries, the incidence of cancer, and respectively volume of cancer surgery in patients older than 65 years, increases steadily. In Europe, it has been estimated that this age group accounts for approximately 58% of all cancers, and 69% of cancer deaths [1]. Surgery, as the "state of the art" therapy for most solid tumors, must now be performed in elderly patients, a major risk group with the highest perioperative mortality rate, ranging from 7% to 15% [2,3], prone to prolonged hospital stay [4,5]. Postoperative morbidity and mortality is associated with an immense socio-economic burden, making prevention of postoperative complications, improved quality of care, and reduced length of in-hospital stay a permanent interest of research [6]. As early as in 1958, Janis described that successful emotional inoculation could be achieved in patients facing severe stress by giving them preparatory information containing accurate warnings about what to expect [7]. Since then many studies investigated the influence of psycho-educational interventions on postoperative recovery [8–10]. Frequently analyzed outcomes were anxiety, pain and health-related quality of life [8,10]. In summary, providing pre-operative education as a form of patient empowerment, including the provision of preparatory information about the postoperative limitations, could improve the outcome, although evidence for this is still inconclusive [6,10–12]. Further, the studies hitherto published were conducted only for minor surgical procedures or cardiac bypass surgery comprising only small patient populations [6]. To our knowledge, the present trial is the first such study focusing on major onco-surgery in elderly patients. This RCT was aimed at investigating whether the short- and long-term outcome of elderly onco-surgical patients can be improved by patient empowerment, with specifically designed information, compared to a standard-of-care control group. Primary clinical outcomes were in-hospital length of stay (short-term perioperative risk) and global health-related quality of life (HRQoL, long-term perioperative risk). Secondary outcomes included perioperative quality of care such as postoperative stress, e.g. level of pain and postoperative complications. BODY.MATERIALS AND METHODS.ETHICS STATEMENT: The study PERATECS ("Patient empowerment and risk-assessed treatment to improve outcome in the elderly after gastrointestinal, thoracic or urogenitary cancer surgery") was conducted in compliance with the Helsinki declaration. The Institutional Review Board of Charité-Universitaetsmedizin Berlin approved the study (EA 2/241/08). The study is registered at ClinicalTrials.gov (NCT01278537). All participants provided written informed consent. BODY.MATERIALS AND METHODS.STUDY DESIGN: The study was performed as a randomized, controlled, open-label, clinical prospective double-center interventional study, at two tertiary medical care university hospitals in Germany (Charité, Universitätsmedizin Berlin (3200 Beds), Klinikum der Universität München (KUM) (2244 beds)). BODY.MATERIALS AND METHODS.DATA COLLECTION AND INCLUSION AND EXCLUSION CRITERIA: From February 2011 to September 2012, all patients older than 65 years scheduled for surgery for gastro-intestinal, genitourinary, gynecological or thoracic cancer were screened for eligibility. Follow-ups were conducted 3 and 12 months after surgery. Last patient last visit was completed in January 2014. Inclusion criteria were age ≥ 65 years, major onco-surgery, proficiency in the German language, a Mini Mental Score (MMSE) of 24 points or higher, as well as written informed consent. Exclusion criteria included, two or more concurrent carcinomas, emergency surgery, life expectancy of less than 2 months, as well as current participation in another trial. Patients lacking proficiency in the German language, living in a closed institution due to an official or judicial order, as well as patients unable or unwilling to provide informed consent could not participate (Fig 1). 10.1371/journal.pone.0137824.g001Fig 1Study flow diagram. BODY.MATERIALS AND METHODS.STUDY GROUPS: After screening and enrollment in the study, patients were randomly allocated to either the intervention group or the control group using block randomization with a block size of four. All participants in the trial received standard care, consisting of two separate visits from the surgeon and anesthetist one day before surgery. During these visits, the surgeon and anesthetist would respond to specific concerns of the patient or their family and obtain informed consent for the proposed surgery and general anesthesia.Several parameters were recorded daily for the first five postoperative days. Patients in the intervention group received additional information regarding the perioperative period. BODY.MATERIALS AND METHODS.INTERVENTION.INFORMATION BOOKLET AND PATIENT DIARY: The patients allocated to the intervention group, i.e. patient empowerment, received a booklet with additional information and a diary at least one day before surgery. The informational booklet and the diary were designed and reviewed by psychologists, anesthesiologists, geriatricians, and nurses, and consisted of 32 illustrated pages of large print (font size 14). In a pilot study, patients were asked to evaluate the information package and the diary, with their comments considered for the final version [13]. The booklet contained information about surgery, anesthesia and perioperative management, such as fasting time and premedication. Information was also included about acute postoperative pain therapy, mobilization, and nutrition, stressing the importance of being actively involved in the rehabilitation process. Life-style-related risks following discharge from the hospital, such as lack of physical activity, unhealthy nutrition and substance abuse were described. Furthermore, the booklet contained concrete information regarding, support groups and home care institutions offering specific assistance after discharge. Patients were repetitively encouraged to play an active role in their rehabilitation process and to ask questions regarding medication and therapy. The diary was kept daily for seven days, starting the day before the operation and continuing until five days after. The patients were encouraged to become more aware of their situation, including pain, mobilization, nutrition, etc. They were encouraged to ask for analgesics if their pain was not properly addressed, and to become more active on their own, to get out of the bed as far as possible, and were motivated to ask questions regarding their recovery and discharge. BODY.MATERIALS AND METHODS.CONTROL GROUP: Patients in the control group were only provided with the standard information regarding surgical and anesthesiological risks and procedures. BODY.MATERIALS AND METHODS.HEALTH-RELATED QUALITY OF LIFE (HRQOL): Quality of life as assessed via HRQoL scores from the European Organisation for Research and Treatment of Cancer 30-Item Core Quality of Life Questionnaire, version 3.0 (EORTC QLQ-C30), a validated questionnaire [14]. Patients completed questionnaires prior to surgery, as well as 3 and 12 months after surgery, using questionnaires sent by mail. The EORTC QLQ-C30 questionnaire is a 30–item questionnaire incorporating nine multiple-item scales and six single items. Global health-related quality of life is a two-item score using a 7-point Likert scale. The score was linearly transformed to a score from 0 to 100 and missing items were handled as in the manual described [15]. Higher scores in global HRQoL imply a higher health-related quality of life. A difference of 5–9 point in the scores represents a small change, 10–20 points a moderate change, and more than 20 points a large change in HRQoL [16]. The EORTC QLQ-C30 is a reliable and valid instrument for assessing HRQoL in cancer patients [14,17]. In 2013, Waldmann et al.[18] published reference data for health related quality of life assessed by EORTC QLQ C-30 for the German population. BODY.MATERIALS AND METHODS.PRE-OPERATIVE DATA COLLECTION: Demographic and clinical data were recorded at baseline. The collected data included age, gender, marital, socio-demographical status, and comorbidities. Comorbidities were evaluated with the Charlson Comorbidity Score (CCS) [19]. Furthermore, the following data were documented: Eastern Cooperative Oncology Group (ECOG) performance status [20], pre-operative risk assessment according to the classification of the American Society of Anesthesiologists (ASA) [21], severity of surgery measured by the Physiological and Operative Severity Scoring system for enUmeration of Mortality and morbidity (POSSUM) [22], cancer site and presence of metastases. The registered intra-operative parameters included duration of surgery, type of anesthesia and length of anesthesia. Additional screening scores for pre-operative risk assessment were elements of the Pre-operative Assessment of Cancer in the Elderly (PACE) [23]. The PACE was developed from the Comprehensive Geriatric Assessment (CGA), integrating a battery of validated instruments for the geriatric assessment, i.e. the Activity of Daily Living (ADL) [24], Instrumental Activities of Daily Living (IADL) [25], the Geriatric Depression Scale (GDS) [26], the Brief Fatigue Inventory (BFI) [27], the ECOG performance status [20], and the Mini Nutritional Assessment (MNA) [28]. Cognitive assessment included the Folstein Mini-Mental State Examination (MMSE) [29]. These measurements were amended by tests assessing the physical function of the patients: the Timed Up and Go test (TUG) [30], hand grip strength, and the Tinetti Balance and Gait test, which determines the fall risk [31]. The staff performing the interviews included study doctors, medical students and study nurses. A senior physician for geriatrics trained the staff in geriatric assessment before the start of the study, with regular refresher courses over the study period. Similarly, a psychologist trained the staff in assessment of postoperative cognitive dysfunction, including completion of the specific questionnaires. The time spent for the interviews ranged from 60 to 120 minutes. BODY.MATERIALS AND METHODS.PERIOPERATIVE TREATMENT: Anesthesia, surgery, perioperative treatment, as well as cancer treatment, were carried out according to the standard operating procedures in the respective university hospitals [32]. BODY.MATERIALS AND METHODS.POSTOPERATIVE DATA COLLECTION: Patients in both groups received daily study-related visits up to the fifth postoperative day. Complications were assessed daily, including operational revision and intensive care transfers. Perioperative complications were defined as any event occurring during hospital stay requiring treatment measures that are not routinely applied following the specific surgery. The recorded complications were revised by two independent reviewers in accordance with the Clavien scale [33], in which a grade of III to V were defined as major complications [34]. Postoperative stress was defined as occurrence of postoperative pain, and postoperative nausea and vomiting (PONV). Postoperative pain was assessed using the numeric rating scale (NRS) [35]. Pain therapy was documented daily, as well as occurrence of PONV, time of first enteral nutrition, intestinal paralysis, and start and duration of mobilization. Furthermore, delirium screening was conducted daily using the Confusion Assessment Method for Intensive Care Units (CAM-ICU) [36] for patients on ICU or the Nursing Delirium Scale (NUDESC) [37,38] for delirium assessment in all other patients. Also recorded were the duration of hospitalization (LOS), defined as time in hospital after surgery, as well as morbidity and in-hospital mortality. At discharge, length of hospital stay, length of ICU stay, and discharge mode were recorded. The Case Report Forms (CRF) for complications was also completed, as well as the HRQoL, GDS, handgrip strength, and TUG. The completeness of the diaries was also documented. At discharge, patients were asked if the information package and/or the diary were useful for their rehabilitation, and answers were given using a four point Likert scale. BODY.MATERIALS AND METHODS.FOLLOW-UP: Follow-up time was 1 year. All patients were contacted per mail 3 and 12 months after surgery. Survival status 12 months after surgery was recorded for all patients. The participants received questionnaires including the EORTC QLQ C30 by mail. If patients did not answer within two weeks, they were contacted by telephone. If the patients could still not be reached, their general practitioner (GP) was contacted and asked about patients' survival status. Further data about mortality was obtained from the clinical cancer registry of the Charité Comprehensive Cancer Center, Charité-Universitätsmedizin Berlin, Germany. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS.SAMPLE SIZE CALCULATION: The short time primary endpoint was the time to fulfill hospital discharge criteria. Postoperative length of hospital stay (LOS) was defined as the time interval between the date of surgery and the date of discharge. We hypothesized that an assumed LOS of 12 days could be reduced to 10 days due to fewer postoperative complications in the intervention group. The long time primary endpoint was the global health-related quality of life of elderly patients at 12 months follow-up. Criterion for improved HRQoL was an increase of at least 5 points in the global health-related quality of life scale of the EORTC QLQ C30 within twelve months in the intervention group. The sample size calculation was conducted with respect to the long-term primary endpoint in a worst-case scenario. Bonferroni-adjusted error of the 1st kind α = 0.025 two-sided, power 80%, and the following quantities [39]: Global health-related quality of life score according to EORTC QLQ C-30 [mean (SD)]: 68.82 (15.51) [before intervention], 73.12 (13.73) [after 12 months follow-up]. Assuming the nonparametric Wilcoxon (Mann-Whitney) rank-sum test, a sample size of 230 patients per group was to be included in the study. With 12 days versus 10 days (time to fulfill discharge criteria from the hospital) and an effect size of 0.5 (according to own studies) for the first primary, a sample size of 83 patients per group would follow (α = 0.025 two-sided, power 80%, calculations using nQuery Advisor® Release 7.0, Stat. Solutions Ltd. & South Bank, Crosse's Green, Cork, Ireland). The global HRQoL values after 12 months of both groups were tested for significant differences using the Mann-Whitney U-test. Due to the high impact of gender on global HRQoL, the data were also analyzed separately for both genders. Values of baseline and 12 months global HRQoL were descriptively compared to published normative data for the German population [18]. Categorical variables are presented as numbers and percentages. Continuous variables are presented as mean and standard deviation (SD) and, when not normally distributed as median (interquartile range, IQR). For categorical variables, differences between groups were tested using Fisher's exact test. Differences between groups in normally distributed continuous variables were evaluated using the students' t-test respectively the Mann-Whitney U-test for continuous non-normally distributed variables. Odds ratios (OR) and regression coefficients with 95% confidence intervals (CI) were determined in robust and logistic regression analysis. To identify predictors of prolonged LOS, univariate robust regressions followed by a multiple robust regression with the endpoint postoperative length of stay were performed. Multiple robust regressions were adjusted for patient age (years), gender, tumor site (genito-urinary vs. gastrointestinary), intervention vs. control, occurrence of major complications (yes vs. none), TUG (<20 sec vs. >20 sec), length of anesthesia (minutes), educational degree (<high school vs. > high school), nutritional state (normal vs. malnutrition), and severity of surgery (minor/moderate vs. major). Beside variables of particular clinical interest, only variables associated with a univariate significant impact (p<0.10) on outcome were introduced in the multivariate models. After calculation with all the chosen variables (full model), most relevant characteristics were identified by backward feature selection. Predictors for one year global HRQoL were identified by multiple linear regressions. Included variables were: intervention vs. control, gender, age (years), ASA state (I/II vs. III/IV), Charlson Comorbidity Score (points), tumor site (genito-urinary vs. gastrointestinary), nutrition state (MNA: normal/risk for malnutrition vs. malnutrition), Timed Up and Go-test (< 20 sec vs. > 21 sec); Severity of surgery (minor/moderate vs. major), pre-operative global HRQoL (points), major complications (yes vs. none), depressions (none vs. manifest), Fatigue (no/mild vs. severe), Activities of Daily Living (points), Mini Mental State (points).The tests for the primary outcome (postoperative length of hospital stay and global health-related quality of life) have been carried out in a confirmatory understanding, but all other tests for secondary endpoints are to be understood in the area of exploratory data analysis; therefore no adjustments for multiple testing have been made. For analysis of the course of global HRQoL from pre- to postoperative, the difference from baseline HRQoL to 1 year HRQoL for each patient was calculated. Means of the difference score between intervention and control group were tested for significance using the Mann-Whitney U-test. The data were also analyzed separately for both genders. Survival data were estimated according to the Kaplan-Meier methods and compared univariately with log rank statistics. Statistical significance was defined as p < 0.05. All statistical tests were two tailed. Statistical analyses were performed using SPSS software (version 22.0 SPSS, Inc., Chicago, IL, USA) and R 3.0.3. BODY.RESULTS.PATIENT RECRUITMENT: The outline of patient recruitment and follow-up is shown in Fig 1. The final sample consisted of 652 patients. Of these, 326 patients were assigned to the intervention-group and 326 patients were assigned to the control-group. After 12 months, questionnaires were sent to the patients. 103 patients (15.8%) had died. We received 450 questionnaires out of 549 (79%). Loss to follow-up was 15.2% (n = 99) after 12 months. Follow-up after 3 months was of no concern for the primary endpoints and is not considered in this analysis. BODY.RESULTS.BASELINE CHARACTERISTICS: The socio-demographic and clinical characteristics of the patients are listed in Tables 1 and 2. The mean age of all patients was 72 ± 4.9 years. There were more men (68.6%) than women (31.4%). Distribution of tumor site is also shown in Table 1. There were no differences regarding age, gender and tumor distribution between both groups (p = 0.5; p = 0.24 and p = 0.74 respectively). All other demographic and clinical data were comparable in intervention and control group (Table 1). 10.1371/journal.pone.0137824.t001 Table 1 Demographic and clinical baseline characteristics of the study population. Intervention No Intervention P N = 326 N = 326 Mean Age (SD) 71.6 (4.6) 72 (5.1) 0.50 € Male (%) 216 (66.3%) 231 (70.9%) 0.24 # BMI, mean (SD) 26 (4.2) 26 (3.6) 0.49 € Tumor site Genito-urinary 226 (69.3%) 221 (67.8%) 0.74 # Abdomino-thoracic 100 (30.7%) 105 (32.2%) Recurrence of cancer 47 (14.4%) 44 (13.5%) 0.82 # Severity of surgery $ Moderate 9 (2.8%) 9 (2.8%) 0.97 # Major 183 (56.1%) 186 (57.1%) Major+ 134 (41.1%) 131 (40.2%) Pre-operative Chemotherapy 40 (12.3%) 34 (10.4%) 0.54 # Pre-operative Radiotherapy 11 (3.4%) 16 (4.9%) 0.43 # ASA I/II 219 (67.2%) 206 (63.2%) 0.32 # ASA III/IV 107 (32.8%) 120 (36.8%) Performance State ECOG 0 315 (96.6%) 311 (95.4%) 0.65 # 1 8 (2.5%) 12 (3.7%) 2/3 3 (0.9%) 3 (0.9%) Comorbidities § , Median (IQR) 2 (2; 4) 2 (2; 4) 0.42 ⌘ Nutrition state (n = 629) & Normal 222 (70.9%) 230 (72.8%) 0.84 # Risk for malnutrition 83 (26.5%) 77 (24.4%) Malnourished 8 (2.6%) 9 (2.8%) Mean hand grip strength [kg] (SD) Women 23.3 (5.3) 23.1 (5.6) 0.83 ⌘ Men 39.2 (8.5) 38.9 (8.5) 0.92 ⌘ Living alone at home 64(20.8%) 62 (20.5%) >0.99 # Living at home with public help 16 (5.4%) 11 (3.7%) 0.33 # School degree (%; n = 589) >High school degree 109 (37.2%) 100 (33.83%) 0.39 # Surgery: median length [minutes](IQR) 165 (105; 250) 170 (105; 260) 0,42 ⌘ Anesthesia: median length [minutes](IQR) 215 (140; 330) 220 (150; 330) 0,56 ⌘ SD = Standard deviation, BMI = Body mass index $ POSSUM: Physiological and Operative Severity Score for the enUmeration of Mortality and Morbidity, IQR = Interquartile Range, MMSE = Mini Mental State, EORTC = European Organisation of Research and Treatment of Cancer, ASA = American Society of Anesthesiologists, ECOG =: Eastern Cooperative Oncology Group § CCS = Charlson Comorbidity Score & Mini Nutritional Assessment (MNA)/BMI € : Students’ t-test # : χ2-Test (Fisher’s Exact test) ⌘ : Mann Whitney U-Test 10.1371/journal.pone.0137824.t002 Table 2 Elements of the pre-operative Comprehensive Geriatric Assessment. Intervention No Intervention P N = 326 N = 326 ADL, Median (IQR) 100 (100; 100) 100 (100; 100) 0.95 ⌘ ADL < 100 (%) (n = 645) 58 (18.0%) 57 (17.7%) 0.99 # IADL, Median (IQR) 8 (8;8) 8 (8;8) 0.81 ⌘ IADL < 8 (%) 53 (16.3%) 54 (16.6%) 0.95 # MMSE, median (IQR) 29 (28; 30) 29 (28; 30) 0.49 ⌘ MMSE <27 (%) 28 (8.6%) 32 (9.8%) 0.69 # Fatigue (BFI) (n = 575) Mild fatigue 136 (47.6%) 113 (39.1%) 0.10 # Moderate fatigue 53 (18.5%) 68 (23.5%) Severe fatigue 7 (2.4%) 14 (4.8%) Depression (GDS) (n = 586) No depression 256 (88.0%) 265 (89.8%) 0.51 # Risk for /Manifest depression 35 (12.0%) 30 (10.2%) Timed up and go test (TUG) (n = 619) < 10 sec 231 (74.5%) 229 (74.1%) 0.92 # 11–20 sec 64 (20.6%) 68 (22.0%) 21–30 sec 7 (2.3%) 6 (1.9%) > 31 sec 8 (2.6%) 6 (1.9%) TUG >10 sec 79 (25.5%) 80 (25.9%) 0.93 # TUG >20 sec 15 (4.8%) 12 (3.9%) 0.70 # Risk of falls (Tinetti Score) (n = 617) High (≤18 pts.) 9 (2.9%) 12 (3.9%) 0.56 # Moderate (19–23 pts.) 15 (4.8%) 20 (6.5%) Low (≥24 pts.) 287 (92.3%) 274 (89.5%) ADL = Activities of daily living, IADL = Instrumental activities of daily living, IQR = Interquartile Range, TUG = Timed up and go, BFI = Brief fatigue inventory, GDS = Geriatric Depression Scale, SD = Standard Deviation. #: χ2-Test (Fisher’s Exact test) ⌘: Mann Whitney U-Test Furthermore, there were no differences regarding functional and cognitive tests assessed by the geriatric assessment (Table 2). In the intervention group, 193 (59%) patients rated the empowerment package as helpful or very helpful. A narrow majority of 186 (57%) kept the diary for at least 50% of the planned days, whereas 82 (25%) patients kept the diary for less than 50% of the planned days. In 58 cases (17.8%) information about diary keeping is entirely missing. BODY.RESULTS.PRIMARY SHORT-TERM OUTCOME.POSTOPERATIVE IN-HOSPITAL STAY: Fig 2 shows the postoperative in-hospital stay: Duration ranged from 0 to 139 days, with a median of 9 days (Interquartile Range, IQR: 7) for all patients. There was no significant difference between both groups (p = 0.99). 10.1371/journal.pone.0137824.g002Fig 2Median postoperative length of in-hospital stay. (A) The median length of postoperative in-hospital stay was 9 (IQR 7) days in the intervention group and 9 (IQR 9) days in the control group (p = 0.99). BODY.RESULTS.PRIMARY LONG-TERM OUTCOME.HEALTH-RELATED QUALITY OF LIFE AT 12 MONTH: At baseline mean global health-related quality of life in the intervention group was 53.95 (95% CI 49.12–58.77) in women and 68.79 (95% CI 65.90–71.69) in men. In the control group, the global HRQoL was 50.41 (95% CI 44.88–55.93) in women and 65.37 (95% CI 62.23–68.52) in men. There were no statistical differences between the groups (woman: p = 0.294; men: p = 0.196). After 12 months, questionnaires were sent to the patients. In the intervention group, 229 questionnaires were sent back, versus 221 questionnaires in the control group. The EORTC QLQ_C30 questionnaire was completed in n = 227 in the intervention group and n = 216 in the control group. Mean global HRQoL after 12 months was 69.27 (95% CI 66.45–72.14) in the intervention group and 69.79 (95% CI 66.84–72.74) in the control group (p = 0.74) (Fig 3). The global HRQoL after 12 months of women in the intervention group was 61.07 (95% CI 55.42–66.71) compared to 63.27 (95% CI 58.28–68.25) in the control group (p = 0.54). Males in the intervention group had a mean global HRQoL after 12 months of 72.49 (CI95% 69.34–75.65) compared to 71.71 (95% CI 68.21–75.20) in the control group (p = 0.94). There was no significant difference between the global HRQoL 12 months after surgery in the intervention and in the control group (all patients: p = 0.74; women: p = 0.54, men: p = 0.94). 10.1371/journal.pone.0137824.g003Fig 3Health-related global quality of life after 12 months for intervention and control groups.HRQoL 12 months after surgery was comparable in intervention and control groups (all patients p = 0.74). Compared to age and gender-adjusted reference values for the German population [18], men showed better global health-related quality of life than the reference population. In contrast, women quoted a lower score of global HRQoL without clinical relevance (< 5 points) (Fig 4). 10.1371/journal.pone.0137824.g004Fig 4Course of HRQoL.Comparing the baseline and follow-up values for health-related quality of life only for patients who survived and answered the 12 months questionnaire (n = 418), there were neither clinical relevant nor statistical significant differences between baseline and 12 months for HRQoL (intervention women: p = 0.58; men: p = 0.49; control women: p = 0.16; men = 0.29). Compared to age and gender-adjusted reference values for the German population [18], men showed better global health-related quality of life than the reference population with clinical relevance. In contrast, women quoted a lower score of global HRQoL but without clinical relevance. BODY.RESULTS.SECONDARY OUTCOMES.POSTOPERATIVE MORBIDITY: In total, 479 (74%) patients had at least one complication within postoperative hospital stay (Fig 5 and Table 3). Of these, 157 (24%) were classified as major complications according to the Clavien Scale (Fig 5). 10.1371/journal.pone.0137824.g005Fig 5Perioperative complications.Overall complications occurred in 74% of the patients. There was no difference between intervention and control group (p = 0.79). Major (Clavien Grade III and higher) perioperative complications occurred in 24% of the patients. There was no difference between the groups (p = 0.41). 10.1371/journal.pone.0137824.t003 Table 3 Short-term postoperative outcomes. Surgical outcomes Intervention No Intervention P N = 326 N = 326 Any complications 238(73%) 241 (73.9%) 0.86 # Delirium 36 (11.0%) 30 (9.2%) 0.52 # Other neurological complications 7 (2.1%) 8 (2.5%) 1.00 # Acute kidney failure, oliguria 21 (6.4%) 13 (4.0%) 0.22 # Infections and sepsis 51 (15.6%) 46 (14.1%) 0.66 # Cardiopulmonary 104 (31.9%) 97 (29.8%) 0.61 # Hemorrhage requiring transfusion 22 (6.7%) 8 (2.5%) 0 . 01 # Anemia requiring transfusion 53 (16.3%) 46 (14.1%) 0.45 # Anastomotic leakage 15 (4.6%) 11 (3.4%) 0.42 # Intestinal obstruction 7 (2.1%) 9 (2.8%) 0.61 # Wound rupture abdominal 16 (4.9%) 8 (2.5%) 0.09 # Postoperative pain 1st postoperative day (n = 628) 234 (75.2%) 261 (82.3%) 0 . 03 # ICU stay 132 (40.7%) 124 (38.6%) 0.63 # ICU stay > 1 day 93 (29.6%) 89 (28.6%) 0.79 # Return to the OR 36 (11%) 39 (12.0%) 0.81 # Depression at discharge (GDS) n = 386 No depression 159 (83.2%) 161 (82.6%) 0.86 # Risk for depression/Manifest depression 32(16.8%) 34 (17.4%) OR: operating room, LOS: length of hospital stay, cardiopulmonary: respiratory insufficiency, angina, myocardial infarction, arrhythmia, lung edema, pulmonary embolism; excluded: pneumonia; SD: standard deviation, IQR: interquartile range. #: χ2-Test The most frequent complications were cardiopulmonary complications (30.8%), followed by anemia (15.2%) and infections (14.9%) (Table 3). Occurrence and severity of complications were comparable in both groups, although severe hemorrhage occurred significantly more often in the intervention group (6.7% vs. 2.5%; p = 0.01). Delirium screening was positive in 66 (10.1%) patients with no difference between the intervention and control group (p = 0.52). Due to postoperative complications, 75 (11.5%) patients had to return to the operating theatre. BODY.RESULTS.SECONDARY OUTCOMES.POSTOPERATIVE STRESS: MOBILIZATION, PONV AND POSTOPERATIVE PAIN: Patients in the intervention group reported less pain on the first postoperative day (75.2% vs. 82.3%, p = 0.03) (Table 3). There were no differences regarding mobilization within the first 24 hours (69.2% vs. 70.4%, p = 0.73), or PONV within the first five days (52.8% vs. 56.4%, p = 0.39). Further, there was no difference in the GDS between intervention and control groups at discharge (p = 0.86). BODY.RESULTS.SECONDARY OUTCOMES.READMISSION: The readmission rate within 90 days was slightly higher for patients in the intervention group (62/245 = 25.3% vs. 59/248 = 23.8%, p = 0.70). In-hospital length of stay at readmission was shorter than in the standard of care group without reaching statistical significance (Median LOS 6.5; IQR: 11 days vs. 10; IQR: 10 days; p = 0.22). BODY.RESULTS.SECONDARY OUTCOMES.MORTALITY: Ten patients (1.5%) died before hospital discharge. Seven of these (1.1%) died within 30 days after surgery. In the intervention group, 6 (1.8%) patients died compared to 4 (1.2%) in the control group (Fig 6). At follow-up one year after surgery, 103 patients had died (15.8%). 10.1371/journal.pone.0137824.g006Fig 6Mortality within 30 days and within one year after surgery for intervention and control groups (p = 0.75 and p = 0.19 respectively).Within 30 days after surgery, 4 patients in the intervention and 3 patients in the control group had died (p = 0.75). One year after surgery, 45 patients in the intervention group compared to 58 patients in the control group had died (p = 0.19). The overall mortality did not differ significantly between the two groups (Log-Rank-test p = 0.197) (Fig 7). 10.1371/journal.pone.0137824.g007Fig 7Kaplan Meier Estimation of Survival in intervention and control groups.Patients were followed up to one year after surgery. After 12 months 103 (15.8%) patients had died. Mortality did not differ between intervention and control groups (log rank p = 0.197). BODY.RESULTS.SECONDARY OUTCOMES.PREDICTORS FOR LENGTH OF IN-HOSPITAL STAY: The intervention was not significant in robust regression analysis for primary endpoint LOS (-0.15; 95% CI -10.5–0.76; p = 0.75), whereas the pre-operative malnutrition,a delayed Time Up and Go test, increased length of anesthesia, severity of surgery, as well as postoperative major complications had a significant influence on postoperative LOS (S1 Table). BODY.RESULTS.SECONDARY OUTCOMES.PREDICTORS FOR HRQOL AT 12 MONTH: The intervention was not significant in multivariate analysis for primary endpoint global HRQoL (EORTC QLQ C30), whereas the pre-operative global HRQoL, CCS, ASA, MMSE, severity of surgery, as well as postoperative major complications had the most significant influence on the one-year HRQoL (S2 Table). BODY.RESULTS.SECONDARY OUTCOMES.COURSE OF HRQOL: Completed baseline and 12 months follow-up EORTC QLQ C-30 questionnaires were available for 418 patients. In women, the mean difference from baseline global HRQoL to global HRoL 12 months after surgery was -2.63 ± 27 in the intervention group, and – 6,16 ± 25 in the control group (p = 0.37). In men, the mean difference score was -0.48 ± 24 in the intervention group compared to -1.62 ± 25 in the control group (p = 0.71). Between the groups, there were no clinical or statistical significant changes. Merely the women in the control group showed a small clinical decline (5–9 points difference) [16]. Detailed information of clinical changes (small, moderate, large) is provided in S1 Fig. BODY.DISCUSSION: To our knowledge, this was the first randomized controlled trial to assess the effect of patient empowerment in elderly patients undergoing major cancer surgery. In the present study, patient empowerment did not affect postoperative LOS or long-term HRQoL but did affect the patients' short-term well-being, such as postoperative pain. In prior investigations providing detailed information about surgical and anesthetic procedures pre-operatively could diminish perioperative fear and anxiety, and was associated with a shorter in-hospital LOS in particular study settings [8,40,41]. However, previous trials on patient education prior to surgery have provided conflicting results: Shuldham et al.[12] and Guo et al. [42] both showed an even longer LOS in cardiac-surgical patients receiving pre-operative patient education in terms of information. Other trials found a reduction in length of stay after pre-operative patient education in terms of physical training before knee and hip replacement surgery [40]. However, Cochrane meta-analyses could not find strong evidence to support the recommendation for pre-operative interventions as patient education [8,10,43,44]. In the present study, a reduction in postoperative pain on the first postoperative day was found. Postoperative pain management may reduce the surgical stress response, organ dysfunction, and improves gastrointestinal motility, thereby facilitating early mobilization [45]. Furthermore, effective postoperative pain relief without use of opioids may reduce the incidence of postoperative nausea and vomiting followed by lower incidence of wound ruptures, as well as facilitating mobilization. Postoperative delirium is also induced by postoperative pain and/or stress [46,47]. The failure to reach significance may be due to the overall short LOS in our study population. We hypothesized a reduction of in-hospital stay from 12 to 10 days due to patient empowerment. In reality, the median LOS for patients was surprisingly low with median 9 days in both groups. During the last decades, in-hospital LOS has been reduced due to various reasons, not the least based on economic pressure [48]. Occurrence and frequency of postoperative complications did not differ between the intervention and the control groups. As anticipated, complications influenced the postoperative LOS significantly. Postoperative complications influence clinical outcomes including patients' well-being and the postoperative mortality [49]. Further, they have important effects on long-term survival and the patients' ability to recover to pre-operative levels of independency [44]. The high rate of overall and major complications in this study is comparable to other study results with elderly cancer patients [49,50]. We believe that this is due to the comprehensiveness of our record: we prospectively recorded all events requiring treatment beyond the routine as complications by asking patients, nurses, physicians, as well as patient records. Postoperative delirium is a seldomly recognized complication [49], and in this study, the postoperative delirium rate of 10% was lower than expected. However, both centers participating in this study have well established postoperative delirium screening and therapy protocols, which has been shown to reduce delirium rates [51]. Furthermore, at enrollment all patients had a Mini Mental State of 24 or higher, and might have been less vulnerable for cognitive disturbances than frail patients with pre-existing cognitive impairment [52]. Despite the high rate of complications, the mortality rate within the first 30 days was surprisingly low. In 2012, the perioperative mortality rate in the EUSOS study was as high as 4% for an unselected inpatient surgery within the first seven days following surgery [53]. For Germany, the in-hospital rate was still 2.5%. In the present study, the patient cohort is a selected study population 65 years old or older in addition to a severe disease, i.e. cancer followed by major onco-surgery. Thus, the anticipated mortality rate was higher than the observed rate of 1.1%. Aside from a selection bias, possible reasons might include the participation in the present RCT, which was combined with daily visits of all patients. Patient Reported Outcomes (PRO) are established outcome parameters [54]. Empowering patients can achieve a better health-related quality of life in patients [55,56]. In the present study, both study groups showed an improvement in global HRQoL close to the 5-point improvement we assumed for this study. The control group had a trend for higher decline. The intervention showed no influence on the HRQoL one year after surgery. Identifying other determinants of global as well as functional and symptom related parts of HRQoL would be subject of a sub analysis. Regarding hospital LOS, the present study revealed that amongst others, pre-operative nutritional state and physical functional capacity were independent in elderly cancer patients [57]. Many factors are associated with in-hospital LOS after elective onco-surgery: age, severity of surgery, blood loss, surgical volume, and surgical complications have been previously described as influencing variables [49,58,59]. Interestingly, impairments in functional capacity were not reflected in ASA scores or in the performance status. This strengthens the assumption that the routinely used pre-operative assessments are unsatisfactory for elderly patients, although published data concerning this issue are still conflicting [49,52,60,61]. A pre-operatively conducted geriatric assessment could identify patients who are functionally impaired, and interventions for improving their physical status may be indicated in order to enhance their postoperative recovery [49]. BODY.DISCUSSION.LIMITATIONS: Additional to the limitations already mentioned above, there might be a selection bias in our study population. The study protocol and battery of questionnaires, including cognitive and physical tests, were time consuming and sometimes even stressful for the participants. Further, the intervention started a day prior to surgery. Several study participants were hesitant to be enrolled in a study due to the severity of their diagnosis and the impending major surgery. A considerable number of patients expected to be completely healed after surgery and therefore saw no need in further interventions. Even more patients felt sometimes too weak, or were simply not able to keep their diary due to their postoperative physical and/ or cognitive condition. In particular, patients transferred to the ICU postoperatively were often not able to keep the diary. Hence, contrary to other studies showing beneficial effects of diary keeping [62], perioperative patient empowerment is limited in elderly patients. The patients that refused to participate could differ significantly from the study population in terms of mental and physical health, leading to another possible limitation of this study. We cannot eliminate the possibility that the daily visits and close interaction of study-staff with the participants influenced the primary endpoints, compared to patient settings in the daily routine. Due to the study protocol there was no reference group to facilitate direct comparisons. Further, the EORTC QLQ-C30 Questionnaire was not specifically designed for elderly patients. The now available EORTC QLQ –C15-PAL, which is specifically designed for elderly patients, did not exist when the present study was designed and conducted [63]. Alternatively, the study has several strengths: the prospective design, the homogenous patient collective, the high recall in long-term follow-up, and the reasonable size of the study population. BODY.CONCLUSION: Patient empowerment in terms of additional pre-operative information failed to shorten postoperative in-hospital stay or improve global HRQoL in elderly patients undergoing major onco-surgery. Postoperative length of stay in elderly cancer patients is mainly influenced by pre-operative nutritional, cognitive and functional state, magnitude of surgery, length of general anesthesia, as well as postoperative complications. Patient empowerment can enhance quality of care in regards to pain, and since over-treatment of pain is particularly harmful for elderly patients, patient safely can thus be improved. Pre-operative information was received well by patients who were cognitively and physically fit. Pre-operative comprehensive geriatric assessment might be beneficial for this high-risk patient group, helping to provide a tailored perioperative management aimed at reducing cognitive and physical barriers, and ultimately improving postoperative outcome. BODY.SUPPORTING INFORMATION: S1 CONSORT ChecklistCONSORT Checklist.(PDF)Click here for additional data file. S1 FigChanges in global HRQoL from baseline to follow-up.Detailed clinical changes in global HRQoL from pre-operative to 12 months follow-up. There were no differences between both groups (p = 0.17). The majority showed no clinical relevant changes in global HRQoL.(TIF)Click here for additional data file. S1 ProtocolClinical Trial Protocol (English).(PDF)Click here for additional data file. S2 ProtocolClinical Trial Protocol (German).(PDF)Click here for additional data file. S1 TableRobust regression analysis with respect to the primary endpoint postoperative length of hospital stays.Variables entered: Intervention no vs. yes, gender, age in years, tumor site (genito-urinary vs. gastrointestinary, nutrition state (MNA: manifest malnutrition vs. normal/risk for malnutrition), Timed up and go: > 21 sec vs. < 20 sec; Severity of surgery§ (major+ vs. moderate/major), major complications (no vs. yes), length of anesthesia in minutes, school degree (> high school vs < high school). Pre-operative malnutrition and a delayed time up and go test, increased length of anesthesia and severity of surgery as well as postoperative major complications had a significant influence on postoperative LOS.(TIF)Click here for additional data file. S2 TablePerioperative predictors of global health-related quality of life (EORTC QLQ C30) one year postoperative.(Variables entered on step 1: Intervention yes vs. no, gender, age in years, ASA state I/II vs. III/IV, Charlson Comorbidity Score per point, tumor site (genito–urinary vs. gastrointestinary), nutrition state (MNA: normal/risk for malnutrition vs. manifest malnutrition), Timed up and go: < 20 sec vs. > 21 sec; Severity of surgery§ (moderate/major vs. majorplus), pre-operative global health-related quality of life per point, major complications (no vs. yes), Depressions (none vs. manifest), Fatigue (no/mild vs. severe), Activities of daily living per point, Mini Mental State per point). Pre-operative global HRQoL, CCS, ASA, and MMSE, intraoperative severity of surgery as well as postoperative major complications had the most significant influence on the one-year HRQoL.(TIF)Click here for additional data file.

TITLE: Effect of Oral Pre-Meal Administration of Betaglucans on Glycaemic Control and Variability in Subjects with Type 1 Diabetes ABSTRACT: We conducted a double-blind placebo-controlled crossover pilot study to investigate the effect of oat betaglucans (β-glucan) on glycaemic control and variability in adults with type 1 diabetes (T1D; n = 14). Stomacol® tablets (1.53 g of β-glucan) or placebo (Plac) were administered three times daily before meals for two weeks. Glucose levels were monitored during the second week by continuous glucose monitoring (CGM). There was an increase in basic measures of glycaemic control (maximal glucose value 341 ± 15 vs. 378 ± 13 mg/dL for Plac and β-glucan, p = 0.004), and average daily risk range (62 ± 5 vs. 79 ± 4 mg/dL for Plac and β-glucan, p = 0.003) favouring Plac over β-glucan, but no increase in the M-value (the weighted average of the glucose values) or other more complex measures. Basic measures of glucose variability were also slightly increased during β-glucan treatment, with no difference in more complex measures. However, glycaemic variability increased between the first and last two CGM days on Plac, but remained unchanged on β-glucan. In conclusion, in this pilot study we were unable to demonstrate a general positive effect of β-glucan before meals on glucose control or variability in T1D. BODY.1. INTRODUCTION: Betaglucans (β-glucan) are non-digestible fibres that form a viscous gel in the gastrointestinal tract [1]. They are approved by the US Food and Drug Administration for lowering low density lipoprotein-cholesterol at a dose of 3 g daily [1]. The mechanism for this effect has been suggested to involve sequestering of bile acids, analogous to regular bile acid sequestrates [2,3]. Meanwhile, the gelling ability due to the high viscosity of β-glucans has also been suggested to positively influence postprandial glucose, and although data have been inconclusive, this ability is also recognised by the European Food Safety Authority [4,5]. Oat β-glucan has a higher molecular weight than barley β-glucan [6]. Its high viscosity even at low concentrations (1%) is believed to delay gastric emptying, slow intestinal transit, and postpone glucose and sterol absorption, all of which may contribute to attenuated postprandial plasma glucose and insulin levels, as has been observed in subjects with type 2 diabetes [7]. Postprandial glucose excursions and glucose variability are substantial challenges to people with diabetes, particularly those on insulin treatment, in part due to variable gastric emptying and intestinal transit time that can be secondary to either variable glucose levels themselves or to diabetic neuropathy [8,9]. Here we aimed to test the hypothesis that oat β-glucan might positively affect post-meal glucose excursions due to their gelling ability in subjects with type 1 diabetes (T1D). BODY.2. MATERIALS AND METHODS .2.1. STUDY SET-UP AND SUBJECTS: We conducted a double blind placebo-controlled crossover study in 14 adult patients with T1D at the Department of Endocrinology, Skåne University Hospital (Malmö, Sweden), as shown in Figure 1. In this pilot study both patients with and without previous problems with variable glucose excursions were informed and invited to participate during routine out-patient visits to the clinic. A diagnosis of T1D, insulin treatment, and informed consent were the only inclusion criteria. However, none of the study participants were taking any kind of medication that would influence gastric emptying or intestinal transit, including glucagon-like peptide-1 agonists or sodium-glucose co-transporter-2 antagonists. One female subject was pre-menopausal. It is unknown weather this could influence glucose variability but there was no difference in her glucose readings between the two periods. The study was designed to be as pragmatic as possible with respect to both timing of dose and the dose itself where tolerability/abdominal discomfort is the most common limitation. One Stomacol® (Brainbridge Commerce AB, Gothenburg, Sweden) tablet (1.53 g β-glucan) or placebo (Plac) was administered three times daily before main meals for two weeks. Patients were instructed to chew on the tablets (2.9 g total weight, 7.8 × 20 mm diameter) until dissolved, and to then swallow them with a glass of water prior to having breakfast, lunch, and dinner. There was no formal control of compliance but patients were asked whether they took the tablets regularly and there was no report of missing doses unless patients ended the study prematurely (one patient). β-glucan and Plac were kind gifts from Brainbridge Commerce AB (Gothenburg, Sweden). The first two-week period was followed by an at least two-week long washout period before the second two-week period on crossover treatment. The patients were asked not to change their daily life habits or eating, but to measure plasma glucose and adjust insulin doses as appropriate just as they normally would. Primary endpoints were glycaemic control and variability. Glucose levels were monitored during the second week by continuous glucose monitoring (CGM; Medtronic iPro2TM with the Enlite® (Medtronic, Minneapolis, MN, USA) sensor; a kind gift from Medtronic AB, Solna, Sweden). Both patients and health care providers were blinded to these readings and patients were instructed to carry on living as previously (including reporting hypoglycemia and adjusting insulin dose according to routine finger prick glucose measures). According to the rules of the ethical committees posted by the local committee at Lund University (Lund, Sweden), all forms of treatment quality assurance are exempt from ethical committee approval. Since this research involved comparing blood glucose between groups with different intake of a substance normally found in the diet of a person with diabetes without introducing anything that could be considered outside of the normal diet, the study was considered to fulfil the criteria of quality assurance of blood glucose control rather than being a study of the medical effects of a new treatment. BODY.2. MATERIALS AND METHODS .2.2. CALCULATION OF INDICES FOR CONTINUOUS GLUCOSE MONITORING (CGM) DATA ANALYSIS: The indices of glycaemic control [10,11,12,13] describe to what extent the glucose data tend to remain near a target value or in a target range. There are both basic indices of descriptive statistics, and more complex indices. As regards to the former, the calculated indices were glucose mean, maximum, minimum, 50th percentile (median) as well as other percentiles, and percentages of glucose values in a target range (4.4–11.1 mmol/L, i.e., 80–200 mg/dL), and below and above a target value (4.4 and 11.1 mmol/L, respectively). More complex indices were also measured. One was the glycemic risk assessment diabetes equation (GRADE), where glucose values were transformed to yield a continuous curvilinear response with a nadir of 5.5 mmol/L and high adverse weighting to hyperglycemia and hypoglycemia using the equation GRADE = 425 × {log10[log10(Glucn)] + 0.16}2(1) with Glucn in mmol/L. Then, the average value was taken. Another measure was the M-VALUE, a weighted average of the glucose values with progressively larger penalties for more extreme values. The corresponding equation is M-VALUE = |10 × log10 (Glucn/IGV)|3(2) where IGV is the ideal glucose value, typically assumed, as in this study, as equal to 6.7 mmol/L (120 mg/dL). Again, an average value was then taken. A third measure was the hypoglycemia index, which is the weighted average of hypoglycemic values. If the blood glucose value is lower than a given threshold, the index is: Hypo_index = (LLTR − Glucn)2.0/30(3) with Glucn and LLTR (Lower Limit of the Target Range) in mg/dL (typically, LLTR = 80 mg/dL). The hyperglycemia index, also used, is the weighted average of hyperglycemic values. If the blood glucose value is higher than a given threshold, the index is: Hyper_index = (Glucn − ULTR)1.1/30(4) with Glucn and ULTR (Upper Limit of the Target Range) in mg/dL (typically, ULTR = 140 mg/dL). Other measures were the index of glycaemic control (IGC), which is the Hypo_index + Hyper_index, and the low blood glucose index (LBGI), which is a transformation that normalizes the blood glucose scale: LBGI = 1.509 × [(loge(Glucn))1.084 − 5.381](5) for blood glucose values less than 112.5 mg/dL. Then, a risk value is assigned to each blood glucose reading as follows: Risk(LBGI) = 10 × LBGI2(6) and finally, the average value is taken. The high blood glucose index (HBGI) was also obtained. Similar to the LBGI, this is a transformation to normalize the blood glucose scale for blood glucose values higher than 112.5 mg/dL. The expression of HBGI is the same as for LBGI. Finally, the average daily risk range (ADRR) is LBGI + HBGI, calculated with the minimum and the maximum glucose value, respectively. The indices of glycaemic variability [10,11,12,13] measure to what extent CGM data tend to oscillate. The higher the variability, the higher the value of such indices. Some basic indices that we calculated were the glucose standard deviation (SD), the coefficient of variation, and the total and the interquartile range. More complex indices included the J-INDEX, which is a combination of information from mean and SD of all glucose values, calculated as J-INDEX = 0.001 × (mean + SD)2(7) and continuous overlapping net glycaemic action (CONGAn), which is the SD of the difference between values obtained exactly n minutes apart. Typically, n is equal to 60 min (or its multiples), but in this case we performed the analysis over the glucose data available, despite the fact that the time interval between consecutive values was typically lower than one hour. Also used was the lability index:(8)LI=∑n=1N(Glucn+1−Glucn)2hn+1− hn where Glucn (in mmol/L) is the n-th glucose value, and hn is the time when that value was collected (similarly to Glucn+1 and hn+1); N is the total number of readings. Typically, hn+1 and hn are at least one hour apart, but we again exploited all the CGM data available. We also computed the mean, instead of the sum. Another index was the mean amplitude of glycaemic excursion (MAGE), which is the arithmetic mean of the glycaemic excursions that are greater than one SD. MAGE (pos) and MAGE (neg) consider the positive and the negative excursions, respectively. The shape index is based on the calculation of the point-by-point second-order derivative of the glucose curve; then, absolute value of each derivative is taken, and the average over all values is calculated. The autocorrelation index considers to what extent the glucose values tend to repeat or change during time, and the autocorrelation sequence is computed as (9)Ad(m)=1N−|m|∑i=1N−|m|x(i)⋅x(i+m) where x in this case is glucose, N is the total number of samples and m is the time lag expressed as number of samples; then the sequence is normalized to Ad(1), and average value is calculated to get the autocorrelation index. BODY.2. MATERIALS AND METHODS .2.3. STATISTICAL ANALYSIS: After testing for normality of distribution, values of the indices were logarithmically transformed, and possible differences between the groups were assessed by paired Wilcoxon test. p < 0.05 was considered statistically significant. Values are reported as mean ± standard deviation (SD) or standard error (SE), unless otherwise specified. BODY.3. RESULTS: One patient stopped the study prematurely and the glucose sensor reading of another failed, leaving 12 evaluated patients: 4 females and 8 males, aged 47 ± 12 years, with age at onset of T1D 25 ± 17 years and T1D duration 22 ± 16 years (mean ± SD). In general, β-glucan and Plac were well tolerated. Table 1 summarizes the CGM data from the last 7 days of the study periods on Plac and β-glucan, respectively. There were no general improvements in favour of β-glucan over Plac. Instead, there was a significant increase in some basic measures of glycaemic control such as maximal glucose and average daily risk range, but none in glycaemic risk assessment diabetes equation or M-value, which are more complex measures of glycaemic control as measured by CGM. Basic measures of glucose variability such as standard deviation were also slightly increased during β-glucan, with no difference in more complex measures (Table 1). Since participation in a study might result in improved glycaemic control and lesser glycaemic variability by itself, for instance due to more frequent glucose measuring and careful insulin dosing, we were also interested to see any trends over time. Indeed, whereas glucose variability tended to increase between the first and last two CGM days on Plac (coefficient of variation from 34.3 ± 1.9 to 39.1 ± 3.0%; p < 0.05), it remained unchanged on β-glucan (from 38.2 ± 3.2 to 38.9 ± 2.1%; p = ns (non-significant)), suggesting a time dependent positive effect in maintaining a stable glucose level. BODY.4. DISCUSSION : From this small-scale pilot study on the effect of pre-meal β-glucan administration on glycaemic control and glucose variability we conclude that the ingestion of 1.5 g β-glucanthree times daily for two weeks by patients with T1D was safe and well tolerated. However, with regards to the primary outcome of glycemic control and glucose variability there was clearly no benefit in favour of pre-meal β-glucan. The study dose, potentially being too low, dosing timing, potentially being too close to the meals, study duration, being too short, and lack of controlling for a range of factors that occur in real life might be important factors to consider for future interventions. With regard to the quality of the β-glucan preparation, it also seems important that a high viscosity gel be produced to affect post-prandial glycaemia [14]. Soluble dietary fibres have been shown to attenuate the postprandial rise in blood glucose levels and even reduce the risk of type 2 diabetes and cardiovascular disease [1,2,3,4,15,16]. This effect seems to be related to its rheological properties including viscosity, which in turn depends on processing methodology with enzymatic methods being more advantageous than aqueous methods [12]. The increase in viscosity of the stomach contents is thought to delay gastric emptying and reduce the mixing of food with digestive enzymes, which in turn would retard glucose absorption. An alternative explanation to an effect on glycaemia per se could be an effect on satiety and dietary intake [17,18]. Our study was not designed to investigate such alternatives. While our findings were not as straightforward, a recent pilot in healthy volunteers suggests that pre-meal β-glucan ingestion might be a valid strategy to control postprandial glucose excursions [19]. A major difference between our studies is the lack of normal glucose regulation by endogenous insulin in the subjects participating here. In addition, rather than looking at a test-meal situation we investigated the potential effect on 24-h glycaemia by CGM. From our data, we conclude that for most patients with T1D, the effect of pre-meal β-glucan will not be as predictable as it would in a person with normal glucose tolerance and/or insulin and incretin secretion [20]. It is important to stress a major similarity in the studies being the instruction to ingest β-glucan prior to eating to allow sufficient time for gelling since β-glucan has otherwise been shown to be ineffective in this respect [21,22]. This concept of "pre-load" has also been extensively studied in type 2 diabetes [23,24,25]. There are several other important limitations to our study. It was small-scale and short-term, and we included a general population of patients with T1D. This means that there is limited power and that one should be careful with the generalizability of our findings. In fact, one particular observation points to that longer observation times may be advisable for future studies. Comparing the two first and two last of the seven days with CGM there was a significant increase in glucose variability on placebo, as opposed to no change on β-glucan. Given the fact that the placebo effect of participating in a study on glucose control where so many different factors contribute is expected to be fairly strong, this finding is not surprising and may point to a positive long-term effect of pre-meal β-glucan. Another limitation of the study is the choice of β-glucan dose. In fact, 1.5 g three times daily was chosen as a trade-off between expected tolerability on the one hand and the effect on cholesterol on the other, but it is quite possible that a higher dose, e.g. related to body mass or meal carbohydrate content but still limited by the expected gastrointestinal discomfort, may be more effective [1,5]. From a technical point of view there is also the choice of CGM to evaluate the effect of β-glucan. The length of the study did not allow evaluation of classical long-term parameters such as hemoglobin A1c but there are other emerging techniques one may consider [26]. BODY.5. CONCLUSIONS: In conclusion, pre-meal ingestion of 1.5 g β-glucan three times daily for a two-week period was well tolerated and safe in subjects with T1D but did not lead to general improvements in glycaemic control or glucose variability in this small-scale pilot study.

TITLE: The Effects of Aromatherapy on Intensive Care Unit Patients' Stress and Sleep Quality: A Nonrandomised Controlled Trial ABSTRACT.BACKGROUND: Stress has both physiological and psychological effects and can negatively impact patients' treatment and recovery. We examined whether the aromatherapy alleviated patients' stress and improved their sleep quality and provided data that can be utilized in clinical settings. ABSTRACT. METHODS: This was a nonrandomised controlled experimental study. Participants included lucid adult patients who were admitted to the intensive care unit and had spent more than two nights there. The experimental treatment required participants to engage in deep breathing with essential oils as part of the aromatherapy. The control group was instructed to go to sleep without receiving the lavender aroma oil. ABSTRACT. RESULTS: The experimental group and control group showed a significant difference in perceived stress (F = 60.11, p < .001), objective stress index (F = 25.65, p < .001), systolic blood pressure (F = 9.09, p < .001), diastolic blood pressure (F = 2.47, p = .046), heart rate (F = 5.71, p < .001), and sleep quality (F = 109.46, p < .001). ABSTRACT. CONCLUSIONS: The results revealed that aromatherapy alleviated stress and improved sleep quality in intensive care unit patients after 2 days of the experimental treatment. These results demonstrate that aromatherapy affects stress and sleep quality, thus indicating its value in nursing interventions. This trial is registered with KCT0002344. BODY.1. INTRODUCTION: Aside from the stress that accompanies illness, hospitalization is a stressful life event that brings about changes in one's daily life and the activities that they engage in. Consequently, patients often encounter psychological and social stress [1]. Stress manifests itself because of the interaction between one's internal propensities and external stimuli that is determined by their environment. This determines an individuals' coping response. Humans exhibit various symptoms in stressful situations, including an increase in physiological symptoms such as headaches and exhaustion; emotional symptoms such as anger, sadness, and helplessness; and behavioural symptoms such as crying and criticizing [2]. Most patients admitted into the intensive care unit (ICU) experience more severe symptoms and a higher likelihood of death. This increases their nursing needs and requires the patient to undergo continuous and intensive observation. The ICU is surrounded by medical teams and a variety of mechanical devices. The fear of being an ICU patient, uncertainty about the future, isolation from family, financial pressure, and exposure to an unfamiliar environment result in severe emotional imbalances in patients [3, 4]. In the ICU, a hospital's entire medical capacity is concentrated on providing patients with life-threatening illnesses with a chance at recovery. In the ICU environment, most patients experience stress due to anxiety about their prognosis; the unfamiliarity of the ICU and its treatment; limited visiting hours; the pressure of the inspection process; and cognitive, emotional, and behavioural stress depending on the dispositions of medical team members. This can cause the patient to resort to inappropriate coping methods [5]. Relatives of ICU patients report high levels of anxiety, depression, and feelings of panic, chaos, and a need for constant vigilance [4]. Psychological instability typically occurs when exhaustion starts to accumulate from stress, and the sympathetic nervous system, which maintains the body's state of equilibrium, is activated. This results in problems such as increased blood pressure, heightened tension, and sleep disorders [6]. It is unclear how stress affects sleep; however, the intimate temporal relationship between the stress of sleep architecture and the hypothalamo-pituitary-adrenal axis plays a vital role [7]. This axis and sympathetic nervous system activity are positively correlated with the total amount of rapid-eye-movement sleep [8]. Sleep is a basic human need, and maintaining good sleep quality is extremely important in preserving a healthy lifestyle [9]. Intensive care unit patients may result in problems such as decreased cognitive function, irritability, aggression, and disruptions in the sleep-wake cycle, which are associated with symptoms of disorientation and are reported to lead to the development of ICU syndrome [10]. There is a high correlation between stress and sleep quality; therefore, there is an urgent demand for nursing intervention to decrease stress and increase sleep quality in ICU patients with weakened immune systems [11]. Until now, stress studies addressed effect of inhalation of lavender essential oil on vital signs in open-heart surgery ICU [12], the effect of providing advanced information on the ICU environment and its impact on anxiety and environmental stress in open-heart surgery patients [4], and effect of inhalation aromatherapy with lavender essential oil on stress and vital signs in patients undergoing coronary artery bypass surgery [13]. Studies such as giving information as nursing interventions claim that these are partially effective for treating anxiety and stress. However, these studies were limited because they only focus on the effects of anxiety and stress on sleep. Studies on sleep quality in ICU patients have attempted to determine the phenomenon of sleep itself by examining sleep patterns, sleep disorder characteristics and factors [14], and factors related to sleep among ICU patients. However, there have been virtually no studies that attempt to improve sleep quality in these patients. ICU patients who are in a lucid state experience elevated levels of stress resulting from fear about survival due to their illness, concerns about their family and finances, limited movement, and the noise of the ICU. They also experience a loss in sleep quality. Because of the severity of their illness, however, patients are more interested in their condition than their basic need for quality sleep. A method to enhance sleep quality must be developed since a loss in sleep quality leads to increased stress. Nurses who have the most direct and continuous contact with ICU patients can play a key role in alleviating patients' stress and improving sleep quality. Aromatherapy has recently gained traction as an alternative therapy. It perceives humans as holistic beings and focuses on their balance and harmony as components of nature [15]. Aromatherapy is a type of complementary alternative treatment that promotes physical, emotional, and psychological health by using the therapeutic elements of essential oils found in the flowers and leaves of various natural plants, stems, and roots [16]. Aromatherapy is a combination of aroma, treatment, and therapy that uses aroma essential oils to achieve balance in the patient by calming the mind, body, and spirit. It is also used in treatments to instil vitality [17]. In contrast to chemicals, aromatherapy is a relatively effective and safe treatment that does not accumulate in the body, but is discharged from the through the respiratory system, liver, and kidneys [18]. Aromatherapy is a noninvasive treatment that directly affects the brain, and individuals can self-administer the treatment regardless of time or place [19]. In addition, depending on the oil's characteristics, aromatherapy has been recognized to have antibacterial, wound-healing, immune enhancing, antidepressant, and calming curative effects [20]. Studies on the clinical effects of aromatherapy have shown that it alleviates stress and increases sleep quality. This includes studies on stress responses and sleep in hospitalized elderly patients [1], sleep and pain in cancer patients [21], sleep in children with autism, and quality of sleep in heart disease patients of hospital [22, 23]. Studies on ICU patients have examined the effects of aromatherapy on sleep in heart disease patients [24], high blood pressure [11], stress, and anxiety. However, these studies [11, 24] examined patients who had undergone treatment for only 1 day and took place only in coronary care units. Studies that examine stress and sleep quality in ICU patients for more than 2 days are lacking. Therefore, this study examined ICU patients who underwent aromatherapy treatment for more than 2 days and verified its effects of alleviating stress and improving sleep quality. The explicit purposes of this study are to investigate the effect of aromatherapy on ICU patients' stress, blood pressure, heart rate, and sleep quality. The research hypotheses were as follows. (1) Primary Hypothesis. There will be significant differences in stress between the experimental group that undergoes aromatherapy treatment and the control group that does not. (2) Secondary Hypothesis. There will be significant differences in quality of sleep between the experimental group and the control group. BODY.2. METHODS.2.1. STUDY DESIGN: This was an experimental study that used a nonrandomised pre- and posttest design that compared stress and sleep quality between 2 groups of ICU patients: those who underwent aromatherapy and those who did not. To prevent diffusion and contamination in the experimental treatment between the 2 groups, data were first collected from the control group (n = 32) from July to August 2016 and then from the experimental group (n = 32) from September to October 2016 (Figure 1). BODY.2. METHODS.2.2. STUDY SETTING: This study was conducted at the intensive care unit in the university hospital. Participants included lucid adult patients who were admitted to the intensive care unit and had spent more than two nights there. BODY.2. METHODS.2.3. PARTICIPANTS.2.3.1. PARTICIPANT SELECTION: This study was conducted for ICU patients who had been admitted via emergency room, hospitalized in ICU of E University Hospital located in D city, for more than 2 nights from July 1st, 2016, to October 15th, 2016. This study was conducted under the consent of a doctor of pulmonology for patients suffering from asthma or chronic obstructive pulmonary disease. Participants who met the following inclusion criteria were selected as participants: age > 18 and <70 years, being lucid and fully capable of communicating, being in the ICU for >2 nights, understanding the study's purpose, being permitted to participate in the study, and providing written consent. For patients aged > 60 years, a guardian provided written consent when applicable. Participants were excluded if they met the following criteria: having a mental illness, taking antianxiety or sleep aid medication, having side effects or allergies to aroma essential oils, being admitted to the ICU between 7 p.m. and 5 a.m. to make the subject's condition the same, having a systolic blood pressure below 100 mmHg (lavender essential oil can lower blood pressure), and having an arrhythmia. BODY.2. METHODS.2.3. PARTICIPANTS.2.3.2. SAMPLE SIZE CALCULATION: We calculated sample size using G. Power Analysis [25]. Assuming a significance level (α) of .05 statistical power (1 − β) of .80, and an effect size of 0.75 calculated in earlier studies [26], we sought to include 29 participants in each group. Data were collected from 32 participants in each group considering a 10% dropout rate. Excluding 2 participants with arrhythmia, 1 participant who refused to participate during the experiment, and 1 participant who was moved to another ward, 30 participants in each group were included in the final analysis (Figure 2). BODY.2. METHODS.2.4. EXPERIMENTAL TREATMENT: The experimental treatment in this study required patients who had spent more than 2 nights in the ICU to undergo aromatherapy before and during sleep. BODY.2. METHODS.2.4. EXPERIMENTAL TREATMENT.2.4.1. SELECTION AND STORAGE OF AROMA OILS: A prescription was received from one of the researchers with an aromatherapy certificate, which had a calming effect when it was used on a predetermined neural network. Lavender [27] was the first essential oil chosen for treating sleeplessness, and it was stored in a refrigerator when not being used. BODY.2. METHODS.2.4. EXPERIMENTAL TREATMENT.2.4.2. APPLICATION OF AROMATHERAPY : The experimental treatment was aromatherapy, which was applied within 1 hour of hospitalization and at 8 p.m. after the pretest, prior to the experiment. Perceived and objective stress, blood pressure, heart rate, and average sleep quality were measured within 1 hour of admittance to the ICU. At 8 p.m. on the same day, perceived and objective stress, blood pressure, and heart rate were measured. The experimental treatment began at 9 p.m. after a pretest, which was administered at 8 p.m. Three drops of lavender essential oil were applied to an aromastone and patients were directed to breathe deeply 10 times.After the oils were inhaled through deep breathing, an aromastone was hung on the centre of the bedside railing within 10 cm and participants were instructed to go to sleep (Aromatherapy 1).The aromastone was removed at 8 a.m. after participants had inhaled the oils and finished sleeping.On the second day of hospitalization, perceived stress, blood pressure, and heart rate were measured in the ICU; then, the experimental group was given an aromastone with 3 drops of lavender essential oil on it and instructed to breathe deeply 10 times.After the oils were inhaled through deep breathing, an aromastone was hung on the centre of the bedside railing within 10 cm, and participants were instructed to go to sleep (Aromatherapy 2).The aromastone was removed at 8 a.m. after participants had inhaled the oils and finished sleeping. BODY.2. METHODS.2.5. MEASURES.2.5.1. STRESS MEASUREMENTS: (i) Perceived Stress Using a Numeric Rating Scale. The scale ranged from 0 (no stress at all) to 10 (severe stress); participants self-reported felt stress. (ii) Stress Index. The stress index is a value that quantifies stress levels with standard derivations. This measurement was based on heart rate variability, which was measured with Canopy9 professional 4.0 (IEMBIO, USA), an automatic nervous system measuring device. This index measures miniscule changes in periodic heart rate on a scale of 1–10. (iii) Blood Pressure. The Philips Intellivue MP50 patient monitor was used to measure blood pressure in participants. The monitoring system was placed on the upper arms of the patients at the same height as the heart, and the lower cuff covered 2 cm of the elbow. Systolic and diastolic blood pressure were then measured and expressed as mmHg units according to the measurement guidelines of the automatic sphygmomanometer. (iv) Heart Rate. The Philips Intellivue MP50 patient monitoring system was used to measure heart rate. After participants rested, heart rate was recorded and expressed as beats/minute. BODY.2. METHODS.2.5. MEASURES.2.5.2. SLEEP QUALITY MEASUREMENTS: We used scores from the Verran & Snyder-Halpern Sleep Scale, which measured sleep quality over the course of 2 days at 8 a.m. when patients awoke. Kim and Kang [28] translated this tool. The scale comprises 9 questions including the number of times one wakes up during the night, how much one tosses and turns, total hours spent sleeping, the depth of sleep, how long it takes to fall asleep, how one feels when waking up, how one awakes from sleep, and level of satisfaction with sleep. The final question is a short-answer question. Excluding the short-answer question, the remaining 8 questions ranged from 0 to 10 points with a lowest possible score of 0 points and highest possible score of 80 points. Higher scores indicated higher quality of sleep. The reliability of this tool was measured using Cronbach's alpha: pretest, .87; first night of sleep, .97; second night of sleep, .98. BODY.2. METHODS.2.6. DATA COLLECTION: Data collection was conducted after explaining the collection procedures to the hospital's nurses and relevant departments, requesting cooperation, and receiving permission. The purpose of the study was explained to those who met the inclusion criteria, and data were collected from participants who agreed to participate.Data were first collected from the control group to avoid diffusion of the experimental treatment.Room instructions, room arrangements, the type of patient monitoring system, and its uses were all identical for patients being admitted. The researcher read the questionnaire and recorded the answers during the survey and checked whether a cannula was inserted or not for stability of patients.All participants were hospitalized by way of the emergency room, and no additional tests were conducted after the initial inspection. The treatment patients received and their environment in the ICU were identical. Blood pressure and heart rate measurements were taken every hour in identical intervals, and the number of patient monitoring devices was identical for each patient. BODY.2. METHODS.2.7. DATA ANALYSIS: The collected data were analysed with using SPSS 23.0. Participants' general characteristics were analysed as percentages and means. The homogeneity of general characteristics between the experimental group and control group was analysed with a chi-square test and a t-test. A t-test, repeated measures analysis of variance (ANOVA), and analysis of covariance were used to analyse differences in perceived stress, the stress index, blood pressure, heart rate, and sleep quality before and after the experimental treatment in both groups. Before we analysed the data with repeated measures ANOVA, we tested whether the sphericity assumption was satisfied. If the assumption of sphericity assumption was not satisfied, we performed multivariate analysis. The reliability of the tool used to measure sleep quality was analysed with Cronbach's α. BODY.2. METHODS.2.8. ETHICAL CONSIDERATIONS: Approval was granted after submitting a research plan to E University Hospital's Institutional Review Board (EMC 2016-01-003-004).Patients who had spent more than 2 nights in E University Hospital's ICU were selected as participants, and a pulmonologist's consent was received for any patients who were using a respirator. The experiment was conducted after explaining the study's purpose and methodology and receiving consent from the nurses and relevant medical teams.Participation was voluntary; before agreeing to participate, participants and guardians were provided with a thorough explanation of the study.The explanation form included information about the participants' right to participate or withdraw from the experiment, potential side effects and treatment methods for such side effects, and compensation.To protect personal information, a serial number-based identification was provided based on guidelines for personal information processing.As a gesture of gratitude, a small gift was provided to the participants after the data were collected.This study was conducted in accordance with the Declaration of Helsinki. BODY.3. RESULTS: This study included a total of 60 subjects, 30 of whom were placed in an experimental group and 30 of whom were placed in a control group. The groups were then used to test the effects of aromatherapy on stress and sleep quality in ICU patients. BODY.3. RESULTS.3.1. HOMOGENEITY TEST: The results of a homogeneity test regarding questions on demographic elements and health are depicted in Table 1. There was no significant difference between groups in the demographic element and health related characteristics of the subjects. The prehomogeneity test results concerning the dependent variables are depicted in Table 1. There were no significant differences between groups in initial systolic or diastolic blood pressure readings, heart rate, or perceived sleep quality. Regarding perceived stress and the objective stress index, the experimental group was significantly different than the control group. In scores measuring perceived stress, the experimental group and control group had scores of 8.10 and 6.27, respectively, showing a significant difference between the groups (t = −4.294, p < .001). In the objective stress index, the experimental group scored 7.73, while the control group scored 6.17, again showing a significant difference between the groups (t = −2.396, p = .020). In review, the prehomogeneity test results on dependent variables between the groups showed no significant differences in systolic blood pressure, diastolic blood pressure, heart rate, sleep quality scores, and attained homogeneity. Test results, however, did show a significant difference between groups in the perceived stress and objective stress index (Table 1). BODY.3. RESULTS.3.2. THE EFFECT OF AROMATHERAPY ON STRESS AND VITAL SIGNS IN ICU PATIENTS: The measurement results over two days to verify the effect of aromatherapy on perceived stress are shown in Table 2. In an initial homogeneity test, perceived stress (D0 Adm), which showed a significant difference, was processed as a covariate. The results of this showed that, on the first morning (D1 8am) when patients had awoken after undergoing aromatherapy the previous night, perceived stress in the experimental group was 5.33 and 7.80 in the control group (F = 120.481, p < .001). Before going to sleep the same evening (D1 8pm), the experimental group had a score of 5.47 and the control group had a score of 8.23 (F = 167.557, p < .001) in perceived stress. On the second morning after undergoing aromatherapy the previous night (D2 8am), the experimental group had a score of 3.73 and the control group had a score of 8.50 in perceived stress, depicting a significant difference between the groups (F = 390.022, p < .001). The results of repeated measurements of perceived stress over the course of 2 days after processing perceived stress (D0 Adm) as a covariate showed that there was a significant difference based on time (F = 2.99, p = .033). There was also a significant difference between the two groups in their respective scores for perceived stress (F = 148.43, p < .001). Lastly, there was a significant difference in the results of the reciprocal interaction of time based on the group (F = 60.11, p < .001) (Table 2). The measurement results over two days to verify the effect of aromatherapy on objective stress index are shown in Table 2. In a homogeneity test, the objective stress index (D0 Adm) was processed as a covariate. The results of this showed that, on the first morning (D1 8am), the experimental group and control group had objective stress index scores of 3.90 and 7.60 (F = 44.866, p < .001), respectively. Measurements on the second morning showed an objective stress score of 4.37 in the experimental group and 8.00 in the control group. These results show a significant difference between the groups (F = 74.309, p < .001). The results of repeated measurements of objective stress index over the course of two days after processing objective stress before the experimental treatment (D0 Adm) as a covariate are as follows. There was a significant difference in the results of the reciprocal interaction of time based on the group (F = 25.65, p < .001) (Table 2). The measurement results over the course of two days to verify the effects of aromatherapy on blood pressure in the experimental group and control group are shown in Table 3. The results of repeated measures of ANOVA on blood pressure showed that on the first morning (D1 8am) blood pressure in the experimental group and control group was 116/71 mmHg and 135/74 mmHg, respectively. On the same evening before patients went to sleep, blood pressure in the experimental group was 119/70 mmHg and 129/71 mmHg in the control group. In the morning of the second day (D2 8am), blood pressure in the experimental group was 113/68 mmHg and 134/72 mmHg in the control group. The results of repeated measurements of systolic blood pressure over the course of two days showed that there was a significant difference based on time (F = 13.53, p < .001) and a significant difference in blood pressure scores between the two groups (F = 10.51, p = .002). The results of the reciprocal interaction with time based on group also showed a significant difference (F = 9.09, p < .001) (Table 3). The results of repeated measures of ANOVA on diastolic blood pressure over the course of two days showed that there was a significant difference based on time (F = 7.35, p < .001) and the reciprocal interaction of group and time (F = 2.47, p = .046). However, there was no significant difference between the diastolic blood pressure results between the groups (F = 0.076, p = .783) (Table 3). The changes in heart rate measurements between both groups are shown in Table 3. There was a significant difference based on time and group. There was also a significant difference in the results of reciprocal interaction of time based on the group (F = 5.71, p < .001) (Table 3). BODY.3. RESULTS.3.3. THE EFFECT OF AROMATHERAPY ON SLEEP QUALITY IN ICU PATIENTS: The Verran & Snyder-Halper (VSH) measurement tool was used in order to verify the effect of aromatherapy on sleep quality. Measurements were taken within 1 hour (D0 Adm) of hospitalization, on the first morning (D1 8am) after patients had awoken, and on the second morning after patients had awoken (D2 8am). The sleep quality of the experimental group and the control group within 1 hour (D0 Adm) of hospitalization had scores of 65.13 and 61.03, respectively (t = −0.640, p = .524). Sleep quality measurements were taken again on the first morning (D1 8am); sleep quality scores were 52.90 for the experimental group and 33.56 for the control group (t = −11.638, p < .001). Sleep quality on the second morning after patients had awoken (D2 8am) was 57.73 points for the experimental group and 25.80 points for the control group (t = −19.577, p < .001). The results of the repeated measure of ANOVA on sleep quality over the course of two days showed that there was a significant difference based on time (F = 294.60, p < .001) and that there was a significant difference in results based on the group (F = 221.12, p < .001). There was also a significant difference between the group and reciprocal interaction of time (F = 109.46, p < .001) (Table 2). BODY.4. DISCUSSION: This study investigated the effect of aromatherapy on stress and sleep quality in patients who had been admitted to the ICU and were being treated there. The experimental group consisted of participants (n = 30) breathing in lavender essential oil, which began after conducting a pretest. BODY.4. DISCUSSION.4.1. AROMATHERAPY AND STRESS: Perceived stress and objective stress index were measured before and after the aromatherapy treatment to verify the effect of aromatherapy on stress. The results showed that perceived stress and the objective stress index in the experimental group that received aromatherapy decreased on both the first and second days. Conversely, perceived stress and the objective stress index in the control group increased on both the first and second days. Stress typically increases in patients when they are admitted to the ICU; however, stress decreased in the experimental group. This result demonstrates the antistress effect of lavender. This study verified that lavender aromatherapy is a significantly effective method for alleviating perceived and objective stress in ICU patients. This result is in line with preceding research that claims aromatherapy is effective in alleviating stress in heart disease patients [29]. The results of the perceived level of stress and objective stress index measured as heart rate variability in patients were identical, and the objective stress index was an indicator of perceived stress. BODY.4. DISCUSSION.4.2. AROMATHERAPY AND BLOOD PRESSURE: Systolic blood pressure decreased in both the experimental and control group between the time of admittance to the ICU and before bedtime at 8 p.m. This may reflect the initial stress of hospitalization, which caused systolic blood pressure to increase, and the effect of half a day's bedrest, which decreased stress and lowered systolic blood pressure. The experimental group used lavender and exhibited decreased systolic blood pressure on the first and second mornings. Conversely, the control group experienced an increase in blood pressure during this same period. In addition, systolic blood pressure in the experimental group increased before bedtime without the lavender; however, it decreased by roughly 10 mmHg after using the lavender in the morning. Blood pressure typically undergoes periodic changes, and there is a tendency for blood pressure to rise due to a morning surge [30]. Therefore, a rise in blood pressure reflects the morning surge that occurs before 8 a.m. The experimental group, however, exhibited low blood pressure readings in the morning, which may demonstrate the effect of lavender in lowering blood pressure. These results show that aromatherapy using lavender is effective and is consistent with preceding research showing that systolic blood pressure drops after inhaling aroma essential oils [11]. As stress in the experimental and control groups decreased after hospitalization, diastolic blood pressure also decreased after a half day's rest. There was no dramatic change in diastolic blood pressure on the first and second days of the experiment that followed. These results are similar to previous results where systolic and diastolic blood pressure decreased in concert [11, 31]. This may show that autonomic arousal is suppressed with the use of lavender [26]. BODY.4. DISCUSSION.4.3. AROMATHERAPY AND HEART RATE: Both groups exhibited a decrease in heart rate over time, which may be a result of resting their bodies after admittance to the ICU. The experimental group showed a continuous decrease in heart rate on the first and second days after the application of aromatherapy. These results also show the effectiveness of aroma inhalation. BODY.4. DISCUSSION.4.4. AROMATHERAPY AND SLEEP QUALITY: Subjective sleep quality scores were measured to verify the effect of aromatherapy on sleep quality. The intensive care unit has various external stimuli such as consciousness evaluation, blood pressure measurement, and blood sampling. Therefore, in this study, objective sleep quality was not measured and subjective sleep quality was measured. It is very meaningful to measure the quality of sleep that the patient feels during the period of admission treatment in ICU. The results of this study show that there was no difference between groups in the pretest, which examined average sleep quality; however, sleep quality decreased in both groups following hospitalization. This is likely a natural result of the hospitalization itself affecting sleep quality. Sleep quality in the control group dropped by 50% in both the first and second nights of hospitalization. Sleep quality in the experimental group, however, decreased only by roughly 10% after the experimental treatment. This indicates that lavender prevents extensive reductions in sleep quality in hospitalized patients and is consistent with preceding research that demonstrates lavender's effectiveness in promoting sleep quality [28]. BODY.4. DISCUSSION.4.5. STRENGTHS: This study has three meaningful strengths:We verified the effect of aromatherapy on stress and sleep quality in ICU patients from a variety of aspects.We used aromatherapy to measure and verify its effect on stress and sleep quality throughout patients' treatment, rather than just measuring its effects on day-to-day stress and sleep quality.We presented subjective and objective data when measuring stress in patients. BODY.4. DISCUSSION.4.6. LIMITATIONS: This study has some limitations. First, we collected data from the control group first to prevent diffusion and contamination of the experimental treatment. As such, the study could be affected by a difference in data collection periods. In addition, it was difficult to completely hide the information regarding which group the subject belongs to because of the aroma-like nature of the experimental procedure. Lastly, the intervention was only performed at one hospital, which limits the generalizability of the results. More research is necessary on whether aromatherapy is effective throughout the treatment process for ICU patients required to undergo long-term treatment. There also needs to be further studies on stress relief and improvements in sleep quality in both ICU patients and patients who are moved to other wards. BODY.5. CONCLUSION: This experimental study used a nonrandomised, pre- and posttest design to address the effect of aromatherapy on stress and sleep quality among ICU patients. The results showed significant differences in perceived stress, objective stress index, blood pressure, heart rate, and sleep quality between the experimental group who received aromatherapy treatment and the control group who did not. Overall, the above research results demonstrate that aromatherapy, which was administered over two days, reduced stress and improved sleep quality in ICU patients.

TITLE: Post-marathon wearing of Masai Barefoot Technology shoes facilitates recovery from race-induced fatigue: an evaluation utilizing a visual analog scale ABSTRACT.PURPOSE: To investigate the potential benefit of post-race wearing of unstable shoes (Masai Barefoot Technology [MBT]) on recovery from marathon race–induced fatigue. ABSTRACT.PATIENTS AND METHODS: Forty-five runners who participated in a full marathon race were divided into three groups: 1) MBT shoes, 2) trail running shoes, and 3) control (CON). Participants ran a full marathon with their own running shoes, and then put on the assigned shoes immediately after the race. They continued to wear the assigned shoes for the ensuing 3 days. The CON group wore their usual shoes. Estimates of post-race fatigue were made by the participants on questionnaires that utilized a visual analog scale. Estimates were made just after the race, as well as for the next 3 days. ABSTRACT.RESULTS: The subjective fatigue of the MBT group was lower than that of the CON (P<0.05) or trail running shoe groups (P<0.05) on day 3. ABSTRACT.CONCLUSION: MBT shoe intervention can promote recovery from the fatigue induced by running a full marathon. BODY.INTRODUCTION: Long-distance running has recently become popular all over the world, and a large number of people participate in full marathons and even longer races.1,2 Completing a full marathon gives rise to profound muscle fatigue.3 Post-race fatigue can be disruptive to both work efficiency and overall quality of life.3 A fast recovery from post-race fatigue also enables athletes to stay in good condition for training and performance. Therefore, there is a large demand for an effective, simple method for optimizing recovery from the exhaustive fatigue that follows the completion of a full marathon. In this study, we focus on the footwear worn by runners in their everyday life, following severe exercise. We particularly evaluated the shoes produced by Masai Barefoot Technology (MBT; MBT USA, Inc., St Louis, MO, USA). These shoes were developed as a training aid that was said to produce a positive effect during standing or walking while wearing the shoes.4 The shoes were developed about 20 years ago, utilizing the concept that a benefit could be derived by causing the wearer to increase their range of motion (ROM) and to control the instability produced by a rounded sole. The producers claim that the MBT shoes: 1) improve balance ability, 2) increase muscle activity, and 3) reduce lower back pain.4 Some evidence does exist that indicates that wearing MBT shoes produces instability during standing5,6 and walking,7 and that prolonged wearing of MBT shoes can improve static balance ability.8 In addition, reduction of joint moment9–11 and altered kinematic changes of the ankle joint6,12,13 during walking have been confirmed. An increase in muscle activity while wearing MBT shoes has also been noted in many studies.4,6,9,10,13,14 There is also evidence that wearing MBT shoes can reduce the pain of knee osteoarthritis8 and that of the lower back.15 In addition to these effects, a number of MBT shoe users have reported that MBT facilitates recovery from exhaustive muscle fatigue (information from Evernew Corporation; also marketing claim of MBT shoes). To this point, there is no scientific evidence that supports these observations. The purpose of this study is to evaluate whether wearing MBT shoes facilitates recovery from the muscle fatigue that follows the completion of a full marathon. BODY.MATERIALS AND METHODS.SHOES: Two types of shoes were assigned to the participants to wear after a full marathon race. One type was the MBT shoe (Figure 1). The other type assigned (as a stable shoe) was the sports shoe, which is commonly used for trail running (TR). This shoe has a flat sole and is not unstable. Both MBT shoes and sports shoes are distributed by Evernew Corporation in Japan. The control group was told to wear what they normally wore after a race. BODY.MATERIALS AND METHODS.SUBJECTS: The subjects were openly recruited from two club teams of Waseda University through recruitment information on the team websites and mailing lists. The inclusion criterion for participating in this study was that the subject's foot size fit the test shoes available for the experiment (22.5∼26.0 cm). Forty-five university students who registered for the Second Fujisan Marathon (42.195 km; November 24, 2013) participated in this study. They were divided into the three groups described earlier. Based on pre-questionnaires, the groups were matched for foot size and targeted race time. All groups put on their assigned shoes immediately after the race and wore them for the next 3 days of their everyday life. Group 1 was the MBT group (mean age 21±1 years; mean BMI [body mass index] 20.3±1.4; n=15 [seven males and eight females]). Group 2 was the TR shoe group (mean age 21±2 years; mean BMI 19.5±2.1; n=14 [seven males and seven females]) and group 3 was the control (CON) group (mean age 21±1 years; mean BMI 21.0±1.3; n=16 [seven males and nine females]). No subject in any of the groups had prior knowledge of the MBT or TR shoe. Before the experiment, written informed consent was obtained from all subjects. The study was approved by the Human Research Ethics Committee of Waseda University (2013–216). BODY.MATERIALS AND METHODS.PROTOCOL: Subjects ran the race in their own running shoes. Immediately after finishing the race, subjects of the MBT and TR groups changed into the MBT or TR shoes. In addition, subjects of the MBT group received a short lecture on how to use the MBT shoes. After the race, subjects of the MBT and TR groups wore the assigned shoes in their daily life except when they were in their own or in others' houses (the Japanese custom is to not wear shoes in the home) or during sports activities. The only requirements of the subjects were to wear the assigned shoes and to answer the questionnaires. BODY.MATERIALS AND METHODS.SUBJECTIVE MUSCLE FATIGUE WITH QUESTIONNAIRES: The subject's degree of muscle fatigue was expressed on a 12 cm visual analog scale (VAS)16 where 0 cm indicated 'comfortable with no fatigue' and 12 cm represented 'too exhausted to do anything'. Each participant answered the questionnaires five times in total. On the day of the marathon, the participants answered the questions before the race (Pre) and immediately after finishing the race (Post). For the following 3 days, the questionnaires were completed in the morning. In addition to the estimates of fatigue, race performance (derived from the official records of the race), total walking time for the assigned condition, and length of time spent wearing the assigned shoes (we did not get this information for the CON group, because the subjects of the CON group did not have to wear shoes) during each day (except day 3) were reported. BODY.MATERIALS AND METHODS.DATA ANALYSIS: The estimates on the VAS were rescaled as percentages of the possible maximum (0 cm =0%, 12 cm =100%). For statistical analyses, the time course utilized was: Post, day 1, day 2, and day 3 after the race. The second factor was the group: MBT, TR, and CON. A two-way analysis of variance (ANOVA) (4 days ×3 groups) was performed on the above factors. In addition, differences in fatigue among the three groups on day 3 were also examined by unpaired t-tests. A comparison of the fatigue in the Pre with that on day 3 was also done by utilizing paired t-tests. The walking time for each of the 3 days was averaged for each day, and then a one-way ANOVA (three groups) was performed. The times recorded for the race were also analyzed by a one-way ANOVA (three groups). The length of time spent wearing the assigned shoes in the MBT and TR groups was compared by utilizing a paired t-test. Prior to performing parametric statistical analyses, the normality and equality of all data were confirmed by Shapiro–Wilk and Levene's tests, respectively. BODY.RESULTS: All subjects were able to complete the full marathon race. There was no main effect of group in the times recorded for the race (F[2, 42] =0.73, P=0.49; Table 1). The time spent walking for the 3 days also did not show a main effect of group (F[2, 42] =1.69, P=0.20; Table 1). The length of time spent wearing the assigned shoes in the MBT and TR groups did not statistically significantly differ (P=0.80). For the feeling of fatigue evaluated utilizing the VAS, a two-way ANOVA indicated a significant main effect of day (F[3, 167] =54.15, P<0.001) as well as a significant main effect of group (F[2, 167] =3.19, P<0.05). No significant interaction was found (F[6, 167] =0.57, P=0.80). On day 3, the feeling of fatigue was significantly lower for the MBT group than for the CON group (P<0.05) or the TR group (P<0.05). No difference between the CON and TR groups was found (P=0.64; Figure 2). Lastly, comparisons of the fatigue experienced before the race (Pre) and on day 3 showed a difference between TR_Pre and TR_day 3 (P<0.001), and CON_Pre and CON_day 3 (P<0.001), but no difference between MBT_Pre and MBT_day 3 (P=0.31). BODY.DISCUSSION: The objective of this study was to investigate the potential value of MBT shoes on the recovery from exhaustive fatigue induced by running a full marathon. The results of this study indicated that the MBT shoes promoted recovery from perceived fatigue. While subjective fatigue was similar among the three groups before and immediately after the race, subjective fatigue in the MBT group was less than that of the CON and TR groups on day 3. The lack of a difference in subjective fatigue of the MBT group between the Pre and day 3 indicated that the fatigue was largely gone by day 3. On the other hand, significant differences in subjective fatigue for the CON and TR groups between the Pre and day 3 values indicate that fatigue still remained on day 3 for these groups. In addition, the lack of a difference between the CON and TR groups at any time during recovery demonstrates that wearing a new type of shoe did not affect the results (no placebo effect). Also, the absence of a difference in the marathon performance between the three groups indicated that running abilities were similar for the three groups. In addition, there was no difference in the daily length of time spent walking between the three groups nor in the difference in wearing time for the assigned shoes. Therefore, the race performance or exercise volume in daily activity during the recovery period were not important factors in determining the speed of recovery after the race. Thus, wearing the MBT shoes had a positive effect on recovery from severe fatigue following exhaustive exercise. How did the wearing of MBT shoes promote the recovery from fatigue? It has been suggested that wearing MBT shoes increases activity in lower limb muscles, including the triceps surae,10,13 tibialis anterior,6 quadriceps,10 and other small muscles.4,5 Maximal forces that could be exerted by the knee extensor and plantar flexor decrease after marathon running.17–19 This suggests that, minimally, the knee extensor and plantar flexor muscles show compromised function as well as subjective fatigue following the race. One possibility is that wearing the MBT shoes increased the activity of the fatigued muscles. This activity may aid in an 'active recovery'. Generally, exercises that involve a slightly higher intensity (active recovery) after exhaustive exercise promote a more effective recovery from fatigue as compared with passive recovery, which involves no specific exercise.20 In addition, since walking with MBT shoes increases the angle of dorsiflexion,6,13 a stretching of the plantar flexor muscles might also occur. Thus, the wearing of MBT shoes would be expected to promote stretching of the lower limb muscles and thus enhance the activity in those muscles. This would result in increased muscle blood flow, which would lead to an increased removal of lactic acid. Lactic acid concentration is generally utilized as a biochemical marker of muscle fatigue.21 Although an enhanced recovery by wearing MBT shoes was clearly indicated by this study, some limitations on the findings exist. First, since the fatigue evaluation involved only a subjective questionnaire, various types of personal bias could not be completely removed. When utilizing only a questionnaire, it is difficult to determine the physiological mechanisms involved in the fatigue recovery provided by the MBT shoes. Second, we did not obtain data on the time and behavior spent while not wearing the shoes. Therefore, we cannot thoroughly eliminate the effect of factors other than wearing of the shoes on the present results. Third, we should have utilized a device like an acceleration meter to objectively measure the subject's physical activity. Lastly, we did not constrain the subject's behavior after the race except for requiring the wearing of the assigned shoes. Some subjects might have trained hard even after the marathon race, while others might have performed little or no physical activity, which might explain the large variability seen in the data. Such limitations are almost unavoidable in a clinical study such as this one. Further experimentation which would include the measurement of biochemical and biomechanical markers in a laboratory setting will be expected to elucidate the physiological underpinning of the recovery effect elicited by wearing MBT shoes. Such results, if positive, would also enhance the validity of this study. Nevertheless, this study clearly supports the usefulness of MBT shoes in clinical applications. Results of the present study suggest the usefulness of MBT shoes in conditioning not only for athletes, but also for recreational use. Recreational activities can also involve exhaustive exercise and can produce strong fatigue. Recovery from this fatigue could likely be speeded up simply by wearing MBT shoes during everyday life. BODY.CONCLUSION: Using MBT shoes during everyday activities promoted recovery from the exhaustive fatigue induced by running a full marathon. This finding has significant clinical implications for both athletes and lay people.

TITLE: A randomized, double-blind, prospective, placebo-controlled study of the efficacy of a diet supplemented with curcuminoids extract, hydrolyzed collagen and green tea extract in owner’s dogs with osteoarthritis ABSTRACT.BACKGROUND: We have previously demonstrated that a mixture of Curcuminoids extract, hydrolyzed COllagen and green Tea extract (CCOT) inhibited inflammatory and catabolic mediator's synthesis by bovine and human chondrocytes. A randomly allocated, double-blind, prospective, placebo-controlled study was performed to evaluate the efficacy of a diet containing this CCOT mixture on dogs with naturally occurring osteoarthritis (OA). Therefore, 42 owner's dogs with OA were randomly assigned to receive for 3 months an experimental diet (control) or the same diet supplemented with CCOT. ABSTRACT.RESULTS: Ground reaction forces did not show statistical differences between groups. After 3 months of feeding, there was a significant reduction of pain at manipulation in the CCOT group, but not in the control group. The evolution for pain at manipulation depended on the diet. The three other parameters evaluated by veterinary subjective assessment (lameness, pain at palpation and joint mobility) did not show statistical differences. Concerning owner subjective assessment, pain severity score worsened in the control group but remained stable in CCOT group. The evolution for pain severity depended on the diet. No statistical difference was found for pain interference, except for the ability to rise to standing from lying down, which was significantly improved in the CCOT compared to the control group. Serum OA biomarkers did not show statistical differences. ABSTRACT.CONCLUSIONS: Objective variables measured, such as ground reaction forces and OA biomarkers, did not show statistical differences. However, indicators of pain appeared reduced in dogs receiving CCOT mixture for 3 months. The difference of evolution between groups suggests that a greater number of dogs may be necessary to reach a stronger effect on other parameters. BODY.BACKGROUND: Osteoarthritis (OA) is a chronic, painful, degenerative and inflammatory condition that affects the synovial joints. It is highly prevalent in dogs [1, 2] with 20% of the canine population over one year old affected [3, 4]. This musculoskeletal disease is related to chronic pain, lameness, loss of joint function and mobility, functional disability and reduced quality of life [5]. The management of OA in dogs is a lifetime commitment, involving a multimodal approach. The main recommendation is to control symptoms by reducing pain, improving mobility and hence quality of life; whilst protecting joints from OA [6]. To decrease pain and inflammation associated with OA, non-steroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed [5]. Indeed, in clinical practice, many dogs suffering from OA are long term treated with NSAIDs such as carprofen [6]. NSAIDs act mostly by inhibiting cyclo-oxygenase and thus reducing the concentration of pro-inflammatory prostaglandins. Unfortunately, the use of NSAIDs may be associated with adverse effects, especially gastrointestinal tract ulcerations [7, 8]. Corticosteroid injection in dogs is usually reserved for severe end stage OA and for cases that have become refractory to other treatments [5]. Beside pain relief, preventing cartilage degradation is an important objective for treatment. This requires the long term use of safe therapies, while the absence of any cure reinforces the importance of prevention [9]. Such prevention and alternative solutions could come from nutrition or from dietary supplements. Indeed, these latter present the advantage of having few or no known side effects. In a preliminary in vitro study [10], we have demonstrated that a mixture of curcuminoids extract, hydrolyzed collagen and green tea extract (CCOT) inhibited inflammatory and catabolic mediator's synthesis by bovine and human chondrocytes. These findings suggest a scientific rationale for the evaluation of these natural ingredients in a clinical trial. Curcumin is the major component of turmeric, a yellow spice derived from the rhizomes of the plant Curcuma longa. Evidence has been published for its potency to target multiple inflammatory diseases [11]. The main characteristic of hydrolyzed collagen is its amino acid composition, which is identical to collagen, thus providing high levels of glycine and proline, two amino acids essential for the stability and regeneration of cartilage [12, 13]. Green tea contains polyphenolic fractions called catechins and, among them, epigallocatechin-3-gallate, which exhibits anti-oxidant, anti-tumoral and anti-mutagenic activities [14]. This randomly allocated, double-blind, prospective, placebo-controlled clinical trial aimed to evaluate the effects of a diet containing CCOT mixture on client owned dogs with OA using objective variables such as force plate analysis and serum OA biomarkers (Coll2–1 and Coll2–1 NO2) as well as subjective variables such as orthopedic evaluation and owner assessment [15]. BODY.METHODS.DOGS: Dogs with OA were recruited among the patients of the Veterinary Hospital of the University of Liege, or through advertisements in pet stores, veterinarians', dog magazines, daily papers, websites with animal news, grooming salons and pet associations. The (potential) participants were informed about the purpose and design of the trial. Inclusion criteria were the presence of clinical (such as lameness) and radiographic (such as the presence of osteophytes, subchondral bone sclerosis) signs of OA on at least one limb, to be older than 18 months and weigh over 10 kg with a body condition score lower than 8 (on a 9-point scale) [16], without evidence of systemic disease identified by history and results of physical examination, serum biochemical analysis and urinalysis. Non-inclusion criteria were as follows: signs of lumbosacral disease or neurologic deficit, acute traumatic injuries (including OA acute crises), treatment with NSAIDs, corticosteroids or antimicrobials within 14 days before enrolment, surgery on any joint within 6 months before enrolment, aggressive behavior, and pregnancy or likelihood of becoming pregnant during the study. For ethical reasons, analgesics (tramadol, 2–5 mg/kg, 2–3 times a day) were allowed, except within 48 h before the evaluation. Dogs were excluded from the study for the following reasons: development of an adverse reaction, injury or illness that required treatment or surgical intervention, excessive pain or other complications as determined by the investigator, lack of owner compliance with study restrictions, and death of the dog because of natural causes or owner-elected euthanasia. BODY.METHODS.STUDY DIETS: The 2 study diets were an experimental diet (control) or the same diet supplemented with CCOT mixture (CCOT) (Table 1). Both dry dog food products had similar nutritional and energy content (3515 kcal/kg) and similar visual aspect. Energy requirements for dogs were based on the equation published by the National Research Council in 2006: 95 kcal/kg0.75 body weight [17, 18]. Both diets were supplied by the manufacturer in identical neutral bags but differentiated by their code names. The diet with the CCOT mixture contained 0.43 g curcuma extract per 1000 kcal, 4.27 g hydrolyzed collagen per 1000 kcal and 0.85 g green tea extract per 1000 kcal. Curcuma extract (Indena, Paris, France) contained between 18 and 22% of curcuminoids and was associated with a phosphatidylcholine complex to increase its bioavailability [19]. Peptides constituting hydrolyzed collagen (Gelita, Eberbach, Germany) were composed of 30 amino acids peptides. Glycine and proline represented more than 35% of total amino acids content. Green tea extract (Naturex, Avignon, France) contained 25% polyphenols, which represented 12.5% catechins representing more than 9.3% epigallocatechin-3-gallate.Table 1Composition of both study dietsUnitControlCCOTMoisture%12,212,1Protein%20,520,5Fat%11,711,7Ash%6,05,9Crude fiber%4,04,0Total dietary fiber%9,09,0Nitrogen free extract%40,239,9Glycine%0,71,0Hydroxyproline%0,0020,2Proline%1,11,2Curcuminoids extract%00,15Hydrolyzed collagen%01,5Green tea polyphenols%00,3 BODY.METHODS.STUDY PROTOCOL: The study was designed as a 3-month double-blind, randomly allocated, prospective, placebo-controlled clinical trial, adhering to the CONSORT guidelines [20]. All owners received a detailed written description of the protocol and provided written informed consent before the inclusion of their dog in the study. The protocol was approved by the Institutional Animal Care and Use Ethics Committee of the University of Liège (reference 12–1330) and by the Royal Canine Ethics Committee. They adhere to a high standard of veterinary care. Investigators collected blood samples at the first screening visit for serum biochemistry which validated dog eligibility for the study. Eligible dogs were randomly assigned (using Microsoft Office Excel 2013) to receive either the control diet or the CCOT supplemented diet. A statistician generated the random allocation sequence. Block size was 10. Allocation ratio was 1:1. The allocation sequence was not concealed but neither pet owners, neither veterinarians nor investigators had knowledge of the diet to which dogs were assigned. A veterinarian enrolled dogs. Upon enrollment in the study, pet owners were instructed to feed their dogs on a transition diet (same composition as control but kibble of a different shape) over 14 days. In this way, all eligible dogs received the same diet (without any potential active compound included within the diet) before starting the study. Feeding guidelines were provided to owners with the intent for dogs to maintain a constant body weight and condition as well as to be fed according to their usual feeding regimen (free choice or meal). An investigator assigned dogs to interventions. Dogs were weighed each month. BODY.METHODS.ADVERSE EVENTS: During the study, all adverse events were reported to the investigator, who noted the event characteristics including severity, occurrence and suspected association with the food. BODY.METHODS.OBJECTIVE MEASUREMENT: FORCE PLATE ANALYSIS: Ground reaction forces were measured with kinetic analysis using biomechanical force platforms (Kistler, Winterthur, Switzerland) at the Laboratory of Human Motion Analysis at the University of Liège. At study start (T0 = inclusion +14 days of transition diet), the limb most affected by lameness, as shown by peak vertical force (PVF) gait analysis and orthopedic examination, and with radiographic OA lesions was defined as "the most affected limb" and followed throughout the study. Dogs were acclimated to the force plate before data collection. Data for the right and left sides were collected from separate passes across the plate. The limb most affected by lameness, as shown by PVF gait analysis and orthopedic examination, and with radiographic OA lesions was considered as the most affected limb and its contralateral limb was considered as less affected limb (compared to its contralateral most affected limb). Ground reaction forces were recorded at T0 and after 3 months of diet (T3). The owner trotted dogs across the force plate, and an investigator observed and filmed each pass to confirm foot strikes and gait. A test was considered valid when only one limb landed on the force plate at a time, while the dog was trotted across it at a velocity of 1.8 to 2.2 m/s and acceleration-deceleration variation of ±0.5 m/s2. The dog's forward velocity was measured with a 3D sensor (Charnwood Dynamics, Rothley, United Kingdom) placed on the back of the dog. Five valid tests were obtained for each limb. PVF, braking and propulsive peak forces, vertical impulse, braking and propulsive impulses, and loading and unloading rates were measured and analyzed using the software CODAmotion (V6.78.1) (Charnwood Dynamics). All parameters were normalized by body weight (BW) (Newton) and expressed in % of BW. Data from five valid tests of each most affected and less affected limb were averaged as previously described [21]. PVF has been defined as the primary outcome of our study. Based on this outcome, we determined the positive and negative responder's rates for control and COT groups [22]. We used the minimal detectable change, at the 95% level (MDC95), which reflected a real change. A change of at least 3.6% BW in PVF measurement, when expressed relatively to baseline values, at the 95% level, needed to occur to be confident [22]. BODY.METHODS.SUBJECTIVE MEASUREMENT: ORTHOPEDIC EVALUATION: At inclusion visit and T3, dogs were examined by a veterinarian specialized in orthopedic surgery. Two orthopedists participated in the study. One orthopedist evaluated 24 dogs, and another 18 dogs. The same veterinarian performed a dog's assessment at T0 and at T3. Lameness (1 to 5), pain at manipulation (0 to 10), pain at palpation (1 to 5) and joint mobility (1 to 5) were evaluated as previously described [23, 24]. The scoring system is described in Table 2. Pain at manipulation was evaluated on a scale from 0 to 10, 0 corresponding to no pain, and 10 corresponding to extreme pain [24]. Pain upon limb manipulation was evaluated by animals' vocalization or other observations of pain during the extension and flexion of all four limbs for a period of several minutes.Table 2Clinical scoring system for assessing dogs with osteoarthritisCriterionGradeClinical evaluationLameness1Walk normally2Slightly lame when walking3Moderately lame when walking4Severely lame when walking5Reluctant to rise and will not walk more than five pacesPain at palpation1None2Mild signs; dog turns head in recognition3Moderate signs; dog pulls limb away4Severe signs; dog vocalizes or becomes aggressive5Dog will not allow palpationJoint mobility1Full range of motion2Mild limitation (10 ∼ 20%) in range of motion; no crepitus3Mild limitation (10 ∼ 20%) in range of motion; crepitus4Moderate limitation (20 ∼ 50%) in range of motion; ± crepitus5Severe limitation (>50%) in range of motion; ± crepitus BODY.METHODS.SUBJECTIVE MEASUREMENT: OWNER ASSESSMENT: Owners evaluated their dog's condition at T0 and T3 by completing a validated Canine Brief Pain Inventory (CBPI), a questionnaire assessing pain severity (PS) and pain interference (PI) [15]. The questionnaire was translated in French. The same owner evaluated his/her dog at T0 and T3. The 4 PS questions (worst pain in the last 7 days, least pain in the last 7 days, average pain in the last 7 days and pain right now) were scored on a numeric scale from 0 (no pain) to 10 (extreme pain). The scores of each question were averaged to generate the global PS score. Each criterion was considered with the same weighing. The 6 PI questions (i.e., how much the pain interfered with the dog's typical function: general activity, enjoyment of life, ability to rise to standing from lying down, ability to walk, ability to run and ability to climb up) were scored on a numeric scale from 0 (does not interfere) to 10 (completely interferes). The scores of each question were averaged to generate the global PI score. Each criterion was considered with the same weighing. BODY.METHODS.OBJECTIVE MEASUREMENT: OA BIOMARKERS: Blood samples were collected each month (T0, after 1 month of diet (T1), after 2 months of diet (T2) and T3) and OA biomarkers (Coll2–1 and Coll2–1 NO2) were quantified by competitive immunoassays (Artialis SA, Liège, Belgium). All Coll2–1 and Coll2–1NO2 tests have been performed by Artialis in collaboration with Bone and Cartilage Research Unit. These assays are competitive immunoassays utilizing a synthetic peptide pre-coated onto the ELISA plate for the quantification of the corresponding antigen in samples. A binding competition between the immobilized peptide and the peptide contained in the standards or samples takes place upon addition of the antibodies. After removal of the unbound peptide, a peroxidase-conjugated goat anti-rabbit antibody is added into each well to detect and quantify the level of competitive binding. After washing of the unbound detection antibody, the antibody-antigen complex is detected by a chromogenic reaction with 3, 3′, 5, 5′-tetramethylbenzidine (TMB). The reaction is stopped by adding acid to give a colorimetric endpoint that is subsequently determined spectrophotometrically. Coll2–1 was quantified in dog sera by competitive ELISA in triplicate with polyclonal rabbit antisera (ab-Coll2–1; Artialis) using buffers specifically developed to measure these fragments in dog serum (confidential composition). A 6-fold dilution of samples has been applied. Intraassay coefficient of variation (CV) was 3.6% and interassay CV was 9.8%. Coll2–1-NO2 was quantified in dog sera by competitive ELISA according to the methodology cited above, in triplicate, with polyclonal rabbit antisera (D37; Artialis) using buffers specifically developed to measure these fragments in dog serum (confidential composition). A 2-fold dilution of samples has been applied. Intraassay CV was 5.8% and interassay CV was 4.7%. BODY.METHODS.STATISTICAL ANALYSIS: The number of dogs needed, on a statistical point of view, was calculated from a previous study evaluating the effect of an extract of turmeric in OA dogs on PVF [9]. Using the related effect size, this study was powered at 80% with an alpha risk at 5%. A two-tailed test was performed. Including a 10–20% margin for fallout during the study, the optimal initial number of dogs was determined between 46 and 50. There was no change to trial outcomes after the trial commenced. PVF was calculated in terms of PVF evolution as Δ T3-T0. PVF was assessed with a mixed model including diet, limb effect (most/less affected), and interaction between diet and limb effect as fixed effects. As given that BW influenced PVF, BW was entered as covariate in PVF statistical analysis. Lameness, pain at manipulation, pain at palpation and joint mobility were also assessed using a mixed model including time, diet, and the related interaction between time and diet as the main effects. Dog was defined as a random term in mixed models. PS and PI were assessed using generalized linear model. Diet, defined as the main effect, was investigated on Δ T3-T0. Generalized linear model or mixed model were analyzed using SAS 9.3. According to data features and residuals distribution of each model, the outcomes were previously rank transformed or not. Homoscedasticity was checked on residuals with a white test at a level of 1%. All data were expressed as mean ± standard deviation. The analysis was two-sided. A p-value ≤0.05 was considered as statistically significant. BODY.RESULTS.DOGS: One hundred and fifty five dog owners responded to the advertising. After a phone interview to get additional information, 115 owners were invited with their dog to the first screening visit. Of those 115 screened dogs, 48 were found eligible for the study and were randomly assigned to receive CCOT (n = 23) or control (n = 25) diet for 3 months. Two dogs in the CCOT and 4 in the control groups were excluded from the study for the following reasons: development of an illness that required treatment (such as NSAIDs) or surgical intervention (3 control dogs), appearance of neurologic deficit (1 CCOT dog), lack of owner compliance with study restrictions (1 control dog), and death of the dog because of owner-elected euthanasia due to deterioration of general condition of the dog (1 CCOT dog). Consequently, 42 dogs completed the study, including 21 from the CCOT group and 21 from the control group (Fig. 1). Only dogs that completed the study were included in the analysis (per-protocol population). Two dogs in the control group needed rescue analgesia during the study: one for 8 days and one for 7 days. The periods of recruitment and follow-up lasted for more than two years (from February 2013 to April 2015). The trial ended because between 46 and 50 dogs were enrolled in the study.Fig. 1Flow diagram through study There were no significant differences concerning population characteristics between control and CCOT groups at T0 and T3 (Table 3). In the control group, the following breeds were represented: mixed breed (n = 6), German Shepherd Dogs (n = 5), Bernese Mountain Dogs (n = 2), Border Collies (n = 2), and 1 each of Australian Shepherd, Bordeaux Mastiff, Braque d'Auvergne, German Shorthaired Pointer, Golden Retriever, Rottweiler. In the CCOT group, the following breeds were represented: Golden Retrievers (n = 5), Border Collies (n = 2), Cockers (n = 2), German Shepherd Dogs (n = 2), Labradors (n = 2), Newfoundland (n = 2) and 1 each of Airedale Terrier, Bernese Mountain Dog, German Mastiff, mixed breed, Pitbull-type dog, Saint-Bernard.Table 3Characteristics of dogs which completed the studyCharacteristicsControlCCOT p-valueTotal number of subjects2121NAMale/female8/1310/11NACastrated male/sterilized female/intact2/9/106/7/8NAAge at T0 (years)7.50 ± 2.857.56 ± 3.070.952Body weight at T0 (kg)34.91 ± 11.6636.43 ± 12.630.563Body weight at T3 (kg)35.17 ± 11.6036.13 ± 12.950.717Peak vertical force at T0 (% BW)69.65 ± 19.8661.81 ± 11.560.618Pain at manipulation at T03.65 ± 2.504.19 ± 2.400.988Pain severity at T02.46 ± 2.082.67 ± 1.820.680Pain interference at T03.44 ± 2.713.26 ± 2.470.898Most affected joint at T0 Carpus10NA Elbow52NA Stifle26NA Hip1313NAData were expressed as distributions (number of dogs) for categorical characteristics and as mean ± SD for continuous characteristics T0 study start, T3 after 3 months of diet, study end, NA not applicable, BW body weight BODY.RESULTS.TOLERANCE: The CCOT diet was well tolerated. There was no significant change in dogs' body weight nor evidence of side effects over the duration of the study. Three dogs in CCOT group and 2 dogs in control group had mild diarrhea but it was minor and transient. BODY.RESULTS.OBJECTIVE MEASUREMENT: FORCE PLATE ANALYSIS: We recruited only few dogs affected by front and hind limbs OA, and those dogs were « clinically » lame only on one leg (usually the front limb). We did not recruit any dogs with lameness on both front and hind limbs. PVF values for the most severely affected limb were not significantly different between control and CCOT groups at T0 (p = 0.618) and T3 (p = 0.953). There was no significant PVF change with time in control group (T0: 69.65 ± 4.33% BW; T3: 69.63 ± 3.18% BW; p = 0.999) and in CCOT group (T0: 61.81 ± 2.52% BW; T3: 67.44 ± 3.32% BW; p = 0.283). The PVF change (Δ T3-T0) was not different between groups (p = 0.319) (Fig. 2).Fig. 2Mean ± SD for Δ PVF in OA dogs (n = 21 control +21 CCOT). PVF = peak vertical force Different levels of change in PVF measurement were observed in dogs. The positive responder's rate was greater in CCOT group (47.6%) than in control group (42.9%). Moreover, the negative responder's rate was greater in control group (23.8%) than in CCOT group (14.3%). Indeed, among the 21 dogs in CCOT group, 13 (61.9%) had clinically meaningful changes, which were positive in 10 (47.6%) or negative in 3 (14.3%) dogs. Among the 21 dogs in control group, 14 (66.7%) had clinically meaningful changes, which were positive in 9 (42.9%) or negative in 5 (23.8%) dogs (Fig. 3).Fig. 3Individual changes in PVF measured at T3 in OA dogs. Changes were the difference between T3 and T0. Dashed lines represent the minimal detectable change, when expressed relatively to baseline values, at the 95% level. T0 = study start; T3 = study end There were no significant differences between groups for the vertical impulse, the braking and propulsive peak forces, the braking and propulsive impulses and the loading and unloading rates changes between T0 and T3 (data not shown). BODY.RESULTS.SUBJECTIVE MEASUREMENT: VETERINARY EVALUATION: Pain at manipulation was significantly decreased in the CCOT group (T0: 4.19 ± 0.52; T3: 2.86 ± 0.51; p = 0.037) but not in the control group (T0: 3.65 ± 0.56; T3: 3.7 ± 0.4, p = 0.999). Furthermore, the evolution for pain at manipulation depended on the diet (p = 0.036) (Fig. 4). There was no significant difference between groups for lameness, pain at palpation and joint mobility at T0 and T3 (Table 4).Fig. 4Mean ± SD for pain at manipulation at T0 and T3 in OA dogs (n = 21 control +21 CCOT). T0 = study start; T3 = study end; *p < 0.05 Table 4Mean ± SD for lameness, pain at palpation and joint mobility of the most severely affected limb in OA dogsT0T3Time*diet p-valueLameness Control (n = 21)1.8 ± 0.771.95 ± 1.050.244 CCOT (n = 21)2.19 ± 0.871.86 ± 0.96Pain at palpation Control (n = 21)2.45 ± 0.942.55 ± 0.940.195 CCOT (n = 21)2.62 ± 0.922.1 ± 0.94Joint mobility Control (n = 21)2.3 ± 1.082.75 ± 1.250.815 CCOT (n = 21)2.71 ± 1.422.7 ± 1.22 T0 study start, T3 after 3 months of diet BODY.RESULTS.SUBJECTIVE MEASUREMENT: OWNER ASSESSMENT: Regarding owner's assessment, PS did not change in the control (T0: 2.46 ± 0.45; T3: 3.58 ± 0.51; p = 0.071) or the CCOT group (T0: 2.67 ± 0.4; T3: 2.42 ± 0.38: p = 0.35). The PS change (∆T3-T0) was significantly different between CCOT and control groups (p = 0.009) (Fig. 5a). There was no significant difference for PI change between CCOT (T0: 3.26 ± 0.54; T3: 2.96 ± 0.45; p = 0.101) and control groups (T0: 3.44 ± 0.59; T3: 3.86 ± 0.66; p = 0.633) (p = 0.063) (Fig. 5b). However, when each question of the PI score was analyzed separately, PI change (∆ T3-T0) on the ability to rise to standing from lying down was significantly improved in CCOT group compared to control group (p = 0.029).Fig. 5Mean ± SD for Δ PS (a) and Δ PI (b) in OA dogs (n = 21 control +21 CCOT). PS = pain severity; PI = pain interference; **p < 0.01 BODY.RESULTS.OBJECTIVE MEASUREMENT: OA BIOMARKERS: Coll2–1 and Coll2–1 NO2 serum concentrations were not significantly different between control and CCOT groups at T0 and T3. No significant changes over time in Coll2–1 and Coll2–1 NO2 serum concentrations were observed in both groups. BODY.DISCUSSION: The results of this study suggest that there is some benefit in feeding symptomatic OA dogs with a diet containing the CCOT mixture, even if the primary outcome (PVF, objective variable) was not improved. Interestingly, our study showed that the veterinary subjective assessment pain at manipulation was decreased by more than 30% by CCOT diet. These data are consistent with that of a study evaluating the effects of curcuma extract in owner's dogs [9]. Another study evaluating the effect of undenatured type II collagen also demonstrated a decrease in pain upon limb manipulation [25]. Pain improvement was not associated with lameness improvement. Indeed, constraints and movement amplitudes are different. This may also be explained by the fact that manipulation investigates pain coming from the peri-articular tissue while lameness could be partially generated by pain triggered by subchondral bone loading. Assessments by owners are subjective measures but, when such measures are used, a composite score is more effective than individual scores [26]. In the present study, we used the validated CBPI which includes two composite scores: PS and PI [15]. Regarding owner's assessment, the evolution for PS, as assessed by ΔT3-T0, was significantly different between CCOT and control groups, with the CCOT group staying stable and the control group worsening. In contrast, no significant difference was observed for PI between CCOT and control groups. This suggests that CCOT could affect pain but not limb function. Nevertheless, when questions corresponding to PI were analyzed separately, we found that change (ΔT3-T0) in the ability to rise to standing from lying down was significantly improved in the CCOT compared to the control group. This is consistent with a study showing that fish oil induced a significant improvement on the ability to rise from a resting position, to play and to walk in OA dogs [27]. It has been shown that the CBPI was not correlated with changes in force plate data [21]. Pain mechanism during the weight bearing is different. Moreover, owners considered that showing improvements in quality of life (performing activities of daily living) is much more important than demonstrating enhancements in force plate data and increased or decreased use of a single limb at a walk or trot [21]. Gait analysis has the limitation of only evaluating an animal at one specific time point, outside of its normal environment. On the contrary, CBPI quantifies the owners' assessment of clinically relevant chronic pain–related behaviors with the dog in its routine environment over an extended period of time as well as how their dog is doing "right now" [21]. However, PVF is often recognized as the most appropriate measure for assessing the effects of therapeutic modalities in OA dogs and is frequently used in clinical trials [26, 28, 29]. PVF was increased by a diet supplemented with fish oil omega-3 fatty acids [26, 30], with type II collagen [24] and with green lipped mussel [31] but not by an extract of curcuma [9]. PVF presents some weaknesses, however. It is less reliable when used in owner's dogs. The fact that data for the most and less affected limbs were mostly collected from separate runs could also bring a bias. Minimal body weight of included dogs was 10 kg. It may have been better to start with higher body weight of dogs (e.g. 20 kg). Historically the weight limitation was related to the ability for the force plate to record a single stride (the size of the plate was too big to have only one leg striking the plate at the same time). Indeed, a small dog will have a faster joint angular velocity than a big dog. That is why we compared the difference between T0 and T3 on the same dog, at the same velocity. We were based on a study in which minimal body weight was 11.4 kg [26]. Additionally, the gait analysis data combined thoracic and pelvic limbs. This was problematic because PVF for the front limb is higher than PVF for the hind limb. There was a higher number of front limb OA dogs in control group (n = 6) than in COT group (n = 2). Therefore PVF was calculated as Δ T3-T0 but this heterogeneity may add a bias to our study. Moreover, PVF decrease has been demonstrated to be more important in dogs affected by stifle OA than by hip OA [32]. So we can speculate that the low ratio of stifle OA in our population explains in part the absence of effects of CCOT on PVF. Indeed in our study, 19% of dogs had stifle OA while 62% had hip OA. Our population was heterogeneous in terms of OA location. Some dogs also had indirect signs of OA (such as a decreased range of motion or discrete discomfort on manipulation) in multiple limbs. An improvement in one limb could hidden pain in others limbs. This is a limitation of this study. However heterogeneous groups of dogs mimics the real clinical situation in veterinary medicine. One could consider this heterogeneity as a strength of our study. The owner was the leash-driver for kinetics gait analysis. This is good for lowering stress associated with the manipulation for the dog. But this could be another source of inter-individuals variability, which may be controlled by the same manipulator for each dog. Before the beginning of the study, we compared kinetic analysis when dogs were guided by their owners or by the manipulator. We concluded that dogs were less troubled when they were guided by their owners. It was the same owner who guided his/her dog at T0 and at T3. Indeed, the dog response to a foreign manipulator is another inter-individual variability. Anyway, all the trials were recorded at the same velocity, acceleration (with the dog not turning its head). Dogs were also guided by their owners in other similar studies [30, 33]. Because mean changes often obscure the individual change, we presented individual changes in PVF for both control and CCOT groups. We also calculated positive and negative responder's rates. The positive responder's rate was greater in CCOT group (47.6%) than in control group (42.9%) whereas the negative responder's rate was greater in control group (23.8%) than in CCOT group (14.3%). Reporting the percentages of dogs which met the MDC95 requirements provided additional insightful interpretations other than considering only the overall mean change scores [22]. The MDC95 that reflected a real change in PVF measurement has been established in a recent study [22]. The MDC95 was found at 2% BW, indicating that any change in PVF for OA dog would be considered as measure noise below 2% BW, and a clinically significant change over 2% BW. However, when expressed relatively to baseline values, the MDC95 was found to be 3.6% BW [22]. As given that the change in PVF was measured between T0 (baseline) and T3, we choose this limit (3.6%) to calculate responder's rates in our study. Chronic pain is complex to measure. Even if the force plate is considered as one of the most appropriate measure of outcome as it is the most objective, so far, it is unknown if it is the most sensitive (pertinent) for chronic OA. To our knowledge, this is the first time that serum OA biomarkers were measured to assess the effects of a diet supplemented with CCOT mixture in OA dogs. Collagen degradation is one of the main features of cartilage breakdown during OA. Coll2–1 and Coll2–1 NO2 serum concentrations were correlated with the macroscopic and histological changes in dogs with OA induced by transection of the anterior cruciate ligament [34]. We failed to observe an effect of CCOT on Coll2–1 and Coll2–1 NO2 serum levels. This may be explained by the higher heterogeneity of biomarker values in our population than in a model of surgically induced OA dogs. No age effect was observed (data not shown) on the outcomes of interest but the study was not designed for this purpose. Our study presented some limitations such as its short duration and the number of dogs included. Even though the number of dogs needed was statistically calculated a priori based on the PVF data, the lack of information to fully represent targeted population variability (i.e. breed effect, gender effect...) might have underestimated required sample size. Additionally, higher numbers are required for subjective veterinary and owner assessments compared to gait analysis. A larger number of dogs and a reduced inter-individual variability could increase the benefit of the CCOT diet and subsequently reinforce the likelihood of measurable improvements of PVF and OA biomarkers serum concentrations. Moreover, the fact that the bags were clearly marked with codes that differentiated the groups and that the randomization sequence was not concealed could add bias to the study. BODY.CONCLUSIONS: Objective variables measured, such as ground reaction forces and OA biomarkers, did not show statistical differences. Regarding the objective outcome PVF, the positive responder's rate was greater in CCOT group than in control group whereas the negative responder's rate was greater in control group than in CCOT group, albeit not statistically tested. The study reveals that dogs receiving diet supplemented with CCOT mixture for 3 months showed less pain at manipulation. The three other parameters evaluated by veterinary subjective assessment (lameness, pain at palpation and joint mobility) did not show statistical differences. Regarding the subjective owner assessment, the evolution of PS showed significant difference between CCOT and control groups. No statistical difference was found for PI, except for the ability to rise to standing from lying down, which was significantly improved in the CCOT compared to the control group. These results suggest that, even if the CCOT diet does not seem to improve lameness (on kinetics and clinical rating) in our studied OA dogs, it could present some benefits on chronic pain and its impact on activities of daily living in OA dogs.

TITLE: Shear Bond Strength of Superficial, Intermediate and Deep Dentin ABSTRACT.BACKGROUND:: This in vitro study is intended to compare the shear bond strength of recent self-etching primers to superficial, intermediate, and deep dentin levels. ABSTRACT.MATERIALS AND METHODS:: All teeth were sectioned at various levels and grouped randomly into two experimental groups and two control groups having three subgroups. The experimental groups consisted of two different dentin bonding system. The positive control group consisted of All Bond 2 and the negative control group was without the bonding agent. Finally, the specimens were subjected to shear bond strength study under Instron machine. The maximum shear bond strengths were noted at the time of fracture. The results were statistically analyzed. ABSTRACT.RESULTS:: Comparing the shear bond strength values, All Bond 2 (Group III) demonstrated fairly higher bond strength values at different levels of dentin. Generally comparing All Bond 2 with the other two experimental groups revealed highly significant statistical results. ABSTRACT.CONCLUSION:: In the present investigation with the fourth generation, higher mean shear bond strength values were recorded compared with the self-etching primers. When intermediate dentin shear bond strength was compared with deep dentin shear bond strength statistically significant results were found with Clearfil Liner Bond 2V, All Bond 2 and the negative control. There was a statistically significant difference in shear bond strength values both with self-etching primers and control groups (fourth generation bonding system and without bonding system) at superficial, intermediate, and deep dentin. There was a significant fall in bond strength values as one reaches deeper levels of dentin from superficial to intermediate to deep. BODY.INTRODUCTION: Adhesive restorations have been widely accepted for both anterior and posterior use in restorative dentistry. Patient's demands for esthetic restorations have caused a recent increase in the use of tooth colored restorative materials. To achieve clinical success with such restorations, good adhesion between restorative materials and tooth substrates is of crucial importance in order to ensure good marginal sealing, reinforcement of the tooth structure, and longer life of the restoration. The adequate hybrid layer formation is believed to be essential to create a strong and durable bond between resin and dentin. It is very well understood that the density of dentinal tubules varies with dentinal depth and as well as the water content of dentin is lowest in superficial dentin and highest in deep dentin. This is probably because the amount of hypermineralized peritubular dentin increases at about the same rate as the amount of intertubular dentin decreases leaving average mineral content very similar, in superficial and deep dentin. The amount of collagen per unit volume of dentin decreases from superficial to deep dentin. This is probably due to the fact that deep dentin has larger tubules than superficial dentin and hence has less intertubular collagen than superficial dentin. The permeability of bonding substrates to monomers and the monomer diffusibility into the substrate are essential factors for the hybridization of resins in dental substrates. The permeability increases in an apical coronal direction and from superficial to deep dentin. While the permeability of inner radicular dentin is only 1:5 of the crown, this value descends to 1:20 in the outer radicular dentin. As of today no studies are available to indicate the effectiveness of new generation self-etching primers against superficial middle and deep dentin. The aim of this study was to test the hypothesis that there is a correlation between dentin depth permeability and bond strength of adhesive resins. BODY.MATERIALS AND METHODS: Forty-eight freshly extracted human molar teeth, non-carious and without having cracks were collected. All teeth were sectioned at various levels using a carborundum disc: Superficial dentin: Dentin within 0.5-1 mm of dentin-enamel junctionIntermediate dentin: Dentin 0.5-1 mm deeper to superficial dentinDeep dentin: Dentin within 0.5 mm of the pulp chamber The specimens were grouped randomly into two experimental groups having three sub groups consisting of four teeth each and two control groups having three subgroups consisting of four teeth each. The occlusal surfaces of teeth were ground on a water-cooled trimming wheel to prepare flat dentin surfaces (Figure 1). Figure 1Prepared Samples. Dentin bonding agents: Clearfil Liner Bond 2V (Kuraray) – light and self cure dental adhesive (Figure 2) Figure 2Clearfil Liner Bond 2V. Xeno III (Dentsply) – Self etching primer (Figure 3) Figure 3Xeno III. All Bond 2 (Bisco) – Dual cured universal dental adhesive (Figure 4) Figure 4All Bond 2. Filtek Z350 (3M) – Nanotechnology light cure composite (Figure 5) Figure 5Filtek Z350 Composite. Spectrum 800 (Dentsply) – Light curing device (Figure 6) Figure 6Spectrum 800 Light Cure Unit. Each group was subdivided into three subgroups of 4 teeth each: BODY.MATERIALS AND METHODS.EXPERIMENTAL GROUP: BODY.MATERIALS AND METHODS.CONTROL GROUP: BODY.MATERIALS AND METHODS.METHODOLOGY.GROUP I: Xeno III was applied to 12 specimens and left undisturbed for 20 s and the excess solvent was removed with a gentle stream of air. Light curing was done for 20 s using a visible light curing unit. After curing the bonding agent, nanocomposite resin was placed, and light cured layer by layer. BODY.MATERIALS AND METHODS.METHODOLOGY.GROUP II: Clearfil Liner Bond 2V was used, the primer liquid A and B were mixed and applied onto 12 samples. It was then dried with the mild air stream and left for 30 s, the primer was not washed. Bonding agent was applied, and light-cured for 20 s. After curing the bonding agent, nanocomposite resin was placed. BODY.MATERIALS AND METHODS.METHODOLOGY.GROUP III: All Bond 2 was applied to 12 samples; etchant was applied for 15 s and washed. It was then dried with mild air stream and primer was applied and left for 30 s. Then primer was not washed. Bonding agent was applied, and light-cured for 20 s. After curing the bonding agent, nanocomposite resin was placed and cured according to manufacturer's instructions. BODY.MATERIALS AND METHODS.METHODOLOGY.GROUP IV: No bonding agent was applied, and light-cured for 20 s. Nanocomposite resin was placed and cured according to manufacturer's instructions. All the 48 specimens were transferred to the Instron testing machine individually and subjected to shear bond strength study. BODY.RESULTS: According to the results analyzed: Comparing the shear bond strength values of Group I, II and Group III (positive control), All Bond 2 (Group III) demonstrated fairly higher bond strength values at different levels of dentin (Graph 1) (P < 0.05). Generally comparing All Bond 2 with the other two experimental groups revealed highly significant statistical results (Graph 1). Graph 1Comparison of shear bond strength. At the superficial level with the All Bond 2, we could record a mean shear bond strength value of 37.34 MPa while at the intermediate and deep dentin shear bond strength value reduced substantially to 26.86 and 20.86 MPa respectively (P = 0.007) (Tables 1 and 2). Table 1Shear bond strength in MPa among different levels of dentin. Table 2Shear bond strength of bonding agent. With the Clearfil Liner Bond 2V, we could record shear bond strength values at the superficial up to 31.79 MPa while at intermediate and deep dentin level we could record only up to 18.73 and 15.99 MPa respectively (P = 0.01).With Xeno III the other experimental group, a self-etching primer, shear bond strength values at the superficial dentin was 25.02 MPa and at intermediate and deep dentin reduced down to 21.72 and 15.17 MPa respectively (P = 0.038) (Table 1). BODY.DISCUSSION: Adhesion to acid-etched enamel was proposed by Buonocore in 1955. Long-lasting adhesion between enamel and resin has been proven by in vitro studies and clinical researches for over five decades. However, bonding to dentin is far more challenging compared to enamel bonding of composite resin, which has a long track record. The enhanced bonding between resin and dentin was established by dentin hybridization. Basically, the hybrid layer formation creates the composite structure of resin, collagen and hydroxyapatite or the functionally graded material between the resin and the dentin. Dentin is a dynamic tissue. It represents a challenge as regards resin based adhesives while the bond strength of enamel has been studied extensively, bonding to dentin with the generation of bonding systems has Tagami et al. (1990)1 attributed this either to differences in chemical composition or regional differences in wetness (dentin permeability). Thus there are several factors that may contribute to high coefficient of variation that is often reported in dentin shear bond strength studies. Pashley et al. (1993)2 studied the bond strengths to superficial, intermediate and deep dentin in vivo with four dentin bonding systems and demonstrated higher bond strengths in superficial dentin and progressively lower bond strengths in deep dentin. Tao and Pashley (1988)3 investigated shear bond strengths to dentin: effects of surface treatment, depth, and position. They demonstrated that there were significant differences in bond strengths at different depths of dentin after etching the surface but not if the smear layer was left undisturbed. Tagami et al. (1990)1 attempted to establish a relationship between dentin permeability and dentin depth and confirmed the results of the previous work on human dentin that permeability increases as dentin becomes thinner. This is possibly due to an increase in tubule diameter and increase in tubular dentin as dentin is thin toward the pulp chamber. The relationship between shear bond strengths of All Bond 2 system and self-etch primers against different levels of dentin depth provided interesting observations. There was a strong correlation between the All Bond 2 and self-etch primer group and dentin depth. Suzuki and Finger (1988)4 studied the efficacy of dentin bonding systems based on the site of dentin with reference to the observation of Causton that bond strengths to deep dentin were considerably lower than those to superficial dentin. This study has confirmed the observation of Causton that the efficacy of dentin adhesives depends on the dentin surface from superficial to deep dentin in the tooth tested. Different from etch and rinse adhesives, self-etch adhesives do not require a separate etching step as they contain acidic monomers that simultaneously condition and prime the dental substrate. Consequently, this approach has been claimed to be user-friendlier and less technique sensitive, thereby resulting in a reliable clinical performance though this appeared very product dependent. User friendlier because of shorter application time and less steps and less technique sensitive because no wet bonding but simple drying. Comparatively with the self-etch adhesives there is a lower incidence of post-operative sensitivity experienced by the patient. This should to a great extent be attributed to the less aggressive and thus more superficial interaction with the dentin leaving tubules largely obstructed with smear layer. Van Meerbeek et al. (2011)5 observed that at enamel, an etch and rinse approach using phosphoric acid remains the choice of preference since it not only guarantees the most durable bond to enamel but also seals and thus protects the more vulnerable bond to dentin against degradation. The most recent research investigated to which extent this compromised enamel bonding obtained with mild self-etch adhesives could be attributed to enamel during cavity preparation. The lower bonding effectiveness of self-etch adhesives to enamel should be ascribed most likely in the first place to less potential for micro-mechanical interlocking but also to a lower chemical reactivity. Triolo and Swift (1992)6 in their in vitro study tested the shear bond strengths of nine third generation dentin bonding systems and concluded that bond strengths are generally less to deep dentin than superficial and intermediate dentin. Pegado et al. (2010)7 compared the effect of different bonding strategies on adhesion to deep and superficial dentin and concluded that bond strength obtained in superficial dentin was significantly higher than that in deep dentin for all adhesives tested. They further concluded that the bond strengths of dentin bonding agents at any depth is dependent on the area occupied by resin tags, the area of intertubular dentin that is infiltrated by the resin and the area of surface adhesion. Van Meerbeek et al. (2011)5 recommended that for further optimization of the self-etch approach, synthesis of functional monomers tailored to exhibit good chemical bonding potential following a mild self-etch approach. The approach appears to guarantee the most durable bonding performance at dentin provided that it deals adequately with the debris smeared across the surface by the bur. Micromechanical interlocking is still the best strategy to bond to enamel. Selective phosphoric acid etching of enamel cavity margins is therefore today highly recommended followed by applying a self-etch procedure to both the earlier etched enamel and un-etched dentin. Such mild self-etch adhesives should contain functional monomers with a high chemical affinity to hydroxyapatite. BODY.CONCLUSION: In vitro investigation was conducted to determine the bond strength of superficial, intermediate and deep dentin with self-etching primers (Clearfil Liner Bond 2V and Xeno III) and fourth generation bonding system (All Bond 2) and single composite resin. The final conclusions were drawn: In the present investigation with the fourth generation (All Bond 2), higher mean shear bond strength values were recorded compared with the self-etching primers (Clearfil Liner Bond 2V and Xeno III).When intermediate dentin shear bond strength was compared with deep dentin shear bond strength, statistically significant results were found with Clearfil Liner Bond 2V, All Bond 2 and the negative control.There was a statistically significant difference in shear bond strength values both with self-etching primers and control groups (fourth generation bonding system and without bonding system) at superficial, intermediate and deep dentin.There was a significant fall in bond strength values as one reaches deeper levels of dentin from superficial to intermediate to deep.

TITLE: Intravitreal ziv-aflibercept for macular edema following retinal vein occlusion ABSTRACT.AIM: To report the efficacy of intravitreal ziv-aflibercept injections in eyes with macular edema due to retinal vein occlusions (RVOs). ABSTRACT.METHODS: Consecutive patients with persistent or recurrent macular edema (central macula thickness >250 μm) due to RVO were enrolled in this prospective study. Study eyes received intravitreal injections of ziv-aflibercept (1.25 mg/0.05 mL) at baseline. Patients were reassessed monthly for 4 months and given additional injections pro re nata for worsening best-corrected visual acuity (BCVA), intraretinal edema or subretinal fluid seen on spectral domain optical coherence tomography, or central macular thickness (CMT) measurements >250 μm. The primary endpoint was improvement in mean CMT at 4 months. Secondary endpoints included improvement in mean BCVA, and ocular and systemic safety signals. ABSTRACT.RESULTS: Nine eyes (five central and four branch RVOs) of nine patients were enrolled. The mean ± standard deviation CMT decreased from 604±199 μm at baseline to 319±115 μm (P=0.001) at 1 month and to 351±205 μm (P=0.026) at 4 months. The mean BCVA did not improve significantly from baseline (1.00 LogMAR) to the 1-month (0.74 LogMAR; P=0.2) and 4-month (0.71 LogMAR; P=0.13) visits. No safety signals were noted. ABSTRACT.CONCLUSION: In this small prospective study, intravitreal ziv-aflibercept significantly improved mean CMT in eyes with persistent or recurrent macular edema due to RVOs. Prospective, randomized trials comparing ziv-aflibercept with standard pharmacotherapy are needed to better define efficacy and safety. BODY.INTRODUCTION: Macular edema due to retinal vein occlusions (RVOs), the second most common retinal vascular condition, frequently results in significant loss of best-corrected visual acuity (BCVA). Intravitreal pharmacotherapy with corticosteroids and drugs that bind vascular endothelial growth factor (VEGF) usually resolves edema and improves BCVA better than observation in eyes with central retinal vein occlusion (CRVO)1–4 and better than laser photocoagulation in eyes with branch retinal vein occlusion (BRVO).5,6 Although most of these eyes respond favorably to first-line intravitreal pharmacotherapy, improvements are usually transient7,8 and some eyes experience no improvement with available therapy. Achieving and maintaining a satisfactory clinical response usually becomes an expensive, long-term process that often challenges patient compliance. Aflibercept (Eylea®, Regeneron, Tarrytown, NY, USA) is the newest anti-VEGF drug that has been approved for the treatment of macular edema due to RVO. This recombinant fusion protein, composed of extra-cytoplasmic, native-receptor VEGF-binding sequences from VEGF receptor (VEGFR) 1 and VEGFR2,9 binds VEGF165 100 times tighter than either bevacizumab or ranibizumab, and also binds isoforms of VEGF-B and placental growth factor.10 The VIBRANT trial demonstrated that aflibercept was superior to laser photocoagulation in eyes with BRVO,6 and the COPERNICUS and GALILEO trials showed that aflibercept was superior to sham in eyes with CRVO.3,4 Aflibercept's strong VEGF-binding affinity may provide a therapeutic advantage over other VEGF-binding drugs when treating vascular conditions with high intravitreal VEGF concentrations such as diabetic macular edema (DME), proliferative diabetic retinopathy, and RVOs, but very few randomized, multicenter, double-masked trials have directly compared aflibercept with bevacizumab and ranibizumab. DRCR.net Protocol T showed that for eyes with DME, aflibercept produced superior 1-year improvements in BCVA compared to bevacizumab and ranibizumab,11 though statistical superiority was not maintained through 2 years.12 The ongoing SCORE-2 trial compares aflibercept with bevacizumab for primary treatment of macular edema due to RVOs, with aflibercept also serving as rescue therapy for eyes that fail to respond favorably to bevacizumab (https://clinicaltrials.gov/ct2/show/NCT01969708). Aflibercept use in developing countries is limited due to its high cost and narrow regulatory approvals. Our group recently reported the short-term safety of intravitreal ziv-aflibercept (Zaltrap®; Regeneron, Tarrytown, NY, USA), which has the identical molecular structure as aflibercept, in eyes with neovascular age-related macular degeneration (nAMD).13–15 The purpose of the study described in this manuscript was to evaluate the 4-month safety and efficacy of intravitreal ziv-aflibercept in eyes with RVO that had previously received intravitreal pharmacotherapy. BODY.METHODS: This two-center, open-label, nonrandomized, single-arm, prospective study enrolled patients between April 2015 and October 2016. Institutional Review Board approval was granted at LV Prasad Eye Institute, Hyderabad, India and Rafic Hariri University Hospital, Beirut, Lebanon and the study adhered to the tenets of the Declaration of Helsinki. Written informed consent to participate in the study was given by each patient after learning about the study methodology and the possible risks and benefits. The clinical trial registry number is NCT02486484. BODY.METHODS.INCLUSION AND EXCLUSION CRITERIA: Key inclusion criteria included previously treated (intravitreal bevacizumab, ranibizumab, dexamethasone insert, or laser photocoagulation) macular edema due to RVO that either recurred since the last treatment or never satisfactorily resolved after at least 3 months of therapy. A central macular thickness (CMT) of at least 250 μm on spectral-domain optical coherence tomography (SD-OCT) was required for enrollment. Exclusion criteria included uncontrolled systemic vascular disease (including systemic arterial hypertension and diabetes mellitus), glaucoma, vitreomacular interface disorders in the study eye that could limit resolution of edema and improvement in BCVA, and previous vitrectomy. BODY.METHODS.CLINICAL EVALUATIONS AND TREATMENTS: Clinical examinations included determination of best- corrected Snellen visual acuity, intraocular pressure measurement with applanation tonometry, slit-lamp examination of the anterior segment, biomicroscopic and indirect ophthalmoscopic evaluation of the fundus, and SD-OCT scanning of the central macula. CMT was measured with the 3D OCT-2000 FA plus Topcon® (Topcon, Tokyo, Japan) in Lebanon and with the Cirrus® (Carl Zeiss Meditec, Dublin, CA, USA) in India. All CMT measurements taken with the Topcon scanner were converted to Cirrus measurements by adding 23.6 μm, consistent with the normalization proposed by Lammer et al.16 Intravenous fluorescein angiography was performed at baseline. Ziv-aflibercept was prepared and injected according to standardized protocols.13 After instillation of topical anesthesia and povidone–iodine solution onto the conjunctiva, a sterile eyelid retractor was positioned. Ziv-aflibercept (1.25 mg/0.05 mL) was injected through the sclera (3.5–4 mm posterior to the limbus) into the mid-vitreous. All study eyes received ziv-aflibercept injections at baseline. Patients received comprehensive eye examinations and SD-OCT macula scans monthly for 4 months. Additional injections of ziv–aflibercept were performed on a pro re nata basis if the BCVA worsened, intraretinal edema or subretinal fluid was noted, or the CMT measured >250 μm. BODY.METHODS.OUTCOMES: The primary outcome measure was the change in mean CMT from baseline to month 4. Secondary outcomes included any change in the mean BCVA at 4 months and ocular and systemic safety signals. BODY.METHODS.STATISTICAL ANALYSES: Statistical analyses were performed using SPSS version 22 (IBM Corporation, Chicago, IL, USA). Snellen BCVA values were converted into LogMAR for statistical analysis. Changes in CMT and BCVA were assessed with the Wilcoxon-signed rank test and P-values of <0.05 were deemed statistically significant. BODY.RESULTS.BASELINE CHARACTERISTICS: Nine eyes (five eyes with CRVO and four eyes with BRVO) of nine patients were enrolled in this study. Baseline characteristics of the patients and study eyes are listed in Table 1. The mean ± standard deviation age of patients in the cohort was 64±8 years and eight of the nine (89%) patients were males. Diabetes mellitus and systemic arterial hypertension were each found in six patients (67%), coronary artery disease in two (22%), and hyperhomocysteinemia in one (11%). None of the patients had features of diabetic retinopathy in the non-study eyes. Prior to enrollment in this study, the patients had fluorescein angiography and had previously been treated with laser photocoagulation and intravitreal injections of bevacizumab, ranibizumab, triamcinolone, or dexamethasone implant (Table 1). The median number of prior intravitreal injections was 5 (range: 2–24) and the mean duration since the last injection was 11±20 months (range: 1–63 months). BODY.RESULTS.STUDY TREATMENTS: The mean number of ziv-aflibercept injections administered during the 4-month study period was 2.3±0.9 (range: 1–4). The mean treatment-free interval after switching to ziv-aflibercept was 1.9±0.2 months (range: 1–2 months). Figure 1 shows the treatment response of a representative case. BODY.RESULTS.VISUAL ACUITY: The mean BCVA at study entry was 1.0 LogMAR (Snellen equivalent: 20/200) with a range of 0.4 to 1.7 LogMAR (Snellen equivalent: 20/50 to 20/1,200). At 1 month, the mean BCVA improved to 0.74 LogMAR (Snellen equivalent: 20/120; P=0.2) and at 4 months to 0.71 LogMAR (Snellen equivalent: 20/100; P=0.13). Visual acuity improved in most eyes (eight of nine) at 4 months (median improvement from 20/160 to 20/80), with greater changes seen in the eyes that were last treated within 3 months (improvement in median BCVA from 20/180 to 20/90) compared to those last treated >12 months previously (median improvement from 20/100 to 20/80). BODY.RESULTS.MACULAR THICKNESS: The mean CMT at baseline (603±199 μm) decreased to 319±115 μm (P=0.001) at 1 month and to 351±205 μm (P=0.026) at 4 months. Significant improvements in CMT occurred both in eyes that had received intravitreal pharmacotherapy only 1 to 3 months previously (mean improvement in CMT from 587 μm to 370 μm) and also in eyes that had last been treated over 1 year prior (mean improvement in CMT from 637 to 361 μm). Changes in BCVA and CMT throughout the study period are shown in Figure 2. BODY.RESULTS.SAFETY: One eye (Case 9) developed minimal anterior chamber flare 2 days after the fourth injection. Cases 1–8 did not develop intraocular inflammation. None of the patients experienced systemic adverse events that could be attributed to intravitreal ziv-aflibercept. The mean intraocular pressure did not change from baseline (13.2±3.3 mmHg) to month 4 (13.4±2.3 mmHg). BODY.DISCUSSION: In this study of patients with previously treated macular edema due to RVOs, we found that switching to ziv-aflibercept resulted in significant improvements in macular edema and modest improvements in BCVA, most likely due to the chronicity of the disease at presentation. The Phase III registration trials demonstrated that intravitreal ranibizumab and aflibercept are superior to standard-of-care for the treatment of macular edema due to BRVO and CRVO.2–6 Bevacizumab has not been studied with the same rigor as ranibizumab and aflibercept but bevacizumab regimens appear to produce comparable improvements in macular edema and BCVA.17,18 Most eyes with RVO respond well to bevacizumab or ranibizumab but a small proportion fail to achieve satisfactory responses with initial therapy or experience an unacceptably early or frequent recurrence of edema. Small retrospective studies reported visual gains after switching to aflibercept but since standardized procedures and control arms were lacking, the significance of these findings is unclear.19,20 Nonetheless, switching incomplete responders to aflibercept appears to be a reasonable treatment option. Surgeons in many developing countries cannot use aflibercept to treat RVOs because it may be excluded from the formularies of insurance plans or national health care plans and its high cost ($1,850 US per dose) prevents many patients from paying out-of-pocket. In countries such as India, aflibercept has been approved for the treatment of nAMD but not yet for RVOs. Just as off-label bevacizumab became an accepted, affordable treatment for chorioretinal vascular disorders, ziv-aflibercept could become a similar option in countries with limited access to aflibercept. Ziv-aflibercept was approved by the US Food and Drug Administration in August 2012 for the intravenous treatment of advanced colorectal carcinoma.21 Ziv-aflibercept is packaged in a single use 4 mL vial (1.25 mg/0.05 mL) and costs ($512 US) substantially less than aflibercept and even slightly less than bevacizumab (4 mL vial costs $660). Buffering solutions in ziv-aflibercept produce a high osmolality (1,000 mOsm/kg) that is toxic to most cells.22 Concerns regarding hyperosmolality-induced retinal toxicity due to intravitreal ziv-aflibercept have been largely dispelled by recent publications.23 Case reports describing ziv-aflibercept treatment of nAMD, polypoidal choroidal vasculopathy, and CRVO identified no safety concerns.13,14,24 Mansour et al noted no ocular toxicity with intravitreal ziv-aflibercept in patients with DME and nAMD, and they posited that the small volume of high osmolality ziv-aflibercept (0.05 mL) is adequately diluted by the relatively large vitreous cavity (4 mL).14 Chhablani et al noted no clinical toxicity and detected no electroretinographic abnormalities in 12 eyes that received single injections of ziv-aflibercept for treatment-resistant nAMD.13 Most eyes in our study, whether last treated within the previous 3 months or more than a year prior, experienced decrease in macular edema. Visual acuity improvements favored eyes treated within the past 3 months but our small cohort is insufficiently powered to draw firm conclusions. Larger RVO trials suggest that even brief periods of under-treatment (as short as 6 months) may result in poorer long-term visual outcomes.2,4 Nonetheless, our results support the notion that RVO patients should be followed closely so that recurrent edema can be treated promptly. The optimal dose of intravitreal ziv-aflibercept is not known. We injected 0.05 mL (1.25 mg) of undiluted ziv-aflibercept so that the small volume would minimize acute elevations in intraocular pressure. The approved dose of aflibercept (2 mg/0.05 mL) delivers 60% more drug than was used in our trial but smaller doses of aflibercept have not been reported in eyes with RVOs. Patients treated with 0.5 mg aflibercept in the VIEW trials achieved comparable results to those receiving the 2 mg dose, suggesting that dose size may not be a critical determinant of clinical response.25 Dose range determinations with ziv-aflibercept continue as a higher dose (2 mg/0.08 mL) is being evaluated. Our study has several strengths and weaknesses. To the best of our knowledge, this is the first prospective study to evaluate the safety and efficacy of ziv-aflibercept for macular edema due to RVOs. The study reports data gathered from more than one site but the small cohort prevents us from detecting statistically significant visual acuity changes. Because of the small cohort, we elected to analyze eyes with central RVO and branch RVO together, even though these conditions often have different baseline characteristics and responses to treatment. The mixed composition of the cohort – patients who were actively being treated for RVO and others whose last injections were at least 1 year prior – makes it difficult to use the data to create firm indications for switching patients to ziv-aflibercept. The 4-month primary endpoint is brief and longer follow-up periods are needed. Despite the shortcomings of this pilot study, we believe that these data support the use of ziv-aflibercept in selected patients with RVOs. This study adds to the growing body of data supporting the safety of intravitreal ziv-aflibercept in eyes with chorioretinal vascular conditions. Whether intravitreal ziv-aflibercept would be an acceptable option in countries where aflibercept has already been approved for the treatment of RVOs is unclear, but in lower income countries that have not approved aflibercept, ziv-aflibercept could become a cost-effective alternative to other anti-VEGF drugs. Larger, multicenter, prospective, controlled studies are needed to better define the efficacy of ziv-aflibercept in patients with macular edema due to RVOs.

TITLE: A 12-Week Commercial Web-Based Weight-Loss Program for Overweight and Obese Adults: Randomized Controlled Trial Comparing Basic Versus Enhanced Features ABSTRACT.BACKGROUND: The development and use of Web-based programs for weight loss is increasing rapidly, yet they have rarely been evaluated using randomized controlled trials (RCTs). Interestingly, most people who attempt weight loss use commercially available programs, yet it is very uncommon for commercial programs to be evaluated independently or rigorously. ABSTRACT.OBJECTIVE: To compare the efficacy of a standard commercial Web-based weight-loss program (basic) versus an enhanced version of this Web program that provided additional personalized e-feedback and contact from the provider (enhanced) versus a wait-list control group (control) on weight outcomes in overweight and obese adults. ABSTRACT.METHODS: This purely Web-based trial using a closed online user group was an assessor-blinded RCT with participants randomly allocated to the basic or enhanced 12-week Web-based program, based on social cognitive theory, or the control, with body mass index (BMI) as the primary outcome. ABSTRACT.RESULTS: We enrolled 309 adults (129/309, 41.8% male, BMI mean 32.3, SD 4 kg/m2) with 84.1% (260/309) retention at 12 weeks. Intention-to-treat analysis showed that both intervention groups reduced their BMI compared with the controls (basic: –0.72, SD 1.1 kg/m2, enhanced: –1.0, SD 1.4, control: 0.15, SD 0.82; P < .001) and lost significant weight (basic: –2.1, SD 3.3 kg, enhanced: –3.0, SD 4.1, control: 0.4, SD 2.3; P < .001) with changes in waist circumference (basic: –2.0, SD 3.5 cm, enhanced: –3.2, SD 4.7, control: 0.5, SD 3.0; P < .001) and waist-to-height ratio (basic: –0.01, SD 0.02, enhanced: –0.02, SD 0.03, control: 0.0, SD 0.02; P < .001), but no differences were observed between the basic and enhanced groups. The addition of personalized e-feedback and contact provided limited additional benefits compared with the basic program. ABSTRACT.CONCLUSIONS: A commercial Web-based weight-loss program can be efficacious across a range of weight-related outcomes and lifestyle behaviors and achieve clinically important weight loss. Although the provision of additional personalized feedback did not facilitate greater weight loss after 12 weeks, the impact of superior participant retention on longer-term outcomes requires further study. Further research is required to determine the optimal mix of program features that lead to the biggest treatment impact over time. ABSTRACT.TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR): 12610000197033; http://www.anzctr.org.au/trial_view.aspx?id=335159 (Archived by WebCite at http://www.webcitation.org/66Wq0Yb7U) BODY.INTRODUCTION: The prevalence of overweight and obesity among adults is increasing worldwide [1]. Therefore, effective treatment programs with a large reach are required. Web-based weight-loss programs have emerged in response to the exponential growth in Internet access, as well as increasing use of the Internet as part of daily life and improved accessibility. In the United States, 66% of households have access to broadband Internet [2], while 72% of Australian households have access to the Internet [3]. Furthermore, many adults (61% in the United States) use the Internet to access information about health, nutrition, physical activity, and weight loss [4]. The multimedia capabilities of the Internet allow up-to-date, interactive, and individualized lifestyle programs to be provided, which endeavor to emulate traditional face-to-face consultations [5]. These programs also overcome several barriers of attending face-to-face consultations, such as poor accessibility [6], lack of anonymity [7], and participant burden associated with attendance. However, Web-based weight-loss programs are an underevaluated treatment medium. A recent systematic review and meta-analysis examined the efficacy of 12 randomized controlled trials (RCTs) of Web-based weight-loss programs [8]. The results suggest that, in general, participants in these programs achieve similar weight-loss outcomes to control or minimal intervention groups. In addition, it has been suggested that Web-based programs with enhanced features (eg, tailored information and counseling) achieve greater weight loss than those that focus on information alone, although these studies are highly heterogeneous [8]. Further studies are required, as it has not yet been possible to establish the overall efficacy of Web-based interventions or the superiority of those with more enhanced features, due to the heterogeneity of study designs and therefore the small number of comparable studies. Of the small number of Web-based programs that have been evaluated using an RCT, remarkably few are available to the public. Commercial Web-based weight-loss programs are likely to be the most accessible to consumers [9] but have rarely been independently evaluated [10]. Of the two RCTs of eDiets, a commercial Web-based weight-loss program in the United States, one found that after 12 months those using eDiets lost significantly less weight than those following a self-help manual (–1.1% vs –4.0%) [11], while the second compared eDiets with a structured behavioral Web-based program [12] and found the behavioral program achieved significantly greater weight loss after 12 months (–2.8% vs –5.5%). Overall, limited evidence exists for the efficacy of commercial Web-based programs as a viable obesity treatment option. Therefore, examining the efficacy of commercially available Web-based weight-loss programs on weight-related outcomes is warranted to increase the treatment options for people seeking to engage in commercial treatment programs, especially those who have limited options in their region. The primary aim of this study was to determine whether there was a significant difference in body mass index (BMI) posttreatment among participants randomly assigned to a standard (basic) 12-week commercial Web-based weight-loss program versus a version of this program with additional online features and personalized e-feedback and reminder calls (enhanced) versus a wait-list control (control). We hypothesized that the reduction in BMI would be greater in the basic and enhanced groups than in the control group, with the BMI reduction greater in the enhanced than in the basic group. BODY.METHODS: The present study was an assessor-blinded RCT with a 12-week follow-up. The methods have been published in detail [13]. Briefly, overweight and obese adults were recruited offline and enrolled by research assistants at the University of Newcastle from October to December 2009 from the Hunter community in New South Wales, Australia. Eligibility criteria were age 18 to 60 years, BMI 25 to 40 kg/m2, not participating in other weight-loss programs, passing a health screen [14], being available for in-person assessments, and having access to a computer with email and Internet services, although neither computer nor eHealth literacy was assessed. Exclusion criteria were pregnancy or trying to conceive, major medical problems, orthopedic problems, recent weight loss of ≥4.5 kg, and medications affecting or affected by weight loss. Trial sample size calculations have been previously published [13]. BODY.METHODS.STRATIFICATION AND RANDOMIZATION: Once written consent was obtained and baseline assessments completed, participants were stratified by sex and BMI category (25 to <30; ≥30 to <35 or ≥35 to 40 kg/m2). They were randomly allocated to one of the three groups between October and December 2009 (Figure 1) using a stratified randomized block design with variable blocks length (either 3 or 6) generated by the statistician. A researcher not involved in data collection distributed sequentially numbered sealed envelopes with allocation details and a login code. Participants were given an instruction sheet and the Web address and asked to set up their own login. We also gave them a toll-free number to call if they experienced any difficulties in logging in. No training on program use was provided to participants in order to mirror the program engagement that commercial users would experience and to increase external validity. The groups were (1) control: a wait-list control group who were not provided with access to the weight-loss program website and were asked to refrain from participating in other weight-loss programs for 12 weeks, (2) basic: who were provided with free access to the basic (standard) Web-based program that was commercially available at that time and did not change, and (3) enhanced: who were provided with free access to an enhanced version of the Web-based program that was provided in a closed test environment. Both basic and enhanced group participants were advised to use the online diary a minimum of 4 times per week to record their dietary intake and physical activity, and to enter a weekly weight. Participants were blinded to allocation for the basic and enhanced groups, but not to the control. Research assistants were blinded to allocation for all groups. At each time point all were reminded to not discuss group allocation at assessments. Figure 1Flow of participants through the three groups (control, basic, enhanced) of a web-based weight-loss randomized controlled trial. BODY.METHODS.STRATIFICATION AND RANDOMIZATION.WEB-BASED WEIGHT-LOSS PROGRAMS: The Web-based program was underpinned by social cognitive theory [15] and targeted key mediators of behavior change, including self-efficacy, goal setting, and self-monitoring of weight, body measurements, exercise, and diet; outcome expectations (knowledge-based Web components); modeling (interactive website features and demonstrations); and social support (forums, blogs, feedback, email, and telephone contact). The interventions were Web based and delivered for 12 weeks, with new program content provided weekly by SP Health Co Pty Ltd, a commercial Web-based weight-loss program provider in Australia, under the name The Biggest Loser Club. Participation was in a quasi-anonymous manner. The reach [16], retention [17], and weight loss achieved by real-world participants [18] in the basic program have been previously evaluated. The basic program had the following features: individualized daily calorie targets to facilitate 0.5–1 kg of weight loss per week; goal-setting options; Web-based food and exercise diary; weekly calorie-controlled, low-fat menu plans and grocery list; weekly physical activity plan based on exercise preferences; weekly educational tips and challenges; Web-based community forums; daily and weekly calculations of energy balance and nutrition summary compared with recommended nutrient targets; weekly email newsletters with alerts to new relevant content; self-monitoring of weight, and waist and hip girths; graphical display of changes in body measurement data and body (BMI) silhouette; and automated weekly reminders to enter weight. The enhanced program included access to the Web-based program described above plus (1) personalized, system-generated enrollment reports that suggested appropriate weight-loss goals and key behavior changes required for success, (2) weekly automated, system-generated, personalized e-feedback for key elements of diet and physical activity based on diary entries, usage patterns for website features, and level of success with weight loss, and (3) an escalating reminder schedule to use the diary, visit the program site, and enter a weekly weight, which was as an initial reminder email, then a short message service text message if they did not respond, then a courtesy reminder phone call if a weekly weight was still not entered. Participants accessed the website using their usual Internet connection, at any time of the day or night that suited them. BODY.METHODS.STRATIFICATION AND RANDOMIZATION.OUTCOME MEASURES: Participant assessments were conducted at baseline and 12 weeks in the Human Performance Laboratory at the University of Newcastle, Callaghan campus [13]. Assessors of the main outcome measures, including those performing anthropometric and blood pressure measurements and blood collection, were blinded to participant group allocation at baseline and 12 weeks. We asked participants not to inform assessors of their group allocation. The primary outcome, BMI, was calculated as weight (in kilograms) divided by height (in meters) squared. Height was measured to 0.1 cm using the stretch stature method on a Harpenden portable stadiometer (Holtain Limited, Croswell, Crymych, Pembrokeshire, UK). Weight was measured in light clothing, without shoes, on a digital scale to 0.01 kg (CH-150kp; A&D Mercury Pty Ltd, Adelaide, Australia). Waist circumference was measured to 0.1 cm using a nonextensible steel tape (KDSF10-02; KDS Corporation, Osaka, Japan) at two points: (1) level with the umbilicus, and (2) at the narrowest point between the lower costal border and the umbilicus. Systolic and diastolic blood pressure and heart rate were measured using an automated blood pressure monitor (NISSEI/DS-105E digital electronic blood pressure monitor; Nihon Seimitsu Sokki Co Ltd, Gunma, Japan) under standardized conditions. Blood samples were collected after an overnight fast and analyzed for lipids (total cholesterol, low-density lipoprotein and high-density lipoprotein cholesterol, and triglycerides), glucose, and insulin using standard automated techniques at a single National Association of Testing Authorities-accredited pathology service. Dietary intake was assessed using the Australian Child and Adolescent Eating Survey, a 135-item semiquantitative food frequency questionnaire used previously in Australian youth [19] and currently being validated in adults. We asked participants to self-report frequency of consumption over the previous 6 months using the paper-based tool at baseline and over the previous 12 weeks at the follow-up assessment. Frequency options ranged from never up to ≥4 times per day. Completed food frequency questionnaires were scanned and nutrient intakes computed using FoodWorks (version 3.02.581; Xyris Software, Highgate Hill, Australia) using the Australian AusNut 1999 database (All Foods) revision 14 and AusFoods (Brands) revision 5 (Food Standards Australia New Zealand, Canberra, Australia) to generate individual mean daily nutrient intakes. We used the paper-based Three-Factor Eating Questionnaire-R18 (TFEQ-R18) to measure cognitive restraint, uncontrolled eating, and emotional eating [20]. Quality of life was assessed using the paper-based SF-36, version 2.0 (QualityMetric Incorporated, Lincoln, RI, USA), a multipurpose, generic, short-form health survey consisting of an 8-scale profile of functional health and well-being scores and psychometrically based physical and mental health summary measures [21]. We used the International Physical Activity Questionnaire (short form) paper-based questionnaire to estimate total metabolic equivalents (METs) in minutes/week [22]. Pedometers were used to objectively measure steps per day for 7 consecutive days (Yamax Digi-Walker SW-700; Yamasa Tokei Keiki Co Ltd, Tokyo, Japan). The step counts were adjusted for additional physical activity self-reported by participants when the pedometers were not worn (eg, contact sports and swimming) or problematic (eg, cycling). We added 1000 steps for every 10 minutes of moderate activity and 2000 steps for every 10 minutes of vigorous activity. BODY.METHODS.ETHICS: The procedures followed were in accordance with the ethics standards of the University of Newcastle Human Ethics Research Committee. We obtained written informed consent from all participants. Institutional affiliations were displayed on paper-based information and consent forms, but not on the Web-based program materials. BODY.METHODS.STATISTICAL ANALYSIS: Analysis of covariance was used to test for differences in weight loss between groups at 3 months after adjusting for baseline values. Analysis was on an intention-to-treat basis with participants who did not use the application included in the analysis, while baseline observation carried forward was used those lost to follow-up. The model was fitted using linear regression with BMI at 12weeks as the outcome variable, treatment group as the predictor variable of interest, and BMI at baseline included as a covariate. We based statistical significance of the primary efficacy analysis on Hochberg multiple testing procedures with the familywise error rate held at 2.5% because there will be an additional analysis at 18 months. All secondary hypothesis tests were performed using a 2-sided 5% significance level. BODY.RESULTS.BASELINE CHARACTERISTICS: Of the 591 people assessed for eligibility, 309 (129 men) were randomly allocated into the trial (Figure 1). Baseline characteristics did not differ between groups for any variable, other than general health scale (Table 1). Most of the participants were overweight (108, 35.0%) or in obese category 1 (122, 39.5%), most (280, 90.6%) were Australian born, and few (33, 11%) had ever smoked. Table 1 Baseline characteristics of participants in a 12-week Web-based weight-loss (WL) program by intervention group. Characteristic Treatment group P value a Control (n = 104) Basic WL (n = 99) Enhanced WL (n = 106) Total (N = 309) Sex, n(%) Men 44 (42%) 41 (41%) 44 (42%) 129 (42%) .99 Women 60 (58%) 58 (59%) 62 (58%) 180 (58%) BMI group strata (kg/m 2 ), n (%) 25 to <30 36 (35%) 34 (34%) 38 (36%) 108 (35.0%) .99 30 to <35 42 (40%) 39 (39%) 41 (39%) 122 (39.5%) 35 to <40 26 (25%) 26 (26%) 27 (25%) 79 (26%) Current or previous smoker, n (%) Never smoker 93 (91%) 85 (87%) 95 (90%) 273 (89%) .59 Current/former smoker 9 (9%) 13 (13%) 11 (10%) 33 (11%) Highest level of education, n (%) School 27 (26%) 32 (32%) 31 (29%) 90 (29%) .78 Trade/diploma 37 (36%) 31 (31%) 43 (41%) 111 (36%) University degree 26 (25%) 23 (23%) 19 (18%) 68 (22%) Higher university degree 13 (13%) 13 (13%) 13 (12%) 39 (13%) Weekly household income (A $), n (%) <$700 10 (10%) 9 (10%) 6 (6%) 25 (8.6%) .72 $700 to <$1000 6 (6%) 3 (3%) 7 (7%) 16 (6%) $1000 to <$1400 12 (12%) 15 (16%) 9 (9%) 36 (12%) $1500 or more 68 (69%) 62 (67%) 75 (75%) 205 (70%) Don’t know/no answer 3 (3%) 4 (4%) 3 (3%) 10 (3%) Country of birth, n (%) Australia 92 (89%) 90 (91%) 98 (92%) 280 (90.6%) .73 Other 11 (11%) 9 (9%) 8 (8%) 28 (9%) Age (years), mean (SD) 41.7 (9.4) 42.0 (10.9) 42.2 (10.2) 42.0 (102) .94 Physical measurements, mean (SD) Height (cm) 1.7 (0.1) 1.7 (0.1) 1.7 (0.1) 1.7 (0.1) .65 Weight (kg) 93.6 (13.9) 94.9 (15.4) 93.4 (14.6) 94.0 (14.6) .75 Body mass index (kg/m 2 ) 32.2 (3.9) 32.3 (3.6) 32.3 (4.3) 32.3 (4.0) .98 Waist circumference (umbilicus) (cm) 107.2 (10.4) 106.9 (9.8) 106.6 (12.5) 106.9 (10.9) .92 Waist circumference (narrowest) (cm) 98.2 (11.4) 98.6 (11.5) 97.7 (11.7) 98.2 (11.5) .86 Waist (umbilicus) to height ratio 0.63 (0.07) 0.63 (0.06) 0.63 (0.08) 0.63 (0.07) .91 Waist (narrowest) to height ratio 0.58 (0.06) 0.58 (0.06) 0.57 (0.06) 0.58 (0.06) .98 Physiological measurements, mean (SD) Systolic blood pressure (mmHg) 122 (16) 121 (13) 121 (11) 122 (13) .81 Diastolic blood pressure (mmHg) 79 (10) 80 (11) 79 (10) 79 (10) .75 Pulse rate (beats/minute) 70 (10) 68 (9) 68 (10) 69 (10) .55 Blood tests, mean (SD) Total serum cholesterol (mmol/L) 5.1 (0.8) 5.2 (1.0) 5.0 (1.1) 5.1 (1.0) .64 LDL b cholesterol (mmol/L) 3.0 (0.6) 3.1 (0.8) 3.0 (0.9) 3.0 (0.8) .38 HDL c cholesterol (mmol/L) 1.3 (0.3) 1.3 (0.3) 1.3 (0.3) 1.3 (0.3) .85 Triglycerides (mmol/L) 1.8 (1.9) 1.6 (0.8) 1.8 (1.2) 1.7 (1.4) .54 LDL to HDL ratio 2.40 (0.79) 2.54 (0.80) 2.38 (0.79) 2.44 (0.79) .35 Glucose (mmol/L) 5.0 (1.4) 4.8 (0.6) 4.8 (0.6) 4.8 (0.9) .20 Insulin (mIU/L) 10.5 (7.5) 11.8 (13.1) 11.2 (13.1) 11.1 (11.5) .73 SF-36 scores, mean (SD) Physical functioning 85.4 (15.2) 86.1 (14.2) 82.5 (19.8) 84.6 (16.7) .27 Role physical 87.1 (19.9) 88.8 (16.8) 86.3 (19.5) 87.4 (18.8) .63 Bodily pain 60.9 (28.4) 61.4 (27.3) 61.2 (29.8) 61.2 (28.4) .99 General health 63.9 (18.5) 69.8 (16.8) 72.8 (18.8) 68.7 (18.5) .004 Vitality 78.1 (70.6) 69.3 (14.8) 81.4 (45.3) 76.8 (51.4) .32 Social functioning 81.4 (23.8) 84.5 (20.6) 85.0 (21.7) 83.5 (22.2) .51 Role emotional 86.5 (17.4) 89.9 (21.7) 89.4 (15.9) 88.5 (18.2) .41 Mental health 74.0 (17.3) 78.9 (16.5) 79.2 (14.8) 77.2 (16.4) .06 Physical activity, mean (SD) Total (MET d minutes/week) 2724 (2732) 3024 (3029) 2846 (3127) 2863 (2964) .80 Step count/day 7971 (3511) 8664 (3773) 8680 (3752) 8427 (3677) .34 Three-Factor Eating Questionnaire-R18 scores, mean (SD) Cognitive restraint scale 13.0 (3.0) 13.3 (2.9) 13.1 (2.8) 13.2 (2.9) .70 Uncontrolled eating scale 20.9 (5.1) 20.9 (4.7) 20.8 (4.8) 20.9 (4.8) .97 Emotional eating scale 7.7 (2.5) 7.6 (2.2) 7.8 (2.6) 7.7 (2.4) .86 Total energy intake (kJ/day), mean (SD) 10,311 (3229) 9958 (3223) 10,250 (3257) 10,175 (3229) .71 a P values are from analysis of variance for continuous measures and from a chi-square test for categorical measures. b Low-density lipoprotein. c High-density lipoprotein. d Metabolic equivalent. BODY.RESULTS.RETENTION AT 12 WEEKS: The percentage of participants who attended the 12-week follow-up assessment was significantly different between treatment groups (P = .003). Participants randomly assigned to the basic group (74/99, 75%) were less likely (P = .001) to attend the 12-week visit than those in the control group (96/104, 92%), with the percentage attending from the enhanced group (90/106, 85%) not significantly different from either the control (P = .09) or basic condition (P = .07) (Figure 1). BODY.RESULTS.CHANGES IN WEIGHT, BMI, AND WAIST CIRCUMFERENCE: Participants in the basic and enhanced groups lost significant amounts of weight whether expressed as BMI or kilograms lost (Table 2, Table 3), with the increase in weight in controls not statistically significant. Consequently, those randomly assigned to the basic and enhanced groups had statistically significant reductions in the primary outcome, BMI (kg/m2), compared with those in the control group. Waist circumferences decreased significantly more in the basic and enhanced groups than in the control group, and waist circumference measured at the narrowest point decreased significantly more in the enhanced group than in the basic group (Table 2, Table 3). Waist-to-height ratios decreased in the basic and enhanced groups compared with the control group. BODY.RESULTS.SECONDARY OUTCOMES: After 12 weeks we observed a statistically significant improvement in total serum cholesterol and systolic and diastolic blood pressures in those randomly assigned to the enhanced condition compared with control, with a nonsignificant benefit in those in the basic condition. There were no differences between groups in changes in any of the other plasma variables, including triglycerides, glucose, and insulin. There was a trend toward a greater reduction in pulse rate in the enhanced compared with control group (P = .06). There was no significant change in total physical activity METs (minutes/week), with the average step count per day decreasing in the controls but increasing in the basic and enhanced groups, and a significantly greater increase in enhanced relative to control (P = .005). While all groups decreased their energy intake (kJ/day), those in the enhanced group decreased theirs more than those in the control group (P = .03). There was no change in most of the subscales of the SF-36 quality-of-life questionnaire, with the exception of the general health scale, which improved more in the enhanced than in the control group (P = .03). Within the TFEQ-R18, the scales of cognitive constraint and uncontrolled eating also improved. Both basic and enhanced participants increased eating restraint and reduced uncontrolled eating compared with controls (P < .001). Table 2 Mean (SD) change in variables from baseline to 12 weeks in each treatment group. Variable Total n Treatment group Control Basic Enhanced Weight (kg) 309 0.36 (2.33) –2.14 (3.32) –2.98 (4.05) Percentage weight loss (%) 309 0.44 (2.44) –2.29 (3.51) –3.26 (4.31) Body mass index (kg/m 2 ) 309 0.15 (0.82) –0.72 (1.07) –0.98 (1.38) Waist circumference at umbilicus (cm) 309 0.26 (3.10) –2.63 (3.99) –3.18 (5.00) Waist circumference at narrowest point (cm) 309 0.46 (3.02) –1.96 (3.47) –3.17 (4.69) Waist to height ratio at umbilicus 309 0.00 (0.02) –0.02 (0.02) –0.02 (0.03) Waist to height ratio at narrowest point 309 0.00 (0.02) –0.01 (0.02) –0.02 (0.03) Systolic blood pressure (mmHg) 308 –1.09 (10.90) –3.56 (9.35) –4.95 (10.08) Diastolic blood pressure (mmHg) 308 –0.35 (7.04) –2.09 (7.74) –3.02 (8.57) Pulse rate (beats/minute) 306 –0.86 (6.54) –0.99 (6.47) –2.52 (6.28) Total serum cholesterol (mmol/L) 309 0.08 (0.55) –0.05 (0.51) –0.17 (0.56) LDL a cholesterol (mmol/L) 271 0.09 (0.49) 0.03 (0.40) –0.05 (0.43) HDL b cholesterol (mmol/L) 309 –0.00 (0.13) 0.01 (0.15) –0.01 (0.16) Triglycerides (mmol/L) 309 –0.22 (1.50) –0.17 (0.58) –0.23 (0.67) LDL to HDL ratio 271 0.06 (0.40) 0.00 (0.36) –0.03 (0.34) Glucose (mmol/L) 307 –0.45 (0.91) –0.35 (0.53) –0.33 (0.55) Insulin (mIU/L) 309 –0.76 (5.11) –1.53 (12.76) –1.55 (6.04) Physical functioning (SF-36) 301 0.45 (14.27) 1.79 (22.64) 4.86 (17.22) Role physical (SF-36) 301 1.92 (21.74) 2.04 (21.74) 3.07 (16.39) Bodily pain (SF-36) 300 –2.08 (27.65) 0.20 (25.40) 0.85 (34.34) General health (SF-36) 303 3.02 (11.90) 3.72 (12.17) 6.75 (12.42) Vitality (SF-36) 297 14.11 (68.04) 2.36 (22.48) 12.91 (59.23) Social functioning (SF-36) 298 1.36 (24.42) 0.26 (14.47) 4.48 (21.77) Role emotional (SF-36) 303 1.32 (20.51) 2.47 (13.55) 3.07 (16.48) Mental health (SF-36) 299 2.28 (15.66) 2.55 (15.68) 4.86 (13.26) Total physical activity MET c (minutes/week) 274 341.8 (3116) 151.4 (1946) 491.6 (2601) Average step count per day 263 –61 (2480) 153 (2095) 867 (2947) Cognitive restraint scale (TFEQ-R18) d 296 0.28 (2.50) 1.16 (2.48) 1.78 (3.34) Uncontrolled eating scale (TFEQ-R18) 302 0.05 (3.03) –1.58 (3.53) –1.81 (3.74) Emotional eating score (TFEQ-R18) 304 –0.32 (1.37) –0.47 (1.38) –0.63 (1.64) Total energy intake (kJ/day) 304 –734 (2129) –1003 (2498) –1465 (2470) a Low-density lipoprotein. b High-density lipoprotein. c Metabolic equivalent. d Three-Factor Eating Questionnaire-R18. Table 3 Absolute and least square mean (LSM) differences between groups at 12 weeks (intention-to-treat population). Variable Total n Treatment group LSM (95% CI a ) Group effect ( P value) Basic vs control Enhanced vs control Enhanced vs basic Difference 95% CI Difference 95% CI Difference 95% CI Weight (kg) 309 2.48 1.38 to 3.58 3.34 2.26 to 4.42 0.86 –0.23 to 1.95 <.001 Percentage weight loss (%) 309 2.73 1.57 to 3.89 3.70 2.55 to 4.84 0.97 –0.19 to 2.12 <.001 Body mass index (kg/m 2 ) 309 0.87 0.51 to 1.24 1.13 0.77 to 1.50 0.26 –0.11 to 0.63 <.001 Waist circumference at umbilicus (cm) 309 2.90 1.54 to 4.26 3.45 2.11 to 4.79 0.55 –0.81 to 1.91 <.001 Waist circumference at narrowest point (cm) 309 2.40 1.16 to 3.64 3.65 2.43 to 4.87 1.25 0.02 to 2.49 <.001 Waist to height ratio at umbilicus 309 0.02 0.01 to 0.03 0.02 0.01 to 0.03 0.00 –0.01 to 0.01 <.001 Waist to height ratio at narrowest point 309 0.01 0.01 to 0.02 0.02 0.01 to 0.03 0.01 –0.00 to 0.01 <.001 Systolic blood pressure (mmHg) 308 2.82 –0.17 to 5.81 4.24 1.31 to 7.17 1.42 –1.55 to 4.39 .003 Diastolic blood pressure (mmHg) 308 1.41 –0.96 to 3.78 2.54 0.22 to 4.87 1.13 –1.23 to 3.49 .04 Pulse rate (beats/minute) 306 0.31 –1.77 to 2.39 1.92 –0.12 to 3.95 1.60 –0.47 to 3.67 .06 Total serum cholesterol (mmol/L) 309 0.11 –0.06 to 0.28 0.25 0.08 to 0.42 0.14 –0.03 to 0.31 .003 LDL b cholesterol (mmol/L) 271 0.04 –0.11 to 0.20 0.14 –0.01 to 0.29 0.10 –0.06 to 0.25 .09 HDL c cholesterol (mmol/L) 309 0.01 –0.04 to 0.05 0.01 –0.03 to 0.06 0.02 –0.03 to 0.07 .59 Triglycerides (mmol/L) 309 0.07 –0.15 to 0.28 0.03 –0.18 to 0.25 0.03 –0.18 to 0.25 .78 LDL to HDL ratio 271 0.05 –0.08 to 0.18 0.09 –0.03 to 0.22 0.05 –0.08 to 0.17 .23 Glucose (mmol/L) 307 0.00 –0.17 to 0.17 0.03 –0.14 to 0.20 0.03 –0.14 to 0.21 .86 Insulin (mIU/L) 309 0.21 –2.07 to 2.50 0.46 –1.78 to 2.71 0.25 –2.02 to 2.53 .89 Physical functioning (SF-36) 301 1.78 –3.51 to 7.06 2.65 –2.54 to 7.83 0.87 –4.37 to 6.11 .47 Role physical (SF-36) 301 0.72 –4.83 to 6.26 0.05 –5.38 to 5.48 0.77 –4.72 to 6.26 .94 Bodily pain (SF-36) 300 2.91 –5.63 to 11.45 2.69 –5.66 to 11.04 0.22 –8.22 to 8.66 .67 General health (SF-36) 303 1.25 –2.63 to 5.13 4.24 0.45 to 8.04 2.99 –0.84 to 6.82 .03 Vitality (SF-36) 297 10.38 –5.56 to 26.32 1.12 –14.5 to 16.71 9.26 –6.49 to 25.01 .24 Social functioning (SF-36) 298 0.15 –6.07 to 6.36 2.05 –4.02 to 8.13 1.90 –4.26 to 8.07 .68 Role emotional (SF-36) 303 2.74 –2.02 to 7.51 1.37 –3.29 to 6.02 1.38 –3.34 to 6.10 .40 Mental health (SF-36) 299 1.02 –3.56 to 5.60 2.30 –2.18 to 6.78 1.28 –3.25 to 5.82 .48 Total physical activity MET d (minutes/week) 274 96.51 –776 to 969.4 190.3 –661 to 1042 286.8 –568 to 1141 .72 Average step count per day 263 436 –485 to 1357 1225 339.3 to 2111 789 –130 to 1707 .005 Cognitive restraint scale (TFEQ-R18) e 296 1.05 0.16 to 1.93 1.57 0.72 to 2.43 0.52 –0.36 to 1.40 <.001 Uncontrolled eating scale (TFEQ-R18) 302 1.65 0.55 to 2.76 1.89 0.82 to 2.96 0.23 –0.86 to 1.33 <.001 Emotional eating score (TFEQ-R18) 304 0.18 –0.30 to 0.65 0.30 –0.16 to 0.77 0.13 –0.35 to 0.60 .30 Total energy intake (kJ/day) 304 397 –291 to 1084 782 100 to 1463 385 –304 to 1074 .03 a Confidence interval. b Low-density lipoprotein. c High-density lipoprotein. d Metabolic equivalent. e Three-Factor Eating Questionnaire-R18. There were significant between-group differences in the proportions of participants who lost 5 to <10% (control: 3%, basic: 18%, enhanced: 17%) or ≥10% of their baseline weight (control: 0%, basic: 18%, enhanced: 28%), or gained weight (control: 53%, basic: 22%, enhanced: 17%) (all P < .001). BODY.DISCUSSION: The primary aim of this study was to determine whether using a commercial Web-based weight-loss program, with or without enhanced online features including personalized e-feedback and reminder calls, would lead to a greater reduction in BMI compared with each other or with a wait-list control group. This study demonstrated that participation in either version of the 12-week commercial Web-based weight-loss program (standard or enhanced with feedback) resulted in statistically significant and clinically important objectively measured weight loss. Many desirable improvements in secondary risk markers for chronic disease were achieved, irrespective of the program version used. This suggests that the fundamental elements of the Web-based program were the drivers of the behavior change. However, the enhanced program version achieved a greater retention rate, which in the longer term is critical for maximizing program reach and opportunity to facilitate maintenance of lost weight. There were advantages for those in the enhanced program group related to the extent of improvements in several secondary outcomes compared with the controls, including decreases in waist circumference, plasma total cholesterol, blood pressure, energy intake, and steps per day. Overall, both program versions provided important reductions in several risk factors for cardiovascular disease, as well as improvements in domains of quality of life and eating behavior. In the only previous RCT of a commercial Web-based weight-loss program [11,12], the commercial Web-based program was found to be less effective than a self-help manual [11] and a more structured Web-based behavioral weight-loss program [12]. The eDiets commercial Web-based program had some similar program features to The Biggest Loser Club program, such as weekly self-monitoring of weight, meal and exercise plans, educational materials, and social support via a discussion board. Although eDiets also offered alternative sources of social support such as Web-based meetings and chat rooms, it did not provide a tool for participants to monitor and receive feedback on their dietary intake and physical activity, nor were participants reminded to use the program, which differs from the program in the current study. Therefore, we hypothesized that the mean weight losses for the basic and enhanced versions after 12 weeks in this study would be greater than those from the 2004 RCT using eDiets (–0.9% after 16 weeks) [11]. Interestingly, the 12-week weight loss in the current study is similar to the results of a more recent RCT using eDiets, which reported a mean weight loss of –3.6% after 6 months [12], which may indicate that developments in online capabilities facilitate program effectiveness. This is the first RCT to examine the efficacy of a commercial program that specifically sought to include enhanced features (provision of additional personalized e-feedback, reminders, and phone calls) compared with the basic commercial program version without these features. We have shown that participants using the enhanced program did not lose significantly more weight after 12 weeks than did those in the basic program. This finding is not consistent with previous RCTs comparing basic versus enhanced versions of Web-based programs, as these have typically demonstrated greater weight loss in the enhanced study arm [12,23-27]. However, the basic programs in these previous studies had fewer program features and typically did not include all of the key components of Web-based interventions reported to be essential, such as being based on a theoretical framework, providing diet and physical activity feedback to participants, and having interactive features [28,29]. For example, Rothert et al's basic Web-based program provided information only [27], while the enhanced features in other studies commonly used more human interventions, such as individualized human e-feedback generated by a therapist [24-26] or Web-based chat sessions conducted by a trained therapist [12]. The lack of difference between the basic and enhanced program in the current study, along with the magnitude of the weight lost after 12 weeks, suggests that either format of the program can produce clinically important initial weight loss. However, it also suggests that the enhanced program may require a greater number or intensity of enhanced features to be more effective than the basic version, or that the enhanced features provided are not necessary for many participants. Although no significant difference in weight loss between basic and enhanced groups was observed, the enhanced group achieved significantly greater improvements in waist circumference than the basic group. They also demonstrated greater improvements in blood pressure, plasma total cholesterol, steps per day, measures of general health, and reduction in total daily energy intake than the control group, whereas the basic group did not. This suggests that the enhanced program offers additional benefit to participants in terms of risk factor reduction and in achieving behavior change that may assist with long-term maintenance of lost weight. Furthermore, the basic group were more likely to drop out of the study (25%) than the enhanced group (15%). The impact of this should not be overlooked because weight-loss success is associated with greater adherence to the prescribed treatment plan, and retention within treatment is the primary component of program adherence. Further follow-up of these participants will determine whether greater initial improvements in indicators of health status, quality of life, dietary intake, and physical activity, as well as higher initial retention, affect weight-loss outcomes in the long term and whether improvements are sustained over time. This will be important in evaluating the long-term efficacy of the basic program compared with the enhanced program. This is one of the first RCTs of a Web-based weight-loss program to comprehensively assess secondary outcomes and to capture the potential of the program to have significant health benefits, irrespective of the weight loss achieved. To our knowledge, no other RCT evaluating Web-based weight-loss studies have assessed changes in lipids or insulin levels, and only one has evaluated changes in glucose levels [26]. The current study also demonstrated significantly greater reductions in blood pressure (systolic and diastolic) among the enhanced program users. Only two other RCTs evaluating Web-based weight-loss programs have assessed changes in blood pressure [30-32], with one of these finding greater reductions in systolic blood pressure among male participants only. Also, these improvements were demonstrated after 12 months [31], but not after 3 or 6 months [32]. Therefore, this is the first Web-based weight-loss RCT to demonstrate significant improvements in plasma total cholesterol and blood pressure after 12 weeks via participation in a Web-based weight-loss program that provides comprehensive personalized feedback and reminders to engage with the program. Only a few Web-based weight-loss RCTs have reported changes in dietary intake and physical activity, and assessed difference in change across study arms [23-26,32-35]. To date, no study has demonstrated significant differences in dietary intake or physical activity change among participants of Web-based weight-loss programs compared with control groups, other treatment formats (eg, face-to-face), or enhanced Web-based programs. The current study found significantly greater reductions in mean total daily energy intake and increases in steps per day in the enhanced program users, which suggests that additional personalized feedback and reminders to use the program had positive influences on food and physical activity behaviors. However, further detailed investigation is needed to examine how participants change their food and physical activity behaviors in response to Web-based intervention. Further, no previous Web-based weight-loss studies have measured components of appetite and hunger using the TFEQ-R18. We have demonstrated improvements in both intervention groups compared with a control group in the domains of cognitive restraint and uncontrolled eating, but no significant improvements in emotional eating scores. Previous research has highlighted the association between appropriate eating patterns, such as higher dietary restraint and less emotional eating, and long-term weight-loss maintenance [36], and therefore this is an area where the current program could be refined. Longer-term follow-up of participants will demonstrate whether these initial improvements in eating patterns can be sustained or improved, and whether this affects weight-loss maintenance. One other Web-based weight-loss study has measured quality of life. McConnon and colleagues found significant improvements in quality of life in Internet and usual-care groups with no significant difference between groups [33]. Therefore, the finding in the current study of significant improvements in quality of life, namely the general health domain of the SF-36, after 12 weeks' participation in the enhanced version of the program is important. A limitation of the current study is that all participants did receive human contact when they attended the clinical research center for clinical assessments. However, they were assessed by blinded assessors. Further, we gave them no advice on how to log in or engage with the program, other than giving them their login details. This was to simulate real-world engagement and use of the program, making the results generalizable to the overweight population of adults using commercial weight-loss programs. Due to the inclusion of a control group, the study also did not consider the potential differential impact of the Web-based programs as a result of participants' varying levels of website usage. Study strengths include the use of a control group compared with two versions of the Web-based program, as well as the robust study design and the use of predominantly objective measures. The results of this study demonstrate that Web-based weight loss can be efficacious across a range of weight-related outcomes and lifestyle behaviors, and that commercial providers can deliver effective Web-based programs targeting this important public health issue. Further study is needed to examine longer-term outcomes and whether Web-based programs with enhanced program features, including provision of personalized feedback, can retain people in the long term and lead to a greater treatment impact over time.

TITLE: Treatment of Rockwood type III acromioclavicular joint dislocation using autogenous semitendinosus tendon graft and endobutton technique ABSTRACT.BACKGROUND: The aim of this study was to evaluate the therapeutic effect of autogenous semitendinosus graft and endobutton technique, and compare with hook plate in treatment of Rockwood type III acromioclavicular (AC) joint dislocation. ABSTRACT.METHODS: From April 2012 to April 2013, we treated 46 patients with Rockwood type III AC joint dislocation. Patients were randomly divided into two groups: Group A was treated using a hook plate and Group B with autogenous semitendinosus graft and endobutton technique. All participants were followed up for 12 months. Radiographic examinations were performed every 2 months postoperatively, and clinical evaluation was performed using the Constant–Murley score at the last follow-up. ABSTRACT.RESULTS: Results indicated that patients in Group B showed higher mean scores (90.3±5.4) than Group A (80.4±11.5) in terms of Constant–Murley score (P=0.001). Group B patients scored higher in terms of pain (P=0.002), activities (P=0.02), range of motion (P<0.001), and strength (P=0.004). In Group A, moderate pain was reported by 2 (8.7%) and mild pain by 8 (34.8%) patients. Mild pain was reported by 1 (4.3%) patient in Group B. All patients in Group B maintained complete reduction, while 2 (8.7%) patients in Group A experienced partial reduction loss. Two patients (8.7%) encountered acromial osteolysis on latest radiographs, with moderate shoulder pain and limited range of motion. ABSTRACT.CONCLUSION: Autogenous semitendinosus graft and endobutton technique showed better results compared with the hook plate method and exhibited advantages of fewer complications such as permanent pain and acromial osteolysis. BODY.INTRODUCTION: Acromioclavicular (AC) joint dislocations account for 9% of shoulder girdle injuries, with a higher incidence in athletes.1 AC joint dislocations are mainly caused by direct trauma, for example, a blow to the lateral shoulder pushing the acromion away from the distal clavicle.2 The AC ligament is affected first, and the effect is cascaded to the coracoclavicular (CC) ligament, and finally to the deltoid and trapezius fascia, with increasing severity.3 Therefore, treatment for AC joint dislocation is focused on reconstructing AC and CC ligaments. There is no established standard of care for AC joint dislocations, although many surgical techniques have been reported. The hook plate was commonly applied in the treatment of acute AC joint dislocation with successful results.4–7 However, persistent pain, infection, delayed wound healing, arthritis, acromial impingement, and clavicle erosion and redislocation were reported in treated patients.7–9 As graft alone is not sufficient to prevent translation at the joint, generally a supplemental fixation is required.10 Therefore, endobutton was applied to avoid redislocation and complications. This study presents a surgical technique using autogenous semitendinosus graft and endobutton to treat Rockwood type III AC joint dislocation. The purpose of the study was to evaluate the short-term results of the technique and compare it with the hook plate in the treatment of Rockwood type III AC joint dislocation. BODY.MATERIALS AND METHODS: This was a prospective, randomized, double-blind study in a hospital setting. Patients visiting the hospital from April 2012 to April 2013 with acute type III complete AC joint dislocation were considered for inclusion in the study. Diagnosis was based on the clinical and radiological results of the patients. The study was conducted after obtaining approval from the relevant Institutional Review Board. The present study was also approved by the ethics committee of the People's Hospital of Huangpi District. Written informed consent was obtained from all patients before they entered the study. The inclusion criteria were as follows: 1) acute type III AC joint dislocation and 2) clinical and radiological follow-up for a minimum period of 6 months. Exclusion criteria were as follows: 1) previous shoulder joint pathology on the dislocated shoulder and 2) chronic AC dislocations or other acute AC dislocations. The patients were divided into two groups. Group A received hook plate treatment and Group B received autogenous semitendinosus graft and endobutton treatment. Randomization was performed by an independent researcher using computer-generated numbers. All investigators, research staff, and participants were blinded to the treatment allocation. BODY.MATERIALS AND METHODS.SURGICAL TECHNIQUE: In Group A, the patients underwent surgery in a beach-chair position under general anesthesia. The surgical technique was performed according to the method adopted by Wolter and Eggers.11 Removal of the hook plate was scheduled after 12 weeks. Patient positioning in Group B were identical to the hook plate group. CC ligament was reconstructed according to the method of Struhl.12 The semitendinosus tendon harvesting was performed before AC joint exposure using a tendon stripper. The semitendinosus tendon graft was weaved and sutured with No 2 Ethibond suture (Ethicon Inc., Somerville, NJ, USA). Two drill holes were made with the diameter matching the graft from the 2.0 cm medial portion in the distal clavicle and on the coracoid. The prepared semitendinosus tendon was passed through two holes and sutured on the coracoid. Another drill hole was made from the 4.0 cm medial portion in the distal clavicle using a 2.5 mm drill bit. The double endobutton fixed by No 5 Ethibond sutures (Ethicon Inc.) was introduced through the third hole and the coracoid hole. According to the postoperative protocol, the shoulder was immobilized using an abduction sling for 2 weeks. Active motion of the elbow, wrist, and hand was recommended. Passive shoulder motion was encouraged after 4 weeks postoperatively. In the follow-up period, patients were interviewed for any complaint, general physical examination was performed, and radiographs were taken. The function of the repaired joint was evaluated using the Constant–Murley scale at the last follow-up. BODY.MATERIALS AND METHODS.FUNCTIONAL OUTCOMES: According to the Constant–Murley score,13 four variables were used to assess the function of the shoulder. The two subjective variables included pain and limitation of activities of daily living, and the two objective variables included range of motion and strength of the shoulder. The subjective part of the test was self-completed by the patient. The absence of pain was allocated a maximum score of 15; no limitation of activities of daily living like sleep, work, recreation, or sport, was allocated a maximum score of 20. Range of motion (maximum 40 points) and strength (maximum 25 points) were measured. BODY.MATERIALS AND METHODS.RADIOGRAPHIC OUTCOMES: Radiographs of the AC joint were obtained every 2 months postoperatively. The radiographic studies were examined for signs of loss of reduction, or other complications such as fracture or distal clavicular osteolysis. The maintenance of AC joint reduction was defined as follows: 1) maintained reduction, no side-to-side difference on anteroposterior (AP) radiographs; 2) partial loss of reduction, a side-to-side difference of less than the width of the clavicle; and 3) complete loss of reduction, a side-to-side difference in excess of the clavicle width.14 BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Categorical and continuous variables were described (ratios, means, and standard deviations). The statistical analyses of differences in continuous variables between the two groups were tested with unpaired Student's t-test and differences in categorical variables with χ2 test. Nonparametric tests of K-independent samples were performed on the equality of the medians for the continuous variables without normal distribution. All analyses were performed with SPSS version 21.0 (SPSS, Chicago, IL, USA). Differences were considered significant at P<0.05. BODY.RESULTS: Forty-six patients (30 men, 16 women) were included in the study. Table 1 shows the baseline characteristics of the patients. The mean age of the participants was 33.4 years (range 30–46 years) at enrollment. The treatment groups were well balanced in terms of baseline characteristics. BODY.RESULTS.CLINICAL OUTCOMES: Clinical evaluation was performed using the Constant–Murley score13 at the final follow-up. The patients acquired an average of 90.3 points (maximum 100 points) in Group B, better than 80.4 points in Group A in the Constant–Murley score (P<0.05). Table 2 shows that patients treated with the autogenous semitendinosus graft and endobutton technique showed higher constant scores than the hook plate-treated patients with regard to pain (P=0.002), activities (P=0.02), range of motion (P<0.001), and strength (P=0.004). During the follow-up period, moderate pain was reported by two (8.7%) hook plate-treated patients and mild pain was reported by eight (34.8%) hook plate-treated patients. Mild pain was reported by one (4.3%) patient treated with autogenous semitendinosus graft and endobutton technique. BODY.RESULTS.RADIOGRAPHIC FINDING: On final follow-up, all the patients of Group A maintained complete reduction in the AP plane, except 2 (8.7%) who experienced partial reduction loss. None of the patients experienced complete loss of reduction. There were 2 (8.7%) patients in Group A with acromial osteolysis on the latest radiographs, which included a 38-year-old woman and a 45-year-old man (Figure 1A and B). Their constant scores were 59 and 55, respectively. Moderate shoulder pain and limited range of motion were reported by both of them. There were no infections or fractures in patients who were included in the study. However, all patients in Group B maintained complete reduction (data not shown). BODY.DISCUSSION: AC joint is an articulation of the lateral clavicle and the acromion. As an amphiarthrodial joint, it plays an important role in maintaining the strength and movement of the whole upper limb. Garretson and Williams2 classified the degree of displacement in AC joint dislocations into six types based on the severity of involvement of AC and CC ligaments. The initial treatment of an acute type III dislocation of AC joint remains controversial.6,15–17 Though the surgical treatments have improved in recent years, a high incidence of complications has also been reported in some studies, and thereby failing to establish a gold standard. However, in our clinical study, we found that the surgical treatment always receives the better effects compared to the conservative treatment, according to our 5 years of follow-up. Therefore, we explored the therapeutic effects of surgical treatment over the conservative treatment. In this study, we evaluated the effect of surgery using autogenous semitendinosus graft and endobutton technique for the treatment of Rockwood type III AC joint dislocation. The previous studies and our clinical practice have been indicated that neither the autogenous semitendinosus graft technique nor the endobutton technique can therapy the Rockwood type III AC joint dislocation efficiently.6,18–22 Therefore, in this study, we added the semitendinosus graft to the endobutton technique. Furthermore, we also observed several types of dislocations in clinical, however, therapy of the type III dislocation with the best outcomes. Therefore, we selected the type III dislocation as the study type. Higher mean constant score was obtained in patients treated using this technique (90.3 points) compared with hook plate-treated patients (80.4 points). Radiographic findings showed that all patients in Group B achieved reconstruction in the AP plane anatomically, while 8.7% (2 of 23) patients in Group A experienced partial reduction loss. Moreover, two cases (8.7%) of acromial osteolysis with moderate shoulder pain and limited range of motion were observed in Group A. Gstettner et al6 reported a high possibility of subluxation in 24.7% of patients using a modified endobutton technique compared with our study. This may be probably because of the fact that autogenous semitendinosus graft was applied together with endobutton. Martetschlager et al18 reported four cases of graft ruptures in 46 shoulders using the tendon graft technique. The four graft ruptures encountered were associated with the insufficient strength of the graft. Mazzocca et al19 suggested that autograft is not sufficient to prevent anterior–posterior translation at the joint. Therefore, modifications to augment the autograft with a fixation material around the base of the coracoid process were essential to prevent graft ruptures. In another study using a double-button and fixation technique, Milewski et al20 reported ten cases involving shuttling the graft through an additional tunnel in the coracoid base. In addition to the chronic dislocations included in the series, a higher rate of coracoid fractures associated with coracoid tunnel techniques indicated that modifications, including the location and diameter of drill holes, should be considered to prevent fractures. In our study, the diameter of the hole matched with the graft to avoid the fractures. Currently, endobutton and hook plate are widely used. The mobile side of the hook plate underneath the acromion may cause symptoms of persistent pain and restriction in movement by acromial erosion and damaging the supraspinatus tendon.9,21 Long-term retention of the plate may lead to acromial osteolysis or fracture, which implies that a second surgery is required to remove the plate after 12 weeks when the ligaments are healed. Kienast et al7 achieved successful results using the hook plate in 313 patients of Rockwood type III–V AC joint dislocation, while all patients reported pain or discomfort with the hook plate. After removal of the hook plate, a high incidence of complications of superficial soft tissue infections and fracture of the acromion and redis-locations were reported. Endobutton is an appropriate technique to reproduce the course of the conoid portion of the CC ligament. The technique was first introduced by Struhl. He had performed only a small incision with minimal soft tissue dissection. The strength and stiffness of the device exceeded the native ligament complex by approximately 40%.12 The deforming forces are distributed on the surface of the double endobutton plates, thereby reducing the effect on the suture material and minimizing the chance of soft tissue reaction to the suture material. The modified device added a second endobutton to create a knotless fixation. The objective was to prevent complications, especially shoulder pain by minimizing recurrent displacement and joint irritation, and to avoid soft tissue reactions and traumatic arthritis.19,21 Because of the biocompatibility of the device, there is no need to remove the device in a second surgery, which can avoid potential injuries.22 This study demonstrated the open technique and exhibited advantages of few complications such as permanent pain and acromial osteolysis. During the surgery, attention should be paid to the diameter and location of the hole during drilling. Minor diameter and accurate location are of paramount importance. To achieve better stability, the distance of the insertion site of the clavicle and acromion should be approximate to the anatomical position of 5.2 mm (women) and 7.6 mm (men).23 Future studies are warranted to explore the long-term therapeutic results of this technique. Actually, the other factors could also affect the clinical outcomes, such as rehab. The limitation of this work is that it only investigated the single-factor-dependent clinical outcomes. Therefore, in the following studies, we would also explore the effects of rehab on the clinical outcomes. BODY.CONCLUSION: This described technique shows better short-term results than the hook plate method and can be used effectively in the treatment of Rockwood type III AC dislocation.

TITLE: Recombinant Erythropoietin in Humans Has a Prolonged Effect on Circulating Erythropoietin Isoform Distribution ABSTRACT: The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2)% (p<0.00001) and 45.2 (7.3)% (p<0.00001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8)% (p<0.00001) and 46.1 (10.4)% (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4)% (p = 0.029); low-dose Epoetin beta: 73.1 (17.8)% (p = 0.039)). In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal. BODY.INTRODUCTION: The use of recombinant human erythropoietin (rhEPO) for enhancement of sports performance was banned in 1990 by the International Olympic Committee. Still, however, doping by blood manipulation remains a problem in elite athletic competitions in spite of ever-increasing anti-doping efforts [1]–[4]. The performance gain following the increase in hemoglobin mass is maintained for at least three weeks [1], [2]. This in combination with short elimination half-lifes of most rhEPO isoforms makes it possible to accommodate doping practices in relation to competition events so as to avoid direct detection of a former use of rhEPO [1], [5]. Moreover, rhEPO isoforms differ only slightly from the endogenous isoforms by their posttranslational glycosylation pattern [3], [6]. Several tests have been developed to distinguish between specific forms of EPO glycosylation but valid and sensitive tests are lacking [1], [3], [7]. Because of the shortcomings of direct tests, indirect marker methods, referred to as 'Blood Passport' or the World Anti-Doping Association's (WADA) 'Athlete Biological Passport', have been introduced [5]. These methods are based on personalized monitoring of haematological parameters, but testing in controlled trials has showed a high incidence of false-negative results [8]–[10]. A new promising approach to anti-doping testing has been the introduction of the membrane-assisted isoform immunoassay (MAIIA) which is a combination of lectin affinity chromatography and lateral flow immunoassay in a combined test-strip [11]. For identification of EPO isoforms, the method uses a non-selective lectin (wheat germ agglutinin (WGA)). In the next step, the addition of buffers containing a competing monosaccharide (N-acetylglycosamine (GlcNAc)) allows a separation of EPO isoforms in fractions according to the specific glycosylation: at high concentrations of GlcNAc (>100 mM), all EPO isoforms are eluted from the lectin. At lower concentrations of GlcNAc (<20 mM), however, only fractions are released from the WGA zone, the amount depending on their glycosylation. Eluted EPO is then bound to an immobilized anti-EPO antibody and quantified by the use of a second anti-EPO antibody labelled with carbon black nano-strings. By testing the same plasma sample at both a high GlcNAc concentration (total desorption of all isoforms) and a low GlcNAc concentration (fractional specific desorptions of given isoforms), the method allows calculation of the 'percent of migrated isoforms' (PMI) [12]. PMI is the amount of EPO desorbed at a low GlcNAc concentration as a percentage of the amount desorped at the high GlcNAc concentration [7], [12]. In example, with the use of 15 mM and 300 mM GlcNAc, PMI in a normal plasma sample with only endogenous EPO isoforms average 70–80% [12]. In contrast, PMI in a solution of Epoetin beta, a form of rhEPO synthesized in Chinese hamster ovary cells, is around 30 % [12]. In samples from subjects that were injected with Epoetin beta (5,000 IU) every second day for 14 days and then again at days 21 and 28, PMI decreased to a nadir of around 30 % at day 16 [13]. Seven days after the last injection, when the plasma concentration of EPO had returned to normal, PMI was still decreased compared with baseline values [13]. Interestingly, studies suggest that the resolution between PMIs for EPO subpopulations can be optimized by the use of different low concentrations of GlcNAc. PMI from normal plasma is best distinguished from samples with Epoetin beta at GlcNAc concentrations of 15 mM, whereas lower concentrations may be used to distinguish endogenous EPO isoforms [12]. Thus, MAIIA may provide a sensitive measure of changes in the EPO isoform distribution pattern caused by former use of recombinant EPO even after normalization of total EPO concentrations in plasma. Such a change may be secondary to prolonged suppression by rhEPO of the endogenous renal synthesis of EPO [14], [15] and also remaining small amounts of rhEPO may alter the distribution profile of circulating EPO. In this placebo-controlled, double-blinded, cross-over study, we tested whether MAIIA may be used as an indirect marker method to detect former administration of Epoetin beta. BODY.METHODS AND MATERIALS.SUBJECTS: Sixteen healthy male volunteers (age 25.4 (3.6) years, (mean (SD)), height 183.5 (6.6) cm, body weight 76.9 (7.3) kg and body mass index 22.9 (2.7) kg/m2) were included in the study. Before inclusion each subject underwent a medical examination and had to fulfill the following inclusion criterias: male gender, age between 20 and 40 years, non-smoking, arterial systolic blood pressure below 140 mmHg and diastolic blood pressure below 90 mmHg, no actual medication, and body mass index <25. Exclusion criterias were participation in other studies, history of elite athletic performance, history of neoplastic diseases, polycythemia, epilepsy or allergy to rhEPO, and/or exposure to altitude (>1,500 metres above sea level) within three months prior to the study. BODY.METHODS AND MATERIALS.EXPERIMENTAL PROTOCOL: The effect of both low and high doses of rhEPO (Epoetin beta, NeoRecormon, Roche, Welwyn Garden City, UK) and placebo was evaluated in each subject by a randomized, double-blinded, placebo-controlled, cross-over design with a 5 week washout interval between the series: Low dose rhEPO: 5,000 IU (∼65 IU.kg−1) of subcutaneously administered Epoetin beta every second day for two weeks (days 1, 3, 5, 7, 9, 11 and 13; placebo on day 2).High dose rhEPO: 30,000 IU (∼390 IU.kg−1) of subcutaneously administered Epoetin beta every day for three days (days 1, 2 and 3; placebo on days 5, 7, 9, 11 and 13).Placebo (sodium chloride, isotonic 9 mg/ml, B.Braun, Melsungen, Germany) administered subcutaneously on days 1, 2, 3, 5, 7, 9, 11 and 13. The primary endpoint in all series was changes in percent migrated isoforms (PMI) at days 4, 11 and 25 as a measure of EPO isoform distribution. Randomization for the entire study (all three test series) was done before the beginning of the experiment. An independent investigator (NVO) generated a restricted randomization list and after inclusion each subject where given a number and a code, unique for subject and intervention. It was not possible for the subjects to visually distinguish between placebo or study medication. Randomization lists were sealed and not available for persons involved in endpoint registration. NVO did not participate in subject inclusion, study management, end-point evaluation or data analysis. The study was approved by the Regional Committee on Biomedical Research Ethics, Committee B (protocol no. H-2-2011-068) and the Danish Medicine Agency (EudraCT 2001-005137-39). The study was conducted according to the principles of Good Clinical Research (GCP), monitored by the GCP Unit at Copenhagen University Hospital (Bispebjerg Hospital) and registered on www.clinicaltrials.gov (NCT01584921). All subjects were informed verbally and in writing before the beginning of the study and gave written informed consent to participate. All experiments were done at the Copenhagen University Hospital (Rigshospitalet), Denmark. All injections were administered subcutaneously between 08.00 and 11.00 a.m. The subjects did not receive any iron supplementation as normally administered during treatment for anemia or in doping so as to avoid confounding effects regarding the effect of Epoetin beta per se. Three days before each test day subjects refrained from excessive salt intake, according to written instructions from a dietitian. The subjects refrained from alcohol, caffeine-containing drinks and extensive exercise 24 hours before each test day. These measures were due to concomitant metabolic and renal clearance studies reported elsewhere. Plasma samples were collected on day 4, 11 and 25. Blood samples were obtained from an anticubital vein after at least 180 min of rest in a sitting position using EDTA tubes and before administration of rhEPO. Immediately after centrifugation at 3000 g for 10 minutes, the plasma were stored at −80°C until analysis. BODY.METHODS AND MATERIALS.TOTAL EPO CONCENTRATION: Plasma EPO concentrations were measured by means of the commercially available Quantikine IVD ELISA kit (R&D Systems Europe, Ltd., Abingdon, UK) according to the manufacturer's protocol which is based on the double-antibody sandwich method. Assay results were measured spectrophotometrically at 450 nm using a microplate reader to determine the optical density. Duplicate readings were averaged for each standard, control and specimen. The log of erythropoietin concentration was plotted versus the log of optical density for the standard curve. Concentrations are given in mIU/ml; samples with EPO concentrations above the range of the assay (2.5–200 mIU/mL) were diluted ten-fold before a second analysis. BODY.METHODS AND MATERIALS.EPO ISOFORM DISTRIBUTION: The change in EPO isoform distribution was measured by membrane-assisted isoform immunoasssay (MAIIA) [13]. The MAIIA kit is based on affinity chromatography and lateral flowimmunoassay on a strip with two important zones: a wheat germ agglutinin (WGA) zone that interacts with EPO via its sugar moieties and, downstream from this, an anti-EPO zone that immobilizes EPO. The strip was immersed in a well containing the sample, which migrates in the strip due to capillary forces. N-acetyl glucosamine (GlcNAc) in different concentrations were used to release the different EPO isoforms from the WGA interaction. With high GlcNAc concentration (300 mM) all EPO melocules are released from the WGA zone but with lower concentrations (5, 10, 15 or 20 mM) different isoforms of EPO are released depending on their glycosylation pattern [11]–[13]. Calculation of the percentage of migrated isoform (PMI) was done by analysing each sample with several strips using both high and low GlcNAc concentrations. For detection and quantification of EPO the anti-EPO zone was used in combination with a secondary anti-EPO antibody and carbon black nano-strings. The MAIIA kit (EPO purification kit, MAIIA Diagnostics, Uppsala, Sweden) was used to purify EPO plasma samples. The procedure was done according to the directions of the manufactor; 1.0 ml of plasma was purified on single use anti-Epo columns and eluted into a final volume of 55 μl and stored at −20°C until analysis. A standard curve was made and the optimal concentration of W elution buffer low was determined. The standards had concentrations of Epoetin beta of 0, 10, 30, 100, 300 and 600 ng/l and were all analysed at 300 mM (high) GlcNAc. The final standard curve (Figure 1) represents a mean of four standard curves and the standard curve parameters were obtained by the use of a four parameter logistic curve fitting routine to fit the "Delta Blackness per Pixel" (DBpB). The parameters were entered in an excel template provided by MAIIA and used for calculation of PMI. For optimization of W elution buffer low (Figure 2), three sets of samples were used: 1) a solution of Epoetin beta (300 ng/l); 2) a plasma sample from a normal subject (CL); and 3) arterial plasma samples obtained from umbillical cord (N = 6), as described previously [16]. All sets of samples from the three specimen were tested with GlcNAc concentrations of 5 mM, 10 mM, 15 mM, 20 mM, and 300 mM of GlcNAc. Samples were diluted 50, 20, 10 or 5 times in a dilution buffer, aming for a sample concentration of EPO within the range of the assay (10–500 ng/l). Based on the results from the optimization analyses (Figure 2), all plasma samples from days 4, 11 and 25 were analysed in duplicates and under standardized conditions regarding temperature, humidity, scanner and reagent lot number at 15 mM and high GlcNAc. In addition, plasma samples from days 11 and 25 were analysed in duplicate at 5 mM and high GlcNAc. 10.1371/journal.pone.0110903.g001Figure 1Standard curve.Concentrations of rhEPO (Epoietin beta) were 0, 10, 30, 100, 300 and 600 ng/l. All solutions were tested at 300 mM of GlcNAc. Values are means ± SD. N = 6. 10.1371/journal.pone.0110903.g002Figure 2Optimization curve.For optimization of W elution buffer low, three samples were used: 1) UC, umbilical cord sample; 2) Plasma sample form a normal subject; and 3) rhEPO, epoetin beta (300 ng/l). All samples from the three specimen were tested with GlcNAc concentrations of 5 mM, 10 mM, 15 mM, 20 mM, and 300 mM. Values are means (SD). N = 6 for UC, N = 5 for plasma and N = 5 for rhEPO. BODY.METHODS AND MATERIALS.STATISTICAL ANALYSIS: Statistical analyses were done by the use of the statistical SPSS software (IBM SPSS Statistics, version 20.0.0). Entries into the database of all data were verified by the GCP Unit at Copenhagen University Hospital. To assess the effect of rhEPO treatment on total EPO concentration and PMI values a linear mixed-effects model (MIXED) for repeated measurements was used. (SPSS: Analyze>Mixed Model>Linear). Repeated measures and pairwise comparisons versus baseline were corrected for multiple comparisons (Bonferroni). Results are expressed as means (SD). All calculations regarding plasma EPO concentrations and PMI values on day 4, 11 and 25 were done comparing each treatment and day against the mean of placebo values on day 4, 11 and 25. No significant carry-over or period effect was found for any of the endpoints. The assumption of normal distribution was evaluated by visual inspection. If necessary, data was log10 transformed before analysis. Two-sided significance tests were used with a significance level of 5 %. BODY.RESULTS: All subjects received 100 % of the planned injections. One subject was excluded due to illness commencing during the first 5-week wash-out period between the first and second series. It was not characterized as an adverse effect by the GCP unit, and the subject's data from the first series are included in the statistical calculations. BODY.RESULTS.TOTAL PLASMA EPO CONCENTRATION (: 10.1371/journal.pone.0110903.t001 Table 1 Total plasma EPO concentration (mIU/ml). Day 4 Day 11 Day 25 Placebo 8.1 (2.6) 11.3 (3.9) 10.1 (3.0) Low-dose EPO 29.7 (11.2) ** 25.5 (8.6) ** 8.1 (3.8) High-dose EPO 470.3 (202.0) ** 15.4 (6.2) * 8.3 (4.9) N = 15. Values are means (SD). Data was log 10 transformed before analysis. * p = 0.004; **p<0.00001 compared with placebo. Total plasma EPO concentration increased after administration of both low-dose and high-dose Epoetin beta. Compared with placebo, low-dose Epoetin beta increased the total plasma EPO concentration by 3-fold on days 4 (p<0.00001) and 11 (p<0.00001) whereas values on day 25 did not differ from placebo (p = 0.116). Concentrations after low-dose Epoetin beta did not differ between days 4 and 11 (p = 0.65) but on both days values were higher compared with day 25 (p<0.00001). High-dose Epoetin beta increased the total plasma EPO concentration by 48-fold (p<0.00001) and two-fold (p = 0.004) at days 4 and 11 whereas values on day 25 did not differ from placebo (p = 0.121). There was significant difference between days 4, 11 and 25 within the high-dose intervention (p<0.00001). BODY.RESULTS.HAEMATOLOGICAL VARIABLES (: 10.1371/journal.pone.0110903.t002 Table 2 Haematological parameters after 4, 11 and 25 days of either placebo, low-dose rhEPO, or high-dose rhEPO. Day 4 Day 11 Day 25 Hematocrit (%) Placebo 42.9 (1.9) 40.7 (2.5) 41.4 (2.7) Low-dose EPO 42.8 (2.0) 43.0 (2.1) 43.5 (2.6) * High-dose EPO 43.0 (1.5) 43.3 (2.4) * 42.6 (2.8) Hemoglobin (g⋅dl −1 ) Placebo 8.9 (0.4) 8.6 (0.5) 8.6 (0.5) Low-dose EPO 9.0 (0.4) 8.9 (0.4) 8.9 (0.5) High-dose EPO 9.0 (0.4) 9.0 (0.5) 8.8 (0.6) Reticulocytes (×10 9 ⋅l −1 ) Placebo 31.1 (10.2) 37.9 (11.8) 35.5 (12.4) Low-dose EPO 40.5 (6.1) 78.7 (19.9) ** 26.3 (6.5) High-dose EPO 68.4 (15.6) ** 84.4 (18.0) ** 27.9 (8.8) Ferritin (µg⋅l −1 ) § Placebo 85.2 (64.1) 70.3 (48.0) 50.9 (33.9) Low-dose EPO 68.7 (71.8) * 32.4 (38.3) ** 73.6 (65.9) High-dose EPO 43.9 (36.8) ** 30.2 (17.9) ** 73.2 (53.5) Transferrin saturation (%) § Placebo 30.9 (12.7) 27.2 (14.1) 28.7 (11.0) Low-dose EPO 20.2 (8.1) ** 12.6 (5.9) ** 35.2 (13.8) High-dose EPO 9.7 (3.2) ** 12.1 (5.1) ** 31.2 (17.9) N = 16. Values are means (SD). § Data was log 10 transformed before analysis. * p<0.05; **p<0.01 compared with placebo. Both low-dose and high-dose Epoetin beta caused small, albeit statistically significant, increases in haematocrit, which with low-dose Epoetin beta extended onto day 25. Also Epoetin beta increased reticulocyte counts. The protocol did not include supplements of iron and as expected both doses of Epoetin beta decreased plasma levels of ferritin and transferrin saturation. All values, except the haematocrit value after low-dose Epoetin beta, had return to normal at day 25. BODY.RESULTS.FARMACOKINETICS: The elimination half-life of Epoetin beta is between 8 to 28 hours if subcutaneously administered [17], [18] and a maximal plasma concentration is obtained about 15 hours after injection [19]. The distribution volume corresponds to 1–2 times the plasma volume [18]. The terminal elimination rate constant β was calculated as ln2/t1/2. Assumed that the elimination rate constant β is a first order rate constant describing drug elimination from the body and Epoetin beta follows first order of kinetics in degradation, the expected contribution of Epoetin beta to the total plasma EPO concentration was estimated to be 386 mIU/ml, 155 mIU/ml, and 0.01 mIU/ml, respectively, with low-dose Epoetin beta at days 4, 11 and 25. Corresponding values with high-dose Epoetin beta were 3104 mIU/ml, 4 mIU/ml, and 0.00001 mIU/ml, respectively, at days 4, 11 and 25. BODY.RESULTS.PERCENTAGE OF MIGRATED ISOFORMS (PMI): In order to distinguish Epoetin beta from endogeneous EPO, the optimization curves demonstrates that the maximal deviation of PMI values was best obtained with 15 mM of GlcNAc as the W elution buffer low (Figure 2). With 15 mM of GlcNAc, PMI for EPO isoforms originating from normal plasma and umbilical cord plasma was 78.5 (3.4) % and 86.0 (9.4) %, respectively, whereas the PMI for Epoetin beta (300 ng/l) was 34.1 (7.8) (Figure 2). Both low-dose and high-dose administration of Epoetin beta caused a significant decrease in PMI values on all three days (Figure 3). PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % and remained unchanged at all days. High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2) % (p<0.00001) and 45.2 (7.3) % (p<0.00001), respectively. PMI differed significantly between all days within the high-dose intervention (p<0.0001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8) % (p<0.00001) and 46.1 (10.4) % (p<0.00001), respectively. PMI after low-dose Epoetin beta did not differ between day 4 and 11, but were on both days significantly lower compared with day 25 (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased compared with placebo (Figure 3); high-dose Epoetin beta: 72.9 (19.4) (p<0.029) %; low-dose Epoetin beta: 73.1 (17.8) % (p<0.039). 10.1371/journal.pone.0110903.g003Figure 3The percent migrated isoform at 15 mM GlcNAc.PMI after 4, 11 and 25 days of either high-dose rhEPO, low-dose rhEPO, or placebo. N = 15. Values are means with 95 % confidence intervals. *p<0.05; ** p<0.00001 compared with placebo. With 5 mM of GlcNAc, PMI of Epoetin beta (300 ng/l) was 4.8 (1.9) %, of normal plasma 17.2 (3.7) %, and of umbilical cord plasma 68.1 (16.0) % (Figure 2). PMI with placebo, reflecting the endogenous EPO isoforms, averaged 27.3 (8.5) % and 27.7 (8.4) % on day 11 and 25, respectively (Figure 4). On day 11, low-dose Epoetin beta and high-dose Epoetin beta decreased PMI to 10.5 (3.7) % (p<0.00001) and 9.5 (2.1) % (p<0.00001), respectively (Figure 4). On day 25, PMI did not differ between study days. 10.1371/journal.pone.0110903.g004Figure 4The percent migrated isoform at 5 mM GlcNAc.PMI after 11 and 25 days of either high-dose rhEPO, low-dose rhEPO, or placebo. N = 15. Values are means with 95 % confidence intervals. * p<0.00001 compared with placebo. BODY.DISCUSSION: This prospective, double-blinded study demonstrates that the MAIIA test is able to detect a change in the distribution of EPO isoforms three weeks after the last injection of three daily high doses of Epoetin beta and 12 days after termination of a series of low dose Epoetin beta administrations. Noteworthy, the disruption of PMI was seen even though plasma EPO concentration and haematological parameters had normalized. The data suggest that plasma analysis by MAIIA may provide a sensitive measure of former intake of Epoetin beta. The membrane assisted isoform immunoassay (MAIIA) assay was used in accordance with the principle outlined by Lönnberg et al. [11], [12], [20]. The MAIIA method was first described by Lönnberg et al. [11] as a rapid method for quantitative determination of low-abundance protein glycoisoforms in different types of biological samples. Compared with other methods, the sensitivity is high with a detection limit for EPO of 0.035 ng/l [21], [22]. Distinguishing between isoforms is based on the fact that differences in glycosylation between EPO isoforms in the presence of a low concentration of the competing GlcNAc solution results in fractionated release of isoforms from the WGA [7], [12], [22]. When expressed as percentage of migrated isoforms (PMI) in relation to the total amount of EPO that can be desorbed by a high GlcNAc solution, the method provides information about the relative distribution of circulating EPO isoforms. Because rhEPO isoforms bind more tightly to the WGA than endogenous isoforms, presence of rhEPO in a plasma sample results in a lower PMI than that measured in normal plasma. Even administration of very small doses of rhEPO has been reported to decrease PMI [23], [24]. In our subjects, PMI in normal plasma (placebo) averaged around 80 %, similar to values obtained in previous studies [12], [13]. By the use of samples from an earlier study [9], Lönnberg & Lundby [13] reported that a 28-days injection period of low-dose Epoetin beta significantly decreased PMI in plasma during the injection period. PMI had returned to normal values at 14 and 21 days after the last injection of Epoetin beta but was still significantly lower compared with baseline at day 7. The present data agree with this but indicate that the interval with significant alteration of PMI is at least 12 days. We added a series with short-term, high-dose rhEPO because this regime has been tested previously for its effects on central fatigue and cognition [25]–[27]. After three daily administration of high-dose Epoetin beta, PMI was still altered after 22 days, indicating a dose-dependent effect of Epoetin beta on circulating distribution of EPO isoforms. The discrepancy between studies may be due to the use of different concentrations of GlcNAc since the use of 10 mM of GlcNAc in the former study [13], as can be seen in Fig. 2, may result in lesser diversity between plasma with and without Epoetin beta. The optimization curves (Fig. 2) demonstrate that PMI, reflecting fractional specific desorptions of isoforms, depend upon the concentration of GlcNAc used in the low concentration. With 15 mM of GlcNAc, around 80 % of the EPO in normal plasma samples are desorbed, whereas PMI in soluted Epoitin beta was around 40 %, indicating that discrimination between endogenous EPO isoforms and Epoitin beta is best obtained with a GlcNAc 15 mM solution. Furthermore, the subjects in the present study served as their own control which eliminated inter-individual variation. Thus, our data suggest that optimization of MAIIA may favorably prolong the time span in which alteration of PMI can be used as an indirect marker of former use of Epoetin beta. The main challenge in revealing doping with rhEPO is that the target effect, the increase in haemoglobin mass, is maintained for weeks after elimination of rhEPO [1], [2]. Even with the development of direct tests with higher sensitivity and specificity than available today, rhEPO in plasma and urine cannot be expected to be detectable for more than a few days after the last administration. As an alternative to direct testing, specific changes in haematological variables induced by former administration of rhEPO may emerge as changes in the 'Athlete Biological Passport' [4], [5], [28], [29]. Initial trials of the concept, however, reported that these indirect screening methods were able to correctly indicate rhEpo injections in only 58 % of the subjects [8]. Injections of very small doses of rhEPO sufficient to increase haemoglobin mass did not result in alteration of measures provided by the Athlete Biological Passport software [10]. In contrast, analysis with MAIIA after this injection regime of rhEPO demonstrated a reduction in PMI for up to 72 h after a microinjection [23], [24]. In the present study, plasma values of haemoglobin, ferritin, and tranferrin saturation and reticulocyte counts had all normalized at day 25, corresponding to 12 and 22 days after the last administration of low-dose and high-dose Epoetin beta, respectively. At the same time, PMI decreased in all subjects, suggesting that MAIIA provides a sensitive measure of the presence of an aberrant EPO isoform pattern at time points when circulating levels of Epoetin beta have fallen below the detection limit of direct tests. MAIIA does not provide an exact molar measure of specific EPO isoforms but merely an indication of the relative binding to the lectin WGA in the actual pool of EPO isoforms. Presence of rhEPO isoforms in the sample will tend to lower the PMI, but the exact dose-response relationship remains unknown. Presumably, the present decrease in PMI is caused by remaining traces of Epoetin beta in plasma. In view of the farmacokinetics of Epoetin beta, however, it seems unlikely that there should be a significant contribution to the total EPO left at 22 and 12 days after last injection of high-dose Epoetin beta and low-dose Epoetin beta, respectively. The total EPO concentration on day 25 did not differ between series, also arguing against a contribution from earlier administred Epoetin beta. The N-glycans of both endogenous and recombinant EPO show a wide molecular variation so that the abundance of EPO consists of a mixture of isoforms [6], [30], [31]. Thus, another explanation for the suppression of PMI is that Epoetin beta causes a prolonged alteration in endogenous EPO synthesis and the isoform pattern. Previous studies have suggested that administration of rhEPO may trigger a negative feed-back mechanism that decreases the synthesis of renal EPO [14], [15], [32]. Epoetin beta causes a prompt decrease in plasma levels of renin and aldosterone, and renal clearance studies suggest that Epoetin beta decreases renal proximal tubular reabsorption rate leading to activation of the tubuloglomerular feedback mechanism and a fall in glomerular filtration rate [15]. Thus, treatment with rhEPO may result in suppression of endogenous EPO production through a decrease in intrarenal oxygen consumption [15], [32]. On the other hand, Epoetin beta also decreased PMI with the use of 5 mM of GlcNAc (Figure 4). At this concentration of GlcNAc, PMI in plasma from umbilical cords clearly deviates from values measured in normal plasma from adults (Figure 2) [16]. Whereas solutions of Epoitin beta consistently lowered PMI in relation to normal plasma, values in umbilical cord plasma were higher compared with normal plasma in adults. Most of the fetal EPO derives from the liver [33], [34] and EPO in umbilical cord plasma have be shown to differ from EPO in adults by the glycosylation pattern [35]. Thus, with 5 mM of GlcNAc, an increased hepatic production of EPO relative to the renal production should be expected to increase PMI [16]. The present finding that Epoetin beta decreased PMI at GlcNAc concentrations of both 5 and 15 mM argues against an effect of Epoetin beta on the relative contribution from endogenous sites of EPO synthesis. Taken together, the current study suggest that Epoetin beta causes an alteration in the EPO isoform pattern but the mechanisms remain unknown. Further studies are warranted to clarify the effect of rhEPO on endogenous EPO synthesis. The administration time of low dose Epoetin beta was of shorter duration than is normally used to produce an increase in hemoglobin mass [36]. Furthermore, we did not include supplementation with dietary iron. Thus, the present results are not readily comparable with common practices in doping. Exposure to high altitude hypoxia did not change the EPO glycoform heterogeneity [16], but the effect, if any, of iron supplementation or autologous blood transfusion remains unknown. The present cross-over design, however, allowed a controlled evaluation of the effects of Epoetin beta per se on PMI obtained by MAIIA, and the effect of more prolonged administration of low dose Epoetin beta is likely to be enhanced. Still, it remains unknown whether other EPO related medical products can be traced by MAIIA and the results from this experimental and translational study cannot be extrapolated to an anti-doping setting. Although the detection sensitivity of MAIIA is superior to the currently accredited isoelectric focusing (IEF) for testing of athletes [12], [13], further large-scale studies are needed to establish the exact cut-off values and the true false positive and false negative rates. In conclusion, this placebo-controlled, double-blinded, cross-over study suggest that administration of Epoetin beta in healthy subjects alters the EPO isoform distribution and leaves a footprint for up to three weeks after the last administration of rhEPO. BODY.SUPPORTING INFORMATION: Data S1 Raw data used in this study. (XLSX)Click here for additional data file.

TITLE: Are frail elderly patients treated in a CGA unit more satisfied with their hospital care than those treated in conventional acute medical care? ABSTRACT.OBJECTIVES: Our aim was to study whether the acute care of frail elderly patients directly admitted to a comprehensive geriatric assessment (CGA) unit is superior to the care in a conventional acute medical care unit in terms of patient satisfaction. ABSTRACT.DESIGN: TREEE (Is the TReatment of frail Elderly patients Effective in an Elderly care unit?) is a clinical, prospective, controlled, one-center intervention trial comparing acute treatment in CGA units and in conventional wards. ABSTRACT.SETTING: This study was conducted in the NÄL-Uddevalla county hospital in western Sweden. ABSTRACT.PARTICIPANTS: In this follow-up to the TREEE study, 229 frail patients, aged ≥75 years, in need of acute in-hospital treatment, were eligible. Of these patients, 139 patients were included in the analysis, 72 allocated to the CGA unit group and 67 to the conventional care group. Mean age was 85 years and 65% were female. ABSTRACT.INTERVENTION: Direct admittance to an acute elderly care unit with structured, systematic interdisciplinary CGA-based care, compared to conventional acute medical care via the emergency room. ABSTRACT.MEASUREMENTS: The primary outcome was the satisfaction reported by the patients shortly after discharge from hospital. A four-item confidential questionnaire was used. Responses were given on a 4-graded scale. ABSTRACT.RESULTS: The response rate was 61%. In unadjusted analyses, significantly more patients in the intervention group responded positively to the following three questions about the hospitalization: "Did you get the nursing from the ward staff that you needed?" (p=0.003), "Are you satisfied with the information you received on your diseases and medication?" (p=0.016), and "Are you satisfied with the planning before discharge from the hospital?" (p=0.032). After adjusted analyses by multiple regression, a significant difference in favor of the intervention remained for the first question (p=0.027). ABSTRACT.CONCLUSION: Acute care in a CGA unit with direct admission was associated with higher levels of patient satisfaction compared with conventional acute care via the emergency room. BODY.INTRODUCTION.BACKGROUND: Frailty is a biological syndrome reflecting vulnerability to stressors and reduced physiological reserves.1 It is associated with functional decline, activity limitations, prolonged recovery, and a high risk of being institutionalized and dying.2–5 Among older persons, the prevalence of frailty is estimated to be 10%–60%, depending on the frailty assessment instrument used and the population studied.6,7 Most of these patients have multiple chronic diseases and recurring episodes of acute illness. Frail elderly individuals constitute a high percentage of emergency patients in every hospital. The current organization of acute care is often poorly adapted to the specific needs of these patients. To meet these needs, in 2008, the NU (NÄL-Uddevalla) hospital group in the Västra Götaland Region of Sweden introduced two acute elderly care comprehensive geriatric assessment (CGA) units (MÄVAs).8 The basic aims were to optimize the clinical treatment and thereby utilize health care resources in the best way. This form of care is characterized by direct admission and a structured, systematic multi- and interdisciplinary CGA and care performed in the ward, including an early rehabilitation strategy involving physicians, occupational therapists, physiotherapists, and nurses as active team members. Previous clinical studies have indicated that frail elderly patients could benefit from a CGA.9–14 Patient satisfaction is an important patient-reported experience measure, and it can also be seen as an outcome in itself and as an integrated part of the results of every type of health care organization. It has been described as crucial for the evaluation of health care quality.15,16 In accordance with the consumer model, satisfaction can be regarded as a difference between expectations and assessed performance.17 On the other hand, it has been argued that it might be problematic to treat elderly patients as consumers.18 Patient satisfaction can also be defined as measuring the needs and wants,19 and it influences patient compliance as well as continuity of care.20,21 BODY.INTRODUCTION.IMPORTANCE: In acute care settings, there is, however, a lack of knowledge regarding the effects of CGA on health-related quality of life (HRQoL) and even more so on patient satisfaction. In the TREEE study (Is the TReatment of frail Elderly patients Effective in an Elderly care unit?), the results on HRQoL, activities of daily living (ADLs), mortality, and re-hospitalizations were favorable for the CGA units as described previously.14 In severely frail elderly patients with poor prognosis who need emergency care, the satisfaction with the received hospital care may be particularly important. BODY.INTRODUCTION.GOALS OF THE INVESTIGATION: Our aim in this follow-up of the TREEE study was to evaluate whether the acute care of frail elderly patients directly admitted to a CGA unit is superior to conventional acute medical care in terms of patient satisfaction. We hypothesized that a higher proportion of patients cared for in a CGA unit would report satisfaction compared with patients treated in a conventional care unit. BODY.PATIENTS AND METHODS.STUDY DESIGN AND SETTING: This study on patient satisfaction used data from the TREEE study, a clinical, prospective, controlled intervention trial with two parallel groups performed at the NU hospital group between March 2013 and July 2015. Details of TREEE have been described earlier.14 The total primary population of the NU health care system is 280,000 inhabitants. The TREEE study was approved by the Independent Ethics Committee at the Sahlgrenska University Hospital in Gothenburg (8883-12, 20121212) and registered at the Swedish National Database of Research and Development (identifier: 113021). Written informed consent was obtained from the patients or from a member of their next of kin. All data were computerized and handled in accordance with legislation and Good Clinical Practice. BODY.PATIENTS AND METHODS.SELECTION OF PARTICIPANTS: TREEE included patients aged ≥75 years, in need of in-hospital treatment, and who fulfilled the foundation of frailty in accordance with the recently validated FRESH (FRail Elderly Support researcH group) screening instrument,22 that is, two or more of the following criteria: tiredness from a short walk, general fatigue, frequent falls/anticipation of falls, dependence in shopping, and three or more visits to the emergency ward during the last 12 months. Patients were excluded if they were clearly suited for care in a conventional acute medical care unit due to the type or severity of the acute illness. When the ambulance nurse, or the primary care physician, had identified a patient who could fulfill the inclusion criteria, a MÄVA doctor was contacted via telephone. If he/she agreed, and there was a bed available at MÄVA, the patient was allocated to the intervention group and admitted directly to MÄVA. If bed was not available, the patient was allocated to the control group and admitted to a conventional acute medical care unit through the emergency room. In the TREEE study, 408 patients were included between March 2013 and July 2015. For practical and resourcing reasons, we could not start the distribution of the questionnaire on patient satisfaction until February 2014. Between February 2014 and July 2015, 229 patients were consecutively considered to be eligible in this part of the TREEE study (Figure 1). BODY.PATIENTS AND METHODS.INTERVENTION AND CONTROL.INTERVENTION GROUP: In addition to care in accordance with national and international guidelines, the CGA units are characterized by a structured, systematic interdisciplinary CGA and care via direct admittance to the wards (Table 1). The CGA involves physicians, nurses, occupational therapists, and physiotherapists as active team members. This implies a broad focus on physiological, psychological, and social factors via validated instruments, focusing on somatic and mental health, medication review, functional and activity ability including early rehabilitation, social situation, and early discharge planning. This also includes care guidance, adjusted to the needs of frail elderly patients. In order to individualize the assessment and treatment, the team has a person-centered approach. There are educational sessions in the intervention, including advice regarding physical exercise after discharge. There is a specially trained nurse responsible for the planning before discharge, with established contacts in primary care, home health care, and other types of support facilities. A formal discussion with the patient and relatives and a total medicine treatment evaluation are included in the routine. BODY.PATIENTS AND METHODS.INTERVENTION AND CONTROL.CONTROL GROUP: Patients are admitted to a conventional acute medical care unit via the emergency room, using standard clinical care and procedures in accordance with national and international guidelines. For both the groups, bedside medical examinations, electrocardiograms (ECGs), medical referrals, laboratory testing, X-ray examinations, blood transfusions, nutritional therapy, and drug treatment are included. BODY.PATIENTS AND METHODS.METHODS AND MEASUREMENTS: The data collection has previously been described in detail14 and is therefore only briefly repeated here. BODY.PATIENTS AND METHODS.METHODS AND MEASUREMENTS.CLINICAL AND DEMOGRAPHIC CHARACTERISTICS: These data were collected at the index hospitalization: age, gender, housing, diabetes mellitus, renal function, heart failure, other comorbidities, mini nutritional assessment (MNA-short form), and numbers of in-hospital care days. BODY.PATIENTS AND METHODS.METHODS AND MEASUREMENTS.THE CHARLSON INDEX SCORE: Charlson's comorbidity index (CCI) is a measurement of the total morbidity.23 It consists of 19 comorbidities, each of which is given a severity weighting depending on the risk of dying associated with this condition. The CCI was completed from medical records by the assessing physician. BODY.PATIENTS AND METHODS.METHODS AND MEASUREMENTS.THE FRESH SCREENING INSTRUMENT: Frailty was assessed using the FRESH screening instrument,22 which is a validated screening instrument for frailty shown to be appropriate in an emergency hospital setting. It consists of five questions (see the "Selection participants" section). The FRESH screening instrument was performed by a physician or a nurse at the time of inclusion. BODY.PATIENTS AND METHODS.METHODS AND MEASUREMENTS.ADL: ADL independence/dependence was assessed by using the ADL Staircase before discharge.24 This instrument consists of five personal ADL (PADL) items (ie, feeding, transferring, going to the toilet, dressing, and bathing), extended by four instrumental ADL (IADL) items (ie, cooking, shopping, cleaning, and transportation). The ADL Staircase was administered through interview and, if possible, observation. BODY.PATIENTS AND METHODS.METHODS AND MEASUREMENTS.THE SATISFACTION QUESTIONNAIRE: From February 2014 to July 2015, the patients were provided with a confidential satisfaction questionnaire to be filled in shortly after discharge from hospital by the patient alone, or with the help of relatives. The patient satisfaction questions were based on the experience with physicians and other team members in the MÄVA units and on questions and topics often brought up by patients and relatives. The aim was to create simple, clinically relevant questions, easy to respond to by ticking one of four options. The questions were carefully discussed internally, and we consulted broadly with geriatric expertise. Questions of the same kind have been used before,25–27 but not previously been formally validated in an acute care context. The questionnaire included four questions regarding patient satisfaction during the hospitalization period: Did you get help from the doctors with the medical problems you came in for? (1)Did you get the nursing from the ward staff that you needed? (2)Are you satisfied with the information you received on your diseases and medication? (3)Are you satisfied with the planning before discharge from the hospital? (4) The alternatives for answers to the questions were: (1) Great help, fairly great help, little help and very little help; (2) Yes always, yes often, no not often, and no seldom; (3) and (4) Very satisfied, fairly satisfied, fairly unsatisfied, and very unsatisfied. BODY.PATIENTS AND METHODS.OUTCOMES: The primary outcome was the level of satisfaction on each question reported by the patient after the index hospitalization. BODY.PATIENTS AND METHODS.ANALYSIS: For the TREEE study, it was estimated that 200 evaluable patients should be included in each study group.14 The two first response alternatives to each of the four questions were considered as positive, the remaining two responses to each question were considered as negative, that is, for each question, the responses were dichotomized before analysis. The data were computerized and analyzed using the Statistical Package for the Social Sciences (IBM SPSS Statistics for Windows, Version 22.0; IBM Corporation, Armonk, NY, USA). Student's t-test was used to compare the continuous, parametric data between the groups, and the χ2 test or Fisher's exact test was used to compare the categorical data. Adjustments for possible differences at baseline were made, using a logistic regression model including age, gender, and CCI score as covariates. BODY.RESULTS.CHARACTERISTICS OF STUDY SUBJECTS: In this follow-up to the TREEE study, 229 frail patients were consecutively identified to be eligible. Of these patients, 139 patients being allocated to the CGA unit group (n=72) or conventional ward group (n=67) responded to the questionnaire and were included in the analysis. The response rate was 61%. Baseline clinical and demographic characteristics are presented in Table 2. The two groups did not differ significantly in terms of age, gender, scores of ADL and MNA, and percentage living in residential care (all p>0.05). The intervention group presented with higher scores of frailty (p=0.046) and Charlson comorbidity index (p<0.001). Both the groups were heavily affected by diseases, particularly cardiovascular disease. Furthermore, dependence or difficulty carrying out PADL or IADL were common among the participants in both the groups. BODY.RESULTS.MAIN RESULTS: The responses to the four questions of the satisfaction questionnaire are reported in Table 3. For each question, the proportions of chosen alternatives on the 4-graded scale are referenced. In the intervention group, the responses were given by the patient alone in 36 cases, a next-of-kin in 11 cases, and a combination in 25 cases. In the control group, the responses were given by the patient alone in 28 cases, a next-of-kin in 11 cases, and a combination in 28 cases. Unadjusted analysis of the dichotomized responses of the questionnaire is shown in Table 4. Significantly more patients in the intervention group responded positively to the following three questions: "Did you get the nursing from the ward staff that you needed?" (p=0.003), "Are you satisfied with the information you received on your diseases and medication?" (p=0.016), and "Are you satisfied with the planning before discharge from the hospital?" (p=0.032). There was no difference between the groups in response to the question "Did you get help from the doctors with the medical problems you came in for?" (p=0.120). After adjusting for age, gender, and Charlson's index in multiple regression analyzes, a statistically significant difference in favor of the intervention remained for the second question "Did you get the nursing from the ward staff that you needed?" (p=0.027). Neither regarding the question "Are you satisfied with the information you received on your diseases and medication?" (p=0.059), nor for the responses of the other two questions (both p>0.05), there were any statistically significant differences in favor of the intervention group. For the questions "Are you satisfied with the information you received on your diseases and medication?" and "Are you satisfied with the planning before discharge from the hospital?" there was a correlation between satisfaction and higher age (both p<0.05). BODY.DISCUSSION: Our study shows that acute care of elderly, frail patients directly admitted to a CGA unit was associated with significantly higher levels of patient satisfaction compared with conventional acute care for three of the four components registered. This difference in favor of the intervention remained partly also after adjusting for age, gender, and comorbidity with a statistically significant difference for the question "Did you get the nursing from the ward staff that you needed?" In both the study groups, a broad majority of the patients reported overall positive satisfaction scores. Furthermore, for two of the questions, there was an association between positive answers and high age. This harmonizes with previous studies, indicating an association between patient satisfaction and age,28,29 particularly among female elderly patients.19 In the current study, the mean age was 85 years, and the majority were females. There is a growing evidence in favor of CGA units for frail elderly patients. The CGA and related care can be considered a complex intervention. Consequently, there may be several critical differences compared to conventional care, which may interact and benefit frail elderly patients with accompanying higher patient satisfaction. The previously reported results of the broader study indicates that well-structured team-based acute care for frail elderly patients in a CGA unit is superior to the care in a conventional acute medical care unit in terms of several clinically meaningful outcomes. The satisfaction results might be linked to these positive outcomes from the TREEE study.14 Moreover, in recent studies, it has been emphasized that patient involvement, participation, and communicating with patients about motivations and expectations might lead to higher satisfaction.19,30,31 A person-centered approach in order to individualize the assessment and treatment, which was applied by the team in the intervention group, might have facilitated these values. Furthermore, patients in the intervention group were directly admitted to the CGA care unit. In a previous study in an emergency context, long waiting times at the reception were associated with dissatisfaction.32–34 It can also be hypothesized that longer hospital stay in the intervention group made it possible to optimize the medical treatment, to inform the patients and relatives in more detail and to perform a more extensive care planning in cooperation with other care-givers. This might be another part of the explanation for the higher satisfaction in the CGA group. Elderly patients are so severely affected by frailty and multi-morbidity as our study patients generate a large part of everyday hospital care consumption in most western countries. There is therefore a particular need to build evidence relating to the treatment and care of this important patient group. Patient satisfaction is an integrated and important part of that work. This study was integrated in the standard daily clinical context and included a wide spectrum of diagnoses, which enhances the generalizability of the study results. Moreover, we studied very frail elderly patients in need of acute care, which adds complementary knowledge to former studies focusing on patients in a more stable and chronic phase. The MÄVA form of care can serve as one example of how acute care for frail elderly people can be organized. More research is needed to identify the most appropriate organizational forms adapted to the varying needs of the frail elderly patients, for example, stable chronic disease and acute illness. This further research should include evaluations of primary and municipal care, ambulant geriatric care, and specialized hospital care. In every such study, the evaluation of patient satisfaction should be an important part. BODY.LIMITATIONS: Our patient satisfaction study has some limitations, the main one being that the questions used in this survey are not formally validated. However, we wanted to take the opportunity to capture the opinions of our patients on these few, short, and clinically relevant questions. In addition, we did not find any validated instrument that fulfilled the needs of the TREEE study. The questions were based on available literature and intense discussions and broad consultations with experts. Another limitation is the low number of patients. However, the response rate of 61% is in the range of what has previously been reported in this kind of population. BODY.CONCLUSION: Acute care in a CGA unit with direct admission was associated with higher levels of patient satisfaction compared with conventional acute care via the emergency room.

TITLE: Impact of Exercise and Moderate Hypoxia on Glycemic Regulation and Substrate Oxidation Pattern ABSTRACT.OBJECTIVE: We examined metabolic and endocrine responses during rest and exercise in moderate hypoxia over a 7.5 h time courses during daytime. ABSTRACT.METHODS: Eight sedentary, overweight men (28.6±0.8 kg/m2) completed four experimental trials: a rest trial in normoxia (FiO2 = 20.9%, NOR-Rest), an exercise trial in normoxia (NOR-Ex), a rest trial in hypoxia (FiO2 = 15.0%, HYP-Rest), and an exercise trial in hypoxia (HYP-Ex). Experimental trials were performed from 8:00 to 15:30 in an environmental chamber. Blood and respiratory gas samples were collected over 7.5 h. In the exercise trials, subjects performed 30 min of pedaling exercise at 60% of VO2max at 8:00, 10:30, and 13:00, and rested during the remaining period in each environment. Standard meals were provided at 8:30, 11:00, and 13:30. ABSTRACT.RESULTS: The areas under the curves for blood glucose and serum insulin concentrations over 7.5 h did not differ among the four trials. At baseline, %carbohydrate contribution was significantly higher in the hypoxic trials than in the normoxic trials (P<0.05). Although exercise promoted carbohydrate oxidation in the NOR-Ex and HYP-Ex trials, %carbohydrate contribution during each exercise and post-exercise period were significantly higher in the HYP-Ex trial than in the NOR-Ex trial (P<0.05). ABSTRACT.CONCLUSION: Three sessions of 30 min exercise (60% of VO2max) in moderate hypoxia over 7.5 h did not attenuate postprandial glucose and insulin responses in young, overweight men. However, carbohydrate oxidation was significantly enhanced when the exercise was conducted in moderate hypoxia. BODY.INTRODUCTION: Obesity leads to multiple metabolic disorders, including insulin resistance and postprandial hyperglycemia. Exercise is an important 'therapy' in the treatment of postprandial hyperglycemia because muscle contraction stimulates glucose uptake by skeletal muscle [1]–[3]. Furthermore, we revealed that 4 weeks of endurance training under hypoxic conditions resulted in greater improvement of postprandial hyperglycemia than training under normoxic conditions [4]. In a study by Mackenzie et al., 2011 [5], type 2 diabetic patients rested or exercised for 60 min under hypoxic conditions. Blood glucose concentrations were significantly lower after 60 min of rest or exercise under hypoxic condition compared with baseline values. Although the mechanism of glucose lowering by hypoxic exposure is not fully understood, changes in substrate utilization patterns may be involved because acute exercise under hypoxic conditions increases carbohydrate oxidation [6]–[8]. In particular, enhanced glucose uptake by skeletal muscle is thought to account for increased carbohydrate oxidation under hypoxic conditions. Several studies have demonstrated that severe hypoxia stimulates the translocation of glucose across the plasma membrane [9], [10]. Kelly et al., 2010 [11] reported that plasma glucose response to a 75 g glucose load was significantly attenuated under severely hypoxic condition (a simulated altitude of 4300 m) in healthy adults. In an animal study, increased glucose uptake by the soleus muscle was observed after 1 week of exposure to severe (FiO2 = 10.0%) hypoxia [12]. Thus, severe hypoxic stimulation (a simulated altitude of> 4000 m) probably has a beneficial effect in preventing postprandial hyperglycemia through promoting glucose uptake by skeletal muscle. However, because the use of severe hypoxia would not be appropriate, due to the risk of acute mountain sickness (e.g., headache, nausea, and anorexia), exposure to moderate hypoxia (a simulated altitude of <3000 m) is more practical in terms of application for obese and overweight people. Although several studies reported that greater improvement of glucose tolerance by chronic staying or hiking at moderate or severe (altitude of 2200–4000 m) altitude was found [13]–[15], information about the influence of acute exposure to moderate hypoxia on glycemic regulation is still limited. Furthermore, no reported study has investigated the effect of rest and exercise under moderately hypoxic conditions on metabolic responses over a day in overweight subjects. Given this context, the purpose of the present study was to examine the effects of rest and exercise under 7.5 h of moderately hypoxic conditions on substrate utilization and postprandial metabolic responses in overweight men. We hypothesized that the combination of rest and exercise under moderately hypoxic conditions would increase carbohydrate oxidation and attenuate postprandial blood glucose response over the day. BODY.METHODS.SUBJECTS: Eight sedentary, overweight men participated. Their ages, physical characteristics, and fitness levels are presented in Table 1. The subjects were not participating in any training programs at the start of this study. All subjects were informed about the purpose of the present study and experimental procedure, and provided written informed consent. The study was approved by the Ethics Committee for Human Experiments at Ritsumeikan University, Japan. 10.1371/journal.pone.0108629.t001 Table 1 Physical characteristics and fitness. Characteristics Age (yrs) 27±3 Height (cm) 174.6±2.1 Body weight (kg) 87±1 BMI (kg/m2) 28.6±0.8 Percentage fat (%) 28.4±2 Waist hip ratio 0.92±0.1 Waist circumference (cm) 97.0±2.1 VO 2 max/BW (ml/min/kg) 37.9±1.9 mean ± SE. BODY.METHODS.EXPERIMENTAL DESIGN: Subjects visited the laboratory six times throughout the experimental period. During the first and second visits the subject's maximal oxygen uptake (VO2max) was assessed using a graded power test on an ergometer (828E, Monark, Stockholm, Sweden) under normoxic (first) and hypoxic (second) conditions. These tests were conducted on different days at least a week before experimental trial. The first load began at 60 W, and the load was increased progressively in 30-W increments every 2 min until exhaustion. The test was terminated when the subject failed to maintain the prescribed pedaling frequency of 60 rpm or reached an oxygen consumption (VO2) plateau. Respiratory gases were collected and analyzed using an automatic gas analyzer (AE300S, Minato Medical Science Co., Ltd, Tokyo, Japan). The data collected were averaged every 30 s. HR was measured continuously during the test using a wireless HR monitor (Acculex Plus; Polar Electro Oy). The test to measure VO2max was repeated under hypoxic conditions (FiO2 = 15.0%) on a separate day to determine the workload during an exercise trial under hypoxic conditions. Four experimental trials were subsequently carried out in a randomized crossover design. Each trial was separated by ≥ 7 days. The experimental trials consisted of four different scenarios: a rest trial under normoxic condition (FiO2 = 20.9%, NOR-Rest), an exercise trial under normoxic condition (FiO2 = 20.9%, NOR-Ex), a rest trial under hypoxic conditions (FiO2 = 15.0%, HYP-Rest), and an exercise trial under hypoxic conditions (FiO2 = 15.0%, HYP-Ex). These four trials were selected to determine the influence of the independent effect of "moderate hypoxic exposure only" or "rest and exercise under moderately hypoxic conditions" on metabolic responses over a day. We selected 15.0% (a simulated altitude of 2700 m) of FiO2 as a "moderate hypoxia" because previous studies reported that endurance training under this level of hypoxia further improved glycemic regulation compared with the same training under normoxic conditions [4], [16]. All trials were completed in an environmental chamber. The temperature and relative humidity in the environmental chamber were maintained at 24°C and 40%, respectively. The experimental protocol is shown in Figure 1. The four experimental trials were performed from 8:00 to 15:30 following an overnight fast (at least 10 h). In the rest trials (NOR-Rest and HYP-Rest), subjects rested on a chair, reading books or watching DVDs throughout each trial. In the NOR-Ex and HYP-Ex trials, they conducted three bouts of 30 min pedaling exercise at 60% of VO2max at 8:00, 10:30, and 13:00, and rested during the remaining periods in the chamber. Standard meals were provided at 8:30, 11:00, and 13:30, and subjects were instructed to consume the meals within 7 min. The meals consisted of 64% carbohydrate, 10% protein and 26% fat (864 kcal/meal). 10.1371/journal.pone.0108629.g001Figure 1Overview of the study design. BODY.METHODS.MEASUREMENTS ON EXPERIMENTAL TRIAL DAYS: Following an overnight fast, the subjects visited the laboratory at 7:30 in the morning and rested before the first blood collection. After a 30 min rest, a polyethylene catheter was inserted into an antecubital vein and a baseline blood sample was collected. Subsequently, respiratory gas, heart rate (HR), and percutaneous oxygen saturation (SpO2) were recorded. During experimental trials, blood samples were collected at baseline and immediately before each meal (at the end of exercise), and at 20, 40, 60, and 120 min after each meal (16 points in total; Fig. 1). Respiratory gas was collected at 10 points (baseline, immediately before, and at 60 and 120 min after each meal) to determine VO2, carbon dioxide production (VCO2), and ventilatory volume (VE). All respiratory variables were averaged in each 3-min period. The respiratory exchange ratio (RER), determined from the VO2 and VCO2 measurements, was used to estimate the relative contribution of carbohydrate (%carbohydrate contribution) and fat oxidation (%fat contribution) to total energy production [17]. HR and SpO2 were recorded at the same time points as respiratory gas sampling. Rate of perceived exertion (RPE) was monitored at the end of each exercise session. BODY.METHODS.BLOOD ANALYSIS: Blood glucose, lactate, serum insulin, free fatty acid (FFA), and glycerol were measured from the whole-blood and serum samples obtained. Serum samples were obtained from blood by centrifugation for 10 min, and were stored at −80°C until analysis. Blood glucose and lactate concentrations were measured immediately after blood collection. Concentrations of blood glucose and lactate were determined using an automatic glucose analyzer (Free Style; Nipro Corporation, Osaka, Japan) and a lactate analyzer (Lactate Pro 2; Arkray Inc. Kyoto, Japan), respectively. The samples for glucose concentrations were analyzed in duplicate. The intraclass coefficient for duplicate measurements in the analysis was 0.99. Serum insulin and FFA concentrations were measured using chemiluminescent enzyme immune assays (Fujirebio Inc., Tokyo, Japan) at a clinical laboratory (SRL Inc., Japan). Serum glycerol concentrations were measured in duplicate using an enzyme-linked immunosorbent assay (Cayman Chemical Company, Ann Arbor, MI, USA). The intra-assay CVs were as follows: 3.3% for serum insulin, 2.2% for serum FFA, and 1.2% for serum glycerol measurements. BODY.METHODS.STATISTICAL ANALYSIS: Data are expressed as means ± SE. A two-way analysis of variance (ANOVA) with repeated measures was used to test the interaction (trial × time) and main effect (trial, time). When ANOVA revealed a significant interaction or main effect, a Tukey-Kramer test was performed as a post hoc analysis to identify differences. For all tests, P values <0.05 were considered to indicate statistical significance. BODY.RESULTS.SPO: Figure 2 shows the time-course of changes in SpO2 and HR over 7.5 h. There were significant interaction (trial × time) and main effects for time and trial (P<0.05) for SpO2. In NOR-Rest and NOR-Ex trials, SpO2 did not change at any time point over the experimental period. The HYP-Rest and HYP-Ex trials showed significantly lower values of SpO2 than the NOR-Rest and NOR-Ex trials at all time points (P<0.05). 10.1371/journal.pone.0108629.g002Figure 2Time-course changes of SpO2 (A) and HR (B) over 7.5 h.Each period of exercise is indicated by the shaded boxes. The arrow indicates the time of meal consumption. a; P<0.05, NOR-Rest vs. HYP-Rest. b; P<0.05, NOR-Ex vs. HYP-Ex. c; P<0.05, NOR-Rest vs. NOR-Ex. d; P<0.05, HYP-Rest vs. HYP-Ex. There were significant interaction (trial × time) and main effects for time and trial (P<0.05) for HR. HR was significantly higher in the HYP-Rest trial than in the NOR-Rest trial after the second meal. In NOR-Ex and HYP Ex trials, HR was elevated significantly during the exercise period (P<0.05). However, HR responses did not differ significantly between the NOR-Ex and HYP-Ex trials over 7.5 h. BODY.RESULTS.BLOOD GLUCOSE AND SERUM INSULIN RESPONSES: Figure 3 shows the time-course of changes and area under the curve (AUC) over 7.5 h for blood glucose and serum insulin concentrations. There were significant interaction (trial × time) and main effects for time and trial (P<0.05) for the blood glucose response. There was no significant difference in fasting glucose concentration at baseline among the four trials. Although blood glucose concentrations increased significantly after each meal in all trials (P<0.05), there was no difference in blood glucose concentrations between the NOR-Rest and HYP-Rest trials at any time point. In the NOR-Ex and HYP-Ex trials, although each exercise reduced blood glucose concentrations relative to the values before exercise, blood glucose responses to the second and third meals (postprandial 20, 40, 60 min) were significantly greater than those in the NOR-Rest and HYP-Rest trials (P<0.05). When time-course changes in blood glucose concentrations over 7.5 h were compared using area under the curve (AUC), the AUC values did not differ significantly among the four trials. 10.1371/journal.pone.0108629.g003Figure 3Time-course changes and area under the curve of blood glucose (A) and serum insulin (B) concentrations over 7.5 h.Each period of exercise is indicated by the shaded boxes. The arrow indicates the time of meal consumption. c; P<0.05, NOR-Rest vs. NOR-Ex. d; P<0.05, HYP-Rest vs. HYP-Ex. For serum insulin responses, there were significant interaction (trial × time) and main effects for trial and time (P<0.05). Fasting serum insulin concentrations did not differ significantly among the trials. Although serum insulin concentrations increased significantly after each meal in all trials (P<0.05), there was no difference in serum insulin concentrations between NOR-Rest and HYP-Rest trials at any time point. In the NOR-Ex and HYP-Ex trials, serum insulin concentrations declined significantly during exercise relative to the values before exercise (P<0.05). When time-course changes in serum insulin concentrations over 7.5 h were compared using AUC, the AUC values did not differ significantly among the four trials. BODY.RESULTS.SERUM FFA, GLYCEROL, AND BLOOD LACTATE RESPONSES: Figure 4 presents the time-course changes in serum FFA, glycerol, and blood lactate concentrations. There were significant interaction (trial × time) and main effects for time and trial (P<0.05) in serum FFA concentrations. Fasting serum FFA concentrations did not differ significantly among the four trials. Serum FFA concentrations declined significantly after each meal in all trials (P<0.05). In the NOR-Ex and HYP-Ex trials, FFA concentrations increased significantly after each exercise (P<0.05). However, FFA responses did not differ significantly between the NOR-Ex and HYP-Ex trials over 7.5 h. 10.1371/journal.pone.0108629.g004Figure 4Time-course changes of FFA (A), glycerol (B) and lactate (C) concentrations over 7.5 h.Each period of exercise is indicated by the shaded boxes. The arrow indicates the time of meal consumption. c; P<0.05, NOR-Rest vs. NOR-Ex. d; P<0.05, HYP-Rest vs. HYP-Ex. There were significant interaction (trial × time) and main effects for time and trial (P<0.05) in serum glycerol concentrations. Fasting blood glycerol concentrations did not differ significantly among the four trials. In the NOR-Rest and HYP-Rest trials, serum glycerol concentrations did not change at any time point over the 7.5 h. In the NOR-Ex and HYP Ex trials, glycerol levels increased significantly during exercise (P<0.05). However, glycerol responses did not differ significantly between the NOR-Ex and HYP-Ex trials over the 7.5 h. For blood lactate responses, there were significant interaction (trial × time) and main effects for time and trial (P<0.05). Fasting blood lactate concentrations did not differ significantly among the four trials. Blood lactate concentrations increased significantly after the first meal in NOR-Rest and HYP-Rest (P<0.05). In the NOR-Ex and HYP-Ex trials, each exercise period significantly increased blood lactate concentrations relative to baseline values (P<0.05). These concentrations returned gradually toward resting levels after consumption of each meal. However, lactate responses did not differ significantly between the NOR-Ex and HYP-Ex trials over the 7.5 h. BODY.RESULTS.RELATIVE CONTRIBUTIONS OF CARBOHYDRATE AND FAT OXIDATION: Time-course changes in relative contributions of carbohydrate (%carbohydrate contribution) and fat oxidation (%fat contribution) to total energy expenditure are shown in Figure 5. The %carbohydrate contribution showed significant interaction (trial × time) and main effects for time and trial (P<0.05). At baseline (after entering the chamber), %carbohydrate contribution was significantly higher in the hypoxic trials (HYP-Rest and HYP-EX) than in the normoxic trials (NOR-Rest and NOR-Ex; P<0.05). However, no difference between NOR-Rest and HYP-Rest trials was apparent at the remaining points over the experimental period. Although exercise promoted carbohydrate oxidation in the NOR-Ex and HYP-Ex trials, %carbohydrate contribution during exercise was significantly higher in the HYP-Ex trial than in the NOR-Ex trial (P<0.05). During the post-exercise period, %carbohydrate contribution remained higher in the HYP-Ex trial than in the NOR-Ex trial, and a significant difference was observed at 120 min after the second exercise period (50.2±5.9% in the HYP-Ex vs. 39.7±3.6% in the NOR-Ex, P<0.05). 10.1371/journal.pone.0108629.g005Figure 5Relative contribution of carbohydrate (A) and fat (B) oxidation over 7.5 h.Each period of exercise is indicated by the shaded boxes. The arrow indicates the time of meal consumption. a; P<0.05, NOR-Rest vs. HYP-Rest. b; P<0.05, NOR-Ex vs. HYP-Ex. The time-course changes in %fat contribution showed significant interaction (trial × time) and main effects for time and trial (P<0.05). These changes were the opposite of those in %carbohydrate contribution, and values in %fat contribution were significantly lower in the hypoxic trials (HYP-Rest and HYP-Ex) than in the normoxic trials (NOR-Rest and HYP-Ex; P<0.05). At baseline, %fat contribution was significantly lower in the hypoxic trials (HYP-Rest and HYP-Ex) than in the normoxic trials (NOR-Rest and NOR-Ex; P<0.05). However, there was no significant difference in %fat contribution between NOR-Rest and HYP-Rest trials at the remaining points during the experimental period. The %fat contributions during each exercise period were significantly lower in the HYP-Ex trial than in the NOR-Ex trial (P<0.05). Lower levels of %fat contributions in the HYP-Ex trial were maintained during the post-exercise period, and a significant difference was observed at 120 min after the second exercise period (49.8±5.9% in the HYP-Ex vs. 60.3±3.6% in the NOR-Ex; P<0.05). BODY.DISCUSSION: This is the first study to investigate the combined effects of rest and exercise under moderately hypoxic conditions on substrate utilization and endocrine responses in overweight men. Postprandial blood glucose responses in hypoxic trials (HYP-Rest and HYP-Ex) did not differ compared with the normoxic trials (NOR-Rest and NOR-Ex) over 7.5 h. However, the HYP-Ex trial showed significantly higher carbohydrate oxidation than the NOR-Ex trial over the day. The novel finding of the present study was that neither rest alone nor rest and exercise in moderate hypoxia attenuated postprandial glucose responses, despite exercise in moderate hypoxia markedly promoted carbohydrate oxidation over 7.5 h. AMPK is an intracellular energy-sensing enzyme that promotes blood glucose transport with stimulation, such as exercise or hypoxia [10]. Because skeletal muscle is a major site for postprandial glucose disposal, the AMPK-related glucose transport pathway is considered to play a predominant role in maintaining glucose homeostasis in the whole body [18]–[20]. Thus, we hypothesized that greater glucose uptake via augmented AMPK activation would be observed in the hypoxic trials (HYP-Rest and HYP-Ex) compared with the normoxic trials (NOR-Rest and NOR-Ex). However, the blood glucose response was not significantly different among the trials over 7.5 h. Overweight or obese individuals generally show metabolic abnormality. In fact, AMPK activation has been reported to be impaired in overweight or obese people [21]. Because we selected overweight subjects in the present study, impaired AMPK activation in the present subjects may explain, at least in part, the lack of difference in the postprandial glucose response between the hypoxic and normoxic trials. As shown in Figure 3, postprandial glucose responses to second and third meals were significantly greater in the exercise trials (NOR-Ex and HYP-Ex) than in the rest trials (NOR-Rest and HYP-Rest). We did not expect these results because exercise has been shown to promote glucose uptake into skeletal muscle [22] and to attenuate postprandial blood glucose responses [23], [24]. However, several previous studies indicated that the postprandial glucose response was aggravated by conducting exercise immediately before a meal [25]–[27]. Aggravated glucose response by prior exercise may be associated with temporal insulin resistance immediately after exercise, characterized by impaired insulin secretion [28] and reduced hepatic glucose uptake [29] in response to the meal consumed. In support of this, insulin responses to the second and third meals in the NOR-Ex and HYP-Ex trials were smaller than in the NOR-Rest and HYP-Rest trials. In future studies, optimal timing of exercise to prevent increased postprandial glucose responses should be examined. A unique component of the present study was the comparison of time-course changes in substrate utilization patterns between normoxic and hypoxic conditions over 7.5 h in overweight men. In baseline data, evaluated after entering the environmental chamber, RER was significantly higher in the hypoxic trials than in the normoxic trials (P<0.05, data not shown). It is well known that acute hypoxic exposure induces hyperventilation [30], [31]. This may cause an overestimation of CO2 production [32]–[34] and affect the validity of calculating RER due to the elevated CO2. However, there was no significant difference in VE at baseline between the normoxic trials and hypoxic trials. Thus, it seems unlikely that hypoxia-induced hyperventilation affected the results of the present study. However, the higher levels of %carbohydrate contribution at baseline in hypoxic trials were temporary responses because there was no significant difference in %carbohydrate contribution between NOR-Rest and HYP-Rest trials at the remaining measurement points. We think that moderate hypoxic exposure alone did not have a strong impact on metabolic or endocrine responses. The relative contribution of carbohydrate oxidation (%carbohydrate contribution) during the exercise and post-exercise periods was significantly higher in the HYP-Ex trial than in the NOR-Ex trial, consistent with several previous reports. Peronnet et al., 2006 [8] and Katayama et al., 2010 [35] reported that carbohydrate oxidation during prolonged exercise was significantly higher in hypoxic conditions than in normoxic conditions. Brooks et al., 1991 [6], 1992 [36] and Roberts et al., 1996a [37], b [38] suggested that enhanced reliance on plasma glucose oxidation for energy production under hypoxic conditions leads to the promotion of carbohydrate oxidation during exercise. Data from the present study indicate that "moderate hypoxia" and "exercise" have synergistic effects in promoting carbohydrate oxidation during exercise and post-exercise periods. However, blood glucose and insulin responses did not differ between NOR-Ex and HYP-Ex trials over 7.5 h. Additionally, blood lactate concentrations during exercise did not differ significantly between the NOR-Ex and HYP-Ex trials. Thus, increased carbohydrate oxidation in the HYP-Ex trial was apparently not sufficient to affect blood variables regarding glucose metabolism. The relative contributions of fat oxidation (%fat contribution) during exercise and the post-exercise period were significantly lower in the HYP-Ex trial than in the NOR-Ex trial. The reduced fat oxidation under hypoxic conditions agrees with a previous study that reported impaired adipose tissue lipolysis after prolonged exposure to hypoxia [39]. However, in the present study, there was no significant difference in serum FFA or glycerol concentrations during exercise between the NOR-Ex and HYP-Ex trials, suggesting that exercise-induced adipose tissue lipolysis was not attenuated by moderate hypoxic exposure. An inconsistent result between fat oxidation and lipolysis has been reported previously. Ohkawara et al., 2013 [40] indicated that an increased meal frequency decreased FFA concentrations over a day, in contrast to fat oxidation, which was unchanged. We understand that some limitations should be carefully considered for interpretation of the present study. First, subjects in the present study were overweight men, but they did not have any metabolic disorder, which may affect the present results. Further studies using severely obese people or people with type 2 diabetes are necessary to extend our understanding. Second, substrate levels in blood do not correspond correctly to substrate utilization because substrate levels such as glucose, lactate or FFA are influenced by the rates of appearance and disappearance. This will partly explain for lack of attenuations of glucose and insulin responses in hypoxia, in despite of augmented carbohydrate oxidation. BODY.CONCLUSION: Neither rest alone nor rest and exercise under moderately hypoxic conditions attenuated postprandial glucose responses. Additionally, rest alone under moderately hypoxic condition did not affect the substrate oxidation pattern markedly. However, carbohydrate oxidation was enhanced significantly over 7.5 h when three bouts of submaximal exercise were incorporated under moderately hypoxic conditions.

TITLE: Comparative Efficacy of Tamsulosin Versus Tamsulosin With Tadalafil in Combination With Prednisolone for the Medical Expulsive Therapy of Lower Ureteric Stones: A Randomized Trial ABSTRACT.PURPOSE: To compare the safety and efficacy of tamsulosin and tamsulosin with the phosphodiesterase-5 inhibitor tadalafil in combination with prednisolone as medical expulsive therapies for lower ureteric stones. ABSTRACT.MATERIALS AND METHODS: Between July 2011 and December 2012, 62 adult patients presenting with distal ureteric stones sized 5 to 10 mm were randomized equally to treatment with tamsulosin (group A) or tamsulosin with tadalafil (group B). Therapy was given for a maximum of 6 weeks. In addition, patients in groups A and B were given 5-mg prednisolone once daily (maximum 1 week). The stone expulsion rate, time to stone expulsion, analgesic use, number of hospital visits for pain, follow-up and endoscopic treatment, and adverse effects of the drugs were noted. Statistical analyses were done by using Student t-test and chi-square test. ABSTRACT.RESULTS: There was a higher expulsion rate (83.9% in group B and 74.2% in group A) and a lower time to expulsion in both treatment groups than in historical controls used in earlier studies. However, these results were not statistically significant (p=0.349, p=0.074, respectively). Statistically significant differences were noted in hospitalization for colic and analgesic requirement, which were less in group B than in group A. There were no serious adverse events. Another important finding was improvement in erectile function in group B. ABSTRACT.CONCLUSIONS: Medical expulsive therapy for distal ureteric stones using tamsulosin and tadalafil with prednisolone is safe and efficacious. Also, the prescription of tadalafil in cases of erectile dysfunction with the development of lower ureteric stones may provide additional advantages. BODY.INTRODUCTION: The incidence of urinary stones has been increasing day by day. This may partially be attributed to better quality of life. Stone incidence also varies with race, ethnicity, and geographic region. Men are affected twice as commonly as women, with peak incidence being at 30 years of age. Ureteral stones contribute to 20% of all urinary tract stones, 70% of which are located in the distal ureter. Fifty percent of patients have a recurrence of renal colic within 5 years of the first episode. Urolithiasis is a chronic disease with substantial economic consequences and great public health importance [1]. Medical expulsive therapy developed after an understanding of the various physiologic and pathophysiologic bases for urinary stones. The ureter is lined by smooth muscle cells with alpha-1 adrenergic receptors, especially in the distal third. Receptor blockade inhibits both basal smooth muscle tone and hyperperistaltic uncoordinated frequency in order to maintain tonic propulsive contractions. Ureteric calculi can induce ureteric spasms that interfere with expulsion; thus, muscle relaxation while maintaining normal peristaltic activity may facilitate passage [2,3]. Therefore, alpha-1 adrenergic receptor antagonists work by creating an increased pressure gradient around the stone, which propels distal ureteral stones out of the ureter. Tamsulosin has a proven role in increasing the stone expulsion rate and in decreasing expulsion time [4,5]. Finally, it has been shown that ureteral calculi induce intense inflammatory changes and submucosal edema in proximity to a stone that may worsen ureteric obstruction, thus increasing the risk of impaction and retention. Thus, steroids can facilitate stone expulsion by reducing the submucosal edema. Recently, tadalafil, which is a phosphodiesterase-5 (PDE5) inhibitor, was shown to act by a nitric oxide/cyclic guanosine monophosphate (cGMP)-signaling pathway, resulting in increased levels of cGMP, leading to smooth muscle relaxation in the ureter [6]. Owing to its smooth muscle relaxation property, tadalafil received approval from the Food and Drug Administration for lower urinary tract symptoms associated with benign prostatic hyperplasia and erectile dysfunction. Daily dosing with 10 mg has shown better results and tolerance than 20 mg per day [7]. By combining drugs acting through different mechanisms, we can achieve better ureteric relaxation and reduction in intramural pressure, which will facilitate stone passage. This was our main aim in studying the use of tadalafil along with tamsulosin. BODY.MATERIALS AND METHODS: The study was performed in a tertiary care institute in Chandigarh, India, after the study investigators received clearance from the institutional ethics committee. Between July 2011 and December 2012, all patients older than 18 years of age with a ureteral stone 5 mm to 10 mm in size situated below the common iliac vessels as diagnosed by noncontrast computed tomography were included in the study if their pain was relieved with diclofenac injection within 1 day. Patients with fever, hydronephrosis, acute or chronic renal failure, multiple ureteral stones, a history of open surgery or endoscopic procedures in the urinary tract, diabetes, peptic ulcer, or concomitant treatment with β-blockers, calcium antagonists, or nitrates; pregnant or lactating mothers; and patients who demanded urgent stone removal were excluded. Sample size was calculated a priori with the alpha level set at 0.05, an anticipated effect size (Cohen's d) of 0.65, and a desired statistical power level of 0.8. The required sample size per group was 30. Unpaired t-test tests and chi-square tests were used for the analysis of the variables and categorical data. Differences were considered significant at a p-value of less than 0.05. Seventy patients were enrolled in the study, of whom 64 were studied, as the rest did not satisfy the inclusion criteria. Written informed consent was obtained and the patients were simply randomized into two equal groups of 32 patients by use of a computer-generated table. The randomization table was stored centrally and the group assigned to each patient was conveyed to the author. Patients in group A were given tamsulosin 0.4 mg once daily, and those in group B were given tamsulosin 0.4 mg and tadalafil 10 mg once daily. In addition, patients in groups A and B received prednisolone 5 mg once daily for 1 week. In both groups, drugs were continued until stone expulsion or for a maximum of 6 weeks. During the study, one patient in each group dropped out. All patients were evaluated by physical examination; serum creatinine; urine culture; plain x-ray of the kidneys, ureters, and bladder (KUB); ultrasonography; and noncontrast computed tomography of the KUB region. All patients presenting with ureteral colic were given pain relief with intramuscular diclofenac. Patients were instructed to filter their urine by using a standard mesh net to detect stone expulsion. The expulsion time; analgesic use; number of hospital visits for pain, follow-up, and endoscopic treatment; and adverse effects of drugs were noted. The maximum duration of follow-up was 6 weeks, after which patients underwent semirigid ureterorenoscopy for removal of stones that were not expelled. The primary outcome studied was the stone expulsion rate. Secondary endpoints were stone expulsion time, number of pain episodes, analgesic use, and self-reported side effects related to medical therapy. Expulsion of the stone was confirmed with plain x-ray, ultrasonography, or noncontrast computed tomography. Discrete variables were taken as counts (or frequencies) and were evaluated by chi-square test. Continuous variables with normal distributions were presented as mean±standard deviations and were compared by unpaired Student t-tests. Data were entered into a Microsoft Excel worksheet (Microsoft Co., Redmond, WA, USA) and were analyzed by using SPSS ver. 17 (SPSS Inc., Chicago, IL, USA). A p-value<0.05 was considered statistically significant. BODY.RESULTS: All patients completed the study. No statistically significant differences were observed between the groups regarding age, gender, or stone size distribution (Table 1). The stone expulsion rate was 74.2% in group A and 83.9% in group B. Although the stone expulsion rate was on the higher side in group B, the difference was not statistically significant (p=0.349). The mean expulsion time trended toward a lower value in group B (15.15±5.5 days) than in group A (18.9±8.7 days), but this difference was also not significant (p=0.074). The average number of hospital visits for colicky pain were comparatively fewer in group B (0.45±0.67) than in group A (2.90±0.90), and this difference was highly significant (p=0.000). Also, the mean analgesic requirement was significantly less in group B (1.87±1.38 times) than in group A (2.90±0.90 times) (p<0.0001). Although side effects such as headache, dizziness, orthostatic hypotension, and backache occurred more often in group B patients (p>0.05), these were not significant enough to exclude the patients from the study. Abnormal ejaculation was observed in 19.4% of patients in group A and 12.9% of patients in group B, which was not a significant difference (p=0.489). Another important finding to note was that none of the patients in group A experienced any changes in erectile function, whereas 12.9% of the patients in group B experienced improvement (Table 2). BODY.DISCUSSION: Urolithiasis is one of the most common urologic diseases. Among all urinary tract stones, 20% are ureteral stones, of which 70% are found in the lower third of the ureter [8]. The factors influencing spontaneous expulsion are stone location, size, number, and structure; ureteral spasm; mucosal edema or inflammation; and ureteral anatomy. Therefore, the use of medical therapy is justifiable to reduce edema, reduce spasm, and relax the smooth muscles for stone expulsion [9,10]. Current therapeutic options for distal ureteral stones include active intervention as well as conservative wait and watch approaches. The efficacy of mini-invasive therapies, such as extracorporeal shock wave lithotripsy and ureterorenoscopy, has been proven by several studies [11,12]. Although such procedures are effective, they are not free from risk or inconvenience and have consequent implications such as lowering the quality of life, high cost, and suspension of regular activities [13]. According to data in the literature, the distal ureteric stone expulsion rate with the watchful waiting approach is 25% to 54% with a mean expulsion time of greater than 10 days and considerable analgesic requirement, even for stones <4 mm. To increase the expulsion rate and reduce the analgesic requirement, there is a great deal of enthusiasm for adjuvant pharmacological interventions [14]. Conservative therapy is considered, especially in cases of distal ureteral stones. In 2005, Sigala et al. [15] found that α-1D and α-1A adrenoceptors are present in significantly larger amounts than α-1B adrenoceptors in the human ureter. Therefore, clinical studies have been conducted to investigate the effect of the combined α-1A- and α-1D-selective antagonist tamsulosin on distal ureteral stone expulsion. Most of these studies showed that tamsulosin treatment improves the expulsion rate of medium-sized (3-10 mm) stones. We also observed an expulsion rate of 74.2% with tamsulosin, which was better than the expulsion rates in historical controls used in earlier studies of 43% and 30.2% [16-19]. Thus, tamsulosin represents a noninvasive and cost-effective alternative to interventional approaches. Although medical expulsive therapy has become a standard treatment option, it is still underused by physicians in emergency departments [20]. We decided to use tadalafil on the basis of reports by Gratzke et al. [21] who demonstrated the role of phosphodiesterase inhibitors in relaxation of ureteric muscles in the rank order of vardenafil>sildenafil>tadalafil [22,23]. Because tadalafil is more selective than sildenafil for PDE5 than PDE6 receptors, which are present in the retina, visual problems are less likely. Tadalafil has the longest duration of action (-36 hours with a half-life of 17.5 hours) among the current PDE5 inhibitors, and its activity is unaffected by meals. Vardenafil has a structure similar to that of sildenafil, but the structure of tadalafil is quite different [24,25]. To keep adverse effects to a minimum, we used tadalafil in smaller doses (10 mg). Another reason to choose tadalafil with tamsulosin was because Kloner et al. [26,27] demonstrated that the combination of tamsulosin+ tadalafil did not show significant hemodynamic changes. This combination had also been used for the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia by Bechara et al. [28] and has shown significant improvements in pain by the relaxation of the bladder, urethra, and prostate and was the basis of this pilot study. We also combined prednisolone (5 mg) in both groups because of its antiedemic property. With regard to the primary end point of our trial, both groups compared in our study proved superior to the historical controls who were treated by the watchful waiting approach. We did not use a placebo or control group in the present study, because our objective was to prospectively compare the efficacy of these two groups, which included drugs that may modulate the motility of the obstructed ureter. We observed an apparently higher expulsion rate and lower expulsion time in group B than in group A; however, these results were not statistically significant [83.9%, 15.15±5.4 days (range, 7-27 days) compared with 74.2%, 18.9±8.7 days (range, 15-35 days); p=0.349 and p=0.074]. The possible explanation for these better results may be the combined spasmolytic effect of these medications on the ureter, whereas prednisolone reduces edema and decreases inflammation. Our results did not reach statistical significance, probably because of the small sample size. This study was undertaken as a pilot project. Thus, because such studies have not been conducted earlier, we could not perform a formal sample size calculation. Colicky pain in ureteral stones occurs owing to an increase in intraureteral pressure above the site of ureteral obstruction. Kinnman et al. [29] found that α-blockade may relieve ureteric colic by blocking the C-fibers responsible for mediating pain. Use of α-blockers for expulsion of ureteric stones probably decreases the analgesic requirement in two ways: expulsion of stones and blockade of C-fibers. Thus, it is difficult to assess which of these may be primarily responsible for decreasing the analgesic requirement. In our study, the analgesic requirement in group B was significantly less than that in group A (p=0.001). This excellent pain control observed in group B patients was also demonstrated by the lesser need for hospitalization for colic during the study. These effects of the combined use of tamsulosin and tadalafil on the ureter were probably due to a decrease in the frequency and amplitude of the phasic peristaltic contractions that accompany ureteric obstruction, i.e., an improved antispasmodic effect. The reported side effects were minimal in our study, probably because of the younger study population and the lack of any associated comorbidity. The use of a near physiological dose of prednisolone and the careful exclusion of patients with contraindications to steroids may explain the lack of significant side effects related to steroid use [30]. The 5-mg dose of prednisolone is nearly five times lower than the 30-mg deflazacort dose used in previous studies. Although abnormal ejaculation was observed in 19.4% of patients in the tamsulosin and prednisolone group A and 12.9% of patients in the tadalafil group B, this difference was not significant (p=0.489). Even though this study was not designed to demonstrate the association of drugs for the treatment of erectile dysfunction, we found improvement in erectile function in 12.9% of patients (i.e., 4 patients) in group B, whereas none of the patients in group A experienced any change. In the data analysis, we found that these patients were in the age group of between 46 and 48 years. The limitation of our study was the small sample size, but the study is still valuable as a pilot study. Furthermore, to our knowledge, this is the comparison study of tamsulosin with tamsulosin and tadalafil and produced some insightful results that should be tested in future studies. BODY.CONCLUSIONS: The results of this study indicate that the addition of tadalafil with tamsulosin and prednisolone increases the ureteric stone expulsion rate, although not significantly so, and provides significant control of pain, a significantly lesser analgesic requirement, and fewer hospital visits. Also, the prescription of tadalafil in cases of erectile dysfunction with the development of lower ureteric stones may provide an additional advantage in the expulsion of stones.

TITLE: The comparison of extemporaneous preparations of omeprazole, pantoprazole oral suspension and intravenous pantoprazole on the gastric pH of critically ill-patients ABSTRACT.BACKGROUND:: Stress-related mucosal disease occurs in many critically ill-patients within 24 h of admission. Proton pump inhibitor therapy has been documented to produce more potent inhibition of gastric acid secretion than histamine 2 receptor antagonists. This study aimed to compare extemporaneous preparations of omeprazole, pantoprazole oral suspension and intravenous (IV) pantoprazole on the gastric pH in intensive care unit patients. ABSTRACT.MATERIALS AND METHODS:: This was a randomized single-blind-study. Patients of ≥ 16 years of age with a nasogastric tube, who required mechanical ventilation for ≥ 48 h, were eligible for inclusion. The excluded patients were those with active gastrointestinal bleeding, known allergy to omeprazole and pantoprazole and those intolerant to the nasogastric tube. Fifty-six patients were randomized to treatment with omeprazole suspension 2 mg/ml (40 mg every day), pantoprazole suspension 2 mg/ml (40 mg every day) and IV pantoprazole (40 mg every day) for up to 14 days. Gastric aspirates were sampled before and 1-2.5 h after the drug administration for the pH measurement using an external pH meter. Data were analyzed using SPSS (version 21.0). ABSTRACT.RESULTS:: In this study, 56 critically ill-patients (39 male, 17 female, mean age: 61.5 ± 15.65 years) were followed for the control of the gastric pH. On each of the 14 trial days the mean of the gastric pH alteration was significantly higher in omeprazole and pantoprazole suspension-treated patients than in IV pantoprazole-treated patients (P < 0.001). ABSTRACT.CONCLUSION:: Omeprazole and pantoprazole oral suspension are more effective than IV pantoprazole in increasing the gastric pH. BODY.INTRODUCTION: Stress-related mucosal damage can be turned up in almost 100% of the patients admitted to intensive care units (ICUs) and may be developed within 24 h after admission.[12] The incidence of clinically important gastrointestinal (GI) bleeding, indicated as overt bleeding complicated by hemodynamic instability, low hemoglobin, and/or need for blood transfusion from stress-related mucosal disease (SRMD) is 3.5% in the ICU patients who are mechanically ventilated for ≥ 48 h.[3] In addition, this type of ulceration is accompanied by increasing the risk of mortality. Moreover, it prolongs the length of stay in the ICU.[3] Although ischemia of the gastric mucosa leads to SRMD, the significant role of gastric acid in the development of mucosal damage and bleeding could not be ignored.[456] Thus, early preventive prophylaxis of the probable GI bleeding, by means of acid-reducing agents, in these patients is rational. Proton pump inhibitors (PPIs) are the most potent and long-lasting medications used for this purpose. It is now generally believed that the aim of acid suppression as the prophylaxis of SRMD is to maintain gastric pH above 4.[7] Omeprazole, which is the first member of the PPI class, is commercially available as a delayed-release capsule and is formulated as enteric-coated granules to be protected against acid degradation. This formulation of oral PPIs put constraints on their usage in critically ill-patients who are NPO and unable to swallow the solid forms of drugs and those who experience an alteration in GI function after major surgery.[8] An alternative method of delivery that allows an aqueous administration, while protecting the intact drug from acid degradation, is omeprazole suspension. Omeprazole suspension, which is administered to critically ill-patients on mechanical ventilation, has been shown to prevent upper GI bleeding as well as maintaining gastric pH above 5.5.[1] The anti-secretory effect of intravenous (IV) pantoprazole, another drug of this family, has been shown in several studies.[91011] Pantoprazole is chemically more stable than other PPIs in higher pH conditions. It also provides earlier healing and superior pain relief in peptic ulcer and gastroesophageal reflux disease compared with omeprazole or histamine 2 receptor antagonists (H2RAs).[1213] The main objective of this study was to evaluate the effect of an extemporaneously formulated pantoprazole suspension, omeprazole suspension and commercially available IV pantoprazole on the gastric pH of critically ill-patients. The secondary objective was to assess the incidence of upper GI bleeding and ventilator-associated pneumonia in the ICU patients. BODY.MATERIALS AND METHODS.SUBJECTS: The inclusion criteria were as follow: Patients older than 16 years old who were admitted to medical and surgical ICUs of National Research Institute of Tuberculosis and Lung Diseases (NRITLD) with an anticipated stay of longer than 72 h. In addition, those who required mechanical ventilation for more than 48 h, had an Acute Physiology and Chronic Health Evaluation (APACHE II) score of bigger than 11 at baseline,[14] and had a nasogastric or orogastric tube in place. Participants were eligible for GI prophylaxis based on up to date defined risk factors.[15] Other acid-reducing agents, including H2RAs and antacids, which may alter the gastric pH, were removed from the patient's medications before the trial. Patients were excluded from the study if any of the following criteria were met: (1) A status of "no cardiopulmonary resuscitation;" (2) delay longer than 48 h from the time of initial eligibility; (3) known hypersensitivity to PPIs including omeprazole or pantoprazole; (4) history of gastric surgery; (5) active GI bleeding; (6) significant risk of swallowing blood; (7) admission for upper GI surgery; (8) inability to take a suspension by nasogastric tube; and (9) end-stage liver disease. If any of the following events were the case, the trial would stop: (1) Removal of a nasogastric tube (2) death; and (3) discharge from the unit [Figure 1]. Figure 1Reasons of discontinuation in treatment groups BODY.MATERIALS AND METHODS.STUDY DESIGN: The study was approved by the NRITLD Ethics Committee. Fifty-six patients were enrolled into this study. The study was a randomized, single-blind, uni-center study between October 2012 and September 2013. Patients were randomly placed in three groups based on a random number table. Group A received immediate-release omeprazole oral suspension 2 mg/ml (40 mg daily), Group B received immediate-release pantoprazole oral suspension 2 mg/ml (40 mg daily) and Group C received IV pantoprazole (40 mg daily) for at least 24 h and up to 14 days depending on their survival, length of stay and removal of nasogastric tube. Omeprazole and pantoprazole suspension were administered via a nasogastric tube. BODY.MATERIALS AND METHODS.EXTEMPORANEOUSLY PREPARATION OF THE SUSPENSION: A 20 mg omeprazole capsule was opened, and the granules were added to 10 ml of 8.4% sodium bicarbonate to achieve a final concentration of 2 mg/ml. The enteric-coated omeprazole granules were allowed to disintegrate with gentle agitation, suspending the omeprazole in the sodium bicarbonate solution.[16] For the preparation of pantoprazole suspension, a similar procedure was followed. A 40 mg pantoprazole enteric-coated tablet triturated into a homogeny powder. The powder added to 20 ml of 8.4% sodium bicarbonate solution resulting in a concentration of 2 mg/ml.[16] BODY.MATERIALS AND METHODS.GASTRIC PH ASSAY: Monitoring of gastric acidity (pH) began immediately before and 1-2.5 h after the drug administration in every trial day by means of an external pH meter (AZ 86502).[17] Due to the intermittent enteral feeding via the NG tube, enteral feeding was held for 2 h before the drug was given. If gastric aspirate contained "coffee-grounds" material, the sample was tested with gastroccult. The primary end point included the mean gastric pH on each trial day. Additional end points were assessment of the incidence of upper GI bleeding and nosocomial pneumonia. For the diagnosis of nosocomial pneumonia, we used Clinical Pulmonary Infection Score (CPIS) described by Pugin et al.[18] CPIS consists of six easily obtainable clinical and laboratory variables: (1) Body temperature, (2) blood leukocyte count and number of band forms, (3) character of tracheal secretions (purulent or not) and quantity of tracheal aspirates, (4) microscopic examination (Gram-stain) and semi-quantitative culture results of the bronchial secretions, (5) ratio of arterial oxygen tension and inspiratory fraction of oxygen (PaO2 /FiO2), (6) interpretations of chest X-ray, and the use of antibiotics. According to Pugin et al., a CPIS score ≥ 6 is considered as an excellent diagnostic tool for nosocomial pneumonia. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: For statistical analysis, raw data acquired of 56 patients (18 patients per omeprazole suspension and IV pantoprazole and 20 patients per pantoprazole suspension group) was entered into SPSS (version 21.0). Comparisons of categorical data were done using the Chi-square test, and parametric numerical data were analyzed by the ANOVA test. P < 0.05 was considered as statistically significant. Gastric pH was compared in all three groups utilizing mean difference of gastric pH alteration before and after treatment by Generalized Linear Model. BODY.RESULTS: The "baseline demographics" and the "baseline clinical characteristics" of 56 participants represented in Tables 1 and 2 respectively. Table 1 Baseline demographics Table 2 Clinical characteristics at baseline There was no statistically significant difference in the baseline demographics and clinical characteristics among the groups. The mean (standard deviation [SD]) age for a total population was 61.50 (15.65) years, which 48.21% were older than 65 years. For the whole population, baseline mean (SD) APACHE II score was 23.55 (7.40). All the participants had acute respiratory failure as a risk factor whereas none of them met trauma and shock. The majority of patients (48.21%) had sepsis as a presenting risk factor. Other risk factors included acute renal failure (35.71%), coagulopathy (28.57%), and surgery (5.36%). The baseline gastric pH had no statistically difference between the treatment groups with a mean (SD) pH for the total population of 5.52 (1.06). Table 3 presents the results of our primary end point-mean gastric pH after drug administration-on each trial day. Due to the important effect of the baseline pH on the gastric pH after the drug administration, we also calculated the mean of the gastric pH alteration. Table 3 Mean gastric pH after the drug administration On every 14 days, the mean gastric pH alteration values were significantly higher in omeprazole and pantoprazole suspension group after prophylaxis with each of the medications compared to IV pantoprazole-treated patients (P < 0.001, all days) [Figure 2]. Figure 2Mean of pH BODY.RESULTS.AVERAGE TIME TO ACHIEVE THE TARGET PH: We considered mean of gastric pH alteration ≥ 1 as a target. Average time to get this pH for the total population was 1.24 ± 0.61 days (1.35 ± 0.79 days in Group A, 1.17 ± 0.51 days in Group B and 1.2 ± 0.52 days in Group C). In addition, 83.6% of patients achieved the target pH after the first dose administration (76.5% in pantoprazole suspension group, 88.9% in omeprazole suspension group and 85% who received pantoprazole suspension). There was no statistically significant difference between three groups. BODY.RESULTS.INCIDENCE OF UPPER GASTROINTESTINAL BLEEDING: Protocol defined upper GI bleeding occurred in 3 (5.6%) patients from the total population. Two patients (11.1%) were in the IV pantoprazole-treated patients, and one of them (5.6%) was in the omeprazole suspension group. No patient in pantoprazole suspension-treated group was identified with upper GI bleeding. BODY.RESULTS.INCIDENCE OF PNEUMONIA: Sixteen (88.9%) patients from IV pantoprazole treated group, 14 (77.8%) patients from omeprazole suspension-treated group and 17 (85%) patients who had received pantoprazole suspension were diagnosed as having nosocomial pneumonia. BODY.DISCUSSION: This study demonstrated that extemporaneously prepared omeprazole and pantoprazole oral suspension produced higher mean gastric pH values than IV pantoprazole. There was statistically significant difference between omeprazole and pantoprazole oral suspension and IV pantoprazole (P < 0.001) [Table 3]. Phillips et al. studied a 20 ml dose of 2 mg/ml omeprazole suspension (containing 40 mg of omeprazole) initially, followed by 20 ml dose administered 6-8 h later, then 10 ml (20 mg) dose for stress-related mucosal damage in 75 patients undergoing mechanical ventilation who had at least one additional risk factor for upper GI bleeding. They observed that the omeprazole suspension increased gastric pH more than 7.1, maintained gastric pH > 5.5 and prevented clinically significant upper GI bleeding without increasing the incidence of nosocomial pneumonia.[8] These findings are completely in line with Lasky et al. study on 60 participants who assessed the effect of omeprazole suspension in preventing clinically significant upper GI bleeding and in preventing stress ulcers in mechanically ventilated trauma patients.[19] In a randomized, double-blind trial, Conrad et al. found that immediate-release omeprazole oral suspension is more effective than a continuous infusion of cimetidine in increasing gastric pH and in preventing upper GI bleeding of critically ill patients.[17] In a smaller, un-blinded, randomized trial Levy et al. compared omeprazole with ranitidine in 67 patients who had risk factors for stress ulcer-related bleeding. Results showed significantly more clinically important upper GI bleeding in the ranitidine group than in the omeprazole group due to the inadequate pH control in the ranitidine-treated patients. This satisfying control of gastric pH by omeprazole and pantoprazole oral suspension, without facing increased rate of pneumonia; which was previously reported by other studies,[2021] could be due to the presence of sodium bicarbonate in the extemporaneous formulation. Although enteric-coating is dissolved by bicarbonate, the alkaline properties of omeprazole suspension appear to protect omeprazole by passing through the stomach and actually aid in the initial control of gastric pH by acting as an antacid. The sodium bicarbonate with pH of 8.4 may stimulate the activation of parietal cells. Such stimulation may improve the pharmacodynamics of the drug by synchronizing the contact time of omeprazole with parietal cells activation.[8] We used nonsterile hospital pharmacy manufacturing service at Pharmaceutical Care Department, NRITLD in order to prepare a prescribed medication (omeprazole or pantoprazole as their commercially available dosage form) for individualized patients who are NPO. Thus, they are unable to swallow the solid forms of the drugs. An interesting finding of the current study is the control of gastric pH after the first dose of the administered drug in the majority (83.6%) of patients. Average time to achieve mean pH alteration bigger than 1 was 1.24 ± 0.61 days for the total population. It can be concluded from the recent data that there might be no need to pH monitoring after the day 1. The current study used an expanded definition for upper GI bleeding. Despite broadening the range of the definition application, which includes even minor self-limited bleeding and bleeding-related to a nasogastric tube trauma, only three patients (5.36%) experienced protocol-defined upper GI bleeding. Two of those who met our secondary end point of upper GI bleeding were among IV pantoprazole-treated patients, and the other one was among the omeprazole suspension group. No significant difference in preventive upper GI bleeding has been indicated between three groups. It has been noted that raising gastric pH ≥ 4 could be a risk factor for the development of nosocomial pneumonia. Increasing intragastric pH may allow bacterial (especially Gram-negative bacilli) proliferation in the duodenum and subsequently endotracheal colonization.[21] However, the clinical trials do not seem to support this hypothesis. A double-blind multi-center study that assessed the effect of sucralfate and ranitidine in 1200 critically ill-patients revealed no significant increase in nosocomial pneumonia with ranitidine although it showed a significant decrease in upper GI bleeding with the H2RA.[22] Despite higher mean gastric pH values in omeprazole and pantoprazole suspension group in comparison with IV pantoprazole-treated patients, we found no significant difference in the rate of nosocomial pneumonia among three treatment groups. Our results are in accord with the finding: As yet, no relation between the type of acid-reducing agent and the incidence of nosocomial pneumonia has been found. BODY.DISCUSSION.LIMITATIONS OF THE STUDY: (1) Due to several inclusion criteria to meet, we were not able to expand the sample size. (2) Restricted enteral feeding protocol for critically ill patients limited us to hold the enteral feeding for more than 2 h before and 1 h after the sampling. (3) Inefficient method of gastric acid measurement (aspiration technique) used in the study put constraints on sampling, especially in patients with low gastric secretions. (4) High mortality rate of patients at ICUs that was the second important reason causes the patients to stop the trial before the day 14. BODY.CONCLUSION: These findings indicate that extemporaneous preparations of omeprazole and pantoprazole oral suspension are more effective than IV pantoprazole in increasing gastric pH in critically ill-patients without no significant difference in the rate of nosocomial pneumonia between the three treatment groups.

TITLE: Enhancing needle visualization during parasagittal approach in paravertebral block for patients undergoing simple mastectomy using in-plane, multiangle ultrasound needle guidance system ABSTRACT.BACKGROUND:: Ultrasound-guided paravertebral blocks during breast surgeries with in-plane needle approaches can be challenging due to difficult needle visualization. The purpose of this study was to assess the usefulness of using a needle guide while performing in-plane parasagittal approach paravertebral block for breast surgery. ABSTRACT.PATIENTS AND METHODS:: Eighty patients, American Society of Anesthesiologists physical status I-III, aged 20-40 years with breast mass scheduled for simple mastectomy surgery, were involved in prospective, controlled, randomized study, and were randomly divided by closed envelope method into two groups: Group I (n = 40): Scheduled for ultrasound-guided paravertebral block or group II (n = 40): Scheduled for ultrasound-guided paravertebral block using the needle guide. Both techniques compared as regards: (i) Needle visibility and block performance time; (ii) number of needle passes; (iii) duration of the block; (iv) doctor and patient satisfaction; and (v) incidence of complications. ABSTRACT.RESULTS:: Needle visibility score was better in group II (2.92 ± 0.26 vs. 1.9 ± 0.44, P < 0.0001). Block performance time was shorter in group II (90.92 ± 15 vs. 128.25 ± 16s, P < 0.0001). A number of needle passes were less in group II (1.27 ± 0.45 vs. 2.2 ± 0.68, P < 0.0001). Doctor and patient satisfaction were better in group II (P = 0.015). No differences were found regarding the duration of the block and incidence of complications between groups. ABSTRACT.CONCLUSION:: A needle guide can help reduce the time needed to perform a parasagittal in-plane thoracic paravertebral block, with a significant reduction in the block performance time, the number of needle passes, better needle visibility and better doctor and patient's satisfaction. However, there was no significant difference regarding the duration of the block or incidence of complications. BODY.INTRODUCTION: Promoting patient safety and increasing health care quality have dominated the health care landscape during the last 15 years. Health care regulators and payers are now tying patient safety outcomes and best practices to hospital reimbursement. Many health care leaders are searching for new technologies that not only make health care for patients safer but also reduce overall health care costs. New advances in ultrasonography have made this technology available to health care providers at the patient's bedside. Point-of-care ultrasound assistance now aids providers with real-time diagnosis and with visualization for procedural guidance.[1] Thoracic paravertebral block (TPVB) is the technique of injecting local anesthetic adjacent to the intervertebral foramina, resulting in unilateral somatic and sympathetic nerve blockade. Previous studies have reported its effectiveness for thoracic surgery including breast surgery and relief of postoperative and chronic pain of unilateral origin from the chest and abdomen. The technique is relatively easy to learn and safer than a thoracic epidural. Its clinical advantages include the inhibition of stress and pressor responses to surgical stimuli, maintenance of hemodynamic stability, low incidence of complication, long duration of analgesia, and few contraindications. Recent advances in ultrasound technology can further increase the effectiveness and the safety of TPVB although identification of the nerve and needle is not still possible.[2] Needle visualization is important for safe and successful ultrasound-guided peripheral nerve block. However, accurate and consistent visualization of the needle tip can be difficult to achieve.[3] It is also a fundamental skill required for competency in ultrasound-guided regional anesthesia.[4] Ultrasound-guided regional anesthesia with in-plane needle approaches can be challenging due to difficult needle visualization. A needle guide can help reduce the time needed to complete a simulated nerve targeting procedure and enhance needle visualization for the novice sonographer in a phantom gel simulation.[5] This study aimed to outline a solution to the technical challenge associated with impaired needle visualization while performing paravertebral block for breast surgeries. BODY.PATIENTS AND METHODS: A prospective, controlled, randomized study was carried out at Kasey El-Aini Hospital in the period from July 2014 to February 2015, after obtaining permission from the Local Ethical and Research Committee and an informed consent from the patient. Eighty patients, American Society of Anesthesiologists physical status I-III, aged 20-40 years with breast mass scheduled for simple mastectomy surgery. Patients with suspected difficult paravertebral block due to obesity (body mass index [BMI] >30 kg/m2), back deformities or having a contradiction for the block were excluded. Patients were exposed to routine preoperative evaluation including history, examination, and investigations. No premedication was given to the patients since full cooperation during block performance was required. On arrival to the operating room, an intravenous (IV) catheter was placed in the upper limb contralateral to the surgical site, and saline solution started at 2 mL/kg/h. Standard anesthesia monitors (electrocardiography, oximeter, noninvasive blood pressure) were applied. Supplemental oxygen (via nasal prongs at 4 L/min) was also applied. Patients were randomly assigned to either group I (n = 40): Scheduled for ultrasound-guided paravertebral block or group II (n = 40): Scheduled for ultrasound-guided paravertebral block using the needle guidance system (CIVCO, Ultra-Pro IITM) [Figure 1]. Figure 1A low-frequency ultrasound transducer (CH5-2, 2-5 MHz) with the needle guidance system attached and the needle trajectory in a two different angles Randomization was carried out as follows: Prior to perform the block, the experienced anesthesiologist blindly chosen a slip of paper from a dark envelope that contained two slips, with each slip marked with one of the two patient groups. Whichever group was written on the paper determined the block technique that was to be used in that patient. The patient (after being positioned) in a lateral position, the region was prepared and draped, The spinous process of the fourth thoracic vertebra was located, then local infiltration of the skin with Lidocaine 2% to raise a bleb over the selected point of entry had been done. An ultrasound machine (SiemensTM, Acuson X300) A low-frequency ultrasound transducer (CH5-2, 2-5 MHz) was placed in the axial (transverse) plane at the selected level, with the transducer positioned just lateral to the spinous process [Figure 2]. The transverse processes and ribs were visualized as hyperechoic structures. The transducer was moved slightly caudad into the intercostal space between adjacent ribs to identify the thoracic paravertebral space (PVS) and the adjoining intercostal space. The PVS appears as a wedge-shaped hypoechoic layer demarcated by the hyperechoic reflections of the pleura below and the internal intercostals membrane above. The needle (B. Braun, Stimuloplex® Ultra, 22 gauge and 120 mm) was introduced in-plane with the transducer until a pop was felt as the needle penetrates the internal intercostal membrane, where the needle was, at that point, positioned in the PVS. Bupivacaine 0.25% in a dose of 20 ml was injected. Downward displacement of the pleura was seen with the injection, indicating proper spread of the local anesthetic. Figure 2An ultrasound image of a needle approaching the paravertebral space while using the needle guidance system Our primary outcome was the needle visibility score which was recorded on a scale from 1 to 3 (1 = poor, 2 = good, 3 = excellent). As we considered visualization of the whole needle or at least the tip of the needle adjacent to the target as an excellent, visualization of "non" of the needle as poor and the rest of partial views of the needle as good.[5] Other variables were also recorded, including procedure performance time (time from applying the probe to the skin, till finishing the injection of the local anesthetic), number of needle passes, and incidence of complications. Then induction of general anesthesia was accomplished using fentanyl 2 μg/kg, propofol 2-3 mg/kg, and rocuronium 0.6 mg/kg to facilitate endotracheal intubation. Anesthesia was maintained using isoflurane 1.5% in O2 /air mixture (FiO2 0.4). If mean blood pressure or heart rate exceeded 20% of the preoperative value, increments of fentanyl 25 μg had been given IV. Incremental doses of ephedrine (5 mg) were used to treat hypotension (mean arterial blood pressure <60 mmHg). After the completion of the surgery, patients received neostigmine and atropine for reversal of the muscle relaxant and had been extubated when the nerve stimulator showed a train-of-four ratio >0.75. The quality of sensory block was assessed by bilateral application of ice over the breast area at 30 min postprocedure once the patient was alert and oriented in the recovery room. Bilateral assessments were done to rule out the epidural spread. Sensory level of analgesia was recorded and patient satisfaction with analgesia documented on a scale from 0 to 3 (0 = terrible, 1 = poor, 2 = good, 3 = excellent). Pain intensity was measured using visual analog scale (VAS) (1-10), starting 30 min after transfer to the recovery room and then repeated every hour till the patient requested analgesia, and then every 2 h in the first 24 h postoperatively. If VAS was >3, the patient had been given: 1 g paracetamol infusion over 20 min, and if the pain persisted after 20 min, 0.05 mg/kg morphine IV was given. If pain persisted after 20 min, another dose of morphine 0.025 mg/kg would be given. BODY.PATIENTS AND METHODS.SAMPLE SIZE REPORT (POWER ANALYSIS): Sample size was calculated with a statistical power of 80%, alfa error of 5%, the confidence interval of 95%, and significance when P < 0.05. According to similar studies, the minimum sample size for each of the two study groups was 37 patients. We enrolled 40 cases per group to accommodate for dropouts. Statistical analysis was done using the SPSS for Windows, version 15 (IBM, SPSS Inc., Chicago, IL, USA). Categorical data were expressed by number and percentages, while continuous data were expressed by mean ± standard deviation categorical data were compared using Chi-square or Fisher's exact test as appropriate. Continuous data were first tested for normality by Kolmogorov-Smirnov test. Comparison of continuous data was done using a unpaired t-test. A P < 0.05 was considered statistically significant. BODY.RESULTS: Our study included 80 patients with breast mass scheduled for simple mastectomy surgery, with 40 patients in each group. Patient characteristics and procedural data of the study population were shown in Table 1. Table 1 Patient characteristics and procedural data of the study population The two groups showed no statistically significant differences regarding age or BMI [Table 2]. Table 2 Comparison between the two study groups Group II showed statistically significant better needle visibility score than group I (2.92 ± 0.26 vs. 1.9 ± 0.44) with a P < 0.0001 [Table 2 and Figure 3]. Figure 3Needle visibility score among the two study groups Group II was also associated with significant shorter block performance time than group I (90.92 ± 15 vs. 128.25 ± 16s) [Table 2]. Furthermore, the use of a needle guidance system while performing in-plane paravertebral block associated with a lesser number of needle passes (1.27 ± 0.45 vs. 2.2 ± 0.68) with a P < 0.0001 [Table 2]. Doctor and patient satisfaction were better in group II when compared to group I with a P < 0.0001 and = 0.001, respectively. No differences were found regarding duration of the block (4.64 ± 0.61 vs.4.62 ± 0.63, P = 1.00). And incidence of complications (2 [2.5%] vs. 0, P = 0.49) between the two groups [Table 2]. BODY.DISCUSSION: Needle visualization is important for safe and successful ultrasound-guided peripheral nerve block. However, accurate and consistent visualization of the needle tip can be difficult to achieve.[3] Our study shows that a needle guide can help reduce the time needed to perform a parasagittal in-plane TPVB, with a significant reduction in the block performance time, the number of needle passes, and better doctor and patient's satisfaction. In a study done by Kaur et al., evaluating a novel ultrasound machine with an electromagnetic-based needle guidance system for the placement of TPVB. The Sonix GPSTM needle guidance system (Ultrasonic, Richmond, BC, Canada) uses sensors in the needle and transducer to provide a real-time display of needle shaft and tip position relative to the ultrasound beam based on the needle trajectory. They concluded that the novel needle guidance technology provides an additional margin of certainty of needle and needle tip positioning during performance of TPVB, as with the ultrasound needle guidance system, real-time TPVBs were performed accurately and without clinical complications such as pleural puncture using in-plane and out-of-plane approaches.[6] But the unavailability and the high cost of that system make it unfeasible. Another study by Tsui describing in vitro demonstration of the potential use of a readily available laser-line unit to assist with in-plane needle alignment with the ultrasound plane in order to ultimately improve needle visibility during ultrasound-guided peripheral nerve block. It requires minimum specialized training and may allow for maximum flexibility with freehand needle insertions in a sterile fashion as this optical guide provides a clear visual indication of precise needle-beam alignment, and may prove useful in teaching and developing bimanual coordination in novices. However, a portion of the needle shaft has to protrude from the skin surface at all time to allow alignment with the laser.[7] But that may require the use of longer block needles that can be more difficult to manipulate. This method is also unsuitable for continuous catheter techniques that require the probe (and laser) to be encased in a sterile sleeve. Another study by Gupta et al., trying to improve needle visualization by novice residents during an in-plane ultrasound nerve block simulation using an in-plane multiangle needle guide, concluded that a needle guide can help reduce the time needed to complete a simulation nerve targeting procedure and enhance needle visualization for the novice sonographers in a phantom gel simulation.[5] But the limitations of this study that it had been performed in-veto (on a phantom gel) by inexperienced residents. In a review article by Chin et al. they tried to sort out challenges and solutions for needle visualization in ultrasound-guided regional anesthesia. They concluded that needle-beam alignment, echogenic needle design, needle manipulation, needle bevel orientation, surrogate markers of needle tip location, and ultrasound imaging technology can affect needle visualization.[3] BODY.DISCUSSION.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.DISCUSSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: An Electromyographic Evaluation of Subdividing Active-Assistive Shoulder Elevation Exercises ABSTRACT.BACKGROUND:: Active-assistive range of motion exercises to gain shoulder elevation have been subdivided into gravity-minimized and upright-assisted exercises, yet no study has evaluated differences in muscular demands. ABSTRACT.HYPOTHESIS:: Compared with gravity-minimized exercises, upright-assisted exercises will generate larger electromyographic (EMG) activity. Compared with all active-assistive exercises, upright active forward elevation will generate more EMG activity. ABSTRACT.STUDY DESIGN:: Controlled laboratory study. ABSTRACT.METHODS:: Fifteen healthy individuals participated in this study. The supraspinatus, infraspinatus, and anterior deltoid were evaluated. The independent variables were 11 exercises performed in random order. The dependent variable was the maximum EMG amplitude of each muscle that was normalized to a maximal voluntary isometric contraction (MVIC). ABSTRACT.RESULTS:: Each muscle demonstrated significant differences between exercises (P < .001), with upright active forward elevation producing the greatest EMG for all muscles (95% confidence interval [CI], 12% to 50% MVIC). The orders of exercise varied by muscle, but the 5 gravity-minimized exercises always generated the lowest EMG activity. The upright-assisted exercises (95% CI, 23% to 42% MVIC) for the anterior deltoid generated more EMG activity than did the gravity-minimized exercises (95% CI, 9% to 21% MVIC) (P < .05). The infraspinatus and supraspinatus demonstrated increasing trends in EMG activity from gravity minimized to upright assisted (P > .05). ABSTRACT.CONCLUSION:: The results suggest a clear distinction between gravity-minimized exercises and upright-assisted exercises for the anterior deltoid but not for the supraspinatus and infraspinatus. Between the 2 types of assisted exercises, the results also suggest a clear distinction in terms of active elevation of the arm for the supraspinatus and anterior deltoid but not for the infraspinatus. ABSTRACT.CLINICAL RELEVANCE:: Muscle activation levels increase as support is removed, but subdivision of active-assistive range of motion to protect the supraspinatus and infraspinatus may not be necessary. BODY: Improving active elevation commonly constitutes a large part of the rehabilitation associated with many shoulder conditions6,12,21 and is typically achieved with a combination of active-assistive and active exercise.12,19,20 However, objective evidence to guide exercise selection and progression for this level or exercise is limited.23,28 Active-assistive range of motion (AAROM) exercise programs to regain active elevation have been described as starting with gravity-minimized exercises and progressing to inclined- and upright-assisted elevation exercises.6,19,21,22 For patients with rotator cuff repairs, Levy et al21 initiated exercises with supine gravity-eliminated activities, which advanced to semisitting exercises and then, finally, standing elevation against gravity. Krupp et al19 recommended a progression from side-lying active shoulder flexion to supine active reaching to progressive upright reaching as a way to achieve upright active elevation following a biceps tenodesis or tenotomy. Bohmer et al6 used a sling suspended overhead to support the arm during gravity-eliminated active elevation exercises, progressing to upright-assisted exercises to reestablish active elevation for patients with impingement. It appears that these studies logically assumed that gravity-minimized elevation exercises should be less demanding than upright-assisted elevation exercises on the shoulder girdle muscles. No study has measured muscular activity with electromyography (EMG) to evaluate this effect of subdividing AAROM elevation exercises into gravity-minimized and assisted elevation. There is an assumption of increased muscular demand based on a biomechanical rationale but no empirical data to document the magnitude of change during this exercise progression. Therefore, the purpose of this study was to evaluate the EMG activity of the supraspinatus, infraspinatus, and anterior deltoid during active-assistive forward elevation exercises to determine if subdivisions of active-assistive exercise exist. We hypothesize that upright-assisted exercises will generate more EMG activity in the supraspinatus, infraspinatus, and anterior deltoid than that of gravity-minimized exercises. Secondarily, compared with all the AAROM exercises, upright active forward elevation will generate more EMG activity in these same muscles. BODY.MATERIALS AND METHODS.PARTICIPANTS: Fifteen participants volunteered for the study: 7 women and 8 men (mean age, 23.9 years; range, 22 to 28; height, 173.4 ± 10.6 cm; weight, 74.4 ± 15.9 kg). Before participation, all had the study explained to them, and all read and signed a consent form approved by the university's institutional review board. The criteria necessary to be included in the study was an age of 18 to 40 years, no previous shoulder injury to the dominant arm (fracture, dislocation, chronic pain, or surgery), no current shoulder girdle pain, and the ability to demonstrate full active range of motion. BODY.MATERIALS AND METHODS.REHABILITATION EXERCISES: The 11 exercises that we studied represent AAROM exercises typically used in our clinical practice to regain patients' active forward elevation. The exercises are described as they were demonstrated to the participants (Table 1). Participants randomly picked exercises from an envelope to minimize the effects of fatigue and order biasing. The patient was given a visual demonstration of each exercise and the opportunity to practice no more than 6 repetitions before data collection started. The patient then performed 5 repetitions of the exercise, controlling arm speed by following an electronic metronome (Seiko, Bloomfield, Connecticut) adjusted to keep a consistent 30 degrees per second for the 11 exercises. Table 1. Exercise descriptions and categorization by type of elevation exercise. Type of Exercise Description Gravity minimized  Dusting center Participant positioned sitting in front of an adjustable table with table at elbow height and elbow at midline of body. Participant placed hand on a towel and then slid towel directly forward and backward in sagittal plane until elbow was fully extended while keeping body still. (See Figure 1 .)  Dusting lateral Dusting exercise repeated 45° lateral to first exercise along plane of the scapula.  Dusting medial Dusting exercise repeated 45° medial to first exercise. Glenohumeral joint moved through an arc of approximately 0° to 45° for each of first 3 exercises.  Side-lying elevation Lying on nondominant side, dominant hand rested on ironing board top that was adjusted to height that dominant arm positioned near plane of the scapula with hand at chest level, elbow flexed humerus near the trunk. Participant instructed to slide towel on board with weight of hand resting on board, effectively elevating arm overhead to approximately 140° and then lowering hand to starting position. (See Figure 2 .)  Supine forward elevation with elastic resistance from 90° (supine band) Participant supine, feet resting flat on plinth, nondominant hand holding red Theraband at waist level. Exercise started with dominant shoulder flexed 90° and elbow extended. Participant actively moved arm into forward elevation of approximately 160°, thereby lengthening band. Dominant arm then lowered slowly back to starting position. No slack and only minimal tension allowed in band at starting position. Dominate arm maintained in external rotation throughout exercise. (See Figure 3 .) Upright assisted  Rope and pulley Starting with dominant arm resting at side, participant raised dominant hand by allowing nondominant hand to primarily assist arm elevation in plane of the scapula. Participant then lowered dominant arm back to original position primarily using nondominant arm. Arm traveled an arc of approximately 0° to 160° during exercise.  Wall walk Participant stood 20 cm to 40 cm from wall with dominant hand resting at shoulder level on wall. Participant instructed to walk hand up and down wall using index and middle fingers into forward elevation within an arc of approximately 30° to 160°. Performed taking 4 beats to walk up and down at a rate of 40 beats per minute.  Ball roll Participant stood 1 arm length from wall with a tennis ball against wall at shoulder height. Participant asked to roll ball up and down wall 1-hand length (approximately a 20° arc) while keeping elbow extended.  Standing T-bar active-assistive forward elevation In standing, participant instructed to primarily use nondominant arm to raise and lower dominant arm into elevation. Dominant arm grasped 1-in. PVC bar with thumb pointing up and moved through an approximately 160° arc. (See Figure 4 .)  Standing T-bar assistive elevation with active lowering Exercise performed exactly as standing T-bar active-assistive forward elevation except upon lowering dominant arm, participant released bar and under volitional control, actively eccentrically lowered dominant arm to starting position keeping elbow extended. Upright active  Active forward elevation While standing, participant asked to actively raise and lower dominant arm from side to full overhead motion through approximately a 160° arc. Movement performed in the plane of scapula, elbow extended, thumb pointed up. Elevation exercises were divided into 3 types: gravity minimized, upright assisted, and upright active. For this study, we defined gravity-minimized elevation exercise as any exercise in which (1) gravity provides assistance to the participant to complete the concentric portion of the exercise or (2) the primary movement is perpendicular to gravity with the weight of the arm supported. In this study, gravity-minimized exercises included dusting center (Figure 1), dusting medial and lateral, side-lying elevation (Figure 2), and supine forward elevation with elastic resistance from 90° (Figure 3). The second type of elevation exercise, upright assisted, is any exercise performed in the upright position that elevates the arm against gravity during the concentric phase of motion with assistance. In this study, assistance was provided (1) by the hand being supported by an object during movement or (2) by the hand moving on an object. Upright-assisted elevation exercises included the rope and pulley, wall walk, ball roll, standing T-bar active-assistive forward elevation (Figure 4), and standing T-bar assistive elevation with active lowering. The third type of elevation exercise, upright active forward elevation, was performed unsupported against gravity to serve as a comparison with the active-assistive exercises. Figure 1.A, starting position for dusting center; B, ending position. Figure 2.A, starting position for side-lying elevation; B, ending position. Figure 3.A, starting position for supine forward elevation with elastic resistance from 90°; B, ending position. Figure 4.A, starting position for standing T-bar active-assistive forward elevation; B, ending position. BODY.MATERIALS AND METHODS.PROCEDURES: Surface and fine wire EMG data were collected from 3 muscles: anterior deltoid (surface), supraspinatus (fine wire), and infraspinatus (fine wire). The rationale for using surface on the anterior deltoid is that surface electrodes have a larger pickup area and are more representative of the muscle's activity.3,7 The supraspinatus is deep to the upper trapezius, so fine wire is necessary. The fine wire instrumentation was used to reduce cross-talk from other musculature for the infraspinatus.3,8 The dominant arm, designated as the throwing arm, was instrumented for the study. Before surface electrode placement, the participants' skin was prepared by abrasion with fine sandpaper and vigorously cleansed with isopropyl alcohol wipes to decrease electrical impedance.3 Bipolar Ag/AgCl disposable surface electrodes (Medicotest, Olstykke, Denmark) with a 2-cm interelectrode distance were applied to the anterior deltoid one-third the measured distance from anterior acromion to the deltoid tuberosity.29 Electrodes were aligned parallel to the underlying muscle fiber orientation with the ground electrode placed on opposite acromion.13 Two muscles, supraspinatus and infraspinatus, were instrumented with indwelling fine wire electrodes (California Fine Wire, Grover City, California). The fine wire electrodes, sterilized in a 27-gauge needle, were inserted into the respective muscle bellies using a 2-needle technique with the interelectrode distance of 1 cm.14 Electrode placement was visually confirmed to ensure placement and minimal cross-talk during active motions. An electronic goniometer (Biometrics Ltd, Ladysmith, Virginia) was adhered to the spine of the scapula and the midline of the humerus with the arm abducted to 90°. The middle of the electronic goniometer was aligned with the center of the posterior glenohumeral joint. The electronic goniometer was monitored during data collection to ensure that it stayed securely affixed during all exercises (Figure 5). The electrical signal from the goniometer indicated the initiation and completion of the exercise and was used during EMG data reduction to define a trial. Figure 5.Demonstration of the electronic goniometer and electrode placement during the supine forward elevation exercise with elastic resistance from 90°. Two trials of 5-second maximal voluntary isometric contractions (MVICs) were performed for each muscle using manual muscle-testing techniques previously described.15,16 Anterior deltoid and supraspinatus were tested with the humerus in plane of the scapula, flexed to 90° in neutral rotation. The infraspinatus was tested with the humerus abducted and elbow flexed to 90° and with the shoulder internally rotated 30°.15 The highest 500-ms root mean square amplitude recorded over the two 5-second trials represented 100% EMG activity for each muscle.2 A 5-second resting baseline was recorded for each participant while standing with one's hands at the side, given that the majority of the exercises were performed in an upright position. The root mean square amplitude for 1 second of this resting data was calculated to subtract out electrocardiographic and background noise signal from the amplitude data recorded during the exercises.25 The maximal electrical activity collected during the exercises is represented as a percentage of the MVIC, for statistical comparison. The rationale for evaluating only maximal activity was to estimate the greatest muscle activity likely to be generated during an exercise. These exercises are typically used during rehabilitation when healing tissues are relatively weak. The greatest demand was important to identify to protect healing tissues. BODY.MATERIALS AND METHODS.EMG DATA REDUCTION: The electrodes and electric goniometer were connected to a Myopac amplifier belt unit (Run Technologies, Mission Viejo, California). The kinematic data gain and sampling were set at 1000 Hz. The amplifier has a common mode rejection ratio of 90 dB at 60 Hz. The raw indwelling EMG data were collected with 1000 gain at a rate of 2000 Hz and band pass filtered at 10 to 1000 Hz, whereas the surface EMG data were collected with 2000 gain at rate of 1000 Hz and band pass filtered at 20 to 500 Hz. A 30-ms root mean square centering algorithm was applied to all EMG data.26 The amplified signals were transmitted to a PC-type computer through a 12-bit A/D board (Measurement Computing, Norton, Massachusetts). Datapac software (Run Technologies, Mission Viejo, California) was used to process, analyze, and store all analog data. Five exercise trials were identified for each exercise with the electric goniometer. The maximal amplitude for each trial was determined as a percentage of the respective muscles' MVIC. The average of the 5 maximal amplitudes recorded from each muscle and each exercise was used for statistical analysis. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Each maximal muscle activity was analyzed separately because of the differing electrode pickup area between surface and indwelling electrodes. Three separate repeated measure analyses of variance were used to determine if differences between exercises existed to determine an exercise progression. The level for significance was set at P < .05, and if found, a Bonferroni post hoc analysis was used to control for family-wise error rate and to minimize for a type I error due to the multiple comparisons (adjusted significance, P < .00091). A Greenhouse-Geiser correction was applied to all analyses of variance to correct for assumption of compound symmetry. The intratrial reliability of the EMG data (based on an intraclass correlation model; ICC2,3) and the standard error of measurement were determined.9 All statistical analysis was performed with SPSS (version 16). BODY.RESULTS: Tables 2 to 4 present the descriptive data of the average maximal EMG activity for the 3 muscles studied. The analysis of variance with Greenhouse-Geiser correction demonstrated significant differences between exercises for the supraspinatus, F(3.3, 46.7) = 19.5, infraspinatus, F(3.2, 42.1) = 7.8, and the anterior deltoid, F(4.3, 60.4) = 34.5, muscles (P < .001). Bonferroni post hoc analysis for the supraspinatus revealed that upright active forward elevation (29% ± 13% MVIC) was significantly greater than all other exercises (Table 2). All gravity-minimized exercises were not significantly different from one another, and all upright elevation exercises were significantly larger than side-lying elevation (7% ± 5% MVIC). Wall walk (21% ± 10% MVIC) was the only upright-assisted elevation exercise that was significantly greater than all gravity-minimized exercises for the supraspinatus muscle. Table 2. Average maximal supraspinatus muscle activity. a % MVIC No. Type Exercise Mean ± SD 95% CI SEM ICC Post Hoc 1 GM Side-lying elevation 7 ± 5 4, 9 2 .89 2 GM Supine band 8 ± 6 5, 11 6 .63 3 GM Dusting medial 12 ± 6 8, 15 3 .90 4 GM Dusting center 12 ± 7 8, 16 3 .83 5 GM Dusting lateral 13 ± 7 9, 17 2 .94 6 UA T-bar active-assistive forward elevation 16 ± 9 11, 21 6 .69 1, 2 7 UA Ball roll 16 ± 9 11, 21 2 .96 1, 2 8 UA Rope and pulley 17 ± 10 11, 23 4 .89 1 9 UA T-bar active low 20 ± 11 13, 26 3 .96 1, 2 10 UA Wall walk 21 ± 10 16, 27 2 .94 1-5 11 A Active forward elevation 29 ± 13 22, 37 4 .95 1-10 a Organized in ascending order for the average maximal supraspinatus muscle activity (percentage maximal voluntary isometric contraction; % MVIC) with associated standard deviation and 95% confidence intervals (CIs). The intratrial reliability (intraclass correlation coefficient; ICC) and standard error of measure (SEM) are reported as well as the type of exercise: gravity minimized (GM), upright assisted (UA), or upright active (A). Bonferroni post hoc analysis revealed multiple significant differences between exercises ( P < .05). Exercises that are significantly less than a particular exercise are indicated by exercise number; for example, ball roll was significantly larger than side-lying elevation (No. 1) and supine band (No. 2). When no significant differences exist between exercises for a muscle, no number is presented. Table 3. Average maximal infraspinatus muscle activity. a % MVIC No. Type Exercise Mean ± SD 95% CI SEM ICC Post Hoc 1 GM Dusting medial 7 ± 5 4, 10 1 .96 2 GM Dusting center 8 ± 5 5, 10 2 .91 3 GM Dusting lateral 9 ± 6 6, 13 2 .92 4 GM Side-lying elevation 10 ± 7 6, 14 3 .92 5 GM Supine band 13 ± 8 9, 18 6 .51 6 UA T-bar active-assistive forward elevation 13 ± 10 8, 19 9 .72 1, 2 7 UA Rope and pulley 14 ± 8 9, 18 1 .98 1, 2 8 UA Ball roll 18 ± 11 11, 24 3 .96 1, 2 9 UA T-Bar active low 18 ± 13 11, 26 3 .98 1, 2 10 UA Wall walk 19 ± 13 11, 27 2 .98 11 A Active forward elevation 21 ± 15 12, 30 2 .98 1 a Organized in ascending order for the average maximal infraspinatus muscle activity (percentage maximal voluntary isometric contraction; % MVIC) with associated standard deviation and 95% confidence intervals (CIs). The intratrial reliability (intraclass correlation coefficient; ICC) and standard error of measure (SEM) are reported as well as the type of exercise: gravity minimized (GM), upright assisted (UA), or upright active (A). Bonferroni post hoc analysis revealed multiple significant differences between exercises ( P < .05). Exercises that are significantly less than a particular exercise are indicated by exercise number; for example, rope and pulley was significantly larger than dusting medial (No. 1) and dusting center (No. 2). When no significant differences exist between exercises for a muscle, no number is presented. Table 4. Average maximal anterior deltoid muscle activity. a % MVIC No. Type Exercise Mean ± SD 95% CI SEM ICC Post Hoc 1 GM Dusting lateral 14 ± 9 9, 19 3 .94 2 GM Supine band 15 ± 5 12, 18 4 .83 3 GM Dusting center 16 ± 8 11, 20 2 .92 4 GM Dusting medial 16 ± 7 12, 20 3 .67 5 GM Side-lying elevation 16 ± 8 12, 21 7 .82 6 UA Ball roll 27 ± 8 23, 32 6 .73 1-5 7 UA T-Bar active-assistive forward elevation 30 ± 10 25, 36 5 .84 1-5 8 UA Rope and pulley 31 ± 13 23, 38 11 .59 1-4 9 UA Wall walk 32 ± 9 27, 36 3 .79 1-5 10 UA T-bar active low 35 ± 11 29, 42 8 .59 1-5 11 A Active forward elevation 45 ± 9 40, 50 10 .57 1-10 a Organized in ascending order for the average maximal anterior deltoid muscle activity (percentage maximal voluntary isometric contraction; % MVIC) with associated standard deviation and 95% confidence intervals (CIs). The intratrial reliability (intraclass correlation coefficient; ICC) and standard error of measure (SEM) are reported as well as the type of exercise: gravity minimized (GM), upright assisted (UA), or upright active (A). Bonferroni post hoc analysis revealed multiple significant differences between exercises ( P < .05). Exercises that are significantly less than a particular exercise are indicated by exercise number; for example, rope and pulley was significantly larger than dusting lateral (No. 1), supine band (No. 2), dusting center (No. 3), and dusting medial (No. 4). When no significant differences exist between exercises for a muscle, no number is presented. Post hoc analysis for the infraspinatus revealed that upright active forward elevation (21% ± 15% MVIC) generated the most EMG activity but was significantly greater than only the dusting medial exercise (7% ± 5% MVIC), which generated the least EMG activity (P = .00089) (Table 3). Only 2 gravity-minimized exercises, dusting medial and center, resulted in significantly lower EMG activity than 4 of the upright-assisted elevation exercises, rope and pulley, ball roll, and both T-bar exercises (Table 3). Bonferroni post hoc analysis for the anterior deltoid activity revealed that active forward elevation (45% ± 9% MVIC) was significantly greater than all other exercises (Table 4). Upright-assisted elevation exercises were found to generate significantly more anterior deltoid activity than that of all gravity-minimized exercises (Table 4); one exception to this result was the rope and pulley exercise (30% ± 12% MVIC), which was not different from side-lying elevation exercise (16% ± 7% MVIC) but approached significance (P = .00092). BODY.DISCUSSION: Relative muscular demand classifies EMG activity into 4 categories: low (< 20%), moderate (20% to 40%), high (41% to 60%), and very high (> 60%).10 According to the present results, all gravity-minimized exercises are low and would thus be used in the earliest stage of a rehabilitation continuum to regain active motion. The EMG levels for these exercises have not been previously reported, making comparisons difficult. Mean EMG values have been reported for the supraspinatus and infraspinatus during continuous passive motion and therapist-assisted passive elevation and did not exceed 10% MVIC.11 This is similar to the maximal EMG activity (range, 7% to 13% MVIC) found during gravity-minimized exercises. Even though maximal EMG activity was calculated and indwelling electrodes were used in the current study (which differ from the mean amplitudes and surface electrodes used by Dockery11), it appears that all gravity-minimized exercises have muscle activity similar to that of previously reported passive elevation exercises. Therefore, gravity-minimized exercises may be a good first step in progressing from passive range of motion to AAROM exercises. Upright-assistive exercises fall into the low and moderate categories, and their levels of muscular activity appear to agree with results of other studies.11,23 Dockery et al11 found that during rope and pulley elevation, average muscle activity of the supraspinatus, infraspinatus, and anterior deltoid were 18% ± 8% MVIC, 10% ± 5% MVIC, and 25% ± 15% MVIC, respectively (standard deviations estimated from figure), which aligns with the current study's findings. As expected, active elevation was categorized as moderate demand and served as reference to differentiate active from assistive exercises. The current study's results support a significant difference between active and assistive exercises for the anterior deltoid and supraspinatus but not for the infraspinatus. Kronberg et al18 reported higher activation levels than those found in this study during forward flexion for the infraspinatus and anterior deltoid (55% MVIC and 75% MVIC, respectively). In both the current study and Kronberg's study,18 maximum supraspinatus muscular activity was 30% MVIC. Unfortunately Kronberg et al18 did not report standard deviations nor the speed of movement during EMG recording. Alpert et al1 documented increased speed of movement and increased EMG amplitudes. Our slow exercise speed (30 degrees per second) may partially account for the lower EMG values observed. A progression of AAROM elevation exercises has been suggested, beginning with gravity-minimized exercises and progressing to upright-assisted elevation exercises,6,19,21-23 as based primarily on a biomechanical rationale. Gravity-minimized elevation exercises provide minimal resistance to the forward elevation movement and are typically performed supine (to decrease the lever arm) or side lying (to support the weight of the arm).6,19,21,22 Inclined active exercise or upright-assisted elevation exercises increase gravity's resistance to the forward elevation by positioning the patient in more upright positions and are theorized to be less demanding than fully upright active elevation exercise because of the reduced gravitational resistance.6,19,21,22 Before this study, the assumption of increased demand was based on the biomechanical rationale—namely, that assisted movement directly against gravity would be more demanding than movement in a gravity-minimized position—but without empirical data to document the magnitude of muscular activity change between these exercise types. Although the average maximal EMG activity level reflects a gradual progression, beginning with gravity-minimized and progressing to upright-assisted elevation exercises for both the supraspinatus and the infraspinatus, a strong distinction between the 2 exercise types for both muscles was not found. Post hoc analysis revealed statistically significant differences between only selected exercises (Tables 2 and 3) and a large overlap in the 95% confidence interval; therefore, our hypothesis was rejected that upright-assisted elevation exercises generate more EMG activity than that of gravity-minimized exercises for the supraspinatus and infraspinatus. For protection of the supraspinatus and infraspinatus, it appears that a subdivision of AAROM elevation exercises into gravity-minimized exercises and upright-assisted elevation exercises is not necessary. Therefore, progression of exercise should be based on other factors, such as available range of motion, pain tolerance, and presence of substitution patterns. Because a clear distinction between gravity-minimized and upright-assisted elevation exercise was found for the anterior deltoid, subdivision of these AAROM exercise types is warranted in the rare situations where deltoid protection is necessary, such as an open rotator cuff repair or deltoidplasty. The current study identified 2 exercises worthy of further comment. The wall walk, which is commonly prescribed to gain active and passive elevation, was found to generate more EMG activity than that of all gravity-minimized AAROM exercises for the anterior deltoid and supraspinatus. The EMG data suggest that it should not be considered a passive range of motion exercise and that it is most appropriately used late in the AAROM progression. The "supine forward elevation with elastic resistance" exercise generated comparable EMG activity as the other gravity-minimized exercises and the passive elevation exercises reported by Dockery et al.11 The current study's results suggest that this resistance exercise is similar to a beginning AAROM exercise and highlights the importance of body positioning in determining muscle activity for lower level forward elevation. This study's results are limited in their interpretation because no direct relationship has been established between dynamic EMG activity and tension in the respective musculotendinous structures. Only in isometric contractions has a moderate correlation been found between muscle tension and EMG activity.4 All surface EMG research is subject to cross-talk from surrounding musculature, and the possibility of other muscular activity may have contributed to the electrical signal recorded. This was minimized by using small electrodes with a 2-cm interelectrode distance near the midsection of the muscles studied.3 Indwelling EMG data do not depict the muscular activation of the entire muscle.17 Therefore, the indwelling and surface EMG data were analyzed and reported separately. The results are also limited owing to a study population of healthy young participants instead of older participants with shoulder girdle pathology. The EMG amplitudes and activation patterns during particular motions may be altered by the effect of pain or altered movement patterns because of range of motion restrictions in a patient population. Previous EMG studies have used similar populations in making recommendations for shoulder exercises.5,24,27 In conclusion, a clear distinction in EMG activity level between active elevation and both types of assistive elevation exercise was found for the supraspinatus and anterior deltoid but not for the infraspinatus. In comparing EMG activity levels for gravity-minimized exercises and upright-assisted elevation exercises, a clear distinction between exercise types was found for only the anterior deltoid. No widespread differences were observed for the supraspinatus and infraspinatus. The results of this study do not support the biomechanical rationale of assumed increased muscle demand to the supraspinatus and infraspinatus with AAROM elevation exercises that progressively exercise the arm against gravity. It appears that subdividing AAROM elevation exercises based on muscle activity is not necessary to protect the supraspinatus or infraspinatus.

TITLE: Trial of Tramadol Plus Gabapentin for Opioid Detoxification ABSTRACT.BACKGROUND:: Substance abuse or drug addiction is one of the most important health issues in every society, which can lead to physical and mental problems. ABSTRACT.OBJECTIVES:: This study aimed to compare the efficacy of tramadol plus gabapentin versus methadone use in the treatment of opiate withdrawal. ABSTRACT.PATIENTS AND METHODS:: Consenting male subjects who fulfilled the DSM-4 criteria for opiate dependence syndrome (opium, residue, and heroin) were randomly assigned in two groups to receive tramadol plus gabapentin or methadone. Assessment tools were Adjective Rating Scale for Withdrawal (ARSW), Clinical Opiate Withdrawal Scale (COWS) and Visual Analogue craving Scale (VAS). Fifty-nine subjects were enrolled and evaluated on days 1, 2, 3, 4, 6, and 8 during their 10 days of admission. Twenty-nine participants received methadone and the other 30 received tramadol plus gabapentin for their treatment. ABSTRACT.RESULTS:: Mean (SD) age of the patients in methadone group and tramadol plus gabapentin group were 33.9 (7.1) and 32.4. (8.1), respectively (P = 0.462). The overall ARSW (P value = 0.263) and COWS (P = 0.862) scores between the two groups were comparable. The differences in the VAS score for craving between the two groups was marginally significant (P = 0.057). The highest VAS score was at the third day of admission in both groups and it was generally higher in methadone group. ABSTRACT.CONCLUSIONS:: The severity of withdrawal syndrome in two groups was not significantly different. The craving was higher in the group receiving methadone from the second day of admission even though the usage amount was higher in the tramadol plus gabapentin group. The findings of this study suggest that the combination of tramadol plus gabapentin is an efficient method for opioid detoxification. BODY.1. BACKGROUND: Substance abuse or drug addiction is one of the most important health issues in every society, which can lead to physical and mental problems (1). Detoxification is the first step after a patient is willing to go for the treatment and it has to be done in the most efficient and convenient way. The detoxification method has to overcome the withdrawal syndrome and help the patient to reduce the signs and symptoms as much as possible through the process (2, 3). Opioid withdrawal syndrome is a set of symptoms that occurs 6-8 hours after discontinuation of opioid class drugs. Different methods have been introduced for the treatment of opioid withdrawal syndrome, which include the opioid and non-opioid medications. They consist of opioid antagonists (naloxone, naltrexone); complete and partial opioid agonists (buprenorphine, methadone, levo-α-acetylmethadol), and α2 adrenergic agonists (clonidine, lofexidine) (3, 4). Also, their efficacy and success rate has been reported from 10% to 90% (5, 6). Tramadol is an atypical analgesic with a dual mechanism of action: serotonin and norepinephrine reuptake inhibition and modest μ-opioid agonist. A prospective human laboratory study demonstrated that oral tramadol doses of 200 or 400 mg produced modest opioid withdrawal suppression among opioid-dependent research volunteers. These participants were maintained on subcutaneous morphine (60 mg/d) who experienced opioid withdrawal (3). Another drug is methadone, which is a synthetic narcotic analgesic compound developed in Germany just prior to World War II. After the war, methadone was studied and found to have effects similar to those of morphine but with longer duration (7). Different studies have shown that gabapentin is effective in the reduction of withdrawal syndrome (8, 9). Another study suggests that gabapentin reduces opioid use during a 10-day buprenorphine detoxification procedure (10). Rapid detoxification, which usually takes less than 5 days is more appropriate for patients using heroin (6). It is recommended that we avoid using anesthesia-assisted rapid opioid detoxification (AAROD) because of its mortality and morbidity in favor of evidence-based options for opioid dependence treatment (11). Transcutaneous electrical acupoint stimulation (TEAS) is an acceptable, inexpensive adjunctive treatment that is feasible to implement on an inpatient unit and may be a beneficial adjunct to pharmacological treatments for opioid detoxification (12). A retrospective cohort control study suggests that tramadol may be comparable to buprenorphine in the management of mild to moderately severe heroin withdrawal (13). Another study indicated a few clinical differences between parenteral buprenorphine and oral tramadol protocols when used in the management of acute heroin withdrawal. As a consequence, tramadol shows some promise as an opioid withdrawal management medication (14). However, in another research, tramadol was found to have limited detoxification efficacy in moderate to severe opioid withdrawal and substantial risk of seizures as compared to buprenorphine (15). In another study, tramadol compared to clonidine (in the management of heroin withdrawal) was more effective in preventing most withdrawal symptoms (16). A study that used the objective opioid withdrawal scale (OOWS) to compare the efficacy and safety of tramadol versus methadone showed that tramadol may be as effective as methadone in the control of withdrawal syndrome (17). Buprenorphine and methadone appear to be the most effective detoxification treatment compared to α2-adrenergic agonists (18). BODY.2. OBJECTIVES: This study aimed to compare the efficacy of tramadol plus gabapentin versus methadone in treatment of the opiate withdrawal. BODY.3. PATIENTS AND METHODS: This clinical trial was conducted on male opiate dependent subjects admitted to Shahid Beheshti Hospital in Kerman (Iran) in 2013. Shahid Beheshti is a state-run psychiatric hospital with almost 200 beds. Inclusion criteria consisted of opioid dependency diagnosis based on DSM-IV-TR, substance abuse for at least a year, and age range from 18 to 65 years. The eligible patients were enrolled consecutively. And the exclusion criteria were consumption of other medications such as BZD, TCA, corticosteroids, clonidine and codeine, iatrogenic dependency, critical medical conditions like diabetes, acute hepatitis, and other liver diseases. There were no subjects who met the exclusion criteria. Using census method, all eligible subjects were enrolled in the study. The study was approved by the Ethics Committee of Kerman University of Medical with the registration number of k90/530. Written informed consent was signed by all participants after a thorough explanation of the study by the physician. They were assured that their information would be kept confidential and they can leave the study whenever they want. A demographic questionnaire was then filled out at the first day of admission and the participants were randomly assigned (using random digit numbers) in two groups by a nurse. The physician was unaware of the patients group. The sample size was calculated to find out a 20% difference in withdrawal symptoms considering type I error of 0.05 and 80% power. The control group received 10 to 30 mg of methadone (syrup) in the first day and it was reduced 2.5 to 5 mg each day from the second day. The other group received 200 to 300 mg of tramadol (tablet) at the first and second day for patients using opium and residue and 300 to 400 mg for patients using heroin divided in 3 doses, 100 mg was added in the third day and was reduced to 50 mg each day from the fourth day. Nine hundred miligram of gabapentin (capsule) was given to them each day divided in three doses. The patients were visited every day and the withdrawal syndrome and craving were assessed at the first, second, fourth, sixth, and eight day of admission using the COWS, ARSW and VAS (in Persian language), which their reliability were confirmed (19). The physician assessing was blind to the treatment group. However, the patient was aware of the treatment and all 59 subjects were enrolled in the study. Statistical analysis was done using the SPSS software (v19). The independent t test and chi-square were used to compare nominal and quantitative variables (Mann-Whitney U for non-parametric data). 2-way repeated measure analysis of variance (ANOVA) was used to compare the COWS, ARSW and VAS (which was converted to a number from 0 to 10). Because the data distribution was normal, we used square root transformation. BODY.4. RESULTS: Out of 59 subjects, 29 patients took the methadone treatment and 30 patients received the tramadol and gabapentin treatment protocol. As presented in Table 1, the mean age of the patients (33.9 years in methadone group and 32.4 years in tramadol and gabapentin group) and years of dependency (5.5 years in methadone group and 5.9 years in tramadol and gabapentin group) were comparable in both groups; however, the pre-intervention drug consumption was higher in tramadol and gabapentin treatment group (P = 0.02). Marital status, degree of education and occupation were not significantly different in the two groups. As demonstrated in Figure 1, the square of ARSW score has been compared between two treatment groups at the first, second, fourth, sixth, and eight day of admission. The differences in the ARSW score between two groups were not significant (P value = 0.263). As demonstrated in Figure 2, the square of COWS score has been compared between two treatment groups at the first, second, fourth, sixth, and eight day of admission. The differences in the COWS score between two groups were not significant (P value = 0.862). As demonstrated in Figure 3, the square of VAS score has been compared between two treatment groups at the first, second, fourth, sixth, and eight day of admission. The differences in the VAS score between two groups were nearly significant (P = 0.057). In methadone group an upsurge of clinical signs was seen in the third day of treatment, whilst in tramadol and gabapentin treatment group a smooth decrease in severity of clinical signs was seen from the onset of treatment. Table 1 . The Mean Age of the Patients and Years of Dependency Were Comparable in Both Groups Characteristics Methadone Tramadol + Gabapentin P Value Age, y a 33.9 ± 7.1 32.4 ± 8.1 0.462 Years of dependency a 5.5 ± 6.2 5.9 ± 4.8 0.538 Usage amount (converted to methadone), mg/d a 99.1 ± 112 163.2 ± 147 0.02 Marital status 0.937 Single 8 8 Married 21 22 Type of drug 0.003 Opium 21 18 Residue 8 3 Heroin 0 9 Degree of Education 0.165 None 1 5 Some education 17 10 High school diploma 9 12 University degree 2 3 Occupation 0.155 Unemployed 0 3 Part-time 25 21 Full-time 4 6 a Data are presented as Mean ± SD. Figure 1.The Square of Adjective Rating Scale for Withdrawal Score has Been Compared in the Two Treatment Groups at the First, Second, Fourth, Sixth, and Eight Day of Admission Figure 2.The Square of Clinical Opiate Withdrawal Scale Score has Been Compared in the Two Treatment Groups at the First, Second, Fourth, Sixth, and Eight Day of Admission Figure 3.The Square of Visual Analogue Craving Scale Score has Been Compared in the Two Treatment Groups at the First, Second, Fourth, Sixth, and Eight Day of Admission BODY.5. DISCUSSION: This clinical trial was conducted on 59 male opiate dependent subjects, which were treated in two different groups. Twenty-nine subjects received methadone and 30 subjects received tramadol and gabapentin for treatment. All the patients were aware of their medications. Although the usage amount (which was much higher in the tramadol plus gabapentin group, P value = 0.02) and type of drug abused (P value = 0.003) was significantly different in the two groups but the differences in the overall ARSW (P value = 0.263) and COWS (P value = 0.862) scores between the groups were not significant. The mean square of ARSW and COWS score at days 1 to 8 were similar. These data suggest that the severity of withdrawal syndrome in the two groups was not significantly different. A similar study, which used the OOWS instrument for assessment of withdrawal syndrome to compare the efficacy and safety of tramadol versus methadone for treatment of opiate withdrawal showed that tramadol may be as effective as methadone in the control of withdrawal syndrome (17). The differences in the VAS score between two the groups were near significant (P value = 0.057). And the craving is higher in the group receiving methadone from the second day of admission even though the usage amount was higher in the tramadol and gabapentin group. The highest craving was at the third day of admission in the methadone group (Figure 3). In this clinical trial, we added gabapentin to tramadol to reduce the risk of seizure by tramadol and to reduce the withdrawal syndrome intensity as been noticed in different studies (8, 9). Another study suggests that gabapentin reduces opioid use during a 10-day buprenorphine detoxification procedure (10). The tramadol plus gabapentin protocol used reduced craving compared to methadone in our study. Most of the studies that compared tramadol with buprenorphine suggested that tramadol may be as effective as buprenorphine in the management of opioid withdrawal syndrome (13-15). In another study, tramadol has been compared to clonidine in the management of heroin withdrawal and tramadol was more effective in preventing most withdrawal symptoms (16). The findings of our study suggest that the combination of tramadol with gabapentin is an efficient method for opioid detoxification. The strong point of the study is the use of 3 different assessments (ARSW, COWS, and VAS) in the two groups. The weak point of the study is the significant differences in respect to the types and amount of drug abuse in two groups. It is recommended for further studies to assess the patient's long-term substance use after the detoxification period. The limitation of the study was that the two groups had significant differences in respect to the types and amount of drug abuse and the variables were not adjusted in the final analysis.

TITLE: Allowing for non-adherence to treatment in a randomized controlled trial of two antidepressants (citalopram ABSTRACT.BACKGROUND: Meta-analyses suggest that reboxetine may be less effective than other antidepressants. Such comparisons may be biased by lower adherence to reboxetine and subsequent handling of missing outcome data. This study illustrates how to adjust for differential non-adherence and hence derive an unbiased estimate of the efficacy of reboxetine compared with citalopram in primary care patients with depression. ABSTRACT.METHOD: A structural mean modelling (SMM) approach was used to generate adherence-adjusted estimates of the efficacy of reboxetine compared with citalopram using GENetic and clinical Predictors Of treatment response in Depression (GENPOD) trial data. Intention-to-treat (ITT) analyses were performed to compare estimates of effectiveness with results from previous meta-analyses. ABSTRACT.RESULTS: At 6 weeks, 92% of those randomized to citalopram were still taking their medication, compared with 72% of those randomized to reboxetine. In ITT analysis, there was only weak evidence that those on reboxetine had a slightly worse outcome than those on citalopram [adjusted difference in mean Beck Depression Inventory (BDI) scores: 1.19, 95% confidence interval (CI) –0.52 to 2.90, p = 0.17]. There was no evidence of a difference in efficacy when differential non-adherence was accounted for using the SMM approach for mean BDI (–0.29, 95% CI –3.04 to 2.46, p = 0.84) or the other mental health outcomes. ABSTRACT.CONCLUSIONS: There was no evidence of a difference in the efficacy of reboxetine and citalopram when these drugs are taken and tolerated by depressed patients. The SMM approach can be implemented in standard statistical software to adjust for differential non-adherence and generate unbiased estimates of treatment efficacy for comparisons of two (or more) active interventions. BODY.INTRODUCTION: Antidepressants are often prescribed in primary care as the first-line treatment for depression. In England in 2011, 46 million prescriptions for antidepressants were issued at a cost of £270 million (HSCIC, 2012). Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed (54% of prescriptions in 2011), with tricyclic antidepressants (TCAs) accounting for a further 29% of prescriptions issued (HSCIC, 2012). Data on the comparative effectiveness of the various antidepressants suggest that there is little difference between different antidepressants (Freemantle et al. 2000; Cipriani et al. 2009). Two meta-analyses suggest that reboxetine may be less effective (Cipriani et al. 2009; Eyding et al. 2010) but others have reported no such differences (Papakostas et al. 2008). Reboxetine is a selective noradrenaline reuptake inhibitor (NaRI), and is the only drug of this class of antidepressants currently licensed in the UK. It is prescribed infrequently (0.1% of total prescriptions for antidepressants in 2011) (HSCIC, 2012). Notably, meta-analyses have highlighted a lower adherence to treatment with reboxetine compared with other antidepressants (Cipriani et al. 2009; Eyding et al. 2010). This differential non-adherence poses problems when examining the results of randomized controlled trials (RCTs) comparing two active treatments because commonly used methods to handle missing data may lead to biased estimates of effectiveness. In the meta-analysis by Cipriani et al. (2009), it was assumed that those patients who were missing outcome data had not responded to treatment. However, as reboxetine was less well tolerated than SSRIs, this imputation has the potential to introduce bias such that the outcome for those on reboxetine may seem less favourable. Similarly, meta-analysis of trials that have used a last observation carried forward (LOCF) approach to handling missing outcome data (Eyding et al. 2010) may be biased in a similar direction. However, neither study explored the potential for bias based on their approach to dealing with missing data. Importantly, these meta-analyses have focused on treatment effectiveness, that is the average outcome of the 'offer' of treatment obtained from intention-to-treat (ITT) analyses, irrespective of adherence to the allocated treatment. However, once it has been established that a medication can be tolerated by a patient, clinicians are often interested in knowing the benefit conferred by that drug when taken as prescribed. There is therefore clinical utility in estimating the efficacy of the drug under 'ideal conditions' (Last, 1995), which includes full adherence to treatment. Estimates of treatment efficacy from 'per-protocol' analyses may be biased (Fleming, 2008), and are further complicated in trials of two (or more) active interventions when there is differential adherence to the allocated treatments. A structural mean modelling (SMM) approach to deal with the issue of non-adherence in trials of two active treatments has been proposed by Fischer et al. (2011). The current study had two aims. First, to test whether two commonly used approaches to dealing with missing data introduce bias in estimates of effectiveness derived in the presence of differential non-adherence between treatment arms. Second, to use data from the GENetic and clinical Predictors Of treatment response in Depression (GENPOD) trial (Lewis et al. 2011; Wiles et al. 2012) to illustrate how to adjust for differential non-adherence in an RCT of two active interventions and hence to derive an unbiased estimate of the efficacy of reboxetine compared with citalopram in the treatment of primary care patients with a new episode of depression. BODY.METHOD.THE GENPOD TRIAL: The GENPOD trial (Thomas et al. 2008) was designed to test two primary hypotheses regarding (1) genetic and (2) clinical predictors of response to antidepressant medication. There was no evidence that the genetic serotonin polymorphism 5-HTTLPR (Lewis et al. 2011) or severity of depression (Wiles et al. 2012) was associated with response to antidepressant medication. Secondary analysis of these trial data can provide information on the comparative efficacy of an SSRI (citalopram) and an NaRI (reboxetine). BODY.METHOD.PARTICIPANTS: Following agreement that an antidepressant should be prescribed, general practitioners (GPs) referred patients to the research team. Those eligible were aged 18–74 years, had a Beck Depression Inventory (BDI; Beck et al. 1996) score of ⩾15 and met ICD-10 criteria for a depressive episode (F32) using the computerized Clinical Interview Schedule – Revised (CIS-R; Lewis et al. 1992; Lewis, 1994). Those who gave written informed consent were randomized to receive either the SSRI citalopram (20 mg daily) or the NaRI reboxetine (4 mg twice daily). Patients with psychosis, bipolar disorder or major substance or alcohol abuse problems were excluded, as were those who had taken antidepressants in the 2 weeks prior to baseline or who could not complete self-administered questionnaires. BODY.METHOD.BASELINE MEASURES: In addition to age, gender, BDI score and CIS-R score, the following data were recorded at baseline: ethnicity, marital status, employment status, financial strain [based on questions from the Breadline Britain survey (Gordon et al. 2000) and a single question asking about how they were managing financially (five response options)], details of home ownership (home owner, tenant, other), whether they had any longstanding illness, disability or infirmity, total number of physical symptoms (based on a list of 28 symptoms), history of depression (self/family) and prior treatment for depression, personality – conscientiousness [Big Five Inventory (BFI); John et al. 1991], Hospital Anxiety and Depression Scale (HADS; Zigmond & Snaith, 1983) score, life events, social support, alcohol use (Alcohol Use Disorders Identification Test for Primary Care, AUDIT-PC; Piccinelli et al. 1997), and scores on the 12-item Short Form Health Survey (SF-12) mental and physical subscales (Jenkinson & Layte, 1997). BODY.METHOD.RANDOMIZATION PROCEDURE: Randomization was conducted by means of a computer-generated code, administered centrally and communicated by telephone and hence concealed from the recruiting researcher. Allocation was stratified by severity of overall symptoms (CIS-R score < 28 or ⩾28) and centre. The researcher gave the allocated medication to the participant. Neither patients nor researchers were blind to treatment allocation. BODY.METHOD.ALLOCATED TREATMENTS: Patients randomized to citalopram were prescribed 20 mg daily. Citalopram taken at this dose has been shown to occupy about 80% of serotonin transporter reuptake sites, which is reported to be the level of occupancy needed to produce reliable antidepressant effects (Meyer et al. 2001). Those randomized to reboxetine were advised to start on 2 mg twice daily and increase to 4 mg twice daily after 4 days. This stepped approach to starting reboxetine treatment was used on the advice of psychopharmacologists to minimize problems with lack of tolerance of this drug. Acute doses of 4 mg of reboxetine increase cortisol levels indicative of increased noradrenergic function (Hill et al. 2003) and this dose of drug also produces peripheral autonomic effects consistent with noradrenaline reuptake blockade (Szabadi et al. 1998). GPs could increase the dose of either allocated treatment if deemed clinically appropriate. BODY.METHOD.MEASURES OF TREATMENT ADHERENCE: Participants were asked about their use of antidepressant medication in the follow-up questionnaires (six closed response options: I have not taken any of my tablets; I have taken hardly any of my tablets; I have taken less than half of my tablets; I have taken more than half of my tablets; I have taken nearly all my tablets; I have taken my tablets every day). BODY.METHOD.OUTCOME MEASURES: Self-reported outcome data were collected 6 and 12 weeks after randomization. For the purpose of this study, which demonstrates the approach to adjusting for differential non-adherence between the two treatments, we used the 6-week outcome data. The (original) primary outcome was the total BDI score at 6 weeks. Secondary outcomes were the HADS total and subscale scores and the SF-12 mental and physical subscale scores. BODY.METHOD.DATASET: The 6-week follow-up was completed by 91% of participants (n = 546) [citalopram: 274/298 (92%) and reboxetine: 272/303 (90%)]. Younger individuals, those with more life events and less social support were more likely to have missing data (Lewis et al. 2011). Adjustment for these variables made no difference to the main trial findings (Lewis et al. 2011) and there was no evidence that these factors were associated with adherence to medication (data not shown). Therefore, for the present analyses, the dataset comprised the 546 participants with 6-week follow-up data (complete cases). BODY.METHOD.STATISTICAL ANALYSIS: All analyses were conducted in Stata version 11.1 (Stata Corporation, USA). To compare the data from the GENPOD trial with the previous literature on the comparative effectiveness of antidepressants (Papakostas et al. 2008; Cipriani et al. 2009; Eyding et al. 2010), we first conducted analyses on the effectiveness of reboxetine versus citalopram according to the ITT principle. We then examined the effect of two approaches to handling missing data that have been used in the previous meta-analyses to illustrate the potential for bias in such estimates of effectiveness in the presence of differential non-adherence. Finally, we focused on the application of the novel SMM approach to estimating treatment efficacy in the presence of differential non-adherence. BODY.METHOD.ESTIMATES OF EFFECTIVENESS: The primary comparative ITT analysis compared the BDI score at 6 weeks between the two groups as randomized, with adjustment for baseline BDI score and the stratification variables. To estimate treatment effectiveness, data from all participants followed up at 6 weeks were included in these analyses, irrespective of adherence to the allocated medication. BODY.METHOD.EFFECT OF IMPUTING MISSING OUTCOMES AS ‘NON-RECOVERY’ OR USING AN LOCF APPROACH TO HANDLING MISSING OUTCOME DATA ON ESTIMATES OF EFFECTIVENESS: Previous studies comparing outcomes for those taking citalopram and reboxetine (Cipriani et al. 2009; Eyding et al. 2010) analysed data on an ITT basis but either: (1) assumed that those who were missing outcome data (which frequently equates to all those who had stopped the trial medication in psychopharmacology trials) had not responded to treatment (Cipriani et al. 2009) or (2) summarized data from publications that used an LOCF approach to handle missing data (Eyding et al. 2010). The effect of these two different approaches to handling missing data was examined by artificially constraining the GENPOD dataset such that only those who had continued to take their medication at 6 weeks were regarded as having outcome data. BODY.METHOD.ADHERENCE-ADJUSTED EFFICACY ESTIMATES: The final set of analyses generated unbiased estimates of treatment efficacy in the presence of differential non-adherence between treatment arms. The SMM method assumes that the mean outcomes in the two arms would be equal in the absence of treatment, and that each treatment has a (separate) linear causal effect on outcome. To estimate the two causal effects of treatment, the approach developed by Fischer et al. (2011) relies on identifying baseline variables that predict adherence differently in the two arms (i.e. they interact with a randomized group in a model for adherence) but that do not predict the causal effect of treatment (i.e. they do not interact with treatment in a causal model for clinical outcome). Baseline variables that predict adherence and/or outcome (as main effects) are also useful in improving precision. The following procedure was used to identify these baseline variables. BODY.METHOD.ADHERENCE-ADJUSTED EFFICACY ESTIMATES.IDENTIFYING PREDICTORS OF OUTCOME: (1) All baseline variables that were possible predictors of outcome [age, gender, ethnicity, marital status, employment status, housing status, financial strain, history of depression (self/family), prior treatment for depression, longstanding illness, disability/infirmity, social support, life events, alcohol score, BDI score, HADS total/anxiety/depression subscale scores, SF-12 mental and physical subscale scores, and number of physical symptoms] were examined in univariable linear regression models with the BDI score at 6 weeks as a continuous outcome. Those variables that were identified as predictors of outcome at p < 0.20 were entered into a multivariable model. The most parsimonious model was identified using backwards selection and the likelihood ratio test until all remaining variables were retained at p < 0.10. Any variables not selected in the initial phase (univariable model: p ⩾0.20) were included in the final multivariable model one by one and retained if p < 0.10. This modelling process was repeated for each of the additional outcomes (HADS total and subscale scores and SF-12 mental and physical subscale scores). All models were adjusted for stratification variables and treatment allocation to improve precision. This liberal modelling approach ensured that all potentially influential variables were included. Omission of a potentially important predictor of outcome from the SMM model would result in a loss of precision. BODY.METHOD.ADHERENCE-ADJUSTED EFFICACY ESTIMATES.IDENTIFYING PREDICTORS OF ADHERENCE: (2) GENPOD relied upon self-reported use of antidepressant medication. A quantitative measure of adherence is required for the SMM approach. Therefore, a pragmatic decision was made to rescale the six response options using increments of 0.2 to generate an adherence score scaled from zero to one, where zero represented total non-adherence and one indicated 'perfect' adherence. This rescaling of the adherence measure assumed that a 0.2 point increase in adherence had the same meaning across the scale. The following baseline variables were possible predictors of adherence: sociodemographic factors (age, gender, ethnicity, marital status, employment status, housing status, financial strain), social support, history of depression (self/family)/prior treatment for depression, longstanding illness/disability/infirmity, personality – conscientiousness, life events, alcohol use, SF-12 physical subscale score, and eight physical symptoms (rapid heartbeat, agitation, dry mouth, sweating, constipation, diarrhoea, daytime drowsiness, and hot flushes). The total number of physical symptoms at baseline was excluded from the list because it was thought that individual physical symptoms may be more relevant to the question of adherence. For example, if someone was already experiencing a dry mouth, taking a drug likely to affect this may differentially affect adherence. The possible predictors of adherence were initially examined in univariable linear regression models with adherence score as the outcome, with adjustment for treatment allocation and predictors of outcome (identified using the process described earlier). All variables that were identified as predictors of adherence (either as a main effect or an interaction with treatment allocation in the univariable models at p < 0.20) were entered into a multivariable model with the variable specified in the appropriate form (main effect or main effect and interaction). Interactions were evaluated one at a time using the likelihood ratio test. Those variables for which the main effect or interaction was significant at p < 0.10 were retained in the final multivariable model. In GENPOD, the primary hypotheses were about differential response to antidepressant treatment dependent on severity of depression and genotype. To be consistent with this hypothesis, it was deemed inappropriate to examine severity as a predictor of adherence to medication because severity may have predicted the effect of treatment other than through adherence. Therefore, all measures of severity of depressive symptoms (CIS-R, BDI, HADS and SF-12 mental subscale score) were excluded from the list of potential predictors of adherence. BODY.METHOD.ADHERENCE-ADJUSTED EFFICACY ESTIMATES.GENERATING ADHERENCE-ADJUSTED ESTIMATES: (3) The SMM approach (Fischer et al. 2011) was implemented using an instrumental variable (IV) model approach in Stata [ivregress command: two-stage least-squares (2sls) approach] for each of the outcomes (BDI, HADS and SF-12 mental and physical subscale scores). Each model was specified in the following format:where y = outcome, x1 = list of predictors of outcome (identified in stage 1), x2 = list of predictors of adherence (identified in stage 2), x3 = stratification variables (centre and CIS-R severity stratum), c1 = adherence score for those randomized to treatment group 1 (citalopram), c2 = adherence score for those randomized to treatment group 2 (reboxetine), r = treatment allocation, and * denotes an interaction, e.g. r*x1= interaction between treatment allocation and predictors of outcome. The SMM method requires identification of baseline variables that predict adherence differentially in the two arms (Fischer et al. 2011). These variables were included in x2 and not in x1, so the interaction r*x2 was an essential part of the model specification whereas the interaction r*x1 is unlikely to be important and could be omitted. Variables that may modify the causal effect of treatment should not be included in x1 or x2. Taking outcome as BDI score at 6 weeks as an example, the IV model estimated the causal effects of full adherence to the two treatments (citalopram and reboxetine); that is, the difference in mean BDI scores for full adherence with the treatment compared to no adherence with any treatment. The difference between the two treatments was then tested formally using the lincom command (lincom c2 – c1), which estimates an adherence-adjusted difference in mean BDI scores between the two treatment groups and its 95% confidence interval (CI). Sensitivity analyses were conducted removing predictors of adherence from the list of x2 variables one by one to examine the robustness of the findings from the SMM IV approach for each of the outcomes. BODY.RESULTS.TRIAL PARTICIPATION AND FOLLOW-UP: The Consolidated Standards Of Reporting Trials (CONSORT) flowchart and baseline comparability of the randomized groups have been published previously (Lewis et al. 2011). In total, 601 participants were randomized to receive either citalopram (n = 298) or reboxetine (n = 303). The mean age of participants was 38.8 years (s.d. = 12.4) and 68% (n = 408) were female. More than 90% of participants had moderate (n = 305) or severe depression (n = 245) according to ICD-10 criteria. The 6-week follow-up was completed by 91% (n = 546) of participants (citalopram: n = 274 and reboxetine: n = 272). BODY.RESULTS.ADHERENCE TO, AND DOSE OF, MEDICATION: Of those randomized to citalopram, 90% (n = 246) were still taking their medication at the time of the 6-week follow-up, compared with 72% (n = 195) of those randomized to reboxetine (difference: 18.4%, 95% CI 12.0–24.8, p < 0.001). At the 6-week follow-up, 149 (55%) of those randomized to receive citalopram reported having taken their tablets 'every day', 90 (33%) had taken 'nearly all' their tablets, and 34 (12%) had taken 'less than half', 'hardly any' or none of their tablets. The comparable figures for those randomized to receive reboxetine were 113 (42%), 89 (33%) and 70 (26%). As reported previously (Lewis et al. 2011), the dose of the allocated medication was increased by the GP only for a minority of participants [citalopram: n = 55 (20%); reboxetine: n = 13 (5%)] during the trial. BODY.RESULTS.ESTIMATES OF EFFECTIVENESS: Among the 546 participants who completed the 6-week follow-up, their mean BDI score at baseline was 33.6 (s.d. = 9.7). The corresponding figures by trial arm are given in Table 1. In an ITT analysis (Table 2), there was only weak evidence to suggest that those randomized to reboxetine had a worse outcome. On average, those on reboxetine scored one point higher on the BDI, although the 95% CI included no difference between groups. The results of the effectiveness analyses for the other mental health outcome measures (HADS total and anxiety/depression subscales; SF-12 mental subscale) were consistent with this (Table 2). Hence, those randomized to receive reboxetine had, on average, a higher score on the HADS (total and subscales) and a lower score on the SF-12 mental subscale, indicative of a worse outcome. Indeed, for the SF-12 mental health subscale, those randomized to reboxetine had a mean score that was, on average, two points lower compared to those randomized to receive citalopram. The CI surrounding this estimate excluded the possibility of no difference. There was little evidence for any difference in outcome in terms of physical health (SF-12 physical subscale score) between those randomized to receive reboxetine compared to citalopram (Table 2). Table 1.Baseline and 6-week follow-up scores on the outcome measures according to allocated treatment group, in those who completed the 6-week follow-upOutcomeTime pointCitalopram (n = 274) a Reboxetine (n = 272)Mean s.d. Mean s.d. BDIBaseline34.29.333.110.06 weeks18.910.819.611.1HADS TotalBaseline26.16.025.56.46 weeks17.28.318.08.4HADS AnxietyBaseline13.33.413.13.86 weeks9.34.29.74.4HADS DepressionBaseline12.83.812.44.16 weeks7.94.98.35.0SF-12 MentalBaseline23.97.624.37.86 weeks39.211.937.212.4SF-12 PhysicalBaseline47.211.547.411.16 weeks48.210.647.810.2BDI, Beck Depression Inventory; HADS, Hospital Anxiety and Depression Scale; SF-12, 12-item Short-Form Health Survey; s.d., standard deviation.an = 273 for SF-12 scores. Table 2.Differences in outcomes at 6 weeks from analysis of treatment effectiveness and estimates of efficacy from SMM models that account for differential non-adherence to allocated treatmentEstimates of effectiveness from analyses conducted according to the ITT principleAdherence-adjusted efficacy estimatesOutcome n Adjusted difference in means a 95% CIp value n Adjusted difference in means a 95% CIp valueBDI5461.19−0.52 to 2.900.17541−0.29−3.04 to 2.460.84HADS Total5461.16−0.16 to 2.470.095431.23−0.71 to 3.170.22HADS Anxiety5460.56−0.09 to 1.210.095430.57−0.43 to 1.570.27HADS Depression5460.60−0.17 to 1.370.135430.62−0.50 to 1.740.28SF-12 Mental545−2.22−4.21 to –0.220.03543−2.53−5.55 to 0.500.10SF-12 Physical545−0.54−1.86 to 0.770.42543−0.61−2.52 to 1.310.53ITT, Intention-to-treat; BDI, Beck Depression Inventory; HADS, Hospital Anxiety and Depression Scale; SF-12, 12-item Short-Form Health Survey; CI, confidence interval.aAdjusted for centre, baseline severity strata (Clinical Interview Schedule – Revised, CIS-R) and baseline score for outcome measure.Difference is reboxetine minus citalopram. A positive difference for BDI and HADS (and a negative difference for SF-12 outcomes) indicates that those on reboxetine have a worse outcome than those on citalopram. BODY.RESULTS.EFFECT OF IMPUTING MISSING OUTCOMES AS ‘NON-RECOVERY’ OR USING AN LOCF APPROACH TO HANDLING MISSING OUTCOME DATA ON ESTIMATES OF EFFECTIVENESS: There was little evidence of a difference in the binary outcome of 'recovery' (BDI score < 10 at 6 weeks) using observed data collected (irrespective of adherence to allocated medication) for 91% of GENPOD participants at 6 weeks when data were analysed using an ITT approach (Table 3). Table 3.Examining the effect of different approaches to handling missing outcome data on the difference between treatment groups (estimates of effectiveness) in the presence of differential adherence to treatmentOutcomeAnalysisCitalopramReboxetineDifference between groups OR (95% CI) a p value N n (%) N n (%)'Recovery' (BDI score < 10)ITT on observed data (n = 546)27460 (21.9)27261 (22.4)0.95 (0.62 to 1.44)0.79Using observed outcome data for those who were continuing to take their allocated medication at 6 weeks and assuming that those who had stopped their allocated medication had not recovered (n = 546)27455 (20.1)27244 (16.2)0.70 (0.45 to 1.10)0.12As above and assuming those with missing outcome data were also non-responders (n = 601)29855 (18.5)30344.9 (14.5)0.70 (0.45 to 1.09)0.11 N Mean (s.d.) N Mean (s.d.)Difference in means (95% CI) a BDI scoreITT on observed data (n = 546)27418.9 (10.8)27219.6 (11.1)1.19 (−0.52 to 2.90)0.17Using observed outcome data for those who were continuing to take their allocated medication at 6 weeks and using an LOCF approach to carry forward the baseline BDI score for those who had stopped their allocated medication (n = 546)27420.2 (11.8)27222.6 (12.0)3.01 (1.18 to 4.85)0.001BDI, Beck Depression Inventory; ITT, intention-to-treat; LOCF, last observation carried forward; OR, odds ratio; CI, confidence interval; s.d., standard deviation.aAdjusted for centre, baseline severity strata (Clinical Interview Schedule – Revised, CIS-R) and baseline BDI score.Difference is reboxetine minus citalopram. An OR < 1 for 'recovery' or a positive difference for differences in BDI scores indicates that those on reboxetine have a worse outcome compared to those on citalopram. Applying the assumption that those who stopped their medication had a poor outcome to the GENPOD data demonstrated that differential adherence to medication between arms introduced bias such that the outcome for those randomized to reboxetine appeared worse [odds ratio (OR) for response 0.70, 95% CI 0.45–1.10)]. Additional imputation of a poor outcome for those individuals not followed up at 6 weeks had little effect (Table 3). Similarly, using an LOCF approach to impute missing outcome data for those who had stopped their medication at 6 weeks suggested that, on average, the outcome for those randomized to reboxetine was three points higher on the BDI (more depressed) compared with those randomized to citalopram. Analysis of the observed outcome data at 6 weeks provided only weak evidence for a difference in outcome between the groups (Table 3). BODY.RESULTS.ADHERENCE-ADJUSTED EFFICACY ESTIMATES: The analyses identified several predictors of outcome and adherence within the GENPOD dataset (see the online Appendix). As expected, for all outcomes, the strongest predictor of outcome was the baseline measurement. In terms of predictors of adherence, those from a non-white ethnic background were less likely to adhere to medication, whereas those who reported a rapid heartbeat were more likely to adhere to medication. Interactions with treatment allocation were found for three variables: marital status, prior history of depression and the personality trait of conscientiousness. Those who were married, those with a previous history of depression and those who were more conscientious were less likely to adhere to reboxetine. The full specification of the IV models that generated the adherence-adjusted estimates can be found in the online Appendix. The adherence-adjusted differences in mean outcomes between the treatment groups are presented in Table 2. There was weak evidence that reboxetine was less efficacious than citalopram in terms of outcome on the SF-12 mental subscale, although the CI included the possibility of no difference. However, there was no evidence of a difference in efficacy between the two treatments based on the other outcomes including the BDI. BODY.RESULTS.SENSITIVITY ANALYSES FOR THE ADHERENCE-ADJUSTED EFFICACY ESTIMATES: The results of the sensitivity analyses examining the effect of removing predictors of adherence from the final SMM IV models for all outcomes are summarized in Table 4. Although the adjusted difference in means between treatment groups varied according to the list of predictors of adherence included in the SMM model (for some outcomes more than others), the estimates were broadly consistent when the CIs were compared. Table 4.Sensitivity analyses around adherence-adjusted instrumental variable (IV) efficacy estimates of the mean difference in outcome between treatment groupsOutcome 〈Y〉Method n Adjusted difference a in mean 〈Y〉 score at 6 weeks95% CIp valueBDI'Adherence-adjusted' efficacy estimate541−0.29−3.04 to 2.460.84 Excluding predictors of adherence from list of x2's one by one Ethnicity541−0.24−3.03 to 2.540.87Rapid heart beat541−0.17−2.94 to 2.600.90Marital status541−0.77−3.88 to 2.330.62History of depression541−0.46−3.34 to 2.420.76BFI conscientiousness score5410.29−2.59 to 3.160.84HADS Total'Adherence-adjusted' efficacy estimate5431.23−0.71 to 3.170.22 Excluding predictors of adherence from list of x2's one by one Ethnicity5431.34−0.64 to 3.310.18Rapid heart beat5431.13−0.82 to 3.080.26Marital status5430.94−1.17 to 3.040.38History of depression5431.32−0.67 to 3.320.19BFI conscientiousness score5431.29−0.76 to 3.340.22HADS Anxiety'Adherence-adjusted' efficacy estimate5430.57−0.43 to 1.570.27 Excluding predictors of adherence from list of x2's one by one Ethnicity5430.66−0.35 to 1.680.20Rapid heart beat5430.52−0.48 to 1.520.31Marital status5430.49−0.58 to 1.560.37History of depression5430.57−0.47 to 1.600.28BFI conscientiousness score5430.55−0.50 to 1.600.31HADS Depression'Adherence-adjusted' efficacy estimate5430.62−0.50 to 1.740.28 Excluding predictors of adherence from list of x2's one by one Ethnicity5430.63−0.50 to 1.760.28Rapid heart beat5430.56−0.57 to 1.690.33Marital status5430.43−0.77 to 1.630.48History of depression5430.72−0.44 to 1.870.22BFI conscientiousness score5430.64−0.54 to 1.830.29SF-12 Mental'Adherence-adjusted' efficacy estimate543−2.53−5.55 to 0.500.10 Excluding predictors of adherence from list of x2's one by one Ethnicity543−2.80−5.89 to 0.290.08Rapid heart beat543−2.29−5.32 to 0.750.14Marital status543−2.85−6.11 to 0.410.09History of depression543−2.97−6.10 to 0.170.06BFI conscientiousness score543−2.01−5.21 to 1.190.22SF-12 Physical'Adherence-adjusted' efficacy estimate543−0.61−2.52 to 1.310.53 Excluding predictors of adherence from list of x2's one by one Ethnicity543−0.83−2.80 to 1.130.41Rapid heart beat543−0.65−2.57 to 1.270.51Marital status5430.13−1.95 to 2.210.90History of depression543−0.96−2.97 to 1.050.35BFI conscientiousness score543−0.82−2.84 to 1.200.43BDI, Beck Depression Inventory; HADS, Hospital Anxiety and Depression Scale; SF-12, 12-item Short-Form Health Survey; BFI, Big Five Inventory; CI, confidence interval.aAdjusted for centre, baseline severity strata (Clinical Interview Schedule – Revised, CIS-R) and baseline score for outcome measure. There was no evidence to support an interaction between severity of depression (or genotype) and response to antidepressant in the GENPOD trial (Lewis et al. 2011; Wiles et al. 2012). Therefore, excluding severity as a predictor of adherence may be questioned. However, there was no evidence for an interaction between severity of depression and adherence to medication (for test of equality of coefficients: interaction between severity and adherence to citalopram/reboxetine, p = 0.27). BODY.DISCUSSION: We have demonstrated how to implement the SMM approach described by Fischer et al. (2011) in a standard statistical software package to obtain an unbiased estimate of treatment efficacy for a trial comparing two active treatments. Analysis was straightforward once suitable covariates for the SMM approach were identified. Data from the GENPOD trial of the two antidepressants citalopram and reboxetine were used as an exemplar. The results of an effectiveness analysis (conducted according to the ITT principle) found only weak evidence that those randomized to reboxetine had a slightly worse outcome than those randomized to citalopram in terms of depressive symptoms (on the BDI/HADS). This is in contrast to previous meta-analyses (Cipriani et al. 2009; Eyding et al. 2010) that suggested that reboxetine was less effective than other antidepressants. It is common practice in psychopharmacology trials for participants who stop taking their allocated medication not to be followed up. Outcomes are then imputed by assuming that those who stopped their allocated medication had a poor outcome or by carrying forward an earlier observation (LOCF). When we applied these approaches to the GENPOD data, by artificially assuming that outcomes were observed only for those who continued on their medication, we found stronger evidence of a poor outcome for those randomized to reboxetine compared with the results of analyses using all observed data. This clearly demonstrates that these common approaches to handling missing data may generate biased estimates of effectiveness when there is differential non-adherence between treatment arms. Using the SMM approach to account for differential non-adherence to treatment between trial arms, we found no evidence of a difference in efficacy in terms of depressive symptoms (BDI) between reboxetine and citalopram at 6 weeks. The adherence-adjusted estimate (based on the difference in causal effects for full adherence to the treatment) was close to the null. There was weak evidence for a difference in efficacy between treatment with reboxetine and citalopram for the SF-12 mental subscale. In discussing these differences, it is important to consider whether these are clinically relevant. Although there is no consensus regarding a 'minimum clinically important difference' on these outcome scales, a change of 0.33 s.d. is often used as the target difference in primary care depression trials (Baxter et al. 2010). Hence, we would regard a three-point change in BDI score, a two-point change in HADS score (one point on subscales) and a three- to four-point change in SF12 scores to be clinically important. The differences and CIs observed in terms of estimates of efficacy from analyses using the SMM approach are smaller than these and, except for the results for the SF-12 mental subscale, we can therefore exclude the possibility of a clinically important difference between citalopram and reboxetine in those who can tolerate the medications. BODY.DISCUSSION.STRENGTHS AND LIMITATIONS: The SMM approach used depends on finding baseline covariates that predict adherence differently in the two randomized groups but that may be assumed not to modify the causal effect of treatment. Bias would occur if the latter assumption failed. In addition, it is assumed that the average outcome does not depend on treatment assignment (the 'exclusion restriction'). In a non-blinded trial such as GENPOD, there is a theoretical possibility that this assumption could be violated given prior beliefs about the treatment. However, there is little evidence to suggest that patients had different expectations of outcome for the two antidepressants. Predictors of adherence were removed from the final SMM IV models one at a time to examine the robustness of the findings. The results of these sensitivity analyses show that the estimates were broadly consistent with the final SMM model incorporating all predictors of adherence. GENPOD relied upon a self-report measure of adherence to medication. Use of electronic monitoring bottles would provide a more accurate measure of adherence. Such data would also provide a continuous adherence score as required for application of the SMM methodology. We rescaled the self-report adherence data to generate a continuous measure of adherence to apply this methodology, albeit therefore introducing some modelling assumptions. At the same time, there was no reason for participants to be motivated to mislead the researchers about their use of medication and we therefore have no reason to suppose that this measure was biased. In total, 601 participants were recruited into the GENPOD trial, making this one of the largest primary care depression trials conducted. Nonetheless, despite its large size, it is of note that estimates obtained from models based on instrumental variables methods remain imprecise. BODY.DISCUSSION.COMPARISONS WITH EXISTING LITERATURE FOR COMPARATIVE EFFECTIVENESS OF ANTIDEPRESSANTS: Meta-analyses have suggested that reboxetine may be less effective than other antidepressants (Cipriani et al. 2009; Eyding et al. 2010). However, in effectiveness analyses of data from the GENPOD trial, we found only weak evidence of very small differences in mental health outcomes (that were unlikely to be clinically significant) at 6 weeks for those randomized to reboxetine compared with citalopram. Both meta-analyses (Cipriani et al. 2009; Eyding et al. 2010) reported that patients randomized to reboxetine were more likely to discontinue treatment compared with those randomized to SSRIs, which is consistent with the findings from GENPOD. However, as we have demonstrated, the assumption that individuals with missing outcome data have not responded to treatment may introduce bias in estimates of effectiveness, such that those on reboxetine seem to do worse. It is therefore important to continue to follow-up trial participants to collect outcome data even if they stop taking the trial medication. BODY.DISCUSSION.EXTENSIONS TO THE SMM METHODOLOGY: We have described the SMM approach for estimating efficacy for a singly-measured quantitative outcome. For a repeated-measured quantitative outcome, a structural nested mean model could be used (Robins, 1994). For a binary outcome, the SMM approach can be used to estimate risk differences, but if interest lies in risk ratios or ORs then a multiplicative SMM or a generalized SMM is needed (Vansteelandt & Goetghebeur, 2003). For time-to-event outcomes, rank-preserving structural nested failure time models could be used (Robins & Tsiatis, 1991). The methods we have described are especially appropriate for equivalence and non-inferiority trials because ITT analysis is known to be anti-conservative in such trials (Jones et al. 1996) whereas per-protocol analyses are potentially biased (Fleming, 2008). An alternative approach to handling non-adherence is the complier average causal effect (CACE; Dunn et al. 2003) model, but this is not well defined in trials comparing two active treatments and also requires adherence to be binary. Dichotomizing a continuous adherence measure is usually undesirable (White et al. 2011). BODY.DISCUSSION.IMPLICATIONS AND FURTHER RESEARCH: It is common practice in RCTs of pharmacological interventions for participants not to be followed up if they stop taking the trial medication. Such a policy is at odds with conducting primary trial analyses according to the principle of ITT, and assumptions that are then made regarding missing data frequently bias estimates of effectiveness. Differential non-adherence between treatment arms presents a particular challenge for trialists. However, as illustrated, it is possible to implement the analytical methods described (Fischer et al. 2011) in a standard statistical software package to take account of non-adherence to treatment when comparing two (or more) active interventions. Such methods will generate an unbiased estimate of the difference in treatment efficacy that is of value to the clinician in terms of describing the likely outcomes when drugs are both taken, and tolerated, by patients. BODY.SUPPLEMENTARY MATERIAL: Supplementary MaterialSupplementary information supplied by authors.Click here for additional data file.

TITLE: Vitamin-D status and neurodevelopment and growth in young north Indian children: a secondary data analysis ABSTRACT.BACKGROUND: Vitamin-D deficiency has been linked with impaired development in animal studies; however, the evidence from human studies is scanty. Evidence as to whether vitamin-D deficiency during early childhood affects growth is also limited and conflicting. We examined the extent to which vitamin-D deficiency (<10 ng/ml) is associated with neurodevelopment and physical growth in young children. ABSTRACT.METHODS: We used data from a randomized controlled trial (RCT) of daily folic acid and/ or vitamin B12 supplementation for six months in children aged 6 to 30 months conducted in Delhi, India. We measured vitamin-D status and neurodevelopment by the Ages and Stages Questionnaire-3 (ASQ-3) at 12 to 36 months of age. Multiple logistic and linear regressions were used to examine the association between vitamin-D deficiency at baseline and neurodevelopment and growth 6 months follow-up. ABSTRACT.RESULTS: 25-hydroxy-vitamin-D (25OHD) concentration was measured at baseline for 960 (96%) children. Of these, 331 (34.5%) children were vitamin-D deficient. The total and subscale (except for the Personal social scale) ASQ-3 scores, were not different between the vitamin-D deficient and non-deficient children. Vitamin-D deficiency was also not associated with physical growth at baseline and at follow -up. ABSTRACT.CONCLUSION: Our data do not support the hypothesis that vitamin-D deficiency is associated with poor growth and neurodevelopment. ABSTRACT.TRIAL REGISTRATION: NCT00717730 and CTRI/2010/091/001090. Date of registration: 08 October, 2010 BODY.BACKGROUND: Vitamin-D deficiency is considered to be one of the most common nutritional deficiencies and a commonly undiagnosed medical condition in the world. [1] The prevalence of vitamin-D deficiency is 50–90% in the Indian subcontinent. [2] Vitamin-D is primarily produced in the skin after exposure to ultraviolet radiation and less than 10% is derived from dietary sources. [3] Poor maternal vitamin-D status during pregnancy has been linked to impaired neurodevelopment among adult offspring and to structural changes in the brain such as enlarged lateral ventricles, thinner cortex, and more cell proliferation in animal studies. [4, 5] Human studies have shown that poor vitamin-D status prenatally is associated with adverse neuropsychiatric outcomes including schizophrenia and child autism. [6, 7] Two recent longitudinal studies showed a link between maternal vitamin-D status in early pregnancy and delayed neurocognitive development including language impairment, mental development, and psychomotor development in early childhood. [8, 9] To our knowledge, there are few studies on the association between vitamin-D status in children and neurodevelopment. Vitamin-D is required for normal calcification of the growth plate and bone mineralization. [10] The major role of vitamin-D in maintaining bone health is to ensure normal calcium and phosphate levels in the blood. Children have higher calcium demands than adults; they require a positive calcium balance to assure adequate calcium for the mineralization of growing bone. [11] It is therefore important to ensure adequate vitamin-D status in order to enhance normal calcification of the growth plate and bone mineralization. We undertook a randomized controlled trial (RCT) of daily supplementation with folic acid and/or vitamin B12 or placebo for six months in 6 to 30 months old children and measured the 25OHD cocentration in the blood specimens at baseline and at 6 months follow-up. We took the opportunity to estimate the association between baseline and at 6 months follow -up vitamin-D status and neurodevelopment measured by the Ages and Stages Questionnaire-3 (ASQ-3) after 6 months as well as the association between vitamin-D deficiency and physical growth (wasting, stunting and underweight) at baseline and 6 months later. BODY.METHODS: The study was conducted from January 2010 to February 2012 in the low-to-middle socioeconomic neighborhoods of Tigri and Dakshinpuri in New Delhi, India. The total population was around 300,000; details of the population have been described previously [12]. The current analysis has been done within the framework of a randomized double-blind placebo-controlled trial (NCT00717730 at https://clinicaltrials.gov/ct2/show/NCT00717730) involving supplementation with folic acid and/or vitamin B12 or placebo for six months to 1000 children who were 6 to 30 months old at enrollment [12]. Vitamin-D status was available for 960 children at baseline and in 243 at 6 months follow-up. For baseline, our analysis for neurodevelopment is restricted to the 401 children in whom ASQ-3 [13] was administered, for physical growth to 960 children in whom anthropometry data were available at baseline and to 919 children in whom anthropometry data were available 6 months later. Of the 243 children, neurodevelopment data were available for 92 children and anthropometry data were available for all. BODY.METHODS.ASSESSMENT: Neurodevelopment was assessed 6 months after enrollment using the ASQ-3 which is a developmental screening tool constructed in the USA. [13] The ASQ-3 consists of age-appropriate questionnaires, all containing 30 items divided into five subscales: Communication, Gross motor, Fine motor, Problem-solving and Personal-social, summing up to five subscale scores (range 0 to 60) and a total score (range 0 to 300). The construct and convergent validity of the translated ASQ-3 forms for the current setting were excellent, and our multiple models were able to explain more than 30% of the variability of the ASQ-3 scores. [14] Details of the Hindi translation and process of validation of ASQ-3, training and standardization methods have been described previously. [15] Three trained field supervisors administered the ASQ-3 directly to the child at the study clinic in the presence of caregivers. The examiners elicited the relevant skills from the child during sessions using standardized materials. The caregiver served as an important contributor in supporting the child, eliciting behaviors and gave relevant information of the child's development when necessary. During the 11 days of training, the field supervisors were standardized in performing the procedure, and they reached a high inter-observer agreement both during training and in the 10% quality control checks throughout the study. To assess the caregiver's promotion of child development two questions were selected from the standardized assessment tool Home Observation for Measurement of the Environment (HOME) that were asked the caregivers during the session. [16] One question was on "Mother's belief that child's behavior can be modified" and one was on "Mother's encouragement of developmental advances". Trained field supervisors measured weight and length at baseline and after six months of supplementation. Weight was measured to the nearest 50 g using electronic scale (Digitron scale). Length was measured using locally manufactured infantometers reading to the nearest 0.1 cm. BODY.METHODS.ANALYTICAL PROCEDURES: Blood samples were obtained at baseline from all children; 3 mL blood was collected in an evacuated tube containing EDTA (Becton Dickinson). The plasma was centrifuged at ~450×g at room temperature for 10 min, separated, and transferred into storage vials and stored at −20 0 C until analyzed. Plasma concentration of vitamin-D was measured by quantitative electro-chemiluminescence binding assay, with detection of 25 OHD, the hydroxylated forms of vitamin-D2 (Roche Diagnostics, Mannheim, Germany) [17] at the Department of Biochemistry, Christian Medical College, Vellore, India. BODY.METHODS.STATISTICAL ANALYSIS: Proportions, means (SD) or medians (IQR) were calculated for categorical and continuous variables by vitamin-D status at baseline. Though The Institute of medicine concluded that for maximum bone health a blood level should be at least 20 ng/mL and the Endocrine Society's Practice Guidelines recommended for maximum bone health a level should be above 30 ng/mL, we considered vitamin-D deficiency was defined at <10 ng/mL (25 nmol/L). [18] We also ran a sensitivity analysis classifying baseline vitamin-D status as <10, 11–20, 21–29 and > = 30 ng/mL. We used multiple regression and a "purposeful selection of covariates method" to identify variables that were associated with vitamin-D deficiency and our predefined outcomes. [19, 20] These variables were used as adjustment variables in the multiple models where vitamin-D deficiency was the exposure variable. We also examined whether the predefined associations were modified by other variables using interaction terms (on a multiplicative scale) in the multiple regression models. Multiple linear and logistic regression analyses were used to compare the total ASQ-3 and subscale-scores between the vitamin-D deficient and the vitamin-D non-deficient groups at baseline and at 6 months followup. In logistic regression models the total and subscale ASQ-3 scores were categorized at the 25th percentile. In these models, we adjusted for age of child, mother's years of schooling, father's years of schooling, log transformed annual family income, family structure, number of toys in the family, whether or not the family owns books, number of children in the family, hours of play with other children during the week, mother's belief that child's behavior can be modified, mother's encouragement of developmental advances, weight-for-height Z score, weight for-age Z scores and intervention group. We used multiple linear and logistic regression analyses to measure the association between vitamin-D deficiency and childhood physical growth at baseline and at 6 months follow-up. In logistic regression models, physical growth was categorized as wasting (< −2 Z scores weight-for-height/length), stunting (<−2 z score height/length-for-age) and underweight (< −2 Z scores weight-for-age). In the linear regression models, we used the Z scores of weight-for-height/length (WHZ), height/length-for-age (HAZ), weight-for-age (WAZ) as dependent variables. In these models, we adjusted for age, sex, breastfeeding status, family structure, log transformed annual family income, mother's years of schooling, father's year of schooling, baseline level of vitamin B12, folate and anemia status for baseline physical growth as well as, intervention group (placebo, folic acid, vitamin B12, or both) for 6 months later physical growth. Statistical analyses were performed using STATA version 14 (Stata Corporation, College Station, TX). We used generalized additive models in the statistical software R version 3.1.2 (The R Foundation for Statistical Computing, Vienna, Austria) to explore nonlinear associations between the vitamin-D status at baseline and HAZ score at baseline after adjustment for potential confounders [21]. We also used generalized additive models to explore nonlinear associations between vitamin-D status at baseline and total ASQ-3 score after 6 months of follow - up. BODY.RESULTS: A total of 1000 children were included in the main trial. Vitamin-D level was analyzed in baseline samples for 960 (96%) children. Of these, 331 (34.5%) were vitamin-D deficient (<10 ng/ml). The mean (SD) and median (IQR) of vitamin-D level were 14.82 (8.7) ng/ml and 13.15 (8.31, 19.2) ng/ml. The baseline characteristics of the population by vitamin-D status are presented in Table 1. Approximately half of the enrolled children were boys and almost all (98%) were ever breast fed. Approximately 70% of the children were anemic (Hb < 11 g/dl).Table 1Baseline characteristics of vitamin-D deficient and non-deficient children aged 6–30 months included in the analysisCharacteristics n = 1000Number of children for whom samples for vitamin-D were available at baseline960 (96.0%)Proportion of children Deficient (< 10 ng/ml)331 (34.5) Non-deficient (≥ 10 ng/ml)629 (65.5)DeficientNon-deficient n = 331 n = 629Infant characteristics Age at enrollment, months (mean, SD)16.9 (7.1)15.8 (7.0) Boys162 (48.9)328 (52.2) Ever breastfed325 (98.2)622 (98.9) Anemia (Hb < 11 g/dl)244 (73.7)424 (67.4) Folate, nmol/L (Median, IQR)9.1 (6.1 to 16.7)12.5 (6.9 to 21.8) Vitamin B12, pmol/L (Median, IQR)266 (181 to 406)266 (172 to 410)Socio-demographic characteristics Mother's age, years (Mean, SD)26.3 (5.8)25.6 (4.1) Mother's schooling, years (Median, IQR)8 (5,10)7 (0,10) Literate mother257 (77.6)466 (74.1) Father's schooling, years (Median, IQR)10 (7,12)9 (6,12) Annual family income, US dollar(Median, IQR)a 1108 (923to 2215)1292 (923 to2123)Figures are number (percentages) unless stated otherwise a1 US Dollar = INR 65 The mean (SD) of the total ASQ-3 and subscales scores by vitamin-D status are shown in Table 2. The overall ASQ-3 score was not significantly lower in the vitamin-D deficient group [mean difference − 6.54 (95% CI: -16.15 to 3.08)] compared to the vitamin-D non-deficient group. The Personal social subscale score was significantly lower in the vitamin-D deficient group [mean difference − 2.63 (95% CI: -5.00 to −0.25)], whereas the other subscale scores were not. There was no interaction on a multiplicative scale between intervention (folic acid and/or vitamin B12 or placebo) and vitamin-D deficiency on the total ASQ-3 score or for the subscales scores.Table 2The association between vitamin-D and total ASQ-3 and subscale scoresDeficient(Vitamin-D status< 10 ng/ml)Non-Deficient(Vitamin-D status≥ 10 ng/ml)Mean (SD) n = 165Mean (SD) n = 236Unadjusted Mean Diff (95% CI)Total ASQ-3229.98 (49.76)236.52 (47.06)−6.54 (−16.15 to 3.08)Subscale Communication49.04 (15.19)49.26 (15.11)- 0.22 (−3.22 to 2.8) Gross motor46.36 (14.39)48.29 (13.41)−1.93 (−4.69 to 0.82) Fine motor46.73 (14.90)48.89 (12.09)−2.15 (−4.81 to 0.50) Problem-solving46.42 (13.97)46.03 (13.45)0.39 (−2.33 to 3.12) Personal social46.42 (12.04)49.05 (11.84)−2.63 (−5 to −0.25)**p < 0.05 We repeated the analyses using logistic regression after dichotomizing the outcomes at the 25th percentiles. After adjusting for potential confounders, we found a significant difference (OR: 1.63; 95% CI: 1.03 to 2.58) in the Personal-social subscale. The overall ASQ-3 score and the remaining subscales were not associated with vitamin-D deficiency. (Table 3).Table 3Odds Ratios for the lower quartile of total ASQ-3 and subscale scores compared with Non-deficient vitamin-D status (≥ 10 ng/ml) adjusting for confoundersa Non-Deficient(Vitamin-D status ≥ 10 ng/ml)Deficient(Vitamin-D status < 10 ng/ml)OROR95% CITotal ASQ-311.360.79 to2.31Subscale Communication11.580.97to 2.59 Gross motor11.270.80 to 2.03 Fine motor11.310.81to 2.11 Problem-solving11.330.84to 2.11 Personal - social11.631.03to 2.58**p < 0.05 aAdjusted for: age of child, mother's years of schooling, father's years of schooling, log transformed annual family income, family structure, number of toys in the family, whether or not the family owns books, number of children in the family, hours of play with other children during the week, mother's belief that child's behavior can be modified, mother's encouragement of developmental advances, weight-for-height Z score, weight for-age Z scores, anemia status at baseline and intervention group Tables 4 and 5 show the distribution of the proportion of children stunted, wasted and underweight according to vitamin-D status at baseline and 6 months later respectively. At baseline in the vitamin-D deficient group, the proportion of stunted, wasted and underweight children was 35.4%, 10.6% and 30.5% respectively. 6 months later, in the vitamin-D deficient group the proportion of stunted, wasted and underweight children was 37.8%, 13.9% and 32.8% respectively. Vitamin-D deficiency was not associated with stunting nor wasting nor underweight at baseline or 6 months later in this population.Table 4The association between baseline Vitamin-D status and growth (at baseline) among childrenDeficient(Vitamin-D status< 10 ng/ml)n = 331Non-Deficient(Vitamin-D status≥ 10 ng/ml)n = 629Adjusted β coefficient(95% CI)a Z scores: Mean(SD) HAZ−1.56 (1.24)−1.63 (1.16).05 (−0.10 to 0.20) WHZ−0.86 (0.92)−0.89 (0.94).02 (−0.10 to 0.14) WAZ−1.46 (1.06)−1.52 (1.05).03 (−0.10 to 0.17)Adjusted OR (95% CI)a Stunted, n (%)117 (35.4)233 (37.0)0.84 (0.63 to 1.13) Wasted, n (%)35 (10.6)68 (10.8)0.99 (0.64 to 1.54) Underweight, n (%)101 (30.5)197 (31.3)0.91 (0.67 to 1.23) aAdjusted for: age, sex, breastfeeding status, log transformed annual family income, family structure, mother's years of schooling, father's years of schooling, baseline levels of vitamin B12, folate, anemia status at baseline Table 5The association between baseline Vitamin-D status and growth (6 months later) among childrenDeficient(Vitamin-D status< 10 ng/ml) n = 323Non-Deficient(Vitamin-D status≥ 10 ng/ml) n = 596Adjusted β coefficient(95% CI)a Z scores: Mean(SD) HAZ−1.69 (1.17)−1.81 (1.12)0.11 (−0.20 to 0.43) WHZ−0.93 (0.97)−0.95 (0.92)0.20 (−0.05 to 0.46) WAZ−1.55 (1.08)−1.62 (1.00)0.20 (−0.06 to 0.47)AdjustedOR (95% CI)a Stunted, n (%)122 (37.8)265 (44.5)0.81 (0.61 to 1.07) Wasted, n (%)45 (13.9)67 (11.2)1.35 (0.90 to 2.04) Underweight, n (%)106 (32.8)209 (35.1)0.97 (0.72 to 1.29) aAdjusted for: age, sex, breastfeeding status, log transformed annual family income, family structure, mother's years of schooling, father's years of schooling, baseline level of vitamin B12, folate, anemia status at baseline, intervention group No substantial or significant differences were found when we did these analyses using HAZ, WHZ and WAZ scores as outcome variables both at baseline and 6 months later i.e. vitamin-D deficiency is not associated with any of the physical growth parameters on a continuous scale. We estimated vitamin D status at end study in only 243 children, and the prevalence of deficiency was 38.3%. Of the 243 children neurodevelopment data were available for 92 children. None of the neurodevelopment outcomes (Total ASQ-3, sub scales of ASQ-3 i.e. Communication, Gross motor, Fine motor, Problem - solving, Personal- social), nor any of the grwoth outcomes were significantly associated with vitamin D status after 6 months (Not shown in table). We also did a sensitivity analysis by classifying baseline vitamin-D status as <10, 11–20, 21–29 and > = 30 ng/mL. 34.6% children were <10, 42.4% were 11–20, 17% were 21–29 and 6% were above 30 ng/ml. None of the neurodevelopment outcomes (Total ASQ-3, subscales of ASQ-3 i.e. Communication, Gross motor, Fine motor, Problem solving, Personal- social) and growth outcomes (wasting, stunting and underweight at baseline and end study) were significantly associated with any of these quartiles. The association between vitamin-D status at baseline and HAZ score at baseline and total ASQ-3 score after 6 months are depicted in Figs 1 and 2. There was no association.Fig. 1Associations between vitamin-D level at baseline and HAZ score at baseline. The graphs were constructed using generalized additive models in R, the solid line depicts the association of vitamin-D level at baseline and HAZ score at baseline. The shaded area spans the 95% confidence interval of this association Fig. 2Associations between vitamin-D level at baseline and total ASQ-3 score after 6 months of follow up. The graphs were constructed using generalized additive models in R, the solid line depicts the association of vitamin-D level at baseline and total ASQ-3 score. The shaded area spans the 95% confidence interval of this association BODY.DISCUSSION: Except for the Personal - social scale, we did not find any significant differences in the total ASQ-3 and subscale scores between vitamin-D deficient and non-deficient children. Nor did we find any association between growth indices and vitamin D deficiency. Our findings are consistent with those from a recently conductedcohort study in India, where vitamin-D status was not associated with the gross motor subscale of ASQ-3 among school- aged children [22]. A recent prospective study also suggests an inverted-U–shaped relation between neonatal vitamin-D status and neurocognitive development in toddlers [23]. Recent studies have shown an association between prenatal vitamin-D deficiency with delayed language development in early childhood [24, 25]. However, the association between vitamin-D deficiency and the Personal social subscale may very well be a chance finding considering the number of outcomes examined. It should also be noted that the personal social scores is the domain with poorest psychometric properties. [15] The ASQ-3 is a screening tool for the assessment of developmental delay constructed in the US with binary cut-offs. It has however been used to measure developmental status on a continuous scale in the present study as in several other studies. [13] Alpha values indicated questionable internal consistency in a few subscales and age categories. Poor internal consistency can be due to constant items (lack of variability of the responses) or random error which may result in false negative results (type II error). However, in another publication from this study we also found that the ASQ-3 scores were associated with linear and ponderal growth, the incidence of diarrhea and pneumonia, as well as socioeconomic status and stimulation and learning opportunities. In fact, these variables explained more than 30% of the variability of the ASQ scores which demonstrates that the translated ASQ-3 test had good convergent validity. [14] Thus, the lack of findings of an association between vitamin-D and ASQ-3 scores is probably not due to poor psychometric properties of the test. In the translated ASQ-3 version, the standardized alphas for the total ASQ-3 scores were strong, indicating an overall acceptable internal consistency. Type II errors can also be due to low sample size, negative confounding, and/or weaknesses with the immunological vitamin –D assay. We used an immunological method to measure vitamin-D concentration. It should be noted that immunoassays can overestimate 25OHD [26] because it is lipophilic which makes it vulnerable to matrix effects in the protein binding assays. [27] We found no association between vitamin-D deficiency and physical growth at baseline and 6 months later. Similar findings have been described in pre-school children in Nepal and HIV exposed but uninfected infants in Africa, where vitamin-D deficiency was not associated with stunting and underweight, although wasted children have been found to be more commonly vitamin- D deficient. [28, 29] However, a recent trial in India showed that weekly administration of 1 RDA of vitamin-D supplementation for six months among LBW infants significantly increased weight and length, and decreased the proportion of children with stunted growth. [30] In addition, an observational study of Canadian infants found higher vitamin - D concentrations during infancy to 3 years of age were associated with leaner body composition. [31] Although the major role of vitamin-D in maintaining bone health is to ensure normal calcium and phosphate levels in the blood, [32] we did not find any association between vitamin-D deficiency and ponderal or linear growth. There may be deficiencies other growth-limiting macro and micronutrients such as calcium, zinc, vitamin –B12 etc. Poor quality of food i.e. lower proportion of animal source protein may also contribute to poor growth. The influence of vitamin D might be small in the light of other growth limiting factors. To our knowledge this is the first analysis to examine the association between vitamin-D status and several domains of the ASQ-3. The strengths of our study are that the data are from a well conducted study with very low attrition rates. [33] More comprehensive assessment tools, such as the Bayley scales, or tools for social emotional functioning, could have added a broader picture of the children's skills and abilities yielding different results. There was a need for follow up assessments since 6 months may not be sufficient to assess the associations between vitamin- D status and development and growth. Finally, more advanced neuroimaging techniques may have identified unique changes to the developing brain in early childhood that could have been linked to vitamin-D deficiency. BODY.CONCLUSION: The results from this analysis do not support that vitamin-D deficiency in early childhood is important for growth and neurodevelopment.

TITLE: Antihistamine Pretreatment to Reduce Incidence of Withdrawal Movement After Rocuronium Injection ABSTRACT: The purpose of this study was to determine the effectiveness of antihistamine therapy for withdrawal movements caused by rocuronium injection. One hundred seventy one ASA I-II adults undergoing elective surgery were randomly assigned to one of two groups. Patients in the control group (Group C) were premedicated with 2 mL normal saline, and those in the antihistamine group (Group A) were pre-medicated with 2 mL (45.5 mg) pheniramine maleate. After the administration of thiopental sodium 5 mg/kg, rocuronium 0.6 mg/kg was injected. Withdrawal movements were assessed using a four-grade scale. The administration of antihistamine reveals lower grade of withdrawal movement after rocuronium injection. BODY.INTRODUCTION: Rocuronium bromide is a non-depolarizing aminosteroidal drug characterized by rapid onset and intermediate duration of action. Pain is a common side effect of rocuronium injection, reported to occur in 50-80% of patients (1-3). Conscious patients administered sub-paralyzing doses often suffer severe burning sensation on injection of the drug (4). Even after loss of consciousness, rocuronium injection causes a withdrawal movement of the injected hand or arm. The pathophysiologic mechanism of pain caused by the intravenous (IV) administration of rocuronium injection remains unclear. Changes in plasma histamine concentration after administration of rocuronium are not significant (5). However, analysis of dermal microdialysis-induced mast cell stimulation reveals that high concentrations of rocuronium lead to significant increases in histamine and tryptase release (6, 7). Histamine is widely known for causing pruritus, rarely provokes pain (8), but high concentrations of histamine may activate polymodal nociceptors, resulting in pain production (5, 9). Peripheral veins are innervated with polymodal nociceptors that mediate the pain response to the injection of rocuronium (6, 10). There are no published studies on the incidence and degree of movement against rocuronium injection pain after pretreatment with antihistamines. This randomized, double-blind, placebo-controlled study was designed to evaluate the effectiveness of antihistamine pretreatment in reducing withdrawal movements associated with the administration of rocuronium. BODY.MATERIALS AND METHODS: A minimum sample size of 127 cases was required in this study by a statistical power analysis (conducted with α=0.05, power=0.80, and determined by the estimated relative proportions in each category for each group using our observations). After institutional ethics committee approval and informed patient consent were obtained, 171 patients (ages 18 to 65 yr, ASA physical status I or II, undergoing general anesthesia for elective surgery) were recruited for a prospective, randomized, and blinded study. Exclusion criteria are; patients with neurological deficits, opioid or local anesthetic allergies, recent exposure to antihistamines or antidepressants, asthma, pregnancy, those who received analgesics within the previous 24 hr, those with difficult venous access, and those requiring rapid sequence induction. All patients were premedicated with glycopyrrolate 0.2 mg and midazolam 2 mg intramuscularly 30 min before induction of anesthesia. Patients were randomly allocated into two groups using a sealed envelope system. The control group (n=79, group C) received 2 mL of saline, and the antihistamine group (n=92, group A) received pheniramine maleate 2 mL (45.5 mg) at the time of premedication. Patient age, sex, height, weight, body mass index (BMI), and IV site were recorded. All patients were monitored with electrocardiogram, pulse oximeter, and non-invasive arterial pressure on arrival at the operating room. Anesthesia was induced with 2.5% thiopental sodium 5 mg/kg. Just after loss of consciousness, rocuronium 0.6 mg/kg was injected over 5 sec. Crystalloids were then administered at maximum gravity flow. Eight patients in group C and 12 patients in group A were excluded from the study since they received succinylcholine instead of rocuronium, owing to poor mask ventilation or complaints of pain during thiopental sodium administration. To estimate the incidence and degree of the withdrawal movements associated with rocuronium, patient response was graded by the investigator according to the following scale proposed by Ahmad and colleagues (1) (Table 1). Mean arterial pressure (MAP) and heart rate (HR) were recorded before the administration of thiopental sodium (baseline), 1 min after injection of rocuronium, and 1 min after tracheal intubation. Statistical analyses were performed using a statistical package (SPSS 15.0 for Windows, SPSS Inc., Chicago, IL, U.S.A.). Data was expressed as mean±standard deviation (SD) or as number of patients. Demographic data was analyzed using a Student's t-test. The incidence and degree of movements, and site of IV cannula were compared using a chi-square test for trend. The incidence of withdrawal movement according to the site of the IV cannula was analyzed using chi-square test for trend. Hemodynamic variables were analyzed using a one-way repeated measures ANOVA. P<0.05 was considered significant. BODY.RESULTS: No significant differences were noted in terms of demographic variables of patients in Group C and Group A (Table 2). The overall incidence of withdrawal was 84.5% (60/71) in Group C and 70% (56/80) in Group A, and there were no significant differences between two groups. The incidence are greater than expected in grade 3/4 withdrawal movement, and lesser than expected in grade 1/2 withdrawal movement in Group C. Group C reveals lesser incidence than expected value in grade 3/4 withdrawal movement (P=0.008) (Table 3). The overall incidence and grade of withdrawal responses were independent of the site on the IV cannula (Fig. 1). MAP and HR during anesthesia induction were not significantly different between two groups (Table 4). BODY.DISCUSSION: This study demonstrates that the degree of withdrawal movement associated with the administration of rocuronium injection can be diminished by pretreatment with an antihistamine. The incidence of pain associated with rocuronium injection is high; reports suggest that 50-80% of patients suffer burning sensation (1-3). Withdrawal movements associated with rocuronium injection may negatively affect patient outcomes. Withdrawal movements may induce pulmonary aspiration secondary to gastric regurgitation in unconscious patients (11), cause dislocation or displacement of the IV catheter, or lead to an emergency situation. The exact mechanism of rocuronium-induced pain remains unclear, but it has been reported that the pain may be due to polymodal nociceptor stimulation caused by the osmolality or pH of the solution, and/or by the release of endogenous mediators, such as histamine, bradykinin, kinin and other substances mediating inflammation (12). Rocuronium is supplied as an isotonic solution of pH 4, and pain is known to be induced by low pH injections (13). However, absence of pain in patients receiving 0.9% NaCl adjusted to pH 4 is inconsistent with this hypothesis (12). Several methods have been attempted to reduce this withdrawal movement and pain, with variable results: pretreatment with ondansetron, lidocaine, tramadol, and fentanyl (3), remifentanil (14) and injection of a mixture of rocuronium and sodium bicarbonate (4, 15). Cheong and Wong (2) found that both 10 mg and 30 mg of lidocaine given before the administration of rocuronium significantly reduced the incidence and severity of pain on rocuronium injection in adults, and the larger dose was more effective in adult patients. However, side effects after the pretreatment is possible, such as anaphylaxis (16), coughing (17), chest rigidity, hypotension and bradycardia (18). The purpose of this study was to determine the effectiveness of antihistamine pretreatment in reducing pain withdrawal associated with rocuronium administration. Pheniramine maleate is an alkylamine antihistamine of which mode of action is achieved by reversible and competitive inhibition of the interaction of histamine with H1 receptors on cells, preventing histamine effects on target organs (8). Several studies have also reported that the analgesic effect of antihistamines (19-21). Although antihistamine has a similar structure to lidocaine, allergic cross reactivity has not been reported. Antihistamine has been recommended as an alternative analgesics for the patients who have allergy to lidocaines (22). After IV administration of pheniramine maleate, peak plasma concentrations are achieved at 15 min (23), with terminal half-lives estimated to range between 8 and 17 hr. We administrated pheniramine maleate at the time of premedication, 30 min before induction of anesthesia. Although Dalgleish (24) demonstrated that the use of a large vein in the antecubital fossa is likely to minimize the discomfort caused to the patient, discomfort did not depend on the site of IV cannulation in either group in our study. Antihistamine administration caused no significant changes in hemodynamic variables, such as MAP and HR. In conclusion, we demonstrated that the degree of withdrawal reaction can be attenuated by treatment with an antihistamine before administration of rocuronium . Patients who were pretreated with antihistamine were less likely to suffer pain than those in the control group.

TITLE: Consumption of cranberry polyphenols enhances human γδ-T cell proliferation and reduces the number of symptoms associated with colds and influenza: a randomized, placebo-controlled intervention study ABSTRACT.BACKGROUND: Our main objective was to evaluate the ability of cranberry phytochemicals to modify immunity, specifically γδ-T cell proliferation, after daily consumption of a cranberry beverage, and its effect on health outcomes related to cold and influenza symptoms. ABSTRACT.METHODS: The study was a randomized, double-blind, placebo-controlled, parallel intervention. Subjects drank a low calorie cranberry beverage (450 ml) made with a juice-derived, powdered cranberry fraction (n = 22) or a placebo beverage (n = 23), daily, for 10 wk. PBMC were cultured for six days with autologous serum and PHA-L stimulation. Cold and influenza symptoms were self-reported. ABSTRACT.RESULTS: The proliferation index of γδ-T cells in culture was almost five times higher after 10 wk of cranberry beverage consumption (p <0.001). In the cranberry beverage group, the incidence of illness was not reduced, however significantly fewer symptoms of illness were reported (p = 0.031). ABSTRACT.CONCLUSIONS: Consumption of the cranberry beverage modified the ex vivo proliferation of γδ-T cells. As these cells are located in the epithelium and serve as a first line of defense, improving their function may be related to reducing the number of symptoms associated with a cold and flu. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01398150. BODY.INTRODUCTION: Cranberries and cranberry juice are associated with promoting urinary tract health [1,2] However, although the responsibility of immune cells is to survey their environment and prevent bacterial and viral infections from overwhelming the body, much of the literature regarding cranberry research has focused on adherence of bacteria [3-6] rather than modification of immune function. Four studies have shown the effects of cranberry on immune function in diverse ways: in a rabbit model of infection-induced oxidative renal damage, cranberry reduced inflammation [7]; consumption of a cranberry beverage in a human intervention study resulted in a reduction in pathogen in 42% of the subjects without altering normal vaginal microbiota [6]; lower levels of urinary IL-6 were found in pregnant women after drinking cranberry juice for at least 3 days [8]; and an enhanced generation of anti-lymphoma antibodies was detected in an immuno-competent mouse model of lymphoma [9]. Recent studies have shown effectiveness of cranberry in reducing reoccurrence of urinary tract infections [10-12]. Recent clinical interventions with cranberry have focused on pyuria and bacteriuria [13,14], but did not examine the influence on systemic immunity. These studies suggest that systemic immunity is modified by the bioactive compounds in cranberries, but have not directly assessed it. An immune cell particularly suited to surveillance of the genitourinary tract is the γδ-T cell, which is strategically located in the epithelium of both the intestine and the reproductive tract. We have shown in human consumption studies with an encapsulated dried fruit and vegetable juice fraction [15], two compounds derived from tea [16], and Concord grape juice [17], that various phytochemicals modify ex vivo γδ-T cell proliferation. Phytochemicals and proanthocyanidins from herbal preparations also interact with γδ-T cells in vitro [18-20]. Cranberry polyphenols and proanthocyanidins, then, would seem to be potential candidates for modifying human immunity. Our objective was to determine if a polyphenol-containing fraction of cranberry would have immuno-modulating activities in humans. Our primary outcome was proliferation of γδ-T cells ex vivo; however, the proliferation of other immune cells was also examined. Our secondary outcome was the evaluation of illness symptoms during the 10-week study. BODY.MATERIALS AND METHODS.SUBJECTS: A total of 54 healthy subjects (17 men and 37 women), ranging in age from 21 to 50 years, with a body mass index between 18 and 30 kg/m2, were recruited by posted advertisements to participate in a 10-wk, double-blind, randomized, placebo-controlled, parallel trial. The University of Florida Institutional Review Board approved the study protocol, and informed written consent was obtained from each subject. Subjects were required to be generally healthy, and exclusion criteria were: taking immunosuppressive drugs, recent or chronic antibiotics, antioxidant supplements or probiotics, or any flavonoid-containing supplements; lactating or being pregnant or on hormone therapy; being a chronic user of non-steroidal anti-inflammatory drugs; having an ongoing infection or hypertension that required medication; consuming more than 14 alcoholic beverages per week, eating more than 7 fruits and vegetables per day or following a vegetarian or strict vegan diet. Participants were not allowed to begin the study if they were ill at the time of the first blood draw. Participants were in contact with the enrolling research assistant by e-mail and telephone, weekly or more often, throughout the study. The predefined primary outcome was proliferation of γδ-T cells in ex vivo culture. Power analysis, (α = 0.05 and power = 0.80), based on data where a difference would be significant if the proliferation of γδ-T cells was double that of the placebo group with an expected standard deviation of 1.74, indicated that 13 subjects would be needed per group to detect a statistical difference. A predefined level of compliance was consumption of 80% of the allotted juice. BODY.MATERIALS AND METHODS.STUDY DESIGN: The study was conducted between March and May of 2009, to coincide with normal cold and influenza season in the Southeast (CDC, http://www.cdc.gov/flu/; accessed October 2011). The CDC weekly report of influenza activity in the state of Florida that year was as follows: March, widespread to regional activity; April, local to sporadic activity; May, regional to local activity. Subjects arrived to the Food Science and Human Nutrition building, for an initial baseline fasting blood draw (Day 0) and were randomly assigned, by drawing cards from an opaque envelope that were numbered either 246 or 638, to receive the experimental treatment [cranberry beverage (CB)] or a placebo beverage (PB), both provided by Ocean Spray Cranberries, Inc. (Lakeville, MA). Both subjects and investigators were blinded regarding the treatment groups. The CB contained cranberry components from juice, filtered water, sugar, natural flavors, citric acid, and sucralose. The PB was a color-(Red 40 and Blue 1), calorie-, and sweetener-matched beverage without cranberry components. The experimental beverage is not commercially available, but was formulated to contain a level of polyphenols similar to that found in commercially available cranberry juice cocktails. The fraction of cranberry components used, derived from the juice of cranberries, was prepared and analyzed by the manufacturer (Table 1). High-performance liquid chromatography analysis of the fraction used to prepare the beverage was conducted (manuscript submitted). Table 1 Chemical characterization a of the cranberry treatment and placebo beverages   Cranberry beverage Placebo beverage Proanthocyanidins, % dwb b 65-77% 0-1% Sugars, % dwb 0.77-1.12% 0% Anthocyanins, % dwb 6.8-11.3% Not detected Organic acids, % dwb 0.5-0.9% 0.1-0.2 Phenolic acids, % dwb 7.1-7.5% Not tested Flavonols, % dwb 6.8-10.0% Not tested Total Solids,% dwb 87.0-107.8% 0.1-1.2% Sucralose (μg/mL) 152 149 Vitamin C Not detected Not detected ORAC c (μM AA/g) 62.1 Not detected Colorant (Red 40/Blue 1) None 1% Brix by refractometry ( o ) 0.25 0.23 a Characterization of the beverages was by standard methodology and performed by the manufacturer of the beverages. Values were assessed in the lab of SSP by standard methodology and were computed against an ascorbic acid (AA) standard curve. b dwb, dry weight basis c ORAC, oxygen radical absorbance capacity. Subjects were given bottles containing 450 ml (15 oz) of beverage and instructed to drink one bottle throughout the day, each day, for 70 days (10 wk). Participants were supplied with more bottles than were needed to complete the study, in case scheduling conflicts delayed the second blood draw. To prevent bottles from being discarded, subjects were instructed to bring back any remaining bottles at the end of the study. Participants were also given a daily illness log to record any cold and influenza symptoms (listed below) they experienced during the 10-wk experimental period. Each day they had to answer this question: Did you have any illness symptoms today? If they answered yes, then they were asked to check the box with each of the symptoms they had that day. The primary outcome of this study was defined, prospectively, as physiological changes to γδ-T cell proliferation in ex vivo culture. Illness symptoms were considered as secondary outcomes because of the small number of subjects, and because they were self-reported. These outcomes were defined as incidence (number of people reporting an illness per group and the number of illnesses per group), duration (total number of days with at least two symptoms and average number of days per group), and total number of symptoms per group. Symptoms listed in the daily diary were: runny or congested nose, cough, sneezing, fever and/or chills, sore throat, headache, wheezing, and intestinal distress (nausea, vomiting, diarrhea, and/or abdominal cramps). Allergy symptoms were not included in the analysis of symptoms. Subjects were instructed as to the different manifestations of colds, influenza and allergies: Colds–symptoms occur one at a time, generally last for 5-7 days, yellow/greenish nasal discharge, may or may not be accompanied by a fever, with slight body aches and pains; Influenza–symptoms occur rapidly, last up to 14 days, no nasal discharge, often a high fever and sometimes chills, with severe body aches and pains, including headache; Allergy–symptoms occur rapidly and all at once, last as long as the allergy-causing agent is present, clear and watery nasal discharge, not associated with a fever, and no body aches or pains. The record of the medications taken during an illness also helped to distinguish allergy from colds and influenza. Subjects were asked to report if they missed class or work, whether they sought medical treatment, if they were prescribed any medications as a result of seeking treatment, which over-the-counter medications they took, and whether they had a significant decrease in normal activities due to illness symptoms. At 10 wk, study participants returned for a final blood draw and to complete an exit questionnaire. The exit questionnaire included questions to determine if subjects experienced any side effects from the beverage, their estimate of compliance and if they generally adhered to the inclusion/exclusion criteria. To determine efficacy of blinding, subjects reported whether they thought they had received the CB or the PB and were asked why they believed that. Study compliance was assessed by comparing the number of bottles of beverage returned at the end of the study (primary assessment of compliance) with the number they should have returned. BODY.MATERIALS AND METHODS.BLOOD COLLECTION AND PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) ISOLATION: Blood was obtained from fasting subjects on Day 0 (baseline) and at 10 wk. Fasting required eating no food after midnight of the previous day. Blood was collected into one 10 ml sodium heparin tube for PBMC isolation, and one 10 ml SSTTM tube (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) to obtain serum. All tubes were processed within 2 h of blood collection. Serum was removed from SSTTM tubes after centrifugation (2,000 × g, 10 min, 4°C) and used as autologous serum in culture media. Aliquots of serum were frozen at-80°C for antioxidant analysis. Whole blood was diluted 1:1 with 0.9% NaCl and layered on a gradient (Lympholyte® H Cell Separation Media, Cedarlane Laboratories Ltd., Burlington, NC) to separate PBMC by centrifugation (800 × g, 20 min, 20°C). The mononuclear cell layer was removed and washed twice with RPMI 1640 (Cellgro, Mediatech, Inc., Manassass, VA) complete medium (100,000 U/L penicillin; 100 mg/L streptomycin; 0.25 mg/L fungizone; 50 mg/L gentamicin; 2 mmol/L L-glutamine; 25 mmol/L HEPES buffer). Cell pellets were resuspended in RPMI 1640 complete medium and counted on a hemocytometer. BODY.MATERIALS AND METHODS.CULTURE OF PBMC FOR PROLIFERATION AND CYTOKINE PRODUCTION: On Day 0, 1.0 × 106 PBMC in RPMI 1640 complete medium containing 50 μM 2-ME and 10% autologous serum were seeded into two wells of duplicate 24-well tissue culture plates (Costar, Corning Incorporated, Corning, NY). Phytohemagglutinin (PHA-L from Phaseolus vulgaris) at a final concentration of 10 μg/ml, was added to one set of wells on each plate, while the other set was brought to volume with RPMI 1640 complete medium. The plates were incubated at 37°C in a humidified 5% CO2 atmosphere. After 24 h, supernatant fluids from one plate were harvested and frozen at-80°C for future cytokine analysis. On Day 3, human recombinant IL-2 (BD Biosciences, San Jose, CA), at a concentration of 1 ng/ml, was added to all wells of the second plate, which was incubated until Day 6 when cells were harvested and processed for flow cytometry. BODY.MATERIALS AND METHODS.FLOW CYTOMETRY: PBMC were analyzed by flow cytometry on Days 0 and 6 of culture, using cell surface markers for identification. Specific cell population numbers were expressed as a percentage of total cells [B cells, monocytes and natural killer (NK) cells] or as a percentage of the CD3+ population (αβ-T cells and γδ-T cells). To determine proliferation changes of the various cell types at both blood draws, the fold change of individual subjects was calculated as the ratio of numbers of cultured cells to uncultured cells (Day 6/Day 0). If no proliferation occurred during that time, the ratio would equal 1.0. Then, the fold change before beverage consumption (baseline) was subtracted from the fold change after consumption (10 wk). Antibodies were obtained from eBioscience, Inc. (San Diego, CA) and used to detect specific cell types, as follows: γδ-T cells (PE-CD3 and FITC-γδ-TCR); αβ-T cells (PE-CD3 and FITC- αβ-TCR); NK cells (PE-CD314 and FITC-CD56); B cells (PE-CD19) and Monocytes (FITC-CD14). After staining, cells were washed and fixed with 1% paraformaldehyde. Data was acquired on a BD Biosciences FACSort flow cytometer with CellQuest Pro software (BD Biosciences, San Jose, CA) within 48 h. FlowJo Analysis Software, version 7.5 (Tree Star, Inc., Ashland, OR) was used for data analysis. BODY.MATERIALS AND METHODS.CYTOKINE LEVEL DETERMINATION IN CELL CULTURE SUPERNATANT FLUIDS: Levels of cytokines (IFN-γ, IL-1α, IL-1β, IL-13, MIP-1β, and TNF-α) secreted by PBMC in culture for 24 h, were quantified using a Human Cytokine Multiplex Immunoassay kit, according to the manufacturer's directions (Millipore Corp., Billerica, MA). Standards and controls were provided with the kit. The beads were analyzed on a Luminex® 200 instrument (Luminex Corporation, Austin, Texas) with xPONENT 3.1 software. In addition, levels of IL-17 were determined by ELISA according to manufacturer's directions (eBioscience, San Diego, CA) with kit standards and controls. The fluids were thawed on ice and used undiluted in the assay. Final absorbance was measured at 450 nm on a SPECTRAmax 340PC plate reader and data analyzed using SOFTmax® Pro 5.2 (Molecular Devices, Sunnyvale, CA). All values are expressed as pg/ml. BODY.MATERIALS AND METHODS.SERUM OXYGEN RADICAL ABSORBANCE CAPACITY (ORAC): The ORAC assay consisted of monitoring the inhibition of decay of fluorescein in the presence of the peroxyl radical generator 2,2′azobis 2-amidinopropane dihydrochloride (AAPH). The rate of fluorescence decay was monitored over time by calculating the area under the fluorescent decay curve and quantified using a standard curve of Trolox (0.312-2.5 μM, Fluka Chemical/Sigma). Antioxidant activity in protein-free sera was determined using the ORAC assay, as previously described [21,22] for a 96-well microplate reader, with some modifications. Briefly, at collection sera were diluted 1:5 in 6% meta-phosphoric acid and frozen at -80°C. After thawing, they were centrifuged to precipitate protein (12,000 × g, 5 min, 4°C) and diluted 1:10 in phosphate buffer. Then, 50 μl of each sample were pipetted into duplicate wells. Serum samples for quality control were prepared in our laboratory and used in each assay plate to determine variability. Plate to plate variability was 7.1%. Fluorescein solution (20 nM) was added to all wells. The plate was mixed for 3 minutes, and then equilibrated for 7 minutes to 37°C in a SPECTRAmax GeminiXPS fluorescent plate reader (Molecular Devices, LLC, Sunnyvale, CA). Freshly prepared AAPH free-radical solution (140 mM in phosphate buffer) was added (50 μl), and the FITC fluorescence decay was monitored at 32-sec intervals for 40 min (excitation, 485 nm; emission, 538 nm; cutoff, 530 nm). ORAC values were calculated as the area under the curve using SOFTmax® Pro 5.2 Software (Molecular Devices, LLC, Sunnyvale, CA), and the data expressed in μmol Trolox equivalents. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: For the cytokine and immune cell measures, a non-parametric Wilcoxon/Kruskal-Wallis Rank sum test was performed on the differences in levels before and after supplementation. Illness and symptom data was analyzed by logistical regression with a generalized linear model and an exponential distribution. An over-dispersion parameter was used, due to larger-than-expected variance (JMP, version 8, Cary, NC). A nominal alpha level of 0.05 was used to declare statistical significance. A sub-group analysis of all primary indices was performed to determine and to address blinding issues, comparing those who guessed correctly (n = 5) with those who did not. BODY.RESULTS: Of 68 people assessed for eligibility, 54 individuals enrolled in the study (Figure 1). During the study, 9 people withdrew: six because they were unable to return for the final blood draw, two because of the taste of the beverage, and one with no explanation. Of the 54 subjects enrolled, 45 (83%) completed the study. Overall compliance, determined by returned bottle count, was 96.1% ± 3.7% bottles of beverage (Table 2). All participants consumed at least 80% of their allocated beverage; therefore, no one was excluded from data analysis based on noncompliance. Figure 1Flow diagram: Study participant eligibility assessment, enrollment, group allocation and analysis. People recruited for the study were assessed and those deemed eligible were enrolled. Subjects were randomized into one of two beverage groups. Bottles of the two beverages, cranberry and placebo, were received from Ocean Spray Cranberries, Inc. and labeled either #638 or #246. Subjects and investigators were blinded regarding the treatment groups. Statistical analysis was performed on data from all subjects completing the study. Investigators were unblinded (cranberry beverage: #638; placebo beverage: #246) following completion of the data analysis. Table 2 Demographics and cold and influenza symptoms Characteristic Placebo beverage Cranberry beverage P Demographic n 23 22   Age (y) ± SD a 24.0 ± 3.3 24.9 ± 5.8   Male 5 9   Female 18 13   BMI b , initial 21.5 ± 2.7 23.2 ± 4.0 0.103 Change in BMI (kg/m 2 ) ± SD -0.095 ± 0.52 -0.063 ± 0.37 0.810 c No. blinded/guessed correctly (%) 23/23 (100) 5/22 (23) <0.001 Compliance d (%) ± SD 96.3 ± 4.6 95.9 ± 3.7 NSD e Cold and Influenza Symptoms n f 20 15 0.624 Total incidence g 31 21 0.282 h Total cold and influenza symptoms 354 297 0.031 h Incidence of intestinal distress 35 15 0.021 h Total days missed work/school 29 20 0.329 h Total times reported a decrease in activity 28 16 0.154 h a SD, standard deviation b BMI, body mass index c p value derived from t -test d Based on bottle count e NSD, not significantly different f Number of people who reported symptoms g Number of colds and cases of influenza reported. Some individuals had more than one illness during the 10 wk period h P value derived from z -test of proportions. Regarding blinding, all subjects (100%) in the placebo group thought they were drinking the PB, while only five of the 22 people in the experimental treatment group (23%) guessed they were drinking the CB (Table 2). As there was a significant difference between the proportion of people guessing correctly versus the proportion of people guessing incorrectly (p < 0.0001, Fisher exact test), with nearly everyone in the study believing they were on the placebo, bias toward treatment was not anticipated to influence outcome. A subgroup analysis indicated that there were no significant differences in any of the parameters, but one, when comparing who guessed correctly with who guessed incorrectly. Those in the group that guessed correctly had slightly higher MIP1α levels at baseline compared to 10 week, or compared to either time point of those who did not guess correctly. Interactions between guessing correctly and time were not significant. Since this was observed in the baseline value, before anyone started the study, the biological significance of this statistical difference is unknown. The total incidence of colds and influenza were not statistically different between the two groups (Table 2). However, the proportion of the total number of symptoms was statistically lower in the CB drinking group (p = 0.031). Since only 11.1% of the subjects thought they were consuming the active treatment, a bias by what they thought they were drinking was not suspected. The report of intestinal distress as a symptom was statistically greater (p = 0.021) in the PB group, as compared to the CB group. No other symptom showed any statistical difference. However, because subjects in the placebo group reported a few more symptoms in each category, the total number of symptoms was proportionally greater in that group compared to the CB group, and total symptoms were statistically different between the groups. The percentage of specifically labeled PE+-FITC+ cells in the lymphocyte population (FSC/SSC) was determined at the blood draws before and after consumption, in both freshly isolated PBMC and in PBMC that were cultured for six days. Proliferation of γδ-T cells was significantly improved after CB consumption (Table 3) compared to the placebo. NK cell proliferation did not achieve significance when treatments were compared. There was no effect of CB on αβ-T cells, B cells or monocytes. The antioxidant activity of the serum was determined in de-proteinated samples, but the groups were not statistically different (data not shown). Table 3 Immune cell proliferation a fold change b after cranberry beverage consumption Cell type Placebo beverage Cranberry beverage P γδ-T cell c 1.20 ± 0.26 f 3.86 ± 0.50 <0.001 NK d cell e 0.15 ± 0.12 0.33 ± 0.19 0.068 αβ-T cell c -0.16 ± 0.09 -0.16 ± 0.08 0.602 Monocyte e -0.11 ± 0.29 -0.30 ± 0.45 0.883 B cells e 0.81 ± 0.16 1.32 ± 0.40 0.235 a After isolation (Day 0), cells were placed into culture in RPMI-1640 with autologous serum and PHA-L, IL-2 and IL-15 for 6 days b A ratio of cultured cell numbers (Day 6/Day 0) was used to calculate proliferation fold changes. Baseline fold change values were subtracted from values obtained at 10 wk c Percentage of the CD3 + population determined by 2-color staining d NK, natural killer e Percentage of total cells f Wilcoxon/Kruskal-Wallis rank sums test was used to determine significant differences. Values were calculated for each subject and the mean ± SEM determined. Cytokine levels in the supernatant fluids of 24 h PBMC cultured with PHA-L were determined, resulting in an observation of variability among subjects. Therefore, the change between cytokine secretion at 10 wk and secretion at baseline was compared between the placebo and treatment groups (Table 4). The ability of PBMC to secrete interferon-γ (IFN-γ) was significantly increased after CB consumption (p = 0.041). Other measured cytokines were not statistically different. Table 4 Cytokines secreted by PBMC a during 24 h culture b Cytokine Placebo beverage Cranberry beverage P c IL-1β -519.7 ± 115.5 d -187.8 ± 107.1 0.088 IFN-γ -24.8 ± 33.4 148.1 ± 80.2 0.041 TNF-α 189.7 ± 155.9 367.2 ± 140.2 0.452 IL-17 16.8 ± 11.3 26.2 ± 16.7 0.973 IL-1α -36.5 ± 20.0 -27.9 ± 29.7 0.496 MIP-1β 968.3 ± 533.1 1120.0 ± 470.3 0.395 IL-13 -3.1 ± 7.1 -11.5 ± 10.6 0.156 a PBMC, peripheral blood mononuclear cells b On Day 0, PBMC were cultured in RPMI-1640 with autologous serum and PHA-L, IL-2 and IL-15 c P values are two-tailed based on the Wilcoxon/Kruskai-Wallis Rank sum test d Values reported are the mean changes ± SEM of the 10-wk data minus the baseline data. BODY.DISCUSSION: The primary predefined outcome measure was a change in the ability of γδ-T cells derived from peripheral blood immune cells to proliferate in ex vivo culture. The γδ-T cells showed an improved ability to proliferate in culture with PHA-L, after the CB was consumed. The cranberry fraction powder was prepared from the juice of cranberries and one serving of the low calorie CB (450 ml) contained polyphenol levels comparable to a serving of cranberry juice cocktail (250 ml). The proanthocyanidin fraction contained both A-and B-type linkages. Cranberries are known to have high antioxidant activity, yet serum antioxidant activity was not different between the groups or from baseline to 10 wk (data not shown). Pharmacokinetic studies that measure immediate accumulation of polyphenols in blood show that the turnover is rapid; about 2-4 hours [23-25]. Thus, a lack of change in serum antioxidant activity is not surprising when measured in the serum of fasting subjects, as we did in this study. We propose that γδ-T cell proliferation ex vivo is a surrogate marker for immune function. We have shown in previous studies that their proliferation can be modified by diet [15-17]. The issue of bioavailability must be addressed since many studies show that polyphenols do not accumulate in the blood, but are rapidly metabolized. Intestinal immune cells are able to interact with the contents of the lumen because of their location in the Peyer's patches and the intra-epithelium. Migration of intestinal immune cells is well documented [26,27] and migration patterns have been shown to be influenced by diet [28,29]. Although data specifically regarding γδ-T cells is lacking, we hypothesize that we measure changes in blood immune cells because they migrate after they have interacted with luminal bioactive compounds in the intestine. In addition, larger molecules, at times, are able to translocate across the intestine and find their way to cells in the lamina propria and the mesenteric lymph nodes. Bacterial toxins are found intact in the blood or lymph [30,31]. Proteins are absorbed intact [32,33] and procyanidin dimers are transferred to the serosal side of enterocytes in the isolated small intestine [34] or in a Caco-2 cell model [35]. Jutila's group has shown that the proanthocyanidin content of herbs interacts with receptors on the γδ-T cell and primes that cell [36]. After priming, the cell is able to respond faster, and to a greater extent, than if it were not primed. Direct interaction of dietary components in the lumen, or translocation of dietary components into the lamina propria, could result in priming of immune cells. Finally, changes detected in systemic blood cells may also occur because of the fermentation of unabsorbed polyphenols in the large intestine, resulting in bioactive compounds that are subsequently absorbed. The study was not designed to distinguish among these different mechanisms. Changes in immune function may be responsible for a reduced number of cold and influenza symptoms. Although the incidence of colds and influenza were similar between the two groups, the total number of symptoms was lower after consuming the CB. Combining 'missed work' and 'lower ability to perform a normal daily routine' resulted in a p value of 0.056. This study was not powered on cold and influenza symptoms; it does not achieve statistical significance, so the idea that cranberry consumption has an effect on a health outcome is only a suggestion. Furthermore, the data on cold and influenza symptoms were self-reported and physicians did not confirm the presence of cold or influenza pathogens. However, Macintyre and Pritchard demonstrated that self-reported symptoms, as well as symptom severity, was highly correlated with assessments made by physicians [37], therefore we feel confident that the symptoms reported by the subjects accurately reflect the symptoms they had. We examined cytokines based on previous in vitro data (manuscript submitted) in which PBMC from human donors were incubated with various cranberry fractions. For evaluation in this study, we chose those cytokines that were secreted in response to the addition of the cranberry fractions. The results from participants in this study showed the response of the cells to ex vivo stimulation was extremely variable. Drawing firm conclusions about the role cranberry plays in cytokine production is premature, yet the data suggest that cranberry contributes to an anti-inflammatory effect. Cranberry proanthocyanidins have been suggested as being anti-inflammatory in in vitro studies [38,39] and in rabbits [7]. One human intervention study showed that the level of urinary IL-6 was reduced in pregnant women who consumed cranberry juice compared to a placebo [8], yet other cytokines measured in that study did not achieve statistical significance, perhaps due to the small n of each group. BODY.CONCLUSIONS: In summary, daily consumption of a cranberry beverage containing cranberry polyphenols and proanthocyanidins at levels similar to commercially available juice for 10 wk, was effective in increasing the proliferation response of γδ-T cells and perhaps NK cells. Overall, the improved proliferation response was coupled with lower production of an inflammatory cytokine. Improvement of functional immunity, particularly of a cell located in the epithelium and responsible for barrier protection, might be another mechanism by which cranberry is able to maintain urinary tract health. These physiological changes could be, in part, responsible for our beneficial health outcome, a reduction of the number of illness symptoms. BODY.ABBREVIATIONS: AAPH: 2,2′azobis 2-amidinopropane dihydrochloride; CB: Cranberry beverage; ELISA: Enzyme-linked immuosorbent assay; FITC: Fluorescein isothiocyanate; NC: Not calculated; NK: Natural killer; NSD: Not significantly different; ORAC: Oxygen radical absorbance capacity; OSC: Ocean Spray Cranberries, Inc.; PB: Placebo beverage; PBMC: Peripheral blood mononuclear cell; PHA-L: Phytohemagglutinin; PE: Phycoerythrin; TCR: T-cell receptor. BODY.COMPETING INTERESTS: CK is an employee of OSC. SSP receives no other compensation from OSC outside of the funding for this research. The other authors have no conflicts of interest. BODY.AUTHORS’ CONTRIBUTIONS: The study was conceived by SSP and CK, and SSP was responsible for the study design. MPN, CAR, CM, and RC contributed to the design of the study and were responsible for coordinating the intervention, performing the assays and writing some of the paper. JC helped with the statistical analysis. CK was responsible for the preparation, analysis and randomization of the beverages. SSP interpreted the data, wrote the paper, and is responsible for final content of the manuscript. All authors read and approved the final manuscript.

TITLE: Nitroglycerine, esmolol and dexmedetomidine for induced hypotension during functional endoscopic sinus surgery: A comparative evaluation ABSTRACT.BACKGROUND AND AIM:: Induced hypotension limits intra-operative blood loss to provide better visibility of the surgical field and diminishes the incidence of major complications during functional endoscopic sinus surgery (FESS). We aimed at comparing nitroglycerine, esmolol and dexmedetomidine for inducing controlled hypotension in patients undergoing FESS. ABSTRACT.MATERIAL AND METHODS:: One hundred and fifty American Society of Anesthesiologists physical status I or II adult patients undergoing FESS under general anesthesia were randomly allocated to three groups of 50 patients each. Group E received esmolol in a loading and maintenance dose of 1 mg/kg over 1 min and 0.5-1.0 mg/kg/h, respectively. Group D received a loading dose of dexmedetomidine 1 μg/kg over 10 min followed by an infusion 0.5-1.0 μg/kg/h, and group N received nitroglycerine infusion at a dose of 0.5-2 μg/kg/min so as to maintain mean arterial pressure (MAP) between 60 and 70 mmHg in all the groups. The visibility of the surgical field was assessed by surgeon using Fromme and Boezaart scoring system. Hemodynamic variables, total intra-operative fentanyl consumption, emergence time and time to first analgesic request were recorded. Any side-effects were noted. The postoperative sedation was assessed using Ramsay Sedation Score. ABSTRACT.RESULT:: The desired MAP (60-70 mmHg) could be achieved in all the three study groups albeit with titration of study drugs during intra-operative period. No significant intergroup difference was observed in Fromme's score during the intra-operative period. The mean total dose of fentanyl (μg/kg) used was found to be significantly lower in group D compared to groups E and N (1.2 ± 0.75 vs. 3.6 ± 1.3 and 2.9 ± 1.1 respectively). The mean heart rate was significantly lower in group D compared to groups E and N at all times of measurement (P < 0.05). The MAP was found to be significantly lower in group D compared to groups E and N after infusion of study drugs, after induction, just after intubation and 5 min after intubation (P < 0.05). The Ramsay Sedation Scores were significantly higher in group D (score 3 in 46%) when compared to group E (score 2 in 50%) and group N (score 2 in 54%) (P < 0.001). The emergence time was significantly lower in group E and group N compared to group D. Time to first analgesic request was significantly longer in group D. ABSTRACT.CONCLUSION:: Dexmedetomidine and esmolol provided better hemodynamic stability and operative field visibility compared to nitroglycerin during FESS. Dexmedetomidine provides an additional benefit of reducing the analgesic requirements and providing postoperative sedation. BODY.INTRODUCTION: Rhino-sinusitis, an important cause of significant discomfort and morbidity is commonly treated with FESS nowadays.[123] However, there can be serious complications associated with this procedure during peri-operative period like orbital cellulitis, optic nerve injuries, meningitis, etc. whose incidence can increase with excessive bleeding during surgery.[45] Hence, it is mandatory to keep the surgical field as free of blood as possible to improve visibility of anatomical landmarks and structures. This can be achieved with the use of topical vasoconstrictors, with local anesthesia or use of controlled hypotension with general anesthesia. Controlled hypotension involves reducing arterial blood pressure 30-40% below its normal range or reducing mean arterial pressure (MAP) to 65 mmHg reversibly and maintaining it at that level throughout the surgery.[6] A variety of medications can be used to induce intra-operative hypotension including vasodilators like sodium nitroprusside,[7] nitroglycerin[8] and hydralazine; inhaled anesthetics like isoflurane[9] and sevoflurane; intravenous anesthetics like propofol; beta adrenergic antagonists like esmolol;[10] trimethaphan, adenosine and α2 agonists. Some of the reported disadvantages with the use of these agents include resistance to vasodilators, tachyphylaxis with nitroglycerin, cyanide toxicity with the use of nitroprusside and delayed recovery from anesthesia with the use of high doses of inhaled anesthetics.[11] Esmolol and nitroglycerine have been frequently compared for controlled hypotension during nasal surgery.[812] Dexmedetomidine has also gained wide acceptance for induced hypotension because of its sedation, analgesia and anxiolysis.[111314] There are no studies comparing the efficacy of these three drugs in achieving controlled hypotension. Therefore, this randomized study was planned using these three drugs for inducing and maintaining controlled hypotension in patients undergoing functional endoscopic sinus surgery (FESS) under general anesthesia. BODY.MATERIAL AND METHODS: This prospective randomized study was carried out after the approval of Institutional Ethics Committee. Hundred and fifty patients belonging to American Society of Anesthesiologists physical status class I or II, aged between 18 and 55 years and posted for elective FESS under general anesthesia were included in the study. Fifty patients were allocated to each of the three groups randomly, based on computer generated numbers. The operating surgeon and the anesthesiologist doing the peri-operative monitoring were blinded to the study drug by wrapping the syringes with number codes. Patients with uncontrolled hypertension, cardiovascular diseases including rhythm disturbances, renal or hepatic dysfunction, coagulation defects or patients on medications affecting coagulation system were excluded from the study. A thorough preanesthetic evaluation was performed and an informed written consent was taken from all the patients by the investigator a day prior to the surgery. The patients received nil per oral instructions as per the standard protocol and were premedicated with alprazolam 0.25 mg and ranitidine 150 mg orally in the night and in the morning of day of surgery. After shifting the patients to the operating room, noninvasive blood pressure, five lead electrocardiography and pulse oximetry were started. Baseline vitals were recorded including heart rate (HR), MAP and oxygen saturation. After securing an intravenous line, preloading was carried out with lactated ringer's solution 5 ml/kg. The patients were randomly allocated by computer generated numbers in to three groups: Group D: Received dexmedetomidine loading dose of 1 μg/kg given over 10 min, followed by a continuous infusion of 0.5-1.0 μg/kg/h. Group N: Received an infusion of nitroglycerine 0.5-2 μg/kg/min. Group E: Received a loading dose of esmolol 1 mg/kg infused over 1 min, followed by a continuous infusion of 0.5-1.0 mg/kg/h. All the infusions were titrated to maintain a MAP between 60 and70 mmHg. The loading doses of dexmedetomidine and esmolol were administered before the induction of anesthesia. Nitroglycerine was started as infusion without any bolus dose. The duration of infusion was constant in all the three groups. The induction of anesthesia was done with thiopentone sodium 5 mg/kg and fentanyl 2 μg/kg, followed by vecuronium 0.1 mg/kg intravenously. After tracheal intubation, anesthesia was maintained with one minimum alveolar concentration isoflurane in nitrous oxide and oxygen mixture (60:40) and top-up doses of vecuronium as and when required. An oropharyngeal pack was kept after the intubation.[15] An additional dose of fentanyl 1 μg/kg was given intra-operatively with an increase in HR and MAP of more than 20% from baseline values. To further reduce the amount of surgical bleeding and for surgeon's convenience, all the patients were positioned in approx. 30° reverse trendelenburg position. Two ml of lignocaine-adrenaline (1:100,000) mixture was infiltrated at the surgical site by the surgeon in all the patients. Heart rate, MAP, SpO2 and EtCO2 were monitored throughout the surgery and recorded at baseline, after loading dose of the study drug, after induction, after intubation, 5 min after intubation, at an interval of 5 min intra-operatively, after reversal, after extubation and 5 min after extubation. HR <45 beats/min was considered as bradycardia, and was managed with 0.5 mg atropine intravenously. MAP <60 mmHg was initially managed with a 50% reduction in the infusion dose of the study drug and further stoppage of the infusion if no response was obtained in 5 min. Mephentermine 6 mg intravenously was administered for the resistant hypotension. The visibility of the operative field was assessed by the surgeon according to the scale proposed by Fromme and Boezaart.[16] Five minutes before the end of surgery, all the study drugs were discontinued. At the end of the surgery, the nasal packing was done keeping a cut piece of small size endotracheal tube to allow the patient to breathe through the nose postoperatively.[17] The residual neuromuscular blockade was antagonized with neostigmine 0.05 mg/kg and glycopyrrolate 0.1 mg/kg intravenously and extubation was done when the patient was fully awake, breathing regularly with adequate tidal volume. Total intra-operative fentanyl consumption, duration of surgery and total anesthesia time were recorded. Emergence time, defined as the interval between discontinuation of the anesthetics to response of eye opening to the verbal command,[18] was also recorded. The postoperative sedation was assessed with Ramsay Sedation Score.[19] The postoperative side-effects such as nausea and vomiting, shivering and dry mouth were observed and recorded. The time for the first analgesic request after the surgery was also recorded. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: Data were compiled analyzed using the Statistical Package for the Social Sciences 15.0 (SPSS Inc., Chicago IL, USA) statistical software package. Pearson Chi-square was used to examine the categorical data. MAP and HR within each group were analyzed using analysis of variance with Bonferroni's correction. Multivariate analysis was carried out to adjust surgeon's biased opinion and other confounding variables. To detect a significant difference of 10 mmHg in the MAP and also the associated simultaneous clarity of the surgical field between the groups, the overall sample size was estimated at 142 with a power of 80% and alpha error of 5%. However, 50 patients were selected for each group keeping in consideration the possible dropouts and for better validation of results. A P < 0.05 was considered significant. BODY.RESULTS: A total of 150 patients were included in the study and were divided randomly into three groups of 50 patients each (n = 50). The demographic variables among the three groups are shown in Table 1 and there was no statistically significant difference among the three groups with regard to demographic variables, duration of surgery and total anesthesia time. The mean total dose of fentanyl in μg/kg used was significantly lower in group D compared to groups N and E (1.2 ± 0.75 vs. 3.6 ± 1.3 and 2.9 ± 1.1 respectively) [Table 1]. Table 1 The demographic variables The mean HR was significantly lower in group D compared to groups N and E at all the times of measurements (P < 0.05) [Table 2]. Table 2 Comparison of mean HR (per minute) in group D, N and E The MAP was significantly lower in group D compared to groups N and E after infusion of study drugs, after induction of anesthesia, after intubation and 5 min after intubation (P < 0.05) [Table 3]. However, the desired MAP for intra-operative induced hypotension could be achieved in all the three groups. There was no statistically significant difference in the Fromme's score in the three groups. None of the patients experienced bradycardia, resistant hypotension or hypertension during the study period. None of them required additional atropine or mephentermine. Table 3 Comparison of MAP (mmHg) in group D, N and E The emergence time was significantly shorter in group E and group N compared to group D [Table 1]. No serious side-effects were observed in any of the three groups. The incidence of nausea and vomiting was comparable in the three groups. The major side-effect observed in group D was dry mouth (26%) [Table 4]. The Ramsay Sedation Scores were significantly higher in group D compared with groups N and E with majority of patients having a sedation score of 3 (46%) in group D while most of the patients had a score of 2 in groups N and E (54% in group N and 50% in group E) [Table 5] two patients in group D had sedation score of 5 and were deeply sedated. The time to first analgesic request was significantly prolonged in group D when compared to other groups [Table 1]. Table 4 Side-effect profile of patients in groups D, N and E Table 5 Degree of sedation (Ramsay Sedation Score) during postoperative period BODY.DISCUSSION: An important technique to reduce bleeding during the surgery is controlled reduction in blood pressure to such levels so that bleeding is minimal, but at the same time perfusion to the vital organs is well-maintained. This is the underlying concept for controlled hypotensive anesthesia.[20] Reduced bleeding in the operative site improves the quality of the surgical field, decreases the number of manipulations as well as the incidence of major complications and shortens the surgical time.[1021] Dexmedetomidine, a selective α2 adrenoceptor agonist, causes reduction in blood pressure, slowing of HR, sedation and analgesia. The fall in blood pressure is mainly due to inhibition of central sympathetic outflow and also due to stimulation of presynaptic α2 adrenoceptors decreasing norepinephrine release.[22] An important advantage is its minimal respiratory depressant effect with potent sedative and analgesic effects compared with opioids and other sedatives. A few studies have shown that dexmedetomidine decreases the bleeding in surgeries within the framework of hemodynamic stability.[11131423] The heart rate was higher in group N due to reflex tachycardia associated with nitroglycerine infusion. Dexmedetomidine caused a lower heart rate due to its sympatholytic effect.[24] The MAP also showed a significant reduction in group D compared to group N and E, but only at three observation times, that is, after induction of anesthesia, after intubation and 5 min after intubation. This observation suggested that dexmedetomidine is effective in blunting the hemodynamic response of stress during laryngoscopy as has been shown by other studies.[2526] The MAP however, was equally lowered in all the three groups suggesting equal efficacy of all the three drugs in lowering the MAP, thereby providing comparable surgical field as suggested by the Fromme and Boezaart's score. Cincikas and Ivaskevicius[27] used nitroglycerine infusion (0.79 ± 0.34 μg/kg/min) to maintain MAP of 50-60 mmHg during endoscopic nasal surgery and observed reduced surgical bleeding and improved surgical view quality. Guven et al.[28] used dexmedetomidine for conscious sedation for FESS and reported better hemodynamic stability and improved surgical field. The analgesic efficacy of dexmedetomidine has been appreciated in diverse settings.[2930313233] Similarly, we found that intra-operative fentanyl requirement was significantly reduced in the dexmedetomidine group as compared to the other two groups. The patients in group D had a longer emergence time, as reported in other studies as well.[1134] We also observed a significant delay in the first postoperative analgesic request in group D as compared to the other two groups. It has been shown that perioperative analgesic requirements are significantly reduced with intra-operative use of dexmedetomidine infusion.[1135] The patients in the dexmedetomidine group had significantly higher sedation scores compared to group N and E. Shams et al.[11] also reported higher postoperative sedation scores with the intra-operative use of dexmedetomidine. The sedative and analgesic sparing effects of dexmedetomidine are mediated through its action in the locus coeruleus and dorsal horn of spinal cord respectively.[36] The postoperative sedation is often desirable, but may sometimes prolong the emergence time.[34] The incidence of postoperative shivering was significantly lower in the dexmedetomidine group, as acknowledged earlier also.[37] The most frequent reported side-effect with dexmedetomidine is dry mouth, which is not bothersome and can be easily managed. BODY.CONCLUSION: Dexmedetomidine and esmolol provided better hemodynamic stability and comparable operative field visibility to nitroglycerine during FESS. Dexmedetomidine provides an additional benefit of reducing the analgesic requirements and providing postoperative sedation.

TITLE: Spectrophotometric analysis of crown discoloration induced by MTA- and ZnOE-based sealers ABSTRACT: Crown discoloration can be induced by root canal sealer remnants following root canal treatment. ABSTRACT.OBJECTIVE:: The aim of this study was to evaluate chromatic alterations in human tooth crowns induced by a Mineral Trioxide Aggregate-based sealer (MTA Fillapex® and a commonly used ZnOE-based sealer (Roth-811). The tested null hypothesis was that the application of the materials did not induce clinically perceptible crown discoloration (Ho: CIE color difference ΔE<3.7). ABSTRACT.MATERIAL AND METHODS:: Forty five fully developed, intact, mandibular third molars were sectioned 1 mm below the cemento-enamel junction. The pulp chambers were chemomechanically debrided via the cervical access. The specimens were randomly assigned into three groups Group 1: MTA Fillapex, Group 2: Roth 811, Group 3: Negative control (unfilled) and immersed in individually marked vials containing distilled water up to the cervix (37±1o C). The spectral reflectance lines were recorded by utilizing a UV-VIS spectrophotometer equipped with integration sphere in the visual spectrum at baseline, 1 week, 1 and 3 months after material placement. Data were transformed into values of the CIE L*a*b* color system and the corresponding ΔE values were calculated. Statistical analysis was performed using two-way mixed ANOVA models, at p=0.05 level of significance. ABSTRACT.RESULTS:: A statistically significant increase in a* and b* chromatic parameters of the MTA Fillapex Group was measured. However, ΔE values did not exceed the human eye perceptibility threshold (set at ΔE<3.7) during the experimental period (ΔEt3=2.88). In Roth-811 Group, a statistically significant decrease in L* and a statistically significant increase in a* and b* chromatic parameters was measured, during all observation periods. Resultant ΔE values exceeded the human eye perceptibility threshold after 1 week (ΔEt1=5.65). ABSTRACT.CONCLUSIONS:: Application of MTA Fillapex in tooth crowns resulted in minimal color alterations, while Roth 811 induced severe discoloration, in vitro. It could be suggested that, in terms of aesthetics, the use of MTA Fillapex appears to be favorable. BODY.INTRODUCTION: Poor aesthetic appearance of endodontically treated teeth remains a challenging issue in clinical dentistry even nowadays. Interestingly, it has been reported that poor aesthetic appearance of a treated tooth significantly affects the patients' quality of life10. A major etiological factor for the occurrence of local intrinsic staining, especially in the cervical and middle third of the crown, is the presence of root canal filling materials in contact with the coronal dentin of the pulp chamber16,17. In the long-term, core materials and sealers interact with dentin. Any change to the optical and chromatic properties of the dentinal structure is likely to cause an alteration in the outward appearance of the crown caused by its light transmitting and reflecting properties16,17. Apart from thorough pulp chamber debridement, a reduction of the coronal aspect of the root canal filling below the clinical cervix is required for the prevention of sealer-induced crown discoloration in the anterior aesthetic zone. However, sealer remnants cannot be always thoroughly removed from the pulp chamber and sometimes are present due to iatrogenic inadequate manipulations. Despite the improvement of physicochemical, biomechanical and biological properties of endodontic sealers, the appearance of coronal discoloration is still evident in daily practice. Several laboratory studies have shown that some categories of sealers including ZnOE and epoxy-based sealers are capable of inducing moderate to severe crown discoloration17,24,25,29. In endodontics, ZnOE sealers are used in clinical practice for many decades and are regarded as a gold standard in several laboratory and clinical studies. These materials are regarded to be clinically satisfactory, providing reasonable seal in the root canal system13. However, after setting reaction, the formation of a weak porous mass leads to dissolution in contact with tissue fluids8. Due to gradual hydrolysis, the release of eugenol leads to long-lasting cytotoxicity and additional potential for sensitization6. Recently, Mineral Trioxide Aggregate (MTA) Fillapex® was introduced as a new generation MTA-based sealer. The main concept for the development of MTA-based sealers is the exploitation of the physical and biological properties of MTA such as bioactivity12, biocompatibility21 and hard tissue conductivity22. A developing amount of research data is becoming available upon MTA Fillapex, with regard to its physical and biological properties2,13,14,23,26. Considering the increasing demands for aesthetics, biomaterials should be chromatically stable, present optical properties similar to dental structures and not exert staining effects to hard dental tissues17. Recent reports showed that both White and Gray MTA formulations are capable of inducing tooth discoloration4,5. From that perceptive, it is mandatory for every new MTA-based material to be tested in terms of aesthetic and color objectives. Currently, there are no available studies investigating the potential of MTA Fillapex to induce color alterations to dental tissues. The aim of this study was to evaluate the chromatic alterations in human tooth crowns induced by MTA Fillapex and Roth 811 root canal sealer. The null-hypothesis (Ho) to be tested was that the application of the materials did not induce clinically perceptible crown discoloration (Ho: CIE color difference ΔE<3.7)19. BODY.PREPARATION OF TEETH: Forty five freshly extracted, fully developed, impacted and semi-impacted mandibular third molars free of cracks, fractures, caries, abrasions and discoloration due to systemic intrinsic causes were collected, according to the guidelines of good clinical practice (Department of Oral and Maxillofacial Surgery, School of Dentistry, Ethical Committee, Aristotle University of Thessaloniki, Greece). Soft tissue was removed and the teeth were sectioned in the coronal third of the root complex, 1 mm below the buccal cemento-enamel junction. Sectioning was performed with the aid of a low-speed diamond-edge rotary saw microtome (Leica RM2255, Leica, Wetzlar, Germany). Access cavity preparation was not performed. Pulps were extirpated with a dental spoon and the internal axial walls of the pulp chambers were chemo-mechanically debrided with Hedstrom files (No. #60-80) and 10 ml of sodium hypochlorite (2.5% w/w), through the apical access. Gentle reaming of the internal axial walls was performed through all directions. Between every file sequence, the pulp chamber was irrigated with 2 ml of sodium hypochlorite (2.5% w/w) (3 file changes x 2 ml=6 ml). A final rinse with 4 ml of sodium hypochlorite (2.5% w/w) was performed at the end of the debridement. The pulp chambers of the specimens were finally washed with 5 ml sterile saline to remove sodium hypochlorite remnants. At the beginning of the experimental period, all crowns were transferred and stored in individually marked polyethlylene tubes containing distilled water up to the cervix of the crown in an incubator at 37±1o C. The teeth (N=45) were randomly assigned in one experimental (n=15), one positive (n=15) and one negative control group (n=15). The materials to be evaluated were MTA Fillapex (Angelus, Londrina, Brazil) (Group 1) and a ZnOE based sealer, Roth 811 (Roth's International, Chicago, IL) (Group 2) (Figure 1). The sealers were mixed and prepared according to the manufacturers' instructions and were placed into the pulp chambers via the cervical access. A finger plugger was used to coat the internal axial walls with the sealers. The apical access was sealed with a thin layer of glass-ionomer cement (Ketac Cem Aplicap, 3M, Espe, Germany) in order to address microleakage and sealers solubility. Negative controls (Group 3) were only instrumented and remained unfilled. Figure 1 Study groups 1-3 Group Sample size (n) Materials under study Manufacturer Composition Group 1 15 MTA Fillapex Angelus, Londrina, Brazil resins (salicylate, diluting, natural), bismuth trioxide, nanoparticulated silica, mineral trioxide aggregate, pigments Group 2 (positive control) 15 Roth 811 Roth's International, Chicago, IL Powder: Zinc oxide, bismouth bicarbonate, barium sulfate, dehydrated tetraboric sodium Liquid: Chemically pure eugenol Group 3(negative control) 15 Unfilled     BODY.MEASUREMENT OF CROWN CHROMATIC ALTERATIONS: A double-beam UV-Vis spectrophotometer equipped with integrating sphere was used (UV-2401PC, Shimadzu Corporation, Kyoto, Japan) and a standardized mounting system was developed, utilizing a previously validated experimental model17. Standard D65 illumination was chosen (Commission Internationale de L'Eclairage 1978), as it corresponds approximately to the spectrum of midday daylight in Western/Northern Europe. The spectrophotometer was linked to a computer, which recorded the spectral reflectance curves of the crowns, in visual spectrum (380-780 nm). The obtained spectral curves in visual spectrum (380-780 nm) were transformed into L*, a* and b* values of the perceptually uniform CIE L*, a*, b* color space using a specialized computer software (Color Analysis UV-2401PC). L* values describe lightness, which range from black (0) to white (100), while a* values represent red (+80a*) to green (-80a*), and b* values represent yellow (+80b*) to blue (-80b*) color variations. Total color differences (ΔE) were calculated according to the equation: ΔE= [(Li-L0*)2+(ai-a0*)2+(bi-b0*)2]1/2 The proposed acceptance for color matching adopted in this study was at 3.7 ΔE units (perceptibility threshold), beyond which the differences are clinically perceptible19. In dental science, when ΔE values are less than 1 unit, then color match occurs and any color differences cannot be identified by independent observers in vitro27. However, color determination in clinical dentistry may become complicated by adjacent anatomic structures and lighting conditions. As a result, the proposed acceptance for color matching in dentistry has been set to 3.7 units (perceptibility threshold), beyond which differences are clinically visible19. A standardized mounting system was developed for the customization and the reproducibility of the crown's position. The cylindrical inner frame (diameter=2 cm, inner height=0.2 cm) of the black bakelite sample assembly that the spectrophotometer was equipped with, was filled with black, non-polychromatic, thermo-plasticized silicone. The lingual surface of each crown was fixed within the silicone mass during its setting phase in order to construct individualized specimen carriers. The same individualized, silicone carrier for each specimen was used for measurements in all time intervals. The specimens were positioned in the in the circular opening of the aperture mask of the integrating sphere with the aid of an aligning system in order to ensure the reproducibility of the measured surface. The dimensions of the polychromatic beam that illuminated the sample were 7x7 mm; thus the majority of the cervix and the crown surface was exposed and measured. The color appearance of the buccal surfaces of the crowns was measured in order to simulate their clinical appearance. Measurements were carried out by the same operator (I.M.) and at ambient temperature of 23±1o C. The spectrophotometer was calibrated at each time interval using BaSO4 reference. Measurements were performed prior to the placement of the materials (baseline: t0) and consecutively 1 week (t1), 1 month (t2) and 3 months (t3) after sealer placement. All measurements were repeated twice and averaged. If the total color difference (ΔE) between 2 measurements taken in a row exceeded the threshold of 1 DE unit, new measurements were obtained. At the end of the 3rd month, three crowns of the experimental groups were randomly selected and longitudinally sectioned vertically to the middle of their mesio-distal dimension, on a bucco-lingual aspect, with the aid of a low-speed diamond-edge rotary saw microtome (Leica RM2255, Leica, Wetzlar, Germany). Digital images of the sectioned crown specimens were taken, in day-light conditions (Macro-Lense 100 mm, Canon EOS 1000D, Tokyo, Japan). BODY.STATISTICAL ANALYSIS: Sample size was calculated after power analysis based on the results of a pilot study, according to the equation n=2(z1-α/2 + zβ)2 σ2 {1+(m - 1)ρ}/mΔ 2 with ρ=0.5, m=4 (repeated measurements), σ2=4, Δ=2.2, alpha=0.05, power=0.8 (the chosen value of Δ was greater than the observed one in order to be close to the 3.7 threshold value and analogously done for the σ 2 )17. Accordingly the sample size to each group was calculated near to n=12 specimens per group. In case of drop out during the experimental period, 3 specimens were added in each group (n=15). Two-way ANOVA with repeated measurements was used for data analysis of the values of CIE L*, a*, b* chromatic parameters and the total color differences (ΔE). The significant effects and interactions of the experimental factors were investigated with pairwise between-group and within-group comparisons, which were conducted with Bonferroni's method. The overall analysis was performed with SPSS software (version 16.0, SPSS Inc., Chicago, Ill, USA). The level of statistical significance was set at p<0.05. BODY.RESULTS: Tables 1 and 2 present the mean L*, a*, b* and ΔE values of all groups, in all time intervals respectively. With regard to L*, a* and b* parameters, MTA Fillapex caused a statistically significant increase in a* (Δa*to-t3=1.043, p<0.001) and b* (Δb* to-t3=2.292, p<0.001) values. L* values remained stable. However, resultant ΔE values indicate that MTA Fillapex induced overall color changes below the human perceptibility threshold, ranging between 2.12-2.88 units. Roth 811 induced severe color changes exceeding the perceptibility threshold from the 1st week of evaluation (ΔE=5.65). A statistically significant increase in a* (Δa*to-t3=-3,335, p<0.001) and b* (Δb* to-t3=-4,014, p<0.001) values was observed, while L* values statistically decreased (ΔL*to-t3=4,926, p<0.001). In Group 3, L*, a*, b* values remained stable in all time intervals. Table 1 CIE L*, a*, b* mean standard deviation values of Groups 1-3, in all experimental periods     baseline 1 st week 1 st month 3 rd month   (CIE) L* parameter           Groups n L 0 L 1 L 2 L 3 MTA Fillapex 15 84.95(3.14) 85.36(2.54) 84.35(3.10) 84.91(2.61) Roth 811 15 84.21(1.98) 81.89(1.18) 79.38(1.82) 79.28(1.71) Negative control 15 84.55(2.22) 84.77(2.26) 84.48(2.34) 84.55(2.17)                             baseline 1 st week 1 st month 3 rd month   (CIE) a* parameter           Groups n a 0 a 1 a 2 a 3 MTA Fillapex 15 0.15(0.68) 0.57(0.67) 0.77(0.68) 1.19(0.65) Roth 811 15 0.01(0.79) 2.03(0.82) 2.71(1.00) 3.34(1.05) Negative control 15 0.42(0.74) 0.39(0.66) 0.38(0.73) 0.41(0.73)                             baseline 1 st week 1 st month 3 rd month   (CIE) b* parameter           Groups n b 0 b 1 b 2 b 3 MTA Fillapex 15 21.60(2.82) 23.36(2.21) 22.64(2.74) 23.89(2.35) Roth 811 15 21.26(2.33) 25.67(2.36) 24.76(2.72) 25.28(2.89) Negative control 15 20.62(3.11) 20.33(2.89) 20.49(2.94) 20.57(3.07) Within-group comparisons: a statistically significant differences when compared to t 0 , b when compared to t 1 , c when compared to t 2 , d when compared to t 3 Between-group comparisons: a statistically significant differences when compared to Fillapex, B when compared to Roth 811 Table 2 Mean standard deviation ΔE values of Groups 1-3 in all experimental periods     1 st week 1 st month 3 rd month Groups n (ΔΕ 1 ) (ΔΕ 2 ) (ΔΕ 3 ) MTA Fillapex 15 2.13(1.18) 2.12(0.72) 2.88(1.49) Roth 811 15 5.65(2.34) 6.70(2.68) 7.37(3.02) Negative control 15 0.51(0.37) 0.61(0.30) 0.51(0.20) Within-group comparisons: a statistically significant differences when compared to ΔΕ1, b when compared to ΔΕ2, c when compared to ΔΕ3 Between-group comparisons: A statistically significant differences when compared to MTA Fillapex, B when compared to Roth 811 The macroscopic examination of the sectioned specimens of Group 1 (MTA Fillapex) showed stability of sealer color within its mass and absence of dentinal staining (Figure 2). On the contrary, in Group 2 (Roth 811) the set sealer displayed a granular, grayish appearance. Dark orange dentinal staining accompanied by varying leaching within tubules was evident, in areas where sealer remnants were in contact with the axial walls of the pulp chamber (Figure 3). Figure 2Appearance of Group 1 (MTA Fillapex) after 3 months. Color stability of MTA Fillapex sealer within its mass and absence of dentinal staining Figure 3Appearance of Group 2 (Roth 811) after 3 months. Granular, grayish appearance of Roth 811 sealer and dark orange dentinal staining accompanied by varying leaching within tubules BODY.DISCUSSION: MTA Fillapex is a newly developed MTA-based root canal sealer. According to the manufacturer, its composition after mixture is basically MTA particles incorporated in a matrix of salicylate resin, natural resin, bismuth and silica. Other proposed MTA-based sealers consist of MTA with other additives, including HEMA, TEGDMA resins (lc-MTA) and water-soluble polymers (ProRoot-Endo sealer)7,11. Recent data reported that MTA Fillapex presents acceptable physical properties, antibacterial properties and intracanal resistance to dislodgement similar to AH Plus2,23. The sealer presents increased cytotoxicity during the setting reaction which decreases over time26. After setting, the sealer is capable of retaining alkaline pH levels due to calcium release, and presents bioactivity stimulating hydroxyapatite crystal nucleation in human osteoblast-like cell cultures26 and mineralization in connective tissue14. The rational for organizing this research study was the increasing publication of case reports, in which the discoloring effect of MTA has been reported. Both Gray and White MTA repair materials were capable of inducing coronal discoloration, when applied in cases of root and furcal perforation, pulpotomy and pulp revascularization procedures4,5. In addition, two recent laboratory studies confirmed the previous clinical observations with the aid of instrumental colorimetry1 and visual spectrophotometry18. It has been proposed that metal oxides, such as iron and manganese, could be responsible for the discoloring effects of Gray MTA18. Moreover, recent data have shown that the discoloring effects of White MTA have been attributed to its progressive mass darkening due the presence of reduced black crystals of bismuth atoms under light conditions28. This laboratory study is the first to report tooth color alterations induced by MTA Fillapex, utilizing the methodology of visual spectrophotometry. Within the limitations of this experimental study, in acceptance of the null hypothesis, MTA Fillapex did not induce clinically perceptible crown discoloration. In rejection of the null hypothesis, Roth 811 induced fast and severe discoloration and exceeded the perceptibility threshold 1 week after sealer placement. The results of this study indicated that the tested sealer had minimum potential to induce color alterations in human teeth in vitro, since the measured color alterations did not exceed the threshold of perceptibility set (ΔE<3.7). The descriptive analysis of the CIE L*, a*, b* chromatic parameters showed a statistically significant color change towards red and yellow after three months, while lightness was not affected. The macroscopic findings confirmed that the sealer did not present color changes within its mass, maintaining its yellowish color throughout the experimental period. Dentinal staining was not evident as well. The increase of CIE a* and b* values of the crowns from the first week of investigation may be attributed to alterations in specimens' optical properties due to the physical presence of the sealer in the internal dentinal surface. Roth 811 sealer induced fast and severe discoloration, which was clinically perceptible 1 week following sealer placement. The chromogenic potential of ZnOE sealers has been attributed to the unstable chemical bond between ZnO and eugenol. Even after the end of the setting reaction, eugenol release leads to self-oxidation and becomes darker with time. The results of this study are in complete accordance with results of previous studies17,25,29. Several methods have been proposed for the evaluation or measurement of sealer-induced discoloration, including visual technique and computer analysis of digital photos24,25,29. Inherent objectivity and standardization difficulties may be improved by the use of a spectrophotometer15. This methodology has been reported as accurate and reliable in dentistry for quantitative tooth color measurements17,20. A major advantage of visual spectrophotometry is that tooth color measurement is based on the measurement of spectral reflectance, which describes the total reflection of a sample in visual spectrum17. Random errors in this study were minimized by strict control of the environmental factors along with multiple measurements and averaging. One of the main limitations of instrumental color measurement, however, is posed by systematic errors that can be attributed to variations in instrument geometrical design, metamerism and calibration techniques27. The comparison of absolute tristimulus values found in that study with other studies is discouraged. Evaluation and comparison of total color differences (expressed by ΔE), however, is well documented and considered as safe since differential measurement is highly reproducible between instruments9. The results of this study are comparable to those of a previous study which was conducted utilizing the same methodology and under the same experimental conditions. After 3 months, non-perceptible total color differences (ΔE) of MTA Fillapex were similar to the ΔE values of Guttaflow (Roeko, Coltene, Whaledent Ltd., Germany) and Epiphany SE (Pentron Clinical Technologies, Wallingford, CT), indicating that they likewise exert minimal chromogenic effects17. The results of this study do not directly represent the in vivo tooth discoloration potential of root canal sealers in good clinical practice. In this laboratory study, the investigation of the discoloration potential of root canal sealers was based on the generation of a "worst case scenario" by leaving a significant amount of sealer in direct contact with the axial dentinal walls and several anatomical features of the pulp chamber17. However, the knowledge of the magnitude of the sealers' chromogenic potential indicates that thorough cleaning measures are essential to prevent discoloration postoperatively. Regardless of the minimal staining effects of new generation root canal sealers including MTA Fillapex, the clinician should always ensure thorough removal of sealer remnants. Apart from basic properties, such as biocompatibility and good sealing ability, it could be suggested that the chromogenic potential of sealers may also play an important role in selecting proper root canal filling materials. BODY.CONCLUSION: Within the limitations of this experimental study, MTA Fillapex did not induce clinically perceptible crown discoloration. Roth 811 induced fast and severe discoloration and exceeded the perceptibility threshold 1 week after sealer placement. Although the incorporated MTA has proved chromogenic potential, MTA Fillapex posed minimal risk for potential staining effects. It could be suggested that, in terms of aesthetics, the use of MTA Fillapex appears to be favorable.

TITLE: Serum lutein concentrations in healthy term infants fed human milk or infant formula with lutein ABSTRACT.BACKGROUND: Lutein is a carotenoid that may play a role in eye health. Human milk typically contains higher concentrations of lutein than infant formula. Preliminary data suggest there are differences in serum lutein concentrations between breastfed and formula-fed infants. ABSTRACT.AIM OF THE STUDY: To measure the serum lutein concentrations among infants fed human milk or formulas with and without added lutein. ABSTRACT.METHODS: A prospective, double-masked trial was conducted in healthy term formula-fed infants (n = 26) randomized between 9 and 16 days of age to study formulas containing 20 (unfortified), 45, 120, and 225 mcg/l of lutein. A breastfed reference group was studied (n = 14) and milk samples were collected from their mothers. Primary outcome was serum lutein concentration at week 12. ABSTRACT.RESULTS: Geometric mean lutein concentration of human milk was 21.1 mcg/l (95% CI 14.9–30.0). At week 12, the human milk group had a sixfold higher geometric mean serum lutein (69.3 mcg/l; 95% CI 40.3–119) than the unfortified formula group (11.3 mcg/l; 95% CI 8.1–15.8). Mean serum lutein increased from baseline in each formula group except the unfortified group. Linear regression equation indicated breastfed infants had a greater increase in serum lutein (slope 3.7; P < 0.001) per unit increase in milk lutein than formula-fed infants (slope 0.9; P < 0.001). ABSTRACT.CONCLUSIONS: Breastfed infants have higher mean serum lutein concentrations than infants who consume formula unfortified with lutein. These data suggest approximately 4 times more lutein is needed in infant formula than in human milk to achieve similar serum lutein concentrations among breastfed and formula fed infants. BODY.INTRODUCTION: Lutein is a carotenoid that selectively accumulates in the macular region of the retina and protects retinal cells required for vision [16]. Humans cannot synthesize lutein and thus all lutein in the human body comes from dietary sources. Studies in adults have revealed that dietary or supplemental lutein leads to variable increase both in serum lutein and in macular pigment [8, 13]. Some epidemiological evidence suggests lutein may reduce the risk of age-related macular degeneration [22, 24]. Lutein may be important well before adulthood. Lutein is present in cord blood at the time of newborn delivery indicating placental transfer to the fetus, and cord blood concentrations are highly correlated to maternal serum concentrations [28]. Lutein is present in human donor eyes as early as 17–22 weeks gestation [1, 9]. After birth, breastfed infants receive lutein from mother's milk [2]. At 1 month of age there are differences in serum lutein concentrations between breastfed and formula-fed infants [14]. This finding is plausible because there are higher concentrations of lutein in human milk than in infant formulas. Infant formulas contain a small amount of innate lutein contributed by ingredients in the formula. A next step in understanding lutein nutrition in early life is to determine serum lutein concentrations of exclusively breastfed and formula-fed infants. The aim of this study was to examine serum lutein concentrations in infants fed human milk or formulas unfortified and fortified with lutein. This study is unique because it is the first prospective study of serum lutein concentrations in healthy full-term infants consuming different concentrations of lutein in formula in comparison to serum lutein concentrations in human milk fed infants. BODY.METHODS.SUBJECTS: This 31⁄2 month double-masked, parallel, prospective 12 week feeding study enrolled exclusively formula-fed and breastfed infants. Infants were recruited from hospitals and pediatric practices in the Portland, Oregon area. Oregon Health and Science University Institutional Review Board approved the study protocol and the informed consent form. Infant inclusion criteria included: healthy singleton term infants (37–42 weeks gestation) between 9 and 16 days of age, parent or guardian of formula-fed infants agreed to feed assigned study formula ad libitum, and breastfeeding mother's agreed to exclusively breastfeed for duration of study. An inclusion criterion for a breastfeeding mother was a self-reported consumption of 6 or more 1⁄2-cup servings per week of dark green vegetables, which are a rich source of lutein [11]. To verify consumption, each breastfeeding mother at study entry completed a validated, semi-quantitative food frequency questionnaire [4]. Questionnaires were analyzed at the Harvard School of Public Health and lutein intake was calculated from the Harvard University food composition database derived from the USDA Survey Nutrient database plus other sources. BODY.METHODS.STUDY FORMULAS: The lutein fortification concentrations targeted for this study were 25, 100, and 200 mcg lutein per liter of formula. These concentrations were chosen because they are within the range of lutein concentrations observed in the largest, multinational study of breast milk carotenoids [2]. Study formulas were analyzed several times during the trial to monitor actual lutein content in the formulas over time. Lutein was present in the raw material and the innate amount of lutein was ~20 mcg/l, which contributed to the final lutein concentration in the formula. Thus, the four study formulas contained the following average concentrations of lutein as confirmed by analysis: 20 (unfortified), 45, 120, and 225 mcg/l. At enrollment, formula-fed infants were randomized to one of these four study formulas, all of which were based on Wyeth S-26 Gold (Askeaton, Ireland). The source of lutein for the fortified formulas was FloraGLO® lutein 20% liquid in safflower oil (Kemin Industries, Inc., Des Moines, Iowa). The lutein in this material was supplied as purified oleoresin obtained from the extraction, purification, and crystallization of oleoresin from the marigold flower. A saponification step in the processing created free lutein from lutein esters. Study formulas were supplied as ready-to-feed liquid in 250 ml tetrabriks with (per l): 14.3 g protein, 73 g carbohydrate, 36 g fat, and 672 calories. All formulas contained approximately 135 mcg/l of beta-carotene, and other micronutrients did not differ among the formulas. BODY.METHODS.HUMAN MILK AND SERUM SAMPLES: Human milk was collected using an Avent ISISTM breast pump (Philips Avent, London, UK) [5] at mid-afternoon from each breastfeeding mother the day before study visit weeks 4, 8, and 12. A single, mid-afternoon sample of breast milk represents the average 24-h concentration of lutein [6]. The mother expressed the sample into clean bottles and stored it in the home freezer until the study visit the following day. Serum lutein concentrations were determined from infant blood samples collected at baseline and week 12. Whole blood (1–2 ml) was obtained from each infant by venipuncture by a registered pediatric phlebotomist or by heel stick by a pediatric registered nurse. Human milk and serum samples were stored at −80 °C at the study site in a cryobox for light sensitive specimens, shipped on dry ice to a contract bioanalytical laboratory (Craft Technologies, Inc., Wilson, North Carolina), and verified frozen on arrival. Samples were stored at −70 °C before being analyzed. All laboratory lights were covered with ultraviolet filters to protect samples from degradation. Human milk was analyzed for milk lipid concentration determined by creamatocrit [17] and carotenoid composition. BODY.METHODS.LUTEIN AND BETA-CAROTENE ANALYSES: Serum concentrations of lutein and beta-carotene were measured by Craft Technologies using high performance liquid chromatography (HPLC) with multiwavelength photodiode-array absorbance detection as previously described [19]. A 150 μl volume of serum was mixed with an equal volume of buffer and then mixed with two volumes of ethanol containing the internal standard (tocol). Lutein and beta-carotene were extracted from the aqueous phase into hexane. The combined hexane extracts were dried under vacuum. The extract was redissolved in ethyl acetate and diluted in mobile phase. An aliquot was injected onto a C18 reversed phase column and eluted isocratically. The analytes possess absorbance that is proportional to their concentration in solution and therefore these properties were used for quantitative analysis. Lutein and beta-carotene were measured by absorbance at 450 nm. Chromatograms were recorded using a computer data system. Analytes were quantified by external standard quantitation using neat standards to calculate response factors based on the peak area of the analyte. The quantities of analytes were corrected for recovery post-run based on the internal standard. Coefficient of variation was less than 7%. Concentrations of these analytes in human milk were determined after saponification of the samples. An aliquot of milk sample was mixed with potassium hydroxide in methanol (with pyrogallol added as antioxidant) and allowed to stand overnight in a refrigerator to hydrolyze lipids. Each sample was then extracted by use of tetrahydrofuran and hexane; the combined extracts of each sample were evaporated under vacuum and the residue was dissolved and analyzed by reversed-phase HPLC as described above. BODY.METHODS.STATISTICAL METHODS: Human milk and serum lutein concentrations were not normally distributed and data are presented as geometric means and 95% two-sided confidence intervals. Linear regression was performed to analyze the relationship between lutein in the milks and lutein in serum at week 12. Linear regression analyses were performed with and without baseline serum lutein concentrations in the model. Final regression lines do not include baseline serum lutein concentrations as a covariate. BODY.RESULTS: The mean (SD) age at study entry was 13 (±2.0) days. A total of 40 infants were enrolled, including 14 human milk-fed and 26 formula-fed infants (Table 1). Eighty-five percent (34/40) of enrolled infants completed the study. Six infants did not complete the study due to the following reasons: 2 infants were lost to follow-up; 2 families moved out of the area; 1 infant was no longer exclusively human milk-fed, and 1 formula-fed infant withdrew due to an adverse event that was not related to the formula according to the study pediatrician.Table 1 Infant demographic information at enrollmentRandomized formula-fed group Formula lutein concentration (mcg/l)Breastfed group20 [Control] (n = 6)45 (n = 6)120 (n = 7)225 (n = 7)Human milk (n = 14)Age at enrollment (days) Mean ± SD13 ± 1.312 ± 2.713 ± 2.114 ± 1.113 ± 2.1 Median1312141413 Range11–159–1510–1612–159–15Female sex [n (%)]4 (67)3 (50)5 (71)5 (71)7 (50)Race [n (%)] White5 (83)5 (83)6 (86)4 (57)12 (86) Black1 (17)0 (0)0 (0)0 (0)0 (0) Other0 (0)1 (17)1 (14)3 (43)2 (14)Anthropometrics Weight (kg)3.8 ± 0.43.9 ± 0.63.3 ± 0.43.9 ± 0.43.8 ± 0.4 Length (cm)51.1 ± 1.552.5 ± 3.450.1 ± 2.851.4 ± 1.151.8 ± 2.2 Head circumference (cm)35.9 ± 1.236.1 ± 0.834.7 ± 1.236.2 ± 1.336.1 ± 1.2 BODY.RESULTS.LUTEIN CONCENTRATIONS IN HUMAN MILK AND LUTEIN IN THE DIET OF BREASTFEEDING MOTHERS: Geometric mean lutein concentrations in human milk samples were similar at weeks 4, 8, and 12 (Table 2). The mean lipid content of the human milk samples was 41.5 g/l (95% CI 37.4–46.0). The average lutein concentration from the three collections of all breastfeeding mothers was 21.1 mcg/l (95% CI 14.9–30.0) and the average amount of lutein per gram of milk fat was 0.492 mcg (95% CI 0.319–0.761). Analysis of the Food Frequency Questionnaire, which was designed to report dietary intakes of both lutein and its structural isomer, zeaxanthin, indicated that the median lutein plus zeaxanthin in the maternal diet was 3363 mg/day [mean 4286 mg/day (95% CI 2679–5892)].Table 2Lutein concentrations in human milk at different study timesWeek 4n = 13Week 8n = 13Week 12n = 12Averagen = 13Lutein (mcg/l) Geometric mean20.520.119.721.1 95% CI12.8–32.915.5–26.112.3–31.514.9–30.0 Median212021.523 Range6–619–373–507–48Limit of detection of lutein in human milk was 0.4 mcg/l BODY.RESULTS.LUTEIN CONCENTRATIONS IN INFANT SERUM: Human milk-fed infants had higher mean serum lutein concentrations (81 mcg/l) than all formula-fed infants (13 mcg/l) at baseline. Serum lutein concentration increased from baseline in each of the formula groups except in the unfortified group (Table 3). Linear regression model with and without baseline serum lutein indicated baseline serum concentrations did not have a significant effect on final serum concentrations in either the formula (P < 0.001) or human milk fed groups (P = 0.001). After 12 weeks of feeding, the human milk group had a sixfold higher mean serum lutein concentration than the unfortified formula group. Fortification of formula with lutein resulted in a dose-dependent increase in serum lutein concentrations after 12 weeks.Table 3Lutein concentrations in infant serum at study entry and week 12Randomized formula-fed group Formula lutein concentration (mcg/l)Breastfed group20 [Control]45120225Human milkStudy entry n667714 Geometric mean11.912.922.48.581 95% CI6–23.96.9–2411.2–456.3–11.651–128.6 Median1013.526895 Range7–405–258–466–1719–236Week 12 n457513 Geometric mean11.335.9107.5192.169.3 95% CI8.1–15.830.1–42.888–131.4141.7–260.640.3–119 Median11.53710918066 Range9–1431–4282–148148–2787–168Limit of detection of lutein in the serum was 2 mcg/l BODY.RESULTS.RELATIONSHIP BETWEEN DIETARY LUTEIN AND INFANT SERUM LUTEIN CONCENTRATIONS: A positive linear dose-dependent relationship (Fig. 1) was observed between lutein in the formula and lutein in the serum. The slope of the regression equation was steeper for human milk lutein than lutein in formula. For infants fed human milk, serum lutein concentration increased approximately 3.7 mcg/l for every 1 mcg/l increase in human milk lutein concentration, whereas for infants fed formula, serum lutein concentration increased only 0.9 mcg/l for every 1 mcg/l increase in formula lutein concentration.Fig. 1Scatterplot of serum lutein concentrations (mcg/l) at week 12 in infants fed different concentrations (mcg/l) of lutein containing formula or human milk. Solid circles represent infant formula groups and open triangles represent human milk group. Linear regression equation for formula-fed groups was Yi = −4.4 + 0.9 (formula lutein) (r2 = 0.87, P < 0.001) and for the human milk group was Yi = 1.8 + 3.7 (human milk lutein) (r2 = 0.67, P < 0.001). Baseline serum concentrations did not have a significant effect on final serum concentrations (P = 0.001 for human milk; P < 0.001 for formula) and the slopes shown are based on unadjusted data BODY.RESULTS.LUTEIN FORTIFICATION AND SERUM BETA-CAROTENE CONCENTRATIONS: The mean average serum beta-carotene concentration at baseline across all formula-fed groups was 24.4 mcg/l. In the infants fed the 20 (control), 45, 120, and 225 mcg/l lutein containing formulas, the corresponding serum beta-carotene concentrations at week 12 were 128.0 (95% CI 47.3–346.6), 201.2 (95% CI 98.4–411.2), 173.2 (95% CI 125.5–238.8), and 202.3 mcg/l (95% CI 139.5–293.3), respectively. BODY.RESULTS.SAFETY EVALUATION: Infant growth was within normal limits as assessed by mean weight gain in grams per day as well as by means for Z-scores in weight, length, and head circumference (data not shown). There were no adverse events related to the formula and no serious adverse events reported. BODY.DISCUSSION: This is the first randomized, prospective study comparing serum lutein concentrations in healthy term infants consuming different concentrations of lutein in formula. An important finding from this study is that the breastfed infant reference group had approximately 6 times the mean serum lutein concentration of infants consuming unfortified formula during the first several months of life. Fortification of formula with lutein resulted in a dose-dependent increase in serum lutein. Approximately 4 times more lutein is needed in the formula than in human milk to achieve similar serum lutein concentrations among the breastfed and formula fed infants. It is interesting to note that despite the similarity in the lutein concentration of human milk and the unfortified formula, serum lutein concentrations were markedly different in the two groups of infants at week 12 but also at study entry. Two observations may account for this finding. Although we did not measure cord blood lutein concentrations, cord blood lutein did not differ at birth among infants who were ultimately fed human milk or infant formula (unpublished data, Wyeth, Dr. Minervini, 1993). However, infants who receive breast milk immediately after birth most likely consume substantially more lutein from colostrum than infants who consume infant formula unfortified with lutein [23, 25] and would explain the differences in serum lutein at study entry. In our study, the mothers reported at study enrollment that approximately 86% of the infants in the human milk group were exclusively breastfed from birth and 77% of the infants in the formula fed group received exclusively commercial formula. Secondly, our data suggest that the bioavailability of lutein from formula is considerably lower than from human milk and could explain the lower mean serum lutein concentration in the unfortified group at week 12 despite similar lutein concentrations in the human milk and unfortified formula. The ratio of the slopes from the linear regression lines indicates that a fourfold higher lutein concentration was needed in formula compared to human milk to achieve similar lutein concentrations between formula-fed and breastfed infants. The reason why lutein availability was different between human milk and lutein-fortified formula remains unclear. Lutein added to the formula is predominantly free lutein and similar to the dominant form of lutein in human milk. Several findings reveal that availability of lutein from foods would be affected by several factors including the food matrix, fat intake, and nutrient–nutrient interactions [3, 7, 10, 20, 21, 27]. In relation to those points, additional studies are needed to explain the difference in bioavailability. The human milk lutein concentration in this study was similar to the average values in the largest human milk survey published to date [2] reporting human milk lutein data from lactating women in nine countries including the US. Because dietary carotenoid intake influences human milk lutein concentrations, a goal of our study was to enroll breastfeeding mothers who consumed a diet rich in lutein. A study inclusion criterion for breastfeeding mothers was a reported consumption of 6 or more 1⁄2-cup servings per week of dark green vegetables, which are a rich source of lutein [11]. The results of the Food Frequency Questionnaire indicated that our goal of recruiting a population of breastfeeding women with a rich dietary intake of lutein was achieved. When compared to a sample of women from the Women's Health Initiative, the breastfeeding mothers in our study fell between the 60th and 80th percentiles of intakes observed in the much larger study (n = 1698) [18]. There is limited information regarding lutein concentrations in infant formulas. Lutein is currently not added to infant formulas in the US but it has been detected in the ingredients used in infant formula manufacturing. Unpublished data from our laboratory indicate typical skim milk powder and whey protein ingredients contain lutein in amounts from <3.3 to 10.2 mcg/100 g and <3.3 to 15.6 mcg/100 g, respectively. One study [12] reported lutein in formulas and in human milk samples from Northern Ireland mothers and found that most formulas studied had detectable lutein and the range of concentrations in formula was similar to the range of concentrations found in human milk. Serum beta-carotene concentrations were measured in this study because interactions between lutein and beta-carotene have been reported. Competition between lutein and beta-carotene for absorption was observed in healthy adults consuming supplements [15] and vegetable sources [26]. Lutein reduced beta-carotene concentrations in some subjects and enhanced it in others [15]. In our study, all infants were given formula with the same concentration of beta-carotene but the infants who consumed lutein supplemented formula had higher mean serum beta-carotene concentrations than infants who consumed control formula. These data suggest lutein supplementation enhanced absorption of beta-carotene but additional studies are needed to confirm. The strengths of this study include the use of a wide range of lutein concentrations in the formula, measurement of lutein concentrations in the human milk, and the length of time exclusively formula or breastfed. The small sample size for each group could be considered a limitation of the study although a linear dose response relationship was apparent. The exclusion criteria of infants less than 9 days old limits our understanding of the amount of lutein in colostrum and how it might have had an early impact on baseline serum lutein concentrations. However, requiring that the infants be older than a week at study entry was necessary to avoid recruiting into a trial during a time when a mother might be deciding between breast and formula feeding. In summary, infants fed formula unfortified with lutein have serum lutein concentrations approximately one-sixth those of breastfed infants in the first few months of life. An increase in lutein in infant formula leads to an increase in serum lutein in a dose-dependent manner. More lutein is needed in infant formula than human milk to achieve similar serum lutein concentrations among breastfed and formula fed infants. Additional studies are needed to demonstrate whether lutein fortification in infant formula leads to improved eye health or other benefits.

TITLE: Results of Surgical and Nonsurgical Treatment of Aneurysms in a Developing Country ABSTRACT: Background. The impact of invasive methods of treatment on results in developing countries may differ from that in developed countries. Methods. This is a prospective clinical study of consecutive patients with Subarachnoid Haemorrhage (SAH) admitted to the Ghaem Hospital, Mashhad during the period from 2005 to 2009. The initial diagnosis and investigations were carried out by neurologists. The patients were divided into two groups. One received surgical treatment whilst the other group was managed medically. The decision as to the choice of the method of treatment was made by the neurosurgeons. The initial medical treatment was standardised for all the patients. The rate of complications and mortality was compared in both medical and surgical groups. Results. 120 SAH patients (52% females) with a mean age of 50.6 ± 7 years were evaluated. The angiography revealed the presence of an aneurysm in 62 patients. 63.5% of the patients received medical treatment and 37.5% underwent aneurysmal surgery. Difference of rebleeding rate in the two therapeutic groups was not significant; X2 = .014, P = .91. The effect of rebleeding on mortality was not significant; X2 = 2.54, P = .14. Within 62 SAH patients with cerebral aneurysm, the mortality rate in both therapeutic groups was also not significantly different; X2 = .16, P = .77. Conclusion. There is no significant difference in the mortality rate between the "surgical" and non-"surgical" groups of Iranian patients with SAH. This could be due to delay in performance of surgery in Iranian neurovascular centers. BODY.1. INTRODUCTION: The outcome for patients with SAH remains poor, with mortality rates up to 45%, and there is significant morbidity among survivors [1]. The review of literature and prospective cohorts has shown that for untreated, ruptured aneurysms, there is at least a 3-4% risk of rebleeding in the first 24 hours and 1-2% per day in the first month [2]. Urgent investigation and treatment of patients with suspected SAH is therefore recommended [2]. The major complications following SAH are due to ischaemic deficit (27%) and hydrocephalus (12%) [3]. However the most feared complication for survivors of the initial haemorrhage is recurrent bleeding, which occurs in 15–20% of the patients and is associated with a 40–78% mortality [3]. The definitive method for prevention of rebleeding is to secure the aneurysm as soon as possible [2]. Early surgery may not be appropriate for every patient with SAH, but every attempt should be made to secure the aneurysm as soon as possible to prevent rebleeding [3]. Unfortunately, in Iran many patients with SAH are admitted to hospitals without facilities for catheter angiography, aneurysmal coiling, or direct aneurysmal surgery. Even in tertiary care hospitals with these facilities, many of the SAH patients are admitted after 3 days following the ictus, and for this reason the surgical or endovascular treatment is delayed for up to 3 weeks after event. Unfortunately, there is no policy of urgency in the Iranian emergency departments to direct the patients with SAH to appropriate management. Additionally, some of the Iranian SAH patients refuse surgery on cultural or economic grounds. In this paper, we report the first prospective study of aneurysmal surgery in patients with SAH in Iran. BODY.2. PATIENTS AND METHODS: Consecutive patients with subarachnoid hemorrhage (SAH) admitted in the Ghaem Hospital, Mashhad during 2005–2009 were enrolled in a prospective clinical study. Ghaem hospital is a university tertiary care center the in northeast of Iran. Neurologists, neurosurgeons, and radiologists are available 24 hours per day and 7 days per week in Ghaem Hospital. SAH patients who died before arriving to hospital were excluded from this study. The initial Hunt and Hess scale for each SAH patient on arrival to the hospital was recorded in both therapeutic groups. Diagnosis of SAH was based on a brain CT scan. Patients suspected to have a SAH but with normal brain CT scan underwent a FLAIR MRI scan and lumbar puncture [4, 5]. Catheter cerebral angiography is a routine diagnostic investigation in our SAH patients and is performed by general radiologists. SAH patients with an initially normal cerebral angiography usually underwent a second angiography after 3 weeks. Patients who did not have cerebral angiography due to poor medical condition, early death in hospital, or allergy to contrast material were excluded. Positive angiograms for aneurysm were found in 63.5% of our SAH patients. SAH patients were usually admitted for 3 weeks in either neurology or neurosurgery divisions. In our center, cerebral angiography followed by aneurysmal clipping is usually performed on patients with SAH within the first 72 hours after SAH. Patients who arrive to hospital after this time or were initially comatose were usually medically treated, and diagnostic procedures and surgery, if required, were performed in this group of cases after 3 weeks after event. SAH patients who underwent craniotomy and aneurysmal clipping were categorized as a surgical therapeutic group. Aneurysmal clipping with the aid of a microscope is the usual type of surgical procedure in our hospital for these patients. Aneurysmal wrapping is not performed and endovascular coiling is not available. Patients who underwent ventricular shunting for hydrocephalus without aneurysmal clipping were included in the medical therapeutic group. Surgical decision for the patient with aneurysm was made by a neurosurgeon. General medical condition of the patients was assessed by anesthetists and patients with poor cardiopulmonary or medical condition were excluded. SAH patients with Hunt and Hess scale of 5 were also excluded. Despite neurosurgical recommendation, some of our patients refused surgery due to cultural aspects or lack of funds. The medical management received was standardized in both surgical and medical groups of SAH patients. Principles of medical management included analgesia, nimodipine, sedatives, laxatives, control of blood pressure, and 3 litres of normal saline per day [4, 5]. Demographic features, risk factors, cerebral CT findings, clinical manifestations and aneurysm characteristics were evaluated in all patients. Mortality and complications in SAH, including rebleeding, hydrocephalus, and brain infarction due to vasospasm, were recorded in both medical and surgical groups of patients during hospitalization. The time was recorded and comparison made in each therapeutic group from SAH onset to hospital arrival, SAH onset to death, SAH onset to surgery (where applicable), and surgery (where applicable) to death in all patients. Chi-square and Fisher tests were used for statistical analysis, and P < .05 was declared as significant. The research project was approved by the ethics committee of Ghaem Hospital, and an informed signed consent was taken from patients or their first-degree relatives. BODY.3. RESULTS: The results of the treatment of one hundred and twenty SAH patients (63 females, 57 males) with a mean age 50.6 ± 7 years were prospectively evaluated. Patients with exclusion criteria are not considered in these 120 SAH cases. SAH was detected on cerebral CT in 95% of the cases. The patients were divided into two therapeutic groups. Among our SAH patients, 62.5% with mean age 52.4 ± 5 received medical treatment and 37.5% with mean age 49.7 ± 3 were subjected to surgery. Hypertension, smoking, oral contraceptive medication, past trauma, and overdosage of oral anticoagulation therapy were found in 41.6%, 19.1%, 0%, 0%, and .8% of patients, respectively. Fifty-six patients were subjected to surgery, of whom 45 underwent craniotomy and aneurysmal clipping, while 11 cases had CSF shunting without aneurysmal clipping and were included into the medically managed group. Overall mortality was 44.2% of all SAH patients (60.4% of females and 39.6% of males). There was no statistically significant difference in the death rate between the two therapeutic groups (X2 = 1.54, df = 1, P = .11) and no significant difference between the females and males (X2 = .73, df = 1, P = .39). The mean SAH onset to admission time in whole of our SAH patients and in their medical and surgical groups was 66 ± 4, 84 ± 1, and 24 ± 7 hours, respectively. The overall mean timing from the onset of SAH to death was 14.1 ± 2 days. In the surgical group, the mean length of time from onset of SAH to surgery was 8.4 ± 3 days, and for those who died, the mean length of the time between surgery and death was 5.9 ± 3 days. Table 1 compares the characteristics between surgical and medical groups. The effect of therapeutic type of aneurysm management on mortality was not significant; X2 = 0.16, P = .77. Rebleeding occurred in 4.4% of patients in the surgical group and 4% in the medical group and the difference was not statistically significant; X2 = .014, P = .91. Among 5 SAH patients with rebleeding, 2 had an anterior communicating artery aneurysm and 3 had a "normal angiography." The influence of rebleeding on the overall mortality was not statistically significant; X2 = 2.54, P = .14. None of the patients studied had rebleeding before admission to hospital. However, rebleeding might have occurred in patients who died before arriving to the hospital, and these were not included in this study. Hydrocephalus was found in 17 patients, and its frequency was significantly different in the two therapeutic groups; X2 = 5.58, P = .03. Out of 17 SAH patients with hydrocephalus, 13 (76%) died, and the effect of hydrocephalus on the mortality of these patients was significant; X2 = 7.93, P = .007. Cerebral infarction due to vasospasm occurred in 7 (5.8%) patients (4.4% of the surgical and 6.7% of the medical group). The effect of aneurysmal therapeutic strategy on frequency of cerebral infarction was not significant; X2 = 0.25, P = .71. Out of seven patients with brain infarction due to vasospasm, three cases died. The effect of cerebral infarction on the overall mortality of SAH patients was not significant; X2 = 0.005, P = 1. Table 2 represents the distribution of complications in two therapeutic groups of our patients. Aneurysms were found in the angiography of 62 patients (45 in the surgical and 17 in the medical group). The distribution was as follows: anterior communicating artery 41.9%, middle cerebral artery 23.1%, internal carotid artery 14.5%, basilar artery 4.8%, anterior cerebral artery 4.8%, posterior communicating artery 6.4%, and multiple aneurysms 3%. Among 62 SAH patients with aneurysm on angiography, 45 patients (72.6%) underwent aneurysm surgery and 17 cases (27.4%) received only medical management. Death was recorded in 48.9% of SAH patients with aneurysm who underwent aneurysm surgery (22/45) and 41.2% of patients with aneurysm who only received medical management (7/17). High Hunt and Hess scale, poor general medical condition, and refusal of patients constituted reasons of excluding these 17 cases with aneurysm from surgical group in 58.8%, 29.4%, and 11.7%, respectively. Details of Hunt and Hess scales of these seventeen cases are presented in Table 1. The difference in the mortality rate in 62 SAH patients with angiographically confirmed aneurysms in two therapeutic groups was not statistically significant; X2 = .16, P = .77. Table 3 illustrates causes of death in surgical and medical groups of our SAH patients. BODY.4. DISCUSSION: This study concerned SAH patients admitted alive; thus, the prehospital mortality is unknown in our SAH patients. Our hospital-based study revealed an incidence of negative angiography in 58/120 (48.3%). In other study from Iran, the incidence of negative angiography among SAH patients was reported to be 35/108 (32.4%) [6]. This is strikingly different from findings in Europe, Japan, and the USA, where studies have reported a 15–20% negative angiography result in SAH [1]. In aneurysmal surgery, delay in treatment is associated with increased rates of preoperative rebleeding, in both retrospective and prospective studies [2]. Recently it has been associated with higher rates of poor outcome [2, 7]. The International Cooperative Study on the Timing of Aneurysmal Surgery analysed management of 3521 patients, of whom 83% underwent surgical repair of the ruptured aneurysm [8]. The timing of surgery after SAH was significantly related to the likelihood of preoperative rebleeding. Patients who underwent early surgery had a significantly lower preoperative rebleeding rate than those who underwent later surgery (3% versus 11%) [8]. The mean time of onset to surgery in our SAH patients was 8 days which is longer than that reported in western countries [7]. This significant delay in the timing of aneurysmal surgery in our patients could be the main reason for failure to decrease mortality in our patients. During this period of delay, a number of complications may occur in the surgical group. In recent years, there has been a trend towards early surgery for ruptured aneurysms, especially in good- and moderate-grade patients [2, 7]. In addition, early surgery facilitates the possibility of aggressive therapy of vasospasm [2]. However, it is also reported that there were no overall differences in outcome in patients operated on early (0–3 days after SAH) or late (11–14 days after SAH) [7]. Surgical mortality was higher with early surgery due to brain swelling, disturbed autoregulation, and haemorrhage [7]. The treatment was most hazardous between days 7 and 10 due to the combined risks of rebleeding and vasospasm [7]. A prospective study, from three centers, indicates that despite attempts to do early surgery, rebleeding is still a significant problem, because only one half of the patients were operated on within 72 hours, and 35% of the patients with poor outcome had suffered rebleeding [9, 10]. In addition, some SAH patients with acute hydrocephalus may benefit from early placement of a ventricular drain in the hospital [11]. Acute hydrocephalus is more frequent in patients with poor clinical grade. The clinical significance of acute hydrocephalus after SAH is uncertain because many patients are apparently asymptomatic and do not deteriorate [2]. There was no statistical significance between type of management, mortality, and complications in our patients. The impact of rebleeding despite presence of hydrocephalus on mortality was not significant in our patients. This is inconsistent with results in developed countries [12]. The late presentation of the SAH patients in our center, compared to developed countries, is a local issue, and this leads to late treatment. The logistic multivariate regression test is necessary, to access the exact influence of the therapeutic subtype of aneurysm, on in-hospital death in SAH patients. However, this type of analysis needs more than five hundred SAH cases, which is out of our reach in the near future. Meanwhile, we consider our work the first pilot study conducted in Iran. Management of SAH patients in Iran is still suffering from major difficulties. Whether the obstacles are situated mainly on the diagnostic, technical, or intensive care level should be investigated in next studies.

TITLE: Randomized study of continuous high-dose lenalidomide, sequential azacitidine and lenalidomide, or azacitidine in persons 65 years and over with newly-diagnosed acute myeloid leukemia ABSTRACT: Therapy of acute myeloid leukemia in older persons is associated with poor outcomes because of intolerance to intensive therapy, resistant disease and co-morbidities. This multi-center, randomized, open-label, phase II trial compared safety and efficacy of three therapeutic strategies in patients 65 years or over with newly-diagnosed acute myeloid leukemia: 1) continuous high-dose lenalidomide (n=15); 2) sequential azacitidine and lenalidomide (n=39); and 3) azacitidine only (n=34). The efficacy end point was 1-year survival. Median age was 76 years (range 66–87 years). Thirteen subjects (15%) had prior myelodysplastic syndrome and 41 (47%) had adverse cytogenetics. One-year survival was 21% [95% confidence interval (CI): 0, 43%] with high-dose lenalidomide, 44% (95%CI: 28, 60%) with sequential azacitidine and lenalidomide, and 52% (95%CI: 35, 70%) with azacitidine only. Lenalidomide at a continuous high-dose schedule was poorly-tolerated resulting in a high rate of early therapy discontinuations. Hazard of death in the first four months was greatest in subjects receiving continuous high-dose lenalidomide; hazards of death thereafter were similar. These data do not favor use of continuous high-dose lenalidomide or sequential azacitidine and lenalidomide over the conventional dose and schedule of azacitidine only in patients aged 65 years or over with newly-diagnosed acute myeloid leukemia. (clinicaltrials.gov identifier: 01358734). BODY.INTRODUCTION: The incidence of acute myeloid leukemia (AML) increases dramatically with age. Survival of older people with AML is poor;1–3 in the US, fewer than 50% of subjects with AML over 65 years of age receive therapy within three months of diagnosis despite considerable data indicating therapy improves their survival.4–6 Reasons for not treating older patients with AML include poor performance score, co-morbidities, frailty, an antecedent hematologic disorder (AHD), presumed therapy-related leukemia, and/or adverse risk biological features.4,7 One study of more than 3000 subjects reported a significant adverse impact of increasing age on outcomes even after adjusting for these variables, suggesting age is an independent prognostic variable associated with poor outcomes.8 Therapy recommendations for older subjects with AML vary with no standard-of-care.7 Commonly-used treatments include intensive therapy (typically cytarabine and an anthracycline) and less intensive approaches such as low-dose cytarabine or DNA hypo-methylating drugs including azacitidine and decitabine.9 Azacitidine was recently approved in Europe in subjects 65 years and over with AML and more than 30% bone marrow blasts judged unfit for a hematopoietic cell transplant.8 Approval was based on a 4-month improvement in median survival compared with conventional therapy or supportive care [10.4 vs. 6.5 months; Hazard Ratio (HR) 0.85, 95% confidence interval (95%CI): 0.69, 1.03]. Considerable data indicate high-dose lenalidomide, an immune-modulatory drug (IMiD®), may be effective in patients with myelodysplastic syndrome (MDS) with excess blasts and patients with AML judged unfit for intensive therapy.10,11 Phase I and II studies report concomitant or sequential azacitidine and lenalidomide is effective in MDS, AML and in chronic myelomonocytic leukemia (CMML).12–15 Several studies of lenalidomide used 50 mg/day continuously without dose modification. Based on these data, we designed a randomized phase II study comparing safety and efficacy of continuous high-dose lenalidomide, sequential azacitidine and lenalidomide or azacitidine only in persons 65 years or over with newly-diagnosed AML. BODY.METHODS: We conducted a phase II randomized, open-label, parallel-group study at 25 sites in North America (clinicaltrials.gov identifier: 01358734). The study was approved by the relevant institutional review boards or an independent ethics committee, and followed guidelines set out in the Declaration of Helsinki. Subjects gave written informed consent. Statistical analyses were carried out by Celgene Corporation, Summit, NJ, USA, and were reviewed by the authors with access to all study data. BODY.METHODS.SUBJECTS: Eligible subjects were 65 years and over with newly-diagnosed AML including those with de novo AML, AML with prior MDS or with presumed therapy-related AML. Subjects could not have previously received lenalidomide, azacitidine, decitabine or cytarabine and were judged ineligible to receive a transplant at study entry. Subjects had to have an Eastern Co-operative Oncology Group (ECOG) performance score ≤2, white blood cell (WBC) count ≤10×109/L (hydroxyurea use was permitted to lower the WBC count), adequate liver and kidney function, and no uncontrolled infection or other cancer within two years of study entry. Subjects who had received prior therapy for AML other than hydroxyurea were not eligible for the study. BODY.METHODS.STUDY DESIGN: Screening procedures were completed no more than 28 days before randomization. Confirmation of AML for study entry was based on local pathology review. A separate central review of all bone marrow aspirates, bone marrow biopsies and blood smears was conducted by a blinded pathologist (Dr. Daniel Arber, Stanford University). Cytogenetic testing on diagnostic samples was carried out locally and reviewed centrally (Prof. Athena Cherry, Stanford University). Subjects were randomized equally to the therapy arms by central interactive voice response system (IVRS). Randomization was stratified for performance score (0–1 vs. 2) and levels of blood myeloblasts (<1 vs. ≥1×109/L). BODY.METHODS.THERAPY: Therapy regimens are shown in Figure 1. Therapy in any arm could be interrupted or delayed because of hematologic or non-hematologic therapy-related adverse events (AEs). Lenalidomide dose-reductions were not allowed in cycles 1–4 in the continuous high-dose lenalidomide cohort or in courses 1 and 2 in the azacitidine and lenalidomide cohort. Dose reductions of lenalidomide could be made thereafter in both cohorts. Dose reductions of azacitidine were permitted as described. Dose reductions of lenalidomide or azacitidine were continued in subsequent cycles unless additional adverse events occurred; in such cases, additional dose reductions were permitted. Subjects were seen weekly for the first 2 courses, every two weeks for the next 2 courses, and on the first day of subsequent courses. Microscope slides of blood and bone marrow samples and cytogenetic testing were obtained pre-randomization, on day 15 of course 1, within seven days of beginning courses 2 and 3, and before starting every third subsequent course. Figure 1.Study design and therapy regimens. Stratification factors were Eastern Co-operative Oncology Group (ECOG) performance score (0–1 vs. 2) and blood blast level (<1 vs. ≥1×109/L). Randomization in the lenalidomide regime was suspended 11th September 2013 and permanently closed on 15th April 2014 as per Amendment 2 because of the high rate of discontinuation of the study treatment in subjects receiving high-dose continuous lenalidomide. AML: acute myeloid leukemia; PO: per oral administration; SC: subcutaneous injection; SPM: secondary primary malignancies. BODY.METHODS.SAFETY AND EFFICACY END POINTS: Safety assessments included vital signs, physical exam, weight, ECOG performance score, complete blood count (CBC) and differential, blood chemistries, urinalyses, coagulation, thyroid function tests and electrocardiograms. AEs were recorded using Common Terminology Criteria for Adverse Events (CTCAE) v.4.0. Study subjects were kept under surveillance for the development of new cancers. Events of interest (EOIs) were recorded and reported as serious AEs (SAEs) and considered medically-important even if not meeting SAE criteria. The primary end point was 1-year survival. Secondary end points included rates of complete remission (CR), complete remission with incomplete recovery of neutrophils or platelets (CRi), and remission duration. Subjects discontinuing the study could receive subsequent therapy during the follow-up interval at their physician's discretion and with their consent. BODY.METHODS.STATISTICAL ANALYSIS: The study was not designed or powered for a formal statistical comparison of the therapy cohorts and a sample size calculation was not made. Target enrollment was 40 subjects per cohort. Preliminary analyses indicated a high rate of discontinuations in the continuous high-dose lenalidomide cohort with 10 of the first 13 subjects receiving less than 56 days of therapy. Based on these data, the Data Safety Monitoring Committee (DSMC) recommended closing randomization into this cohort and the planned study sample size was reduced to 95 subjects. Time-to-death from any cause was defined as the interval from randomization to death. Living subjects were censored at date of last contact, withdrawal of consent, loss to follow up, or study completion. There was no pre-specified statistical plan to compare survival between the cohorts. However, an exploratory analysis was made comparing 1-year survival Kaplan-Meier estimates between the cohorts using the log rank test and which indicated non-proportional hazards of death. Consequently, we estimated HR and 95% confidence intervals for the intervals 0–4 months and >4–12 months using a Cox proportional hazards model. P-values were derived from the Cox model. BODY.RESULTS.SUBJECTS: Enrollment began on 27th April 2012 with database lock on 1st May 2015. Because of poor accrual, only 88 subjects were randomized, all of whom constitute the intent-to-treat (ITT) population including 15 subjects randomized to receive high-dose continuous lenalidomide, 39 randomized to receive sequential azacitidine and lenalidomide, and 34 randomized to receive azacitidine only. Subjects in the high-dose lenalidomide cohort were more likely to be over 75 years of age and to have had prior MDS, had a higher percent of bone marrow blasts, and lower levels of blood neutrophils. This cohort also had fewer subjects with adverse cytogenetics than the other cohorts (Table 1). There were 6 subjects with del(5/5q), including one each in the high-dose lenalidomide and in the azacitidine only cohorts, and 4 in the sequential azacitidine and lenalidomide cohort. Table 1. Baseline variables for the intent-to-treat populations. BODY.RESULTS.EFFICACY: Median therapy duration was six weeks (range 1–48 weeks) for the continuous high-dose lenalidomide cohort, eight weeks (0–78 weeks) for the sequential azacitidine and lenalidomide cohort, and 29 weeks (1–143 weeks) for the azacitidine only cohort. The most common reason for discontinuation was leukemia progression in 5, 11, and 13 subjects (Figure 2). Figure 2.CONSORT study-flow diagram. *Four randomized subjects were not treated because of withdrawal of consent, adverse events, or other reasons, e.g. hospitalization. Percents are based on the intention-to-treat population. One-year survivals were 21% (95%CI: 0, 43%), 44% (95%CI: 28, 60%) and 52% (95%CI: 35, 70%) (Figure 3) in the lenalidomide, azacitidine and lenalidomide and azacitidine only cohorts. Subjects in the high-dose continuous lenalidomide cohort had a higher hazard of death than subjects in the azacitidine cohort [HR 5.73 (1.91, 17.20); P=0.002] or subjects in the sequential azacitidine and lenalidomide cohort [HR 2.19 (95%CI: 0.94, 5.13); P=0.071] (Table 2) in the first four months post randomization. Hazard of death was also higher in the sequential azacitidine and lenalidomide cohort compared with the azacitidine cohort [HR 2.51 (0.89, 7.05); P=0.081]. Hazards of death >4–12 months were similar in the 3 cohorts. Figure 3.Kaplan-Meier estimates of 1-year survival. Subjects in the high-dose continuous lenalidomide cohort had a higher hazard of death in 0–4 months than subjects in the azacitidine alone cohort (P=0.002) and subjects in the sequential azacitidine and lenalidomide cohort (P=0.071). Table 2. Hazard Ratios for death. There were 7 deaths in the continuous high-dose lenalidomide cohort in the first four months. Median duration of therapy of these subjects was 36 days (range 7–84 days). Two subjects discontinued therapy because of an adverse event and 5 because of AML progression. Causes of death were AML and/or its complications such as infection. One subject had a pulmonary infarct. Rates of CR and CRi for the cohorts are shown in Table 3. Interestingly, 2 subjects receiving high-dose lenalidomide only, a non-cytotoxic drug, achieved a complete remission and 3, a complete remission with incomplete recovery of blood levels of neutrophils and/or platelets. There were too few subjects with del(5/5q) in each cohort to compare outcomes of this subset. Table 3. Proportion of subjects with complete remission with (CR) and without (CRi) complete hematologic recovery. BODY.RESULTS.ADVERSE EVENTS: Eighty-four subjects received 1 dose or more of study drug and constitute the safety population including 14 subjects receiving high-dose continuous lenalidomide, 38 receiving sequential azacitidine and lenalidomide, and 32 receiving azacitidine only. Treatment was discontinued within the first 2 cycles in 10 subjects in the high-dose continuous lenalidomide, 19 in the sequential azacitidine and lenalidomide cohort, and 8 in the azacitidine only cohort. The most common reasons for discontinuation of study therapy within 2 courses across the 3 cohorts were adverse events (3, 6 and 2, respectively), death (2, 6 and 1, respectively) and disease progression (3, 2 and 2, respectively). During the study the proportion of treatment-emergent adverse events (TEAEs) resulting in therapy discontinuation was highest in the high-dose continuous lenalidomide cohort (n=4) followed by the azacitidine and lenalidomide cohort (n=7), and lowest in the azacitidine only cohort (n=3). Infections were the most frequent cause of permanent therapy discontinuation and were most frequent in the high-dose continuous lenalidomide cohort (n=4 compared with 3 subjects in the azacitidine and lenalidomide cohort and 1 subject in the azacitidine only cohort). There were 3 new cancers (vulvar cancer, stage 0; lung adenocarcinoma; and central nervous system cancer), all in the azacitidine only cohort. These cancers were diagnosed at approximately, 6, 9 and less than 1 month post randomization. The most frequent TEAEs (any grade) considered drug-related were hematologic and gastrointestinal. The most frequent TEAE grade 3 or over was febrile neutropenia (n=6, 17 and 9, respectively) (Table 4). Frequencies of treatment-emergent serious AEs (TE-SAEs) was higher in the high-dose continuous lenalidomide cohort (n=10) than in the other cohorts (n=16 and 7, respectively). Pneumonia was the most frequent infectious TE-SAE reported in 1, 2 and 0 subjects, respectively. The most common causes of death on therapy were AML progression and infections. Deaths from AML progression were reported in 8, 12 and 11 subjects and deaths from infections were reported in 2, 5 and 2 subjects, respectively. Median hospital days for the 3 cohorts were ten days (range 2–54 days), 11 days (range 2–43 days), and seven days (range 2–24 days). Table 4. Most common (≥3 subjects) grade ≥3 treatment-emergent adverse events. BODY.DISCUSSION: Our randomized phase II study in patients over 65 years with newly-diagnosed AML was designed to numerically compare 1-year survivals between the cohorts. It was not designed nor powered to make statistical comparisons in outcomes. The sequential azacitidine and lenalidomide and azacitidine only regimens were reasonably well-tolerated. Proportions of subjects alive at one year in these cohorts were 52% (35, 70%) and 44% (28, 60%), similar to azacitidine only in a large phase III trial in similar patients.16 However, the HR for death of 2.51 (0.89, 7.05) in the lenalidomide plus azacitidine cohort in months 0–4 suggests azacitidine only may be the best regimen. Lenalidomide at the dose and schedule used in the high-dose continuous lenalidomide cohort was less well-tolerated, perhaps because no dose reduction was permitted, resulting in a high proportion of early treatment discontinuation and in subsequent leukemia progression. Subjects enrolled in this cohort had an increased hazard of death in the first four months of therapy compared with the other cohorts. Hazards of death thereafter were similar but favor the azacitidine cohort. This early difference in hazard of death should be viewed cautiously as these analyses were not specified in the pre-study statistical plan and sample sizes are small. Proportions of subjects achieving a CR/CRi were similar with wide 95% confidence intervals. It is also important to emphasize that the small sample sizes may have resulted in an imbalance in baseline variables. For example, subjects in the high-dose continuous lenalidomide cohort were older and more likely to have prior MDS and severe bone marrow failure but less frequent adverse cytogenetics. This cohort also had a greater proportion of subjects with cardio-vascular disease risk factors (data not shown). None of these differences were tested for statistical significance according to the pre-study statistical plan. Finally, discontinuation rates were generally similar to those in other clinical studies of continuous high-dose lenalidomide in older persons with AML.14 Randomization with or without stratification does not guarantee cohorts have similar distributions of known and latent prognostic variables, especially when there are few subjects in each cohort. Typically, potential imbalances are accounted for by calculating P-values and indicating confidence intervals. Because we did not perform these calculations for outcomes other than for 0–4 and >4–12-month survival intervals between the cohorts, differences in outcomes should not be assumed to result from therapy assignment. Sequential azacitidine and lenalidomide did not increase the frequency of >grade 3 or over TEAEs compared with continuous high-dose lenalidomide. Subjects in the continuous high-dose lenalidomide cohort were scheduled to receive 50 mg/day for 56 days whereas those in the sequential azacitidine and lenalidomide cohort were scheduled to receive the same dose of lenalidomide for 21 days followed by 14 days off therapy. Consequently, there are significant differences in schedule and total dose of lenalidomide between the cohorts. Neither regimen with high-dose lenalidomide, continuous or sequential, was as well-tolerated as azacitidine only. In conclusion, data from unplanned survival analysis indicate high-dose continuous lenalidomide given without dose reduction resulted in a high rate of early discontinuations and an early but not late increased hazard of death compared with sequential azacitidine and lenalidomide and with azacitidine only in subjects 65 years and over with newly-diagnosed AML. Our data also suggest therapy with azacitidine only may be better than sequential lenalidomide and azacitidine based on the 95% confidence interval of the HR for death. Although continuous high-dose lenalidomide is active in AML and sometimes produces prolonged complete remissions,10 we cannot recommend the dose and schedule we tested for subjects 65 years and over with newly-diagnosed AML. Adding lenalidomide to azacitidine did not improve 1-year survival and may have reduced it in the first four months of therapy. However, our study was neither designed nor powered to test this question. Whether a different dose, schedule and/or dose-modification of lenalidomide alone or with azacitidine might be better tolerated and/or more effective than the dose and schedule we studied is unknown. However, the achievement of complete remission with or without complete recovery of blood levels of neutrophils and/or platelets by a non-cytotoxic drug is noteworthy.

TITLE: Alcohol breaks down interhemispheric inhibition in females but not in males ABSTRACT.INTRODUCTION: Alcohol has renowned behavioral disinhibitory properties which are suggested to involve reductions in frontal lobe functioning as a result of diminished interhemispheric connectivity. ABSTRACT.METHODS: To examine sex differences in frontal interhemispheric connectivity in response to alcohol, 12 female and ten male healthy volunteers received a single administration of 0.5‰ alcohol in a placebo-controlled counterbalanced crossover design. Paired-pulse transcranial magnetic stimulation was applied to measure transcallosal inhibition (TCI) between the left and right primary motor cortex (M1). ABSTRACT.RESULTS: Results showed significant reductions in TCI after alcohol administration in female participants exclusively. ABSTRACT.DISCUSSION: These findings provide the first evidence that moderate doses of alcohol differentially affect frontal interhemispheric connectivity in males and females. The present data may shed new light on the physiological mechanisms underlying sex differences in the susceptibility to alcohol. BODY.INTRODUCTION: In the last decades, alcohol consumption steadily increased culminating in 3.2 million yearly deaths either as a direct or indirect result of alcohol worldwide (World Health Organisation 2004). Moderate doses of alcohol (≤0.5‰) can produce feelings of relaxation and release of response inhibition, whereas higher doses of alcohol (1–1.5‰) cause impairments in motor coordination and vision. Extremely high alcohol levels (≥2.5‰) ultimately lead to coma and death (Hieda et al. 2005). Interestingly, the effects of alcohol seem to be more pronounced in females than in males (Mills and Bisgrove 1983; Niaura et al. 1987; Mumenthaler et al. 1999; Hommer 2003). Sex differences in response to alcohol intake can in part be explained by differences in the percentage of body fat (Addolorato et al. 1999) and the availability of the enzyme alcohol dehydrogenase which breaks down alcohol (Baraona et al. 2001). However, other research suggests that females differ from males on the innate physiological susceptibility to the central effects of alcohol (Hommer et al. 1996, 2001; Agartz et al. 2003; Sasaki et al. 2009). For instance, chronic misuse of alcohol leaves female alcoholics with more brain damage than their male counterparts (Hommer et al. 2001; Hommer 2003). Notably, however, moderate drinking habits also affect the female brain more strongly than the male brain (Sasaki et al. 2009). In addition to the general effects on the central and peripheral nervous system, alcohol typically affects frontal cortical functions such as response inhibition and cognitive regulation (Jentsch and Taylor 1999; Lyvers 2000). In fact, even though alcohol diffuses through all biological membranes and is distributed throughout the body, the anterior cortical regions of the brain have been shown to be especially vulnerable to the acute (Weitlauf and Woodward 2008) and chronic (Wobrock et al. 2009) effects of alcohol. Low doses of alcohol already reduce excitability of the frontal cortex as evidenced by transcranial magnetic stimulation (TMS) and electroencephalography (EEG) (Ziemann et al. 1995; Kähkönen et al. 2001, 2003). This reduction in frontal cortical excitability has been argued to relate to binding of alcohol to gamma-amino butyric acid (GABA)-a receptors in addition to alcohol-related inhibition of N-methyl d-aspartate (NMDA) currents (Kähkönen et al. 2003). However, the precise mechanisms that underlie the behavioral effects of alcohol and possible sex differences remain unclear. One possible mechanism may involve (transient) frontal lobe impairments arguably due to reductions in effective frontal interhemispheric connectivity (Kähkönen et al. 2001). Frontal interhemispheric connectivity refers to callosal signal transfer between the left and right frontal cortex (Aboitiz and Montiel 2003). This signal transfer can be measured with paired-pulse TMS wherein the connectivity between the left and right primary motor cortex (M1) is operationalized by applying a conditioning pulse to ipsilateral M1 followed by a test pulse to contralateral M1 10 ms later (Ferbert et al. 1992). Compared to the motor evoked potential (MEP) elicited by the test pulse alone, the conditioning pulse inhibits the MEP of the test pulse by about 50% (Daskalakis et al. 2002). This so-called transcallosal inhibition (TCI) reflects an important physiological mechanism by which the frontal lobes interact and contribute to behavior by providing a noninvasive way to study frontal interhemispheric connectivity (Hofman and Schutter 2009). The aim of the present placebo-controlled counterbalanced cross-over design was to examine sex differences in frontal interhemispheric connectivity in response to alcohol intake as indexed by TCI. We hypothesized that alcohol will reduce TCI in both males and females. In addition, it is expected that reductions in TCI will be more pronounced in females than in males. BODY.MATERIALS AND METHODS.PARTICIPANTS: Twenty-two healthy volunteers (ten males, mean ± SD, 22.5 ± 0.85 years of age) participated in this study. All participants were right-handed, nonsmoking, and free of any psychiatric or neurological disorders and had more than 12 years of education. All female participants used oral contraceptives. Subjects had no history of alcohol abuse or dependence as measured with the Alcohol Use Disorder Identification Test (AUDIT; Berks and McCormick 2008). Written informed consent was obtained, and volunteers were paid for participation. The study was approved by the medical ethical committee of the University Medical Centre Utrecht and in accordance with the Declaration of Helsinki. All participants were naïve to the aim of the study. BODY.MATERIALS AND METHODS.ALCOHOL ADMINISTRATION AND MONITORING: The amount of alcohol which would induce a blood alcohol concentration (BAC) of 0.5‰ was estimated with Widmark's formula: where m is body weight and r refers to the distribution ratio which for men is on average 0.68 and for women 0.55 (Friel et al. 1995). The alcoholic solution (300 ml) consisted of alcohol mixed with orange juice and a few drops of peppermint oil to mask the alcohol. Placebo (300 ml) consisted of orange juice and peppermint oil. Participants' BAC was measured every 5 min using an AlcoMate CORE AL-600 Pro Alcohol Breath Analyzer (AK Solutions USA LLC, Palisades Park, NJ, USA). Measurements were started when BAC approximated 0.5‰. Importantly, TCI was measured at the start of the downward limb of the BAC curve because at this point, BAC values are suggested to be relatively stable (Verster et al. 2002). BODY.MATERIALS AND METHODS.TRANSCRANIAL MAGNETIC STIMULATION: TMS was performed using two biphasic magnetic brain stimulators (maximum output 4,160 A peak/1,750 VAC peak) and an iron core coil (Neotonus, Atlanta, GA, USA) over the left and right primary motor cortex (M1). Bilateral paired-pulse TMS was applied to measure TCI. In this paradigm, the MEP following the single test pulse (unconditioned MEP, uMEP) to M1 is compared to the MEP evoked by the test pulse that is preceded by a conditioning pulse to the contralateral M1 10 ms earlier (conditioned MEP, cMEP; Ferbert et al. 1992). The mean percentage reduction between the unconditioned and conditioned MEP was calculated. Stimulation intensity was set at 120% of the individual motor threshold (MT). To minimize anticipation effects, TMS was applied at a frequency of 0.18–0.2 Hz. The following conditions were applied in random counterbalanced order: (1) a single test pulse to the left M1 (uMEPleft), (2) a conditioning pulse to the right M1 followed by a test pulse to the left M1 (cMEPleft), (3) a single test pulse to the right M1 (uMEPright), and (4) a conditioning pulse to the left M1 followed by a test pulse to the right M1 (cMEPright; Ferbert et al. 1992). BODY.MATERIALS AND METHODS.ELECTROMYOGRAPHIC RECORDINGS: The electromyogram (EMG) was recorded using sintered 11 × 17-mm active Ag–AgCl electrodes with the ActiveTwo system (BioSemi, Amsterdam, The Netherlands) relative to the common mode sense in a belly–tendon arrangement. The MEP was recorded from the left and right abductor pollicis brevis (APB). The active electrode was placed over the muscle belly of the APB. The reference electrode was placed over the proximal phalanx of the thumb. The ground electrode was attached to the wrist. EMG signal was digitized at 16 kHz, low-pass filtered (−3dB cutoff frequency 3,334 Hz; roll-off 30 dB/octave), and offline high-pass filtered (−3dB cutoff frequency 20 Hz; roll-off 48 dB/octave). BODY.MATERIALS AND METHODS.PROCEDURE: In the present crossover design, participants received either a single dose of alcohol or placebo on one of the two occasions in a randomized counterbalanced order. Prior to the testing sessions and on a separate day, participants were invited upon the laboratory. A safety-screening list was administered to check for contraindications to TMS (Keel et al. 2001), and health was checked with a standard interview. In addition, safety issues and experimental procedures were explained to the subject, and written informed consent was obtained. Alcohol abuse and alcohol dependence were indexed with the AUDIT (Berner et al. 2007), and participants with AUDIT scores of 0 or >8 were excluded. Right handedness was assessed with the Edinburgh handedness inventory (Oldfield 1971). Resting MT was determined using the standardized motor threshold estimation procedure (Schutter and van Honk 2006; see Table 1 for details). Table 1Main demographics and characteristics of the participantsMales (mean ± SEM)Females (mean ± SEM)pAge (in years)23.3 ± 1.421.9 ± 1.10.43AUDIT6.4 ± 0.65.8 ± 0.60.53MT left hemisphere47.5 ± 2.849 ± 2.00.66MT right hemisphere45.2 ± 2.747.8 ± 2.20.47Handedness45.5 ± 0.746.3 ± 0.70.44MT motor threshold Participants were instructed to refrain from consuming alcoholic beverages 24 h prior to the testing session, not to consume coffee, tea, or chocolate 5 h before testing and to use their last meal at least 2 h prior to the testing session. To control for circadian rhythms, both testing sessions were conducted at the same time of the day. Each testing session was separated by at least 24 h. Moreover, to minimize the effects of additional hormonal influences, female participants were not tested during the stopping week. Each experimental session started with a BAC measurement to ascertain sobriety after which participants consumed the alcoholic solution or placebo. Participants had 2 min to drink the solution while keeping their nose closed with their hand. The time between administration and paired-pulse TMS was approximately 30 min. During this period, EMG electrodes were attached to the left and right APB, and the optimal target sites for TMS were determined. TCI was measured when the BAC curve approximated 0.5‰ on the descending limb of the BAC curve (Verster et al. 2002). At the end of the final session, participants were debriefed and paid for participation. Both testing sessions took approximately 1 h to complete. BODY.MATERIALS AND METHODS.DATA REDUCTION AND ANALYSIS: MEP amplitude was quantified as the peak-to-peak amplitude of the maximal EMG response. Left-to-right (l-rTCI) and right-to-left transcallosal inhibition (r-lTCI) were expressed according to the formulas [(1 − (cMEPright/uMEPright)) × 100] and [(1 − (cMEPleft/uMEPleft)) × 100], respectively. A general linear model (GLM) for repeated measurements with drug (alcohol versus placebo) and TCI (left to right versus right to left) as within-subjects factor and sex (female and male) as a between-subjects factor was run to examine the alcohol-related effects and possible sex differences on TCI. To rule out possible alcohol-related effects of single-pulse TMS on TCI, an additional GLM for repeated measurements with drug (alcohol versus placebo) and side (left versus right hemisphere) as within-subjects factors and sex (female versus male) as a between-subjects factor was performed. Finally, to exclude effects of baseline EMG activity on TCI, a 2 × 2 GLM for repeated measurements with drug (alcohol versus placebo) and EMG (left versus right hand) as within-subjects factor and sex (female versus male) as within-subjects factor was conducted. The alpha level of significance was set at 0.05 (two-tailed). BODY.RESULTS: BAC values did not differ between males (mean ± SD, 0.69 ± 0.04) and females (mean ± SD, 0.603 ± 0.05; p = 0.17). Our hypothesis that alcohol would reduce TCI in both males and females was not confirmed (F(1, 21) = 2,752, p = 0.113). In contrast, a significant drug × sex interaction (F(1, 21) = 4.95; p = 0.038) was observed. Post hoc analyses demonstrated that alcohol significantly reduced TCI in females (F(1, 11) = 12.52; p = 0.005; ηp2 = 0.53) but not in males (F(1, 9) = 0.10; p = 0.76; Fig. 1). Fig. 1Mean and standard error of the mean of frontal interhemispheric inhibition in the placebo and alcohol condition The observed sex difference in TCI after alcohol could not be explained by reductions in single-pulse TMS as demonstrated by a nonsignificant drug × sex interaction (F(1, 21) = 1.024; p = 0.324). However, a main effect of alcohol on single-pulse TMS (F(1, 21) = 4,517; p = 0.046; ηp2 = 0.184) was observed, replicating prior studies that found alcohol-related reductions in cortical excitability (Ziemann et al. 1995; Kähkönen et al. 2003). Finally, the sex-related effect of alcohol on TCI could not be explained by differences in baseline EMG activity as shown by the nonsignificant drug × sex interaction (F(1, 21) = 0.278; p = 0.604). No main effects of alcohol on baseline EMG was observed (F(1, 21) = 0.270; p = 0.609). For an impression of the raw data, please see Table 2. Table 2Sex-related effect of alcohol on TCI, single pulse, and EMGTCISingle pulseEMGMalesFemalesMalesFemalesMalesFemalesPlacebo (mean ± SEM)57.1 ± 5.956.3 ± 7.21,843.2 ± 235.61,880.8 ± 653.84,769 ± 704.98,574.7 ± 2,403.2Alcohol (mean ± SEM)59.4 ± 7.240.7 ± 8.11,573.2 ± 3361,119.8 ± 396.76,351.9 ± 1,118.28,563.9 ± 1,256.4Single-pulse TMS is expressed in microvoltTCI transcallosal inhibition (expressed in percentage inhibition of single-pulse TMS), EMG electromyogram (expressed in microvolt), SEM standard error of the mean BODY.DISCUSSION: The aim of the present study was to examine alcohol-related sex differences in frontal interhemispheric connectivity and found reductions in frontal interhemispheric connectivity to a moderate dose of alcohol in healthy female but not male subjects. To the best of our knowledge, this is the first study to demonstrate evidence for sex differences in frontal interhemispheric connectivity in response to alcohol that cannot be explained by effects of alcohol on baseline EMG activity or single-pulse TMS. The global reduction in cortical excitability as evidenced by a decrease in MEP size in both sexes replicates earlier findings by Ziemann et al. (1995) and Kähkönen et al. (2003). This reduction in frontal cortical excitability has been explained in terms of increased binding of alcohol to GABA-a receptors and alcohol-related inhibition of NMDA currents (Kähkönen et al. 2003). In measuring TCI with paired-pulse TMS, the conditioning pulse administered over the motor cortex is thought to activate glutamatergic excitatory callosal fibers terminating on local GABAergic interneurons of the contralateral motor cortex (Daskalakis et al. 2002). Excitation of these GABAergic inhibitory interneurons results in inhibition of the pyramidal motor neurons which causes a reduction in the MEP size. Consequently, a decrease in frontal interhemispheric connectivity may relate to the influence of alcohol on either NMDA glutamate receptor functioning or GABAergic inhibitory interneurons. Previous TMS research has demonstrated that alcohol mainly influences GABAergic functioning leaving glutamatergic excitatory transmission relatively unaffected (Ziemann et al. 1995; Conte et al. 2008). In contrast, alcohol has also been shown to attenuate glutamate/NMDA receptor functioning (Lovinger et al. 1989; White et al. 1990; Masood et al. 1994; Weitlauf and Woodward 2008) leaving open the possibility that alcohol may have disrupted the excitatory callosal pathway that terminates on inhibitory interneurons. This would result in less excitation of inhibitory interneurons, subsequently leading to a relatively increased MEP size. However, the crucial point revolves around the question how to explain the alcohol-related sex difference on frontal interhemispheric connectivity, and at present, the precise physiological mechanisms through which alcohol reduces frontal interhemispheric connectivity in females remain to be elucidated. A possible role for steroid hormones in explaining the presently observed sex differences is evidenced by a study in which testosterone was found to counteracts some of the effects of alcohol (Khalil et al. 2005). In this study, testosterone was shown to diminish alcohol-induced deficits in spatial memory (Khalil et al. 2005). Interestingly, acute alcohol administration reduces testosterone production in both males and females (Ylikahri et al. 1980; Valimaki et al. 1984). Together with the notion that men have multiple times more testosterone than females, testosterone may play a role in the currently observed reductions of TCI in females exclusively. In agreement, we recently found evidence that a single administration of testosterone significantly increases frontal interhemispheric connectivity in healthy female subjects (Hoppenbrouwers et al., submitted). In sum, even though sex differences in steroid hormones may provide for an explanation of our findings, it remains elusive whether these sex differences already surface after an acute administration of alcohol. It is suggested that the decrease in frontal interhemispheric connectivity observed in females will most likely involve complex interactions between steroid hormones and the combined action of alcohol on GABAergic interneurons and NMDA glutamate receptor functioning in the corpus callosum. Despite the observed female vulnerability to the effects of alcohol on frontal interhemispheric connectivity, several issues should be mentioned. First, the relationship between alcohol-related effects on frontal interhemispheric connectivity and behavior is of importance. Executive functions including behavioral inhibition and cognitive regulation are normally ascribed to the prefrontal cortex (PFC) rather than M1. Even though both M1 and PFC are part of the frontal cortex, our current findings on M1 interhemispheric connectivity cannot simply be extrapolated to the PFC. Defensibly, there is evidence from recent interleaved TMS–EEG studies showing that M1 and the PFC share similar physiological properties (Daskalakis et al. 2008). Second, the fact that we observed reductions in frontal interhemispheric to alcohol consumption in females only does not in any way imply that men are immune to the effects of alcohol. In other words, males will likely demonstrate similar reductions in frontal interhemispheric connectivity at higher BAC levels. Third, in our sample, all female participants were taking oral contraceptives to abolish fluctuations in steroid levels at the time of testing. However, oral contraceptives have been shown to reduce plasma levels of progesterone metabolites in female rats and women (Follesa et al. 2001). Some of these metabolites (e.g., allopregnanolone) are highly potent endogenous positive modulators of the GABA-a receptor (Mitchell et al. 2008). It has been suggested that the combined effects of endogenous steroids and alcohol modulate GABA-a receptor functioning (Follesa et al. 2004). In contrast, alcohol has also been shown to increase allopregnanolone levels in the cerebral cortex (VanDoren et al. 2000), an increase that has proven effective to potentiate GABA-a receptor-mediated inhibition in the brain (VanDoren et al. 2000) and explain some of the physiological and behavioral effects of alcohol (VanDoren et al. 2000; Izumi et al. 2007). Taken together, these findings suggest that the contraceptive- and alcohol-related increases in allopregnanolone levels may have contributed to the presently observed reductions in TCI. As it stands, the present findings can only be generalized to males and females taking oral contraceptives. Finally, an additional line of evidence consisting of conventional TMS measures of GABA function such as short-interval intracortical inhibition and long-interval intracortical cortical inhibition could potentially has given more direct evidence for the exact mechanisms underlying the observed effect. In conclusion, the present study provides the first evidence for reductions in frontal interhemispheric connectivity in females but not in males following a moderate dose of alcohol. Future research may focus on alcohol dose–response patterns in males and the involvement of steroid hormones to further explain the observed sex differences in interhemispheric connectivity. In addition, to clarify the potential role of contraceptives, future research may also look into the effects of alcohol on interhemispheric inhibition in females not using contraceptives.

TITLE: A Comparison of Betamethasone Valerate 0.1% Cream Twice Daily Plus Oral Simvastatin Versus Betamethasone Valerate 0.1% Cream Alone in the Treatment of Vitiligo Patients ABSTRACT.BACKGROUND:: Vitiligo, a common disorder of depigmentation, is often difficult to treat. Corticosteroids are known to be effective, but with modest results. Although simvastatin has been reported to be effective for immunorelated dermatologic disorders including vitiligo, controlled trials are lacking. This study was conducted to compare the efficacy of topical betamethasone valerate 0.1% cream (as a standard method of treatment for vitiligo) versus a combination of betamethasone valerate plus oral simvastatin in the treatment of vitiligo. ABSTRACT.MATERIALS AND METHODS:: Eighty-eight subjects with symmetric vitiligo who had body surface involvement up to 20% were divided randomly into two groups. Group A were treated with betamethasone valerate 01% cream twice daily and Group B with betamethasone valerate 01% cream twice daily and oral simvastatin 80 mg daily for 12 weeks. Finally, 46 patients completed treatment after 12 weeks in both groups. The results were evaluated by a blind dermatologist using Vitiligo Area Scoring Index (VASI) score at baseline, 4th, 8th, and 12th week of treatment. In a similar way, subjective assessment performed by patients based on photo evaluation at the end of the study. ABSTRACT.RESULTS:: Despite a continuous reduction in VASI score in both groups, according to both physician (P = 0.13) and patient (P = 0.374) assessment oral simvastatin was not statistically more effective than conventional treatment of vitiligo. ABSTRACT.CONCLUSION:: This study indicates that oral simvastatin is not associated with significant impacts in the treatment of vitiligo as compared to other inflammatory dermatologic conditions such as psoriasis. Indeed, other studies should be initiated regarding exact molecular and cellular effects of statins in the treatment of vitiligo. BODY.INTRODUCTION: Vitiligo is an acquired pigmentary disorder, with a word wide distribution.[1] Although it commonly affects 0.5–2% of the population,[1] based on the epidemiologic reports from Gujarat, India, prevalence up to 8.8% has also been recorded.[2] The average age of onset is around 20 years, but any age may be affected.[1] Although men and women are equally involved,[34] the mean peak of involvement in female patients occurs in younger ages.[4] In its common form, vitiligo presents as uniformly depigmented macules or patches with convex borders surrounded by normal skin.[1] The cutaneous macules of vitiligo commonly affect normally hyperpigmented areas of the body, including the face, the dorsal surface of the hands, nipples, axillae, umbilicus, sacrum, and genital regions, but can occur anywhere.[1] Developing of lesions in areas of friction indicates koebnerization is common.[5] Despite the fact that the exact etiology of vitiligo is unknown, the more accepted idea is that vitiligo represents a group of various pathophysiologic etiologies. Among many speculations attempting to demonstrate the pathogenesis of the disease, the autoimmune hypothesis is the most popular.[6] The other suggested etiologies include neural, biochemical, and genetic hypotheses.[7] The common pathway of different etiologic factors end in the reduction of melanocyte survival.[8] Due to the fact that cosmetic disfigurement and psychological burden of the disease is noticeable, patients frequently seek medical treatment. Although numerous modalities have been introduced, the treatment of vitiligo remains frustrating for dermatologists. Among various options, phototherapy, and topical corticosteroid therapy are the cornerstone of treatment. Following treatment with the latter, a reduction in antibody-mediated cytotoxicity against melanocytes has been detected.[9] Regarding the proposed leading immunomodulatory role of statins over the past 2 decades,[1011121314] researches on the potential aspects of the use of these drugs for dermatologic disorders including systemic sclerosis, bullous pemphigoid, alopecia areata, vitiligo, lichen planus, psoriasis, and subacute cutaneous lupus erythematosus has been commenced.[1516] Recently, a randomized control trial showed a remarkable benefit of adding oral simvastatin in the treatment of psoriasis.[17] Despite the suggested therapeutic potential of statins in the treatment of vitiligo, no clinical trial has still been conducted. This study is the first, to our knowledge, to measure quantitatively the added effect of oral simvastatin to the conventional treatment of vitiligo with topical corticosteroids. Both have no significant adverse effects. BODY.MATERIALS AND METHODS: This randomized, controlled trial was conducted on patients with vitiligo referring to the outpatient Department of Dermatology of Alzahra Hospital in Isfahan (Iran), during April 2012 to February 2013. Patients included to in this study were subjects of 20–60 years old, with a body surface area involvement of <20%. The study was approved by the ethical committee of Isfahan University of Medical Sciences. Research Project Number was 39127. The diagnosis was established on the basis of clinical findings. Patients were divided into two different treatment groups, using a table of random numbers. Inclusion criteria included any new case of vitiligo, aged between 20 and 60 years, with a body surface area involvement of <20%. Patients with leucoderma secondary to other causes, segmental or zosteriform vitiligo, lesions unresponsive to treatment after 8th week of treatment, pregnancy and lactation, impaired kidney function, significant liver function abnormalities, hepatitis, cirrhosis, and those on mandatory therapy with medications that inhibit CYP3A4 pathways (erythromycin, clarithromycin, metronidazole, azole antifungals, calcium channel blockers, cimetidine, amiodarone, and selective serotonin reuptake inhibitors) were excluded from the study. Despite the fact that most vitiligo patients are healthy, detailed history was taken from each patient concerning possible associated situations (Graves' disease, Hashimotos' thyroiditis, Addison's disease, pernicious anemia, autoimmune polyendocrinopathy syndrome, halo nevi, alopecia areata, and lichen sclerous). With regard to type I error (alpha) = 0.05, study power = 80%, and expected difference of 30% in response rate, the sample size was calculated as 44 subjects in each group. The study was approved by the ethical committee of Isfahan University of Medical Sciences. Research Project Number was 39217. After an initial screening visit, this double-blind clinical trial was initiated with 88 eligible subjects (aged 20–59 years). The patients were divided into two different treatment groups, using a table of random numbers. Finally, 46 patients with surface area involvement of <20% completed treatment modality. Thirty-nine subjects excluded from the study due to the persistence of lesions after the 8th week of treatment or aggravation of lesions and 3 subjects were not willing to complete the study. In each group, 44 patients for 12 weeks of treatment were enrolled in the study. The patients in Group A received betamethasone valerate cream 0.1% twice daily; and the patients in Group B received betamethasone valerate cream 0.1% twice daily similarly plus simvastatin at the dose of 40 mg two times daily. The duration of treatment was 12 weeks in both groups [Figure 1]. Figure 1Flow chart of the treatment in the 2 groups The severity of vitiligo was evaluated by a physician blinded to treatment identifications before the initiation of the treatment and at the end of 4th, 8th, and 12th week of treatment using Vitiligo Area Scoring Index (VASI). According to this method, VASI is calculated as below: VASI: Σ(Hand units) × (Residual depigmentation [%]) A hand unit is considered the volar hand including fingers.[18] At 100% depigmentation, no pigment is present; at 90%, specks of pigment are present; at 75%, the depigmented aea exceeds the pigmented area; at 50%, the depigmented and pigmented areas are equal; at 25%, the pigmented area exceeds the depigmented area; and at 10%, only depigmentation are present.[19] The patients were advised to report the occurrence of any unwilling adverse effects (redness, burning, itching, and erosion) during the 12 weeks of the study. Evaluation of improvement and side effects were conducted at baseline and 4th, 8th, and 12th week of treatment. In each visit, digital photographs were taken by a facial photo fixture using a Canon PowerShot G12 stand-off camera. In order to evaluate patient satisfaction surveys, assessment of the treatment areas using comparative photographs were conducted by patient based satisfaction, and 12 weeks after starting of treatment (Patient Global Assessment).[20] The improvement of vitiligo was scored by a quartile grading scale – mild: <25%, moderate: 25–50%, good: 51–75%, and excellent response: 76–100%.[19] Laboratory tests including a check of serum levels of lactate dehydrogenase, creatine phosphokinase and also liver function tests performed in all patients at the beginning and the end of the treatment. The statistical analysis was carried out by SPSS version 18.0 for Windows software (SPSS Inc., Chicago, IL, USA) by using Chi-square, Mann–Whitney, and repeated measures ANOVA analyses. The significance level was set at P < 0.05. BODY.RESULTS: Eighty-eight subjects (100%) with a body surface area involvement of <20% were recruited in the study. At the end of study 42 subjects failed to complete the study. Thirty-nine subjects (23 subjects in Group A and 16 subjects in Group B) excluded from the study due to persistence of lesions after 8th week of treatment or aggravation of lesions and 3 subjects (2 subjects in Group A and 1 subject in Group B) gave up the study due to scheduling difficulties. The mean age of patients was 35.7 ± 10.5 years in Group A and 36.5 ± 10.2 years in Group B. The difference in mean ages of the two groups was not statistically significant (P = 0.75). Group A included 24 (45%) female and 20 (54.5%) male patients. Group B included 21 (47.7%) female and 23 (52.3%) male patients. There was no significant difference between sex ratios in the two groups (P = 0.52). By Chi-square test, the distribution of vitiligo patches between two groups (P = 0.776) was not statistically significant between them [Table 1]. Table 1 Patient characteristic of betamethasone valerate versus betamethasone valerate plus oral simvastatin group Patient satisfaction survey at the end of the study revealed that of patients in Group A, 31.6% (6 out of 19) rated themselves as having <25% of overall improvement, 52.6% (10 out of 19) judged themselves as having 25–50% improvement and 15.8% (3 out of 19) rated improvement of 50–75%. Nobody reported improvement of more than 75% (excellent). Both total number of patients with improvement and the number of subjects with more than 50% were increased in Group B [Table 2]. Table 2 Vitiligo parameters betamethasone cream and betamethasone cream plus oral simvastatin group Despite the steadily decrease in mean VASI value in both groups by means of repeated measure ANOVA test, Mann–Whitney analysis revealed no significantly greater improvement of depigmented patches according to clinician's (P = 0.1) and patient satisfaction assessment (P = 0.14) at the end of study in patients of Group B. In a similar way, the results of dermatologist assessment at the end of 4th (P = 0.98) and 8th (P = 0.89) week of treatment confirmed the aforementioned final results. By considering the clinical response with repigmentation >50%, greater number of subjects in Group B have been improved at the end of 4th (7% vs. 0%), 8th (22% vs. 15.4%), and 12th (37% vs. 26.3%) week of treatment. However, Mann–Whitney test is used because of nonnormal frequency distributions. It detected no significant difference between groups in all stages of evaluation. No side effect or any laboratory abnormality has been detected in both groups. BODY.DISCUSSION: Although topical corticosteroids along with phototherapy are the mainstay of treatment of vitiligo, the overall outcome still remains modest.[21] Indeed, an interest for finding a cheap, safe, and common treatment is unstopping. With regard to the more accepted role of autoimmune factors in the pathogenesis of vitiligo, investigators turn their attention to the drugs with immunomodulatory activities. In fact, nonsegmental vitiligo as the more common form of the disease is primarily affected by the autoimmune factors.[6] In this model, the main effector cell is CD8+ T cell that depends on the action of both interferon-gamma (IFN-γ) and chemokine receptors, especially CCR3.[16] Other than inhibiting of 3-hydroxy-3-methyl glutaryl CoA, another convincing mechanisms by which statins are therapeutic, are anti-inflammatory properties.[10] By means of flow cytometry, it was shown that simvastatin are direct inhibitors of IFN-γ. Furthermore, statins downregulate the Th1-type chemokine receptors including CCR5 and CCR3.[22] In the present study to our knowledge, we provide the first clinical evidence that measures quantitatively the effectiveness of oral simvastatin in the treatment of vitiligo. Considering the effectiveness of topical corticosteroids according to current data in vitiligo, betamethasone valerate cream as class III corticosteroid, was given to all patients.[192324] Topical corticosteroids included among the most compelling approaches for small, localized vitiligo.[9] Despite the primary recommendations on the use of Class III and IV of corticosteroids, recent publications propose that even Class I can be used with good results in an intermittent manner to minimize side effects.[1] The reported efficacy of corticosteroid varying from one study to another (ranging from 30% to 80%) indicating a different response.[2325] In cases that there was not response after 2 months of therapy, to minimize the possible side effects, treatment should be halted.[1] In our study, 26.3% (5 out of 19) of our patients in Group A (reflecting only the effect of corticosteroid) revealed >50% regimentation, which is near to the lower limit of response in the statistics of literature. The lower response rate may reflect the small number of subjects in our study. Moreover, by eliminating children from the study, a population group with better responses in comparison with adults, was excluded.[24] Despite the greater number of patients with improvement more than 50% greater in subjects of Group B (37% vs. 26.3%), by statistical analysis no significance difference between two groups at the end of study was detected. In other words, oral simvastatin was not accompanied by remarkable effects in the treatment of vitiligo. Following explanation relevant documents on the immunomodulatory effects of statins in the treatment of dermatologic disease,[16] Wolkenstein et al. has noticed the diminished risk of psoriasis associated with statin intake.[26] In another study Naseri et al., evaluated the efficacy of oral simvastatin on improvement of psoriasis.[17] This paper has shown significantly more reduction of Psoriasis Area Severity Index score in the group received oral simvastatin compared to group who did not (P = 0.001). Concerning vitiligo, only in one case study by Noël et al., a clear regression of skin's depigmentation was observed in a 55-year-old man with vitiligo who received simvastatin. After the cessation of simvastatin, his vitiligo lesions relapsed. He was retreated with simvastatin which resulted in reallevation of his disease.[27] In comparison to aforementioned studies on the beneficial impact of simvastatin in psoriasis and inflammatory conditions, the number of patients with remarkable response in our study was much less. This difference in response could be because of some limitations. It is now proven that vitiligo represent a group of heterozygous pathophysiologic disorders with the same characteristics. Despite the fact that autoimmune mechanisms are considered the main cause of generalized vitiligo, the role of neurohumoral, cytotoxic, and genetic factors is not insignificant.[7] In other words, considering autoimmune factors as the only underlying cause of all forms of generalized vitiligo is not logical. Furthermore, pathway alteration of the Th1/Th2 balance and inhibition of lymphocyte migration are the conjectural actions of simvastatin in vitiligo. However, inhibition of Th17 cell induction and interleukin-17 production, inhibition of mast cell degranulation, and induction of angiogenesis which are attributable to statins,[16] are not considered to be involved in improving vitiligo. In addition, childhood vitiligo better responds to therapy compared with the adult.[23] Excluding children from the study thus have affected the results, and underscores the clinical relevance of the pigmentory induction capacities of the treatment protocol. In determining the limitations of study, if we excluded subjects with rapidly spreading vitiligo, and those with lesions on areas known to be recalcitrant to therapy, fewer subjects would drop out of the study. Moreover, most of the studies regarding evaluation the effectiveness of a given therapy defined for 8 weeks. Considering that lesions unresponsive to corticosteroids after 8th week of treatment should be excluded, if we defined the study for 8 weeks, more subjects would remain at the end of the study. BODY.CONCLUSION: In summary, despite the continuous improvement of disease by therapy, adding of simvastatin was not statistically associated with significant effects. With regard to the discovery of immunomodulatory effects of simvastatin through studies of organ transplant patients, caution should be performed when considering definite immunorelated disorders such as vitiligo. Moreover, parallel experiments should be initiated regarding exact molecular and cellular effects of statins in the treatment of vitiligo. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: The Effect of Breakfast Prior to Morning Exercise on Cognitive Performance, Mood and Appetite Later in the Day in Habitually Active Women ABSTRACT: Pre-exercise nutritional practices for active females exercising for mood, cognitive and appetite benefits are not well established. Results from an initial field pilot study showed that higher energy intake at breakfast was associated with lower fatigue and higher overall mood and alertness post-exercise (all p < 0.05). In a follow-up, randomised, controlled trial, 24 active women completed three trials in a balanced, cross-over design. At 0815 h participants completed baseline cognitive tasks, mood and appetite visual analogue scales (VAS) and were administered a cereal breakfast (providing 118 or 236 kcal) or no breakfast. After 45 min, they completed a 30 min run at 65% heart rate reserve (HRR). Parameters were re-assessed immediately after exercise, then hourly until lunch (~1240 h), immediately post-lunch and at 1500 and 1900 h via a mobile phone. Breakfast enhanced feelings of relaxation before lunch (p < 0.05, d > 0.40), though breakfast was detrimental for working memory mid-afternoon (p = 0.019, d = 0.37) and mental fatigue and tension later in the day (all p < 0.05, d > 0.038). Breakfast was also beneficial for appetite control before lunch irrespective of size (all p < 0.05, d > 0.43). These data provide information on pre-exercise nutritional practices for active females and suggest that a small breakfast eaten prior to exercise can benefit post-exercise mood and subjective appetite ratings. BODY.1. INTRODUCTION: Many people exercise regularly for physical and psychological benefits, which are well documented in the literature (e.g., [1,2]). Acutely, exercise can improve cognitive function [3], psychological state and mood [4,5] and support weight regulation [1]. These variables can also be positively influenced by following healthy dietary practices and it is widely believed that one such practice is the regular consumption of breakfast [6,7,8]. Breakfast is a likely source of pre-exercise nutrition if exercise is undertaken in the morning but is a frequently omitted meal among young females [9,10]. Pilot survey data in an active female population revealed that the main reasons for skipping breakfast before morning exercise were lack of time and avoiding discomfort during exercise (unpublished data), which mirrors findings of a previous study in swimmers [11]. In addition, a common reason for females to exercise is for weight management [9,12], and as exercise in a fasted state can increase fat oxidation [13,14], they may also follow this practice to maximize their weight loss potential. Little is known about how pre-exercise nutrition affects post-exercise cognitive performance, mood and appetite. Specifically, the relationship between breakfast consumption and exercise and the subsequent effects on these parameters has not been explored in depth and preliminary research in this area has yielded conflicting results. In active adult males, breakfast consumption (451 kcals), compared to omission, prior to exercise has been found to reduce mental fatigue [15] and reduce subjective appetite [13] post-exercise. Conversely, other studies have shown no effect of breakfast consumption prior to exercise on cognitive performance [15,16] (451 kcals and 281 kcals, respectively) or mood [11,16] (451 kcals and 200 kcals, respectively). Although there are nutritional guidelines available for athletes to follow with regards to exercise performance, for example, consuming carbohydrate (CHO) following an overnight fast and 2–4 h before exercise [17], these guidelines may not be applicable for those exercising recreationally rather than for competition or performance enhancement. In a recent survey only 23% of individuals (n = 699) who regularly engaged in moderate intensity physical activity reported that they preferred not to have breakfast before exercise. At least 40% of the sample agreed that they had more energy on days that they ate breakfast, that eating breakfast led to increased physical activity, and that they felt they must eat breakfast before they engaged in exercise [18]. It has been suggested that women are more sensitive to changes in mood and appetite than men [19,20], and therefore may be more susceptible to any negative effects of omitting breakfast, particularly prior to exercise. To date, no studies have assessed the effect of consuming a typical breakfast prior to exercise on post-exercise cognitive performance, mood, appetite and energy intake (EI) in recreationally active females; the current paper describes two studies which aimed to explore this paradigm. To begin, a pilot field study was conducted to investigate whether consuming breakfast prior to morning exercise affects cognitive performance, mood, appetite and energy intake (EI) for the remainder of the day. The results from this study suggested that breakfast size may influence post-exercise mood and appetite. Subsequently, a randomized controlled trial was conducted to see if a small breakfast, which was quick to consume and digest and therefore perhaps be suited to this population, could lead to cognitive or mood benefits over no breakfast or consuming a larger breakfast prior to a morning exercise session; this study will provide the main focus of the paper. BODY.2. EXPERIMENTAL SECTION.2.1. PILOT STUDY.2.1.1. METHODS: A preliminary, single-day field study was conducted to assess whether energy intake at breakfast prior to morning exercise influenced cognitive function, mood, appetite and energy intake (EI) for the remainder of the day. BODY.2. EXPERIMENTAL SECTION.2.1. PILOT STUDY.2.1.1. METHODS.PARTICIPANTS: Forty five, healthy, habitually active females (defined as exercising for at least 30 min, 3 times per week for at least the previous 6 months; [21]) were recruited and completed the study. Their mean ± SD age, height, body mass (BM) and body mass index (BMI) score were 21.4 ± 3.3 years, 168.6 ± 0.1 cm, 62.4 ± 7.8 kg and 21.8 ± 2.8 kg/m2 respectively. As well as being habitually active and regularly exercising in the morning, participants also confirmed they were aged 18–35 years, in good health, a non-smoker, free from medication and herbal and dietary supplements and had no history of head trauma, learning difficulties, attention deficit hyperactivity disorder, dyslexia, migraines or gastric problems, a good standard of English and a healthy BMI (>18 and <25 kg/m2) and blood pressure (≤140/90 mmHg). BODY.2. EXPERIMENTAL SECTION.2.1. PILOT STUDY.2.1.1. METHODS.SCREENING AND FAMILIARISATION: Ethical approval for this study was granted by the Ethics Committee of the Faculty of Health and Life Sciences at Northumbria University and was conducted according to the Declaration of Helsinki. Prior to participation volunteers gave written informed consent. They were then familiarised with the mobile phone cognitive tasks, mood and physical state visual analogue scales (VAS; taken from Rogers et al., 2003 [22]) and appetite VAS (for full descriptions of the mobile phone tasks and VAS see Document S1), completing them 3 times to reduce learning effects. They also received detailed instruction on recording food intake using a food diary and completed the Three-Factor Eating Questionnaire (TFEQ) R-18, a measure of dietary restraint [23]; mean ± SD TFEQ score for the sample was 21.6 ± 5.9. BODY.2. EXPERIMENTAL SECTION.2.1. PILOT STUDY.2.1.1. METHODS.EXPERIMENTAL PROCEDURES: Each participant completed a single study day which took place in their own environment where they undertook a morning exercise session (between the hours of 0600 and 1100 h) which was of typical mode and intensity for them. Details of this exercise session (time, type, duration, intensity) were recorded in a log immediately after the session. They also completed the mobile phone cognitive tasks upon waking (baseline), at 1130 h, 1500 h and 2000 h. Food intake and physical activity were recorded from breakfast the day preceding, until breakfast the day after, the study day. BODY.2. EXPERIMENTAL SECTION.2.1. PILOT STUDY.2.1.2. STATISTIC: Food diaries were analysed using Microdiet (Downlee Systems Ltd, Chapel-en-le-Frith, Derbyshire, UK). Scores for each individual task were analysed as "change from baseline" using SPSS 19 (SPSS, Inc., Chicago, IL, USA) and averaged across the 3 post-exercise time points to give overall scores for each variable. Partial correlations were conducted to assess the effect of the size of breakfast consumed prior to exercise on post-exercise scores. Breakfast was defined as anything other than waterconsumed between waking and exercise; non-breakfast consumers were included in the analysis. Ratings for exercise intensity (mm) were multiplied by exercise duration (min) to give each individual an overall exercise workload score which was used as a covariate in all analyses. An alpha level of 0.05 was used for all statistical tests. Significant results are reported. Confidence intervals are reported in square brackets and means and Standard Error Means (SEM) are reported where appropriate. BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.1. METHODS: A randomized, balanced, cross-over study was conducted to compare the effects of two different sized breakfasts and no breakfast consumed prior to morning exercise on cognitive function, mood, appetite and energy intake (EI) for the remainder of the day. BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.1. METHODS.PARTICIPANTS: Twenty five young, healthy, habitually active females took part in the study. One participant withdrew due to time commitments, leaving a final sample of 24. Their mean ± SD age, height, body mass (BM) and Body Mass Index (BMI) were 20.9 ± 2.3 years, 170.0 ± 7.0 cm, 63.0 ± 6.4 kg, 21.9 ± 1.9 kg/m2 respectively. In addition to the general inclusion criteria listed for the pilot study (see Section 2.1.1), participants also confirmed they were habitually active (exercising for at least 30 min, 3 times per week for at least the previous 6 months; [21]), regularly exercised in the morning, were able to run for 30 min continuously at a moderate speed, habitually consumed breakfast prior to a morning exercise session and had a liking for all of the food items provided in the study. Restrained eating was measured at screening using the TFEQ R-18 [23] but not used as an inclusion criterion. All participants but one were restrained eaters, scoring >10 on the TFEQ. Where participants were not using hormonal contraception, both main trials were conducted during the same phase (either luteal or follicular) of their menstrual cycle where possible. BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.1. METHODS.SCREENING AND FAMILIARISATION: Ethical approval for this study was granted by the Ethics Committee of the Faculty of Health and Life Sciences at Northumbria University and was conducted according to the Declaration of Helsinki. Prior to participation volunteers gave written informed consent. Participants completed a screening session to assess eligibility and demographic, anthropometric and blood pressure measures were collected. Familiarisation with the experimental procedures and the computer and mobile phone cognitive tasks and VAS was provided. Participants completed the computerized and mobile phone tasks 3 times before taking part in the study to reduce the chance of learning effects on main trial days. Participants were also fully briefed on how to correctly complete a food diary and were provided with a set of electronic kitchen scales where necessary. BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.1. METHODS.COGNITIVE TASKS: The computerized cognitive tasks were delivered via the Computerised Mental Performance Assessment System (COMPASS, Northumbria University), a programme used to present standard psychometric tests. COMPASS has been used in several previous nutritional intervention studies and has been shown to be sensitive to cognitive enhancement following a variety of nutritional interventions [24,25,26]. The COMPASS tasks comprised of Four Choice Reaction Time, Stroop, N-Back and a Rapid Visual Information Processing task (for full task descriptions see Document S1). Data from studies measuring episodic memory measured using a word recall task provide the most convincing positive evidence for the effect of breakfast on cognitive performance. However, more research is needed focusing on other cognitive domains and tasks before concrete conclusions can be drawn. Furthermore, it has been suggested that null findings in studies investigating the effect of breakfast on cognitive function may be due a lack of sensitivity in tasks measuring cognitive domains other than memory [27]. This advocates further exploration using different tasks, perhaps steering away from the very commonly used word recall task. The mobile phone cognitive tasks consisted of an N-Back task (as used in Kennedy et al., 2011 [28]), an RVIP task and an Arrow Reaction Time task (for full mobile phone task descriptions see Document S1). BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.1. METHODS.VISUAL ANALOGUE SCALES (VAS): Prior to cognitive task completion, VAS were used to measure mood and physical state ("relaxed", "alert", "jittery", "tired", "tense", "headache", "overall mood"), mental fatigue and task difficulty, breakfast and lunch "liking" and exercise enjoyment. Subjective appetite ratings ("hunger", "fullness", "desire to eat" and "satisfaction" were also recorded using VAS, a valid and sensitive method of measuring appetite [29]. All VAS were completed electronically with the exception of the appetite VAS completed during exercise which were pen and paper (for full VAS descriptions see Document S1). BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.1. METHODS.PRELIMINARY EXERCISE TESTS: Participants also undertook two preliminary exercise tests. The first established the relationship between heart rate (HR) and running speed on a flat treadmill using a 16 min test. The test began at a low-moderate speed (between 7–8.5 km·h−1). Every 4 min, the speed of the treadmill was increased by 1 km·h−1 until 16 min had elapsed. HR was recorded during the last minute of each stage. Participants were allowed 5–10 min recovery after the preliminary test before undertaking a maximal exercise test to establish their maximum HR (HRmax). An incremental treadmill test was utilized [30] whereby the speed of the treadmill remained constant (typically 9–11 km·h−1) but the gradient of the treadmill was increased by 1%·min−1 to exhaustion. Verbal encouragement was given towards the latter stages of the test to ensure that subjects worked to exhaustion. Reaching a HR within 10 beats·min−1 of age-predicted HRmax was deemed satisfactory. The running speed equivalent to 65% of each participant's heart rate reserve (HRR) was then determined. HRR is accepted as an accurate method of controlling exercise intensity [31] and is calculated as HRmax—resting HR. Previously, 60%–70% of HRR has been used to achieve exercise of moderate intensity [32]. BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.1. METHODS.PRE-TESTING PROCEDURES: On the day preceding the first trial, participants kept a record of their food intake and physical activity. They were required to replicate these behaviours and diet the day preceding subsequent trials. In the 24 h period prior to each main trial, participants abstained from alcohol, caffeine and vigorous physical activity. Participants consumed a standardized meal the evening prior to each main trial which was provided by the researcher, as cognitive responses to breakfast can be influenced by food consumed the previous evening [33]. This meal consisted of a pack of Uncle Ben's grilled Mediterranean vegetable microwave risotto (412 kcal, 78.8 g CHO, 9.6 g protein, 6 g fat) prepared with water as per the manufacturer's instructions and an Ambrosia low-fat custard pot (134 kcal, 23.2 g CHO, 4.4 g protein, 2.7 g fat). Participants were required to consume this meal before 2000 h the evening before each main trial, and ensure this was the last food they consumed before each of the test sessions. They were also required to fast for 12 h prior to the start of each main trial. BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.1. METHODS.TREATMENTS/TEST MEALS: The nutritional content of the breakfast and lunch provided in the study are detailed in Table 1. In the two breakfast trials, participants were given a breakfast consisting 40 g or 20 g of Special K breakfast cereal (Kellogg's, Manchester, UK) with 166 mL and 83 mL of semi-skimmed milk (Sainsbury's, London, UK) respectively. These amounts were chosen based on the manufacturer's recommendation of 30 g of cereal, which was considered to be an average serving. Participants were given a maximum of 10 min to consume the breakfast. Each portion of the ad libitum lunch was prepared in advance and re-heated in the microwave for 5 min as required. Participants were initially served 400 g of pasta. Approximately every 3–5 min, more pasta was added to the bowl (300 g initially, then 100 g thereafter) until the participant indicated that they were full. Participants were not allowed to completely finish a portion before the bowl was refilled. nutrients-07-05250-t001_Table 1 Table 1 Nutritional content of study foods. kJ/Kcal Carbohydrate (g) Protein (g) Fat (g) Fibre (g) Breakfasts 20 g Special K cereal 321/76 15 3 0.3 0.9 83 mL Semi-skimmed milk 171/42 4 1.5 1.5 <0.5 Total 492/118 19 4.5 1.8 0.9 40 g Special K cereal 642/152 30 6 0.6 1.8 166 mL Semi-skimmed milk 342/81 8 3 3 <0.5 Total 984/236 38 9 3.6 1.8 Lunch 125 g Penne Pasta 1894/446 91 15 2 3 250 g Tomato and Herb pasta sauce 540/128 20 4 3 4 15 g Olive Oil 508/123 <0.5 <0.5 14 <0.5 40 g Cheddar Cheese 648/156 <0.5 10 13 <0.5 Total per 430 g portion 3544/938 111 29 32 7 Total per 100 g 824/218 26 7 7 1.5 BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.1. METHODS.EXPERIMENTAL PROCEDURES (: Each participant completed three trials, in a randomised, cross-over design, where they were administered one of three breakfasts: 40 g cereal + 166 mL milk, 20 g cereal + 83 mL milk or no breakfast. The first trial was undertaken between 48 h and 14 days of the initial screening visit. Trials were separated by ≥48 h and all trials were performed under similar laboratory conditions. Trials began at 0815 h (±15 min). After confirming compliance to the study restrictions, participants completed baseline cognitive tasks and mood and appetite VAS before being administered the test breakfast or remaining fasted. Participants were then required to rest for 45 min to allow for the digestion of breakfast before repeating the cognitive tasks and mood and appetite VAS. Immediately following this, they undertook a 30 min treadmill run (at approximately 65% HRR). Heart rate was monitored throughout the exercise period using telemetry (Polar T31 transmitter, Polar Electro Oy, HQ, Professorintie 5, FIN-90440 Kempele, Finland) and Rate Perceived Exertion (RPE [34]) was measured at 5 min intervals. Participants then completed the cognitive tasks and mood and appetite VAS immediately after, and at 1 and 2 h, post-exercise. They were then administered an ad libitum pasta lunch where they were asked to consume enough food to feel satisfied to a normal level. After lunch, they completed the cognitive battery and mood and appetite VAS for a final time and were then free to leave the laboratory. Water intake was recorded during the first trial and was matched in the subsequent trials. Participants also completed similar cognitive tasks, mood and appetite VAS via a mobile phone at 1500 and 1900 h and completed a food diary for the rest of the day. Figure 1Main study schematic. BODY.2. EXPERIMENTAL SECTION.2.2. MAIN STUDY.2.2.2. STATISTICAL ANALYSIS: A previous study investigating the effect of breakfast prior to exercise on mood, cognitive function and appetite at 4 time-points post-exercise yielded medium-large effects (Cohen's D (d) = 0.5–0.8). Based on this, a power calculation (based on number of cognitive task repetitions) carried out a priori indicated that a total sample size of 24 would provide statistical power to detect large effects above 80% with an alpha level of 0.05 [35,36]. Before the main statistical analysis, a one-way ANOVA was used to compare pre-breakfast baseline data to assess for differences in performance across the main trials. Scores for each individual task outcome completed on the computer (MPSVAS, FCRT, Stroop, N-back, RVIP, mental fatigue VAS and task difficulty VAS) were analysed as "change from baseline". All appetite VAS were analysed as time-averaged AUC, calculated using the trapezoidal method. Data for the breakfast and lunch liking VAS, exercise enjoyment VAS, RPE, EI and the mobile phone tasks were analysed as absolute values. TFEQ scores were used as a covariate in the EI analysis only as it has been previous found that restraint scores calculated using the TFEQ were not associated with appetite sensations [37]. Data were split into four parts for analysis: pre-exercise (not presented in this paper), pre-during-post exercise (appetite only), post-exercise and mobile phone data. A 3 × 4 (breakfast × repetition) repeated-measures ANOVA was used to assess differences in performance and mood during the post-exercise time-points in the laboratory and a 3 × 2 (breakfast × repetition) ANOVA for the mobile phone data analysis. A one-way repeated-measures ANOVA was used when data was collected only at a single time point (for example, pre-exercise). All analyses were carried out using SPSS 19 (SPSS, Inc., Chicago, IL, USA). Planned comparisons (using t tests calculated with the Mean Squares Error) were then employed to show where the ANOVA was significant. An alpha level of 0.05 was used for all statistical tests. All analyses were corrected for multiple comparisons using the Bonferroni correction. Effect sizes for significant results are reported using d. BODY.3. RESULTS.3.1. PILOT STUDY RESULTS: There were no significant correlations between pre-exercise breakfast size and baseline scores, post-exercise cognitive task outcomes or the appetite VAS or workload score. No associations were found between pre or post-exercise EI and BMI. Neither pre-, nor post-, exercise EI correlated significantly with TFEQ scores. Average waking time, exercise start time and exercise duration were 0755 h (range: 0615–0945 h), 0900 h (range: 0645–1015 h) and 62 min (25–135 min) respectively. The average pre-exercise breakfast size was 234 kcal (range: 0–743 kcal). Energy intake at breakfast prior to exercise were significantly related to post-exercise mental fatigue (r (43) = −0.307; p = 0.043) and thirst (r (43) = −0.333; p = 0.027). As EI (kcal) increased, mental fatigue (Figure S1) and thirst ratings decreased. Energy intake at breakfast prior to exercise was also significantly related to post-exercise alertness (r (43) = 0.315; p = 0.037) and overall mood (r (43) = 0.372; p = 0.013). As EI (kcal) increased, alertness and overall mood increased (Figure S1). Energy intake (kcal) consumed at breakfast prior to exercise was significantly related to post-exercise EI (r (43) = 0.421; p =0.004) with a higher EI (kcal) consumed at breakfast, was associated with a higher post-exercise EI (Figure S2). BODY.3. RESULTS.3.2. MAIN STUDY RESULTS.3.2.1. COGNITIVE FUNCTION: No significant results were found for laboratory-based computer cognitive task performance between the different treatment conditions up to 2 h post-exercise (Table S1). A significant breakfast × repetition interaction was observed for RVIP false alarms (errors) [F (2, 16) = 4.86, p = 0.014] when completed on the mobile phone. Comparisons revealed that at 1500 h participants made significantly less false alarms in the no breakfast (NB) condition compared to the 40 g breakfast condition (p = 0.019, d = 0.37). However, at 1900 h there were significantly less false alarm responses in the 20 g breakfast condition compared to the NB (p = 0.001, d = 0.58) and 40 g breakfast (p < 0.0001, d = 0.58) conditions (Figure 2, Table S1). Figure 2The effects of consuming breakfast cereal (20 g or 40 g) or no breakfast (NB) prior to exercise on RVIP false alarms 5 h (1500 h) and 9 h (1900 h) post-exercise, in habitually active females (n = 17). (* p < 0.05, ** p < 0.01, *** p < 0.001). BODY.3. RESULTS.3.2. MAIN STUDY RESULTS.3.2.2. MOOD AND PHYSICAL STATE: A significant breakfast x repetition interaction was observed for relaxed scores [F (2, 23) = 2.51, p = 0.025]. Comparisons revealed that at 1 h and 2 h post-exercise and post-lunch participants were significantly less relaxed in the NB condition compared to the 20 g breakfast condition (p = 0.013, d = 0.40, p = 0.003, d = 0.50 and p < 0.002, d = 0.60 respectively) and 40 g breakfast condition (2 h post-exercise and post-lunch only, p < 0.001, d = 0.75 and p = 0.002, d = 0.57 respectively; Figure 3, Table S2). No other significant differences were observed between conditions for the computer Mood and Physical State Visual Analogue Scales (see Table S2). There were also no significant differences observed between the NB, 40 g and 20 g breakfast conditions for RPE during exercise (10.8 ± 0.3, 10.7 ± 0.3 and 10.6 ± 0.3 respectively) or exercise enjoyment (53.8 ± 2.3, 54.6 ± 3.1, 58.7 ± 2.1 respectively). Figure 3The effects of consuming breakfast cereal (20 g or 40 g) or no breakfast (NB) prior to exercise on feelings of relaxation immediately, 1 and 2 h post-exercise and post-lunch, in habitually active females (n = 24). Points which share a letter are significantly different from one another (* p < 0.05) Significant effects were found for mental fatigue and tension ratings when completed using the mobile phone. A significant main effect of breakfast condition was observed for mental fatigue ratings when completed 5–9 h post-exercise [F (2, 16) = 4.38, p = 0.021]. Comparisons revealed that participants were significantly less mentally fatigued in the NB condition compared to the 20 g breakfast condition (p = 0.018, d = 0.62; Figure 4a, Table S2). A significant breakfast × repetition interaction was also observed for tension scores [F (2, 16) = 3.38, p = 0.046]. Comparisons revealed that at 1900 h participants were significantly less tense in the NB condition compared to the 20 g (p = 0.001, d = 0.55) and 40 g (p = 0.045, d = 0.38) breakfast conditions (Figure 4b, Table S2). BODY.3. RESULTS.3.2. MAIN STUDY RESULTS.3.2.3. APPETITE: Significant main effects of breakfast were observed for pre-during-post exercise hunger [F (2, 23) = 23.88, p < 0.0001], desire to eat [F (2, 23) = 23.11, p < 0.0001], fullness [F (2, 23) = 43.09, p < 0.0001] and satisfaction [F (2, 23) = 39.91, p < 0.0001] AUC ratings. Participant's reported feeling significantly less hungry (p = 0.025, d = 0.68; Figure 5a) and felt they could eat less (p = 0.020, d = 0.59) after consuming the 40 g compared to 20 g breakfast (see Table S1). When they had consumed no breakfast compared to the 20 g and 40 g breakfasts participants reported feeling more hungry (p < 0.0001, d = 1.11 and p < 0.0001, d = 1.86 respectively; Figure 5b) and desired to eat more (p < 0.0001, d = 1.12 and p < 0.0001, d = 1.60 respectively) and felt less full (p < 0.0001, d = 1.63 and p < 0.0001, d = 2.09 respectively) and less satisfied (p < 0.0001, d = 1.67 and p < 0.0001, d = 1.79 respectively). Figure 4The effects of consuming breakfast cereal (20 g or 40 g) or no breakfast (NB) prior to exercise on ratings of (a) mental fatigue 5–9 h post-exercise and (b) tension 9 h post-exercise, in habitually active females (n = 17) (* p < 0.05, ** p < 0.001). Figure 5The effects of consuming breakfast cereal (20 g or 40 g) or no breakfast (NB) prior to exercise on pre-, during and post-exercise hunger in habitually active females (n = 24). Values are (a) absolute and (b) time-averaged AUC (* p < 0.05, ** p < 0.01, *** p <0.001). Significant main effects of breakfast were observed for post-exercise hunger [F (2, 23) = 6.60, p = 0.003], desire to eat [F (2, 23) = 6.49, p = 0.003], fullness [F (2, 23) = 6.20, p = 0.004] and satisfaction [F (2, 23) = 16.41, p < 0.0001; Figure 6]. When no breakfast was consumed compared to 40 g breakfast, participants reported feeling more hungry (p = 0.001, d = 0.78), desired to eat more (p = 0.001, d = 0.79) and felt less full (p = 0.001, d = 0.83 respectively). They were also less satisfied when no breakfast was consumed compared to both the 20 g and 40 g breakfasts (p = 0.001, d = 0.58 and p < 0.0001, d = 0.97 respectively). They also reported feeling that they felt they could eat significantly less (p = 0.044, d = 0.64) and were significantly more satisfied (p = 0.025, d = 0.43; Figure 6) after consuming the 40 g compared to 20 g breakfast (Table S3). There were no significant differences observed between conditions for subjective appetite between 1500 and 1900 h measured using the mobile phone (Table S3). BODY.3. RESULTS.3.2. MAIN STUDY RESULTS.3.2.4. ENERGY INTAKE: There was no significant difference between the conditions for EI during the ad libitum lunch or post-lunch (Table 2). BODY.3. RESULTS.3.2. MAIN STUDY RESULTS.3.2.5. MEAL LIKING: A significant main effect of breakfast was observed for the "breakfast liking" VAS [F (2, 23) = 106.83, p < 0.0001]. However, whereas participants "liked" consuming the 20 g and 40 g breakfast significantly more than consuming no breakfast (p < 0.0001, d = 3.62 and p < 0.0001, d = 3.20 respectively), both the 20 g and 40 g breakfast were liked equally as much (Table 2). There was no significant differences between conditions for the "lunch liking" VAS (Table 2). Figure 6The effects of consuming breakfast cereal (20 g or 40 g) or no breakfast (NB) prior to exercise on hunger, desire to eat, fullness and satisfaction over a 3 h post-exercise period, in habitually active females (n = 24). Values are time-averaged AUC (* p < 0.05, ** p < 0.01, *** p < 0.001). nutrients-07-05250-t002_Table 2 Table 2 Rate of perceived exertion during exercise, exercise enjoyment, meal liking and energy intake (lunch, post-lunch and net) mean values ( n = 24). Measure Condition Mean Value Breakfast Liking (mm) NB 19.4 * , † ± 2.7 20 g 68.9 ± 3.0 40 g 67.9 ± 3.5 Lunch Liking (mm) NB 74.5 ± 3.0 20 g 76.5 ± 2.2 40 g 75.8 ± 2.4 Lunch Energy Intake (kcal) NB 763.2 ± 37.2 20 g 786.2 ± 37.1 40 g 778.7 ± 38.8 Post-lunch Energy Intake (kcal; n = 22) NB 1000.8 ± 76.9 20 g 1055.9 ± 105.6 40 g 1067.5 ± 114.5 Net Energy Intake (kcal; n = 22) NB 1780.14 ± 80.59 20 g 1957.21 ± 114.64 40 g 2088.57 ± 132.30 Means ± SEM are presented; * Mean value was significantly different from 40 g breakfast ( p < 0.05); † Mean value was significantly different from 20 g breakfast ( p < 0.05). BODY.4. DISCUSSION.4.1. PILOT STUDY: A preliminary, single-day field study was conducted to assess whether energy intake at breakfast prior to morning exercise influenced cognitive function, mood, appetite and EI for the remainder of the day. An increase in EI at breakfast was associated with lower mental fatigue, higher alertness and EI and better overall mood post-exercise. The limitations of conducting a field study are obvious; a lack of control over the environment in which the study is completed which may lead to confounding results and the inability to detect subtle effects which may only become apparent in a more controlled environment. Participants notoriously under-report when asked to complete a food diary, decreasing the internal validity of the data [38]. For this reason, collecting additional data on a subsequent day would have been preferable. However, these results did suggest that breakfast size before exercise may influence energy intake and mood in active females, warranting further investigation in a randomised controlled trial as subsequently discussed. BODY.4. DISCUSSION.4.2. MAIN STUDY: Beneficial pre-exercise nutritional practices for females exercising for weight control or mood and cognitive benefits have not been well established. The main study presented in this paper aimed to assess the effect of breakfast size prior to exercise on post-exercise cognitive performance, mood and appetite in healthy, habitually active females. Breakfast consumption compared to breakfast omission prior to exercise improved subjective hunger ratings, irrespective of size. Breakfast was also favourable for some mood benefits in the hours following exercise, but consuming a smaller breakfast (118 kcals) prevented mid-afternoon cognitive decrements associated with consuming a larger breakfast (236 kcals). Feelings of relaxation were enhanced in the initial post-exercise period when either breakfast was consumed, a positive finding given that women sight stress reduction as a main reason for exercise [9]. Consuming breakfast replicated the usual routine of this sample and some evidence suggests that habitual breakfast habits may influence mood responses to acute breakfast consumption [39,40], which may explain this finding. In addition, past research suggests that CHO intake may reduce the effects of energy depletion in the brain by attenuating the synthesis of certain metabolites and neurotransmitters [41]. Previously, a higher concentration of plasma glucose was observed immediately post-exercise when CHO, compared to placebo, had been consumed during a moderate intensity 2 h cycle, which corresponded with increased feelings of pleasure in males [42]. Foster et al., (2007) also found higher blood glucose accompanied increases in alertness and contentment when breakfast was consumed rather than omitted [43]; consuming a meal containing CHO prior to exercise may contribute to post-exercise mood state by increasing pre, and therefore post, exercise glycogen stores [17]. Worse performance on the RVIP task was observed mid-afternoon when the larger breakfast was consumed compared to the smaller breakfast or no breakfast and in the evening fewer RVIP errors were seen following consumption of the smaller breakfast compared to the other conditions. Although previous data suggests working memory is improved by prior breakfast consumption (for review see [27]), our data suggests that when consumed prior to exercise, a smaller, rather than larger, breakfast avoids detriments in working memory later in the day. These results somewhat mirror those found by Nabb and Benton (1996), who reported that memory score negatively correlated with the caloric content of the breakfast administered, attributing this to only minor increases in blood glucose following a smaller meal [44]. Indeed, recently Zilberter and Zilberter (2013) suggested that the belief that consuming breakfast is beneficial for cognitive function may be incorrect [45]. They argued that consumption of a low-CHO, high-fat breakfast or indeed omitting breakfast produces the most stable metabolic response, a factor which appears to contribute positively to cognitive function possibly via a neuroprotective effect. However, even though fasting may lead to a more stable metabolic response acutely, there is evidence that insulin sensitivity and glucose response to a meal are improved when breakfast has been consumed beforehand both acutely [46] and regularly [8]. It is perhaps surprising that cognitive differences were observed between the conditions when tasks were completed on the mobile phone but not when completed on a computer in the laboratory. Whilst the lack of cognitive effects in the initial post-exercise period does mirror previous laboratory based data [15,16] few studies have looked at the effect of breakfast consumption on cognitive function in the evening. A delayed effect of breakfast on cognitive performance, or of course a second-meal effect, whereby the effects of breakfast on cognitive performance only become apparent after the next meal has been consumed, are plausible given that the glycaemic index of an evening meal has been shown to affect cognitive function post-breakfast breakfast the following morning [34]. It has also been suggested that if breakfast is consumed regularly, omitting breakfast occasionally may not have a negative impact on cognitive performance; rather these effects may only be seen if breakfast is omitted over a long period of time [47]. In the current study, consuming breakfast lowered subjective appetite at every time point following breakfast up until immediately after lunch with the greatest differences observed between the NB and 40 g breakfast conditions, as expected. Whilst these results are not considered novel when viewed in isolation, the study also aimed to draw parallels between subjective appetite sensations and mood state and cognitive function. Indeed, the superior appetite profile seen following breakfast concurred with an improvement in at least one facet of mood at most time points throughout the study day; we reported a similar effect in a previous study in male subjects when enhanced appetite control occurred concurrently with lower mental fatigue ratings [16]. Breakfast can also positively influence appetite responses to a second feeding [46] but our data showed no effect of breakfast on appetite beyond immediately post-lunch; any effects were likely masked due to the ad libitum nature of the lunch meal. Research shows that breakfast consumption can also reduce subsequent EI [46,48] although the effect of exercise on this parameter is debatable [49,50]. Previous data from our laboratory [12] revealed that breakfast consumption (451 kcals) compared to omission before exercise improved subjective appetite control, but did not affect EI at lunch following exercise in males. Similarly, lower subjective appetite ratings did not coincide with a reduction in EI at lunch or post-lunch in the current study. This could be due to a lack of power to detect EI effects when administering an ad libitum meal although other explanations should be considered. The majority of the sample in the current study were restrained eaters. The high prevalence of eating restraint in females [51] is consistently highlighted as a problem in this area of research, although there is data suggesting dietary restraint is not a reliable predictor of EI [52,53]. It should also be considered that post-lunch, participants were able to resume their normal diet; there may of course be an interactive effect of breakfast and exercise on subsequent EI, but not one which is robust enough to override everyday habitual behaviours [54]. It is also important to acknowledge here the well-known complications in gathering accurate food-intake information using food diaries [38,55], and this data should be viewed with this in consideration. A limitation this study was the unconstrained environment in which the mobile phone tasks were completed and a variety of nutritional, behavioural and emotional factors may have influenced task performance and mood state at these times; this should be considered when interpreting the apparent negative effect that the smaller breakfast had on tension and mental fatigue in the evening. Nevertheless, this novel method is considered suitable for data collection of this nature [56] and the "free-living" data it provides is lacking in the literature and is useful to guide the direction of research and formulate future hypotheses. The breakfasts administered provided approximately 5% and 10% of the energy needs of a typical adult female, lower than recommended (between 20% and 35% of total daily energy needs [57]). However, the larger of the breakfasts administered in the current study was based on data from the pilot study where the average breakfast consumed was just 234 kcal and therefore perhaps reflects that typically consumed by the population of interest. It should also be noted that the both studies described in this paper used predominately student samples; therefore, this data may not be as valid for other populations. It should certainly be considered that the importance of eating a substantial breakfast regularly for other health benefits has been established and the results from this study which show some benefits of a smaller breakfast may not be applicable to the majority. However, if post-exercise mood and appetite benefits can be attained by eating a small, quick breakfast, which is equally liked compared to consuming a larger breakfast, then those who skip breakfast prior to exercise due to lack of time or to avoid discomfort during exercise may be able to incorporate a breakfast such as this into their morning exercise routine successfully. Testing of this theory in a controlled trial using this particular population would be a logical next step in this area of research. Additional research is needed to explore further how pre-exercise nutrition may impact on post-exercise wellbeing and performance of daily activities. This research could focus on the glycaemic index (GI) or macronutrient content of the breakfast consumed; initial data suggests that the GI of breakfast consumed prior to exercise may affect cognition depending on the domain being observed [58] and that a high-energy CHO drink does not appear to elicit positive mood changes during exercise in women [59]. It would also be interesting to investigate whether a breakfast split between pre and post-exercise would elicit further benefits. BODY.5. CONCLUSIONS: In summary, we believe this to be the first controlled intervention study to demonstrate that breakfast consumption prior to exercise can have a positive, although transient, influence on some aspects of mood and result in superior appetite control after exercise in an active female sample. A pre-exercise breakfast of approximately 118 kcal can improve mood and appetite control post-exercise, although further research is needed before this can be considered a recommendation.

TITLE: Immune-mediated changes in actinic keratosis following topical treatment with imiquimod 5% cream ABSTRACT.BACKGROUND: The objective of this study was to identify the molecular processes responsible for the anti-lesional activity of imiquimod in subjects with actinic keratosis using global gene expression profiling. ABSTRACT.METHODS: A double-blind, placebo-controlled, randomized study was conducted to evaluate gene expression changes in actinic keratosis treated with imiquimod 5% cream. Male subjects (N = 17) with ≥ 5 actinic keratosis on the scalp applied placebo cream or imiquimod 3 times a week on nonconsecutive days for 4 weeks. To elucidate the molecular processes involved in actinic keratosis lesion regression by imiquimod, gene expression analysis using oligonucleotide arrays and real time reverse transcriptase polymerase chain reaction were performed on shave biopsies of lesions taken before and after treatment. ABSTRACT.RESULTS: Imiquimod modulated the expression of a large number of genes important in both the innate and adaptive immune response, including increased expression of interferon-inducible genes with known antiviral, anti-proliferative and immune modulatory activity, as well as various Toll-like receptors. In addition, imiquimod increased the expression of genes associated with activation of macrophages, dendritic cells, cytotoxic T cells, and natural killer cells, as well as activation of apoptotic pathways. ABSTRACT.CONCLUSION: Data suggest that topical application of imiquimod stimulates cells in the skin to secrete cytokines and chemokines that lead to inflammatory cell influx into the lesions and subsequent apoptotic and immune cell-mediated destruction of lesions. BODY.BACKGROUND: Actinic keratosis (AK) are common, cutaneous, precancerous neoplasms appearing as rough, dry, scaly lesions that occur primarily on the sun-exposed skin of middle-aged and elderly people [1-3]. Although the exact mechanism of pathogenesis of AK development is unknown, part of the pathogenesis may involve suppression of the immune response against dysplastic cells [4]. It is believed that prolonged ultraviolet exposure changes the immune surveillance mechanism of the skin, contributing to the tolerance of tumor cells [5]. If left untreated, AK can progress to squamous cell carcinoma, a locally aggressive and occasionally metastatic tumor type [6]. Standard treatment of AK includes various types of surgical and chemical treatments [7,8], which are often associated with scarring and infection, and may not address sub clinical lesions [8]. Toll-like receptors (TLR) are pattern recognition receptors that detect pathogen-associated molecular patterns (PAMPs) and play key roles in the activation of innate and adaptive immune responses [9,10]. Currently, 10 human TLRs have been identified. The natural ligands for all but TLR10 have also been identified [9]. Toll-like receptors are primarily expressed on immune cells such as monocytes, dendritic cells (DCs), and lymphocytes [11], but some TLRs are also expressed on nonimmune cells, including endothelial cells, epithelial cells, and keratinocytes [12]. The role of TLRs in the pathogenesis and treatment of dermatological diseases is increasingly recognized [13]. Imiquimod, a member of a class of drugs termed immune response modifiers has been shown to be a selective TLR7 agonist [[14,15], and unpublished internal data]. Imiquimod is the first TLR-agonist pharmaceutical product approved for human use, and is indicated for the topical treatment of external genital and perianal warts caused by human papilloma virus [16]. Recently, the approved indications have been expanded to include treatment of AK [17] and superficial basal cell carcinoma [18-20]. The antiviral and anti-tumor activity of imiquimod is believed to be due to the activation of the innate immune response, specifically activation of antigen-presenting cells such as monocytes, macrophages and plasmacytoid and myeloid DCs to induce interferon alpha (IFNα) and other cytokines and chemokines [21,15]. Imiquimod also enhances co stimulatory molecule expression important for triggering an adaptive immune response [15]. Topical application of the drug has been shown to induce IFNα and interleukin 6 (IL6) in AK lesions and external genital warts [22,23]. Imiquimod and the chemically related immune response modifier resiquimod have also shown potent vaccine adjuvant effects in mice and man [23-27]. Even though the immune-modulatory activity of imiquimod is well established, the precise molecular changes responsible for the antilesional activity of topically applied imiquimod in AK is not fully understood. The objective of this study was to explore the molecular processes responsible for the antilesional activity of imiquimod in subjects with actinic keratosis using global gene expression profiling. BODY.METHODS AND MATERIALS.INSTITUTIONAL REVIEW BOARD/INFORMED CONSENT: This study was conducted at Loma Linda University School of Medicine/Medical Center, Department of Internal Medicine, Division of Dermatology, Loma Linda, California. The study protocol, subject informed consent documents, and subject information documents were submitted to and received approval from the study center's Institution Review Board. This study was conducted according to the Code of Federal Regulations of the United States Food and Drug Administration (21 CFR Part 56, Institutional Review Boards, and Part 50, Protection of Human Subjects) and the International Conference on Harmonization Edition 6, Guideline for Good Clinical Practice. BODY.METHODS AND MATERIALS.STUDY CONDUCT: This was a phase II, double-blind, placebo-controlled, randomized parallel group study. Randomized subjects had at least 5 clinically visible AK lesions within a 25-cm2 area on the balding scalp. Subjects were randomized to imiquimod or placebo cream in a 3:1 ratio and applied study cream to the treatment area 3 times per week for 4 weeks. Study cream was applied prior to normal sleeping hours and remained on the skin for approximately 8 hours before it was removed. Safety evaluations were made at all treatment and post treatment visits, and included monitoring of adverse events and local skin reactions, as well as photographing the treatment area and reviewing any concomitant medications. At the screening visit, AK lesions were assessed clinically and by confocal microscopy and a representative lesion confirmed by histology. Because AK lesions are in general small in size, histology and gene expression analysis could not be performed on the same biopsy. Therefore, confocal microscopy was performed to establish a correlation of the confocal images and their respective non-sun exposed non-lesional skin, sun exposed non-lesional skin, actinic keratoses lesions, and squamous cell carcinoma. All subjects with lesions histologically identified as having a degree of dysplesia suggestive of squamous cell carcinoma were disqualified from the study. All sites identified as AK lesions were marked and a plastic template of their locations made for exact identification at a later time. Thereafter, changes in AK lesions due to treatment with imiquimod were assessed clinically and by confocal microscopy. Lesions were scored as cleared if the skin exhibited normal epidermis as assessed clinically and by confocal microscopy. Assessment of lesion regression and the results of the confocal microscopy as they relate to aberrant gene expression in AK are discussed in a manuscript submitted for publication (Torres et al, 2006. Micro Array Analysis of Aberrant Gene Expression in Actinic Keratosis: Effect of Imiquimod 5% Cream). At the treatment initiation visit, a shave biopsy was taken for gene expression analysis from an untreated AK lesion, from a sun-exposed non-lesional site on the head and from a non-lesional sun-unexposed site from under the arm area. An additional biopsy was taken at each subsequent study visit, (treatment period weeks 1, 2, and 4) and at 4 weeks post treatment of either a remaining AK, or if no AKs were present, of nonlesional skin at a previously identified lesional site. Thus each biopsy was of a different AK lesion. In an effort to standardize the amount of tissue that was removed at each biopsy, the same size punch was used to score the skin surrounding all the lesions to be biopsied for gene expression studies with an attempt to shave-biopsy the lesion at the papillary dermis level. Biopsies were taken 8 hr to 16 hr after the third treatment of the treatment period. The nonlesional, sun-unexposed site biopsy was used to establish a baseline for comparison of gene expression changes in AK before and after treatment with imiquimod. Shave biopsies were immediately immersed in RNALater (Ambion, Austin, Texas), equilibrated at room temperature for 1 hour, kept at 4°C for 24 hours, and then stored at -20°C prior to RNA extraction. BODY.METHODS AND MATERIALS.RNA EXTRACTION AND PURIFICATION: Total RNA from the biopsy samples was extracted and purified using Qiagen RNeasy Mini Kit Protocol for the Isolation of Total RNA from Heart, Muscle and Skin Tissue (Qiagen, Valencia, California) according to manufacturer's instructions. RNA yield varied from 1.5 to 14 υg. The purity of the RNA was determined by the 260 nm/280 nm absorbance ratio. The median 260 nm/280 nm ratio value for the 119 samples was 2.0 (range 1.9 to 2.2). RNA integrity was determined using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Assay (Agilant Technologies, Palo Alto, CA). All samples gave 28S/18S ratio between 2 and 3 indicating good quality RNA. BODY.METHODS AND MATERIALS.MICRO ARRAY ANALYSIS: Samples for micro array analysis were prepared by 2 rounds of linear target amplification according to the Affymetrix instructions for eukaryotic small sample preparation [28,29]. Briefly, double-stranded cDNA was synthesized from 100 ng of total RNA with oligo(dT)24T7 primer (Affymetrix, Santa Clara, California), followed by 2 cycles of in vitro transcription of cRNA. The first cycle of invitro transcription was performed using a T7 polymerase (MEGAscript T7 Kit, Ambion, Austin, Texas) and the second cycle using Enzo BioArray High Yield RNA Transcript Labeling Kit (Affymetrix). The median value for the biotinylated cRNA yield over the 119 samples was 90 (range 42 to 121). The biotinylated cRNA was hybridized to Affymetrix U133A and U133B GeneChip arrays containing 22,253 probes sets each. Each array was hybridized for 16 hours, washed, then stained with streptavidin-phycoerythrin conjugate and scanned according to manufacturer's instructions. Images were analyzed using Micro Array Suite Version 5 (MAS5). Chips were normalized to a global average intensity of 150 to allow chip-to-chip comparison. The quality of the images was ascertained by monitoring the noise, background, percent transcript present, and the 3'/5' ratio for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which ranged from 1.5 to 5 and beta actin which ranged from 7 to 24. These values are similar to those reported for double amplification protocols [29,30]. BODY.METHODS AND MATERIALS.TAQMAN™ REAL TIME REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION: TaqMan real time RT-PCR was performed for a number of the genes to confirm the micro array results. cDNA was reverse transcribed from total RNA using Invitrogen Superscript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California). Real time RT-PCR was performed using the Applied Biosystems 7900HTTM sequence detection instrument (Applied Biosystems, Foster City, California), and TaqMan low density custom array micro fluidic cards (Applied Biosystems, Foster City, California) as described previously[31]. The micro fluidic cards consisted of 8 ports with 24 different TaqMan primer pair/probe sets arrayed in duplicate in a 384-well micro plate footprint. Each well contained a gene-specific forward and reverse primer, as well as a gene-specific probe, which is labeled at the 5' position with 6FAM (areporter dye) and at the 3' position with minor groove binder/non-fluorescent quencher. Samples were mixed with TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, California), applied to each port of the card, and analyzed by PCR on the 7900HT instrument using Applied Biosystems Sequence Detection System 2.0 software according to the manufacturer's instructions. A total of 46 selected genes associated with the Toll-like receptor pathway, apoptosis, cell cycle, and immune cell infiltration were analyzed using the TaqMan arrays. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize each sample. The TLR copy number was calculated for 2 ng of RNA. BODY.METHODS AND MATERIALS.ANALYSIS OF AFFYMETRIX GENE CHIP DATA: Signals from the gene chip images of biopsy samples for untreated normal skin obtained from sun-unexposed sites were used as a control for the calculation of changes in expression in samples of pretreatment AK lesions and AK lesions during and after treatment (treatment weeks 1, 2, and 4 and 4 weeks post treatment). The statistical algorithm in MAS5 evaluates the image for the expression signal, the absent/present call, and the p-value associated with the signal. It also evaluates the fold change of the sample relative to the designated control sample expressed as the signal log2 ratio, the p-value associated with the fold change, and the direction of change (increased, I; decreased, D; or no call, NC). Using the Affymetrix data mining tool software, the expression data from MAS5 were filtered on the basis of specific criteria to identify differentially expressed genes. A given gene was retained if 1sample from the series passed the following criteria: signal detection p-value ≤ 0.01, signal log2 ratio ≤ -2 or ≥ 2, and a change call designation of 'increased' (I) or 'decreased' (D). A total of 1682 genes passed these criteria. There was a large subject-to-subject variation in the magnitude of changes in expression, as well as the temporal pattern of expression (subject-specific variation in magnitude of response with treatment time) of the 1682 genes. This variation is exemplified in Figure 1, which shows the variation in the temporal expression of IRF7 for selected subjects. Peak expression varied from week 1-treatment to week 4-treatment depending on the subject. This variation may be due to the inherent differences in the responses of subjects and (or) due to variation in the time from treatment to biopsy (range 8 to 16 hr). In order to identify differentially expressed genes in the treatment group without regard to variation in temporal response, the data were reduced in the following manner. The fold change values for samples in the week 1, week 2 and week 4 treatment groups for the 1682 genes for each subject were compared to identify the maximum change in expression (increased or decreased) due to imiquimod or placebo treatment. This value was designated as the treatment-response fold change for imiquimod or placebo and used in the ANOVA described in the Statistical Analysis section. Figure 1Variation in the temporal expression of IRF7 during treatment with imiquimod as determined by real time RT-PCR. 'AK,' designates pretreatment AK lesions. 'WK1 IMIQ', 'WK2 IMIQ' and 'WK4 IMIQ' designate treatment times week 1, 2 and 4. '4 WK Post designates' 4 weeks post end of treatment (WK4 treatment). Fold change was calculated with respect to sun-unexposed, non-lesional skin. S-01, S-02, S-06, S-15 and S-16 are samples from subjects 1, 2, 6, 15 and 16. Cluster analysis was performed with Spotfire DecisionSite-8.1 for Functional Genomics (Spotfire Inc, Somerville, Massachusetts), using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and the Euclidean similarity measure. Functional categorization of genes was based on gene ontology analysis using the Ontology Browser in Spotfire and gene descriptions at the National Center for Biotechnology Information (NCBI) website [32]. The ontology browser calculates a Fisher's Exact Test p-value, which reflects the chance that the gene ontology category is represented by random chance [33]. P-value < 0.05 is considered significant. BODY.METHODS AND MATERIALS.STATISTICAL ANALYSIS: The natural log of the fold change (with respect to sun-unexposed normal skin) from the Affymetrix gene expression and the real time RT-PCR experiments were used in an ANOVA to determine statistically significant changes in expression between sample groups. Due to the small sample size of the vehicle group, 4 subjects, compared to 13 subjects in the imiquimod-treated group, a one way ANOVA comparing vehicle-treated subjects to imiquimod-treated subjects is not expected to yield a reliable determination of imiquimod-response genes. Therefore, a 2-way ANOVA using a blocking factor to account for repeat observations on the same subject was used to compare: (1) the fold change for pretreatment AK (n = 13) and the fold change during treatment with imiquimod (n = 13), and (2) the fold change for pretreatment AK (n = 13) and the fold change for samples taken 4 weeks after the last imiquimod treatment (n = 13). The 'during treatment' fold change is the maximum response fold change value (decreased or increased) selected from week 1, week 2 and week 4 treatment fold changes. Differences between sample groups were considered significant if the p-value for the ANOVA was < 0.05. BODY.RESULTS AND DISCUSSION.DEMOGRAPHICS AND RESPONSE TO TREATMENT: Seventeen white males were randomized to receive treatment (13 to imiquimod and 4 to placebo). The mean age was 75 years (range, 62 to 89 years). All 17 subjects in the study completed the treatment and post treatment portions of the study. The median number of AK lesions at baseline was 10 per subject (range, 6 to 13 lesions). Because of the short duration of the follow-up period (4 weeks for the post treatment period), efficacy was not measured in this study. However, clinical clearance was observed in 25% of the imiquimod-treated subjects 4 weeks after the end of treatment. Imiquimod-treated subjects 1, 2, 4, and 6 and placebo-treated subject 7 were assessed as having clinical clearance of lesions 4 weeks post treatment as determined by return of the lesional site to normal skin. The complete clearance rate in a study where AK subjects were treated for 16 weeks, with a post treatment period of 8 weeks, was 57% [17]. BODY.RESULTS AND DISCUSSION.ANALYSIS OF GLOBAL GENE EXPRESSION USING AFFYMETIX GENECHIPS: GENE ONTOLOGY CLASSIFICATION: A 2-way analysis of variance (ANOVA) was performed comparing the treatment response gene expression fold change (the maximum response value from week 1, week 2 and week 4 treatments) of samples from the imiquimod-treated subjects to the gene expression fold change values of their respective pretreatment AK samples. This comparison resulted in 530 unique genes that had p-values < 0.05 and a median fold change from pretreatment AK <-2 for suppressed genes and >2 for induced genes. Data for the 530 genes is documented in [Additional file 1]. Two-way ANOVA comparing pretreatment AK samples and samples taken 4 weeks after the last imiquimod treatment resulted in 111 unique genes that differentiated the 2 groups with a p-value < 0.05 [see Additional file 1]. The expression of the rest of the genes returned to basal levels 4 weeks post treatment. Of the total number of differentially regulated genes during imiquimod treatment or 4 weeks post treatment, 87% were up-regulated and 13% were down-regulated. Table 1 summarizes the gene ontology classification of imiquimod-modulated genes from the Affymetrix analysis. P-values for representation of ontology categories are given for categories with p-values < 0.05. Of the 530 unique genes whose expression was modulated by treatment, 436 genes had some annotation in the ontology database. Of these, 106 were annotated to the immune response category, 123 to signal transduction, 72 to receptor activity, and 25 to inflammatory response. Specific genes in several molecular function categories are also listed. Other ontology categories that were represented in the data but had p-values >0.05 for representation in the gene ontology category included metabolism (148 genes), development (37 genes) and regulation of transcription (24 genes). The ontology analysis shows that imiquimod treatment of AK results in global gene expression changes impacting various cellular processes, with immune response and signal transduction being the 2 major processes represented. Table 1 Gene Ontology Classification of Imiquimod-Induced Genes in AK Lesions Ontology Genes in the Data 1 Annotated Genes 2 P-value 3 Increased Expression Decreased Expression Description of Processes and Specific Genes Total 436 17479 369 67 Response to stimuli 137 2475 2.13E-19 125 12 defense response, response to external biotic stimuli including, pest and pathogens, antimicrobial, anti-fungal, anti-viral response Defense response 105 1174 1.54E-40 102 3 immune response, inflammatory response Immune response 106 1020 3.05E-40 104 2 cellular defense response, humoral defense response, antigen binding, pattern binding, cytokine synthesis, chemokine synthesis, antigen presentation processing, inflammatory response Response to stress 68 1337 1.77E-12 63 5 response to pest and pathogen, inflammatory response, response to virus, response to wounding Inflammatory response 25 244 4.35E-11 25 0 defensive reaction (by vertebrate tissue) to infection or injury Response to wounding 39 395 3.72E-16 39 0 defensive reaction (by vertebrate tissue) to physical injury Signal transduction 123 3547 4.70E-06 95 17 cytokine signaling, death receptor signaling, G-protein coupled receptor signaling, intigrin binding, MHC protein binding Receptor activity 72 1914 1.80E-07 62 10 chemokine receptors, pattern recognition receptors, immunoglobulin receptors, complement receptors, MHC I\MHCII receptors, scavenger receptors, Hematopoietin/interferon class (D200) receptors Antigen binding 8 91 2.66E-03 8 0 SCF1, IGLV2-14, LAG3, LILRA1, SLAMF1, TRA@, TRGC2, TAP2 Carbohydrate binding 16 326 1.18E-03 14 2 CCL8, CD69, CLEC4A, FCN1, FGFR2, GALNT7, HMMR, KLRC1/KLRC2, KLRF1, LGALS2, LGALS9, POSTN, PTN, SELL, SELPLG, SN Pattern binding 10 159 4.58E-02 8 2 CCL8, CD14, FGFR2, HMMR, POSTN, PTN, TLR2, TLR7, TLR8, TLR4 Cytokine receptors 9 99 1.97E-04 9 0 CCR5, CCR1, CD74, CSF2RB, CXCR4, IL10RA, IL1RL1, IL21R, TNFRSF1B Cytokines 12 257 1.11E-04 11 1 CCL3, CCL5, CCL8, CXCL5, CXCL10, CXCL11, CXCL12, CXCL16, TNFSF10, ECGF1, PTN, SLURP1 Immunoglobulin binding 4 25 1.56E-03 4 0 FCER1A, FCER1G, FCGR3B , 214511_x_at Cystein type endopeptidase 9 169 8.21E-03 9 0 CASP1, CASP8, CTSB, CTSL, CTSC, STSS, LGMN, TNFAIP3, USP18 1 Number of genes in the data for which annotation was found in the Gene Ontology database. 2 Total number of gene with annotation in the Gene Ontology database. 3 P-value for representation of gene ontology category in the data. BODY.RESULTS AND DISCUSSION.VALIDATION OF SELECTED GENES OBSERVED IN THE AFFYMETRIX EXPERIMENT USING REAL TIME REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION: Micro array analysis may not be sensitive enough to capture all changes in gene expression. In comparison, real time reverse transcriptase polymerase chain reaction (RT-PCR) has been shown to be more sensitive for the detection of low abundance transcripts [31]. Therefore, we analyzed AK samples and imiquimod treated AK samples for a selected set of genes using real time RT-PCR. The data is summarized in [Additional File 2] which compares the median fold change values (13 imiquimod-treated subjects) measured using both Affymetrix GeneChip analysis and RT-PCR for a subset of the genes. In general, good agreement was obtained between the 2 methods, with the RT-PCR data showing similar or higher expression than the Affymetrix data. Figure 2 shows a comparison of the 2 methods for the expression of interferon regulatory factor 7 (IRF7). The individual fold change values for the 13 imiquimod-treated subjects during imiquimod treatment were used for the regression analysis (R Square = 0.83). In general, the direction of change in expression for each gene was the same in both assays whereas the magnitude of the fold change values was higher for the RT-PCR analysis than the Affymetrix analysis. Of the 46 genes evaluated on both gene expression platforms, only 3 genes (CD80, MyD88 and TLR6) were identified as having changed in expression in the RT-PCR experiments only (p value ANOVA analysis < 0.05). We did not identify any genes that were differentially expressed by the Affymetrix method that were not also differentially expressed by real time RT-PCR. Thus, overall good agreement was obtained between the 2 methods, with differences between the 2 methods likely due to the increased sensitivity and quantitative nature of the RT-PCR platform. Figure 2Comparison of gene expression data obtained by Affymetrix GeneChip analysis and real time RT-PCR. Linear regression analysis for IRF7 comparing fold change values for individual subjects as measured by Affymetrix analysis and real time RT-PCR analysis. The imiquimod response fold change, which is the maximum response from week 1, week 2 and week 4 treatment times was used. Fold change was calculated relative to sun-unexposed, nonlesional skin samples for 13 subjects treated with imiquimod. The R square value for the comparison was 0.83. BODY.RESULTS AND DISCUSSION.IMIQUIMOD INCREASES EXPRESSION OF PATTERN-RECOGNITION RECEPTORS OF THE INNATE IMMUNE SYSTEM: Since imiquimod is a TLR7 agonist, we sought to determine if TLR7 and other TLRs were expressed in AK lesions and if treatment with imiquimod altered their expression. We analyzed expression levels of various TLRs in biopsy samples of pretreatment AK and after treatment with imiquimod using RT-PCR. We also measured the expression levels of MyD88, an adaptor molecule for various TLRs including TLR7; and IRF7, a transcription factor recently shown to be important in the regulation of interferon gene expression through the TLR7 pathway [34]. Figure 3 shows the median expression levels of the various TLRs, MyD88 and IRF7 for subjects treated with imiquimod (n = 13) during treatment and 4 weeks post treatment. The expression of TLR1, TLR3, TLR6, TLR7, TLR8, TLR9, MyD88 and IRF7 were all increased at statistically significant levels (p-value < 0.05, during treatment [see also Additional File 2]. The increase in TLR4 expression did not reach a statistically significant level (p-value = 0.066) in the imiquimod-treated samples, whereas the Affymetrix data shows a statistically significant increase (p-value of 0.028). The most statistically significant changes observed upon treatment with imiquimod were for TLR3, TLR7, TLR8, and IRF7, with good agreement between the Affymetrix and RT-PCR analysis both for the magnitude of change and for the p-values. There was no change in the expression levels of TLR5 and TLR10 upon treatment with imiquimod. The data are consistent with previous reports of induction of TLRs by various TLR agonists and IFNα [35-37][38,39]. The increased expression of the TLRs may be a result of increased expression of the genes in cells resident in the skin (e.g., DCs, macrophages) or due to the influx of cells with high expression of these genes (e.g., DCs, macrophages, plasmacytoid DCs). The increased expression of TLR3, TLR7, and TLR8 is consistent with increased expression observed in human peripheral blood mononuclear cells upon treatment with imiquimod. The data are also consistent with increased expression of TLR7 observed in imiquimod-treated AK [22]. In summary, treatment of AK lesions with imiquimod results in increased expression of several TLRs and TLR pathway components, thus potentially priming for further amplification of the innate immune system. The increase in the expression of IRF7 is also predicted to amplify the innate immune response by increased expression of type 1 interferons and interferon-inducible genes. Figure 3Basal TLR, IRF7, and MyD88 gene expression in skin biopsies as determined by real time RT-PCR. White bars represent pretreatment AK, black bars represent during imiquimod treatment (maximum response value from week 1, week 2 and week 4 treatment times), and hatched bars represent 4-weeks post treatment. Relative copy number was determined as outlined in Methods and Materials section. Asterisks indicate those genes that had p-values < 0.05 in the ANOVA, comparing expression in pretreatment AK samples to the maximum response expression in samples from subjects (n = 13) during imiquimod treatment. [See Additional file 2]. In addition to the various TLR(s) which recognize viral and bacterial components, an intracellular antiviral pathway which detects viral RNA and results in the induction of type1 interferons has recently been described [40-42]. The central components of this pathway, which are also inducible by type1 interferons [43,44] are DDX58 (RIG-I, retinoid acid inducible gene) and IFIH1 (MDA5, melanoma differentiation antigen 5). Both genes contain helicase domains responsible for detection of double-stranded RNA. They also contain caspase recruitment domains (CARDdomains), which are responsible for signaling through TBK1 (resulting in activation of NFKB and IRF3), as well as induction of type-1 interferons. The activation of this pathway leads to growth inhibition as well as antiviral activity [45]. Figure 4a and 4b show changes in expression of DDX58 and IFIH1 upon treatment with imiquimod as determined from Affymetrix GeneChip analysis. The expression of both genes was increased to statistically significant levels during treatment. The expression of DDX58 remained higher than its pretreatment level (p-value = 0.004), four weeks, post treatment, whereas that of IFIH1 returned to basal level. The induction of several members of the cytoplasmic helicase innate immune pathway, as well as several TLRs, indicates that in addition to activation of the TLR7 pathway, treatment with imiquimod also results in priming of other innate pathways. These pathways may augment other aspects of the innate immune response and may be important for eliminating pre-neoplastic cells. Figure 4Increase in expression of the helicase family of virus-sensing genes upon treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A)DDX58 (RIG-I) and (B)IFIH1 (MDA5). Experimental conditions are as described in the Methods and Materials section. Box plots were generated using MINITAB version 14. 'AK,' 'IMIQ,' and 'Post' designate pretreatment AK, imiquimod-treated skin during treatment (maximum response from week 1, week 2 and week 4), and imiquimod-treated skin 4 weeks post treatment, respectively. Fold change was calculated with respect to sun-unexposed skin. Boxes indicate the median 95% confidence intervals, asterisks designate outliers, and the lines connect median values. P values are given for 2-way ANOVA, comparing the fold change values for pretreatment AK samples and imiquimod-treated samples (n = 13). BODY.RESULTS AND DISCUSSION.IMIQUIMOD INDUCES A LARGE NUMBER OF TYPE I INTERFERON-INDUCIBLE GENES WITH GROWTH INHIBITORY AND IMMUNE-STIMULATORY ACTIVITY: Imidazoquinoline TLR7 agonists such as imiquimod and resiquimod are know to induce various cytokines, including interferon-α, IL6, MCP-1 and IL12 as well as the co-stimulatory markers CD80 and CD86 [15,46]. Type I interferons are known to be powerful regulators of the innate and adaptive immune system through the induction of various genes with antiviral, anti-tumor and immune regulatory functions [43,44,47-51]. In this study, we did not detect increased expression of interferon upon treatment with imiquimod, but observed the increased expression of a large number of IFN-inducible genes (114 genes), [see Additional file 3]. The lack of detection of type 1 interferons after imiquimod treatment in this study may be due to the early induction and degradation of their respective mRNA. Biopsies were taken approximately 8 to 16 hr after application of the drug. We have observed that mRNA for type 1 interferons in human blood mononuclear cells treated with imiquimod peaks in 1 to 2 hours and declines to basal levels 6 to 8 hours post treatment (unpublished internal data). Analysis of gene ontology classification of the interferon-inducible genes increased by treatment with imiquimod identified 46 genes with immune response classification. Figure 5 shows a 2-way hierarchical clustering of the log2 transformed fold changes of all of the 530 imiquimod-induced genes [see Additional file 1]. Figure 6 exhibits clustering of the 46 interferon-inducible genes which classify as immune response genes Two main clusters are apparent in Figure 5. One cluster consists of 8 imiquimod-treated samples. The second and larger cluster consists of all of the pretreatment AK samples, the vehicle-treated samples (designated as placebo in the figure) and five of the imiquimod-treated samples for subjects 02, 05, 09, 12 and 17 (IMIQ-02, IMIQ-05, IMIQ-09, IMIQ-12 and IMIQ-17). The fact that the vehicle-treated samples cluster with pretreatment AK lesions indicates the lack of a significant vehicle-effect in the gene expression profile. In Figure 6, 10 of the imiquimod-treated samples appear in 1 cluster, with IMIQ-02 and IMIQ-05 now as part of the IMIQ-treated cluster. This cluster is characterized by high expression of the interferon-inducible genes such as MX1, IFIT1 and IFIT3. The cluster also contains the samples placebo-03 and AK-11, indicating that these AK lesions were already manifesting spontaneous immune response. The imiquimod-treated samples 09, 12, and 17 are clustered with the pretreatment AK samples in both figures, indicating that Subjects 09, 12, and 17 had little gene expression response to imiquimod. Thus, the 46 interferon-inducible immune response genes segregate the imiquimod-treated samples from pretreatment AK and placebo-treated samples better than the whole set of 530 genes. This group of genes may therefore be good predictors of gene expression response to imiquimod. Collectively, these genes have been reported as 'interferon-signature' genes induced by several IFNα subtypes and IFNβ in monocytes [51]. Figure 5Cluster analysis of genes regulated by imiquimod treatment. Two-way hierarchical clustering was performed as described in the Methods and Materials section. 'AK,' 'IMIQ,' and 'Placebo' designate the fold change with respect to sun-unexposed, nonlesional skin for pretreatment AK samples; for samples during imiquimod treatment (maximum response from week 1, week 2 and week 4), and samples for vehicle-treatment (maximum response from week 1, week 2 and week 4) respectively. Numbers designate subjects. Hierarchical clustering was performed using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and the Euclidean similarity measure. Red, white, and green indicate up-regulated, unchanged, and down-regulated genes, respectively. The color bar insert shows the corresponding expression levels. The cluster consists of 530 imiquimod-responsive genes whose expression was statistically different when comparing the AK group of samples to the IMIQ group of samples. Expression changes for the 530 genes are documented in [Additional file 1]. Figure 6Cluster of 46 immune response genes that are also known to be inducible upon treatment with type 1 interferons as reported in [18, 43, 45, 48, 51, 102] Cluster analysis was performed as described in Figure 5. [See Additional file 3]. Heterogeneity of pretreatment AK lesions was not documented by clinical assessment in this study. However, the gene expression profiles shown in Figure 5 and Figure 6 indicate heterogeneity in lesions. In addition to AK 11 and Placebo 03 in Figure 6 which cluster with the imiquimod treatment group indicating some level of immune response in these samples, other AK lesions also show low levels of expression of interferon inducible genes. For example, AK 06, AK 03, and AK 07 show low levels of expression of several interferon-inducible genes whereas AK 16, AK 17, AK 12 and AK 15 show normal to slightly depressed levels (Figure 6). Interferon-inducible genes regulate diverse cellular processes, such as cell growth and differentiation, cell death, and T-cell co stimulation, activation, and migration. These genes have been reported to possess antiviral [52-54], pro-apoptotic [55-57], and anti-proliferative activities [58,59]. The interferon-inducible genes which increased following imiquimod treatment include those known to be induced by viruses as well as those with known anti-viral activity. These include the 2'5'-oligoadenylate synthetases OAS1, OAS2, OAS3 and OASL, the genes encoding the interferon-inducible proteins with tetratricopepetide repeats, IFIT1, IFIT2, and IFIT3, IFTM1, and other interferon inducible genes such as IFI35, IFI16, MX1, MX2, EIF2AK2 (PRKR), G1P2 (ISG15), G1P3, ISG20, RSAD2 (Cig5), CCL8 (MCP2), CXCL10 (IP10) and CXCL11 (ITAC) [43,44,47,49-52,54,60,61]. Several of the interferon-inducible genes were also increased at statistically significant levels 4 weeks post treatment. These included: IRF7, IFI44, IFIT2, IFIT3, IFITM1, IFI35, RSAD2 (Cig5), G1P2, MX1, OAS1, and OAS2 [see Additional file 1]. In addition to interferon-inducible genes with antiviral activity, several genes known or predicted to possess growth-inhibition and/or cell-differentiating activities [49,50] were induced by imiquimod, including IFI16, AIM2 [62,63], IFIH1 (melanoma differentiation antigen, MDA5) [45], CXCL10 (IP10) [58] and EIF2AK2 (PRKR) [64]. Some of the interferon-inducible genes also possess pro-apoptotic activity, including MX1, TNFSF10 (TRAIL), OAS1, and PRF1 [55,57,65]. Figure 7a and Figure 7b show changes in the expression of the pro-apoptotic genes TNFSF10 (TRAIL) and MX1 with imiquimod treatment as determined from the Affymetrix analysis. The expression of MX1 remained elevated at statistically significant level 4 weeks post treatment, whereas the expression of TNFSF10 returned to basal levels. The data are consistent with the observation of increased expression of IFNα-inducible genes in imiquimod-treated BCC and cutaneous T-cell lymphoma (CTCL) [66,67]. Thus, interferon-inducible genes with pro-apoptotic activity (e.g., MX1, TNFSF10) and growth inhibitory activity (e.g., IFIH1, AIM2, IFI16) may result in growth inhibition of neoplastic cells whereas those with immune-stimulatory activity such as CXCL10 (IP10), CXCL11 (ITAC), and CCL8 (MCP2) may facilitate cell-mediated lesion destruction by recruiting immune cells into the lesions. Indeed, further evidence for the recruitment of immune cells into AK lesions with imiquimod treatment is presented in the following sections. Figure 7Imiquimod treatment is associated with an increase in expression of pro-apoptotic genes as determined by Affymetrix GeneChip analysis. (A) TNFSF10 (B)MX1. Experimental conditions and analysis are as described for Figure 4. BODY.RESULTS AND DISCUSSION.IMIQUIMOD INDUCES THE EXPRESSION OF CHEMOKINES RESPONSIBLE FOR RECRUITMENT OF IMMUNE CELLS TO AK LESIONS: Gene ontology classification (Table 1) shows that 106 genes classified as immune responsive were induced by imiquimod, some of which are also shown to be interferon α/β-inducible [see Additional File 3]. Out of the 106 immune response genes, several were classified as cytokines/chemokines and chemokine receptors. In addition to CXCL10, CXCL11 and CCL8 mentioned above, other chemokines were induced by imiquimod treatment, including CCL3 (MIP1a), CCL4 (MIP1b), CCL5 (Rantes), CXCL12 (SDF1), and CXCL16, and the chemokine receptors CCR1, CCR5, and CXCR4. Figure 8a and 8b illustrate changes in expression of CXCL10 and CXCL11 observed upon treatment of AK lesions with imiquimod. The magnitude of the changes observed in the expression of chemokine genes during treatment ranged from a median fold change value of 1.8 for CXCL12 (SDF1) to 40.8 for CXCL11. The expression of both genes returned to pretreatment levels 4 weeks post treatment. These data are consistent with previous reports of increased expression of chemokines genes and their receptors upon topical treatment of BCC and CTCL with imiquimod [67], as well as in vitro studies of human blood mononuclear cells stimulated with other imidazoquinoline TLR7 agonists showing the induction of CXCL10 and CXCL11 proteins [46]. The cocktail of chemokines up-regulated by imiquimod is consistent with recruitment and/or activation of macrophages, DCs, plasmacytoid DCs, gamma/delta T cells, cytotoxic T cells, and natural killer (NK) cells, and is also consistent with the cell surface markers indicating the presence of these cells upon imiquimod treatment. Indeed, topical treatment of various neoplastic skin conditions with imiquimod cream have shown inflammatory conditions at the site of treatment, indicating the infiltration of immune cells into the site [17,19,20,68,69]. In this study, the gene expression fingerprints that indicate recruitment of various immune cells are further discussed below. Figure 8Increase in expression of chemokines after treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A)CXCL10 (IP10), (B)CXCL11 (ITAC). Experimental conditions and analysis are as described for Figure 4. BODY.RESULTS AND DISCUSSION.IMIQUIMOD INCREASES THE EXPRESSION OF GENES PREDICTIVE OF INFILTRATING MACROPHAGE AND DENDRITIC CELLS: Of the imiquimod-induced genes classified as immune response in gene ontology, several have receptor activity and (or) are hematopoietic cell surface markers (Table 1). The increased expression of these genes indicates the recruitment of various immune cell types to the lesion sites. Macrophage and/or monocyte infiltration of AK lesions upon treatment with imiquimod was indicated by an increase in CD14, CD163, and CLECSF9 (CLECSF4, MINCLE), a C-type lectin found on activated macrophages [70]. The increase in expression of genes of the classical complement pathway, C1QA, C1QB, C3AR1, and C5R1, also indicates an increase in and/or the activation of macrophages [71]. The data are consistent with histologic observation of macrophage and/or monocyte infiltration after application ofimiquimod in the treatment of AK [72] and in lentigo maligna [73,74]. The presence of DCs is shown by increases in the co stimulatory molecules CD86 and CLEC4A (DCIR, CLECSF6), as well as by 3 leukocyte immunoglobulin receptors: LILRB3 (ILT3) and LILRB1 (ILT2) which are expressed in both myeloid and plasmacytoid DCs [75], while ILT7 (LILRA4, CD85g) is restricted to plasmacytoid DCs [76,77]. Figure 9a and Figure 9b illustrate the increase in expression of CD86 and ILT7 with imiquimod treatment. The expression of CD86 remained elevated 4 weeks post treatment. Increased expression is taken as indication of recruitment of these cells to the site of treatment. These observations are consistent with previous studies using topically-applied imiquimod in the treatment of human BCC [69] and melanoma in mice [78], showing recruitment of plasmacytoid DCs into the site of treatment. Figure 9Increase in expression of genes indicating the infiltration of DCs upon treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A)CD86 (B) ILT7. Experimental conditions and analysis are as described for Figure 4. It is interesting to note that CD1C is markedly decreased upon treatment with imiquimod [see Additional file 3]. CD1c is found on Langerhans cells as well as on DCs [79,80]. The decrease in CD1C expression may reflect (although not exclusively) the migration of CD1C+ Langerhans cells out of the dermis. Migration of Langerhans cells out of mouse dermis was observed after topical treatment with imiquimod [78,81]. In the case of the Palamara studies [78] in mouse melanoma, Langerhans cells were observed to return to normal levels in the dermis by day 20. The decrease in CD1C observed upon treatment of AK lesions with imiquimod therefore suggests the activation and migration of Langerhans cells to the lymph nodes and is consistent with previous observations. BODY.RESULTS AND DISCUSSION.IMIQUIMOD INCREASES THE EXPRESSION OF GENES PREDICTIVE OF INFILTRATING CYTOTOXIC T CELLS AND NATURAL KILLER CELLS: Natural killer cells mediate lysis of tumor cells as well as virally-infected cells. Natural killer cells preferentially express several calcium-dependent (C-type) lectins, known as the NKG2 family, which have been implicated in the regulation of NK cell function, and are believed to be important for NK cell-mediated tumor rejection and T-cell mediated immunity [82]. Transcripts for 3 of these C-type lectins, KLRC1/C2, KLRK1 (NKG2D) and KLRF1 were increased in expression in imiquimod-treated samples. In addition, several genes important to the cytolytic function of NK cells and cytotoxic T cells, including TYROBP (DAP12) and CD69 (early T-cell activation antigen), ITGAL (CD11a), ITGB2 (CD18), ICAM1, and the ligands for the NKG2 receptors, MICA and MICB [Additional file 1] [83-88] were increased in expression with imiquimod treatment. Also, genes of the granule products of NK cells and cytotoxic T cells, such as granzymes GZMA, GZMB, GZMK, and GYNYL (granulysin, NKG5), PRF (perforin) and NKG7 (GIG1, GMP-17) were increased in expression, indicating that these cells are cytolytically active [89-93]. Figure 10a and 10b show changes in the expression of GZMA and NKG7 after imiquimod treatment. The expression of both genes returned to pretreatment levels 4 weeks post treatment. Thus, the coordinate increased expression of genes important for NK cell recognition of tumor cells, and for the activation and cytolytic response of NK cells and cytotoxic T cells, suggests that these cells are at least in part responsible for the antilesional activity of imiquimod. Figure 10Increase in expression of genes associated with NK cells, and cytotoxic T cells after treatment with imiquimod as determined by Affymetrix gene expression. (A)GZMA (B) NKG7. Experimental conditions and analysis are as described for Figure 4. BODY.RESULTS AND DISCUSSION.IMIQUIMOD INCREASES THE EXPRESSION OF GENES PREDICTIVE OF THE ACTIVATION OF THE ADAPTIVE IMMUNE SYSTEM: The presence of activated T cells in imiquimod-treated AK lesions is indicated by the expression of several members of the T-cell activation pathway [Additional File 1]). These include the T-cell receptor (TCR) subunits TRD and TRG, TCR-signaling pathway genes such as Fyn, Fyb and LCP2 [94,95], and other genes associated with T-cell activation such as HCK, CD69, PTPRC (CD45) SELL (CD62L, L-Selectin), ITGA4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor), and LAG3. In addition, treatment of AK lesions with imiquimod resulted in a small but significant increase in the expression of CD8β, suggesting that there is an increase in CD8 T cells [96,97]. The presence of CD8 memory T cells in imiquimod-treated subjects is also suggested by increases in expression of SELL, NT5E (CD73), LGALS2 (galectin 2), and LAIR1 (leukocyte-associated immunoglobulin-like receptor 1), which was recently identified to be differentially expressed in mouse memory Tcells [98]. Figure 11a and Figure 11b illustrate the increase in expression of SELL and NT5E respectively. The expression levels of these genes returned to pretreatment levels 4 weeks post treatment. Taken together, the increased expression of Tcell receptor genes, T cell activation marker genes and genes important for T cell co stimulation suggests that treatment with imiquimod results in the infiltration of T cells into AK lesions. The increased expression of genes important for the development of T-cell memory (e.g. SELL, NT5E), as well as genes associated with cytotoxic T cells (e.g. granzymes and perforin) suggests that imiquimod treatment recruits both memory and effector T cells. These results are consistent with the observation of increased CD3, CD4 and CD8 positive cells after topical application of imiquimod to AK lesions [72], as well as infiltration of CD8 T cells in BCC [19,66] and in cutaneous squamous cell carcinoma treated with topical imiquimod [99]. The increased expression of genes important in the activation and co stimulation of cells of the adaptive immune system is consistent with the infiltration of cells important in the development of the adaptive immune system. These observations are also consistent with the reported adjuvant activity of imiquimod and its analog resiquimod [26], and the low recurrence rates observed in animal studies [100] and clinical studies with imiquimod [101]. These same observations suggest that imiquimod may also prevent recurrence of AK lesions as well. Figure 11Increase in expression of genes associated with lymphocyte function after treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A) SELL (CD62L) (B) NT5E (DC73). Experimental conditions and analysis are as described for Figure 4. In summary, the data suggest that the therapeutic effect of imiquimod in the treatment of AK involves the stimulation of both the innate and adaptive immune responses. The role of type 1 interferons in imiquimod's mechanism of action is underscored by the induction of large numbers of IFNα/β-inducible genes with tumor growth-inhibitory and immune stimulatory activity. Data indicate that the antilesional activity of imiquimod is a result of the induction of a strong immune cell-mediated cytolytic and apoptotic gene expression program that leads to destruction of AK lesions and sun-damaged cells. The development of immune memory indicated by this study, as well as the observation of low recurrence in clinical studies, makes imiquimod a unique therapy for AK as well as for other cutaneous neoplasms. BODY.ABBREVIATIONS: AK–actinic keratosis(ses), ANOVA–analysis of variance, BCC–basal cell carcinoma, DC–dendritic cells, IFN–interferon, IL–interleukin, IRF–interferon regulatory factor, NK–natural killer [cells], RT-PCR–Reverse Transcriptase Polymerase Chain Reaction, TLR–Toll like receptors BODY.COMPETING INTERESTS: Abel Torres, MD, JD, has acted as a lecturer for 3M Pharmaceutical and has participated in 3M Pharmaceutical funded research. He also does consulting for 3M Pharmaceuticals. RM, BJB, JJ, SR, and WB are employees of 3M Pharmaceuticals. JL and HB were employees of 3M Pharmaceuticals at the time of the study and manuscript writing. BODY.AUTHORS' CONTRIBUTIONS: AT, LS and MA conducted the clinical study. JL was the clinical coordinator. Gene expression analysis was performed at 3M Pharmaceuticals by WB, BB and JJ. All authors approved the manuscript. BODY.SUPPLEMENTARY MATERIAL: Additional file 1Imiquimod regulated genes. This file summarizes fold change in expression for 530 imiquimod regulated genes before and after treatment with imiquimod. Genes were selected on the basis of an ANOVA analysis comparing pre-treatment AK expression values to expression values during imiquimod treatment or 4-weeks after treatment with imiquimod, with P-values < 0.05Click here for file Additional file 2Comparison of differential expression as measured by Affymetrix gene array and real time RT-PCR. This file summarizes a comparison of differential gene expression as measured by Affymetrix gene chip analysis and real time RT-PCR using low density TaqMan arrays. Expression changes for several toll-like receptors and other selected genes before and after treatment with imiquimod are reported.Click here for file Additional file 3Imiquimod-induced genes with immune response gene ontology category and/or known to be IFNα/β-inducible. This file reports on expression changes before and after treatment with imiquimod, for selected genes with gene ontology category of immune response and (or) are know to be inducible by type 1 interferons.Click here for file

TITLE: ABSTRACT.OBJECTIVES:: This in situ study evaluated the effect of saliva, associated or not with fluoride, on enamel previously submitted to prophylaxis using sodium bicarbonate. ABSTRACT.METHODS:: The study was conducted on enamel blocks submitted to in vitro prophylaxis using sodium bicarbonate. The blocks were randomly divided into 2 groups (G1/G2) and mounted on intraoral appliances wore by 10 volunteers. G1 blocks were directly exposed to saliva in situ, while blocks in G2 were exposed to saliva with fluoride (rinsing with 0.2% NaF solution during the initial minute). Enamel alterations were evaluated using surface microhardness and profilometry. Enamel hardness data were analyzed by ANOVA and Tukey tests and surface wear was evaluated using paired t test (P<.05). ABSTRACT.RESULTS:: No significant differences were found between G1 and G2 for enamel hardness and wear. The wear after prophylaxis was not different from the wear after the in situ stage. Baseline mean values of enamel hardness, after prophylaxis and after the in situ stage were 340±16.6, 329±35.7 and 354±37.8 for G1 and 338±15.6, 312±46.3 and 340±21.8 for G2, respectively. ABSTRACT.CONCLUSIONS:: It was concluded that saliva alone exhibited a similar effect to saliva associated with fluoride; after 4h of in situ remineralization, there was no recovery in height of the enamel structure that had been lost due to the application of sodium bicarbonate. BODY.INTRODUCTION: Dental caries has been widely investigated for decades.1,2 Knowledge of the multifactorial etiology of caries concerning cariogenic diet, frequent ingestion of sucrose, and presence of dental biofilm on the dental surface of a susceptible host for a certain period of time encouraged investigations into preventive methods.2–4 Initially, classical studies demonstrated that dietary control with reduced ingestion of sucrose leads to a lower prevalence of dental caries.5,6 However, in reality, the establishment of this preventive measure is difficult, since it requires changes in dietary habits that consequently demand cultural changes and the need to make individuals realize the value of oral health, which rarely occurs. The use of fluorides has also been widely employed in the last decades.7–9 However, indiscriminate use of systemic fluoride can cause adverse effects, such as the occurrence of dental fluorosis.10 Therefore, other preventive measures are needed. Greater emphasis has been given to biofilm control as a probable preventive measure against dental caries.11–15 Mechanical dental plaque removal may be achieved by self-control measures (toothbrushing and flossing) or by professional prophylaxis. It should be highlighted that self-control measures are subject to the child's and his/her family's limitations in performing oral hygiene. Taking this into consideration, children have been submitted to preventive programs based on regular plaque control by professional prophylaxis, which aims to compensate for poor toothbrushing.11–16 When regularly performed, professional prophylaxis may significantly reduce the progression of dental caries.11–15 One method for performing this procedure is the use of sodium bicarbonate. Investigations comparing the effectiveness of prophylaxis using sodium bicarbonate with that performed using rubber cup and pumice reveal that sodium bicarbonate removes dental plaque more effectively, especially in pit and fissure regions.16,17 Tooth wear could be a possible adverse effect of the regular use of sodium bicarbonate. Thus, several studies have been conducted to quantify the amount of tooth structure removed in each session of professional prophylaxis using sodium bicarbonate.18–20 In general, authors agree that sodium bicarbonate has a slight effect on intact enamel.18–20 However, these studies evaluated the dental surface immediately after the procedure, while doubts still remain concerning the protective capacity of the oral environment due to the presence of saliva and/or fluoride. Oral fluids may offer some protection, even when considering the loss of tooth structure by abrasion caused by the mechanical action of sodium bicarbonate under water pressure. Saliva is rich in minerals and proteins, it is supersaturated with calcium and phosphate ions, and it lubricates the teeth.21 Thus, saliva acts against demineralization and may be able to recover the slight mineral loss of enamel caused by prophylaxis using sodium bicarbonate. Furthermore, fluoride can increase the rate of re-mineralization due to its mechanism of action.8,22 However, it is not known whether fluoride may influence the redeposition of minerals in case of abrasion of enamel surfaces. Considering such aspects, this in situ study aimed to evaluate the effect of saliva, whether associated or not with fluoride, on enamel that had previously been subjected to prophylaxis using sodium bicarbonate. BODY.MATERIAL AND METHODS.EXPERIMENTAL DESIGN: This in situ study involved a crossover, blind design performed in two phases of 4 hours,20 with a wash-out period of 7 days. The groups under study were: G1—treatment with sodium bicarbonate jet in vitro and saliva exposure for 4h in situ, and G2—treatment with sodium bicarbonate jet in vitro, 0.2% NaF rinse during the first minute and saliva exposure for 4h in situ. Each phase corresponded to one group: G1 or G2. Ten healthy adult volunteers living in the same fluoridated area (0.7 ppm) with a mean age of 28 years (range 23–35 years) and normal salivary flow rate took part in this study after signing an informed, written consent, approved by the IRB of Bauru Dental School, University of São Paulo. They wore acrylic palatal appliances, each containing two dental enamel blocks (Figure 1). BODY.MATERIAL AND METHODS.PREPARATION OF THE ENAMEL BLOCKS: Enamel blocks (4x4x3 mm) were prepared from bovine incisors sterilized by storage in 2% formaldehyde solution pH 7.0 for 30 days at room temperature.23 The enamel surface of the blocks was ground flat with water-cooled carborundum discs (320, 600, and 1200 grits of Al2O3 papers; Buehler, Lake Bluff, IL, USA), and polished with diamond spray (1μm; Buehler). BODY.MATERIAL AND METHODS.BASELINE SUPERFICIAL MICROHARDNESS ASSESSMENT: For surface enamel hardness determination, five indentations were done in different regions of the block (25 g, 5 s, HMV-2000; Shimadzu Corporation, Tokyo, Japan). The microhardness tester was calibrated before use and all measurements were conducted by the same person throughout the experiment. Forty blocks that had a mean hardness of approximately 339 KHN were selected. These blocks were randomly assigned to the two groups and the baseline hardness values of the blocks were similar for both groups. Each group contained 20 enamel blocks (duplicates) or 10 experimental units (n=10). These blocks were randomly assigned to the two groups. BODY.MATERIAL AND METHODS: To maintain reference surfaces, two layers of nail varnish were applied on half of the blocks' surfaces.18–20 On the other half, the sodium bicabornate jet was applied (Dabi Atlante Industrias Médico Odontológicas LTDA) at a distance of 5 mm from the block for 10 seconds, at a 90o angle, without interruption.18–20 After this procedure, the nail varnish was removed and the enamel hardness and profilometric analyses were conducted. Ten days prior to the beginning of the experiment, and also during the whole experimental period, the volunteers brushed their teeth with fluoridated dentifrice (Colgate® - 1450 ppm of fluoride). Custom-made acrylic palatal devices were made with 2 cavities in the surface of each appliance. One block was fixed with wax in each of the 2 cavities. In G1, the volunteers wore the appliance inside the mouth for 4h. In G2, the volunteers also wore the appliance for 4h, but during the first minute, with the appliance in place, they rinsed using a 0.2% NaF solution (Pharmácia Específica LTDA, Bauru, SP). BODY.MATERIAL AND METHODS.SURFACE PROFILOMETRY: Enamel wear was determined by profilometry (Hommel Tester T1000, VS, Schwenningen).1,20–22 The tracing parameters were established at Lt: 1.5 mm and Lc: 0.25 mm, and the profilometry accuracy is 0.4 μm. Four readings were performed on each block from the reference to the exposed surface. The average wear depth by volunteer of an experimental unit was computed using 8 readings, four each of two blocks. BODY.MATERIAL AND METHODS.SUPERFICIAL MICROHARDNESS ASSESSMENT: After the profilometric measurements were made, the blocks were once again subjected to microhardness tests in the same manner as described above. The nail varnish was reapplied on half of the blocks' surfaces. BODY.MATERIAL AND METHODS.PROFILOMETRY MEASUREMENTS AND SUPERFICIAL MICROHARDNESS ASSESSMENT: The nail varnish was removed and the microhardness and profilometry measurements were made as described above. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: The assumptions of equality of variances and normal distribution of errors were checked for the response variables tested. Since the assumptions were satisfied, ANOVA and Tukey's tests were carried out for statistical comparisons for superficial enamel hardness data. A t-test was used to compare G1 and G2, taking into account the differences of final and baseline surface profiles (wear). For all tests, the significance level was set at P≤.05. BODY.RESULTS: Concerning the variable surface hardness, statistical analysis revealed no statistically significant difference in the action of saliva compared to saliva associated with fluoride (Table 1). Professional prophylaxis applied on enamel blocks in vitro led to statistically significantly lower surface hardness compared to baseline hardness values and after the in situ stage (Table 1). With regard to wear, there was no significant difference between wear after prophylaxis compared to the in situ situation (Table 1). The wear of G1 was different from that found in G2; however, this difference was also observed between groups before the in situ stage (Table 1). Thus, the paired t test was applied to allow a more correct analysis (P<.05), considering the results of differences in wear (profile after study in the in situ stage minus profile after prophylaxis), so that the initial differences would not be considered. The results reveal that the wear of blocks submitted to saliva (−0.0016) was similar to the wear of blocks submitted to saliva associated with fluoride (−0.0074) (P=0.6196). BODY.DISCUSSION: Most investigations of the wear caused by professional methods for dental plaque removal have an in vitro design 18–20 and do not consider the protective properties of the oral environment, mainly related to the action of saliva. The in situ models suggested by Kolourides et al24 for investigations on dental caries present great advantages compared to laboratory studies. The in situ model may simulate intraoral events and simultaneously standardize the experimental conditions; at a short time period, they allow for the development of enamel alterations or recovery.25 Bovine incisors was used in this study because they have been widely used in dental research,26–28 especially because of their structural similarity to human tooth,29 homogeneous and reproducible surface,30 and lower biological variability,30 since they are not exposed to high concentrations of fluoride.24,25,28 The 4-hour period for duration of each step was based on the findings of Ribeiro et al.20 These authors demonstrated that, after one hour in an in vitro study, saliva can recover previously abraded bovine enamel. Moreover, this period of time is the maximum mean period during which the oral cavity is not subjected to other stimuli after professional prophylaxis, such as contact with food.20 In the present study, after professional prophylaxis with sodium bicarbonate in vitro, there was a reduction in surface enamel hardness. Since this exposed surface was less mineralized, saliva had a remineralizing action, with a significant increase in surface hardness, which was not different from baseline hardness. Similar results were observed by Ribeiro et al,20 who conducted an in vitro study to investigate the action of saliva on enamel previously submitted to professional prophylaxis. They observed an increase in surface hardness after one hour. Conversely, analysis of wear did not reveal any reparative action of saliva. After the in situ stage, during which the blocks were submitted to the action of saliva for 4 hours, there was no significant reduction in wear. On the other hand, during investigations on demineralized enamel by artificial caries solution, the authors20 observed reduction in wear, in agreement with Honório et al.18 These results may be explained by the fact that the enamel with white spot lesions is especially more prone to chemically receiving minerals (calcium and phosphate) from saliva, since the enamel crystals are altered and consequently have lower mineral content, which are still present in the tooth structure, thus allowing recovery and a slight gain in height.36 Conversely, sound enamel submitted to mechanical wear completely loses the surface crystals, precluding recovery and structure gain in height, since there is no matrix for deposition of ions from the saliva. In the present study, care was taken to assure the homogeneity of experimental groups by random division according to initial surface enamel hardness. Notwithstanding, G1 exhibited statistically higher wear than G2 after in vitro professional prophylaxis, which was performed equally for both groups, even though hardness values after prophylaxis were not different between the groups. There is no explanation for such an outcome; however, to overcome this problem, the difference in wear should be considered to allow actual comparison of influence of the in situ stage between G1 and G2. The action of fluoride on a structure previously submitted to professional prophylaxis was investigated because no studies have addressed its effect on exclusively abraded surfaces, even though the remineralizing effect of this element on surfaces previously demineralized by dental caries is well established. The 0.2% NaF solution was selected due to the demonstrated clinical action for caries remineralization and easy utilization.32,33 A solution was employed because utilization of dentifrice might promote further wear, since its utilization is related to toothbrushing (abrasion). The results revealed that the action of saliva (G1) was not different from saliva associated with 0.2% sodium fluoride (G2) for both surface enamel hardness and wear variables. Both promoted an increase in surface enamel hardness (G1, 329 and 354 KHN; G2, 312 and 340 after prophylaxis and after the in situ stage, respectively), yet none could reestablish the enamel in height (G1 −0.0016 and G2 −0.0074 of mean difference). Initially, it was assumed that utilization of fluoridated dentifrice before the in situ stage might promote a residual effect of fluoride, thus masking the effect of fluoride mouth rinse. This phenomenon hardly occurred, since care was taken to assure a one-hour interval between toothbrushing and onset of the in situ stage, to avoid the action of fluoride from dentifrice in the study.34 However, Bruun, Givskov, and Thylstrup35 found residual fluoride in saliva for more than one hour after toothbrushing with fluoridated dentifrice, compared to saliva of the placebo dentifrice. Thus, future studies should ideally evaluate the fluoride in saliva before onset of the in situ stage, increase the washout period of dentifrice, or employ non-fluoridated dentifrice. The type of fluoride may also influence the outcome. High concentration fluorides, such as fluoridated varnishes, have demonstrated greater effectiveness in remineralization.36 In addition, some authors37,38 observed that concentrations above 500 ppm of fluoride did not produce significant increase in remineralization. Similar studies should be conducted to compare several concentrations of fluoride to promote r a better understanding of the action of fluoride on abrasion. Moreover, its action should be evaluated not only on "intact" enamel, but also on previously demineralized enamel. Thus, considering the importance of mechanical biofilm removal as a caries preventive measure, regardless of the effectiveness of the in situ action of saliva associated or not with fluoride to recover the enamel structure lost during prophylaxis with sodium bicarbonate, the wear produced by this procedure was very slight. Therefore, professionals may safely perform prophylaxis without concern regarding the adverse effects of significant loss of intact tooth structure. In the case of high caries risk patients who present dental plaque accumulation, high concentration topical fluoride products may be professionally applied after prophylaxis, enhancing remineralization. BODY.CONCLUSIONS: Based on the results, it was concluded that saliva exhibited a similar effect as saliva associated with fluoride; after 4h of in situ remineralization, there was no recovery in height of the tooth structure lost due to application of sodium bicarbonate on sound enamel.

TITLE: The Effect of a Short-Term and Long-Term Whole-Body Vibration in Healthy Men upon the Postural Stability ABSTRACT: The study aimed to establish the short-term and long-term effects of whole-body vibration on postural stability. The sample consisted of 28 male subjects randomly allocated to four comparative groups, three of which exercised on a vibration platform with parameters set individually for the groups. The stabilographic signal was recorded before the test commenced, after a single session of whole-body vibration, immediately after the last set of exercises of the 4-week whole-body vibration training, and one week after the training ended. The subjects were exposed to vibrations 3 times a week for 4 weeks. Long-term vibration training significantly shortened the rambling and trembling paths in the frontal plane. The path lengths were significantly reduced in the frontal plane one week after the training end date. Most changes in the values of the center of pressure (COP) path lengths in the sagittal and frontal plane were statistically insignificant. We concluded that long-term vibration training improves the postural stability of young healthy individuals in the frontal plane. BODY.INTRODUCTION: The interest in using mechanical vibrations in physiotherapy and sports has clearly grown in the last several years. Although many authors state that vibration training has a positive effect on the human body [1], [2], some published studies have not demonstrated a positive effect [3], [4]. According to Gyulai et al. [5], the reaction to whole-body vibration is training and individual dependent. Whole-body vibration training is an interesting area of research because of its effect on postural stability, and postural stability certainly plays an important role in daily life. Stabilography is a typical procedure that assesses postural control. Postural stability during static conditions is estimated by analyzing center of pressure (COP) movements in the frontal and sagittal planes, which are recorded with a subject standing in a free standing position on the posturographic platform [6]. The center of pressure measurements are usually reported in postural studies. Zatsiorsky and Duarte [7], [8] have reported a method of decomposing stabilograms. The authors have described the movements of the reference point against which postural stability is instantaneously restored (rambling) and COP oscillations around the reference point trajectory (trembling). The computation method that Zatsiorsky and Duarte [7] have proposed increases the efficiency of investigating the postural control mechanisms and addresses more explicitly the dynamic nature of COP. The knowledge of vibration effects on postural stability is still unsatisfactory, and available studies present conflicting results. Researchers apply different vibration parameters [frequency and amplitude] and vibration training procedures. For instance, while Polonova and Hlavacka [9] utilized 40–100 Hz vibration, Gomez et al. [10] selected 85 Hz. Existing studies on postural control systems disturbed by vibration use one of two devices: a vibration platform indirectly producing whole-body vibration [2], [11] or oscillating heads applied directly to the belly of a muscle or a tendon [9]. According to previous research, the effects of vibration are determined by the length of training [11], [12]. A short-term application of vibration to single groups of muscles increases sway [12]. The range and direction of the COP sway depend on the frequency of vibration applied and where the stimulated muscles are located [9]. In the available studies, multiple application of whole-body vibration has either improved the ability to maintain postural balance [13], which indicates improved neuromuscular control [14], or has not changed it significantly [2], [11], [15]. Because studies dealing with the effect of mechanical vibration on human postural stability have yielded diverse results and vibration training methodology has not been standardized, there is a need to conduct more comprehensive research in this field. None of the available publications have used rambling-trembling analysis to study vibration-induced changes in postural stability. According to Zatsiorsky and Duarte [7], [8] supraspinal processes that are involved in controlling the displacement of the instant equilibrium point may be related to the rambling trajectory, whereas trembling is a reflection of the reflexes and mechanical properties of the muscles and joints. Mechanical oscillations are known to affect kinesthetic perception and produce reflex muscle contractions known as a tonic vibration reflex. Additionally, vibration masks short-latency phasic spinal reflexes by increasing presynaptic inhibition [16]. This allows expecting effects on both rambling and trembling after vibration training. Therefore, the main purpose of the current study was to establish the effect of whole-body vibration training utilizing different vibration parameters on the postural stability of young males. To analyze the stabilogram, the rambling-trembling approach was applied. BODY.MATERIALS AND METHODS.SUBJECTS: Twenty eight male subjects (22.4±2.4 years) participated voluntarily in the experiment. The mean body mass was 76.3±7.3 kg and the mean height was 179.63±5.1 cm. Young healthy adults that were not professional athletes were recruited. The subjects were randomly allocated to one of four groups; each group consisted of seven subjects. Subjects in groups I-III participated in 4-week whole-body vibration training on a vibration platform, and the frequency and amplitude of vibration were set individually for each group, i.e. 2 mm/20 Hz for group I, 2 mm/40 Hz for group II, and 2 mm/60 Hz for group III. Vibrations below 20 Hz are not used in vibration training studies because it induces mechanical resonance and has an adverse effect on internal organs [17]. A control group (group IV) also participated. These subjects performed exercises similar to those in the other groups but without the concurrent application of vibration. All participants gave their written informed consent to participate in this study prior to the experiment, which was approved by an ethics committee of the Institutional Review Board. BODY.MATERIALS AND METHODS.VIBRATION TRAINING: The goal of this research was to investigate the impact of whole-body vibration on the stability of the human body. The influence of a single session of whole-body vibration and 4-week whole-body vibration training were assessed. A single vibration session in the study was a series of 5 static exercises on a vibration platform for 1 min, followed by a 1 min break. The study design is presented in Figure 1. 10.1371/journal.pone.0088295.g001Figure 1Flow diagram for study design. Whole-body vibration training was carried out on a vibration platform (Fitvibe 600, Gymna Uniphy N.V.). During the experiment, the vibration groups performed 5 one-minute exercises separated by one-minute breaks, 3 times a week (Monday, Wednesday, and Friday). The exercise and rest times were adapted from the procedures used by other researchers [17]. The test participant was in a static position during the exercises. Briefly, each subject was asked to stand on the platform, loading his feet uniformly, with the knee and femoral joints bent at 90 degrees and the upper extremities stretched horizontally forwards (fig. 2). The range of flexion of the hip and knee joints was measured with a goniometer. Krol et al. [18] reported significantly increased bioelectrical activity of the lower extremity muscles of subjects standing with the knee joints bent at 90°. This position is also safer because the knee flexion reduces the amount of vibration that reaches the head. 10.1371/journal.pone.0088295.g002Figure 2Whole-body vibration. BODY.MATERIALS AND METHODS.STABILOGRAPHIC MEASUREMENT: For the stabilographic measurements, all subjects were asked to maintain a stable neutral comfortable position with their feet shoulders' width apart, arms lying at the side and eyes closed. Five 30-second trials were recorded on four different occasions: before training started, after a single session of whole-body vibration, after the fourth week of whole-body vibration training, and one week after training ended. Hence, human postural stability was evaluated on 4 different occasions. The postural stability of subjects in the control group was measured in the same manner. Forces were recorded with a piezoelectric platform (Kistler 9281C) with a charge amplifier (type 9865B) and a computer. Forces were recorded at a frequency of 100 Hz (25 Hz low-pass filter) using the BioWareTM software (type 2812A1-20). The sampling frequency was 100 Hz (25 Hz low-pass filter). The sampling and filtering frequencies were adopted from the procedures of Zatsiorsky and Duarte [7]. All three components of the ground reaction force (Fz - vertical, Fy - anterior-posterior and Fx - lateral components), as well as the three components of moment of force (Mz, My, Mx) about the center of the platform were registered. The COP trajectory was automatically calculated using the Bioware program, and a formula from the guidelines of the manufacturer's platform and its technical parameters was used to determine the position of COP. The stabilographic signal was obtained using a new method of stabilogram decomposition, proposed by Zatsiorsky and Duarte [7], into two components: rambling (the motion of an instant equilibrium point about which the body's equilibrium is maintained) and trembling (the oscillation of COP around the reference point trajectory). Using the BioWareTM program, the values of the path length of COP and the path length of rambling and trembling were obtained. The results of Słomka et al. [19] indicate the high reliability of the measures of rambling – trembling. The sequence of operations used for stabilogram decomposition is described below [20] (fig. 3). When the horizontal force (Fhor) is zero, the body is in an equilibrium state. These instances were identified in the COP displacement data. The COP positions when Fhor is 0 were located, and the signal between each point was interpolated using a cubic spline function to obtain an estimate of the rambling trajectory. The COP trajectory is compared with the interpolated rambling trajectory. To obtain the trembling trajectory, the deviation of the COP from the rambling trajectory was calculated. 10.1371/journal.pone.0088295.g003Figure 3Representative center of pressure, rambling and trembling trajectories of a single subject along the anterior-posterior (AP) axis. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The Shapiro-Wilk test was used to check the data for normal distribution, while variance homogeneity was investigated using Levene's test. Because some parameters failed to meet the assumption regarding the normal distribution of variables and variance homogeneity, the Wilcoxon matched pairs test was employed to assess the significance of the differences between the means of particular variables in the groups. The influence of the vibration frequencies and amplitudes on the means of particular variables was verified with the Mann Whitney U test. The intergroup statistical analysis used the relative values of the difference variables, which were calculated as the ratio of the difference between the initial and final value of a variable to its initial value: The level of statistical significance for all analyses was considered p<0.05. BODY.RESULTS: Both a single application of vibration and the 4-week whole-body vibration training had an effect on the COP path length values in the sagittal and frontal planes, but most changes were statistically insignificant (p>0.05) (fig. 4, 5). Group I was the only group where the COP path length in the AP plane was significantly increased after the last session of the 4-week whole-body vibration training (p = 0.046) (fig. 4). In contrast, the path length of COP in group III was significantly decreased after the last session of the 4-week whole-body vibration training (p = 0.027) (fig. 4). 10.1371/journal.pone.0088295.g004Figure 4Length of COP in the AP plane (mean ± standard deviation) (mm) after training. 10.1371/journal.pone.0088295.g005Figure 5Length of COP in the ML plane (mean ± standard deviation) (mm) after training. Figures 6 and 7 show the lengths of the rambling and trembling paths in the sagittal plane, and figures 8 and 9 give the same information for the frontal plane. 10.1371/journal.pone.0088295.g006Figure 6Length of rambling in the AP plane (mean ± standard deviation) (mm) after training. 10.1371/journal.pone.0088295.g007Figure 7Length of trembling in the AP plane (mean ± standard deviation) (mm) after training. 10.1371/journal.pone.0088295.g008Figure 8Length of rambling in the ML plane (mean ± standard deviation) (mm) after training. 10.1371/journal.pone.0088295.g009Figure 9Length of trembling in the ML plane (mean ± standard deviation) (mm) after training. A single session of whole-body vibration training did not significantly affect the path lengths of rambling and trembling in the sagittal plane (fig. 6, 7) (p>0.05) or in the frontal plane (fig. 8, 9) (p>0.05). In groups I-III, long-term vibration training significantly shortened the rambling path in the sagittal plane (fig. 6) (p<0.05). In group III, the significantly shorter trembling path in the sagittal plane (p<0.05) after 4 weeks of vibration training is notable (fig. 7). Interestingly, statistical analysis showed significantly shorter rambling and trembling paths in the frontal plane (p<0.05) (fig. 8, 9) for all test groups participating in 4-week vibration training (excluding the control group). This phenomenon turned out to be relatively long lived; statistical analysis showed that the rambling and trembling paths were significantly shorter in all test groups (excluding the control group) one week after the training end date (p<0.05) (fig. 8, 9). To establish the impacts of different vibration frequency and amplitude parameters, test groups I-III were compared with each other. The Mann Whitney U test did not show a significant influence of vibration frequency and amplitude on the path length of the COP and rambling and trembling paths (p>0.05) between the pre-test measurements and those made directly after the first session of whole-body vibration, immediately after the last set of exercises of the 4-week whole-body vibration training and one week after. BODY.DISCUSSION: The complex system of postural control includes a variety of elements: motor synergies, sensory input, anticipatory postural adjustments, and adaptive control [21]. Appropriate postural adjustments, i.e. kinematic and kinetic adjustments, are aimed at keeping the center of gravity (COG) over the base of support [22]. According to Collins and de Luca [23], during quiet standing, the postural control system utilizes open-loop and closed-loop control schemes over short-term and long-term intervals, respectively. All forms of postural activity during normal standing in healthy young males are cyclical in nature and include high and low frequency components [6]. The size of the base of support is an important factor influencing the postural stability [24]. An important aspect of postural control that enables people to move safely in the immediate environment is the processing of information received from four sensory entrances – the labyrinth, the organ of sight, the proprioceptors and the tactile receptors [24]. If sensory receptors receive incorrect information, then postural stability may be affected [25]. Based on the published results, several different types of receptors may be sensitive to mechanical oscillations, for example, extroceptive receptors of the foot (Merkel, Meissner, and Ruffini receptors) [26] and proprioceptors [27]. Primary endings are the most sensitive to vibratory stimulation [28]. Brumagne et al. [27] have demonstrated that local vibration of paraspinal muscles impairs proprioception in healthy humans. In our experiment, proprioceptors were disturbed by mechanical vibrations of different frequencies (20, 40, and 60 Hz) and the same amplitude of 2 mm. Many studies exploring vibration effects on postural stability have been conducted [2], [11], [12], [13], [15], but their results are diverse and inconsistent. There is still a lack of standardization regarding the testing and training protocols for whole-body vibration, which makes it difficult to compare the various research studies. There are differences in the application methods for vibration. Direct vibration is normally locally applied to a muscle or tendon with oscillating heads at high vibration frequency (100–150 Hz) and small amplitude (1–2 mm) for a short period of time (2–15 s). Indirect vibration typically uses vibration platforms producing whole-body mechanical oscillations with lower vibration frequency (25–45 Hz), higher amplitude (2–10 mm) and longer duration time of either continuous (3–5 min) or intermittent exposure (30–60 s) [29]. In the available studies, multiple application of whole-body vibration training has either improved postural stability [13] or has not resulted in significant changes [2], [11], [15]. The improved postural stability in patients might be due to increased muscle strength, improved synchronization of the firing of motor units and improved co-contraction of synergist muscles after whole-body vibration training [17]. Center of pressure (COP) measurements are commonly used output measures of the postural control system, as they are indicative of postural stability. A short-term application of a direct vibration of 60 Hz and an amplitude of 1 mm to single groups of muscles increases sway [12]. Indirect vibration, which is applied to the whole body with frequencies of 20, 40, and 60 Hz and amplitudes of 2 and 4 mm, does not significantly change the sagittal and frontal COP sways in young males [15]. In our experiment, the path length of COP changed in all test groups, and most changes were statistically insignificant (p>0.05). Additionally, the first set of exercises involving the vibration platform changed the lengths of the rambling and trembling paths in the sagittal and frontal planes in test groups I-III, but the differences were not statistically significant (p>0.05). This lack of effect likely results from compensatory postural adjustments. According to Winter [30], there is a certain degree of redundancy that can be applied when one or more of the sensory systems fails or is temporarily lost. Healthy humans use compensatory information from the complementary sensory systems (visual, vestibular and proprioceptive) for postural control [31]. The re-ranking of receptor information as a result of disturbed postural stability has been discussed by many authors [32], [33]. Too much sensory information or malfunctioning of any of the receptors causes the nervous system to reduce the receptor's role in postural control and switches to signals from receptors offering more reliable and noise-free information. The hierarchy of receptors depends, inter alia, on the motor task requirements [33] and receptor efficiency [32]. Dolny and Reyes [34] are of the opinion that exposing young, healthy individuals to whole-body vibration may not be sufficient to induce adaptive changes in their neuro-muscular system. A single session of whole-body vibration in our experiment was most likely too weak of a disturbing stimulus to modify postural sway. According to its authors, a possible explanation of why short-term vibration did not significantly differentiate the path length of COP and the lengths of the rambling and trembling paths between the tested groups, should be found for young subjects going through a period of ontogenetic development, with its relative balance between excitation and inhibition processes. In our experiment, the vibration application time was 1 minute, similar to the studies of other authors [18]. According to Cochrane [35], vibration applied continuously for longer than 1 minute causes muscle fatigue that is known to increase postural instability in the ML and AP directions [36]. As shown by the results of this experiment, five 1-minute static exercises did not induce muscle fatigue in any experimental group. Zatsiorsky and Duarte [7] have proved that the horizontal force and the trembling component are highly and negatively correlated with each other. Each time the COP deviates from the interpolated IEP path, a horizontal force tending to reduce the deviation is triggered [7]. In the experiment, the moderate amount of deviation from the rambling path suggests that the source of corrective force was the apparent intrinsic stiffness of the lower extremity muscles of the subjects [37]. According to Winter et al. [24], reduced postural sway can be explained in terms of the increased stiffness of the ankle joints primarily caused by the tension of the postural muscles that stabilize them. This corrective force is released instantaneously [7]. Tahayor et al. [38] suggest that during peripheral perturbation, different strategies are involved in posture control. These changes in the dynamics are related to adaptive strategies that aim to control postural sway [38]. Repetitive mechanical stimulation might lead to a rearrangement of postural control strategies [39]. Because the cyclical exposure to whole-body vibration frequently causes adaptive changes in the COP signal [40], we acted on the hypothesis that 4-week whole-body vibration training should induce permanent changes in the amount of postural sway. The statistical analysis results proved that this hypothesis was true. In all test groups participating in the whole-body vibration training, the rambling and trembling paths were significantly decreased in the frontal plane after 4 weeks of exposure vis-à-vis the initial measurements (p<0.05). Interestingly, the difference continued for a long period. Statistical analysis showed that the paths were significantly shorter even one week after the training program ended (p<0.05). The changes deserve special attention because the statistical significance obtained for the small groups (n = 7) clearly shows that the result is valid. Shorter path lengths in our experiment may signify that regular, long-term vibration training caused permanent adaptive changes in the postural stability of the subjects. According to many authors, vibration training induces adaptive changes in muscle tissue as well [2], [11]. The long-term vibration training used in our experiment most likely affected the viscoelastic stiffness properties of the muscles [41]. Wierzbicka et al. [41] demonstrated long-lasting dynamical changes in postural stability after local vibrations. According to Duclos et al. [42] these long-lasting alterations might originate from sustained artificial activation of the somesthetic mechanoreceptors, which results in modification of the central integration of this sensory information. The activation of sensorimotor cortical networks by proprioceptive signals, induced by mechanical oscillation, remains after the vibratory stimulation is completed [42]. In a human standing with natural posture, the amount of sagittal sway is greater than the amount of frontal sway [24]. Thomas and Whitney [6] found that in healthy, young adults, the average amplitude of anterior-posterior movement of COP is 0.60 cm. Greater sway in the sagittal plane is shown by the results obtained for all test groups in our experiment (fig. 4, 5). It is assumed that in the sagittal plane, the human body behaves similar to a one-piece, inverted pendulum with the rotation axis going through the ankle joints. In discussing upright posture control, Kuczyński [37] refers to stabilization ensured by springs with absorbers to describe the viscoelastic properties of the muscles. Frontal stability is determined by the bipedal stance and substantially restricted the mobility of the joints of the lower extremities in this plane [24]. In the quiet standing position, with the feet side by side, the ankle strategy is primarily applied in the sagittal plane, whereas a separate hip load/unload strategy is the dominant strategy in the frontal plane [30]. Because the COP measurements were not significantly sensitive to vibration training in this experiment, the rambling-trembling technique may be more suitable, as it allows discrimination between supraspinal and spinal processes. Additionally, rambling-trembling parameters have a higher reliability coefficient than COP parameters [19]. This study aimed to establish the short-term and long-term effects of whole-body vibration training on postural stability. As an experimental study, it has known limitations. A small sample size in each group may not be sufficient to establish full training effects. Healthy, young males most likely have very good balance at baseline. Therefore, it seems worthwhile to conduct multifaceted research among older people and patients with postural balance problems. Comprehensive and reliable studies with an innovative use of vibration platform [43] are still required. BODY.DISCUSSION.CONCLUSIONS: While aware that further studies exploring the influence of various types of whole-body vibration training on postural stability are necessary, current research conclusions suggest the following: Long-term whole-body vibration training significantly shortens the rambling and trembling paths in the frontal plane.Rambling and trembling paths are significantly decreased one week after whole-body vibration training compared with the initial measurements.Rambling – trembling decomposition is a more sensitive method for the analysis of quiet standing than classical stabilometric parameters of COP.

TITLE: Agreement between self-reported and measured weight and height collected in general practice patients: a prospective study ABSTRACT.BACKGROUND: Self-reported weight and height is frequently used to quantify overweight and obesity. It is however, associated with limitations such as bias and poor agreement, which may be a result of social desirability or difficulties with recall. Methods to reduce these biases would improve the accuracy of assessment of overweight and obesity using patient self-report. The level of agreement between self-reported and measured weight and height has not been widely examined in general practice patients. ABSTRACT.METHODS: Consenting patients, presenting for care within four hour sessions, were randomly allocated to the informed or uninformed group. Participants were notified either a) prior to (informed group), or b) after (uninformed group) reporting their weight and height using a touchscreen computer questionnaire, that they would be measured. The differences in accuracy of self-report between the groups were examined by comparing mean differences, intraclass correlations (ICCs), Bland Altman plot with limits of agreement (LOAs) and Cohen's kappa. Overall agreement was assessed using similar statistical methods. ABSTRACT.RESULTS: Of consenting participants, 32% were aged between 18–39 years, 42% between 40–64 years and 25% were 65 years and above. The informed group (n = 172) did not report their weight and height more accurately than the uninformed group (n = 160). Mean differences between self-reported and measured weight (p = 0.4004), height (p = 0.5342) and body mass index (BMI) (p = 0.4409) were not statistically different between the informed and uninformed group. Overall, there were small mean differences (−1.2 kg for weight, 0.8 for height and −0.6 kg/m2 for BMI) and high ICCs (>0.9) between self-reported and measured values. A substantially high kappa (0.70) was obtained when using self-reported weight and height relative to measured values to quantify the proportion underweight, normal weight, overweight or obese. While the average bias of self-reported weight and height as estimates of the measured quantities is small, the LOAs indicate that substantial discrepancies occur at the individual level. ABSTRACT.CONCLUSIONS: Informing patients that their weight and height would be measured did not improve accuracy of reporting. The use of self-reported weight and height for surveillance studies in this setting appears acceptable; however this measure needs to be interpreted with care when used for individual patients. BODY.BACKGROUND: Overweight and obesity affects a large proportion of the population in the developed world [1]. As the access point for health care systems in countries including Australia, Canada and United Kingdom, general practice is a valuable setting to target the reduction of overweight or obesity. GPs have access to a large proportion of the population, with 81% of Australians aged 15 years and above reporting having consulted their GP at least once a year [2]. Both GPs [3] and patients [4,5] perceive weight management to be part of a physicians' role and those who are advised to lose weight by a health care provider are more likely to attempt to lose weight [6]. Self-reported weight and height is commonly used to assess overweight and obesity as it enables the body mass index (BMI) to be calculated. In the general practice setting, self-reported weight and height is often utilised in large-scale monitoring studies, where it may not be feasible to carry out weight and height measurements. For example, the largest ongoing study with general practice patients in Australia (the Bettering the Evaluation and Care of Health (BEACH)) uses self-reported weight and height to provide surveillance data on prevalence of overweight or obesity [7]. While self-report is a relatively cost-effective, practical and less invasive way of obtaining weight and height, this method of assessment is subject to a number of limitations such as bias or poor agreement, which may be a result of social desirability or difficulties with recall [8]. Previous population studies have reported that using self-reported weight and height frequently leads to an underestimation of overweight and obesity when compared to measured values [9,10]. Most studies examining the accuracy of self-report have however been conducted in the general population [9,11-13]. In order to utilise self-report for monitoring of overweight or obesity in this setting, the accuracy of self-reported weight and height in general practice patients' needs to be evaluated. Simple strategies to improve self-reported weight and height could potentially be useful in helping improve surveillance of excess weight in general practice. One strategy that has been used to improve the reporting of socially undesirable behaviours is the bogus pipeline method [14]. Using this method, respondents are given the impression that the accuracy of their responses will be independently checked. It is underpinned by the assumption that people are more likely to tell the truth when they think that their responses will be verified [15]. Black et al. tested the effectiveness of a variation of this method in improving accuracy of self-reported weight and height in volunteers in a shopping mall [16]. Participants in the intervention group were informed that their weight and height would be measured and then asked to report their weight and height; whilst those in the control group reported their weight and height before being told that they would be measured [16]. Participant in the intervention group reported their weight and height significantly more accurately than those in the control group. Despite its' potential to improve accuracy of self-reported weight and height, no other study examining this intervention exists, to our knowledge. This study therefore aimed to test whether advising general practice patients that their height and weight would be measured was effective in improving accuracy of self-report. It also aims to provide an index of reliability and agreement for self-reported weight and height in general practice patients, collected using a touchscreen computer, using mean differences, intraclass correlations (ICC) and Bland Altman plots with 95% limit of agreements (LOAs). The impact of self-report on categorization of underweight, normal weight, overweight and obesity was also assessed using Cohen's kappa. An additional aim was to determine whether mean difference in reporting of self-reported and measured weight, height and BMI varied by age category. BODY.METHODS: This study was conducted as part of a larger study testing the acceptability of using a touchscreen computer questionnaire in twelve general practices in Australia [2]. A subsample of patients from three practices was invited to participate in the current study. Consecutive patients aged 18 years and above, presenting for an appointment to their GP and able to provide informed consent were eligible to participate. Patients were not excluded based on the presence of other health conditions. Research staff recorded the sex of all invited patients in order to assess for consent bias. Participants were randomised to the informed or uninformed group and completed a touchscreen computer questionnaire. Participants' weight and height measurements were obtained after completion of the questionnaire. BODY.METHODS.EXPERIMENTAL GROUPS: General practice sessions (4 hours) were centrally randomised by the researcher to the informed or uninformed group using a random number table. Participants recruited within the one session were all allocated to the same group. Neither practice staff nor patients were aware of group allocation. BODY.METHODS.EXPERIMENTAL GROUPS.INFORMED GROUP: Participants' consent to have their weight and height measured was sought prior to commencement of the questionnaire. After consenting to have their measurements taken, participants provided their self-reported weight and height using the touchscreen questionnaire. BODY.METHODS.EXPERIMENTAL GROUPS.UNINFORMED GROUP: Participants provided their self-reported height and weight as part of completion of the questionnaire. The research assistant asked for consent to obtain weight and height measurements after participants provided their self-reported weight and height. BODY.METHODS.VARIABLES.SELF-REPORT: Participants were asked to provide demographic information including gender and whether they had a government concession health card. Patients were asked to select their age from the following categories: 1 = 18-24; 2 = 25-29; 3 = 30-34; 4 = 35-39; 5 = 40-44; 6 = 45-49; 7 = 50-54; 8 = 55-59; 9 = 60-64; 10 = 65-69; 11 = 70 and above. Participants also reported weight in either kilograms (kg) or stones/pounds and height in centimetres (cm) or feet/inches. All weight responses were converted to kg and height response converted to cm. BODY.METHODS.VARIABLES.MEASURED: Participants' weight was obtained using a digital body fat and muscle weighing scale and height measured with participants head in the Frankfort plane using a mounted stadiometer. Participants were asked to remove their shoes, any heavy outer garments and personal belongings prior to measurement. Weight was measured to the nearest 0.1 kg and height to the nearest 0.1 cm. A trained anthropometrist took patients' weight and height measurements twice. A third measurement was taken if there was more than a 10% variation between the first and second measurement. BODY.METHODS.ETHICAL APPROVAL: Ethical approval was provided by the University of Newcastle Human Research Ethics Committee (H2009-0341) and ratified by the University of New South Wales HREC (HREC 09393/ UN H-2009-0341) and Monash University HREC (2009001860). BODY.METHODS.DATA ANALYSIS: STATA SE version 11.0 (StataCorp, College Station, Tex, USA) was used to perform all statistical analyses. Self-reported values of height larger than 240 cm and smaller than 120 cm and values of weight larger than 250 kg and less than 30 kg were coded as missing as these values were perceived to be errors in self-report. BMI was calculated from both self-reported and measured data using weight in kg divided by metres squared. Consent rates for physical measures were compared between the informed and uninformed groups. Differences between self-reported and measured values were obtained for weight, height and BMI. Mean differences, ICCs and corresponding 95% CIs for height, weight and BMI were tabulated separately for the informed and uninformed groups and compared between groups using student's t-test for mean differences and by comparing 95% CIs for ICCs [17,18]. Bland Altman plots with 95% LOA for height, weight and BMI were generated for both groups. The Bland Altman test is a statistically robust method of assessing reliability and agreement [19]. Additionally, Cohen's kappa statistic and 95% CI for classification of underweight (BMI <18.5 kg/m2), normal weight (BMI ≥ 18.5 kg/m2 and <25 kg/m2), overweight (BMI ≥25 kg/m2and <30 kg/m2) or obesity (BMI ≥ 30 kg/m2) was generated and compared between groups using 95% CIs. The overall level of agreement between self-reported and measured weight, height and BMI was also assessed. Mean differences between self-reported and measured values and corresponding standard deviations for males and females were reported. An ICC for the overall sample was calculated to provide an estimate of reliability. Cohen's kappa was calculated to provide the level of agreement between self-reported and measured classification of BMI categories. The degree of agreement between patient measured and self-reported overweight and obesity was assessed as follows: κ < 0 is none/poor; 0 ≤ κ ≤ 0.20 is slight; 0.21 ≤ κ ≤ 0.40 is fair; 0.41 ≤ κ ≤ 0.60 is moderate; 0.61 ≤ κ ≤ 0.80 is substantial; and 0.81 ≤ κ ≤ 1.0 is almost perfect [20]. Mean differences in self-reported weight and height were reported by age group. An ANOVA test was carried out to compare the mean difference in reporting by age (collapsed as 18–24, 25–44, 45–64 and ≥65 years). BODY.METHODS.SAMPLE SIZE CALCULATION: An initial sample size calculation was calculated to detect a difference of 0.5 kg/m2 in mean BMI between the two groups, with 80% power and 95% significance level, assuming a standard deviation of 1.5. To achieve this, a minimum of 142 participants needed to be recruited into each group (284 patients overall). A sample of this size would allow detection of a difference +/− 0.02 in mean ICCs between groups with 80% power, at 5% significance, assuming a standard deviation of 0.5. For overall agreement, this number of patients would allow estimation of kappa with 95% confidence within +/− 0.1, for a kappa of 0.4 or higher [21]. This sample size would also allow us to detect an ICC of 0.7 or more as being significantly greater than 0.6 [22]. A sample size of approximately 300 (75 per age group) would have at least 80% power, with 5% significance, to detect a difference in the variation between self-reported and measured weight, height and BMI of 0.6 standard deviations. BODY.RESULTS: Overall, 86% of patients consented to completing the questionnaire for the larger study. 355 patients were asked if they were willing to have their weight and height measured and 93% (n = 332) consented. No significant differences in proportion of males and females who consented to and did not consent to being measured were identified (χ2: 1.1304, df = 1; p = 0.288). There was no significant difference in the proportion who consented to being measured between the informed (93%) and uninformed (92%) group (χ2: 0.9213, df = 1; p = 0.337). Eleven participants reported having a height of more than 240 cm or less than 120 cm and/or having a weight of more than 250 kg or less than 30 kg. One participant in the uninformed group was excluded as the height difference tabulated was beyond reasonable error rate. More than half (56%) of consenting participants were female, 25% were aged 65 years and above and 42% had a government subsidised health care card. 14.2% of the Australian population are aged 65 years above [23]. While not directly comparable due to the inclusion of those aged below 18 in the latter population statistics, the current sample had a larger proportion of older people (≥65 years) than would be expected in the general population. This larger proportion of older participants is consistent with that identified in other general practice datasets [7]. Demographic characteristics are presented for the informed (n = 172) and uninformed (n = 160) groups (see Table 1). Table 1 Mean difference and intraclass correlation for weight, height and BMI for informed and uninformed group Group Informed (n = 172)* Uninformed ( n = 160)** Sex n (%)     Male 78 (45) 69 (43) Female 94 (55) 91 (57) Age n (%)     18-39 60 (35) 48 (30) 40-64 67 (39) 74 (46) 65 + 45 (26) 38 (24) Number with concession health care (%) 71 (42) 66 (42) Mean difference a (sd) [95% CI]     Weight −1.0 (4.5) [−9.8, 7.8] −1.4 (3.5) [−8.1,5.4] Height 0.7 (3.9) [−6.9, 8.2] 1.0 (5.0) [−8.7, 10] BMI −0.6 (2.2) [−4.8, 3.7] −0.7 (1.9) [−4.4,3.0] ICC [95% CI]     Weight 0.97 [0.96, 0.98] 0.98 [0.97, 0.99] Height 0.92 [0.90, 0.94] 0.90 [0.87, 0.93] BMI 0.93 [0.91, 0.95] 0.94 [0.93, 0.96] ICC: Intraclass correlation; CI: confidence interval; BMI: Body Mass Index; sd: standard deviation. a mean difference calculated using self-reported weight minus measured weight. *Missing data for informed group: 3 missing for weight, 3 missing for height; subsequently 6 missing for BMI. **Missing data for uninformed group: 2 missing for weight, 6 missing for height; subsequently 6 missing for BMI. There were no significant differences in mean difference of self-reported and measured weight (p = 0.4004), height (p = 0.5342) and BMI (p = 0.4409) and ICCs between the informed and uninformed group (see Table 1). When measured and self-reported BMI categories were examined the percentage agreement was 78% for the informed group and 81% for the uninformed group. The kappa values were 0.68 [95% CI 0.58, 0.78] for the informed and 0.72 [95% CI 0.61, 0.83] for the uninformed group and overlap between 95% CIs indicated no significant differences. The Bland-Altman plots for weight, height and BMI for the informed and uninformed groups are shown in Figures 1, 2 and 3. Figure 1Bland Altman plot for self-reported and measured weight (in kilograms) in informed and uniformed general practice patients. Middle line represents mean difference of methods. Lines above and below represent 95% limits of agreements (LOA), where upper LOA is +1.96 SD and lower line is −1.96 SD from overall mean differences. Figure 2Bland Altman plot for self-reported and measured height (in centimetres) in informed and uniformed general practice patients. Middle line represents mean difference of methods. Lines above and below represent 95% limits of agreements (LOA), where upper LOA is +1.96 SD and lower line is −1.96 SD from overall mean differences. Figure 3Bland Altman plot for self-reported and measured body mass index (in kg/m2) in informed and uniformed general practice patients. Middle line represents mean difference of methods. Lines above and below represent 95% limits of agreements (LOA), where upper LOA is +1.96 SD and lower line is −1.96 SD from overall mean differences. As there were no significant differences in accuracy of self-reported weight and height between the groups, the sample was pooled to assess overall agreement and reliability. Overall mean differences between self-reported and measured values were −1.2 kg (4.0) for weight [males −1.2 kg (3.6), females −1.2 kg (4.4)], 0.8 cm (4.4) for height [males 1.5 cm (3.5); females 0.3 cm (4.9)] and −0.6 kg/m2 (2.0) for BMI [males −0.9 kg/m2 (1.7); females −0.4 kg/m2 (2.3)]. The overall ICCs for self-reported and measured values and their corresponding 95% CIs were 0.97 [0.97, 0.98] for weight, 0.91 [0.89, 0.93] for height and 0.94 [0.91, 0.95] for BMI. The Bland Altman plots and LOA provide an indication of the extent of underreporting and overreporting of weight, height and BMI when compared to measured values (see Figures 1, 2, 3). The overall percentage agreement between self-reported and measured classification of BMI categories was 80% [95% CI 75, 84]. Twenty percent of those who were overweight were categorised as normal weight using self-reported weight and height (see Table 2). Of those who were obese, 22% were classified as overweight using self-reported weight and height. The prevalence of obesity was underestimated by 5% (35% using measured and 30% using self-report) and prevalence of normal weight was overestimated by 5% (27% using measured and 32% using self-report). The kappa for categorisation of BMI was 0.70 [95% CI 0.63, 0.77]; representing substantial agreement [20] and that level of agreement was greater than expected by chance alone (p < 0.001). Table 2 Categorisation of body mass index (BMI) category based on self-reported and measured weight and height   Measured n (%) b Total a Self-report Underweight Normal weight Overweight Obese   Underweight 4 (67) 2 (2.3) 0 (0) 0 (0) 6 (1.9) Normal weight 2 (33) 78 (91) 23 (20) 0 (0) 103 (32) Overweight 0 (0) 6 (7.0) 85 (74) 25 (22) 116 (36) Obese 0 (0) 0 (0) 7 (6.1) 88 (78) 95 (30) Total 6 (1.9) 86 (27) 115 (36) 113 (35) 320 a Total less than overall included due to invalid values in self-reported weight and height. b Percentage reported is proportion of measured for each BMI category. Underweight defined as BMI <18.5 kg/m 2 ; Normal weight defined as BMI ≥18.5 kg/m 2 and <25 kg/m 2 ; Overweight defined as BMI ≥25 kg/m 2 and <30 kg/m 2 ; Obese defined as BMI ≥30 kg/m 2 . There were no significant differences by age, when mean differences in measured and self-reported weight, height and BMI were compared (see Table 3). Table 3 Mean difference between measured and self-reported weight, height and BMI by age categories in Australian general practice patients   Mean difference* (standard deviation) ANOVA test   18 – 24 25 – 44 45 – 64 ≥ 65 F df p-value   ( n   = 33) ( n =   95) ( n =   121) ( n =   83)       Weight −1.6 (0.6) −0.8 (0.4) −1.1 (0.4) −1.6 (0.4) 0.73 3 0.5344 Height −0.004 (1.0) 1.2 (0.6) 0.7 (0.3) 0.9 (0.4) 0.67 3 0.5695 BMI −0.6 (0.3) −0.5 (2.0) −0.6 (0.2) −0.9 (0.2) 0.63 3 0.5950 *Mean calculated using measured self-reported minus measured values. BODY.DISCUSSION: This study demonstrated that informing general practice patients that their height and weight would be measured did not improve accuracy of self-report. This contrasts with Black and colleagues' findings, where those who were informed that they would be measured reported their weight and height significantly more accurate than those who were not informed [16]. This difference in findings could have occurred due to several differences in study methodology, setting, participants and statistical analyses conducted. Black and colleagues recruited their sample for a health screen in a shopping mall whereas the current study recruited participants presenting for general practice care. General practice patients may be more willing to disclose their weight and height compared to volunteers in a shopping mall. Further, Black's study included only participants aged between 18 to 28 years whereas only a small proportion (18%) of our sample was aged between 18 and 29 years [16]. Those in the younger age bracket may be more likely to be affected by cultural ideals regarding weight and height [24], which might have led to attempts to misreport these measures. Inconsistent findings regarding the accuracy of self-reported weight and height in older patients have been identified, with one longitudinal study reporting that only small changes in reporting of weight and height occurred with increasing age [25] and others identifying substantial differences between measured and self-reported weight and height in those older [26,27]. Our study did not find any significant differences in mean reporting of self-reported and measured values with age category; and no pattern of increasing or decreasing difference with age was observed. Black and colleagues also asked participants in the informed group six additional 'body weighing questions' which may have helped with recall of weight [16]. The current study aimed to test solely if informing patients that their weight and height would be measured would improve accuracy of self-report and thus did not include these questions. Given that GPs see a larger proportion of older patients, reporting of weight and height in this group may be less affected by social desirability bias and suggests that misreporting may be attributed largely to recall bias or not having an accurate knowledge of one's current weight and height. Future studies testing this intervention in younger patients may produce different findings. Consistent with findings in other populations [8], participants in the present study tended to underreport their weight and overreport their height. Mean differences between self-reported and measured weight (−1.2 kg in males; -1.2 kg in females) and height (1.5 cm in males; 0.3 cm in females) are within the range of that identified in a review examining accuracy of self-reported weight (−1.9 kg to 0.4 kg in males; -1.6 kg to 0.7 kg in females) and height (−1.3 cm to 2.3 cm in males; -1.7 cm to 2.2 cm in females) in the general population [8]. Only one Australian study was included in the review, however this study did not report mean differences. When compared to other Australian studies, our study had lower mean differences in self-reported and measured values for reporting of height and weight, particularly for females. Taylor et al. identified mean differences of −1.5 kg in males; -1.8 kg in females for weight and 1.4 cm in males; 1.3 cm in females for height [12]. Another study identified mean differences of 2.0 cm in males; 0.8 cm in females for height and −1.4 kg for males and −3.0 kg for females [13]. There is some evidence to suggest that females who had recently consulted a doctor may be able to more accurately recall their weight and height [28]. Additionally, patients presenting for care to their GP may represent a more 'health conscious' sample and thus, may be more aware of their weight and height measurements. Differences could also be attributed to the fact there was no time lag between self-report and measured assessments in our study, whereas an average of 23.5 days between self-reported and measured data was reported in the study conducted by Taylor [12]. Overall, 80% of participants were accurately classified as underweight, normal weight, overweight or obese using self-reported weight and height. The use of self-reported BMI resulted in no difference in prevalence of overweight and only a 5% lower prevalence of obesity when compared to estimates obtained using measured data. These findings are favourable when compared to other studies which indicate that self-reported data underestimated the proportion of participants classified as overweight by 2% to 12% and obese by approximately 7% [12,13]. The present findings however, are comparable to the 2008 Australian National Health Survey, which identified a 6% rate of underestimation of prevalence of overweight or obese when self-reported data was compared to measured data [10]. While the current study identified high reliability between self-reported and measured weight and height, represented by high ICCs (>0.9) for weight, height and BMI, the estimated Bland Altman LOAs suggests that accuracy of individuals' self-report may vary. When compared to measured weight, inaccuracies in self-reported weight ranged from overestimation of 6.7 kg to underestimation of 9.1 kg. Similarly, inaccuracies in self-reported height ranged from an overestimation of 8.6 cm to underestimation of 7.1 cm. This subsequently led to overreporting of BMI by 3.3 kg/m2 and underreporting of up to 4.6 kg/m2. BODY.DISCUSSION.STRENGTHS AND LIMITATIONS: A high consent rate was achieved, with 93% agreeing to have their weight and height measured. This high consent rate may be due to the use of the touchscreen computer which could have provided participants with a more private way of reporting weight and height. There was no time lapse between provision of self-report and actual measurement of weight and height, thus reducing potential error attributed to weight change during the time lapse. The use of ICCs and Bland Altman plots with LOAs provide a more robust examination of agreement compared to the more traditionally used Pearson's correlation coefficients as it provides an indication of variability and agreement rather than association. The ICC however treats self-reported and measured values as exchangeable (i.e. method of measurement is assumed to be a random effect). When systematic differences between methods of measurement occur, high ICCs may not necessarily imply high agreement. Some of the variation between self-reported values and measured values may be accounted for by the way in which participants report their weight and height (e.g. end-digit preferences [12] and reporting in imperial units rather than metric). A large proportion of participants included in this study were aged 65 years and above. However, when mean differences in self-reported and measured values were compared, no differences were identified between older and younger participants. This study was conducted in only three practices, potentially limiting the generalizability of findings. No significant differences in participant's sex was observed when compared to a larger Australian general practice dataset (BEACH), which included 95,839 patient encounters recruited by 958 GPs [7]. However, a difference in distribution of age was observed between our sample and the BEACH dataset [7]. BODY.CONCLUSION: Informing general practice patients that their weight and height would be measured did not significantly improve accuracy of self-report. Testing this strategy in subgroups likely to be affected by cultural ideals regarding weight (i.e. younger, female) may be beneficial in helping identify ways to improve accuracy of self-report for these groups. Self-reported weight and height provides relatively accurate estimates of BMI in Australian general practice patients. Thus, in circumstances where population trends are of interest such as in large surveillance studies, self-report is likely to be an accurate alternative. While the average bias of self-reported weight and height as estimates of the measured quantities is small, the LOAs indicate that there is a need for these values to be interpreted with caution in individuals. BODY.COMPETING INTEREST: The authors declare no competing interest. BODY.AUTHORS’ CONTRIBUTIONS: SY, MC, CD and RSF all participated in conception of the study and survey design. SY conducted all data collection and initial data analysis. SY, MC and CD had input into the statistical analysis. All authors offered critical comments on the draft of the manuscript and approved the final submitted version. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2288/13/38/prepub

TITLE: Tolerability and efficacy of a combination of paracetamol and caffeine in the treatment of tension-type headache: a randomised, double-blind, double-dummy, cross-over study versus placebo and naproxen sodium ABSTRACT: The main aim of this study was to confirm in an Italian population affected by tension-type headache (TTH) the good profile of safety and tolerability of the combination paracetamol 1,000 mg–caffeine 130 mg (PCF) observed in previous studies, by a comparison with naproxen sodium 550 mg (NAP) and placebo (PLA). A secondary objective was to assess the efficacy of PCF in the acute treatment of TTH. This was a multicentre, randomised, double-blind, double-dummy, crossover, placebo-controlled trial. Tolerability was assessed by recording adverse events by the patient in the 4-h post-dose treatment. To assess the efficacy, the sum of pain intensity differences (SPID) and the total pain relief (TOTPAR) were calculated. Comparing PCF and NAP and PCF and PLA for tolerability, the difference was nonsignificant but the result regarding noninferiority was inconclusive, whilst NAP was noninferior to PLA. As regards SPID and TOTPAR, both PCF and NAP were better than placebo (P < 0.05), but not significantly different from each other. In conclusion, PCF was well-tolerated and effective in the treatment of acute TTH. BODY.INTRODUCTION: Tension-type headache (TTH) is the most prevalent primary headache with a worldwide lifetime prevalence of 46% [1]. In Europe the lifetime prevalence of TTH seems to be even higher: it was as high as 86% in a population-based study in Denmark [2]. Even if in that study most of the patients had episodic infrequent TTH (1 day or less per month), nevertheless 24–37% had TTH several times a month and 10% had TTH weekly. Due to its high prevalence, TTH carries a great (and still partially unexplored) burden for the affected individuals and the society. The results of two Danish studies have shown that the number of work days missed in the population owing to TTH was three times higher than the number for migraine [3, 4]; and a US study has also found that absenteeism due to TTH is considerable [5]. Under the assumption that the indirect costs of TTH far outweigh the direct costs, we can therefore speculate that the cost of TTH is greater than the cost of migraine [1, 6]. Given the high prevalence and costs of TTH, it is of paramount importance to have safe, well-tolerated, and effective drugs to treat TTH at our disposal. Lacking specific drugs, as triptans are for the acute treatment of migraine, NSAIDs are commonly employed [7]. Some NSAIDs have been proved to be superior to placebo in the acute treatment of TTH [8]. However, gastric irritation and occasionally ulceration may complicate treatment with aspirin or other NSAIDs, even when they are used intermittently. The combination of paracetamol and caffeine is an useful alternative to NSAIDs: it was compared with placebo in two high-quality studies, showing its superiority over placebo as well as a good tolerability [9, 10]. Such studies, however, were carried out in Anglo-Saxon populations, with different habits respect to Italian patients, including higher consumption of caffeine, and therefore a different sensitivity to its peripheral and central stimulatory effects. The main aim of this study was to confirm in an Italian population affected by TTH the good profile of safety and tolerability of the combination paracetamol 1000 mg–caffeine 130 mg observed in previous studies [9, 10], by a comparison with naproxen sodium, a NSAID widely used in the acute treatment of TTH [11–13], and placebo. A secondary objective was to assess the efficacy of the combination paracetamol 1000 mg–caffeine 130 mg in the acute treatment of TTH, versus naproxen sodium 550 mg and placebo. BODY.PATIENTS AND METHODS.PATIENTS: In this multicentre study, conducted between December 2004 and May 2007 in eight Headache Centres throughout Italy, outpatient volunteers of both genders (18–65 years) with a clinical history of TTH were recruited if they fulfiled the following inclusion criteria:Diagnosis of episodic tension-type headache according to the ICHD-II criteria [14], modified in the single, following criterion: absence of nausea, vomiting, photophobia and phonophobia (to exclude subjects with migraine headaches),Mean frequency of 4–14 days with TTH per month,History of response to treatment of TTH with over-the-counter pain-killers,Daily consumption of at least two cups of coffee,Adequate contraception in women of fertile age,Medical history and physical examination inconsistent with organic disorders associated with headaches. Subjects were excluded from the study if they had a history of any of the following:Known hypersensitivity or allergy to paracetamol or naproxen,Chronic headache, either recurrent or continuous,Concomitant use/overuse of NSAIDs or other analgesics; treatment with antiplatelet or anticoagulant drugs,Migraine or post-traumatic headache,Alcohol abuse, drug dependency, or psychiatric disease,Coagulation disorders, peptic ulcer disease, pancreatic disease, clinically significant renal or hepatic disease, blood hypertension, mild/moderate kidney or liver failure, Gilbert's syndrome. The study was conducted in accordance with the Declaration of Helsinki (Tokio version 2004) and Good Clinical Practice standards and did not start before independent Ethics Committee approval appropriate for each investigator was obtained. Prior to enrolment the patients gave their written informed consent; they were allowed to terminate participation in the trial at any time, without giving reasons. This trail complies with the Guidelines for trials of drug treatments in TTH of the International Headache Society [15]. BODY.PATIENTS AND METHODS.STUDY DESIGN AND TREATMENTS: This study was designed as a randomised, double-blind, double-dummy, crossover, placebo-controlled trial. After having signed the informed consent form, patients were required to treat the next three consecutive TTH attacks with the investigational study medications, according to a randomised crossover sequence which was computer generated using Microsoft® Access 2003. Each patient was randomly allocated to one of the six sequences illustrated in Table 1, according to a 1:1:1:1:1:1 scheme. Eligible patients were assigned in sequential order of entry. Access to the randomisation code was strictly controlled and the treatment assignment remained unknown to all parties involved in the trial until formal database lock. Subjects in all treatment groups received three identical boxes (numbered progressively from 1 to 3 to indicate the exact order in which they should have been used) containing: one soft gel capsule containing one tablet of placebo and one sachet containing paracetamol 1,000 mg + caffeine 130 mg [Tachicaf®, Angelini Farmaceutici, ACRAF S.p.A., Pomezia (RM), Italy] or one soft gel capsule containing one tablet of naproxen sodium 550 mg (Synflex Forte®, Recordati S.p.A., Milano, Italy) and one sachet of placebo or one soft gel capsule containing one tablet of placebo and one sachet of placebo. The doses corresponded to the maximal individual therapeutic single doses recommended for these analgesics. Blinding was ensured using matched trial supplies, identical in colour, size, shape and taste. At each TTH attack patients would have to take one soft gel capsule and one sachet at the same time. The trial medication was to be taken when the headache occurred, and when the patients would normally have taken their usual analgesic. Other than study medication, patients received rescue medication (ibuprofen 600 mg), to be taken 2 h after the administration of the trial medication, if the pain persisted.Table 1Sequences of study treatments1. PCF–NAP–PLA2. NAP–PLA–PCF3. PLA–PCF–NAP4. PCF–PLA–NAP5. NAP–PCF–PLA6. PLA–NAP–PCFPCF paracetamol 1,000 mg + caffeine 130 mg, NAP naproxen sodium 550 mg, PLA placebo At baseline visit, patients' medical histories and concomitant treatments were recorded, vital signs were registered and physical examination was performed by the investigator. Patients were required to record in a headache diary, the date and time of drug ingestion, pain intensity before treatment and pain intensity, pain relief and adverse events (AEs) after treatment recorded at 1, 2, 3, and 4 h. At the end of 4-h measurement interval or at the time of use of rescue medication, patients had to record the presence and intensity of AEs and to evaluate their impression of the efficacy and tolerability of the study medication. The same procedures were repeated for the two subsequent TTH attacks, provided that they were 48 h apart from each other. Patients had to contact the investigator within 48 h after the third treated episode of TTH for the final visit. The investigator reviewed the completed diary with the patient to ensure that all required information had been registered, recorded vital signs and concomitant treatments and expressed a global assessment of tolerability. Patients were asked to indicate their preference for one of the three treatments, taking into account efficacy and tolerability. BODY.PATIENTS AND METHODS.OUTCOMES: Safety and tolerability were evaluated by comparing vital signs at screening and final visits and by recording AEs. AEs could be recorded by the investigator or by the patient [filling in a symptom check-list (including nervousness, palpitation, insomnia, dizziness, abdominal pain, dyspepsia, nausea, vomiting, drowsiness and fatigue) hourly for 4 h after the study medication ingestion]. AEs intensity was determined by subjective evaluation of the patient and classified as mild (signs or symptoms easily tolerated), moderate (discomfort sufficient to cause interference with normal activities) and severe (incapacitating with inability to do work or undertake normal activity). A global assessment of tolerability was expressed by the patient, using a 5-point Verbal Rating Scale (VRS: 'excellent', 'very good', 'good', 'sufficient' and 'poor'). The investigator expressed a global assessment of tolerability, using the following 5-point VRS: 'very good' (no AEs), 'good' (mild AEs recovered spontaneously without treatment), 'fair' (AEs requiring treatment to recover), 'poor' (AEs requiring withdrawal from study), 'very poor' (serious AEs). To assess treatments' efficacy, intensity of pain (on a 4-point scale: 0 'absent', 1 'mild', 2 'moderate', 3 'severe') and pain relief (on a 5-point scale: 0 'no relief', 1 'little relief', 2 'some relief', 3 'much relief', 4 'complete relief') were evaluated hourly during the 4-h post-dose period. Based on these two variables, the following parameters were calculated:Pain intensity difference (PID). For each patient the sum of pain intensity differences (SPID) was calculated as the sum of differences between pre-dose assessment and every post-dose assessment,Total pain relief (TOTPAR), calculated as the sum of every post-dose assessment. Finally, patients expressed their preference for one of the investigational treatments, answering the following question: "Taking into account both tolerability and efficacy, which of the three treatments would you take again at the next headache attack?" (Options were: (a) none, (b) treatment number 1, (c) treatment number 2, (d) treatment number 3). BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: The study was powered to test the primary hypothesis, namely that paracetamol 1,000 mg + caffeine 130 mg (PCF) would be non-inferior to naproxen sodium 550 mg (NAP) as regards the proportion of patients complaining of AEs in the 4-h post-dose period. Assuming a 7% threshold as the maximum tolerated difference between PCF and NAP, a 7.9% proportion of discordant pairs, 80% power, and one-tailed test with a 0.05 significance level, the required sample size was estimated to be 100 treated patients. In order to take any premature withdrawals into account, the number of patients to be enrolled was set at 150. According to study protocol, three populations have been evaluated: per-protocol (PP, subjects fulfiling all the inclusion criteria who took the three treatments and performed all the tolerability evaluations), randomized patients (RP, patients who took at least one of the treatments), and intention-to-treat [ITT, patients who took the three treatments and performed at least one post-dose evaluation. Data missing for any scheduled evaluation was replaced by the last observation carried forward (LOCF) procedure]. The tolerability endpoints were evaluated using PP and RP populations; ITT population was employed for efficacy analyses. Descriptive statistics on RP population was used for demographic and baseline characteristics. The Mc Nemar test (reported as 90% confidence interval) was used to compare the percentage of patients who recorded AEs after each treatment. Besides those recorded by patients in the 4-h post-dose period, all the AEs were classified by the investigator on the basis of: treatment received, system involved, severity and correlation with the investigational medication. The analysis of variance was used to evaluate the differences of vital signs respect to baseline. The Wilcoxon signed rank test was used to analyse the global assessment of tolerability expressed by patients. The analysis of variance was used to evaluate SPID and TOTPAR. The patient' preference for one of the treatments was reported as a distribution of frequency. The evaluation of the quality and completeness of the data, identification of important protocol deviations and handling of problem cases were performed regularly and finally decided before locking and unblinding the database. BODY.RESULTS.STUDY POPULATION: One-hundred and eleven subjects entered the study, 99 of whom took at least one of the treatments (RP). Twelve patients were excluded from the study, since they did not fulfil the inclusion criteria (n = 2) or did not take any medication (n = 10). Other six patients took 1 or 2 investigational medications only. Therefore PP population included 93 subjects. Reasons why patients did not complete the study were: explicit request to withdraw from the study (n = 8), lack of compliance to study procedures (n = 5), severe nausea (n = 1), unmasking of assigned treatment (n = 1), expiry of investigational medication (n = 1). ITT population included 93 subjects, 91 and 81 of whom were available for the efficacy analyses concerning pain severity and pain relief, respectively. Demographic characteristics and headache history of RP population are shown in Table 2. As regards baseline pain intensity of treated headaches (n = 287), 47 attacks (16.4%) were judged to be mild, 168 (58.5%) moderate and 72 (25.1%) severe.Table 2Demographic characteristics and headache history (randomized patients dataset; n = 99)Gender, n (%) Male40 (40.4) Female59 (59.6)Age, years Mean ± SD35.1 ± 10.19 Range19–64Race, n (%): Caucasian98 (99.0) Other1 (1.0)Headache duration, years Mean ± SD22.2 ± 9.09 Range6–44Mean number of days with tension-type headache per month, n (%) <42 (2.0) 4–1497 (98.0) >14–Usual pain intensity, n (%) Mild21 (21.2) Moderate75 (75.8) Severe3 (3.0) BODY.RESULTS.TOLERABILITY AND SAFETY: Considering the PP population (n = 93), in which all the tolerability assessments were available, the following percentages of patients reported AEs in the 4-h post-dose period: 36.6% with PCF, 31.2% with NAP, and 36.6% with placebo (PLA). Comparing PCF and NAP, the proportion of discordant pairs (that is, patients who reported AEs with one but not the other drug and vice versa) were 19.4 and 14.0%, respectively, with a difference of 5.4% (90% CI, −4.4 to 15.2%). Comparing PCF and PLA, the proportion of discordant pairs was 14.0% for both treatments, with no differences of discordant pairs (90% CI, −9.0 to 9.0%). Since in the comparisons PCF–NAP and PCF–PLA the confidence interval included both Δ (the maximum tolerated difference between treatments) and zero, the difference was nonsignificant but the result regarding noninferiority was inconclusive [16]. Comparing NAP and PLA, the proportion of discordant pairs were 12.9 and 18.3%, respectively, with a difference of −5.4% (90% CI, −14.9 to 4.1%). Therefore NAP was noninferior to PLA. During the 4-h post-dose period, 224 AEs were recorded in the RP population, 76 (33.9%) after PCF ingestion, 66 (29.5%) after NAP and 82 (36.6%) after PLA. Table 3 illustrates the percentage of AEs according to investigational drugs. Most AEs were codified as mild or moderate. Only 21 AEs (9.4%) were recorded as severe: 13 (5.8%) after PLA, 5 (2.3%) after NAP and 3 (1.3%) after PCF. The most frequently observed AEs were nausea, drowsiness, fatigue and nervousness (Table 3).Table 3Adverse events recorded in 4-h period after ingestion of investigational drugs (randomized patients dataset; n = 99)Adverse eventPCF n (%)NAP n (%)PLA n (%)Nervousness11 (14.5)8 (12.1)12 (14.6)Palpitation4 (5.3)2 (3.0)3 (3.7)Insomnia4 (5.3)3 (4.6)3 (3.7)Dizziness6 (7.9)–4 (4.9)Abdominal pain5 (6.6)4 (6.1)5 (6.1)Dyspepsia6 (7.9)7 (10.6)5 (6.1)Nausea15 (19.7)15 (22.7)21 (25.6)Vomiting2 (2.6)3 (4.6)2 (2.4)Drowsiness11 (14.5)14 (21.2)13 (15.9)Fatigue12 (15.8)10 (15.2)14 (17.1)PCF paracetamol 1,000 mg + caffeine 130 mg (n = 98), NAP naproxen sodium 550 mg (n = 94), PLA placebo (n = 98) Besides AEs directly recorded by the patients, other 20 AEs were registered by the investigator. Only two of them (sweating and lips dryness), however, were judged to be possibly correlated to the drug. No serious AEs occurred. One patient dropped from the study because of severe nausea during a headache attack treated with PCF. No significant differences occurred in vital signs recorded at final visit compared to those recorded at screening visit. The global assessment of tolerability expressed by the patient is shown in Table 4. The evaluation was 'excellent' or 'very good' in 45.7% of the patients after PCF, in 51.6% after NAP, and 41.7% after PLA. The difference was statistically significant (P < 0.05) between NAP and PLA.Table 4Global assessment of tolerability expressed by the patient (randomized patients dataset)PCF n (%)NAP n (%)PLA n (%)Excellent17 (18.1)32 (35.2)20 (20.8)Very good26 (27.7)15 (16.5)20 (20.8)Good26 (27.7)23 (25.3)22 (22.9)Sufficient12 (12.8)9 (9.9)14 (14.6)Poor13 (13.8)12 (13.2)20 (20.8)Total94 (100.0)91 (100.0)96 (100.0)PCF paracetamol 1,000 mg + caffeine 130 mg, NAP naproxen sodium 550 mg, PLA placebo The global assessment of tolerability expressed by the investigator on the RP population (n = 97) at the final visit and referring to the sum of investigational drugs, gave the following results: 'very good' or 'good' in 96%, 'fair' in 3% and 'poor' in 1% of the patients. BODY.RESULTS.EFFICACY: Figure 1 reports the time course of PID for the three investigational treatments, whilst the SPID mean values with standard errors are illustrated in Table 5. PID relative to baseline steadily increased over time in both the active treatments compared to placebo. PCF and NAP were significantly better than placebo (P < 0.05), whilst no differences emerged between the two active treatments.Fig. 1Time course of the mean pain intensity difference (PID) for the three investigational treatments (intention-to-treat dataset, n = 91). PCF paracetamol 1,000 mg + caffeine 130 mg; NAP naproxen sodium 550 mg, PLA placeboTable 5Sum of pain intensity differences (SPID) and total pain relief (TOTPAR) in the 4-h observation period (intention-to-treat dataset)PCFNAPPLASPID n919191 Baseline intensity (SE)2.0 (0.06)2.1 (0.07)2.1 (0.08) Mean (SE)4.0 (0.35)4.2 (0.38)2.1 (0.35)TOTPAR n818181 Mean (SE)8.5 (0.60)8.0 (0.63)4.8 (0.60)PCF paracetamol 1,000 mg + caffeine 130 mg, NAP naproxen sodium 550 mg, PLA placebo The time course of TOTPAR during the 4-h observation period is shown in Fig. 2, whilst TOTPAR mean values with standard errors are displayed in Table 5. Both PCF and NAP provided significantly more relief than placebo (P < 0.05), but were not significantly different from each other.Fig. 2Time course of total pain relief (TOTPAR) for the three investigational treatments (intention-to-treat dataset, n = 81). PCF paracetamol 1,000 mg + caffeine 130 mg, NAP naproxen sodium 550 mg, PLA placebo The percentage of subjects using rescue medication was similar for PCF and NAP (4.8 and 3.3%, respectively) and both were less than the 10.0% of subjects who used rescue medication after PLA. As regards the patients' preference for one of the tested treatments (RP dataset, n = 92), 30 patients (32.6%) preferred PCF, 41 patients (44.6%) NAP and 21 patients (22.8%) PLA. BODY.DISCUSSION: In a condition as widespread as TTH is, using the most effective dose of a drug that is well tolerated by a patient (and safe) is a reasonable basis for selecting a medication. Exerting its analgesic activity thanks to a direct effect on the central nervous system [17], at least in part mediated by the serotonergic system [18, 19], paracetamol has some advantages over NSAIDs, due to its scarce inhibition of peripheral cyclooxygenase: it is well tolerated at gastrointestinal level, it is only a weak inhibitor of aggregation of platelets and does not alter the bleeding time [20]. In a recent systematic review on the therapy of acute episodic TTH [7], paracetamol 1,000 mg was significantly more effective than placebo with AEs matching those of placebo. A quantitative analysis of seven studies comparing NSAIDs and paracetamol, however, showed a significant difference in short-term pain relief in favour of NSAIDs [7]. The combination paracetamol 1,000 mg and caffeine 130 mg has been proved to be superior both to placebo and paracetamol 1,000 mg in the treatment of TTH [10], at no significant cost in term of AEs, thanks to the synergistic effect of caffeine. Caffeine has a direct analgesic effect not mediated via effects on mood or on caffeine withdrawal [9]. Suggested, albeit unproven, mechanisms for caffeine analgesia are the antagonistic activity on adenosine receptors [21] and the ability to increase the norepinephrine activity in the central nervous system [22]. Moreover, caffeine has a well-documented adjuvant action, when combined with analgesics [23]. The mechanisms of this analgesic adjuvant effect are not well-established, but could, at least partially, be explained by pharmacokinetic interactions, in terms of increased absorption or reduced metabolism of analgesics induced by caffeine [24, 25]. Whatever the mechanisms are, it has been shown that the addition of caffeine to a NSAID increases its analgesic potency by 40% [26]. Some worry as far as Italy is concerned could be raised by the fact that the studies on the 'high-dose' paracetamol–caffeine combination (i.e., paracetamol 1,000 mg and caffeine 130 mg) were carried out in Anglo-Saxon populations, with a consumption of caffeine higher than that found in Italians, and therefore theoretically less prone to the stimulating effects of caffeine. The results of the present study are absolutely reassuring about the tolerability of the paracetamol–caffeine combination. The difference amongst treatments was nonsignificant, even though the noninferiority of PCF compared to NAP and PLA could not be conclusively demonstrated. Moreover, the percentage of patients complaining of AEs in the 4-h post-treatment period was exactly the same (36.6%) after PCF and after PLA. The AEs theoretically attributable to the stimulating effect of caffeine (nervousness, palpitation and insomnia) were roughly the same with the three investigational medications (Table 3). The high frequency of some, even severe, AEs like nausea after PLA is likely to be due to symptoms accompanying headache not extinguished by the active drugs. It is not a chance that a good evaluation in the global assessment of tolerability expressed by the patient was lower after PLA than after active drugs. As regards the efficacy, this study confirms the good results obtained in previous clinical trials [9, 10], showing a significant superiority of PCF over placebo, and no difference compared to a reference drug as NAP, in both the reduction of pain intensity and the pain relief. The results of this trial may have important implications for clinical practice. Many drugs for the treatment of TTH are expensive and/or have contraindications or relevant side effects [27]. The combination of paracetamol 1,000 mg and caffeine 130 mg seems to be effective and well tolerated, in particular not showing the specific stimulatory effects of caffeine. It is therefore a serious candidate for the first line treatment of acute episodic TTH.

TITLE: Inflammatory and structural changes in vertebral bodies and posterior elements of the spine in axial spondyloarthritis: construct validity, responsiveness and discriminatory ability of the anatomy-based CANDEN scoring system in a randomised placebo-controlled trial ABSTRACT.BACKGROUND: The Canada-Denmark (CANDEN) definitions of spinal MRI lesions allow a detailed anatomy-based evaluation of inflammatory and structural lesions in vertebral bodies and posterior elements of the spine in patients with axial spondyloarthritis (axSpA). The objective was to examine the reliability, responsiveness and discrimination of scores for spinal inflammation, fat, bone erosion and new bone formation based on the CANDEN system and to describe patterns of inflammatory and structural lesions and their temporal development. ABSTRACT.METHODS: 49 patients with axSpA from an investigator-initiated, randomised, placebo-controlled trial of adalimumab underwent spinal MRI at weeks 0/6/24/48. MR images were scored according to the CANDEN system and the Spondyloarthritis Research Consortium of Canada (SPARCC) method. Total scores, and various subscores, were created by summing individual lesion scores. ABSTRACT.RESULTS: The CANDEN spine inflammation score had high responsiveness, similar to the SPARCC MRI spine index (Guyatt's responsiveness index 1.88 and 1.67, respectively), and discriminated between adalimumab and placebo treatment already at 6 weeks' follow-up (P=0.03). Anterior/posterior corner inflammation subscores showed similar responsiveness. Inter-reader reliability for the CANDEN spine inflammation and fat scores was good to very good for status and change scores (intraclass correlation coefficient (ICC)=0.71–0.92). Reliability for CANDEN new bone formation and erosion scores was good to very good for status scores (ICC=0.61–0.75) but, due to minimal progression, poor for change scores (ICC≤0.40). ABSTRACT.CONCLUSIONS: The CANDEN spine inflammation score showed good responsiveness, discrimination between active treatment and placebo and reliability. The CANDEN spine structural scores had good cross-sectional reliability, but longer studies are needed to investigate their sensitivity to change. ABSTRACT.TRIAL REGISTRATION NUMBER: NCT01029847; Results. BODY: Key messagesWhat is already known about this subject?Patients with axial spondyloarthritis (axSpA) have different trajectories of inflammatory and structural spinal lesions during the disease course.Detailed scoring of MR images of the vertebral bodies and posterior elements of the spine is possible using the 2009 Canada-Denmark (CANDEN) definitions for inflammatory and structural spinal lesions.What does this study add?The CANDEN spine inflammation score showed good reliability, responsiveness and discriminated well between active treatment and placebo, and it has inherent good face validity as posterior elements of the spine are also assessed.How might this impact on clinical practice?The CANDEN scores are not intended to be used to diagnose axSpA, but rather to allow detailed monitoring in research settings of changes over time in patients who have already been diagnosed with axSpA. BODY.INTRODUCTION: Axial spondyloarthritis (axSpA) is a chronic inflammatory disease that affects the sacroiliac joints and the spine.1 MRI can detect inflammatory lesions (bone marrow oedema (BME)/osteitis), and structural lesions (erosions, bone spurs, ankylosis and fat metaplasia). The histopathological correlation of MRI inflammation and structural changes are mostly unknown, but BME of the facet joints has been shown to correlate with inflammatory cell infiltrates.2 In 2009, the founders of the Canada-Denmark (CANDEN) MRI working group (RL, WM, MØ, SJP) developed definitions and an atlas of inflammatory3 and structural lesions4 of the spine, and reported the reliability of scoring the individual lesions in a multireader exercise.5 6 The CANDEN approach allows a systematic assessment of MR images of the spine from the perspective of diagnostic ascertainment, and of quantifying both active inflammatory and structural abnormalities according to their precise anatomical locations and their relations over time.7 The definitions were based on sagittal T1-weighted and Short Tau Inversion Recover (STIR) MRI sequences of the vertebral bodies and the posterior elements of the vertebrae (ie, the costotransverse, costovertebral and facet joints, transverse and spinous processes and the adjacent soft tissues). Since then, a score for spinal fat lesions (Fat Spondyloarthritis Spine Score (FASSS))8 and a score for new bone formation,9–11 based on the CANDEN MRI definitions has been developed. Reference images are available online.12 The Spondyloarthritis Research Consortium of Canada (SPARCC) MRI spine index is a well-established MRI scoring method for spinal inflammation. The standard SPARCC 23-discovertebral unit (DVU) and 6-DVU methods cover the vertebral bodies,13 but not lesions located in the posterior elements of the spine or the soft tissues adjacent to the spine. An extended SPARCC scoring method that captured lesions in the posterior elements has been developed,14 but has not been used in clinical trials. The SPARCC method is applied on only three consecutive sagittal slices of each DVU and may therefore not provide information on the total inflammatory load of the patients. In contrast, the CANDEN MRI definitions for inflammatory and structural lesions comprise detailed information on the anatomical location of lesions in the vertebral bodies as well as in the posterior elements (figure 1), and may therefore provide more accurate information on the total inflammatory load and allow detailed analyses of changes in lesions at specific locations over time. Figure 1(A) User interface used for scoring of sagittal images according to the Canada-Denmark scoring system. Twenty-three discovertebral units (DVUs) are assessed. Fat lesions are scored in a similar way as inflammatory lesions, except that the posterior elements (FIL, SP, ST and TP/R) are not assessed for fat lesions. Fat lesions follow the principles of inflammation, except that posterior elements are not assessed. aCIL, anterior corner inflammatory lesion; NIL, non-corner inflammatory lesion; pCIL, posterior corner inflammatory lesion; FIL, facet joint inflammatory lesion; SP, spinous process inflammatory lesion; ST, soft tissue inflammatory lesion; aLIL, anterior lateral inflammatory lesion; pLIL, posterior lateral inflammatory lesion; TP/R, transverse process/rib; aCOBE, anterior corner bone erosion; pCOBE posterior corner bone erosion; NOBE, non-corner bone erosion; aCANK, anterior corner ankylosis; pCANK, posterior corner ankylosis; NANK, non-corner ankylosis; aCOS, anterior corner spur; pCOS, posterior corner spur; NOS, non-corner spur; FANK, facet joint ankylosis. (B) Axial view of inflammatory lesions. Note that as sagittal images are used for scoring, the boundaries illustrated in axial view may vary a few millimeters depending on the exact placement of the sagittal slices, when following the definition of central and lateral slices. The aim of this study was to develop and validate an anatomy-based inflammation score, the CANDEN spine inflammation score and to further develop and validate CANDEN spine scores of erosion, fat and new bone formation. Moreover, we aimed to apply the CANDEN MRI definitions for spinal inflammation, fat metaplasia, erosion and new bone formation in an investigator-initiated, randomised, double-blind, placebo-controlled trial of adalimumab versus placebo to describe the frequencies of inflammatory and structural lesions and their development over time. BODY.METHODS.STUDY DESIGN: Forty-nine patients participated in a randomised, double-blind, placebo-controlled trial of adalimumab versus placebo (ASIM trial, ClinicalTrials.gov NCT01029847). All patients fulfilled the Assessment in Spondyloarthritis International Society (ASAS) criteria for axSpA and had sacroiliitis on MRI and/or radiography. The patients were randomly assigned to adalimumab 40 mg subcutaneously every other week or placebo for 6 weeks, thereafter all patients were treated with adalimumab 40 mg subcutaneously every other week to week 48. The six patients not responding at week 24 had their treatment changed to another tumour necrosis factor (TNF) inhibitor at the discretion of the treating rheumatologist. Further details of this trial, including whole-body MRI outcomes, are described in a separate article.15 The study took place at five rheumatology outpatient clinics in The Capital and Zealand Regions of Denmark from February 2010 to March 2014. All imaging examinations were performed at the Department of Radiology, Copenhagen University Hospital Herlev. BODY.METHODS.IMAGE ACQUISITION AND SCORING: MRI of the spine was performed at baseline and at weeks 6, 24 and 48. The spine was imaged in three parts (separately for cervical, thoracic and lumbar spine). MRI was performed in a single high-field Philips Medical Systems Achieva scanner (3.0 T) by sagittal T1-weighted turbo spin echo (repetition time: 518 ms, echo time: 8 ms, matrix (cerv./thor./lumb.): 200×143/300×213/300×213, field of view (FOV) (cerv./thor./lumb.): 178×150 mm/270×150 mm/270×150 mm, slice thickness 4 mm, interslice gap 0.4 mm) and STIR (repetition time: 4990–8530 ms, echo time: 80 ms and inversion time: 120 ms, matrix (cerv./thor./lumb.): 272×212/272×215/272×215, FOV (cerv./thor./lumb.): 270×230 mm/270×150 mm/270×230 mm, slice thickness 4 mm, interslice gap 0.8 mm). All MRI scans were read in chronological order by an experienced (SJP) and a newly trained (SK) reader blinded to other imaging and clinical data. Images were scored according to the definitions in the CANDEN MRI spine system3–6 and by the 23-DVU SPARCC MRI spine index using a web-based data entry interface (COPECARE SAMRI (Spondyloarthritis MRI module), figure 1). To ensure proper calibration of the two MRI readers for the SPARCC method, one of the developers of the SPARCC system (WM) also scored all images for the 23-DVU SPARCC MRI spine index. Radiographs were obtained at baseline and at 48 weeks and were scored in known chronology by the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) by one experienced reader (SJP) in known chronological order, blinded to all other data. BODY.METHODS.SCORING OF INFLAMMATORY LESIONS: Inflammation, new bone formation and erosion scores were developed from the CANDEN definitions of lesions and scoring,5 6 in a similar way as FASSS. The development and validation of FASSS based on the CANDEN MRI definitions has previously been reported.8 The spine is divided into 23 levels from C2/C3 to L5/S1. Central sagittal slices are the sagittal slices that include the spinal canal; the pedicle may be partially seen but is not continuous between the vertebral body and posterior elements3 5 (figure 1). Lateral sagittal slices are the sagittal slices that are located lateral to the spinal canal, and either the pedicle must be continuous between vertebral body and posterior elements or the slice is lateral to the pedicle. Only BME is scored, except for posterior element soft tissue oedema. To calculate the CANDEN spine inflammation score, a set of scoring rules are defined. BODY.METHODS.SCORING OF INFLAMMATORY LESIONS.VERTEBRAL BODIES: In central sagittal slices, anterior and posterior corner inflammatory lesions are scored as 0 (absent) or 1 (present); in DVUs in thoracic and lumbar spine (T1/T2 and below), a score of 1 is added for large lesions (≥25% of the anteroposterior (AP) diameter of the vertebral endplate and/or height of the vertebral body, perpendicular to the endplate).3 Non-corner inflammatory lesions (BME located between the corners of a vertebral body) are scored as 0 (absent), 2 (present); in DVUs in the thoracic and lumbar spine, a score of 2 is added for large non-corner lesions (≥25% of the height of the vertebral body, perpendicular to the endplate). It was decided to assign the score 2 for non-corner lesions, while 1 for corner lesions (both doubled if large, see definitions), because the area of this anatomical region is typically larger than the corner areas, and because each vertebral endplate contains two corner regions but only one non-corner region, thereby giving the same total weight to the two lesion types in the central sagittal slices. If a corner lesion in any central slice involves >50% of the AP diameter of the vertebra, it is counted as a combined corner and non-corner lesion. In lateral sagittal slices in the thoracic and lumbar spine, BME is scored if located at the anterior or posterior corner; these antero-lateral and postero-lateral inflammatory lesions are scored as 0 (absent) or 1 (present). All lesions are scored based on anatomical location; for example, if a lesion is visible at the anterior corner on a central sagitttal slice and in the anterior half of the vertebral body on a lateral slice, it is scored as the combination of an anterior corner lesion (sagittal slice) and an antero-lateral corner lesion (lateral slice). BODY.METHODS.SCORING OF INFLAMMATORY LESIONS.POSTERIOR ELEMENTS: For each of the 23 levels from C2/C3 to L5/S1: facet joint inflammatory lesions on left and right side, spinous process inflammatory lesions and soft tissue inflammatory lesions (hyperintense signal on STIR at ligaments and entheses at the posterior elements of the vertebrae) are each scored as 0 (absent) or 1 (present). For each of the 17 vertebrae from T1 to L5: rib/transverse process inflammatory lesions are scored as 0 (absent) or 1 (present) on left and right side. Inflammation in the costovertebral joints was not scored separately but as inflammation of rib/transverse process and/or postero-lateral corner inflammation, when present at these locations. The total scoring range for the CANDEN spine inflammation score is 0–582. The range for the vertebral body subscore is 0–456, and the range for the posterior elements subscore is 0–126. BODY.METHODS.SCORING OF STRUCTURAL LESIONS: Fat metaplasia (FASSS) is scored as previously described,6 8 the scoring range is 0–456. The method for scoring erosions is similar to the method for scoring spine inflammation, except that erosions are only assessed in central sagittal slices. Anterior and posterior corner erosions are scored 1, non-corner erosion is scored 2, in DVUs in the thoracic and lumbar spine, a score of 1 is added for large corner lesions and a score of 2 is added for large non-corner lesions. Erosions are not scored in the facet joints or other posterior elements. The scoring range for the CANDEN spine erosion score is 0–320. To obtain the CANDEN spine new bone formation score, at each level, the presence of anterior corner ankylosis, posterior corner ankylosis and non-corner ankylosis are each scored as 0 (absent) or 6 (present), anterior corner, posterior corner and non-corner bone spurs are each scored as 0 (absent) or 2 (present), facet joint ankylosis is scored as 0 (absent) or 1 (present), and all individual lesion scores are summed. The new bone formation scores were chosen to be consistent with the scores used in the radiographic mSASSS system that assigns a score of 2 for bone spur and a score of 3 for ankylosis (in total a score of 6 for two ankylosed corners that are opposing each other). Scoring range for the CANDEN spine new bone formation score is 0–460; the vertebral body subscore has a range of 0–414 and the facet joints subscore has a range of 0–46. The estimated reading time including data entry for obtaining the CANDEN spine inflammation score was not recorded systematically, but judged to be in the range of 10–45 min for all four timepoints depending on the amount of inflammation, similar to the SPARCC method in most cases. Total scoring time for inflammation and structural lesions in the spine were in the range 15–90 min per patient for four timepoints. BODY.METHODS.STATISTICAL ANALYSIS: Data were characterised by descriptive statistics and correlations were assessed by Spearman's correlation analysis. Difference between groups in change from baseline to weeks 6, 24 and 48 was assessed by Mann-Whitney U tests with continuity correction according to the predefined statistical analysis plan. Analysis of covariance (ANCOVA) with group allocation and baseline score as covariates and changes in continuous outcome variables as dependent outcomes were performed as post hoc secondary analyses to take differences in baseline values into account. Standardised response mean (SRM) was calculated as the overall mean change score divided by the SD of the change scores, while Guyatt's responsiveness index (GRI) was calculated as the mean change score in the adalimumab group divided by the SD of the change scores in the placebo group at week 6; values ≥0.8 were judged to represent a large degree of responsiveness.16 Inter-reader agreement was assessed using a two-way random effects single measure model of intraclass correlation coefficient (ICC)1 3 based on absolute agreement. ICCs≥0.6 were considered to represent good reliability, and ≥0.8 very good reliability. Smallest detectable change (SDC) was calculated from baseline to week 24 as 1.96×SDdifference in change scores/(√2√k) for the k=2 readers (SJP and SK). All MRI results at the patient level are reported as the mean score of the two primary MRI readers (SJP and SK). All MRI results at the lesion level are reported for the most experienced primary reader (SJP) for conciseness; largely similar results were obtained for the other reader (SK, data not shown). No imputation of missing data was performed, 39 of 49 patients had complete data at week 48. All statistical analyses were performed using R V.3.4.0. BODY.RESULTS: The patients had mean age 38 years (range 20–61), 51% were males, 73% were HLA-B27 positive, mean symptom duration 12 years (range 0–45) and mean baseline Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) 6.4 (range 4.2–9.8). Twenty-eight patients (57%) fulfilled the modified New York radiographic criteria for ankylosing spondylitis. BODY.RESULTS.THE ANATOMICAL DISTRIBUTION OF INFLAMMATORY LESIONS AT BASELINE AND CHANGES DURING THE STUDY: At baseline, 40 patients (82%) had a CANDEN spine inflammation vertebral body subscore ≥1. Six patients (12%) had a CANDEN spine inflammation posterior elements subscore ≥1. Patients with inflammatory lesions in the posterior elements all had concomitant inflammatory lesions in the vertebral bodies, not necessarily at the same level. Overall, inflammatory lesions were seen at 161 (14%) of 1127 levels (table 1). Fourteen (9%) of the involved levels had inflammatory lesions of both vertebral bodies and posterior elements, 134 levels (83%) had inflammatory lesions of only the vertebral bodies and 13 levels (8%) had inflammatory lesions of only the posterior elements. Inflammatory lesions were most frequently located at the anterior, posterior, antero-lateral and postero-lateral corners of the thoracic or lumbar spine (figure 2). Facet joint inflammation comprised 26 (81%) of 32 inflammatory lesions in the posterior elements. Table 1 The anatomical distribution of inflammatory and structural lesions in the spine Cervical spine (%) Thoracic spine (%) Lumbar spine (%) Total spine (%) Inflammatory lesions  Anterior corner 8 (16) 20 (41) 25 (51) 35 (71)  Posterior corner 2 (4) 14 (29) 9 (18) 21 (43)  Non-corner 2 (4) 7 (14) 5 (10) 10 (20)  Antero-lateral corner –* 11 (22) 14 (29) 20 (41)  Postero-lateral corner –* 16 (33) 4 (8) 19 (39)  Transverse processes/ribs –* 2 (4) 1 (2) 2 (4)  Facet joints 3 (6) 4 (8) 3 (6) 6 (12)  Spinous processes 0 (0) 1 (2) 0 (0) 1 (2)  Soft tissue 0 (0) 0 (0) 0 (0) 0 (0) Structural lesions  Type  Anterior corner   Fat 8 (16) 22 (45) 16 (33) 27 (55)   Erosion 0 (0) 1 (2) 0 (0) 1 (2)   Bone spurs 3 (6) 0 (0) 2 (4) 3 (6)   Ankylosis 1 (2) 1 (2) 3 (6) 5 (10)  Posterior corner   Fat 4 (8) 8 (16) 11 (22) 16 (33)   Erosion 0 (0) 1 (2) 0 (0) 1 (2)   Bone spurs 1 (2) 0 (0) 0 (0) 1 (2)   Ankylosis 0 (0) 3 (6) 1 (2) 4 (8)  Non-corner   Fat 1 (2) 2 (4) 6 (12) 7 (14)   Erosion 1 (2) 4 (8) 2 (4) 5 (10)   Bone spurs 0 (0) 0 (0) 0 (0) 0 (0)   Ankylosis 0 (0) 3 (6) 1 (2) 3 (6)  Antero-lateral corner   Fat –* 8 (16) 9 (18) 14 (29)  Postero-lateral corner   Fat –* 6 (12) 3 (6) 6 (12)  Facet joints   Ankylosis 5 (10) 4 (8) 4 (8) 6 (12) Percentage of patients with a score ≥1 according to the Canada-Denmark spine MRI definitions by anatomical location and lesion category. Cervical spine is defined here as C2/C3 to C7/T1 (6 discovertebral units (DVUs)), thoracic spine as T1/T2 to T12/L1 (12 DVUs) and lumbar spine as L1/L2 to L5/S1 (5 DVUs). *Antero-lateral corner lesions, postero-lateral corner lesions and transverse processes/ribs were assessed only in the thoracic and lumbar spine. Figure 2The distribution at baseline of inflammatory and structural lesions across vertebral bodies and posterior elements at each level from C2/C3 to L5/S1. Bars represent the number of patients with lesion by type and anatomical location. BME, bone marrow oedema; ERO, erosion; FAT, fat lesion; NBF, new bone formation (ankylosis/bone spur); PE, posterior elements; VB, vertebral body. Corner inflammatory lesions of the vertebral bodies were seen in 131 (16%) of 833 thoracic or lumbar DVUs. Fifty-three of these DVUs (40%) had lesions visible on both central and lateral slices, while 60 DVUs (46%) had lesions that were only visualised on central slices and 18 DVUs (14%) had lesions that were only visualised on lateral slices. Most inflammatory lesions present at baseline had disappeared after 24 weeks, while new inflammatory lesions developed very infrequently (table 2). Table 2 The disappearance of inflammatory lesions of the spine between baseline and week 24 during adalimumab therapy and the development of new lesions by anatomical location Lesion type Disappearance: number of lesions no longer present at week 24/number of lesions present at baseline (%) Development: number of lesions present at week 24 but not at baseline/number of baseline zero scores (%) Disappearance: number of patients with disappearance of at least one lesion at week 24 (%) Development: number of patients with development of at least one lesion at week 24 (%) Anterior corner lesions 107/159 (67) 7/1657 (0.4) 23/42 (55) 3/42 (7) Posterior corner lesions 38/54 (70) 3/1766 (0.2) 14/42 (33) 2/42 (5) Non-corner lesions 15/27 (56) 1/1789 (0.1) 6/42 (14) 1/42 (2) Antero-lateral corner lesions 41/53 (77) 3/2571 (0.1) 14/42 (33) 3/42 (7) Postero-lateral corner lesions 58/69 (84) 3/2563 (0.1) 14/42 (33) 1/42 (2) Transverse processes/ribs 3/4 (75) 3/1312 (0.2) 2/42 (5) 2/42 (5) Facet joints 18/26 (69) 3/1794 (0.2) 5/42 (12) 2/42 (5) Spinous processes 1/2 (50) 0/908 (0.0) 1/42 (2) 0/42 (0) Soft tissue 0/0 (NA) 0/910 (0.0) 0/42 (0) 0/42 (0) NA, not available. BODY.RESULTS.CANDEN SPINE INFLAMMATION SCORE: DISCRIMINATION: The mean CANDEN spine inflammation score was 8.8 (SD 12) at baseline. The vertebral bodies subscore had a mean value of 8.2 (SD 11) and constituted the major part of the total score. The posterior elements subscore had mean value 0.6 (SD 1.7). The CANDEN spine inflammation score decreased significantly more in the adalimumab group than in the placebo group at week 6 (table 3, figure 3). At weeks 24 and 48, no significant differences between groups remained (see online supplementary table 1). In post hoc secondary analyses, similar results were found using ANCOVA with baseline score as covariate, except that the difference between groups in SPARCC MRI spine index (23-DVU) at week 6 was highly significant when using ANCOVA (P=0.008) (figure 3). SPARCC results by the SPARCC developer can be found in online supplementary table 2. 10.1136/rmdopen-2017-000624.supp1Supplementary file 1 Table 3 Construct validity as assessed by change in scores from baseline to week 6 in adalimumab group compared with placebo group Adalimumab Placebo Difference in change score from baseline to week 6, P value Baseline (n=25) Change at week 6 (n=24) Baseline (n=24) Change at week 6 (n=23) CANDEN spine total inflammation score 10.2 (13) −5.7 (9.2) 7.3 (10) −0.8 (3.0) 0.03  Vertebral body subscore 9.5 (12) −5.2 (8.3) 6.9 (9.9) −0.9 (2.6) 0.03   Anterior corner subscore 3.9 (5.6) −2.0 (3.8) 3.3 (4.3) −0.3 (1.2) 0.21   Posterior corner subscore 1.5 (2.6) −0.9 (1.8) 1.3 (2.4) 0.0 (0.4) 0.01   Non-corner subscore 1.5 (3.2) −0.3 (0.8) 0.9 (2.4) −0.2 (0.8) 0.21    Lateral corner subscore 2.5 (3.8) −1.9 (3.2) 1.5 (2.5) −0.4 (1.8) 0.03  Posterior elements subscore 0.7 (2.3) −0.5 (1.7) 0.4 (0.8) 0.1 (0.6) 0.16 SPARCC MRI spine index (23-DVU) 11.0 (14) −6.3 (10) 8.7 (13) −1.5 (3.7) 0.05 CANDEN spine erosion score 0.7 (1.7) 0.0 (0.2) 0.6 (0.9) 0.0 (0.1) 0.58 Fat Spondyloarthritis Spine Score 7.8 (11) 0.7 (2.7) 3.2 (5.5) 0.1 (0.6) 0.35 CANDEN new bone formation score 9.3 (26) 0.2 (0.6) 5.7 (12) 0.3 (0.9) 0.92 mSASSS 8.0 (15) NA 6.3 (12) NA NA BASDAI 6.3 (1.2) −2.4 (2.2) 6.4 (1.5) −0.6 (1.8) 0.01 All values as mean (SD). Significance tests for difference in change scores at week 6 between groups were calculated by Mann-Whitney U tests with continuity correction. Two patients in the adalimumab group had missing BASDAI values at week 6 and were excluded from the BASDAI results. MRI scores were the average of the two readers. Lateral corner subscore is the sum of antero-lateral and postero-lateral lesion scores. BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; CANDEN, Canada-Denmark; DVU, discovertebral unit; mSASSS, modified Stoke Ankylosing Spondylitis Spine Score; NA, not available; PE, posterior elements; SPARCC, Spondyloarthritis Research Consortium of Canada. Figure 3Mean values of CANDEN spine inflammation score and for vertebral body and posterior elements subscores, bars represent the SEM; as observed (n=49, 47, 42, 39). For comparison, change in SPARCC MRI spine index (23-DVU) is also shown. P values for difference between group at week 6 were calculated in post hoc secondary analyses by ANCOVA. Bar plots show changes from baseline to week 6 with P values for the difference between change values at week 6 calculated by Mann-Whitney U tests with continuity correction (per protocol); as observed (n=47). MRI scores were the average of the two readers. CANDEN,Canada-Denmark; DVU, discovertebral unit; SPARCC, Spondyloarthritis Research Consortium of Canada. BODY.RESULTS.CANDEN SPINE INFLAMMATION SCORE: RESPONSIVENESS: The CANDEN spine inflammation score demonstrated a large degree of responsiveness at week 6 (GRI 1.88) comparable to the responsiveness of the SPARCC MRI spine index (GRI 1.67). The vertebral body subscore had a higher responsiveness (GRI 1.99) than the posterior elements subscore (GRI 0.84). At week 6, BASDAI (GRI 1.32) was less responsive than the CANDEN spine inflammation score and the SPARCC MRI spine index (table 4). At weeks 24 and 48, the responsiveness of the CANDEN spine inflammation score was moderate (SRM 0.57 and 0.55) and comparable with the SPARCC MRI spine index (SRM 0.57 and 0.55). Table 4 Baseline distribution and responsiveness of Canada-Denmark spine inflammation and structural scores compared with SPARCC and BASDAI Measurement instrument Observed range Mean (SD) Median (IQR) GRI at week 6 SRM at week 6 SRM at week 24 SRM at week 48 Number of patients analysed 49 49 49 47 47 42 39 Canada-Denmark spine total inflammation score (range 0–582) 0–54 8.8 (12) 4 (1–12) 1.88 0.46 0.57 0.55  Vertebral body subscore  (range 0–456) 0–48 8.2 (11) 4 (1–12) 1.99 0.47 0.57 0.55   Anterior corner subscore 0–23 3.6 (5.0) 2 (1–5) 1.64 0.40 0.57 0.59   Posterior corner subscore 0–12 1.4 (2.5) 0 (0–2) 2.56 0.33 0.46 0.45   Non-corner subscore 0–12 1.2 (2.8) 0 (0–0) 0.35 0.30 0.34 0.27   Lateral corner subscore 0–16 2.0 (3.2) 1 (0–3) 1.08 0.45 0.48 0.49  Posterior elements subscore (range 0–126) 0–10 0.6 (1.7) 0 (0–0) 0.84 0.18 0.37 0.35 Canada-Denmark spine erosion score (range 0–320) 0–7 0.7 (1.4) 0 (0–1) 0.20 0.00 0.21 0.19 Fat Spondyloarthritis Spine Score (range 0–456) 0–39 5.5 (8.8) 2 (0–7) 1.25 0.21 0.38 0.38 Canada-Denmark spine new bone formation score (range 0–460) 0–123 7.5 (20) 0 (0–3) 0.22 0.37 0.51 0.48 SPARCC MRI spine index (23-DVU) (range 0–414) 0–52 9.8 (13) 4 (1–14) 1.67 0.48 0.57 0.55 mSASSS 0–61 7.2 (14) 2 (0–6) NA NA NA 0.35 BASDAI (range 0–10) 4.2–9.8 6.4 (1.3) 6.3 (5.4–7.2) 1.32 0.69 1.64 1.96 Median, range and IQR are for all 49 patients. Responsiveness was assessed by SRM and GRI for all patients who had an MRI scan performed at the follow-up timepoint. MRI scores were the average of the two readers. Lateral corner subscore is the sum of antero-lateral and postero-lateral lesion scores. BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; DVU, discovertebral unit; GRI, Guyatt’s Responsiveness Index; mSASSS, modified Stoke Ankylosing Spondylitis Spine Score; SPARCC, Spondyloarthritis Research Consortium of Canada; SRM, standardised response mean. BODY.RESULTS.CANDEN SPINE INFLAMMATION SCORE: CONSTRUCT VALIDITY: The CANDEN spine inflammation score was highly correlated with SPARCC MRI Spine Inflammation Index at baseline (rho=0.98, P<0.001), and changes in these two MRI measures were highly correlated (from week 0 to 6: rho=0.90, P<0.001; from week 0 to 24: rho=0.96, P<0.001). The CANDEN inflammation score correlated weakly with C-reactive protein (CRP) (rho=0.21, P=0.14), with the posterior elements subscore reaching statistical significance (rho=0.37, P=0.008), while the vertebral body subscore did not (rho=0.19, P=0.20). This was similar in magnitude to the correlation between SPARCC MRI Spine Inflammation Index and CRP (rho=0.18, P=0.22). No significant correlations were found at baseline between CANDEN spine inflammation score and visual analogue scale (VAS) pain (rho=−0.13, P=0.37), BASDAI (rho=−0.11, P=0.45), Bath Ankylosing Spondylitis Functional Index (BASFI) (rho=−0.06, P=0.70) or Ankylosing Spondylitis Disease Activity Score (ASDAS) (rho=0.14, P=0.35). Changes in CANDEN spine inflammation score were not significantly correlated with changes in these clinical measures of disease activity and in CRP at week 6 or week 24 (data not shown). BODY.RESULTS.THE ANATOMICAL DISTRIBUTION OF FAT METAPLASIA AT BASELINE AND CHANGES DURING THE STUDY: The most frequent structural lesions at baseline were fat anterior corner lesions in the thoracic and lumbar spine (figure 2). At the patient level, a decrease in CANDEN spine inflammatory score from baseline to week 24 was associated with an increase in the FASSS score (rho=−0.59, P<0.001). The mean increase in FASSS at week 24 was 1.7 (range −4 to 24). Change in FASSS at week 6 tended to be higher in the adalimumab group than the placebo group (table 3). Twenty-four (3%) of 906 DVUs had an increase in fat score at week 24, 21 (88%) of these had a concomitant decrease in BME score, while 3 (13%) had no concomitant decrease in BME. Eighty-three DVUs had a decrease in BME without a concomitant increase in fat score at week 24. A decrease in fat at week 24 was observed in 5 DVUs. At the individual lesion level, 7 (3%) of 233 fat lesions present at baseline had disappeared at week 24, while 51 new fat lesions had developed, 43 (84%) of the new fat lesions were located in exactly the same locations where inflammation had been present at baseline. BODY.RESULTS.STRUCTURAL SCORES: RESPONSIVENESS, CONSTRUCT VALIDITY AND COMPARISON WITH RADIOGRAPHY: FASSS showed large responsiveness (GRI 0.41) at week 6, but low responsiveness (SRM 0.28 and 0.27) at weeks 24 and 48. Responsiveness for the CANDEN spine erosion score and the CANDEN spine new bone formation score was low during 48 weeks of follow-up, similar to mSASSS of radiographs (table 4). The CANDEN spine new bone formation score correlated with clinical examination (Bath Ankylosing Spondylitis Metrology Index (BASMI), rho=0.41, P=0.003) as well as with radiographic scores (mSASSS, rho=0.49, P<0.001). No significant correlations of the CANDEN spine erosion score were found. MRI demonstrated new bone formation in nine (18%) patients at baseline (from C2/C3 to L5/S1). Ankylosis was found in eight (16%) patients; five patients had ankylosis of the vertebral bodies and six patients had ankylosis of the facet joints. In comparison, lateral radiographs showed new bone formation in 26 (53%) patients at baseline (from C2/C3 to C7/T1 and from T12/L1 to L5/S1); 9 patients had ankylosis of the vertebral bodies. Thus, MRI was markedly less sensitive in the lumbar and cervical areas than radiography, while the specificity for any spur/ankylosis by MRI was 99.6%, with radiographs as reference standard. Erosions and new bone formation remained largely unchanged as assessed by MRI. In comparison, two new bone spurs and ankylosis at three DVUs developed between baseline and week 48 as judged by radiographic scoring. BODY.RESULTS.INTER-READER RELIABILITY AND SMALLEST DETECTABLE DIFFERENCE: CANDEN spine inflammation score had good to very good reliability for both status scores (inter-reader ICC of the 4 timepoints mean 0.82) and change scores (from baseline to the 3 follow-up timepoints mean 0.72), similar to that of the SPARCC MRI spine index (ICC 0.82 for status score, 0.76 for change scores). Inter-reader reliability of SPARCC for reader pairs WM-SJP/WM-SK was 0.80/0.79 for status scores and 0.86/0.83 for change scores, which confirms that the two CANDEN readers of this study (SJP and SK) were well calibrated according to the SPARCC method. The FASSS had a very good reliability (ICC mean 0.92 for status score and 0.71 for change score, respectively). Reliability for status score was good to very good for CANDEN spine erosion score (mean 0.61) and CANDEN spine new bone formation score (mean 0.75), while reliability for change scores were poor (all <0.40). At week 24, SDC was 7 points for the CANDEN total spine inflammation score; in comparison SPARCC score had a SDC of 8 points; six patients had an improvement in inflammation ≥SDC using either scoring method. The FASSS had a SDC of 2 points; at week 24, 12 patients had an increase above this threshold. Only limited changes in erosion score and new bone formation score were observed. BODY.DISCUSSION: We have developed and preliminarily validated a new, comprehensive set of anatomy-based MRI outcome measures for spinal inflammation (CANDEN spine inflammation score) and structural changes (CANDEN spine new bone formation score, CANDEN spine erosion score) in patients with axSpA in a randomised controlled trial based on previously published CANDEN MRI definitions. The inflammatory component, the CANDEN spine inflammation score, possessed face, content and construct validity as well as good sensitivity to change and discrimination. Inflammation in axSpA involves different tissues, such as the fibrocartilaginous joints between the vertebral bodies that include the ring apophysis where the annulus fibrosus inserts, the synovial facet joints and costotransverse joints and the complex synovial costovertebral joints where most ribs articulate with facets on the margins of adjacent thoracic vertebral bodies and the intervertebral disc, as well as numerous entheseal sites, especially in the posterior elements. These different sites may respond differently to drugs with various modes of action. Thus, the detailed CANDEN system may allow an increased understanding of how different drugs may modify different aspects of the spinal disease. In contrast to currently used MRI scoring methods of the spine such as the SPARCC system, which is based on assessment of the vertebral bodies on three consecutive slices at each discovertebral unit, and does not record whether inflammation is present as non-corner lesions, lateral lesions or anterior corner lesions, this anatomy-based assessment system provides this information. The system measures inflammation in the entire spine, including different parts of the posterior elements of the spine, providing greater face and content validity. First, the posterior elements constitute a large volume of the vertebrae and have large surface areas with insertions of ligaments and tendons, and there is no reason why inflammation here should be less clinically relevant to the patients. Second, the specificity of inflammation of the costotransverse, costovertebral and facet joints may be greater for axSpA, compared with the corner and particularly the non-corner inflammatory lesions of the vertebral bodies, which may be seen in healthy controls and in patients with degenerative disc disease.17–19 The pattern and prevalence of inflammatory and structural changes at different anatomical locations in a population of patients starting TNF inhibitor treatment was described, and found to be highest in the lower thoracic and lumbar spine. The frequency of posterior findings was lower than expected, as only six (12%) patients had inflammatory lesions of the posterior elements in this trial. Lesions in the posterior elements only gave a minor contribution to the total mean scores. Interestingly, all six patients with inflammatory lesions of the posterior elements were HLA-B27 positive and fulfilled the radiographical criteria for ankylosing spondylitis (AS), and 4 (67%) were clinical responders at week 24. This may suggest that posterior elements are rarely involved in nr-axSpA, but this needs further investigation. Other studies have reported higher prevalences of involvement of the posterior elements. Bochkova et al found inflammatory lesions of the posterior elements in 22 (76%) of 29 patients that all fulfilled the radiographic criteria for AS and almost all (28 (97%) of 29) were HLA-B27 positive.20 They performed additional axial images at painful regions of the spine in case of normal sagittal images, which may increase sensitivity. Maksymowych et al found inflammatory lesions of the posterior elements in 28 (88%) of 32 patients with AS.14 Althoff et al found inflammatory lesions of the posterior elements more often in the AS group (10 (26%) of 39) than in the non-radiographic axSpA group (3 (8%) of 36).21 Larbi et al found inflammatory lesions in 23 of 112 (21%) of recently diagnosed HLA-B27-positive patients with SpA.22 In comparison, in our study 36 (73%) were HLA-B27 positive and 28 (57%) fulfilled the radiographical criteria for AS. Thus, the lower prevalence of posterior inflammatory lesions in our study may be due to a different case mix of patients compared with the other studies. Most vertebral non-corner inflammatory lesions that were present at baseline had disappeared after 24 weeks (56%), and the number of new similar lesions that developed during the trial was minimal. Still, other inflammatory lesions such as anterior and posterior corner lesions disappeared even more frequently during treatment. Non-corner lesions are often seen as a part of endplate lesions similar to a Modic type I lesion, and disc degeneration rather than spondyloarthritis will be a probable cause of some of these lesions. Rennie et al have reported that lesions in 20% of inflamed vertebral bodies were only visible on 'lateral' sagittal MRI slices.23 A comparable result was reached in our study, where 14% of inflamed vertebral bodies were only visualised on the lateral slices. The spatial distribution of syndesmophytes along the vertebral rim has been demonstrated by CT to involve most of the circumference, preferentially the postero-lateral rim,23 and including as much of the rim as possible in the scoring should increase the sensitivity of the instrument. This underlines the need for assessing central sagittal slices, and lateral sagittal slices, if a comprehensive score for all inflammatory lesions in the vertebral bodies is needed. The SPARCC spine method does assess lateral inflammation in the vertebral body, as the three consecutive slices approach can be applied to lesions in the lateral slices in the same manner as for central slices. At baseline, only six patients (12%) had inflammatory lesions in the posterior elements, and all these had also lesions in the vertebral bodies. The GRI for the total CANDEN spine inflammation score and for the vertebral body subscore were almost identical. The addition of posterior elements data to the vertebral body subscore did not improve responsiveness. The low frequency of posterior lesions in this study likely accounts for the lack of impact of including these elements. The responsiveness measures SRM and GRI assume that change scores are normally distributed; many patients in this study had little spinal involvement and therefore no change over time, and these responsiveness measures would likely have been higher if the case mix had consisted of more patients with substantial spinal involvement. Importantly, the responsiveness of the CANDEN spine inflammation score was similar to that of the SPARCC MRI spine index. It is important to note that a method that focuses on assessing presence/absence and size of individual lesions, such as CANDEN, may perform differently than a method that has been designed to assess the total amount of inflammation, independent of location, such as SPARCC, in groups of patients with many spinal lesions as compared to those with few. Consequently, comparison of responsiveness and discrimination will be required in larger cohorts with a broader range of spinal lesions. In this study, the fat metaplasia score (FASSS) tended to increase over time, and week 24 change in FASSS correlated with the disappearance of inflammatory lesions. This temporal association, and the fact that 84% of new fat lesions occurred in areas in which inflammation had been present at baseline, further validates the FASSS score as a measure of structural changes caused by inflammation. Bone spurs and ankylosis of the vertebral bodies were observed more frequently by radiography than MRI, but MRI allowed an assessment of the thoracic spine and facet joints that are difficult to delineate on lateral radiographs and that are not included in the mSASSS score for radiographic changes. Further studies with longer follow-up are needed to clarify the clinical importance of these structural measures as well as finding possible ways to improve their reliable detection by MRI. It is important to note that the CANDEN scores are not intended to be used to diagnose axSpA, but rather to allow detailed monitoring of changes over time in patients who have already been diagnosed with axSpA. The value of the CANDEN system is that it allows more detailed analyses of the link between inflammation and structural changes and their development over time and at the same time may provide a patient-level score for spinal inflammation. Ten patients did not complete the trial,15 but since their baseline characteristics were similar to the other patients (data not shown), the results presented here were likely not impacted. In conclusion, the present study documents the construct validity, responsiveness, discriminatory ability and reliability of the CANDEN spine inflammation score. The method is a promising outcome measure in patients with axSpA, as it both allows assessment of the total spinal inflammation, and also separate analyses of various articular, entheseal and discovertebral components of the disease. Through comprehensive assessment of all parts of the spine, it has inherently good face and content validity. The structural CANDEN spine scores showed good cross-sectional reliability, but further studies with longer follow-up are needed to clarify the validity and utility in clinical trials and longitudinal cohorts.

TITLE: Evaluation of adjunctive systemic doxycycline with non-surgical periodontal therapy within type 2 diabetic patients ABSTRACT.OBJECTIVES:: To evaluate the effects of systemic doxycycline on clinical and microbiological parameters of diabetic subjects with chronic periodontitis. ABSTRACT.METHODS:: This 9-month multi-center, randomized, parallel, single-blinded study was conducted from different hospitals in Riyadh, Saudi Arabia between April 2010 and December 2010. A total of 76 diabetic subjects with chronic periodontitis were randomized into 2 groups: control group (CG) received only scaling and root planing (SRP), and the treatment group (TG) receiving systemic doxycycline during the reevaluation visit 45 days after the completion of SRP. Probing pocket depth, clinical attachment level, gingival index, plaque index, and bleeding on probing were collected at baseline, 45 days after SRP, and one, 3, and 6 months after the use of systemic doxycycline. Microbiological analysis comprised the detection of Tannerella forsythia (Tf), Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Prevotella intermedia (Pi) by polymerase chain reaction method. ABSTRACT.RESULTS:: Sixty-eight (33 CG and 35 TG) subjects completed the study. Greater reduction in the population of Tf, Pg, and Pi were observed in TG compared with CG in the first month after the administration of systemic doxycycline. The TG showed a significant improvement in gingival index scores compared with the CG (p<0.05) by the end of the first and 6 months after the administration of doxycycline. ABSTRACT.CONCLUSION:: Adjunct systemic doxycycline can be associated with a reduction of Tf, Pg, and Pi in the first month after the administration of doxycycline with an improvement in the GI. BODY: The relationship between periodontal diseases (PD) and diabetes are bidirectional,1 which provides an example of systemic disease predisposing to oral infection, established infection, and exacerbating the systemic disease.2 Diabetic patients are known to be susceptible to infectious diseases.3 The influence of diabetes on the onset and development of PD has been widely studied.4,5 The PD is now considered the sixth complication of diabetes mellitus.6,7 Diabetes in itself does not cause PD, but it makes the patients more susceptible to periodontal destruction. Diabetics exhibit greater severity and a faster rate of PD progression.8,9 Treating chronic periodontitis in diabetic patients poses numerous challenges, especially with regards to the control of oral microorganisms. Treatment of PD is directed to the elimination of sub-gingival bacterial infections. More than 700 different bacterial species are found in the sub-gingival environment.10 Several studies have reported that the bacteria involved in periodontitis are usually anaerobic Gram-negative bacteria.11 Aggregatibacter actinomycetemcomitans (Aa), Tannerella forsythia (Tf), and Porphyromonas gingivalis (Pg) were directly implicated in the destruction of periodontal tissues, and the presence of any of these pathogens is considered a risk factor for future periodontal destruction.12 Direct and indirect damage to periodontal supporting tissues are well-documented pathogenic effects of Gram-negative bacteria due to their toxic products and the activation of a series of inflammatory reactions.13 Mechanical means using non-surgical and surgical techniques is the primary approach employed in the treatment of PD. Subgingival scaling and root planning (SRP) is an effective method to slow or arrest the progression of PD.14,15 Even after meticulous SRP, some patients may still experience continuous loss of attachment due to the inability of the therapy to suppress periodontal pathogens to the optimal levels.16 The Aa and Pg, is likely to evade SRP, especially in the subgingival niche, due to limited access to the root surface and the tissue-invading skills of the bacteria.17 The efficacy of SRP may also be compromised by the remaining bacterial virulence factors and ineffective personal plaque control.18 Thus, various pharmacological agents were used as adjunctive therapy to improve the treatment outcome of non-surgical periodontal therapy.19 Adjunctive antimicrobials can be used for local or systemic delivery. The benefits of using various adjunctive antibiotics such as penicillin, tetracycline, amoxicillin, and metronidazole along with SRP have been reported to improve periodontal health.20-22 Studies that evaluated the antimicrobial effect of systemic doxycycline on the periodontal tissues of diabetic patients are scarce. This study aims to investigate the effect of SRP in conjunction with the administration of an antimicrobial dose of systemic doxycycline on the clinical and microbiological parameters in diabetic patients with chronic periodontitis. BODY.METHODS: This 9-month multi-center, randomized, parallel, single-blinded study was conducted between April 2010 and December 2010 at King Faisal Specialist Hospital & Research Center, King Abdulaziz Medical City, Naval Base Hospital, and Sultan Bin Abdulaziz Humanitarian City, Riyadh, Saudi Arabia. A total of 76 diabetic subjects with chronic periodontitis were included in this study. This study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000, and the protocol of this study was approved by the research ethics committee of Sultan Bin Abdulaziz Humanitarian City, Riyadh, Saudi Arabia. Informed written consent was acquired from each subject that agreed to participate voluntarily. BODY.METHODS.STUDY POPULATION: Diabetic subjects with moderate to severe chronic periodontitis were recruited into this study following a screening examination, including full mouth probing and radiographic evaluation. The inclusion criteria were as follows: age between 21 and 80 years, type 2 diabetes, diabetes diagnosed for ≥1 year, good physical condition with no additional serious medical conditions, the presence of at least 16 teeth, and a minimum of 8 sites with pocket depth of >5mm and clinical attachment level >5mm. Subjects were excluded if they: 1) were using any contradicted medications, 2) smoked within the past 5 years, 3) presented with compromised medical condition, 4) had been treated with SRP within 6 months of the baseline visit, 5) used antibiotics within 3 months prior to the study or 6) pregnant or breast feeding. Subjects who failed to appear at any time after the baseline visit or failed to attend the last visit (6 months) were also excluded from the study. The study included 76 diabetic patients with chronic periodontitis who were randomized by a computer-generated system into 2 groups. Of these, 38 subjects were assigned to the control group (CG), for whom only SRP would be provided. Three subjects from this group discontinued the study, and 2 were excluded due to lack of compliance with the medication use and visits. Three out of the 38 subjects assigned to the treatment group (TG) were excluded due to lack of compliance. BODY.METHODS.MICROBIOLOGICAL SAMPLING: Bacterial examination for pathogenic anaerobes: Tannerella forsythia, Aa, Pg, and Prevotellaintermedia (Pi) was performed by using the polymerase chain reaction (PCR) method. For each patient, 4 sites with pocket depths of ≥5 mm were randomly selected. The supragingival plaque was removed for the selected teeth, and the Florida probes were wiped with 70% isopropyl alcohol between measurements to reduce bacterial cross-contamination of the sites.23 After the clinical measurements were recorded, a subgingival plaque sample was taken from each site with separate sterile curettes. Each sample was instantly placed in a sterile micro centrifuge tube containing 0.5 ml Trisethylenediaminetetraacetic acid (EDTA [TE]) buffer (10 mM Tris hydrogen chloride [HCl] [pH 7.6], and 1 mM EDTA [pH 8.0]). For PCR analysis, 90 ml of vortex-mixed subgingival plaque was added to 10 μl of 10 x lysis buffer (100 mM Tris-HCl, pH 8.0, 10 mM EDTA, 10% Triton X-100), and boiled for 5 min, and 5 μl of this lysate was used in each PCR reaction. The species specific primers used for the PCR analyses are shown in Table 1. The PCR amplification was carried out in a reaction volume of 25 μl consisting of 5 μl sample lysate, 20 μl reaction mixture containing 1 x PCR buffer (10 mM Tris-HCl, pH 8.8, 1.5 mM magnesium chloride, 50 mM potassium chloride, 0.1% Triton X-100), 2 units of Taq DNA polymerase (Amersham Pharmacia Biotech Inc, Piscataway, NJ, USA), 0.2 mM deoxynucleotide triphosphates, and 100 pmol of each primer. The PCR cycling was carried out in a DNA thermal cycler PE 480 (PerkinElmer, Covina, California, USA). The cycling conditions for Tf, Aa, and Pg consisted of an initial denaturation for 5 min at 95°C, 35 amplification cycles of denaturation at 95°C for one minute, annealing of primers at 55°C (Aa) or at 60°C (Tf) or 70°C (Pi & Pg) for one minute, and primer extension at 72°C for one to 2 minutes, followed by a final extension step at 72°C for 2-10 minutes. The cycling conditions for Pi were the same as those for Tf, Aa, and Pg, except if the annealing temperature was 58°C. The reaction products were examined immediately after completion of the PCR. Ten microliters of each reaction product was fractionated in a 1.5% agarose gel containing ethidium bromide (0.5 mg/ml) with 50 or 100 bp DNA ladder (Amersham Pharmacia Biotech Inc, Piscataway, NJ, USA) as a size marker, and visualized and photographed using a gel documentation and analysis system (Ultra-Violet Products Ltd, Cambridge, England). Samples from the same sites were collected for bacterial analysis at baseline, reevaluation visit (fourthy-fifth day after SRP), and at one, 3, and 6 months after treatment with systemic doxycycline. Table 1 Primers for polymerase chain reaction-based identification of periodontopathogens. BODY.METHODS.CLINICAL PROCEDURES: During the study, clinical dental examinations for all subjects were carried out in 4 study centers by 4 trained and pre-calibrated examiners (periodontists) who were supported by trained dental assistants. Probing depth (PD), clinical attachment level (CAL), plaque index (PI), gingival index (GI), and bleeding on probing (BOP) were measured on all existing teeth at the mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual areas24 with manual periodontal probes (Florida Probe Corporation, Gainesville, FL, USA). The CAL was calculated by adding PD with the distance between cemento-enamel junction and gingival margin. After baseline data collection, each subject received 6-8 sessions of scaling and root planing with ultrasonic and hand instrumentation until the root surface felt smooth with the tip of a metallic probe. Four trained dental hygienists at 4 centers performed the SRP. The dental hygiene aids were provided for the subjects. Written oral hygiene guidelines were given to all subjects within the different treatment groups at each session, including proper tooth brushing technique. After 45 days of completion of SRP, the patients were recalled for the reevaluation visit and the clinical measurements and sub-gingival plaque samples were taken. At this time, all individuals underwent sub-gingival debridement with an ultrasonic device to disturb the sub-gingival bio-film and allow better action of the drug. Randomization of the subjects into treatment and control groups was performed by using computer-generated random codes. Subjects belonging to the treatment group received an antimicrobial dose of systemic doxycycline, 100 mg per day for 14 days with a loading dose of 200 mg on the first day. The control group received no treatment other than oral hygiene during the study. They were instructed to brush and floss 2 times a day. Clinical measurements were taken at each of the subsequent visits at the first, third, and sixth months. After obtaining the measurements and samples from the subjects, they were provided with professional supragingival plaque control measures, and oral hygiene was reinforced. This was repeated in the following recall visits. BODY.METHODS.DATA ANALYSIS: All statistical analyses were carried out using the Statistical Package for Social Sciences for Windows, Version 17.0 (SPSS Inc., Chicago, IL, USA). The presence or absence of Tf, Pg, Pi, and Aa were evaluated with McNemar's test, and the results are presented in terms of the percentage of sites with the microorganisms. The results of dental parameters (PPD, CAL, GI, PI, and BOP) were analyzed using the 2-way analysis of variance (ANOVA). A value of p<0.05 was considered statistically significant. BODY.RESULTS: Of the 76 subjects included in the study, 68 (33 CG and 35 TG) subjects completed the study. Most of the subjects (61.8%) were male, and the mean age of the participants was 42±6.41 years. On average, the subjects had 27 teeth in the oral cavity. The analysis of the periodontal parameters is shown in Table 2. There were significant (p<0.05) changes in PPD, PI, GI, and BOP for all the follow-up visits when compared with the baseline measurements. The PPD was reduced by 0.93 mm for the treatment and 0.88 mm for the control group in the reevaluation visit, which was statistically significant (p<0.01). The CAL for CG was reduced by 0.84 mm during the reevaluation visit and by 0.96 mm from the baseline measurements by the end of 6 months. For TG, CAL was reduced by 0.39 mm (p=0.09) in the reevaluation visit and by 0.74 mm by the end of 6 months. However, no significant difference was observed between CG and TG in the reevaluation visit or during any of the remaining follow-up visits. Gingival index was the only parameter that showed any significant difference between TG and CG after one, 3, and 6 months after the administration of systemic doxycycline (Table 2). Table 2 Dental parameters of diabetic study subjects at baseline, re-evaluation, one, 3, and 6 months after administering systemic doxycycline. The results showing the effects of different treatments on Tf are described in Figure 1A. In the first month after administering doxycycline, a significantly higher decline in the percentage of sites harboring Tf was observed for TG compared with CG (p=0.02). However, a better reduction was observed in the CG as compared with the TG at the third-month visit, though this reduction was not statistically significant (Table 3). Figure 1Percentage of site harboring A) Tannerella forsythia (Tf), B) Porphyromonas gingivalis (Pg), C) Prevotella intermedia (Pi), and D) Aggregatibacter actinomycetemcomitans (Aa) as measured at the different time intervals among 76 diabetic patients with chronic periodontitis. CG - control group, TG - test group Table 3 Percentage reduction in sites harboring different species at each follow up visit compared to the values obtained in the previous visit among 76 diabetic patients with chronic periodontitis. The results showing the effect of systemic doxycycline on Pg and Pi are shown in Figures 1B & 1C. One month after the administration of doxycycline, both Pg, and Pi showed significant reduction in the percentage of site for TG compared with CG (p<0.01 and p=0.04). A minimal decline was observed in the following visits. There was no significant difference between CG and TG at different time points of the reduction in the percentage of sites with Aa (Figure 1D). The percentage reductions in the sites harboring the different species at each of the follow-up visits are shown in Table 3. BODY.DISCUSSION: Periodontal disease involves complex interactions between microbial factors, and a susceptible host.25 One of the biological mechanisms reported is through the accumulation of glucose-mediated advanced glycation end products (AGEs), which leads to the impairment of the chemotactic and phagocytic functions of polymorphonuclear leukocytes. The bacterial products of the Gram-negative periodontopathic bacteria in periodontal pockets were found to increase the secretion of interleukin-1 beta (IL-1b), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFa), and prostaglandin E2 (PGE2). The TNFa is a pro-inflammatory cytokine that has been implicated in insulin resistance. Adjunctive antimicrobial periodontal treatment has been reported to significantly reduce circulating TNFa and glycosylated hemoglobin (HbA1C) levels.26,27 Also, doxycycline at lower doses has been reported to reduce the periodontal tissue destruction by inhibiting collagenase activity and synsthesis.28 The reduction in HbA1c levels with the use of low dose doxycycline has been previously reported.29 In this study, the clinical and microbial effect of an antimicrobial regimen of systemic doxycycline on the periodontal tissues of diabetic patients with chronic periodontitis was evaluated. The occurrence of periodontal pathogens in subgingival flora in periodontitis is a risk for periodontal disease progression.30,31 Therefore, microbiological diagnostic procedures are justifiably indicated in the detection of pathogens as well as in monitoring of therapeutic success and result of the disease. Specific bacterial species are now considered to be vital in the initiation and progression of periodontitis, and Aa, Pg, Tf, and Pi30,32 are some of the more frequently encountered species. Doxycycline has been reported to substantially reduce or eliminate pathogenic species, especially Gram-negative bacilli.33 Several studies34 have reported that due to the tissue-invading nature of periodontal pathogens such as Pg and Aa, mechanical therapy alone may not be sufficient to eliminate these pathogens. Hence, it may be useful to administer antibiotics that will help in eliminating these pathogens. In this study, the administration of systemic doxycycline was effective in the reduction of Tf, Pi, and Pg. The greatest reduction in the percentage of sites after the administration of doxycycline was observed for Pg. This finding is agreeing with results from a previous study performed on smokers.12 Porphyromonas gingivalis induces a local chronic inflammatory response that results in oral inflammatory bone destruction, which manifests as periodontal disease.35 Eradication of Aa from deep periodontal pockets could be difficult. It is reported that the decrease in Aa is often not as great as that for other bacteria following SRP.36 A study by Muller et al,37 reported that Aa at subgingival and extra-crevicular sites are suppressed with the use of adjunct antimicrobial therapy. Similarly, in this study, the reduction of Aa was observed, although it was less than that observed with other pathogens. Additionally, in the case of Aa and Tf, there was a slight increase in the percentage of sites after 3 months. This is similar to the study by Colombo et al,38 where the frequency of Aa increased after therapy, probably due to an absence of antibiotic protection. Compared with the baseline measurements, all the clinical parameters in both, the CG and TG had significantly improved at the reevaluation visit during the follow up visits. However, with exception to GI, no other clinical parameters showed a significant difference between the CG and TG. The GI significantly improved with the administration of adjunct systemic doxycycline. This could be due to the inhibitory effect of doxycycline that significantly reduces the periodontal tissue destruction by inhibiting collagenase activity and synthesis.28 Many previous studies28,39 administered adjunctive antimicrobial agent along with SRP. However, to understand the effectiveness of an antimicrobial drug, it should be introduced after SRP; however, only microorganisms that were resistant to SRP would be present.40 Also, it is well known that the number and percentage of Tf, Pg, and Pi decrease with decreasing pocket depth with or without treatment.41 Therefore, it would not be possible to state conclusively the role of the antibiotic in the reduction of the microbial population. Hence, during the reevaluation visit, doxycycline was administered 45 days after SRP. BODY.DISCUSSION.STUDY LIMITATIONS: The major limitation of this study was the limited number of patients and limited number of bacteria examined. Further research is needed to support a causal relationship and its clinical implications. In conclusion, the TG showed significant reduction of Tf, Pg, and Pi in the first month after the administration of systemic doxycycline. However, this did not improve the periodontal status of the subjects. There were minimal differences between CG and TG at the 3 and 6-month follow-up visits. Additional long-term studies with larger sample sizes are required to provide a more comprehensive picture of the effects of SRP and systemic doxycycline at clinical and microbial levels in diabetic patients with chronic periodontitis.

TITLE: Exposure to Poverty and Productivity ABSTRACT: We study whether exposure to poverty can induce affective states that decrease productivity. In a controlled laboratory setting, we find that subjects randomly assigned to a treatment, in which they view a video featuring individuals that live in extreme poverty, exhibit lower subsequent productivity compared to subjects assigned to a control treatment. Questionnaire responses, as well as facial recognition software, provide quantitative measures of the affective state evoked by the two treatments. Subjects exposed to images of poverty experience a more negative affective state than those in the control treatment. Further analysis shows that individuals in a more positive emotional state exhibit less of a treatment effect. Also, those who exhibit greater attentiveness upon viewing the poverty video are less productive. The results are consistent with the notion that exposure to poverty can induce a psychological state in individuals that adversely affects productivity. BODY.INTRODUCTION: The state of poverty influences productivity in at least two different ways. On the one hand, financial constraints dampen physical and cognitive performance through nutritional deficiencies [1, 2], low educational quality [3, 4], and poor health conditions [5, 6], which in turn affect productivity. On the other hand, a recent literature underscoring the psychological aspects of poverty has identified additional channels through which poverty affects individual decisions in a way that can become counterproductive. These mechanisms include risk and time preferences [7] or individuals' motivations and aspirations [8, 9]. According to [7], the economic and social conditions under which poor people live may lower their willingness to take risks and to forgo current income in favor of higher future incomes, even though the intrinsic time and risk preferences of the poor may be identical to those of wealthier people. One plausible explanation may be that the poor are more liquidity constrained. Because of this tighter constraint, if a poor individual has the choice between a current and a delayed payment in an experiment, he or she may opt for the current payment. Similarly, the anticipation of future liquidity constraints may also induce an individual to prefer a safe payment over a risky payment. Regarding aspirations, [8] observe that, due to lower access to credit, less influential contacts or less access to relevant information, poverty makes it harder for the poor to achieve a given outcome, ceteris paribus. This exacerbates the adverse effects of a behavioural bias that both poor and wealthier people may have in setting aspirations. As a consequence, the poor are more likely to choose a low aspiration level and effort relative to the best outcome they could achieve. Our focus in this research is on a particular aspect of the psychology of poverty. There may be psychological effects arising from exposure to the poverty of others that an individual is in contact with, which are distinct from those arising directly from one's own experience of poverty. We study whether the affective state associated with exposure to the poverty of others, on its own, leads to lower individual productivity. Such an effect would exist above and beyond the consequences of other difficulties that the poor face. We study the link between exposure to poverty of others and productivity in a controlled setting, where the effect can be isolated from other factors and affect can be precisely measured. We construct an experimental environment designed to induce the affective load associated with exposure to others' poverty, but without the physical, social and economic consequences of one's own poverty. We do so by providing individuals with minimal exposure to conditions of poverty suffered by others. We operate under the assumption that the emotions induced by longer, more intense, and more personal, exposure to poverty than those we create here would be at least as strong. If exposure to the poverty of others reduces work performance, it would suggest that removing poor individuals from such exposure might increase their productivity. Indeed, in a recent paper, [10] show that young children assigned to the Moving to Opportunity program, in which poor American families are given vouchers which allow them to rent housing in more affluent areas, exhibited an increase in college attendance rates and income. These results corroborate some of the findings of [11]. The study finds that these improvements stem from a difference in the childhood experience of those assigned to the program. Among the aspects included in such experience, the authors highlight better education, greater safety, greater neighborhood satisfaction, and a lower incidence of single parent households. While one might presume that emotional factors are at work and contributing to the better outcomes of families in the program, the effect of improved emotional state cannot be distinguished from that of the other resources they have available. We differ from the existing literature in that we study whether the affects associated with mere exposure to the poverty of others, rather than with the experience of one's own poverty itself, have an effect on productivity. The exposure to poverty in our study is brief and not very intense. Nevertheless, we find that mere exposure to a video showing the reality of poverty for seven minutes has an effect on subsequent performance in a relatively simple task. It also induces a more negative emotional state. Detailed analysis of the data, however, suggests that the effect of exposure to poverty of others on performance is cognitive rather than emotional, as the exposure appears to impede the focus of attention on performing the task. Our experiment has two treatments. In the Poverty treatment, subjects watch a video clip that illustrates the conditions faced by a family in a state of poverty. In the Neutral treatment, which serves as a control condition, subjects observe a neutral video, known from other studies to evoke no strong emotional response. To measure individual productivity we use a real effort task. We employ the slider task introduced by [12], which consists of setting as many sliders as possible in the exact middle position of the available range, by moving a cursor with a computer mouse. The advantages of this task for our purposes are that it does not require specialized knowledge, the instructions are easy to follow, the output has no value to the experimenter (so that social preferences with regard to the experimenter are minimized as a consideration in participants' effort decisions), and it has been widely used previously in experimental economics. This last feature facilitates comparison of any effect sizes that we observe to previous and future studies. An individual's productivity is measured as the number of sliders that are correctly aligned in a 20-minute work period. We register psychological affects in two ways. First, we administer the PANAS questionnaire to participants immediately after they view the video clip. This questionnaire provides a subjective self-evaluation of the current intensity of a number of specific emotions [13], and allows for the construction of broader indices describing more general affective states. Second, we use a facial recognition software package, called Noldus FacereaderTM, to identify the intensity of the emotions evoked by the videos. We find that the subjects randomly assigned to the Poverty treatment exhibit lower average performance than those assigned to the Neutral treatment. Moreover, images of poverty evoke higher self-reported scores on measures of negative affect and attentiveness. The physiological data from the facereading software confirms that exposure to poverty induces an emotional state of more negative valence than does the control. Further analysis shows that: i) The difference in productivity between the two treatments is greater for individuals in a less positive emotional state after watching the video, and ii) subjects who display greater attentiveness when viewing the images of poverty display lower productivity. Our paper fits into a recent and active literature that focuses on the psychology of poverty. While previous research documents a relationship between affective state and poverty [7], we show that mere exposure to poverty of others can influence own affective state, which in turn affects productivity. Our work is also consistent with studies that associate positive emotional states with better performance in various tasks [14, 15, 16, 17, 18], in settings unrelated to poverty. It also relates to work that explores the role of poverty on cognition [19, 20, 21]. We find that subjects exposed to images of poverty report being more attentive after watching the video, and that those who were the most attentive displayed lower productivity. This suggests that for some individuals, exposure to poverty of others imposes a cognitive load that hampers their performance. BODY.EXPERIMENTAL DESIGN AND PROCEDURES: Our experiment employs human subjects. Our protocol was not approved by an Institutional Review Board. However, we are fully in compliance with Dutch Law, which does not require social science research to receive prior approval from an IRB. Although Tilburg University does not have an institutionalized IRB, the Director of CentERLab or the Scientific Director of CentER screens and authorizes the content and purpose of all of the experiments taking place in the laboratory. This particular study was reviewed and approved by the Scientific Director of CentER, Professor Geert Duijsters, after the research was conducted. He formally confirmed that the study was conducted according to the principles expressed in the Declaration of Helsinki, and that this work complied with Dutch laws and Tilburg School of Economics and Management's policy regarding the ethical treatment of human subjects. All subjects gave their signed written consent to participate in the study at the beginning of their experimental session, including consenting to be videotaped. Our dataset consists of 15 experimental sessions conducted in June, 2014 at the CentERLab at Tilburg University in the Netherlands. All subjects were students at the university. We used z-Tree [22] to implement the experiment. Subjects were recruited via an online system. On average, a session lasted approximately 45 minutes. Between five and ten subjects took part in each session, and no subject participated more than once in the experiment. There were two treatments, Poverty and Neutral, and upon arrival at the experimental laboratory, subjects were randomly assigned to one of the treatments. A total of 105 participants whose average age was 23, participated in the study, 55 in the Poverty treatment, and 50 in the Neutral treatment. In the Poverty treatment, subjects watched a video clip that depicted the struggles of a poor family living in a garbage dump in Moscow, Russia. In the Neutral treatment, subjects watched a video of the Alaskan landscape that is known not to evoke any emotion or mood, and has been used in psychological research to induce a state of neutrality [23]. We did not film the videos ourselves. They were publicly available online. The Poverty video is available in the following link https://www.youtube.com/watch?v=lDzhufj9GN0. A 2 minute version of the Neutral video is available in the following link https://www.youtube.com/watch?v=rbTCQrNOV_w. Both videos lasted for six to seven minutes. Subjects performed the experiment in individual soundproof cubicles. This allowed us to run both treatments within the same session while having each subject participate in only one treatment, thus avoiding confounds from session fixed effects [24]. After watching one of the videos, subjects had to complete a PANAS positive and negative affects schedule [13]. In this questionnaire, subjects stated the current subjective intensity, on a scale from one to five, of various affects. Ten negative and ten positive affects are included in this schedule. From the responses to these questions, we constructed scales of positive affect, negative affect, self-assurance, attentiveness, hostility, joviality, guilt, hostility, and fear. The questionnaire is reprinted in Supporting Information. After completing this questionnaire, subjects performed a time consuming real effort task. We used the task introduced by [12], known as the slider task. It consists of setting the highest possible number of sliders, which are displayed on the subject's computer screen, in the exact middle point of a pre-specified range, using their computer mouse to move a cursor. The task was unfamiliar to all participants and it entailed a cost of effort in terms of attention and patience. Subjects assigned to either treatment faced the same piece-rate incentive in the slider task. The accurate completion of each slider increased an individual's earnings by 5 Euro cents. Each session was divided into ten periods of 2 minutes each. All periods counted towards the subjects' earnings. Finally, subjects completed a questionnaire to gather information on demographic characteristics such as age, program of study, country of origin, previous exposure to poverty, gender, and indirect questions intended to measure family wealth and socioeconomic status. Previous exposure to poverty captures whether the subject traveled and/or lived in a poor country. We acknowledge that this measure is imperfect. However, we believe that is at least positively correlated with actual previous exposure to poverty. While it is certainly true that there is poverty in every European country, one can expect that ceteris-paribus, a person who has lived in or traveled to the developing world is more exposed to the type of images of poverty in the video, than someone who has not left the developed world. The questionnaire can be found in the supporting information. Identifying information was destroyed after the initial processing of the data and replaced with identifiers that did not permit the identity of a participant to be deduced. Throughout the entire session, subjects were videotaped with their prior consent using the webcams on their computers. The videos were analysed later using the facial recognition software Noldus Facereader. The software locates 530 points on a subject's face, and compares it to a database of several thousand annotated images. Facereader measures the conformity of the subject's facial expression to each of the six universal emotions: Happiness, Anger, Sadness, Disgust, Scare and Surprise, as well as Neutrality. The facial expressions that correspond to the six basic emotions appear to be universal and innate, in that they are common across all cultures and across different primates [25, 26], as well as between blind and sighted humans [26, 27]. Facereader takes a reading every 1/30th of a second. The program also constructs a measure of valence, using the formula Happiness − max(Anger, Sadness, Disgust, Scare). In our analysis, we use the average reading of each emotion over the one minute interval before the video begins, as well as over the one minute interval after the video ends. The effect of the video on an individual is measured as the difference in the average in the earlier and later intervals. BODY.RESULTS.PERFORMANCE: The measure of performance in our experiment is the number of sliders an individual correctly aligns over the course of a ten-period session. On average subjects solved 171.91 sliders with a standard deviation of 46.96 in a session. As illustrated in Fig 1, subjects in the Poverty treatment solved 165.2 sliders as compared to 179.3 sliders in the Neutral treatment. This difference is borderline significant (t(97,151) = 1.534, p = 0.063). A Kolmogorov-Smirnov test rejects the hypothesis that the number of sliders completed in the two treatments are drawn from the same distribution (KS-test, p<0.01). Performance in the Neutral treatment is significantly lower than that observed by [12], where subjects on average solved 222 sliders in 20 minutes. We attribute this difference to the fact that [12] employ tournament incentives that have been proven to increase effort [28]. 10.1371/journal.pone.0170231.g001Fig 1Average performance by treatment. Furthermore, the treatment effect on performance is significant once other sources of variation are controlled for. We estimate a regression of performance on condition dummies, covariates of wealth, and previous exposure to poverty. The estimates of this linear regression are presented in Table 1. The table shows that the effect of being assigned to Poverty is significant once the variables that capture the subject's previous exposure to poverty and wealth are included. Ceteris paribus, a subject assigned to Poverty produces on average 1.54 less sliders in each two-minute round as compared to a subject assigned to the Neutral condition. 10.1371/journal.pone.0170231.t001 Table 1 Linear Regression of Performance on Treatment, Round and Control Variables. (1) (2) (3) Performance Performance Performance Poverty -1.410 -1.547 * -1.544 * (-1.54) (-1.68) (-1.68) Previous Exposure 0.724 0.731 (1.38) (1.38) Wealth -0.076 (-0.12) Round 0.703 *** 0.703 *** 0.703 *** (14.57) (14.56) (14.55) Constant 14.06 *** 13.40 *** 13.45 *** (18.99) (16.03) (13.12) N 1050 1050 1050 R 2 0.133 0.142 0.142 Note: This table presents the estimates of an ordinary least squares regression of the statistical model Performance i = α 0 + α 1 Poverty i + α 2 PreviousExposure + α 3 Wealth + ϵ i 1 . Performance i is the number of sliders solved per round. Previous Exposure is a variable that captures whether the subject traveled and/or lived in a poor country. Wealth is a variable that captures whether the subject’s parents have more than three cars and/or own more than two real estate properties. Clustered standard errors at the individual level. *p<0.1; ** p<0.05; ***p<0.01. Fig 2 illustrates the performance gap between the treatments by round. This figure shows that the average number of sliders in every round is lower for subjects assigned to the Poverty treatment. Moreover, the figure suggests that the effect of the Poverty video on performance persists throughout the entire session. This may be either because the video itself affects performance throughout the session, or that the video has only a short-term effect in early rounds, but the performance in early rounds serves to anchor performance in later rounds. 10.1371/journal.pone.0170231.g002Fig 2Average performance by round and treatment. BODY.RESULTS.AFFECTS.SELF-REPORTED MEASURES: As described in the experimental design and procedures section, we administered the PANAS questionnaire [13] immediately after the subjects watched the video. Based on [29], we constructed, using the subjects responses, six emotional and affective scales, and two general dimension scales. Tables 2 and 3 present the mean and standard deviation of negative and positive affects in each treatment. The tables also report the average values of the affective scales and general dimension scales for both treatments. 10.1371/journal.pone.0170231.t002 Table 2 Average and Standard Deviation of Positive Affect Scales, by Treatment. Affect Poverty Neutral Z-Score Items Interested 3.691 3.100 2.879 *** (1.143) (1.119) Excited 2.145 2.600 -2.089 ** (1.070) (1.021) Strong 2.927 2.620 1.388 (1.190) (1.019) Enthusiastic 2.273 2.780 -2.393 ** (1.136) (1.083) Proud 1.982 2.180 -1.304 (1.259) (1.091) Alert 3.291 2.680 2.258 ** (1.247) (1.393) Inspired 3.055 2.540 2.239 ** (1.243) (1.082) Determined 3.309 2.880 2.155 ** (1.094) (1.014) Attentive 3.418 3.120 1.178 (1.004) (1.108) Active 2.709 3.080 -1.590 (1.217) (.9978) Affective Scales Joviality 4.41 5.38 -2.546 ** (1.877) (1.940) Self-Assurance 4.909 4.8 0.172 (2.076) (1.910) Attentiveness 13.709 11.78 3.006 *** (3.309) (3.180) General Dimension Scale Positive Affects (PA) 28.80 27.58 2.236 ** (7.631) (7.655) N 55 50 Note: This table presents the average score and standard deviation of each of the ten items representing positive affects in the PANAS questionnaire. The standard deviation of each item is presented in parentheses. The scale Joviality is constructed as the sum of the items Excited and Enthusiastic . The scale Self-Assurance is constructed as the sum of the items Proud and Strong . The scale Attentiveness is constructed as the sum of the items Inspired , Determined and Attentive . The row Positive Affects is the summation of the ten items. The column Z-score presents the statistic evaluating the rank sum difference between the two treatments. The significance is evaluated with the following significance levels. *p<0.1; ** p<0.05; *** p<0.01. 10.1371/journal.pone.0170231.t003 Table 3 Average and Standard Deviation of Negative Affect Scales, by Treatment. Affect Poverty Neutral Z-Score Items Distressed 2.509 2.560 -0.126 (1.008) (1.204) Upset 2.782 1.620 4.789 *** (1.247) (1.038) Guilty 2.218 1.420 3.749 *** (1.217) (0.778) Scared 1.709 1.540 0.596 (1.004) (0.830) Hostile 1.818 1.400 2.200 ** (.9934) (0.722) Irritable 2.036 2.040 0.027 (1.010) (1.039) Ashamed 2.182 1.440 3.401 *** (1.178) (0.753) Nervous 1.982 2.100 -0.622 (1.088) (1.064) Jittery 2.145 2.000 0.822 (0.924) (0.939) Afraid 1.655 1.560 0.458 (0.920) (0.829) Affective Scales Fear 5.509 5.1 1.028 (2.167) (2.121) Guilt 4.4 2.86 3.631 *** (2.231) (1.343) Hostility 1.818 1.400 2.200 ** (.993) (0.722) General Dimension Scale Negative Affects (NA) 21.036 17.680 2.728 *** (6.327) (5.859) N 55 50 Note: This table presents the average score and standard deviation of each of each of the ten items representing negative affects in the PANAS questionnaire.The standard deviation of each item presented in parentheses. The scale Fear is constructed as the sum of the items Afraid , Scared and Jittery . The scale Guilt is constructed as the sum of the items Guilt and Ashamed . The scale Hostile is represented by the item Hostility . The row Negative Affects is the summation of the ten items. The column Z-Score presents the statistic evaluating the rank sum difference between the two treatments. The significance is evaluated with the following significance levels. *p<0.1; ** p<0.05; ***p<0.01. These tables show that the Poverty treatment yields a higher score on the general dimension scale of negative affects (referred to as NA from here onward), compared to the Neutral treatment (p<0.01). This difference stems from the higher score of the Guilt scale, composed by the items Guilty and Ashamed (p<0.001) and the higher scores of the items Hostile (p<0.05), and Upset (p<0.001), under Poverty. Moreover, the Poverty treatment also yields a higher score on the general dimension scale of positive affects (PA from here onward), than under Neutral (p = 0.025). This difference between treatments is driven by a higher score on the attentiveness scale, composed of the items Alert, Determined and Interested, in Poverty (p<0.05). Note that even though the Joviality scale, composed of Enthusiastic and Excited, exhibits a lower score under Poverty (p = 0.011), the direction of the difference in PA between treatments shows that this difference is not as large as that displayed by attentiveness. Poverty induces higher average negative affects compared to the PANAS scores obtained under typical natural conditions. The average score of 21.036 in NA after watching the Poverty video is significantly larger than a typical baseline average of NA 14.8 reported by [13]. However, the Poverty treatment does not induce higher average positive affect than 29.7, the score observed by [13]. Taken together, these results show that the Poverty video induces a considerable increase on the score of both positive and negative affects as compared to the Neutral video. On the one hand the Poverty video evokes higher scores on guilt and hostility. On the other hand, the video increases the score related to attentiveness. BODY.RESULTS.AFFECTS.PHYSIOLOGICAL MEASURE OF EMOTIONAL STATE: The Facereader data indicate that before the videos are played, there are no significant differences in the physiological measures of emotions between treatments. We report no significant difference across treatments in neutrality (p = .9873), sadness (p = 0.1591) happiness (p = .757), anger (p = .7411), scare (p = .4343) or disgust (p = .6738). As a consequence and as can be seen in Fig 3, there is no significant difference in the emotional valence before presentation of the videos (p = 0.782). 10.1371/journal.pone.0170231.g003Fig 3Initial value and change in valence from before to after the video was shown. The descriptive statistics of the subject's physiological reaction due to the videos, measured as the difference between the one-minute interval before the beginning of the video and the one-minute interval immediately after it has finished, are presented in Table 4. These statistics, along with Fig 3, show that the Poverty video induces a more negative valence (one sided t-test, p = .055). This finding is in agreement with the self-reported data, in that the Poverty treatment induces a more negative emotional state than the Neutral treatment. This difference is driven by the specific emotions of Scare (one sided t-test, p = .039) and Sadness (one sided t-test, p = .057). These patterns, coupled with the PANAS results, reveal that the Poverty video increases a number of negative emotions such as fear, guilt, sadness, shame and upset. Whether these differences in affects correlate with lower performance is addressed in the next section. 10.1371/journal.pone.0170231.t004 Table 4 Changes in Physiological Measure of Emotions from Before to After the Video, both Treatments. Emotion Poverty Neutral Difference Neutral -.0011 0.048 -.058 (.213) (0.294) (.066) Happy -.068 -0.051 .-017 (.125) (0.122) (.033) Sad 0.037 -0.002 .039 * (0.098) (0.077) (.024) Angry 0.002 -0.002 .004 (0.091) (0.062) (.025) Surprised 0.007 0.027 -.020 (0.097) (0.090) (.022) Scared 0.010 -0.006 .015 ** (0.036) (0.019) (.008) Disgusted -0.003 -0.004 .001 (0.016) (0.010) (.003) Valence -0.111 -0.037 -.073 * (0.185) (0.134) (.045) N 39 21 21 Note: This table presents the difference in average intensity, between before and after the stimulus was presented to the subjects for the 7 emotions analyzed by the facereader software. The standard deviation is given in Parentheses. The row Valence contains the change in average valence from before to after the stimulus was presented to the subjects. Valence is calculated as Happiness − max ( Sadness , Anger , Scare , Disgust ). The column labeled Difference gives the average difference in emotional intensity between the Poverty and Neutral treatments. The significance of Difference is evaluated with a one sided t-test. *p<0.1; ** p<0.05; ***p<0.01. BODY.WHAT MODERATES THE EFFECT OF EXPOSURE TO POVERTY OF OTHERS?: The results reported in the previous section indicate that subjects assigned to Poverty experience lower average performance. Moreover, participants in the two treatments exhibit differences in affective scales: under Poverty they have a more negative, self-reported and physiologically measured, affective state. They also have higher scores on the attentiveness scale, and lower scores on the joviality scale. To investigate whether the treatment difference in performance varies depending on an individual's affective state, we employ a moderation analysis [30]. A variable is said to moderate a treatment effect if higher values of the variable are correlated with a weaker treatment effect. For example, suppose that the sample of all participants is divided into two subsamples. In one subsample are those with a relatively positive prior emotional state, and in the other those in a relatively negative one. If positive emotional valence moderates the treatment effect, the difference between treatments would be greater for the subsample in the relatively negative state than that in the more positive state. Table 5 presents the estimates of the statistical model, Performancei=α0+α1Poverty+α2Ai+α3Ai*Poverty+Γ′X+εi, where Poverty is a dummy variable that equals 1 under the Poverty treatment and Ai is a variable that captures an affective measure for subject i. Throughout the analysis, Ai may represent a single affect or emotion, an affective scale as in [29], physiological valence, or a general dimension scale. We begin the moderation analysis using the responses of the PANAS. Specifically, we estimate the statistical model with the dimension scales NA or PA representing Ai. These composite scores capture the total variation of self-reported affects. The coefficients on the variable Poverty * Ai reveal whether the scale or emotion in question is a moderating factor. The first two columns of Table 5 show that these scales do not moderate the effect of treatment on performance. Hence, the lower performance of those assigned to Poverty does not depend on the variation in the general score of positive or negative affects between treatments. 10.1371/journal.pone.0170231.t005 Table 5 Moderation Analysis, Self-Reported Affects. (1) (2) (3) (4) (5) (6) (7) (8) Performance Performance Performance Performance Performance Performance Performance Performance Poverty 0.530 -5.739 * 0.465 0.046 -0.419 -7.584 *** -3.045 -4.543 (2.905) (2.959) (2.382) (1.932) (2.007) (2.297) (2.098) (3.356) A i 0.0678 -0.174 * 0.141 0.929 0.235 -0.654 ** -0.520 ** -0.389 * (0.097) (0.078) (0.245) (0.880) (0.445) (0.308) (0.254) (0.210) Poverty* A i -0.110 0.154 -0.377 -1.089 -0.339 1.225 ** 0.321 0.275 (0.130) (0.101) (0.371) (1.056) (0.503) (0.436) (0.359) (0.271) Previous Exposure 0.767 0.653 0.764 0.738 0.759 0.801 0.657 0.628 (0.533) (0.555) (0.535) (0.539) (0.525) (0.540) (0.538) (0.540) Wealth -0.0716 -0.198 -0.0478 -0.0768 -0.103 -0.327 -0.179 -0.111 (0.613) (0.636) (0.612) (0.613) (0.618) (0.635) (0.634) (0.620) Round 0.703 *** 0.703 *** 0.703 *** 0.703 *** 0.703 *** 0.703 *** 0.703 *** 0.703 *** (0.048) (0.048) (0.048) (0.048) (0.048) (0.048) (0.048) (0.048) Constant 12.21 *** 18.42 *** 12.68 *** 12.14 *** 12.77 *** 17.10 *** 16.09 *** 18.15 *** (2.218) (2.574) (1.822) (1.438) (1.788) (2.130) (1.676) (2.762) A i NA PA Fear Hostility Guilt Joviality Self-Assesement Attentiveness N 1050 1050 1050 1050 1050 1050 1050 1050 adj. (R 2 ) 0.143 0.165 0.145 0.147 0.142 0.179 0.156 0.164 Note: This table presents the estimates of an ordinary least squares regression of the statistical model Performance i = α 0 + α 1 * Poverty + α 2 * A i + α 3 A i * Poverty + Γ′ X i + ϵ i 1 . Effort i is the number of sliders solved per round. T i is the assignment to the Neutral or the Poverty condition, A i is a composite index of self-reported affects. The matrix X i contains the variables Previous Exposure , which captures whether the subject traveled or/and lived in a poor country and Wealth , which is a variable that indicates whether the subject’s parents had more than 3 cars or/and more than 2 real estate properties. Standard errors are clustered at the level of the individual subject. *p<0.1; ** p<0.05; ***p<0.01. The moderation analysis also shows that the Joviality scale moderates the effect of the Poverty treatment on performance. This can be seen in column 6 of the table. Specifically, subjects reporting higher scores on the items enthusiastic and excited after watching the videos exhibit a smaller difference in performance between treatments. Finally, we report that the self-reported responses representing Fear, Hostility, Guilt, Self-Assessment and Attentiveness do not moderate the lower performance due to the video, even when some of these scales display a significant difference between treatments. Additionally, we investigate the role of the physiological measures of emotions as moderators. We perform the moderation analysis using two different representations of emotional state using the facereader data. The first one is the absolute emotional state before the video is played, and the second one is the change in emotional state from before to after the video is played. Table 6 reports whether the emotional state before viewing the video affects performance. The findings suggest that negative valence before the videos are presented moderate the treatment effect. In other words, subjects in a more negative prior emotional state experience a smaller reduction in performance from the Poverty, relative to the Neutral, condition. In particular, higher measures of happiness and higher values of sadness have a moderating effect. 10.1371/journal.pone.0170231.t006 Table 6 Moderation Analysis, Physiological Measures Taken Before Video is Played. (1) (2) (3) (4) (5) (6) (7) Performance Performance Performance Performance Performance Performance Performance Poverty -0.642 1.687 -4.550 * -1.450 -2.182 -1.834 -0.948 (1.313) (1.421) (2.290) (1.735) (1.741) (1.654) (1.442) A i 12.16 16.29 * -43.55 ** 5.558 -12.71 -36.65 -55.64 (8.352) (8.192) (19.82) (12.646) (20.686) (91.78) (51.489) Poverty*A i -18.28 ** -22.59 *** 46.71 ** -0.0519 14.31 48.81 -205.2 *** (8.486) (8.340) (21.81) (13.567) (21.508) (97.480) (71.652) Previous Exposure 0.644 0.486 0.315 0.553 0.525 0.621 0.705 (0.634) (0.624) (0.775) (0.715) (0.699) (0.720) (0.641) Wealth -1.219 -1.023 -1.675 * -1.139 -1.180 -1.319 -1.622 * (0.867) (0.809) (0.896) (0.909) (0.976) (0.925) (0.845) Round 0.712 *** 0.712 *** 0.712 *** 0.712 *** 0.712 *** 0.712 *** 0.712 *** (0.072) (0.072) (0.072) (0.072) (0.072) (0.072) (0.072) Constant 13.99 *** 12.11 *** 18.14 *** 14.24 *** 15.12 *** 14.89 *** 15.17 *** (1.686) (1.492) (2.833) (2.134) (2.293) (2.082) (1.904) A i Valence Happy Sad Angry Surprised Disgusted Scared N 610 610 610 610 610 610 610 adj. R 2 0.239 0.249 0.205 0.160 0.158 0.157 0.227 Note: This table presents the estimates of an ordinary least squares regression of the statistical model Performance i = α 0 + α 1 * Poverty + α 2 * A i + α 3 A i * Poverty + Γ′ X i + ϵ i 1 . Effort i is the number of sliders solved per round. T i is the assignment to the Neutral or the Poverty condition, A i is a composite index of self-reported affects. The matrix X i contains the variables Previous Exposure , which captures whether the subject traveled or/and lived in a poor country and Wealth , which is a variable that indicates whether the subject’s parents had more than 3 cars or/and more than 2 real estate properties. Standard errors are clustered at the level of the individual subject. *p<0.1; ** p<0.05; ***p<0.01. Furthermore, Table 7 presents the moderation analysis for the change in emotional state due to the videos. The results show that a more positive (or less negative) change in valence from before to after watching the video moderates the treatment effect. Hence, individuals experiencing a less negative change in valence after watching the video, exhibit a smaller difference in performance between treatments. This effect is led by the emotion of fear. For those experiencing relatively high levels of fear, there is a larger difference in performance after viewing the Poverty than the Neutral video. 10.1371/journal.pone.0170231.t007 Table 7 Moderation Analysis, Change in Physiological Measures Due to Video. (1) (2) (3) (4) (5) (6) (7) Performance Performance Performance Performance Performance Performance Performance Poverty 0.0270 0.206 -0.220 -1.824 -2.891 -2.126 -0.675 (1.202) (1.386) (2.155) (1.601) (1.836) (1.624) (1.488) A i -21.27 * 16.96 *** 10.12 5.256 -10.32 -207.2 2171.3 *** (11.427) (5.011) (23.710) (9.906) (10.798) (288.924) (646.210) Poverty* A i 26.60 ** -18.32 ** -19.90 2.871 15.47 119.4 -2176.0 *** (11.076) (8.245) (24.392) (11.884) (11.227) (323.114) (645.029) Previous Exposure 0.135 0.874 0.453 0.591 0.432 0.674 0.713 (0.613) (0.675) (0.653) (0.688) (0.725) (0.640) (0.665) Wealth -1.029 -1.490 * -1.338 -1.377 -1.134 -1.597 * -1.747 ** (0.826) (0.863) (0.837) (0.913) (0.865) (0.872) (0.835) Round 0.703 *** 0.705 *** 0.705 *** 0.705 *** 0.705 *** 0.705 *** 0.705 *** (0.072) (0.070) (0.070) (0.070) (0.070) (0.070) (0.070) Constant 14.00 *** 13.07 *** 14.50 *** 14.82 *** 15.66 *** 15.85 *** 14.33 *** (1.542) (1.639) (2.513) (1.935) (2.198) (2.078) (1.879) A i Valence Happy Sad Angry Surprised Disgusted Scared N 600 620 620 620 620 620 620 adj. R 2 0.247 0.221 0.173 0.167 0.170 0.174 0.186 Note: This table presents the estimates of an ordinary least squares regression of the statistical model Performance i = α 0 + α 1 * Poverty + α 2 * A i + α 3 A i * Poverty + Γ′ X i + ϵ i 1 . Effort i is the number of sliders solved per round. T i is the assignment to the Neutral or the Poverty condition, A i is a composite index of self-reported affects. The matrix X i contains the variables Previous Exposure , which captures whether the subject traveled or/and lived in a poor country and Wealth , which is a variable that indicates whether the subject’s parents had more than 3 cars or/and more than 2 real estate properties. Standard errors are clustered at the level of the individual subject. *p<0.1; ** p<0.05; ***p<0.01. These two results illustrate that subjects experiencing lower emotional valence due to the Poverty video also exhibit lower performance levels, compared to subjects assigned to Neutral. Moreover, these declines in performance worsen with the degree of positive valence that a subject assigned to Poverty exhibits before the video is displayed. BODY.DO AFFECTS AND EMOTIONS MEDIATE THE DECREASE IN PRODUCTIVITY?: In this section we consider whether the effect of the videos on emotional state accounts for some or all of the difference in productivity between treatments. We employ the mediation analysis developed by [31] to evaluate the extent to which affective states induced by the videos mediate the lower performance. To that end, we estimate the following system of equations: Ai=β0+β1Poverty+C′Xi+εi1, Performancei=α0+α1Poverty+α2Ai+D′Xi+εi2. The estimates of this system of equations intend to isolate two effects. The first is given by α1, which measures the direct effect of treatment on performance α1^. In other words, this is the effect on performance of being assigned to a treatment once the relationship between variations in affect and performance is accounted for. The second effect is the indirect effect of treatment on performance via changes in affect δi^=α2^β1^. The assumptions required for this interpretation of the estimates [31], are {Performancei′,Ai}⊥Poverty|Xi, and Performancei′⊥Ai|Xi,Poverty. Where Performancei′≠Performance. The first equation of the assumption states that the treatment assignment is statistically independent of potential performance outcomes and mediators given pre-treatment confounders. In our experiment this assumption holds due to the treatment randomization. The second equation states that the observed mediator is statistically independent given treatment assignment and pre-treatment confounders. This means that the mediator is regarded as if it were randomized over treatments. Evidence that this assumption holds in our sample is provided using the Facereading data before the video is displayed. As stated in Section 3, we find no differences between treatments in Physiological measures before the video was displayed. The total effect of treatment is the sum of the direct and indirect effects. We use the Quasi-Bayesian Monte Carlo approximation of [32] to make statistical inference about δi^. The parameters reported are the average of 100 draws. Tables 8 and 9 present the estimates, using the self-reported scales to represent Ai. The findings suggest that neither of the general dimension scales, NA or PA, mediates the effect of the treatment on performance. Moreover, Table 9 shows that among the affective scales that exhibit significant differences between treatments, only the attentiveness scale is a significant mediator of the treatment effect. Specifically, this scale mediates 31% of the total treatment effect. 10.1371/journal.pone.0170231.t008 Table 8 Results of the Mediation Analysis with the General Dimension Scales PA and NA. (1) (2) (3) Performance Performance Performance Mediator: A i = NA δ i ^ (Mediation Effect) .00048 -.0372 -.0046 α 1 ^ (Direct Effect) -1.338 -1.285 -1.653 * Total Effect -1.338 -1.322 -1.657 * Mediator: A i = PA δ i (Mediation Effect) -.112 -.135 -.170 α 1 ^ (Direct Effect) -1.224 -1.174 -1.482 Total Effect -1.337 -1.310 -1.652 * Wealth NO YES YES Previous Exposure NO NO YES Observations 1050 1050 1050 Note: This table presents the average of 100 draws of a Monte Carlo Simulation using the sampling distribution of α 2 ^ β 1 ^ and α 1 ^ which are estimated through multiple Least Squares of the system equatons composed by A i = β 0 + β 1 Poverty i + C ′ X i + ϵ i 1 and P e r f o r m a n c e i = α 0 + α 1 P o v e r t y i + α 2 ′ A i + D ′ X i + ϵ i 2 . Performance i is the number of sliders solved per round. A i is the total score of the Negative Affects elicited through the PANAS questionnaire in the top panel of the table and the total score of the Positive Affects elicited through the PANAS questionnaire in the top panel of the table. Previous Exposure is a variable that captures whether the subject traveled and/or lived in a poor country. Wealth is a variable that captures whether the subject’s parents had more than 3 cars or/and more than 2 real estate properties. Clustered standard errors at the individual level. *p<0.1; ** p<0.05; ***p<0.01. 10.1371/journal.pone.0170231.t009 Table 9 Mediation Analysis for Self-Reported Affect Scales. (1) (2) (3) Performance Performance Performance Mediator: A i = Joviality δ i ^ (Mediation Effect) .069 .0591 .033 γ i ^ (Direct Effect) -1.401 -1.483 -1.561 ** Total Effect -1.331 -1.424 -1.528 * Mediator: A i = Fear δ i ^ (Mediation Effect) -.0171 -.031 -.035 γ i ^ (Direct Effect) -1.316 -1.395 -1.504 * Total Effect -1.333 -1.427 -1.540 * Mediator: A i = Guilt δ i ^ (Mediation Effect) -.0145 -.022 -.052 γ i ^ (Direct Effect) -1.327 -1.405 -1.500 Total Effect -1.342 -1.427 -1.552 * Mediator: A i = Attentiveness δ i ^ (Mediation Effect) -.501 ** -.549 *** -.559 *** γ i ^ (Direct Effect) -.838 -.788 -1.126 Total Effect -1.339 -1.338 * -1.685 * Wealth NO YES YES Previous Exposure NO NO YES Observations 1050 1050 1050 Note: This table presents the average of 100 draws of a Monte Carlo Simulation using the sampling distribution of α 2 ^ β 1 ^ and α 1 ^ which are estimated through multiple Least Squares of the system of models composed by A i = β 0 + β 1 Poverty i + CX i + ϵ i 1 and P e r f o r m a n c e i = α 0 + α 1 T i + α 2 ′ A i + D X i ′ + ϵ i 2 . Performance i is the number of sliders per round. A i is the guilt scale. A i is the Joviality scale at the top panel, Fear at the top-middle panel, Guilt at the bottom-middle panel and Attentiveness at the bottom panel. Previous Exposure is a variable that captures whether the subject traveled and/or lived in a poor country. Wealth is a variable that captures whether the subject’s parents had more than 3 cars or/and more than 2 real estate properties. Clustered standard errors at the individual level. *p<0.1; ** p<0.05; ***p<0.01. We also study the role of the physiological measures as mediators. We report the estimates of happiness and valence as mediators in Table 10. We find that emotional valence does not mediate the effect of the Poverty video on performance. Nevertheless, we find that the specific emotion of happiness mediates the effect of the treatment, after controlling for some measures of previous exposure to poverty. Happiness mediates nearly 27% of the total effect in one specification, but this mediating effect disappears once we control for the degree of prior exposure to poverty, indicating that the effect is not robust. Finally, we find no evidence that the emotions sadness, scare, disgust, surprise or anger mediate the treatment difference. 10.1371/journal.pone.0170231.t010 Table 10 Mediation Analysis Table for the Physiological Variables of Happiness and Emotional Valence. (1) (2) (3) Performance Performance Performance Mediator: A i = Happiness δ i ^ (Mediation Effect) -.519 -.655 * -.564 γ i ^ (Direct Effect) -.985 -.979 -1.149 Total Effect -1.504 -1.635 -1.714 Mediator: A i = Valence δ i ^ (Mediation Effect) -.395 -.422 -.369 γ i ^ (Direct Effect) -1.093 -.948 -1.336 Total Effect -1.487 -1.370 -1.705 Wealth NO YES YES Previous Exposure NO NO YES Observations 620 620 620 Note: This table presents the average of 100 draws of a Monte Carlo Simulation using the sampling distribution of α 2 ^ β 1 ^ and α 1 ^ which are estimated through multiple Least Squares of the system of models composed by A i = β 0 + β 1 Poverty i + CX i + ϵ i 1 and P e r f o r m a n c e i = α 0 + α 1 T i + α 2 ′ A i + D X i ′ + ϵ i 2 . Performance i is the number of sliders per round. A i is the guilt scale. A i is the Happiness measured by facereader at the top panel and Valence as measured by facereader at the bottom panel. Previous Exposure is a variable that captures whether the subject traveled and/or lived in a poor country. Wealth is a variable that captures whether the subject’s parents had more than 3 cars and/or more than 2 real estate properties. Clustered standard errors at the individual level. *p<0.1; ** p<0.05; ***p<0.01. BODY.CONCLUSION: In this paper, we have presented evidence consistent with the notion that the affective state associated with exposure to the poverty of others can decrease individual productivity. We required participants in our study to view a video and then had them perform a task that required effort and concentration. In the Poverty treatment, the video exposed participants to images of poverty, and in the control treatment, a neutral video was shown instead. Subjects assigned to the Poverty treatment exhibited lower average productivity compared to subjects that were in the Neutral condition. The Poverty video induces a more negative emotional state on the part of viewers, as measured both by self-reports and facial recognition software. The Poverty video increases the self-reported levels of some emotions that form components of a positive scale, attentiveness, but we do not view this as a positive emotional state, as for example we would view joy or satisfaction. Poverty does not increase the physiological measures of positive emotions registered with Facereader. The affective states measured on the PANAS scales of attentiveness, guilt, and hostility are greater under the Poverty than under the Neutral treatment. The facial recognition software reports that the Poverty video evokes greater fear and sadness than the Neutral video. These patterns show that (i) exposure to poverty of others, (ii) own emotional state, and (iii) own productivity, are related. A moderation analysis allows us to consider who is more susceptible to the treatment effect. It reports that those who score higher on the Joviality scale after viewing a video exhibit a smaller difference between the two treatments. Similarly, the physiological data indicate that those in a more positive emotional state after watching the video are less susceptible to the treatment effect. This pattern suggests that an overall positive emotional state is a buffer against the adverse consequences of exposure to poverty of others. A mediation analysis allows us to investigate the causal nature of these relationships. We conclude that the treatment effect of the Poverty video is mediated by self-reported measures of attentiveness. Specifically, those paying relatively more attention to the Poverty video, experience lower subsequent productivity. However, those who seem to avert their attention when being exposed to poverty experience a less detrimental impact from the exposure. This finding is in line with those of [21] and [20], in which individuals in the state of poverty exhibit an impediment in their cognitive function, something that the authors describe as "tunneling". In our framework we provide evidence that exposure to the poverty of others diverts individuals' attention, and this in turn decreases their performance in a subsequent productive task. We view our study as a proof of principle, illustrating that mere exposure to the poverty of others can lead to a decrease in productivity. We have shown that very brief low-intensity exposure can have an effect on a task performed immediately after the exposure. We do not know for sure what would be the effect on productivity if the length and intensity of exposure were both increased to the scales that exist in developing countries or in underprivileged neighborhoods outside the laboratory. However, one can well imagine that the effect could be stronger and more long-lasting, unless individuals become less sensitive to its effect as they receive more exposure. Whether the effect of mere exposure to poverty of others would be strong and durable enough to have a long-term effect on work performance remains to be established in future research. It would be interesting to study the effect of other videos that evoke strong emotions on productivity. They may have a similar effect on output as the video we chose because they induce similar emotions. However, the fact that we also observe a direct effect of our Poverty video on productivity means that emotions are not the only channel whereby the video is exerting its effect. There is a specific effect of the content of the video, once emotions are controlled for. It is certainly possible, and perhaps likely, that videos with different content would also induce a direct effect on productivity. It is certainly not the case that exposure to poverty is the only type of exposure that would have an effect of output. We make no claims that exposure to poverty is the only potential cause of low productivity, there are certainly many influences on productivity, and some of these presumably have emotional substrates as well. The task that subjects have to perform in our experiment is one that requires attention and focus, but it is not cognitively demanding and has a repetitive aspect. Future studies could determine whether the same results hold for tasks that require a stronger application of cognitive abilities. In addition, the subject pool in this experiment was composed by university students who may have a higher or lower proclivity to be affected by images of poverty than other populations. Further studies could explore, by varying the characteristics of the sample of subjects, which groups might be more or less affected. BODY.SUPPORTING INFORMATION: S1 AppendixExperimental instructions.PANAS Questionnaire. Socio-Economic Status Questionnaire.(DOCX)Click here for additional data file. S1 Replication CodeStata codes used for data analysis.(DO)Click here for additional data file. S1 DatasetDataset used for the analysis in Stata.(DTA)Click here for additional data file.

TITLE: Repetitive recombinant human bone morphogenetic protein 2 injections improve the callus microarchitecture and mechanical stiffness in a sheep model of distraction osteogenesis ABSTRACT: Evidence suggests that recombinant human bone morphogenetic protein 2 (rhBMP-2) increases the mechanical integrity of callus tissue during bone healing. This effect may be either explained by an increase of callus formation or a modification of the trabecular microarchitecture. Therefore the purpose of the study was to evaluate the potential benefit of rhBMP-2 on the trabecular microarchitecture and on multidirectional callus stiffness. Further we asked, whether microarchitecture changes correlate with optimized callus stiffness. In this study a tibial distraction osteogenesis (DO) model in 12 sheep was used to determine, whether percutaneous injection of rhBMP-2 into the distraction zone influences the microarchitecture of the bone regenerate. After a latency period of 4 days, the tibiae were distracted at a rate of 1.25 mm/day over a period of 20 days, resulting in total lengthening of 25 mm. The operated limbs were randomly assigned to one treatment groups and one control group: (A) triple injection of rhBMP-2 (4 mg rhBMP-2/injection) and (B) no injection. The tibiae were harvested after 74 days and scanned by μCT (90 μm/voxel). In addition, we conducted a multidirectional mechanical testing of the tibiae by using a material testing system to assess the multidirectional strength. The distraction zones were tested for torsional stiffness and bending stiffness antero-posterior (AP) and medio-lateral (ML) direction, compression strength and maximum axial torsion. Statistical analysis was performed using multivariate analysis of variance (ANOVA) followed by student's t-test and Regression analysis using power functions with a significance level of P<0.05. Triple injections of rhBMP-2 induced significant changes in the trabecular architecture of the regenerate compared with the control: increased trabecular number (Tb.N.) (treatment group 1.73 mm/1 vs. control group 1.2 mm/1), increased cortical bone volume fraction (BV/TV) (treatment group 0.68 vs. control group 0.47), and decreased trabecular separation (Tb.Sp.) (treatment group 0.18 mm vs. control group 0.43 mm). The analyses of the mechanical strength of regenerated bone showed significant differences between treatment group (A) and the control group (B). The bending stiffness anterior-posterior (treatment group 17.48 Nm vs. control group 8.3 Nm), medial-lateral (treatment group 18,9 Nm vs. control group 7.92 Nm) and the torsional stiffness (treatment group 41.17N/° vs. control group 16.41N/°) are significantly higher in the treatment group than in the control group. The regression analyses revealed significant non-linear relationships between BV/TV, TB.N., Tb.Sp. and all mechanical properties. Maximal correlation coefficients were found for the Tb.Sp. vs. the bending stiffness AP and ML with R2=0.69 and R2=0.70 (P<0.0001). There was no significant relation between Connectivity and the compression strength and the maximum axial torque. This study suggests that rhBMP-2 optimizes the trabecular microarchitecture of the regenerate, which might explain the advanced mechanical integrity of newly formed bone under rhBMP-2 treatment. BODY.INTRODUCTION: Recombinant human bone morphogenetic protein 2 (rhBMP-2) has been shown to enhance bone healing1–10 and thereby to improve the mechanical integrity11,12 of bone regenerate at a distinct time point. However, it remains unclear whether rhBMP-2 application just accelerates bone consolidation resulting in terms of a time lead of healing or even induces a microstructural optimized trabecular microarchitecture in long bone healing. To investigate this question we used a sheep distraction osteogenesis (DO) model as a model of controlled bone healing. Specifically we asked, whether rhBMP-2 influences the bone consolidation process. During consolidation the bone remodeling of the callus tissue due to osteoblast and osteoclast activation and the mineralisation of the bone matrix are the main processes. While BMP-2 has been shown to be a potential osteoinductive factor13,14 in the first instance, it has also been shown to stimulate osteoblast and osteoclast differentiation,15–20 and to be involved in late stages of osteoblast development and matrix mineralisation.16,21,22 Therefore a remodeling effect during bone consolidation may be supposed. Further evidence suggests that rhBMP-2 increases the mechanical integrity of the callus, but the responsible mechanism remains unclear since there are conflicting results about the rhBMP-2-effect on the total amount of callus and the callus mineralisation.1–3,6,7,11 For trabecular bone the strength not only depends on the amount of calcified tissue present, but primarily on its spatial distribution.23 Therefore the strength and the fracture resistance of bone are determined by the architecture of its trabecular network and its cortical shell.24,25 However, a potential modification of the trabecular microarchitecture by rhBMP-2 application during DO has been investigated by Ming and Zhu et al., but these studies examined rhBMP-2 in combination with other biological agents. Other studies investigated the effect of rhBMP-2 on bone microarchitecture in anterior lumbar interbody fusion.26,27 In this study the microarchitecture of the bone regenerate was assessed using 3D microcomputed tomography (μCT). We hypothesized that repetitive rhBMP 2-injections improve the microarchitecture of the bone regenerate and thereby increase its mechanical resistance. A second hypothesis was that changes in the trabecular microarchitecture would correlate with the mechanical stiffness of the callus tissue. BODY.MATERIALS AND METHODS.ANIMALS: Twelve skeletally mature female sheep with a mean presurgical weight of 81 kg±13.6 kg were used. Animals were housed indoors in boxes during the experimentation period and fed a standard cereal foodstuff. Animal experiments were conducted under an ethic commission approved protocol according to German federal animal welfare legislation. BODY.MATERIALS AND METHODS.SURGERY AND DISTRACTION: For preanesthetic sedation animals received 5–10 mg propofol intravenous. The sheep were intubated and fixed in a left lateral position. Inhalation anaesthesia was performed with Isofluran for the duration of surgery. A modified external half ring fixator,28 consisting of four half-rings, connected by threaded rods, was applied to the right tibia under radiographic control. Six 5 mm Schanz-screws were inserted in the tibia in a defined order. A middiaphyseal transversal osteotomy was created over a medial approach using an oscillating saw. The periosteum was reapproximated and the wound closed. A postoperative antimicrobial prophylaxis, consisting of penicillin and streptomycin (0.1 mL/kg, Tardomycel, Bayer GmbH, Leverkusen) was applied by subcutaneous injection. To protect the sheep from hurting themselves and each other, a soft plastic cover was fixed on the fixator frame. Unrestricted weight bearing and activity were allowed postoperatively. After a latency period of 4 days the tibiae were distracted at a rate of 1.25 mm daily in two increments over a period of 20 days. The distraction resulted in total lengthening of 25 mm. The distraction period was followed by a consolidation period of 50 days, during which the fixator was hold in place with no distraction. Throughout the study sheep were examined daily for signs of pin-infection and pain. Antero-posterior x-ray views of the lengthened tibiae were performed weekly. BODY.MATERIALS AND METHODS.INJECTION PROTOCOL: The operated sheep were randomly assigned to the treatment group or the control group resulting in 6 animals per group. Treatment groups included: (A) triple injection of 1 mL rhBMP-2/NaCl (4 mg rhBMP-2/ml) (Genetics Institute, MA). The control group had no injection (B). The sheep in treatment group (A) were injected on day 3, 10 and 17. During the injection procedure the sheep were fixed in a dorsal position. Injections were performed at the center of the distraction zone (0.4 mL solution) and 1 cm distal and proximal from the central point (0.2 mL solution each) under fluoroscopic control. Sheep were then sacrificed after 74 days by intravenous injection of propofol and pentobarbital (Eutha 77, Essex Pharma, Germany). Afterwards the external fixator was removed and the right tibiae was resected and frozen at −20°C until μCT analysis were performed. BODY.MATERIALS AND METHODS.STEREOLOGICAL ANALYSIS: To determine 3D stereological histomorphometric parameters indices a complete three-dimensional digitized image of each distraction zone was generated using a microcomputed tomography system (μCT). The system is equipped with a microfocus x-ray tube with a focal spot of 10 mm, producing a cone beam that is detected by a charge-couple device (CCD) 512×512 pixel array. The tibiae were positioned in custom-made fixation device in air for scanning. The whole distraction zone with a diameter of approximately 2,5 cm was scanned. All distraction zones were reconstructed on a 120 μm mesh, so that each 3D data point of the reconstruction (voxel) represented a subvolume of the actual specimen of (90×90×90 μm3). The distraction callus was analyzed for bone volume/total volume (BV/TV), trabecular number (Tb.N.: mm−1), trabecular thickness (Tb.Th.: mm), trabecular separation (Tb.Sp.: mm) and Connectivity, as defined by Parfitt and Odgaard.29;30 The region of interest (ROI) was fit into the distraction zone (1.) as a sphere with the diameter of the distraction zone: total volume; (2.) as two spheres with half of the diameter of the distraction zone: cortical weighted subvolume (CWS) (Figure 1). Both ROI methods were used to determine BV/TV. All other parameters were measured in the cortical weighted subvolume (method 2). A modified algorithm of the Hipp and Simmons method of directed secants was used to compute the structural parameters defined above.31 Figure 1Model of region of interest (ROI): total volume and region of interest: cortical weighted subvolume. BODY.MATERIALS AND METHODS.MECHANICAL TEST: Before biomechanical testing at day 74, the frozen tibiae were thawed at room temperature and kept moist using gauze soaked in 0.9% NaCl solution during the entire test period. The embedded specimens were mounted in specially constructed jigs for compressive, 4-point-bending, or torsional testing in a Material Testing System (MTS [Model 858, MTS Corp., Minneapolis, USA]) (Figure 2). The order of stiffness testing was randomised and measurements were performed on each specimen by using compressive, bending (anteroposterior and mediolateral) and torsional stiffness. The resulting deformation was detected by custom-made compression, torsion and deflection sensors (LVTD and precision potentiometer). For each stiffness testing-procedure, a preconditioning of 10 cycles was conducted before the actual testing in order to assure repeatability. The callus tissue within the specimens was loaded during the different types of testing up to 15 Nm for torsional, to 750 N for compressive and to 6.5 Nm for bending load. During testing, load and deformation were simultaneously recorded in order to determine stiffness, which is defined as the slope of the load-deformation curve. In a final experiment, the specimens were loaded in torsion until failure to determine the load-bearing capacity. The detailed testing protocol was performed as previously described.32 Figure 2Photography of the custom-made jig for bending testing including the specially constructed deflection sensor. BODY.MATERIALS AND METHODS.STATISTICS: The results are presented as mean and SD. For comparison of the structural parameters obtained from the treatment group (A) and the control group (B) data were analyzed using multivariate analysis of variance (ANOVA) followed by student's t-test. The mechanical parameters (ordinate) and the different types of bone parameters as BV/TV, Tb.N. and Tb.Sp. were plotted in a diagram. For each measured parameter an analysis of variance (ANOVA) was performed with SPSS Software (SPSS, SPSS Inc., Chicago, USA). Regression analyses were performed using power functions. A P value of less than 0.05 was considered significant. BODY.RESULTS: The stereological evaluation revealed a significantly increased higher cortical BV/TV in the treatment group A compared the control group (B). The increase was given by 45% (P=0.001). Measured for the total volume of the distraction zone the BV/TV showed no significant difference between the two groups (Figure 3). The trabecular number (Tb.N.) in the treatment group (A) was significantly increased compared with the control group (B). The percentage difference was 46% (P=0.004), which corresponds to an absolute increase of the mean trabecular number (Tb.N.) by 5–6 trabeculae per millimeter. Regarding the trabecular spacing (Th.Sp.), there were significant differences between the treatment group (A) and the control group (B). Treatment group (A) compared with control group (B) had a lower mean trabecular spacing (Tb.Sp.) by 58% (P=0.022), representing an absolute difference of 0.25 mm. For the trabecular thickness (Tb.Th.), there were no significant differences between the treatment group (A) and the control group (B). The maximum trabecular thickness (Tb.Th.) in treatment group (A) reached 0.39 mm compared to 0.35 mm in the control group (Figure 4). Figure 3Comparison of the bone volume fraction (BV/TV) in the total volume and the cortical weighted subvolume. Figure 4Comparison of the trabecular number (Tb.N), trabecular spacing (Tb.Sp.) and trabecular thickness (Tb.Th.) in the cortical weighted subvolume. The analyses of the mechanical strength of the distraction zones indicated significant differences between treatment (A) and control group (B). The comparison showed an increase in torsional stiffness of the treatment group of 248% to controls (P=0.012). The anterior-posterior bending stiffness revealed an increase of 210% (P=0.007) and the medial-lateral bending stiffness showed an increase of 239% (P=0.002). There were no significant differences between the treatment group and the control group in compression and maximum axial torque (Figure 5). Figure 5Comparison of the bending stiffness anterior-posterior and medial-lateral, compression, torsional stiffness and maximum axial torque. The regression analyses revealed significant non-linear relationships between BV/TV, Tb.N., Tb.Sp. and all mechanical properties. The Tb.Sp. showed the highest correlation witch each of the stiffness parameters. Maximal correlation coefficients were found for the Tb.Sp. vs. the bending stiffness AP and ML with R2=0.69 and R2=0.70 (P<0.0001; n=12) (Figure 6). Thereby relationship between trabecular spacing (Tb.Sp.) and maximal torsional stiffness was found to be inversely proportional. Poor correlations (R2 <0.4) were found for Connectivity with all parameters. There was no significant correlation between Connectivity and the compression strength and the maximum axial torque (P>0.05) (Table 1). Figure 6Regression of trabecular separation (Tb.Sp.) vs. bending stiffness in anterior-posterior (AP) and medio-posterior (ML) direction. Table 1Regression (R2) values of the microarchitectural parameters to the multiaxial Mechanical stiffness.Trabecular Spacing (Tb.Sp.)trabecular number (Tb.N)connectivitybending AP0.690.530.31Bending ML0.700.590.26Compression0.530.35n.sign.Torsion0.660.500.39Max. torque0.590.41n.sign.AP, anterior-posterior; ML, and medio-posterior BODY.DISCUSSION: Our results indicate that local rhBMP-2 application induces significant changes of the regenerate microarchitecture in a sheep tibial DO model. The μCT measurements indicated that the trabecular microstructure of the rhBMP-2 treated distraction zones were characterized by an increased cortical bone volume fraction (BV/TV), an increased Tb.N and a decreased Tb.Sp. There were no significant differences found for the Tb.Th. between the rhBMP-2 injected group and the control. While the BV/TV in the cortical weighted ROI is increased for treatment group (A), there were no significant differences found for the BV/TV in the total ROI (Figure 3). As these results suggest, rhBMP-2 does not increase the total amount of newly formed bone, but it enhances the reformation of the cortex and the marrow cavity. These findings were confirmed by previous studies, which investigated the effect of rhBMP-2 on the rate of callus formation and mineralisation.1,5 The authors argue that the amount of bone and cartilage in fracture callus, and therefore its size, is primarily influenced by mechanical factors, while rhBMP-2 accelerates the rate of development of the callus and the cortical union. But this effect may be time dependent. Li et al. investigated the effect of rhBMP-2 on bone consolidation in an rabbit DO model and found a significant higher bone mineral content (BMC) in the rhBMP-2 treated groups 14 days after surgery compared with the control.5 However at 28 days the BMC of the regenerate was similar in all groups. The assimilation of the BMC between control and treatment groups at 28 days was explained by an advanced remodeling of the bone regenerate in the treatment groups, which included a redistribution of mineralized bone tissue from the central region of the distraction zone to the newly formed cortex. Our results have been strengthened by Zhu et al., who found an increased BV/TV, Tb.Th. and Tb.N. and significantly decreased Tb.Sp. in the rhBMP-2 group compared to their control group in a rabbit model. These authors investigated the combined effects of rhBMP-2 and NEL-like molecular-1 (NELL-1) on bone regeneration in rapid distraction osteogenesis.33 Moreover, they showed that the combination of rhBMP-2 and NELL-1 reached the highest value in BV/TV, Tb.Th. and Tb.N. and the lowest value at Th.Sp. in comparison to the control group. This provides an indication that the osteogenesis can be positively influenced by other biological agents. Nevertheless, rhBMP-2 takes an important role, because the trabecular microarchitecture parameters were significant higher in the rhBMP-2-only-treated group than in the NELL-1 group. The accelerating effect on the cortex regeneration as found in the current study may be explained by the fact, that BMP is primarily expressed by chondrocytes during the consolidation, which are typically located in external and periosteal callus.34 A more rapid chondral ossification of the external and periosteal callus, which is induced by external application rhBMP-2, is likely to explain the enhanced cortical bone formation in this study. Box et al. stated that rhBMP-2 does not increase the size of the callus significantly but enhances the callus maturation so that the rhBMP-2 effects during distraction osteogenesis are characterized to be qualitative, not quantitative.1 Some studies have been demonstrated the potential positive influence of rhBMP-2 on the trabecular microarchitecture of bone, however, in conjunction with other biological agents or other body parts. In this study a local application of rhBMP-2 has been shown to induce significant changes in the callus microarchitecture. The microstructural changes in the cortical subvolume: increase of Tb.N and decrease of Tb.Sp. are discussed to be biomechanically highly relevant.23,35 Our results were confirmed by Foldager et al., who investigated the difference in early osteogenesis and bone microarchitecture in anterior lumbar interbody fusion with rhBMP-2, equine bone protein extract and autograft.26 They found that BV/TV and Tb.N. are increased in rhBMP-2 treatment group, but Tb.Th. significantly decreased as in osteoporotic bone morphology after 8 weeks. However, the connectivity density was significantly higher in the rhBMP-2 treated group, suggesting that the trabecular number is more important for the bone strength than the trabecular thickness. Therefore the rhBMP-2 induced microstructural changes, characterized in the present study: increased BV/TV, increased Tb.N. and decreased Tb.Sp. correspond to the microstructural pattern formulated for a biomechanically optimized trabecular microarchitecture. These results coincide with the results of the current study as we found significant correlations between BV/TV, Tb.N., Tb.Sp. and the mechanical parameters like bending stiffness, torsional stiffness, compression and maximum axial torque measured by mechanical testing. These findings were confirmed by Ni et al. who found a higher torsional stiffness and higher maximum torque in the investigation of rabbit DO model with hydroxyapatite/tri-calcium phosphates (HA-TCP biomaterial), rhBMP-2 and alendronate.36 Moreover Sandhu et al. support our findings regarding the increased stiffness in the treatment group when they could show that the rhBMP-2 treated group had a significantly increased bending stiffness in al directions compared to the control group. These 3D structural indices Tb.N and Tb.Sp were shown to have a high ability to reflect the bone strength.37 Our assumption, that repetitive injections of rhBMP-2 may optimize the trabecular microstructure due to an increase of Tb.N. and decrease of Tb.Sp. resulting in an increased bone stiffness is supported by other authors.27 In our own regression analyses we found a strong correlation between microstructure parameters (BV/TV, Tb.N., Tb.Sp.) and the multiaxial stiffness in callus tissue. Thereby the trabecular separation (Tb.Sp.) was observed to be the best predictor of callus stiffness. On the other hand we found a poor correlation between the connectivity in our 3D analysis and the stiffness parameters. This observation may be explained by the presence of isolated bone particles in the distraction zones, which may be interpreted unconnected trabeculae, as they have been characterized during DO(38). Isolated particles in the 3D dataset have been reported to produce negative values of Connectivity.39 Thereby its obvious that isolated bone particles will contribute to the bone volume fraction of the regenerate (BV/TV) but not to its mechanical competence. We are aware of several limitations of our study. First, we were unable to measure weight bearing in the operated and limbs during consolidation, which may influence healing and ultimately, the microarchitecture of the regenerate. Second, the microarchitecture was only assessed in vitro at one timepoint (day 74), so that we ware not able to evaluate the microarchitectural development during the healing process. Nevertheless our data strongly support an effect use of a triple injection of rhBMP-2 to accelerate the consolidation period during. In conclusion this study suggests that a triple injection of rhBMP-2 optimizes the trabecular microarchitecture in the distraction zone, which might explain the advanced mechanical integrity of newly formed bone under rhBMP-2 treatment as found in our study for bending and torsional stiffness.

TITLE: Intervention with Delivery of Diabetic Meals Improves Glycemic Control in Patients with Type 2 Diabetes Mellitus ABSTRACT: The aim of this study was to investigate the effects of a diabetic meal delivery system on glycemic control over a 12 month period in patients with type 2 diabetes. A total of 77 patients with type 2 diabetes were assigned randomly into three dietary intervention groups: group M, diabetic meal delivery; group D, individual dietary counseling; and group C, conventional dietary education. In group M, HbA1c levels decreased significantly from 8.2 ± 1.2% to 7.4 ± 0.8% after 12 months (p<0.05), while in group D, HbA1c levels decreased significantly throughout the entire 12 month period, from 8.5 ± 1.7% at baseline to 7.4 ± 1.1% at the endpoint. Similarly, fasting blood glucose (FBG) levels decreased significantly between 1 and 12 months in group M (p<0.05), and decreased significantly during the entire 12 month period in group D (p<0.01). There were no significant changes in either HbA1c or FBG levels in group C. This study provides evidence that intervention with delivery of diabetic meals to patients with type 2 diabetes can be equally effective for achieving glycemic control as individual dietary counselling by a dietitian. Diabetic meal delivery can therefore be used successfully to provide diabetes education to outpatients. BODY.INTRODUCTION: Numerous studies have demonstrated the beneficial effects of dietary intervention and increased physical activity on hyperglycemia and other cardiovascular risk factors such as high cholesterol and hypertension [1–4]. However, it often proves very difficult for patients with diabetes to change their behavior or their lifestyle, especially their diet. Even if patients succeed in making these changes, many have a tendency to revert to their previous lifestyle and dietary habits [5, 6]. In Japan, patients with diabetes often stay in hospital for 1 or 2 weeks for diabetes education, primarily to learn about diet. However, this is not a convenient method for many patients due to high costs and suspension of work and daily life. Therefore, various other methods for managing diabetes have been designed with the aim of improving the quality and efficiency of self-care in patients. Studies have been carried out to establish educational programs that encourage patients to follow and adhere to practical guidelines. Diabetes care involves managing patients with a chronic disease consistently in outpatient settings. To establish a more efficient system for diabetes care, we devised a diabetic meal delivery system for patients with type 2 diabetes. Using this system, the patients were able to learn about diabetes dietary requirements, with this knowledge making it easier from them to diet without the need for a stay in hospital. The aim of this study was to investigate the effects of our diabetic meal delivery system on glycemic control in patients with type 2 diabetes. BODY.SUBJECTS AND METHODS.PATIENTS AND DIETARY INTERVENTION: The study protocol was approved by the Ethics Committee of the School of Comprehensive Rehabilitation at Osaka Prefecture University and informed consent was obtained from all patients. Participants diagnosed with type 2 diabetes were recruited from outpatients attending the Kajiyama Clinic between 2004 and 2005. Patients were excluded if they had any significant diseases that were likely to affect the outcome or compliance with this study. Such diseases or conditions included heart failure, hepatic dysfunction, renal failure or serious physical and mental conditions. A total of 77 adults with type 2 diabetes who attended the clinic (mean age 63.6 ± 10.8 years (means ± SD), range 36–80 years) were assigned into three dietary groups by the stratified randomization method that considered age, gender, and duration of diabetes. Each group received one of the following diabetic dietary interventions. The diabetic meal delivery group (group M) had diabetic meals delivered to their home every day. The meals were specially designed for diabetes patients and consisted of 1,200 to 1,800 kcal, 16% protein, 60–64% carbohydrates, 20–25% fat, and approximately 9 g of salt. The meals were cooked and chilled immediately to 3°C prior to delivery. If a patient agreed to participate in the study, the subject had three sessions with dietitians at the start of intervention and after 4 months, and 12 months. The sessions were interactive and included a focus on type 2 diabetes, its symptoms, treatment (especially diet), and associated complications. The individual diet counseling group (group D) involved patients having face-to-face consultation sessions (20 to 30 min) with dietitians every month (12 sessions over 12 months). Although the sessions had pre-planned topics such as diabetes risk factors, diet, physical activity and problem solving, the discussions were individualized and focused on specific individual problems, mainly diet. The goal was to equip the subjects with the necessary knowledge and skills to initiate behavioral changes. The subjects were encouraged to make intermediate goals for themselves by thinking about practical things they could use to change in their diet and daily exercise habits. For example, instead of an abstract goal such as "increase fiber intake and daily exercise", a practical goal would be "to eat brown rice and walk 10,000 steps everyday". The dietitians also addressed coping skills for improving psychosocial functions. The participants were also encouraged to measure and record their weight at home on a regular basis, but were not required to record their diet. The family members responsible for shopping and cooking were invited to join the sessions, with the importance of family participation and the support structures provided being emphasized. The conventional dietary education group (group C) involved the patients receiving their usual outpatients management every month. At baseline, the control group was given general information about lifestyle and diabetes risk. This briefly information was provided individually by either a doctor or nurse at every visit with some printed material also being distributed. The message was to reduce weight, increase physical activity and make qualitative changes in dietary habits. BODY.SUBJECTS AND METHODS.LABORATORY ANALYSIS: Fasting blood samples were collected from all participants every month and examined by auto analyzer. HbA1c levels were determined by a latex cohesion method (JCA-BM2250, KYOWA MEDEX, Co., Ltd., Tokyo, Japan). Fasting blood glucose (FBG) levels were examined by the hexokinase method (JCA-BM12, Shino Test, Co., Ltd., Tokyo, Japan). Total cholesterol (T-Ch) and triglyceride (TG) levels were determined by enzyme assay. HDL cholesterol (HDL-Ch) levels by a direct method (Labospect 008K, Bio Majesty JCA-BM 8060, JEOL, Ltd., Tokyo, Japan) and LDL cholesterol (LDL-Ch) levels by an enzymatic method (Bio Majesty JCA-BM 8060, JEOL, Ltd., Tokyo, Japan). BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSIS: Data were expressed as means ± SD. Statistical analyses were performed using SPSS version 14.0 (SPSS Inc., Chicago, IL). Differences between the three groups at baseline were determined by one-way analysis of variance (ANOVA). The Bonferroni/Dunnett post hoc test was used to determine the significant of continuous data and the χ2 test for significance of the categorical variables. Repeated-measures ANOVA was used to determined the difference between the groups and over times. When a significant difference was found by repeated-measures ANOVA, paired t tests with Bonferroni correction were applied to identify specific differences. A p value of less than 0.05 was considered statistically significant. BODY.RESULTS: The clinical characteristics and baseline laboratory data of the three intervention groups are shown in Table 1. There were no significant differences between the three groups with respect to age, body mass index (BMI), diabetes duration and glucose control methods or in terms of laboratory data, including baseline FBG, HbA1c, T-Ch, HDL-Ch, LDL-Ch and TG. Our primary objective was glycemic control, as indicated by HbA1c levels. Fig. 1 shows HbA1c results in the three groups during the intervention period. In group M, HbA1c levels decreased from 8.2 ± 1.2% to 7.7 ± 1.1% after one month (p<0.05) and continued to decrease significantly after two and three months (p<0.05), with a reduction to 7.4 ± 0.8% after 12 month. In group D, HbA1c also decreased significantly overall throughout the 12 month period (p<0.01), from 8.5 ± 1.7% to 7.4 ± 1.1%. In contrast, in group C HbA1c increased slightly from 8.1 ± 1.2% at baseline to 8.3 ± 1.1% at the end of 12 month period. HbA1c levels in groups M and D were significantly lower than in group C at the end of the study (p<0.05 and p<0.01, respectively). Table 2 shows the changes in BMI and laboratory data from baseline to 12 months in the three dietary intervention groups. BMI tended to decrease in groups M and D, but remained unchanged in group C. FBG levels decreased significantly from 167 ± 30 to 128 ± 26 mg/dl in group M (p<0.05) and from 181 ± 70 to 145 ± 45 mg/dl in group D (p<0.01). No significant change was observed in group C. There were no significant changes in serum lipids levels in the three intervention groups, although serum TG levels were tended to decrease in groups M and D. BODY.DISCUSSION: To our knowledge, the present study is the first to evaluate the use of a diabetic meal delivery system. In this study, we showed that delivery of the diabetic meals led to improved glycemic control. Intervention with the diabetic meal delivery had a beneficial effect on HbA1c levels similar to that seen with individual dietary counseling. It is well known that intensive blood glucose control decreases the risk of the micro- and macro-vascular complications [7, 8]. Our results therefore have important clinical implications, given that medical costs for diabetic patients are increasing and self-care for diabetes patients is considered to be of considerable benefit. For example, it costs approximately $2,100 a week to stay in hospital for diabetic education, whereas diabetic meal delivery costs only $740 for 6 dinners for 4 months. In addition, improving glucose control will reduce the economic costs associated with diabetic complications [9, 10]. For example, hemodialysis costs $50,000 a year for one diabetic patient with chronic renal failure. There are several possible explanations for the improved glucose control in the diabetic meal delivery group. Firstly, the patients were able to learn and experience dietary changes as a result of receiving the diabetic meals at their home. In addition, the patients in the diabetic meal delivery group received medical data, with the majority showing improved glucose control soon after intervention, as a consequence of increased self-efficacy. These factors may have stimulated and motivated the patients to control their glucose levels more enthusiastically. After the period of diabetic meal delivery, the majority of patients gained dietary skills and continued to consume a proper diet during the 12 month study period. Our study provides evidence that diabetic meal delivery is as effective as individual counseling for the self-management of diabetes. The method was particularly practical for diabetic outpatients who were not able to receive either regular education and counseling by dietitians or stay in hospital for diabetes education. Secondly, delivery of meals was suitable for patients who were not able to depend on family participation or support. We often experience that it is difficult to motivate lifestyle change, especially diet, unless there is family understanding and support. In our study, all the participants were encouraged, but not required, to record their diet. There were several limitations to our study. The first was the relatively small number of patients, especially in the diabetic meal delivery group. In this study, first we assigned 30 patients in each of the three groups. However, 12 patients in group M were not able to complete intervention with delivery of diabetic meals perfectly. The main reasons were particular food preferences and the cost of the meal delivery. It is therefore necessary to improve the quality, taste and cost of the diabetic meals in order that they are accepted by more patients. The second limitation was that the number of meals delivered each week and the period of meal delivery varied among the patients. Patients were free to choose both the frequency and the period of delivery. This difference may have affected the change in HbA1c levels over the 12 month period. However, we found there was no difference in metabolic levels or behavior modification between patients with different frequencies or periods of meal delivery. Further studies are required to examine how many meals or how frequently the diabetic meals need to be delivered in order to maintain optimal glycemic control and behavior modification in the future. Methods of efficient self-management training are required in public health programs as from 2008 in Japan, all health insurance organizations will be responsible for educating people with metabolic problems or other health problems. In order to educate a larger group of people efficiently, previous studies have shown that a telephone- [11] or an internet-based blood glucose monitoring system are effective for diabetes management [12]. However, many elderly Japanese are not yet familiar with the use of the internet, while education over the telephone, although an effective psychological support may not be a suitable method for teaching facts about diet. The diabetic meal delivery service provides an opportunity for people with diabetes who are not able to receive individual counseling, or who live in rural and medically underserved areas, to have access to high-quality diabetes education. The observed difference in incidence between intervention groups and the usual care control group indicated that the intervention needs to be individualized and performed consistently in order to be effective in self-management, especially diet. During the study it proved necessary to explore methods to respond to increasing demands for individual self-management education. However, the findings presented in this paper need to be confirmed in a study on a larger number of patients. In conclusion, this study provides evidence that intervention using delivery of diabetic meals in Japanese patients with type 2 diabetes is as effective as individual diet counseling for maintaining glycemic control. It is hoped that delivery of diabetic meals makes it easier to maintain an appropriate diet at home and also to motivate and empower underserved patients to start self-management and thereby avoid the serious complications of diabetes.

TITLE: Comparison of Standard and Low Dose Intraoperative Mitomycin C in Prevention of Corneal Hazeafter Photorefractive Keratectomy ABSTRACT.BACKGROUND:: This study aimed to compare two doses of Mitomycin C in reducing haze formation after photorefractive keratectomy. ABSTRACT.METHODS:: 170 eyes of 85 patients enrolled; in each patient one eye randomly assigned to be treated by low dose intraoperative MMC (LDMMC) and other eye by standard dose MMC (SDMMC). Then the patients were followed up to 6 months and refraction, Uncorrected Distant Visual Acuity (UCDVA), Best Corrected Distant Visual Acuity (BCDVA) and haze formation submitted in each postoperative exam. ABSTRACT.RESULTS:: The mean preoperative refraction in LDMMC eyes was −3.08 (SD 1.65) sphere and 0.92 (SD 0.88) cylinder. These values for SDMMC eyes were −3.25 (SD 1.80) sphere and 0.81 (SD 0.84) cylinder. Mean postoperative sphere in LDMMC group was −0.132 (SD 0.503) and −0.138 (SD 0.484) in 3 and 6 months after PRK. These results was −0.041 (SD 0.501) and −0.076 (SD 0.489) for SDMMC group. Mean postoperative cylinder 3 and 6 month after PRK was 0.435 (SD 0.218) and 0.423 (SD 0.255). In LDMMC group and 0.435 (SD 0.247) and 0.426 (SD 0.261) in SDMMC group. In third month 14 eyes in LDMMC group presented with grade 1 score of clinical haze. From these eyes only 2 still had this haze after 6 month. 7 eyes in SDMMC group had grade 1 clinical haze at third month- but no clinical haze was seen at the end of 6th month. ABSTRACT.CONCLUSION:: The results of the two doses of Mitomycin C were not significant. We suggest to use the lower dose to reduce its side effects. BODY.INTRODUCTION: Refractive surgery is an evolving ground in recent ophthalmology and photorefractive keratectomy (PRK) is one of the most popular means to correct refractive error of the eye. Many studies have carried to evaluate long-time stability, complications and causative factors in eyes underwent PRK.[12] Kim et al., showed that myopic regression can occur up to 5 years after surgery and pre-PRK refraction is the most important factor.[1] Alteration in extracellular components after PRK along with generation of myo-fibroblast may reduce corneal transparency which may present as sub-epithelial haze and can be clinically significant in many cases.[3] Applying intraoperative Mitomycin C (MMC) as an effort to reduce the rate of corneal haze has tried earlier and in numerous studies prophylactic use of MMC 0.02% was associated with lower rates of haze formation and better visual outcomes.[45] Majmudar et al., revealed that topical application of MMC (0.02%) may be a successful method of preventing recurrence of subepithelial fibrosis after corneal surgery.[4] Anyway, concerns persist regarding MMC toxicity and long-term complications such as corneal melting and endothelial cell loss.[67] There is one report of corneal edema after phototherapeutic keratectomy (PTK) including intraoperative use of MMC 0.02%. Although evidences of keratocyte apoptosis and myo-fibroblast death which had leaded to reduced corneal cellularity after MMC use is of great concern.[8–11] Effect of Mitomycin C application on endothelial cells has reported in many studies[12] when the ocular surface alteration is also possible.[13] Because the standard dose of intraoperative MMC (0.02%) has driven empirically based on pterygium removal and glaucoma filtering surgery results many controversies persist in dosing and timing of MMC usage.[14] Because timing of MMC application seems less important than the dosage, this study tries to find the effect of low dose MMC (0.01%) intraoperative use in PRK versus standard dose (0.02%) in preventing corneal haze and promoting desirable visual outcome. BODY.METHODS: All the patients with inclusion criteria were informed of the study and written consent obtained. These patients were myopic patients with ages between 20 and 30 years who will to correct their refractive error by PRK. 170 eyes of 85 patients included. Primary evaluation of refraction, uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), slit-lamp examination with fundus evaluation performed and eyes with any disorder that could influence postoperative outcomes were excluded. After all, the eyes divided into two arms, in each patient one eye randomly assigned to be treated by low dose intraoperative MMC (LDMMC) and other eye by standard dose MMC (SDMMC). All operations were done by one surgeon. After scrub by povidone-iodine topical anesthetic drop instilled and eyes were treated by Technolas 217z-100 laser ablation and then MMC applied by Weck-cell spongefor 45 seconds. Then the surface irrigated by balanced salt solution. Postoperative examination done 1 week, 1 month, 3 month and 6 month afterwards, regarding refraction, uncorrected distant visual acuity (UCDVA), best corrected distant visual acuity (BCDVA) and haze formation score, respectively. Haze intensity according to the Frantes scale was graded as no haze, trace, 1, 2, 3 and 4. Then t-test and Mann-Whitney tests were used to analysis the outcome considering 85% power. BODY.RESULTS: 170 eyes of 85 patients underwent photorefractive keratectomy between May and November 2011 (23 men and 62 women). Mean age of the patients in this study was 25.98 years (Range: 20-30 years). The mean preoperative refraction in LDMMC eyes was −3.08 (SD 1.65) sphere and 0.92 (SD 0.88) cylinder. These values for SDMMC eyes were −3.25 (SD 1.80) sphere and 0.81 (SD 0.84) cylinder. Mean postoperative sphere in LDMMC group was −0.132 (SD 0.503) and −0.138 (SD 0.484) in 3 and 6 months after PRK. These results was −0.041 (SD 0.501) and −0.076 (SD 0.489) for SDMMC group. Mean postoperative cylinder 3 and 6 month after PRK was 0.435 (SD 0.218) and 0.423 (SD 0.255) in LDMMC group and 0.435 (SD 0.247) and 0.426 (SD 0.261) in SDMMC group. In third month 14 eyes in LDMMC group presented with grade 1 score of clinical haze. From these eyes only 2 still had this haze after 6 month. 7 eyes in SDMMC group had grade 1 clinical haze at third month - but no clinical haze was seen at the end of 6th month. Comparison of two arms of this study did not disclose any significant difference in preoperative parameters including refraction, UCDVA and BCDVA. t-test study also reveals no meaningful difference in these parameters in postoperative measurements. Comparison of haze formation rate in these two groups by Mann-Whitney test demonstrated no significant difference in results (P = 0.104 3rd month and P = 0.156 6th month after operation). BODY.DISCUSSION: Application of intraoperative MMC is a valuable mean to prevent corneal haze especially in high myopic patients but concerns about its side effects lead us to determine if a lower dose of MMC may be as effective as the standard dose. Midena et al. demonstrated that 0.02% topical MMC has no significant adverse effect on corneal keratocytes.[15] Goldsberry et al. also remarks no side effect of MMC on endothelial quantitative and qualitative parameters.[16] Although Netto et al. reported that MMC may cause keratocyte and myo-fibroblast apoptosis and decrease in anterior stromakeratocyte density may lead to future complications.[17] According to our study there was no significant difference in visual outcome or haze formation by use of lower dose of MMC (0.01%) compared with standard dose (0.02%). BODY.CONCLUSION: Our findings shows that low dose intraoperative MMC has the same effect as standard dose in prevention of corneal haze and visual outcomes have no meaningful difference while it promises lower side effects and long-term complications.

TITLE: Efficacy of Postoperative Continuous Wound Infiltration With Local Anesthesia After Open Hepatectomy ABSTRACT.OBJECTIVES:: Local anesthetic wound infiltration is widely used as an effective adjunct during multimodal postoperative pain management. The aim of this study was to evaluate the effectiveness of continuous wound infusion of ropivacaine in postoperative pain relief, opioid sparing, incidence of nausea and vomiting, and bowel and liver function improvement in patients undergoing open hepatectomy. ABSTRACT.METHODS:: Forty patients undergoing open hepatectomy were enrolled in this prospective, randomized, double-blinded, placebo-controlled trial. Patients were divided into 2 groups: the 0.9% saline continuous infusion group (the control group; n=20) and the ropivacaine continuous infusion group (the Ropi group; n=20). Outcomes measured postoperatively were pain score at rest and on movement, sufentanil consumption, incidence of nausea and vomiting, and sedation score across 48 postoperative hours. Time to bowel recovery, liver function change, mean length of hospitalization, patient satisfaction, and other data after 48 postoperative hours were collected until hospital discharge. ABSTRACT.RESULTS:: Pain scores at rest were lower for the ropivacaine group and reached significance after 8 and 16 hours (P<0.01). Sufentanil consumption (41.50±21.80 vs. 89.70±35.22 μg; P<0.01) after 48 hours, time to bowel recovery (1.80±0.70 vs. 3.15±1.04 d; P<0.01), incidence of nausea and vomiting (1.75±0.72 vs. 2.40±0.68; P<0.05), and mean length of hospitalization (5.6±2.44 vs. 7.35±2.85 d; P<0.01) were significantly reduced, and the sedation score and liver function change were also comparable between the 2 groups. There was no difference with respect to pain scores on movement, nor with respect to patient satisfaction. ABSTRACT.CONCLUSIONS:: Surgical wound infusion with ropivacaine after hepatectomy can improve pain relief at rest and accelerate recovery and discharge. BODY: Control of postoperative pain is of interest to surgeons, anesthetists, hospital administrators, and patients. As a major form of upper abdominal surgery, hepatectomy is always accompanied by significant pain, and patients with severe pain are less likely to cough or move after surgery, which interferes with the return of gastrointestinal and respiratory functions. The most often used analgesic methods for pain alleviation are epidural analgesia and intravenous opioid analgesics through patient-controlled delivery systems.1 Unfortunately, epidural puncture is a relatively complicated process with potential complications. Patient-controlled pain management has the risk of addiction, as large doses of opioids are used for a long period of time. Furthermore, opioids have potentially serious side effects such as nausea, excessive sedation, respiratory depression, slowing down of bowel function, and impairment of liver function.2 Thus, to avoid these potentially serious adverse effects, postoperative pain is often undertreated.3 Although wound infiltration for abdominal analgesia had been used as early as 1986,4 lack of knowledge on the part of the surgeon and material limitation for localized persistent infiltration deterred it from being used to its full potential until the early 1990s. With the recognition of the important role played by parietal nociceptive afferents in the origin of overall pain, local anesthetic wound infiltration is widely recognized as a useful adjunct in multimodality approaches to postoperative pain management.5,6 It has been demonstrated that the pain pump is safe and effective in a variety of procedures, but the efficacy of this technique has been poorly described in hepatectomy patients. After hepatectomy, patients are most likely to develop primary liver cancer or hepatolith, accompanied by liver dysfunction, and the procedure itself may worsen liver dysfunction and even lead to acute liver failure. Ropivacaine, as a widely used amide-type local anesthetic, has comparable efficacy to bupivacaine but lower systemic toxicity and shorter half-life to reduce the risk of plasma accumulation. Therefore, we speculate that wound instillation with 0.3% ropivacaine is an effective and well-tolerated method for postoperative analgesia after hepatectomy. We conducted a prospective, double-blinded, randomized, controlled design study to support our hypothesis. BODY.METHODS: Forty patients undergoing open hepatectomy requiring a subcostal incision >20 cm that was performed by the same experienced surgeon were enrolled into our study. The patients were hospitalized between May 2011 and July 2012. All patients signed a written informed consent form to participate in this study before the operation. Group allocation was done using computer-generated code allocation. Ropivacaine and saline were added to the elastomeric pump by an anesthesiologist not involved in the study. The patients, surgeons and investigator were kept blinded to the assigned treatment groups throughout the study period. Patients were randomized to receive a continuous surgical wound infusion of either 0.3% ropivacaine or 0.9% saline delivered through an elastomeric pump (TJPS120-2-250-5; 200 mL) by means of 2 soaker catheters. All patients were given unrestricted access to sufentanil (Graseby 9300; 200 mL) through a patient-controlled analgesic (PCA) device according to our hospital standard of care for breakthrough pain. Patients with a history of adverse reactions to local anesthetics, obesity (BMI>30 kg/m2), chronic pain, chronic preoperative opioid consumption, or psychiatric or neurological diseases, and those unable to use a PCA device, were excluded from the study. When closing the abdomen, the surgeon inserted two 20-G multirole soaker catheters along the full length of the wound. One catheter was placed between the closed parietal peritoneum and the deep muscular fascia, and the other one was placed among the subcutaneous tissue. Ten minutes before the end of the surgical procedure, 10 mL of 0.5% ropivacaine was injected through each catheter as an initial dose in both groups, and the catheters were connected to an elastomeric pump. After the patients' arrival in the recovery area, continuous instillation of 0.3% ropivacaine or 0.9% saline into the wound was commenced at an infusion rate of 2 mL/h per catheter. For recording data, the time immediately after the patient regained consciousness was considered as time 0. All patients had unrestricted access to sufentanil rescue use through an IV-PCA device (2 mg bolus dose, 5 min lockout time, 40 mg dose limit over 4 h). The patient-controlled analgesia and the wound catheter were removed at 48 hours. The outcomes measured across 48 postoperative hours were: pain at rest and on movement, assessed using a numerical rating score (NRS) for pain (NRS, 0 to 10 scale); sedation, which was evaluated on the basis of Ramsay grades at various postoperative assessment times (1=the patient is cooperative, oriented, and tranquil; 2=the patient can be easily aroused through verbal stimuli; 3=the patient is asleep, but shows brisk response to a light glabellar tap or loud auditory stimulus; 4=the patient is asleep, and shows sluggish response to a light glabellar tap or loud auditory stimulus; 5=the patient is asleep, and shows no response to an external stimulus); sufentanil consumption, which was measured on the PCA device; nausea and vomiting, which was recorded as absent or present (1=no nausea or vomiting; 2=nausea; 3=vomiting); the success of treatment, which was judged by the patients on the day of discharge on a 4-point scale (1=poor, 2=fair, 3=good, 4=excellent); and changes in liver function (aspartate aminotransferase [AST], alanine aminotransferase [ALT], alkaline phosphatase [ALP], total bilirubin [TBIL], direct bilirubin [DBIL], albumin, prothrombin time [PT], and activated partial thromboplastin time [APTT]), which were recorded 3 days after surgery. The first postoperative bowel movement (first flatus as symbol), length of hospitalization, and other adverse events were also recorded until hospital discharge. BODY.STATISTICAL ANALYSIS: On the basis of local data on total sufentanil consumption across 48 postoperative hours, a sample size of 20 in each group would have 88% power to detect a 30% reduction in the total dose. Statistical analyses were performed using SPSS software version 18.0. The data were presented as means±SD for continuous variables. The χ2 test, the t test, and repeated measures ANOVA were carried out where appropriate. Statistical significance was set at P<0.05. BODY.RESULTS: Forty patients were initially enrolled in our study. However, 1 patient was lost because of postoperative bleeding, and thus, finally, 19 patients were included in the ropivacaine group and 20 in the control group. No significant differences in terms of age, height, weight, sex, incision length, and surgical procedures were observed between the 2 groups (Table 1). TABLE 1 Patient Characteristics, Incision Length, and Surgical Procedures Time to bowel recovery (1.80±0.70 vs. 3.15±1.04 d; P<0.01), incidence of nausea/vomiting (1.75±0.72 vs. 2.4±0.68; P<0.05), and mean length of hospitalization (5.6±2.44 vs. 7.35±2.85 d; P<0.01) were significantly reduced. Liver function (AST, ALT, ALP, TBIL, and DBIL levels) recovery was better in the Ropi group than in the control group. There was no difference with respect to patient satisfaction (Table 2, 3). TABLE 2 Time to Bowel Recovery, Nausea/Vomiting, Hospital Stay, and Patient Satisfaction TABLE 3 Liver Function Before Operation and 3 Days After Operation The pain scores at rest are presented in Figure 1. Significant differences in pain scores at rest between the 2 groups were detected at 8 hours (NRS 1.5±0.51 for the Ropi group and 2.45±0.51 for the control group; P<0.01) and at 16 hours (NRS 1.75±0.44 for the Ropi group and 2.1±0.31 for the control group; P<0.01). However, there was no difference in pain score on movement between the 2 groups, as shown in Figure 2. FIGURE 1Mean numerical rating score at rest at each time interval. FIGURE 2Mean numerical rating score on movement at each time interval. Sufentanil consumption calculated over 48 postoperative hours was significantly different between the 2 groups as shown in Figure 3. The mean total sufentanil consumption by the control group at 48 hours was double that of the Ropi group (89.70±35.22 μg for the control group vs. 41.50±21.80 μg for the Ropi group; P<0.01). FIGURE 3Cumulative sufentanil consumption (μg) at each time interval. The sedation scores are presented in Figure 4. It was higher in the control group than in the Ropi group at 0 and 8 hours; after 16 hours the scores became similar for the 2 groups. FIGURE 4Sedation score at each time interval. BODY.DISCUSSION: Control of postoperative pain is of interest to patients. Optimal management of postoperative pain, a fast turnover, and a shorter length of hospital stay after a major surgery have been important concerns among surgeons, anesthetists and hospital administrators. The most important source of postoperative pain is the surgical incision; for example, hepatectomy being a major abdominal surgery often has a subcostal incision >20 cm. The traditional effective approaches are epidural analgesia, oral or injecting nonsteroid anti-inflammatory drugs, and opioids. As epidural puncture is always associated with side effects such as neuraxial hematoma and nerve damages, patients with spinal malformations, infectious puncture site, and anticoagulant therapy are normally precluded to use epidural analgesia. Opioid administration even at lower doses can induce oxidative stress in the liver, leading to hepatocyte apoptosis and liver enzyme elevation.7 Some studies have shown that repeated morphine administration, which is commonly used for pain relief, would interfere with liver antioxidant defense and hepatocyte vitality. Unexpected sensitivity to systemic opioid has been observed after hepatic resection. In addition, most hepatectomy patients experience liver dysfunction preoperatively, and surgery itself would aggravate liver dysfunction or even lead to liver failure. Thus, it would be dangerous for hepatectomy patients to consume continuous doses of opioids. The discovery of new analgesic techniques that work in different phases of analgesia can further improve the quality of medical care for hepatectomy patients. Local anesthetic wound infiltration is now recognized as a useful adjunct in a multimodality approach to postoperative pain management. It has been reported that the method can be used in many types of surgery, such as cesarean delivery, inguinal hernia repair, total hip arthroplasty, and colorectal surgery.1,2,8 However, to the best of our knowledge, the application of local anesthetic wound infiltration in hepatectomy patients has rarely been reported. Chan et al9 first described the use of continuous ropivacaine infusion after open hepatic surgery. Different from theirs, we focus more on important functional measurements such as the return of bowel function, the recovery of liver function, and the duration of hospitalization. We also measured liver enzyme changes instead of indocyanine green elimination rate, which reflect the liver reserve function, to assess liver cirrhosis or estimate surgical risk. In our study, we confirmed a lower NRS score on rest and reduced sufentanil consumption after continuous infiltration of 0.3% ropivacaine into the wound among patients undergoing open hepatectomy. Local anesthetics can relieve pain intensity, shorten the duration of postoperative pain, and reduce analgesic requirements by inhibiting the transmission of noxious impulses from the injured side.10 Moreover, it can also inhibit the local inflammatory response to injury that could sensitize nociceptive receptors and contribute to hyperalgesia.11 In contrast, a systemic effect of ropivacaine administrated through a catheter cannot be ignored. Systemic local anesthetics can be analgesic and this effect has been clearly demonstrated. The potential mechanisms may involve an inhibition of central hyperalgesia and the axonal transportation of inflammatory mediations.11 However, local anesthetic wound infiltration technically had no advantage in pain relief on movement in our study; maybe the infusion rate or concentration of ropivacaine was not enough, and therefore further research is needed. Reduced systemic sufentanil resulted in faster time to bowel recovery, as sufentanil inhibits gut motility and propulsive activity by combining the μ-2 and κ receptors in the gastrointestinal tract, inducing bowel dysfunction.12 In contrast, ropivancaine could accelerate postoperative intestinal motility by reducing the inflammatory response. In our study, we chose to measure the liver serum biochemical index to compare the liver function between the 2 groups. Hepatocellular damage is the predominant pattern of hepatic damage. The Council for International Organizations of Medical Science proposed the evaluation of the levels of ALT or conjugated bilirubin, or a combined level of AST, ALP, and TBIL, when investigating drug-induced liver injury.13 ALT is the most sensitive serum marker in drug-induced liver injury, as AST can reflect the severity of liver injury. Significant changes in liver enzymes (ALT, AST, and ALP) were observed in the Ropi group compared with the control group 3 days after surgery. TBIL and DBIL levels were also comparable after surgery. The bilirubin level decreased significantly in patients with biliary obstruction once the obstruction was relieved surgically. However, there were no differences in albumin, PT, and APTT levels; this may perhaps be because they mainly reflect the synthetic function of the liver and are not sensitive to drug-induced injury, or perhaps because protein and coagulation factors have a longer half-life and hence there were no changes 3 days later. We found an interesting phenomenon in our study. Despite the increasing amount of sufentanil in the saline group, the sedation score was the same in both groups after 16 hours. Unlike other opioids, sufentanil is a highly selective μ opioid receptor agonist, although it exerts a sedative effect by acting on κ receptors, which are located in the central nervous system. Studies have evaluated the dose of sufentanil (0.35 μg/kg) that could have an effect on the central nervous system.14 The consumption of sufentanil every 8 hours was much lower than 0.35 μg/kg, and thus the sedation scores of the enrolled patients were all below 2, and after 16 hours all the patients in both groups became cooperative and oriented (sedation score=1). We choose ropivacaine, a pure levorotatory stereoisomer, as we can reduce its cardiotoxicity in the event of an overdose.15 We did not measure the plasma ropivacaine concentrations in the present study, for it has been reported that the total plasma concentration of ropivacaine remains far below the known toxic threshold when larger amounts of ropivacaine are used.8 Systemic toxicity has been defined by Labaille and colleagues as a serum level >4.0 μg/mL, which was determined on the basis of healthy adult volunteers receiving intravenous infusions of ropivacaine.16 However, the absorption, distribution, and elimination of drugs change in hepatic insufficiency. In Chan's study, the authors had measured plasma ropivacaine concentration (0.25%, 4 mL/h for 68 h) after open liver resection and only 2 patients were seen to have plasma ropivacaine concentrations >3.0 μg/mL.8 Different from the healthy individuals, there may be a reduced free fraction of local anesthetic after surgery owing to rising levels of α-1-acid-glucoprotein, which has the potential advantage of buffering the unbound ropivacaine, providing a protective effect against toxic reactions.17 A significant proportion of postoperative pain comes from superficial structures, but peritoneal pain may also be of importance. To affect the deep muscular-peritoneal component of abdominal pain, we placed 1 catheter between the parietal peritoneum and deep muscular fascia and the other catheter in the subcutaneous tissues. We finally obtained positive results in our trial compared with other studies conducted on only infiltrated skin or subcutaneous tissues. However, there were no differences in patient satisfaction. When we conducted the research, the investigators did not particularly disclose the study aspects of our research and only asked the patients to give a subjective evaluation of the entire hospital process. Maybe Chinese patients are more concerned about the success of the operation, the amount of bleeding, wound healing, postoperative complications, and postoperative care. The wound infiltration technique is easy to administer and follow-up, with no extra training required and no motor nerve blockade. However, its success mostly depends on the effectiveness of the infiltration by the surgeons. Consequently, wound infiltration with local anesthetics not only provides pain control but also affects inflammatory responses and increases catecholamine, which has an added benefit of enhancing wound healing by increasing wound perfusion and oxygenation.18 In our trial, there were no surgical wound infections, which was consistent with Lluis's study in which use of an in-site preperitoneal catheter for postoperative anesthesia did not increase the risk for surgical site infection.19 Hence, we again eliminate the anxiety of surgeons that continuous infusion of local anesthetic through a catheter will increase surgical wound infection. The limitations of our study are the small number of patients included and the elastomeric pump running at a fixed rate of infusion (2 mL/h). A PCA pump may provide better analgesic effects. We did not measure the systemic concentration of ropivacaine in patients with liver dysfunction. We will conduct a study on the systemic concentration of free ropivacaine and α-1-acid-glucoprotein while using a local wound instillation technique in liver or renal insufficiency patients. In our study, 1 patient underwent a reoperation because of bleeding. We found that the tissue planes were difficult to be identified and separated to appropriate position of the catheters. In conclusion, continuous surgical wound infusion with ropivacaine was effective and seemed to improve pain management and accelerate patient's recovery and discharge after open hepatectomy.

TITLE: A randomized, 4-week double-blind placebo control study on the efficacy of donepezil augmentation of lithium for treatment of acute mania ABSTRACT.INTRODUCTION: A significant number of mania patients fail to respond to current pharmacotherapy, thereby there is need for novel augmentation strategies. The results of some early studies showed the effectiveness of cholinomimetics in the treatment of mania. One open case series suggested the efficacy of donepezil in the treatment of bipolar disorder. Our aim was to explore whether an oral cholinesterase inhibitor, donepezil, administered during a 4-week treatment period, would benefit patients with acute mania. ABSTRACT.METHODS: We conducted a 4-week double-blind, placebo-controlled trial of donepezil as an adjunctive treatment to lithium in patients with acute mania. Eligible subjects were randomly assigned to receive donepezil or placebo in addition to lithium. Donepezil was started at 5 mg/day, and increased to 10 mg/day in the first week. Patients were rated with the Young Mania Rating Scale (YMRS) and Brief Psychiatric Rating Scale (BPRS) at baseline, day 1, week 1, week 2, and week 4. ABSTRACT.RESULTS: Out of the 30 patients who were enrolled, 15 were on donepezil and 15 were on placebo. All patients completed the 4-week trial. On the first day, there was a difference of 1.97 units on the psychomotor symptoms scale of the YMRS in the donepezil group as compared to the placebo group (t = 2.39, P = 0.02). There was a difference of 0.57 units (t = 2.09, P = 0.04) in the speech item and a difference of 0.29 units in the sexual interest item (t = 2.11, P = 0.04) in the donepezil group as compared to the placebo group. The total YMRS difference on the first day approached the conventional significance level (1.97 units, t = 1.84, P = 0.07). Over the course of 4 weeks, we failed to find that donepezil produced any significant difference in the YMRS (6.71 units difference, t = −1.44, P = 0.16) or the BPRS scale (1.29 units difference, t = −0.33, P = 0.75) as compared to placebo. Ten subjects (66.67%) in both groups met the criteria for clinical response (Fisher's exact P = 1.00). Five subjects (33.33%) in the donepezil group met the criteria for clinical remission while nine subjects (60.00%) in the placebo group met the remission criteria (Fisher's exact P = 0.27). ABSTRACT.CONCLUSION: Use of the oral anticholinergic donepezil had some benefit in the augmentation of lithium treatment on the first day, but did not provide any significant benefits in the long-term. BODY.INTRODUCTION: Bipolar disorder is a chronic mental illness, and the lifetime prevalence for bipolar I and II disorders is 1.0% and 1.1%, respectively.1 The spectrum of bipolar conditions has a higher prevalence (2.6%–6.5%).2–5 The World Health Organization identified bipolar disorder as the sixth leading cause of disability-adjusted life years worldwide among people ages 15 to 44 years.6 The literature suggests that lithium is useful during the acute manic phase.7,8 However, a significant number of patients fail to respond to the current pharmacotherapy.9–11 A good number of double-blind, randomized, clinical trials supported the efficacy of antipsychotic agents in the treatment of acute mania,7 but concerns about neurological adverse effects, arousal of depressive symptoms, and lack of adherence limited their clinical use. Therefore, there is a need for new effective and well-tolerated approaches for this clinical condition. Treatment of mania with cholinomimetics was first documented in 1889.12 In the 1950s, several observations and studies with organophosphate insecticides and similar cholinesterase inhibitors were conducted.13–15 In 1972, Janowsky et al reported the first double-blind study suggesting the effectiveness of the cholinesterase inhibitor physostigmine in the treatment of mania.16 They found that physostigmine rapidly raised brain acetylcholine and had a dramatic effect on acute mania, starting a few minutes after dosage and lasting for about 30 minutes, which was consistent with the pharmacokinetics of physostigmine.16 The patients were initially very manic; rhyming, punning, hyperactive, talking fast, the patients thoughts were grandiose, and euphoric. Gradually, they transitioned through euthymia and became depressed. As physostigmine dissipated from their system, they became briefly euthymic and then full mania returned. Janowsky later proposed the "cholinergic-adrenergic hypothesis of mania and depression."17 According to the cholinergic–adrenergic hypothesis, cholinergic and adrenergic tones have reciprocating and balancing effects on mood; the adrenergic tone being manicogenic, and the cholinergic tone being depressogenic.17 The main criticism of the cholinergic–adrenergic hypothesis questions the nature of the depressant effects of cholinomimetic drugs. Some researchers have argued that the depressogenic effect after using cholinesterase inhibitors is not associated with changes in mood or manic thought content.18 Some researchers have concluded that the effects on behavior, sleep, and hormones may represent the side effects secondary to peripheral cholinesterase inhibition.19 Some studies found that even without other peripheral side effects, changes in behavior, sleep, and stress-related hormones after cholinesterase inhibitor administration often occurred.20–22 Sokolski and DeMet found that patients with more severe mania had a decreased cholinergic tone, which also supported the cholinergic–adrenergic hypothesis.23 Cholinesterase inhibitors that increase the concentrations of acetylcholine in the synapses have been proposed as antimanic agents.24 Most cholinesterase inhibitors have a short half-life, have to be given by intravenous infusion, and exhibit many peripheral side effects which make them impractical for clinical use. Donepezil is a longer acting, orally available, reversible cholinesterase inhibitor. It has greater selectivity for acetylcholinesterase than it has for butyrylcholinesterase. This leads to increased central versus peripheral cholinomimetic specificity. In an open case series, the addition of donepezil 5–10 mg/day to ongoing mood-stabilizer treatment showed efficacy and safety in treatment-resistant mania.25 A double-blind, placebo-controlled trial did not find a beneficial effect of donepezil augmentation for refractory mania.26 But, the strength of the conclusion of this trial was limited by the possibility of a false-negative result due to the small sample (donepezil group n = 6, placebo group n = 5).26 We conducted a 4-week double-blind, placebo-controlled trial of donepezil as an adjunctive treatment to lithium in patients with acute mania. Our aim was to explore whether an oral cholinesterase inhibitor, donepezil, would benefit acute mania similarly to studies using physostigmine and compare its effect on day 1 and after 4 weeks. We hypothesized that there would be greater mean reduction in manic symptoms with donepezil augmentation of lithium as compared to placebo. BODY.PATIENTS AND METHODS.PATIENTS: Men and women, aged 18 to 65 years, diagnosed with bipolar disorder with acute manic episodes were eligible to participate. Diagnoses were made independently by three experienced psychiatrists. All subjects were recruited from the inpatient department of the Shanghai Mental Health Center, Shanghai, People's Republic of China, between May 2005 and April 2008. Subjects were required to have a score of greater than 20 on the Young Mania Rating Scale (YMRS)27 at screening and at baseline. All subjects were in good physical health as evidenced by medical history, physical examination, blood investigations, and electrocardiogram. Subjects were excluded if they met the following: (1) had a history of allergy to donepezil or lithium, (2) had a history of suicide or homicide attempts or intent, (3) reported active substance abuse in the past 2 weeks or substance dependence in the past 2 months, (4) were administered any investigational drug within 30 days of screening, (5) had a diagnosis of a current Axis I psychiatric disorder other than bipolar disorder, (6) current laboratory tests were clinically abnormal, or were pregnant or lactating, (7) continuous or current use of anticholinergic medications or cholinomimetics, and (8) other factors that the investigators considered not suitable for this study. The study was approved by the institutional review board of the Shanghai Mental Health Center. All subjects provided written informed consent before entry into the study. This trial was registered at http://clinicaltrials.gov, NCT ID: NCT01191918. BODY.PATIENTS AND METHODS.STUDY DESIGN: Eligible subjects were randomly assigned to receive either donepezil or placebo in addition to lithium. All subjects and research staff were blinded to the randomization codes. Study medications were supplied in identical capsules containing either 5 mg of donepezil or placebo. Donepezil was purchased from Eisai Co, Ltd (Suzhou, People's Republic of China). Donepezil was started at 5 mg/day, and increased to 10 mg/day in the first week. On the same day that the study medication was started, patients were also started on lithium, and the dose was titrated as per plasma levels. Plasma lithium levels were obtained at days 1, 7, 14, and 28, and lithium doses were adjusted accordingly to achieve a level of 0.8–1.2 mmol/L. Concomitant use of clonazepam was allowed during the first 3 weeks (maximum 6 mg/day at the first week, 4 mg/day at the second week, 2 mg/day at the third week) as needed for agitation and insomnia. Zolpidem was used for insomnia at the fourth week as needed. Antidepressants, antipsychotic agents, and other anticholinergics were not allowed during the study period. BODY.PATIENTS AND METHODS.ASSESSMENT: Treatment efficacy was assessed at baseline and at days 1, 7, 14, and 28. All subjects were assessed by the YMRS at hours 4, 8, 12, and 24 in the first day of treatment. The primary efficacy measure was mean change in total score on the YMRS from baseline to the end of study (day 28). Clinical response was defined as a 50% or greater decrease in the YMRS score from baseline. Remitters were those who had a YMRS score of 12 or less at the end of study. Psychomotor subscales were the sum of the YMRS items of increased motor activity/energy, sexual interest, irritability, speech (rate and amount), content, and disruptive/aggressive behavior. The secondary efficacy measure was mean change for the Brief Psychiatric Rating Scale (BPRS). A trained psychiatric resident who was blinded to the treatment administered the YMRS and BPRS. All adverse events were recorded by psychiatrists who were in charge of the clinical care of the participants. BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: Patients' demographic and clinical variables were compared between groups by use of independent samples t-tests or Mann–Whitney tests for quantitative variables and Fisher's exact test for qualitative variables. The mean changes in total score on the YMRS and BPRS (from day 1 and day 28 to baseline) were compared by independent samples t-tests. The proportion of participants achieving clinical response or remission was examined by Fisher's exact test. Differences were considered significant at a two-tailed P < 0.05. BODY.RESULTS: Forty five subjects completed the consent process and were screened. Thirty subjects were found eligible and were enrolled and randomized into receiving study medication. All subjects (donepezil: n = 15; placebo: n = 15) completed the 4-week treatment period. The subjects' demographic characteristics are summarized in Table 1. There were no significant differences between the two groups at baseline on the YMRS and BPRS totals or subscale subtotals (see Table 2). The final mean serum lithium level was 0.83 mmol/L in the donepezil group and 0.82 mmol/L in the control group. There were nine subjects in the donepezil group and eight subjects in the control group that used clonazepam in the first 2 weeks. The maximum dose of clonazepam was 4.11 mg/day in the donepezil group and 3.5 mg/day in the control group (t = 0.64, P = 0.53). BODY.RESULTS.EFFICACY: Over the course of the 4 weeks, we failed to find that donepezil produced any significant difference in the YMRS as compared to placebo (see Table 3). Both the donepezil and placebo group produced a significant improvement in total scores on the YMRS from baseline to endpoint (donepezil group: t = 4.54, P < 0.0001; placebo group: t = 9.06, P < 0.0001). Ten subjects (66.67%) in the donepezil group met the criteria for clinical response, and similarly ten subjects (66.67%) in the placebo group (Fisher's exact P = 1.00) met response criteria. Five subjects (33.33%) in the donepezil group met the criteria for clinical remission, while nine subjects (60.00%) in the placebo group met the remission criteria (Fisher's exact P = 0.27). On the first day, the total YMRS difference between the two groups approached the conventional significance level (t = 1.84, P = 0.07). The mean changes in the psychomotor symptoms subscale from YMRS, and specifically the speech and sexual interest items of the psychomotor symptoms subscale in the donepezil group were greater than those in the placebo group (see Table 4). The psychomotor symptoms scale from YMRS in the donepezil group decreased by 1.97 units more than in the placebo group (t = 2.39, P = 0.02). In the donepezil group, the speech items decreased by 0.57 units more than in the placebo group (t = 2.09, P = 0.04). The sexual interest items in the donepezil group decreased by 0.29 units more than in the placebo group (t = 2.11, P = 0.04). We failed to find that donepezil produced any significant difference in the BPRS as compared to placebo at the endpoint (see Table 3). BODY.RESULTS.ADVERSE EFFECTS: Participants in the donepezil group reported 19 mild to moderate adverse events, while participants in the placebo group reported seven mild to moderate adverse events (see Table 5). No subject presented any severe adverse effects. BODY.DISCUSSION: This is, to our knowledge, the first reported randomized, double-blind, placebo-controlled trial to test the augmentation effects of donepezil for acute mania. In this trial, we found that donepezil was significantly superior to placebo in regards to psychomotor symptoms on the first day. But donepezil was not superior to placebo on the primary efficacy measure, which was measured by the change in mean YMRS score from baseline to endpoint. Similarly, donepezil was not superior to placebo on the secondary efficacy measure of change in the mean BPRS score from baseline to endpoint. The response rate was similar in the two groups. The remission rate was worse with donepezil, though it did not reach the level of significance. The incidence of adverse events was higher in the donepezil group than in the placebo group. All the adverse events were mild to moderate in severity. On the first day, the psychomotor symptoms, especially speech and sexual interest, of patients in the donepezil group improved more than of those patients in the placebo group. This is consistent with the findings of Janowsky et al.16 Since donepezil has greater selectivity for acetylcholinesterase than it has for butyrylcholinesterase, these changes should be unassociated with the peripheral side effects of cholinesterase inhibition. Lithium has been used in the treatment of acute bipolar mania for over 50 years, and has demonstrated superiority over placebo in many controlled clinical trials.28–34 In practice, lithium's antimanic action can take up to 10–14 days to take full effect.29,36 This is significantly slower than most atypical antipsychotics and lithium in combination with an antipsychotic.36 Some atypical antipsychotics seemed to induce more extrapyramidal symptoms than placebo. The atypical antipsychotics were also associated with higher rates of somnolence than placebo.35 The available trials indicate that the combinations of an antipsychotic and lithium (or valproate) increased the possibility of weight gain than monotherapy.37 So, the availability and tolerability of atypical antipsychotics and combination therapy are decreased. Our evidence suggests that donepezil had some benefit in augmentation of lithium treatment on the first day. The possibility of adding a cholinergic agent instead of an antipsychotic to improve treatment response time in the short term certainly exists. We recorded treatment effect on the first day and then on day 7. We are unaware of any changes in symptoms during the first week. Therefore, we cannot predict the length of the superior effect after the first day. Further investigations are necessary. Our results show that adding donepezil did not produce any additional benefit over regular treatment at the end of the 4-week treatment period. The duration of most acute mania studies has been 3 weeks,38,39 and about 80% of patients on lithium show at least moderate improvement after 3 weeks.28 Since we did not assess the YMRS and BPRS at day 21, we cannot exclude the possibility that the placebo group caught up with the donepezil group by the end of the 4 weeks. Numerically, YMRS total, remission rates, and BRPS total were worse in the donepezil group at the end of study, but not at a statistically significant level. This result may be due to type II error. We cannot exclude the possibility that donepezil actually led to the worsening of acute mania, or attenuated the improvements that come from lithium monotherapy. BODY.DISCUSSION.LIMITATIONS: This trial involved addition of a cholinesterase inhibitor as augmentation treatment with lithium and was not designed to assess the efficacy of donepezil as monotherapy for acute mania. The duration of this study was only 4 weeks, thus the long-term effects of treatment with donepezil for acute mania still need to be studied. There is a good possibility that the observed beneficial effect with physostigmine was achieved with a greater degree of cholinesterase inhibition than was seen with a dose of donepezil used in this study. We do not regard our results as a definitive negative either for the 24-hour effects or the effects over the course of a month, because we cannot rule out the possibility that a higher dose could be effective. However, with the dose used in this study, we saw no effects except as noted. An additional limitation of this study was not including depression rating scales. We decided not to include scales for measuring depression as we were specifically interested in the antimanic effects of donepezil. We could not observe if any patients developed depressive symptoms during the 4 weeks. We did not obtain laboratory values indicating central and peripheral cholinergic activity (eg, growth hormone levels, salivary gland secretion). These could have substantiated the hypothesis that central cholinergic mechanisms are in action. BODY.CONCLUSION: To summarize, our evidence suggested that the use of an oral anticholinergic, donepezil, had some benefit in augmentation of lithium treatment on the first day, but no benefit in the long-term. The results need to be confirmed by larger sample clinical trials.

TITLE: Effects of telephone follow-up on blood glucose levels and postpartum screening in mothers with Gestational Diabetes Mellitus ABSTRACT: Background: Gestational diabetes mellitus (GDM) is a form of diabetes that occurs in pregnancy. GDM, defined as glucose intolerance, first diagnosed or initiated during pregnancy affects 1-14% of pregnancies based on various studies. Screening and early diagnosis and appropriate glycemic control can improve prenatal outcomes. Telephone follow-up seems to be a reasonable way for pregnant women follow-up. The present study evaluated the effects of telephone follow-up on blood glucose level during pregnancy and postpartum screening. Methods: Eighty mothers with GDM were enrolled in this clinical trial and randomly divided into intervention and control groups. All mothers were asked to check their blood sugar levels fivetimes daily. In intervention group, telephone intervention was performed for 10 weeks. In each follow-up, individuals were followed for insulin injections, diet, clinical tests and reminding the next visit. In control group, three times of telephone call was established to record blood sugar levels. Another telephone call was established at 6 weeks of postpartum in both study groups to evaluate the performance of the screening test for blood sugar. Results: The mean age of mothers was 30.9±5 years in the control and 30.7±5.1 years in the intervention groups In intervention group, mean level of blood glucose, 2 hours after lunch at 28 weeks of pregnancy was significantly lower than the control group (P<0.05). Mean differences in levels of fasting blood glucose between 28 weeks and 32 and between 28 and 36 weeks of pregnancy were significantly higher in the intervention than the control group (P<0.05). Rate of postpartum glucose screening test was significantly higher in the intervention group (P<0.001). Conclusion: The findings of this study demonstrated that telephone follow-up could significantly reduce fasting blood glucose levels in mothers with gestational diabetes and also increased the rate of postpartum screening test. BODY.INTRODUCTION: Gestational diabetes mellitus (GDM) is a form of diabetes that occurs in pregnancy. GDM, defined as glucose intolerance, first diagnosed or initiated during pregnancy (1-3) affects 1-14% of pregnancies according to various studies.The most important risk factors for GDM are high maternal age, family history of type 2 diabetes, overweight before pregnancy and GDM or glucose intolerance in pervious pregnancies (4). Gestational diabetes usually recovers after delivery, but these women are at high risk for type 2 diabetes; according to previous studies, it is around a 7-12 fold increased risk for future type 2 diabetes (5).Many national and international organizations and conferences recommend the screening for type 2 diabetes in women with history of gestational diabetes 6-12 weeks after the delivery (6). Gestational diabetes and glucose intolerance during pregnancy are associated with poor outcomes (7). Macrosomia (birth weight ≥ 4000 g) and related health problems in newborns are more in women with gestational diabetes than other women (8). Screening and early diagnosis of women with gestational diabetes and appropriate glycemic control can improve prenatal outcomes (9,10). Thus, prevention or postponing overt diabetes in women of childbearing age also protects infants from the harmful effects of intrauterine hyperglycemia. These include macrosomia, increased rate of cesarean section, birth defects, and infants predisposed to obesity and diabetes in their lifetime (11,12). However, despite the hyperglycemic complications in pregnancy, increased risk for type 2 diabetes after delivery, and recommendations for follow-up and screening during and after childbirth (13,14), studies have shown that unfortunately the rate of postpartum screenings is still low (7,15). So it seems that it's quite necessary to provide a suitable way for pregnant women follow-up and inform them about the risks and medical requirements. One of these ways is telephone follow-up, which is the application of information technology in clients' health care, administered in self-assessment, monitoring, decision making and advising and is planned based on client needs, when the client is not available.In this method, based on age, sex and health problems, the patients explain their physical performance status for the health care provider and receive the necessary care through the telephone (16). The telephone has a significant potential; relatively inexpensive, widely available, unlimited by geographical barriers, and is approved by large governmental agencies and NGOs with the capacity to provide large-scale interventions (17).Considering the importance of follow-up by midwives in high-risk pregnancies such as gestational diabetes mellitus and lack of a study in Iran in this issue, the present study evaluated the effects of telephone follow-up on blood glucose level during pregnancy, maternal weight changes, newborn weight and postpartum screening in mothers with gestational diabetes BODY.METHODS: Eighty mothers with gestational diabetes mellitus, who were referred to prenatal clinic of Imam Khomeini hospital and signed consent form, were enrolled in this clinical trial, which consisted of a telephone follow-up as the intervention. This trial has been registered in Iranian Registry of Clinical Trials (IRCT ID: IRCT138904143037n3). The Institutional Review Board approval was granted by the Research Ethic Committee of Tehran University of Medical Sciences. Inclusion criteria were single pregnancy, gestational age between 24-28 weeks, access to telephone, not using medications which increase blood glucose such as corticosteroids, no history of chronic diseases such as chronic hypertension, no history of psychiatric disorders, no history of diabetes apart from pregnancy, no history of infertility or using assisted reproductive plan and no history of hearing loss. Exclusion criteria were loss of pregnancy, premature delivery, individuals without telephones and conditions that increase blood sugar such as stress, death of relatives, drugs consumption such as steroids and, etc. we conducted a single- blind randomized clinical trial on 80 mothers with gestational diabetes who were divided into two groups with 40 mothers in each (intervention and control groups). During the study participants did not have any information about telephone follow- up procedure. The list of allocation sequence was created by computer- generated randomization and base on it, each mother with unique identification number was assigned to study group. Demographic information was gathered at enrollment. The mothers were weighed at the time of enrollment, 28 weeks of pregnancy and at the end of the intervention (at 38 weeks of pregnancy). All mothers were asked to check and document their blood sugar levels 5 times daily (fasting, 2 hours after breakfast, 2 hours after lunch, 2 hours after dinner and before sleeping). In the intervention group, telephone intervention was performed for 10 weeks. In each telephone follow-up, individuals were followed for insulin injections, diet, clinical tests and reminding the next visit. Meanwhile, all mothers' questions and concerns about pregnancy and diabetes-related problems were also replied. Ultimately, possible solutions were proposed to the clients in order to solve their problems. Telephone follow-ups were performed every 2 weeks from 28 to 36 weeks of pregnancy and every week from 36 weeks to 38 weeks of pregnancy. The average call duration was 10 to 15 minutes. In control group, 3 times of telephone call was established at weeks 28, 32 and 36 to record blood sugar levels but no consultation was performed through the telephone calls. Another telephone call was established at 6 weeks of postpartum in both study groups to evaluate the performance of a screening test for blood sugar after the delivery. The two study groups were just different in the quality of telephone follow-ups however all other medical cares were exactly identical in both groups. Newborns' birth weight was also documented. BODY.METHODS.STATISTICAL ANALYSIS: Data analyses were performed using SPSS Version 16.0. Descriptive and inferential statistics were used to describe the data. Descriptive statistics including mean and standard deviation and inferential statistics such as chi-square test, Fisher exact test were used. Chi-square test and Fisher exact test were used to compare demographic characteristics, qualitative and nominal variables. T-test was used to compare quantitative variables. P Values of less than 0.05 were considered as significant. BODY.RESULTS: In this clinical trial, mean±SD age of mothers was 30.9±5 years in control group and 30.7±5.1 years in intervention group (p>0.5). At enrollment, both control and intervention groups were not significantly different in the level of education, occupation status, parity, number of born children, history of abortion, history of stillbirth, time of diabetes diagnosis, age at diagnosis, history of gestational diabetes, history of macrosomia and participation in diabetes educational workshop (p>0.05). Mean±SD level of fasting blood glucose at 28 weeks of pregnancy was 104.3±18.7 mg/dl in control and 100.2±21.2 mg/dl in intervention group (p=0.09). Mean±SD level of blood glucose 2 hours after breakfast at 28 weeks of pregnancy was 131.2±20.9 mg/dl in control and 123.2±18.8 mg/dl in intervention group (p=0.058). Mean±SD level of blood glucose 2 hours after lunch at 28 weeks of pregnancy was 137.5±25.7 mg/dl in control and 123±18 mg/dl in intervention group (p=0.008). Mean±SD level of blood glucose 2 hours after dinner at 28 weeks of pregnancy was 131.1±22.1 mg/dl in control and 122.5±19.7 mg/dl in intervention group (p=0.06). Mean±SD level of blood glucose before sleeping at 28 weeks of pregnancy was 124.7±13.9 mg/dl in control and 113.2±15.8 mg/dl in intervention group (p=0.1). Mean±SD level of fasting, 2 hours after breakfast, 2 hours after lunch, 2 hours after dinner and before sleeping blood glucose in both groups at 28 weeks of pregnancy are shown in Table 1. The differences between blood glucose levels at 28 weeks and 32 weeks and between 28 and 36 weeks for intervention and controls are also shown in Table 2 and 3, respectively.As shown in the tables, Mean±SD of differences in levels of fasting blood glucose between 28 and 32 weeks and between 28 and 36 weeks of pregnancy were significantly higher in the intervention than the control group (p<0.05). Table 1 Daily glucose levels at 28 weeks of pregnancy in both groups Time 28 week Group Control Intervention Mean (SD) Mean (SD) p Fasting 104.3 (18.75) 100.2 (21.27) 0.097 2 hours after breakfast 131.2 (20.96) 123.2 (18.85) 0.058 2 hours after lunch 138.5 (25.71) 123 (18.08) 0.008 2 hours after dinner 131.1 (22.13) 122.5 (19.76) 0.065 Before sleeping 124.7 (13.94) 113.2 (15.89) 0.134 Table 2 Differences between blood glucose levels at 28 weeks and 32 weeks in both groups Group Control Intervention Mean (SD) Mean (SD) p Fasting 0.14 (20.32) -8.13 (18.47) 0.013 2 hours after breakfast -2.41 (27.74) -4.89 (22.14) 0.625 2 hours after lunch -4.73 (28.11) -6.02 (14.89) 0.613 2 hours after dinner -1.26 (26.74) -3.45 (19.71) 0.639 Before sleeping 0.65 (16.52) -0.89 (17.74) 0.541 Table 3 Differences between blood glucose levels at 28 weeks and 36 weeks in both Group Control Intervention Mean (SD) Mean (SD) p Fasting -0.365 (18.99‏) -9.11 (19.43‏) 0.023 2 hours after breakfast -4.41 (21.93‏) -5.71 (17.83‏) 0.749 2 hours after lunch -13.82 (25.40‏) -9.37 (17.22‏) 0.413 2 hours after dinner -5.97 (27.41‏) -7.62 (18.34‏) 0.956 Before sleeping -4.21 (17.65‏) -2.74 (16.07‏) 0.734 Mean±SD changes in maternal weight between 28 and 38 weeks of pregnancy was 5.4±3.3 kg in control and 5.3±5.9 kg in intervention group (p=0.1). Mean±SD infants' birth weight was 3482.5±692.4 gr in control and 3307.9±713.4 gr in intervention group (p=0.2) In control group 14 mothers (34.1%) performed postpartum glucose screening test while 37 mothers (94.9%) of the intervention group conducted the screening test and the difference was statistically significant (p<0.001). BODY.DISCUSSION: Gestational diabetes and glucose intolerance during pregnancy are associated with poor outcomes. Recent studies have shown promising results in using telephone as the primary method for presenting a lifestyle and chronic disease management interventions. But most of these studies have been done on type 2 diabetic patients and a few on type 1 diabetes and a small number of studies have been conducted on pregnant women with GDM (18-20). The results of the present study showed that telephone intervention was more effective in lowering fasting blood glucose level in intervention group comparing to control group that may be due to more and better adherence to diet, and drug therapy prescribed by the doctor. Our results were in consistent with Kim &Oh's study (21) which evaluated the effect of telephone follow-up on adherence to treatment recommendations in patients with diabetes. In their study, patients were divided into two telephone and control groups. One of the researchers conducted a telephone intervention which included a weekly call for continuing education, forced diet, exercise, regulation of medications and monitoring blood glucose level. Patients in the telephone group showed a reduction of 1.2% in HbA1c comparing to 0.6% in the control group.The results of our study were also compatible with Mons et al's (22) results that assessed the impact of telephone calling by a nurse (over 12 months) on medical and psycho-social outcomes in type 2 diabetes patients. Intervention group in their study demonstrated reduction in cardiovascular risk factors, quality of life and depression after 6, 12 and 18 months telephone follow-up, which was significant as compared to control group. Also HbA1c decreased significantly from the baseline in both intervention and control group. Dunbar et al (17) evaluated the benefits of the diabetes prevention program and the role of telephone follow-up. In this study, the patients who attended in a 12-month diabetes prevention program were randomly divided into telephone support and self-care groups.The changes between 12-30 months were not significantly different in telephone support group comparing to the self-care group. The positive results obtained in the 12-month program usually last 18 months. Telephone support did not seem to have additional benefits.Their results were inconsistent with the results of our study that may be because of the presence of a relatively long-term educational program in diabetes prevention, which can serve as a useful tool in patient adherence and reducing the benefits of telephone follow-up. In our study, maternal weight changes and newborns' birth weight were not significantly different between control and intervention groups. They may be due to the fact that the researchers did not provide a special educational program in telephone counseling intervention, and the mothers were only followed for doctor's prescribed treatment plan. The results showed that postpartum screening test was more performed in the intervention group. Hunt & Conway (23) stated that postpartum diabetes screening increases by nurses' follow-up for reminding blood sugar level testing and health-care providers can notify patients about the importance of glucose testing and long-term risks of diabetes. Clark et al (24) also evaluated the effect of reminding postpartum screening of diabetes mellitus in women with gestational diabetes. They divided the patients into 4 groups: patient/physician reminder, physician only reminder, patient only reminder, no reminder. They showed that the screening rate was significantly increased in physician/patient reminder group, patient-only reminder group, and physician only reminder group respectively as compared to no reminder group. Their findings correspond to the result of our study. BODY.CONCLUSION: The result of the current study demonstrated that telephone follow-up as an easy and inexpensive supportive program could significantly reduce fasting blood glucose levels in mothers with gestational diabetes and also increased the rate of postpartum screening test. BODY.ACKNOWLEDGEMENTS: This study was funded by Tehran University of Medical sciences, Deputy of research. The proposal code is: 13165-28-01-90.

TITLE: Effectiveness of educational outreach in infectious diseases management: a cluster randomized trial in Uganda ABSTRACT.BACKGROUND: Integrated Infectious Diseases Capacity Building Evaluation (IDCAP) teams designed and implemented two health worker in-service training approaches: 1) an off-site classroom-based integrated management of infectious diseases (IMID) course with distance learning aspects, and 2) on-site support (OSS), an educational outreach intervention. We tested the effects of OSS on workload and 12 facility performance indicators for emergency triage assessment and treatment, HIV testing, and malaria and pneumonia case management among outpatients by two subgroups: 1) mid-level practitioners (MLP) who attended IMID training (IMID-MLP) and 2) health workers who did not (No-IMID). ABSTRACT.METHODS: Thirty-six health facilities participated in the IDCAP trial, with 18 randomly assigned to Arm A and 18 to Arm B. Two MLP in both arms received IMID. All providers at Arm A facilities received nine monthly OSS visits from April to December 2010 while Arm B did not. From November 2009 to December 2010, 777,667 outpatient visits occurred. We analyzed 669,580 (86.1 %) outpatient visits, where provider cadre was reported. Treatment was provided by 64 IMID-MLP and 1,515 No-IMID providers. The effect of OSS was measured by the difference in pre/post changes across arms after controlling for covariates (adjusted ratio of relative risks = a RRR). ABSTRACT.RESULTS: The effect of OSS on patients-per-provider-per-day (workload) among IMID-MLP (aRRR = 1.21; p = 0.48) and No-IMID (aRRR = 0.90; p = 0.44) was not statistically significant. Among IMID-MLP, OSS was effective for three indicators: malaria cases receiving an appropriate antimalarial (aRRR = 1.26, 99 % CI = 1.02-1.56), patients with negative malaria test result prescribed an antimalarial (aRRR = 0.49, 99 % CI = 0.26-0.92), and patients with acid-fast bacilli smear negative result receiving empiric treatment for acute respiratory infection (aRRR = 2.04, 99 % CI = 1.06-3.94). Among No-IMID, OSS was effective for two indicators: emergency and priority patients admitted, detained or referred (aRRR = 2.12, 99 % CI = 1.05-4.28) and emergency patients receiving at least one appropriate treatment (aRRR = 1.98, 99 % CI = 1.21-3.24). ABSTRACT.CONCLUSION: Effects of OSS on workload were not statistically significant. Significant OSS effects on facility performance across subgroups were heterogeneous. OSS supported MLP who diagnosed and treated patients to apply IMID knowledge. For other providers, OSS supported team work to manage emergency patients. This evidence on OSS effectiveness could inform interventions to improve health workers' capacity to deliver better quality infectious diseases care. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article 10.1186/s12889-016-3375-4) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Sub Saharan Africa experiences 37 % of the total deaths attributable to infectious diseases globally [1], but contains only 3 % of the world's health workers [2]. The shortage of physicians in sub Saharan Africa is at critical levels, with only 2 physicians per 10,000 population compared to 28 in high-income regions [3]. This shortage of physicians has led to many physician roles, such as initiation of antiretroviral therapy shifting to mid-level practitioners (MLP) with fewer years of medical training [4], formally [5–7] and informally [8, 9]. Yet as reported in one Ugandan study, many of these MLP have not been trained to take on these tasks [10]. They therefore require capacity building to meet the demands of changing health policies and guidelines [11]. Some studies have shown that with additional training MLP can competently execute physicians' roles and, in some instances, match their performance [12–14]. However, most trainings for MLP are classroom-based, often taking them away from their workstations [15, 16], disrupting workflows and limiting patients' access to care. Several systematic reviews show that classroom-based training may not equip the attendants with sufficient skills to ensure adherence to clinical guidelines [17–19]. There is increasing interest from experts and donors in favor of educational outreach and continuous quality improvement (CQI) approaches [20–24] that integrate across diseases [25–28]. Facility-based educational outreach trainings increase opportunities for team-based interactions during MLP learning [20, 29] and reduces MLP time away from health facilities. Several studies have shown that educational outreach both with and without CQI activities can improve the quality of patient care [5, 30–35]. It may also build capacity in patient care for almost all providers, without increasing workload on the MLP who do not attend trainings. Between 2009 and 2010, the curriculum development and mobile teams of the Integrated Infectious Diseases Capacity Building Evaluation (IDCAP) developed and delivered two novel interventions to build capacity for treatment and prevention of infectious diseases: 1) the Integrated Management of Infectious Disease (IMID) course with distance learning aspects, taught at the Infectious Diseases Institute in Kampala, Uganda, and 2) facility-based on-site support (OSS) visits, an educational outreach intervention with CQI activities [29]. Implemented as a mixed design with pre/post and cluster randomized trial components, IDCAP tested the effect of IMID training and OSS on individual clinician competence [36], practice [37], mortality among children less than five years of age [20, 38], and facility performance [33, 39]. IMID was associated with a statistically significant 9.8 % absolute increase in clinical competence, whereas OSS was not associated with an incremental improvement in the same outcomes. [36]. For clinical practice, IMID was associated with statistically significant improvements in taking a patient's history and physical examination, and OSS was associated with an incremental improvement in these tasks [37]. Child mortality increased in both arms, but the incremental effect of OSS delayed the increase during the intervention [38]. In the main trial analysis of facility performance, [33] Weaver et al. reported the effects of IDCAP's IMID and OSS on 23 facility performance indicators. Facility performance refers to the process of care, and may reflect the contributions of more than one provider at a facility. For example, the proportion of malaria suspects with a malaria test result recorded (Indicator 4), reflects whether or not the clinician ordered the test, and the laboratory professional performed the test and recorded the result. Of the 23 indicators, 12 were based on a data surveillance system with data on each outpatient visit. The other 11 indicators were based on Ministry of Health Registers for HIV care, maternal, child and newborn health and tuberculosis. The 12 indicators based on the data surveillance system are presented in Table 1, and include three indicators for emergency triage assessment and treatment, four indicators for malaria case management, four indicators for case management of respiratory illness, and one indicator for HIV testing.Table 1Definitions of facility performance indicatorsProgram area and performance indicatorDefinitionReferenceEmergency Triage, Assessment and Treatment (ETAT)1Proportion of outpatients triagedNumerator: Number of outpatients triaged, meaning that the patient was classified as emergency, priority, or queue, or an emergency sign was noted in the triage section of the form. Denominator: Number of outpatients[50, 51]2Proportion of emergency and priority patients who were admitted, detained or referredNumerator: Number of emergency and priority patients admitted, detained or referred for care. Denominator: Number of outpatients classified as emergency or priority or an emergency sign was noted in the triage section of the form.In line with World Health Organization guidelines, emergencies were defined as patients with one or more of the ABCDO triage signs (i.e., Airway; Breathing difficulty; Circulation / Coma / Convulsion / Confusion; Dehydration; and Other). Priorities were defined as patients with the 3TPR-MOB priority signs (i.e. Tiny baby (sick child of under 2 months of age); Temperature (child is very hot); Trauma or other urgent surgical condition; Pallor (severe); Poisoning; Pain (severe); Respiratory distress; Restless, lethargy or continuously irritable; Referral; Malnutrition (severe wasting); Oedema of both feet; and Burns) [52].[50, 51]3Estimated proportion of emergency patients who received at least one appropriate treatmentNumerator: Number of emergency patients who received at least one treatment prescribed according to ETAT standards where the standards were reported in Kinoti et al. (manuscript under review in PlosOne). For emergency patients who were prescribed treatment and data on drug availability were missing, we applied the "in-stock" rate for patients with those data. Denominator: Number of outpatients classified as emergency or an emergency sign was noted in the triage section of the form[50, 51]Case management of fever and malaria4Proportion of malaria suspects with a malaria test result recordedNumerator: Number of malaria suspects with a result for a laboratory test or rapid diagnostic test for malaria, where the definition of a malaria suspect was reported in Mbonye et al. [37]. Denominator: Number of malaria suspects[16, 39, 53]5Estimated proportion of malaria cases who received an appropriate antimalarialNumerator: Number of outpatients treated with appropriate anti-malarial(s), where appropriate antimalarial treatments were quinine and four artemisinin-based combination therapies reported in Mbonye et al. [37]. For patients who were prescribed an antimalarial and data on drug availability were missing, we applied the "in-stock" rate for patients with those data. Denominator: Number of outpatients treated for malaria[16, 39, 53]6Proportion of patients with a negative malaria test result who were prescribed an antimalarialNumerator: Number of patients with a negative malaria test result prescribed any antimalarial including appropriate treatments and those that do not comply with Ugandan national guidelines. Denominator: Number of patients with a negative malaria test result[16, 39, 53]7Proportion of patients with a positive malaria test result who were prescribed an antibioticNumerator: Number of patients with a positive malaria test result prescribed any antibiotic(s), where antibiotic treatment refers to the 31 drugs listed in Mbonye et al. [37]. Denominator: Number of patients with a positive malaria test result[16, 39, 53]Case management of respiratory illness8Proportion of pneumonia suspects aged under 5 years assessed for pneumoniaNumerator: Number of child pneumonia suspects with at least one of the three following assessment results recorded: 1) abnormal chest sounds, 2) chest in-drawing, and 3) rapid breaths per minute. A pneumonia suspect was defined as any child aged under five years presenting with cough or who received a diagnosis of "pneumonia" or "cough/cold no pneumonia". Denominator: Number of child pneumonia suspects. Note: The definition of suspect focused on children with cough; difficulty in breathing was inadvertently omitted from the form.[54]9Estimated proportion of patients aged under 5 years diagnosed with pneumonia who received appropriate antibiotic treatmentNumerator: Number of children diagnosed with pneumonia treated with appropriate antibiotic, where appropriate antibiotic treatment referred to six drugs on the revised Medical Form 5: amoxicillin, benzyl penicillin, erythromycin, chloramphenicol, gentamicin, cotrimoxazole, and 11 other drugs that were specified: ampicillin, azithromycin, cefixime, ceftriaxone, cefuroxime, co-amoxiclav, gatifloxacin, levofloxacin, penicillin, phenoxymethylpenicillin, ampiclox (amoxicillin and cloxacillin). For patients who were prescribed an antibiotic and data on drug availability were missing, we applied the "in-stock" rate for patients with those data. Denominator: Number of children diagnosed with pneumonia[54]10Proportion of TB suspects with a first Acid-Fast Bacilli (AFB) smear resultNumerator: Number of TB suspects who get a first AFB smear result, where TB suspect was defined as anyone with a history of: cough for longer than two weeks, cough for less than two weeks and night sweats, cough for less than two weeks and weight loss, TB test ordered, new TB diagnosis, started on initial TB treatment, or referred for TB treatment. For children, the definition extended to anyone who had contact with someone with TB. Denominator: Number of TB suspects. Note: The definition of TB suspect is from the Intensified Case Finding Form for People Living with HIV, contacts of smear positive patients, and HIV care settings [55]. Increasing AFB smears among TB suspects will increase case detection, but this is not the case detection indicator used by the Stop TB program [56].[52, 56–58]11Estimated proportion of patients with AFB smear negative results who received empiric treatment for acute respiratory infectionNumerator: Number of people with AFB smear negative test results who received empiric treatment for acute respiratory infection, including amoxicillin, doxycycline, or erythromycin. For patients who were prescribed an antibiotic and data on drug availability were missing, we applied the "in-stock" rate for patients with those data. Denominator: Number of people with AFB smear negative result[52, 56–58]HIV testing and prevention16Proportion of patients with an HIV test result recordedNumerator: Number of outpatients who were not TB suspects with an HIV test result recorded. TB suspect is defined for Indicator 10. Denominator: Number of outpatients who were not TB suspects. Note: This indicator included anyone who said they knew their HIV status in the protocol, and was revised to comply with the MOH definition based strictly on a laboratory test result on the day of the outpatient visit.[58]Numerator: Number of TB suspects with an HIV test result recorded. TB suspect is defined for Indicator 10. Denominator: Number of TB suspects. Note: This indicator included anyone who said they knew their HIV status in the protocol, and was revised as described above. Abbreviations: AFB Acid-fast bacilli, CQI Continuous Quality Improvement, HIV Human Immunodeficiency Syndrome, MOH Ministry or Health Among those 12 indicators, Weaver et al.'s reported that IMID alone was associated with statistically significant pre/post improvements in three indicators: outpatients triaged (Indicator 1), emergency and priority patients who were admitted, detained or referred (Indicator 2), and pneumonia suspects under age five assessed for pneumonia (Indicator 8). A combination of IMID and OSS was associated with statistically significant improvements in four indicators: outpatients triaged (Indicator 1), emergency and priority patients who were admitted, detained or referred (Indicator 2), 3) estimated malaria cases who received appropriate antimalarial treatment (Indicator 5), and patients with a negative malaria test result who were prescribed an antimalarial (Indicator 6), for which a decrease was an improvement. The incremental effect of OSS was not statistically significant for any of the 12 indicators, but many of the effect sizes were large. We hypothesized that OSS improved facility performance more among the providers who attended IMID training (IMID-MLP) compared to the ones who did not (No-IMID). Weaver et al.'s analysis included all patients, and we suspected that the No-IMID subgroup could have diluted the effects of OSS in the IMID-MLP subgroup. We therefore sought to test the effect of OSS on the 12 indicators based on the data surveillance system in the two subgroups of health providers: IMID-MLP and No-IMID. The analysis was limited to only outpatient visits with data to identify provider cadres, and whether or not the provider attended IMID training. In this article, the term "indicator" refers to the facility performance indicators. Also, during the ethical review of the IDCAP protocol, the School of Medicine Ethics and Research Committee of Makerere University expressed concern that the interventions would affect the distribution of the workload. The data surveillance system data provided an opportunity to examine whether workload increased among the IMID-MLP with OSS, and among the No-IMID health workers with OSS. We therefore tested the effect of OSS on changes in workload in each subgroup of providers. BODY.METHODS.STUDY DESIGN: We conducted a secondary analysis of IDCAP data. As noted above, IDCAP was a mixed design study consisting of pre/post and cluster randomized trial components. It was implemented at 36 health center IVs (HCIV) or comparable health facilities across Uganda. Eighteen of the 36 health facilities were randomized to Arm A and the other 18 to Arm B. In both arms, two MLP from each of the 36 health facilities received IMID training. From April to December 2010, all providers at Arm A facilities received nine monthly OSS visits while Arm B did not. The pre/post component measured the effects of the combination of IMID training and OSS in Arm A, and IMID training alone in Arm B. The cluster randomized trial component measured the incremental effect of OSS as the difference between the pre/post effects in the two arms. Figure 1a presents the design of the main test of facility performance indicators, which is reported as the overall result below. Note that the overall results presented in Table 5 differ from the results of Weaver et al.'s analysis, because only outpatient records with data on provider cadre and whether or not the provider attended IMID training were analyzed. Figure 1b presents the design of the subgroup analysis.Fig. 1 a Design of the overall comparison across arms. OSS refers to On-Site Support, IMID to Integrated Management of Infectious Disease, and MLP to mid-level practitioners. b Design of the across arm comparisons within the IMID-MLP and No-IMID subgroups The process of randomization, blinding, sample size calculation, and recruitment for the cluster-randomized trial were previously reported in Weaver et al. [33]. A The IDCAP protocol is summarized by Naikoba et al. [20]. The full protocol can also be accessed as supplementary file in both Mbonye et al. [39], Weaver et al. [33] and is available as supporting information to this paper in Protocol 1. The CONSORT checklist for this subgroup analysis is available as Additional file 1. BODY.METHODS.SITES, PARTICIPANTS, AND INCLUSION CRITERIA: The 36 study health facilities included 31 health center IV (HCIV) and five small hospitals comparable in size to HCIV. A HCIV serves a population of about 100,000 people providing basic preventive and curative care, and is at the highest referral point for a health sub-district [40]. Two MLP, defined as clinical officers, registered nurses or midwives, from each health facility participated in the IMID training. Inclusion criteria for facilities and IMID participants have been described elsewhere [20, 29]. Briefly, the key inclusion criteria for selecting IMID participants was involvement in daily management of patients in the outpatient clinics, and spending over 80 % of the time seeing patients at the participating health facilities [20]. All health workers including IMID participants were invited to participate in the OSS sessions. All outpatients participated as part of the routine healthcare seeking process. For the subgroup analyses, as noted above, only records of outpatient visits with data on the health provider's cadre and whether or not the health provider attended IMID training were included. BODY.METHODS.INTERVENTIONS: IMID was a three-week core course followed by two one-week booster courses at 12 and 24 weeks after the core course and distance learning [29]. The core course was taught in four sessions at the Infectious Diseases Institute in Kampala, Uganda with two sessions for Arm A in March and April 2010 and two sessions for Arm B in May and June 2010. OSS was a two-day visit to the health facilities in Arm A delivered by mobile teams for nine consecutive months from April to December 2010 [20, 29]. Each mobile team consisted of four members: a medical officer with CQI experience, a clinical officer, a laboratory technologist, and a registered nurse. Each visit focused on one OSS topic as well as follow-up from the previous sessions and was characterized by four distinct activities: the multidisciplinary team training, one-on-one mentorship, breakout sessions, and CQI activities. BODY.METHODS.OUTCOMES: The outcomes were patients per provider used to measure changes in workload, and 12 facility performance indicators based on the data surveillance system whose definitions by numerator and denominator are reported in Table 1, as well as sources of the indicators. Indicator 16, proportion of patients with an HIV test recorded, differs from the earlier report [33], because it excludes registry data from antenatal and tuberculosis clinics. BODY.METHODS.DATA COLLECTION: The data source was the revised Uganda Ministry of Health outpatient medical form (MF5) [41]. Using the MF5, health workers prospectively collected data on every outpatient visit from November 2009 to December 2010. From March 2010, data entry assistants were stationed at each health facility to capture these data electronically in an Epi Info® Version 3.2 (U.S. Centers for Disease Control and Prevention, Atlanta, GA) database. At the end of each month, the data entry assistants transmitted electronic data to the Infectious Diseases Institute where data for all health facilities were merged using Microsoft Excel® 2010 (Microsoft Corporation, Redmond, WA, USA), and further cleaned. BODY.METHODS.DATA ANALYSIS: We used frequencies to describe provider, patients, and outcomes across arms, subgroups, and time periods. The effect of OSS was estimated as a difference in pre/post (Time 0/Time 1) changes between Arm A and Arm B. In Arm A, Time 0 was from November 2009 to March 2010 and Time 1 started after the first IMID training in Arm A and was from April to December 2010. In Arm B, Time 0 was from November 2009 to May 2010 and Time 1 started after the first IMID training in Arm B and was from June to December 2010. During Time 1, nine OSS visits were implemented in Arm A. Using a generalized linear latent and mixed model [42], data on the number of patients per provider were analyzed as continuous dependent variables with main effects for arm, subgroup, time period and their interaction to estimate pre/post differences within each subgroup in each arm and ratios of relative risk (RRR) for the effect of OSS within each subgroup. The unit of analysis was a subgroup-facility-month, and regressions were estimated with clusters for provider and facility with robust standard errors. The RRR for a given performance indicator was estimated with main effects for arm, time period and their interaction, using data for the total sample and for each subgroup and the generalized linear model with a Poisson family and log link as described earlier [33, 39]. The unit of analysis was facility month, and all regression analyses were clustered on the health facility. To adjust for over dispersion and using Poisson rather than binomial family, the regressions were estimated with robust standard errors. Multiple comparisons were addressed by basing the statistical tests on α = 0.01 and all results were presented with 99 % confidence intervals (CI). All regression analyses were adjusted for facility type (public or private not-for-profit), facility level (HCIV or small hospital), data entry assistant on-site, Baylor and previous participation in the national HIV CQI program (see description of strata in [33, 39]). Indicators 1–7 and 10–11 were also adjusted for staffing and patient age (<5, > = 5 years), indicators 8–9 for staffing, and indicator 16 for staffing, TB suspect and patient age group (2–14 months, 18 months – 13 years and ≥14 years) to reflect HIV testing guidelines. All data analyses were conducted using Stata® version 12 (StataCorp, College Station, Texas, USA). BODY.METHODS.ETHICAL CONSIDERATIONS: The School of Medicine Research and Ethics Committee of Makerere University Kampala (reference number 2009–175) and the Uganda National Council for Science and Technology (reference number HS-722) approved the IDCAP protocol. The University of Washington Human Subjects Division determined that IDCAP did not meet the regulatory definition of research under 45 CFR 46.102 (d). This subgroup analysis was exempt from review by the University of Antwerp ethical review board. BODY.RESULTS.PARTICIPANT FLOW: The flow of the participating health facilities, and IMID training and OSS participants is presented in Fig. 2 [33]. All health facilities participated up to the end of the study. Of the 36 individuals who participated in IMID training, one person in Arm A and two in Arm B did not attend one or more boost courses. Attendance of OSS sessions was also not consistent. Out of 513 clinical staff expected to attend multi-disciplinary team sessions, only 440 (86 %) attended at least once; out of 499 clinical staff expected to attend the breakout session, only 344 (69 %) attended at least once; and out of 351 clinical staff expected to attend the clinical mentoring and coaching session, only 186 (53 %) attended at least once. Among those who attended at least one OSS session, the mean number of sessions attended per clinical staff was 4.93 for multi-disciplinary team sessions, 3.64 for breakout sessions and 4.01 for clinical mentoring and coaching sessions.Fig. 2Consort flow diagram – recruitment and randomization A total of 777,667 outpatients visited the 36 health facilities during the study period. Of these visits, 669,580 (86.1 %) had data on the provider's name and cadre, which allowed us to determine whether or not the provider attended IMID training, and were included in this analysis (Table 2). During these visits, patients were seen by 64 IMID-MLP and 1,515 No-IMID providers. Data on 108,087 (13.9 %) visits with missing (91,006), unknown (14,686) or indeterminate (2,395) data on the provider were omitted from the analysis.Table 2Distribution of providers by arm and subgroupProviderArm AArm BTotalPatientsIMID-MLPNo-IMIDIMID-MLPNo-IMIDIMID-MLPNo-IMIDTotal (Percent)1Medical Officer064080014432,994 (4.9)2Clinical Officer241371919344330370,745 (55.4)3Nurse12172823120403162,542 (24.3)4Midwife00101051 (0.008)5Nursing Assistant01100179028962,968 (9.4)6Other less skilled01630186034940,280 (6.2)Total3664628869641,515669,580 BODY.RESULTS.BASELINE.PATIENT VOLUME: In Table 3, baseline data for the number of providers, the number of patients seen, and the estimated number of patients per provider per calendar day is reported by time period, arm and subgroup. The number of patients per provider per calendar day was higher in Arm B with more hospitals than in Arm A in the IMID-MLP subgroup (4.3 vs. 3.7) and similar across arms in the No-IMID subgroup (1.1 vs. 1.1).Table 3Before and during OSS comparison of average number of patients per provider per month by subgroup and armVariableIMID-MLPNo-IMIDArm AArm BArm AArm BPart 1: Patient and provider population Time 0 Total number of patients19,68625,70859,614126,611 Number of providers3528358559 Number of calendar days151212151212 Time 1 Total number of patients54,90426,163174,935181,956 Number of providers3626496641 Number of calendar days275214275214Part II: Patients per provider per calendar day Time 03.74.31.11.1 Time 15.54.71.31.4 Change (Time 1-Time 0)1.820.370.150.28 Difference between Arm A and Arm B1.45−0.13Part III: RegressionaRR (99 % CI), p-valueaRR (99 % CI), p-valueaRR (99 % CI), p-valueaRR (99 % CI), p-value Time 1 – Time 01.15 (0.72, 1.85), 0.4370.96 (0.57, 1.62), 0.8280.99 (0.76, 1.30), 0.9211.10 (0.88, 1.37), 0.264aRRR (99 % CI), p-valueaRRR (99 % CI), p-value Effect of OSS in each group1.21 (0.61, 2.38), 0.4780.90 (0.63, 1.28), 0.443 **Denotes that the effect of OSS was significant at the .01 level and *Denotes that the effect of OSS was significant at the .05 levelThe 99% confidence intervals (CI) are based on the .01 level of significanceAbbreviations: aRR adjusted relative risk, aRRR adjusted ratio of relative risks, IMID Integrated Management of Infectious Disease, OSS On-site support, MLP Mid-level practitioners, RR Relative Risk measured the comparisons in pre/post change from Time 0 to Time 1; RRR Ratio of Relative Risk Measured the effect of OSS in each subgroupEstimates were adjusted for: whether the facility received the on-site intervention from Baylor International Pediatric AIDS Initiative or not, facility was implementing continuous quality improvement prior to IDCAP trial, level of health facility (small hospital or health center IV), facility ownership (public or private-not-for profit) and data entry assistant on-site BODY.RESULTS.BASELINE.PATIENT CARE: In Table 4, baseline data for each indicator are reported by both arm and subgroup. Performance in Arm B was generally higher than in Arm A within both IMID-MLP and No-IMID subgroups for eight out of the 12 indicators. For indicators 6 (Proportion of patients with a negative malaria test result who were prescribed an antimalarial), and 7 (Proportion of patients with a positive malaria test result who were prescribed an antibiotic) a lower percentage indicated better performance, and Arm A had lower percentages than Arm B for both indicators. Performance was highest for indicator 5 (estimated proportion of malaria cases who received an appropriate antimalarial). For the remaining indicators, performance was generally low, did not exceed 64 % in both arms and subgroups, and was as low as 3.1 % for indicator 8 (Proportion of pneumonia suspects aged under 5 years assessed for pneumonia) in the IMID-MLP subgroup of Arm A.Table 4Frequencies and percentages by arm, time period and subgroupInd no.IndicatorArm AArm BTime 0Time 1Time 0Time 1No-IMIDIMID-MLPNo-IMIDIMID-MLPNo-IMIDIMID-MLPNo-IMIDIMID-MLP1Proportion of outpatients triaged15,463 (27.7 %)6,104 (32.8 %)145,205 (84.6 %)49,611 (92.0 %)55,052 (45.4 %)15,894 (63.8 %)127,500 (71.4 %)22,722 (87.8 %)2Proportion of emergency and priority patients who were admitted, detained or referred425 (10.1 %)133 (23.2 %)4,268 (36.1 %)1,931 (40.8 %)2,474 (15.4 %)909 (28.3 %)4,251 (25.8 %)793 (35.2 %)3Estimated proportion of emergency patients who received at least one appropriate treatment544 (23.9 %)82 (36.2 %)1,469 (51.5 %)737 (57.4 %)1,956 (31.3 %)411 (43.4 %)2,357 (41.7 %)177 (22.5 %)4Proportion of malaria suspects with a malaria test result recorded13,727 (37.3 %)5,154 (39.4 %)53,764 (51.0 %)16,698 (51.5 %)24,320 (30.5 %)5,958 (37.5 %)38,615 (33.4 %)6,323 (41.3 %)5Estimated proportion of malaria cases who received an appropriate antimalarial25,438 (83.2 %)8,650 (77.3 %)70,090 (92.5 %)19,463 (93.8 %)59,950 (87.5 %)11,624 (88.8 %)81,959 (85.7 %)9,363 (82.7 %)6Proportion of patients with a negative malaria test result who were prescribed an antimalarial2,904 (42.4 %)1,475 (56.6 %)9,525 (31.9 %)2,308 (23.2 %)6,699 (51.9 %)1,573 (45.7 %)11,976 (50.4 %)1,331 (35.6 %)7Proportion of patients with a positive malaria test result who were prescribed an antibiotic3,052 (44.4 %)1,192 (46.8 %)10,681 (44.6 %)3,107 (46.0 %)5,253 (46.1 %)1,067 (42.4 %)7,629 (51.4 %)965 (37.3 %)8Proportion of pneumonia suspects aged under 5 years assessed for pneumonia343 (3.3 %)214 (3.1 %)5,000 (15.4 %)4,172 (17.6 %)1,274 (5.7 %)970 (8.6 %)5,984 (20.4 %)3,665 (25.5 %)9Estimated proportion of patients aged under 5 years diagnosed with pneumonia who received appropriate antibiotic treatment634 (50.7 %)464 (51.5 %)2,535 (59.3 %)1,771 (53.8 %)2,064 (54.7 %)1,190 (54.1 %)2,996 (62.9 %)1,572 (56.2 %)10Proportion of TB suspects with a first Acid-Fast Bacilli (AFB) smear result283 (7.6 %)220 (7.7 %)1,342 (8.7 %)1,100 (8.9 %)441 (8.9 %)283 (13.1 %)884 (13.8 %)460 (16.4 %)11Estimated proportion of patients with AFB smear negative results who received empiric treatment for acute respiratory infection50 (17.7 %)33 (17.5 %)379 (29.8 %)322 (32.5 %)123 (21.9 %)69 (27.9 %)231 (26.3 %)102 (24.9 %)16Proportion of patients with an HIV test result recorded (all patients ≥2 months)5,483 (5.6 %)1,750 (5.6 %)23,352 (7.7 %)11,044 (11.4 %)7,399 (3.4 %)1,857 (4.4 %)15,637 (4.8 %)2,321 (5.2 %) BODY.RESULTS.OUTCOMES.PATIENT VOLUME: As shown in Table 3, pre/post change in the number of patients per provider per month from Time 0 to Time 1 among the IMID-MLP subgroup was higher in the Arm A (adjusted relative risk (aRR) = 1.15, 99 % CI = 0.72-1.85, p = 0.437) than in Arm B (aRR = 0.96, 99 % CI = 0.57-1.62, p = 0.828), and the effect of OSS in this subgroup was not statistically significant (adjusted RRR (aRRR) = 1.21; 99 % CI = 0.61-2.38, p = 0.478). In the No-IMID subgroup however, pre/post change was lower in Arm A (aRR = 0.99, 99 % CI = 0.76-1.30, p = 0.922) than Arm B (aRR = 0.1.10, 99 % CI = 0.88-1.37, p = 0.264), and the effect of OSS was also not statistically significant (aRR = 0.90; 99 % CI = 0.63-1.28, p = 0.443). BODY.RESULTS.OUTCOMES.FACILITY PERFORMANCE: The effect of OSS on 12 facility performance indicators is reported in Table 5. Overall, the incremental effect of OSS was statistically significant for three indicators; increases in the estimated proportion of emergency patients who received at least one appropriate treatment (Indicator 3, aRRR = 2.00; 99 % CI = 1.11-3.79, p = 0.003), and estimated proportion of malaria cases who received an appropriate antimalarial (Indicator 5, aRRR = 1.15, 99 % CI = 1.01-1.32, p = 0.006), and decrease in proportion of patients with a negative malaria test result prescribed an antimalarial (Indicator 6, aRRR = 0.65, 99 % CI = 0.44-0.98, p = 0.006). Within the IMID-MLP, the incremental effect of OSS was statistically significant for three indicators; increases in estimated proportion of malaria cases who received an appropriate antimalarial (Indicator 5, aRRR = 1.26, 99 % CI = 1.02-1.56, p = 0.005) and estimated proportion of patients with acid-fast bacilli (AFB) smear negative results who received empiric treatment for acute respiratory infection (indicator 11, aRRR = 2.04, 99 % CI = 1.06-3.94, p = 0.005) and decrease in proportion of patients with a negative malaria test result prescribed an antimalarial (Indicator 6, aRRR = 0.49, 99 % CI = 0.26-0.92, p = 0.004). Within the No-IMID subgroup, the incremental effect of OSS was statistically significant for only two indicators; increases in the proportion of emergency and priority patients admitted, detained or referred (Indicator 2, aRRR = 2.12, 99 % CI = 1.05-4.28, p = 0.006), and estimated proportion of emergency patients who received at least one appropriate treatment (Indicator 3, aRRR = 1.98, 99 % CI = 1.21-3.24, p < 0.001). Compared to the No-IMID subgroup, the effect sizes were larger in the IMID-MLP subgroup in seven (3, 5, 6, 8, 10, 11 and 16) of the 12 indicators measured.Table 5Effect of OSS training on facility performance in each of the IMID-MLP and No-IMID groupsInd no.IndicatorOverall (n = 669,580)No-IMID (n = 543,119)IMID-MLP (n = 126,461)aRRR (99 % CI) p-valueaRRR (99 % CI) p-valueaRRR (99 % CI) p-value1Proportion of outpatients triaged*1.67 (0.88, 3.16)0.04*1.68 (0.90, 3.16)0.031.65 (0.63, 4.31)0.182Proportion of emergency and priority patients who were admitted, detained or referred*2.00 (0.93, 4.27)0.02**2.12 (1.05, 4.28)0.0061.31 (0.63, 2.72)0.343Estimated proportion of emergency patients who received at least one appropriate treatment**2.00 (1.11, 3.79)0.003**1.98 (1.21, 3.24)0.000*2.15 (0.83, 5.54)0.044Proportion of malaria suspects with a malaria test result recorded1.21 (0.86, 1.69)0.151.21 (0.81, 1.82)0.221.21 (0.80, 1.81)0.245Estimated proportion of malaria cases who received an appropriate antimalarial**1.15 (1.01, 1.32)0.006*1.12 (0.99, 1.27)0.011**1.26 (1.02, 1.56)0.0056Proportion of patients with a negative malaria test result who were prescribed an antimalarial**0.65 (0.44, 0.98)0.0060.75 (0.48, 1.18)0.10**0.49 (0.26, 0.92)0.0047Proportion of patients with a positive malaria test result who were prescribed an antibiotic0.94 (0.78, 1.13)0.370.92 (0.77, 1.09)0.201.13 (0.66, 1.96)0.558Proportion of pneumonia suspects aged under 5 years assessed for pneumonia1.05 (0.35, 3.15)0.900.94 (0.32, 2.78)0.891.21 (0.37, 4.03)0.689Estimated proportion of patients aged under 5 years diagnosed with pneumonia who received appropriate antibiotic treatment0.95 (0.57, 1.58)0.800.96 (0.59, 1.15)0.830.95 (0.54, 1.69)0.8410Proportion of TB suspects with a first Acid-Fast Bacilli (AFB) smear result1.03 (0.45, 2.38)0.920.94 (0.39, 2.28)0.881.19 (0.44, 3.17)0.6611Estimated proportion of patients with AFB smear negative results who received empiric treatment for acute respiratory infection*1.76 (0.93, 3.32)0.0231.58 (0.82, 3.03)0.07**2.04 (1.06, 3.94)0.00516Proportion of patients with an HIV test result recorded (all patients ≥2 months)1.20 (0.73, 1.96)0.351.10 (0.64, 1.87)0.661.48 (0.84, 2.61)0.07 **Denotes that the effect of OSS was significant at the .01 level and *Denotes that the effect of OSS was significant at the .05 level The 99% confidence intervals (CI) are based on the .01 level of significanceAbbreviations: aRRR adjusted ratio of relative risk, IMID Integrated Management of Infectious Disease, OSS On-site support, MLP Mid-level practitioners, AFB Acid-fast bacilli, HIV HumanImmunodeficiency Syndrome, TB Tuberculosis aRRR adjusted Ratio of Relative Risk measured the effect of OSS in each subgroupEstimates were adjusted for: whether the facility received the on-site intervention from Baylor International Pediatric AIDS Initiative or not, facility was implementing continuous quality improvement prior to IDCAP trial, level of health facility (small hospital or health center IV), facility ownership (public or private-not-for profit) and data entry assistant on-site BODY.DISCUSSION: There have been limited rigorous randomized education trials that demonstrate the effects of health worker training approaches on patient care outcomes in the resource constrained world. In this study, we have evaluated the effect of an educational outreach intervention involving nine monthly on-site support (OSS) sessions on health facility performance in two groups of trainees – those who were provided with five weeks of off-site classroom education and those who did not have this opportunity. The OSS intervention did not significantly increase patient load within the subgroup of health providers who did not participate in the IMID training. Among the mid-level practitioners (MLP) who received both IMID training and OSS, the number of patients per provider per calendar day increased; but the changes in workload attributable to OSS were not statistically significant. The effects of OSS on facility performance indicators were heterogeneous. Patients treated by health providers who attended the IMID training benefited from improved case management of malaria and respiratory infections. Patients treated by providers without the IMID training benefited from improvements in emergency triage, assessment and treatment. The number of patients per provider per calendar day was much lower than we expected. On average, the health providers with IMID training saw 4.64 patients per calendar day, and the other providers 0.83 indicating that the MLP saw 5 times more patients than other providers. This was expected since MLP involved in daily management of patients in the outpatient clinics and spent over 80 % of their time at the health facility seeing patients were prioritized during the selection process [20]. These results for the MLP with IMID training are consistent with those of the Institute for Health Metrics and Evaluation's report that showed health providers saw an average of three to five patients per day [43]. These results also indicate that health workers in Uganda treat very few patients per day, but this finding contrasts with other reports in which complaints of heavy workload have been cited among health workers in Uganda [44] and other sub-Saharan Africa countries [45, 46]. The reported number of patients seen per provider is an average across the estimated working days for each of the study time periods. It is possible that providers did not work on all estimated days and thus treated a higher number of patients on days when they worked [47]. In a review of studies in six countries including Uganda, it was found that 35 % of the health care providers were absent during their assigned shifts [48]. Among many factors, the absence usually arises because health workers are on leave, attending trainings off-site, tending to their gardens, or doing work in private clinics to supplement their income [47]. If health workers are only on duty an average of 65 % of the time, then the average number of patients per provider per day would increase by one-third. It is also possible that the number of patients' visits per day was higher on some days than others. Adding in the fluctuations in patient visits and it possible that some clinicians would manage much higher number of patients on some days. Further analysis is required to determine how the number of patients per day seen by clinicians varies by day and across facilities. Among the health workers without the IMID training, this analysis showed no statistically significant effect of OSS on changes in the volume of patients they managed. In fact, the number of patients seen per provider per calendar day among these health providers remained the same before and during the OSS. Yet a slight increase in volume of patients seen per provider per calendar day among the MLP with IMID training was observed during OSS, although this increase was not statistically significant. This increase in patient load among IMID trainees coupled with no increase among providers who did not attend IMID training, shows a general increase in patient volume overall during OSS, perhaps pointing to an appreciation of better quality services being offered at the health facilities. Rather than shift tasks and workload among cadres, OSS sought to improve facility performance within each cadre's scope of practice. This could explain why there was no significant increase in patient volume among health workers without IMID training and decrease among health workers without the IMID training. Overall, OSS was effective for three of the 12 facility performance indicators in this subgroup analysis, compared to none in the main IDCAP trial results [33]. In this subgroup analysis, only records with complete data on the provider's cadre and IMID attendance were analyzed unlike in Weaver et al.'s [33] analysis that included all patient records. It is plausible that providers with incomplete data also attended fewer OSS or benefited less from them and diluted the effects. We saw evidence of the no-IMID subgroup diluting the OSS effects of the MLP with IMID especially for indicators 11 (patients with AFB smear negative results who received empiric treatment for acute respiratory infection) and 16 (patients with an HIV test result recorded). On the other hand, evidence of the MLP with IMID diluting the effects of the no-IMID subgroup was observed for indicator 2 (emergency and priority patients who were admitted, detained or referred). The effects of OSS among the MLP with IMID tended to be statistically significant for indicators that reflected clinical decision-making, such as prescribing antimalarials and antibiotics. In contrast, the effects of OSS among the no-IMID subgroup were for emergency, triage and assessment indicators that required a broader team effort to identify and manage patients appropriately. Although OSS did not seek to change a cadre's scope of practice, it did seek to empower all providers to triage patients and organize immediate care for emergency and priority patients. The effects of OSS observed in this analysis were heterogeneous across indicators as well as across subgroups. In the overall sample, the average effect size was 34 % and higher than estimates elsewhere. For instance, reviews of continuous medical education and effect of educational outreach visits on health workers' training reported median improvements ranging from 6.9 to 13.9 % [19] and from 5.6 to 21 % [49] respectively. The effect sizes for the emergency, triage and assessment and malaria case management indicators were generally larger than the effect sizes for case management of respiratory infections and HIV testing. The indicators for the first two coincide with the first two OSS. The OSS sessions for the latter two were delivered later in the intervention, an indication that perhaps, the expectation of immediate improvements over a shorter period of time may have been unrealistic. There is need for monitoring these indicators over more months to observe whether facility performance will improve over time. Also future researchers should consider designing their trials for a subgroup analysis. Within resource-limited settings in many sub-Saharan African countries, clinicians and other higher level cadres are the ones usually selected to attend continuous medical education trainings to support them improve the quality of diagnosis and treatment. Allied health workers, nurses and other lower level providers often miss out on such opportunities since they mainly play supporting roles to clinicians in providing patient care. OSS would therefore be a great opportunity to improve capacity of these non-clinician health workers to dispense better quality patient care in the entire patient care process. For clinicians and higher level cadres, OSS would be important in supporting them to apply knowledge and skills in diagnosis and treatment acquired from classroom training. To our knowledge, the IDCAP trial is one of the first randomized trials that evaluated the effects of an integrated educational outreach and quality improvement intervention in sub-Saharan Africa. The results of this study therefore provide important evidence that could inform capacity building interventions and policies for health workers in resource limited settings. BODY.DISCUSSION.LIMITATIONS: As noted in Weaver et al. [33] sample size calculations for the IDCAP trial were based on comparison across arms and an α = 0.05. Consequently, this subgroup analysis may have been underpowered to detect difference-in-difference at α = 0.01 when comparing effect of OSS on facility performance between arms. OSS sessions were delivered gradually for nine months, and its effect may have been underestimated by measuring it during its implementation rather than after. BODY.DISCUSSION.GENERALIZABILITY: This study was conducted in a sample of sub-district level referral HCIVs or comparable small hospitals that met the inclusion criteria. These results can thus be generalized for similar health facilities in Uganda and other countries, and for indicators for which OSS was effective. BODY.CONCLUSIONS: The MLP with IMID training managed thrice as many patients per day compared to other providers. Out of the 12 facility performance indicators, OSS improved three indicators across all patients and among patients managed by MLP with IMID training, but only two indicators among patients managed by providers without IMID training. These results show that OSS supported MLP who diagnosed and treated patients to apply knowledge from IMID. For other providers, OSS supported team work to manage emergency patients. Since provision of patient care is a team effort with clear roles for every health worker, health workers engaged in clinical diagnosis and treatment may need OSS in addition to classroom training, while the nurses and other providers who play a supporting role to the clinicians will need OSS to effectively contribute more to patient care.

TITLE: Yerba Maté ( ABSTRACT: Yerba Maté (YM), has become a popular herb ingested for enhancing metabolic health and weight-loss outcomes. No studies have tested the combined metabolic, satiety, and psychomotor effects of YM during exercise. We tested whether YM ingestion affects fatty acid oxidation (FAO), profile of mood state score (POMS), and subjective appetite scale (VAS), during prolonged moderate exercise. Twelve healthy active females were randomized to ingest either 2 g of YM or placebo (PLC) in a repeated-measures design. Participants rested for 120 min before performing a 30-min cycling exercise corresponding to individuals' crossover point intensity (COP). FAO, determined using indirect calorimetry, was significantly higher during the 30-min exercise in YM vs. PLC (0.21 ± 0.07 vs. 0.17 ± 0.06 g/min, p < 0.05). VAS scores for hunger, prospective eating, and desire to eat were all reduced (p < 0.05). Whereas, POMS measures of focus, energy, and concentration were all increased (p < 0.05). There was no significant time-effect for any of the measured variables, nor was there any interaction effects between YM treatment and time. Combining YM intake with prolonged exercise at targeted "fat-loss"' intensities augments FAO and improves measures of satiety and mood state. Such positive combined metabolic, satiety, and psychomotor effects may provide an important role for designing future fat and weight-loss lifestyle interventions. BODY.1. INTRODUCTION: Nutritional and exercise lifestyle strategies are primary tools for weight and fat loss to prevent major health risks and lifestyle diseases such as obesity, diabetes, and cardiovascular disease. The effectiveness of healthy lifestyle strategies can be maximized with a variety of nutritional aids in the form of functional foods and naturally available herbal ingredients [1,2]. Yerba Maté (YM), the plant Ilex paraguariensis, is traditionally consumed in many South American regions, but its popularity is increasing in North America, Europe, and other regions worldwide [3]. Several anti-atherogenic and weight-loss properties have been associated with the regular consumption of YM [4], including anti-oxidation, vasodilation, reduction in blood lipids, and other anti-mutagenic and anti-glycation benefits [5,6,7,8,9]. These health properties have been attributed to several naturally-present bioactive ingredients, which have been detected in YM including polyphenols and caffeoyl derivatives (caffeic acid, chlorogenic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid and 3,5-Dicaffeoylquinic acid), phytosterols, saponins, some amino acids, vitamins, and minerals [4,5]. Metabolic functions of YM components are thought to be responsible for reductions in serum cholesterol, serum triglycerides, and glucose concentrations; and an improved glycemic control and lipid profile in in high-fat fed mice [7], reduced body fat mass, and distribution and reduced waist to hip ratio in humans, all shown following YM ingestion [10,11]. Recent findings have also indicated that YM metabolic properties may be combined with positive psychomotor and appetite control effects, which complement the YM fat-loss promoting properties. Such effects include suppressed appetite through an increased expression of glucagon-like peptide-1 (GLP-1) and delayed gastric emptying, as seen in mice studies [12,13], and increased ghrelin up to 4.2-fold in rat models following YM ingestion [14]. A trend towards increased satiety, reduced hunger, and improved mood state has also been found using visual analogue scale in human participants who ingested YM combined with other fat-loss ingredients [15,16]. The reported psychomotor effects include improved total mood disturbance score [15], increased focus, alertness and energy, and decreased fatigue in habitual caffeine consumers [16]. Modifying behavioral factors of mood state and appetite control is considered essential for effective weight-loss lifestyle interventions [17,18,19]. Consequently, indications of the positive YM effects combined with exercise on those outcomes should be further investigated, especially given the known positive effects of exercise on mood state and mental health [20]. Along with the nutritional metabolic weight and fat-loss benefits, exercise is known to stimulate fat metabolism, and reverse associated metabolic health risks. YM effects on thermogenesis has been suggested to promote fat-loss by influencing indirect calorimetry measures such as energy expenditure (EE), fatty acid oxidation (FAO), and respiratory exchange ratio (RER) in resting healthy obese participants [21]. However, little is known about such YM metabolic effects during exercise. Our recent work has shown that YM favors FAO as a fuel source during exercise, when either ingested as a single ingredient [22] or combined with other fat-loss compounds in a multi-ingredient supplement [15]. In Alkhatib, 2014 it was found that 1 g of YM can induce an over 20% increase in FAO at the exercise intensity range of 40–70% of peak oxygen uptake (V ̇O2peak), which is considered to be within the low-to-moderate exercise intensity domain [22]. This intensity range of exercise corresponds to maximal fat oxidation (Fatmax) intensity, defined as the exercise intensity where FAO becomes maximal, and the crossover point (COP), defined as the power output when the energy expenditure derived from carbohydrates (CHO) fuels predominates over that from FAO fuels [23,24]. Performing exercise at individually determined Fatmax or COP intensities has been shown to induce favorable metabolic outcomes, such as enhanced FAO ability, improved insulin sensitivity, and enhanced vascular function [25,26,27]. However, to date no study has tested whether the exercise-induced metabolic effects at those effective exercise intensities (i.e., COP intensities) could be augmented with YM ingestion. Given our recent promising findings of YM acute effects on FAO during exercise intensities in the COP range [15,22], and the YM weight-loss postulated effects on satiety and mood state [13,22], our study is important to test whether and how YM affects FAO, satiety, and mood state during prolonged exercise at individuals' COP intensities. This study aims to test the hypothesis that YM ingestion combined with steady state exercise at the COP intensities augments FAO, and improves the measures of satiety and mood state. BODY.2. MATERIALS AND METHODS .2.1. DESIGN AND PARTICIPANTS: The study followed a double-blind repeated-measures crossover placebo-controlled design. All tests were performed at similar laboratory environmental conditions controlling for air temperature, barometric pressure, and relative humidity. The study was approved by the institution's ethical committee. All experimental procedures were carried out in accordance with the ethical guidelines of the World Medical Association Declaration of Helsinki. All study participants provided their informed written consent and were given an explanation of the purpose of the research and experimental procedures. Exclusion criteria were as follows. (1) History of any cardiovascular or respiratory disease, hypertension, liver or kidney disease, musculoskeletal or neuromuscular or neurological disease, autoimmune disease, cancer, peptic ulcers or anemia; (2) Taking medications, including those for heart, pulmonary, thyroid, anti-hyperlipidemic, hypoglycemic, anti-hypertensive, endocrinologic, psychotropic, neuromuscular, neurological, or androgenic conditions, as well as a family history of heart problems, high blood pressure, and/or stroke, and being pregnant or breastfeeding. (3) Consuming any ergogenic aid or above habitual caffeine consumption rate (≥200 mg/day) for at least six weeks prior to the study similarly to a previously used approach [16], based on all types of caffeinated beverages (coffee, energy drinks, soft drinks, caffeine supplements, or medications). Inclusion criteria included females, aged 18–40 years, habitually complete ≥150 min of moderate physical activity per week, BMI < 30.0 kg/m2. Participants were asked to specify the phase of their menstrual cycle (luteal or follicular phase). All participants were eumenorrheic and none of them were taking any oral contraceptive pill or any other forms of hormonal contraceptives. All participants refrained from taking any supplements for the duration of the study and were instructed to refrain from strenuous exercise or alcohol and caffeine consumption for at least 24 h before each test. Each exercise testing trial was performed within one week (between three and seven days) of the previous session. All participants were familiarized with the testing equipment and procedures prior to the start of the experiment. Participants were physically active healthy women volunteers, and were recruited by convenient sampling. A total of 12 females completed the study [Mean ± SD, age = 30.8 ± 7.3 years, stature: 167.2 ± 3.9 m, body mass: 61.5 ± 2.8 kg, BMI = 22.0 ± 1.1 kg/m2, body fat percentage (BF%) 19.8 ± 4.2%]. Nine out of 21 participants who met the inclusion criteria failed to complete all tests, mainly due to lack of time or inability to commit for all testing sessions. Sample size calculations were based on achieving a large effect size based on the least meaningful difference induced by YM supplement on FAO in previous studies [22], and provided a power of 90% for an alpha level significance of 5%. The calculation showed 12 participants were needed. BODY.2. MATERIALS AND METHODS .2.2. EXPERIMENTAL PROCEDURES AND EXERCISE PROTOCOLS: All participants reported to the Physiology Laboratory on three separate occasions followed by 3 h fasting state in the first session, and 10–12 h overnight fasting in the second and third sessions. Each testing session (between 09:30 and 11:30 a.m.) was separated by at least three days within a two-week period. Session one involved the participants being assessed for their body composition and exercise performance variables for all participants, including V ̇O2peak and the individualized intensity associated with COP, which is close to Fatmax intensities [23,28]. Sessions two and three involved the participants being randomized to ingest either 2 g (4 × 500 mg capsules) of YM capsules or a placebo (PLC) empty capsules presented in similar color, size, and appearance. The YM capsules contained a standardized dried ground YM green leaves (Rio Trading Health Company, Brighton, UK), and all YM capsules were obtained from the same batch number (No. 17558), with approved content and safety. A certificate of analysis was obtained and included analyses of aspect, color, aroma, moisture, total and insoluble ash percentage, caffeine content (approximately 1.5%, minimum 0.4%), granulometry, but not the polyphenol content. Each YM capsule weighed approximately 575 mg (total weight of the four capsules 2.3 g), and a typical powder density within the capsule size used was 475–480 mg, the YM weight in the four capsules was approximately 2 g. Participants ingested all four capsules at once from a special weighted mug (292.1 g) with a <1% difference in its weight between treatments. A total of 500 mL of water was consumed over the duration of each testing visit as a standardized amount for all participants and to reduce the risk of dehydration, 300 mL with the supplement and 200 mL over the total remaining testing period. Participants were required to refrain from consuming caffeine for a minimum of seven days prior to the first testing visit and over the duration of the study. Participants also completed a three day (two week days and one weekend day) food diary with details about serving amounts for breakfast, lunch, dinner, snacks, and additional meals. A blank food diary and an example food diary were provided to each participant. Subjects were instructed on how to complete the food diary and asked to provide full details on all food, drinks, and snacks consumed both inside and outside the home including the portion size and the method of preparation. Participants were asked to replicate their recorded food dietary intake for each experimental test. BODY.2. MATERIALS AND METHODS .2.3. BASELINE ASSESSMENTS (VISIT ONE): Body composition was assessed using bioelectrical impedance scale, which also provides body weight, assessed to the nearest 0.1 kg (InBody version 720, Biospace, Seoul, Korea). Height was assessed to the nearest 0.5 cm using a Stadiometer (Seca scales, Hamburg, Germany). The baseline exercise protocol involved the participants completing an incremental exercise test using an electronically braked cycle ergometer (Lode, Excalibur Sport, Groningen, The Netherlands). The saddle height and distance from the handlebar were recorded at the initial visit and re-applied in all subsequent visits. Participants began cycling at 25 watts for three minutes and work rate increased by 25 watts every three minutes until volitional exhaustion. V ̇O2 and V ̇CO2 data were measured breath-by-breath using an Ultima (Medgraphics CPX Ultima, Medical Graphics Ltd., Gloucester, UK). Participants were fitted to wear a nose clip and breathe through a mouthpiece attached to a pneumotach. The volume and gas analyzers of the system were calibrated using a 3-L calibration pump and calibration gases (12%O2; 5%CO2) as per manufacturer's instructions. Heart rate (HR) was recorded every minute using a HR monitor (FS1 Polar electro OY, Kempele, Finland). The rate of perceived exertion (RPE) was recorded during each stage using the (6–20) Borg scale [29]. The breath-by breath cardiorespiratory data of V ̇O2 and V ̇CO2 were averaged over the last minute of each 3-min stage during the incremental test where the RER was <1 (below CHO saturation level). The data was also averaged for the last minute of every time point (5 × 30 min) at the 2-h rest and (6 × 5 min) during exercise the following two steady-state sessions. For each of the stages, EE (Kcal/min), FAO (g/min), CHO (g/min) were calculated using stoichiometric equations and corresponding energy metabolic equivalents, similarly to previous studies [22]. The COP intensity, which was used for the YM and PLC exercise trials, was determined for each individual, as the power output (W) and exercise intensity relative to V ̇O2peak (% V ̇O2peak) corresponding to when the relative EE derived from CHO predominates (50% or higher) over that of FAO [24]. This intensity was used during the steady state exercise in two experimental trials (PLC and YM) for each participant. V ̇O2peak was considered maximal when two of the three following conditions were met; a levelling off V ̇O2peak with further increasing workloads (an increase of ≤2 mL/kg/min), a heart rate (HR) within 10 beats per minute of the age predicted maximum (220 bpm—age) and a respiratory exchange ratio (RER) of >1.05 (8). The last 15 s of the maximal exercise test were averaged to determine V ̇O2peak. Peak power (Ppeak) was calculated as the highest power output for the last completed exercise stage before test termination. If the stage was incomplete Ppeak was calculated based on the completed time fraction of the final stage in seconds, and the power increment as previously described for a similar protocol [22]. BODY.2. MATERIALS AND METHODS .2.4. EXERCISE ASSESSMENT AND SUPPLEMENTATION (VISITS TWO AND THREE): Participants received the supplement immediately before resting for 120 min in a semi-recumbent position during which they remained awake, while not talking and limited their movement. At the end of the resting period, participants were transitioned to a cycle ergometer where they cycled continuously for 30 min at a workload pre-determined from their COP calculated during the baseline visit. The cadence for the exercise protocol was self-selected to fall within a range of 60–80 RPM for the two supplementation trials. V ̇O2, V ̇CO2 and HR data were collected continuously and RPE was assessed every 10 minutes throughout the exercise protocol. BODY.2. MATERIALS AND METHODS .2.5. APPETITE AND MOOD STATE: Appetite and satiety were measured using a visual analogue 100 mm scale (VAS), which recorded perceived hunger, fullness, desire to eat, and prospective food consumption [30]. The scale was anchored at each end with the labels "not at all" (0 mm) and "extremely" (100 mm). Subjects marked a line through the scale between the two extremes of the symptoms being rated which they considered to indicate the degree of the subjective feeling being rated. VAS was completed prior to supplementation, every 30 min during the 120-min resting period, immediately post exercise (0 min), and 30 min post exercise. Profile of mood state (POMS) questionnaire [31] was used to assess participants' mood state (perceived alertness, focus, energy, fatigue, and concentration). Participants were asked to rate their perceived mood on a scale ranging between one and five for all five categories. POMS questionnaires were completed prior to supplementation, every 30-min during the 120-min resting period, immediately post exercise, and 30-min post exercise. BODY.2. MATERIALS AND METHODS .2.6. DATA PROCESSING, ANALYSES, AND STATISTICS: All data are presented as means and standard deviations. FAO, CHO, EE, HR, POMS, and VAS were analyzed using two-way repeated measures ANOVA (YM × Time), with YM supplement as within factors, and six time points during 30-min for the exercise metabolic data (FAO, CHO, EE, HR) as between factors. Bonferroni post hoc test was applied to analyze the differences at each time point. For VAS and POMS the measured time points were analyzed using two-way ANOVA (Treatment × Time) for both rest condition at time points (0, 30, 60, 90, and 120 min), and exercise condition immediately before supplementation (0-min), immediately before exercise (120-min), immediately after exercise (150-min), and post 30-min recovery following exercise (180-min). The area under the curve (AUC) was calculated using trapezoidal method, and was compared between the treatments using a paired t-test. For all statistics SPSS IBM statistics V24 was used and the significance level was set at p < 0.05. BODY.3. RESULTS: The exercise power output at the cross-over point (COP) determined during the baseline assessment was 50.8 ± 22.3 W corresponding to a relative exercise intensity of 37.5 ± 8.0% V ̇O2peak. Peak data determined at baseline assessment were (V ̇O2peak = 38.3 ± 4.7 mL/kg/min, RPEPeak = 19.7 ± 0.5, RERPeak = 1.13 ± 0.06, PPeak = 194.5 ± 11.4 W, HRpeak = 177.1 ± 11.5 BPM). BODY.3. RESULTS.3.1. FAO, CHO, EE, AND HR DURING EXERCISE: The YM treatment elicited significantly higher FAO compared with PLC during the 30-min steady-state exercise (ANOVA treatment effects, p = 0.037), (Figure 1). The difference was also affected by the time of exercise (ANOVA time effect p < 0.001). The highest FAO difference was after 20 min (0.21 ± 0.07 vs. 0.17 ± 0.05 g/min, p = 0.039), and 25 of exercise (0.22 ± 0.07 vs. 0.17 ± 0.06 g/min, p = 0.015) for YM compared with PLC (Figure 1). Calculating AUC showed a higher total AUC for FAO in YM than PLC (5.22 vs. 4.20, p < 0.001). However, there was no significant interaction between YM treatment and exercise time. CHO was not significantly different between the treatments, but there was a time effect (ANOVA time effects, p < 0.001) with the CHO initially increased and then decreased over exercise time in both treatments (Figure 2). No interaction for CHO between treatment and time was found. However, the total AUC for CHO was lower in YM than PLC (13.3 vs. 15.00, p < 0.001). Total Energy Expenditure (TEE) was neither significantly affected by the treatment (YM vs. PLC) nor the exercise time (Figure 3). However, the total AUC for TEE was higher (85.60 vs. 82.13, p < 0.001) in YM than PLC. HR was not different between the treatments, but the time had significant effects (p < 0.001), (Figure 4). No interaction effects were found. RPE, measured at three exercise time points, was also not affected between the treatments (YM vs. PLC) or exercise duration, nor was there any interaction effect between YM and exercise time (Figure 5). BODY.3. RESULTS.3.2. SATIETY MEASURES OF VAS: There were significant treatment effects (YM vs. PLC) on VAS measures of Hunger (p = 0.019), Prospective eating (p = 0.022), and a trend towards a reduced desire to eat (p = 0.079) for exercise (Pre, immediately before, after, and 30 min post exercise). There was no YM effect on fullness score. The time effect was not significant for all VAS satiety measures, and there was no interaction between time and YM treatment (Table 1). When analyzing the resting data only (immediately pre-ingestion 0 to 120 min rest duration), only prospective eating score was slightly reduced (p = 0.046) at YM treatment compared with PLC, but there were no significant YM treatment or rest time effects in any of the remaining VAS measures, nor were there any interaction effects (Table 1). BODY.3. RESULTS.3.3. MOOD STATE MEASURES OF POMS: YM significantly affected POMS measures following exercise by an increased focus (p = 0.022), energy (p = 0.008) and concentration (p = 0.003), and a trend towards an increased alertness (p = 0.066) in the YM treatment compared with PLC (Table 2). No effects were found for fatigue scores. Exercise time had no effect on any of the POMS measures nor was there any interaction between treatment and time (Table 2). When analyzing the resting data only (immediately pre-ingestion 0 to 120 min rest duration) no significant effects were found for YM treatment, rest duration, nor were there any interaction effects (Table 2). BODY.4. DISCUSSION: In this experiment, YM increased FAO during prolonged steady-state exercise and induced positive psychomotor mood state and satiety during and after exercise without affecting the exercise RPE. Augmented FAO was approximately 23% higher in YM compared with PLC during 30 min of steady-state low-to-moderate intensity exercise corresponding to individuals' COP intensity (Figure 1). This increase is comparable with 24% increase found during low to moderate exercise intensities determined using an incremental protocol in our previous study [22]. The present study extended previous findings by determining COP individually, and demonstrated that YM ingestion is effective in enhancing the impact of FAO at the targeted COP exercise intensities. Targeting such intensities with exercise training enhances fat metabolism, and associated "fat-loss" metabolic health outcomes including increased insulin sensitivity [25], enhanced lipolysis and ability to oxidize lipids [32,33], and microvascular activity [26]. Therefore, YM augments such metabolic outcomes when combined with prolonged exercise at such given fat-loss intensities. Previous studies in human participants have shown promising effects of YM ingestion on metabolic rate and RER acutely [21], and after 12 weeks of ingestion, on blood lipid metabolites in healthy obese participants [11,21]. However, these metabolic effects were only tested at rest. YM was also administered, with various metabolic efficacy, in various doses of ≈1 g of proprietary multi-ingredient thermogenic blends containing weight-loss ingredients such as YM and green tea extracts, caffeine anhydrous, guarana, yohimbine HCI, capsicum, ginger and bitter orange extracts, and other proprietary blends [16,22,34]. In two separate studies conducted previously, we tested the exercise-dependent effects on FAO at various intensities with 1 g YM [22] or when YM combined with a proprietary thermogenic blend of 1.5 g dose [15]. Both studies used mixed gender samples and showed an augmented FAO during low-to-moderate intensity exercise of 24% and 26% in YM compared with PLC. This is close to the 23% found for FAO in this cohort of female participants, using a higher ingestion dose of 2 g (Figure 1). The 38% V ̇O2peak intensity used in this study is less than intensities (40–70%) [22] and 44% V ̇O2peak [15] used in those two previous studies, but demonstrated almost equal % difference in FAO, which suggests that supplementing a higher dosage of 2 g could be more effective at higher intensities, and merits further investigation. Such intensity effects of the higher dosage used in the present study could also be attributed to the no significant difference found in the TEE (Figure 3). All three studies used sufficient amounts of resting time of 1–3 h following ingestion and prior to exercise, which is considered sufficient to induce the YM thermogenic effects at rest [21] and during exercise [22]. Other available studies to compare our findings with during exercise are limited to herbs which share some similar active ingredients to YM, especially green tea [4,5]. For example, Gahreman et al. (2015) [35] combined green tea with intermittent exercise and showed an increased FAO, plasma glycerol, and plasma catecholamines at rest and post exercise compared with placebo in healthy active female participants of similar characteristics to the present study. Another study by Hodgson et al. (2013) [36] found that a drink containing 1.2 g of green tea affected the metabolic profile (3-β-hydroxybutyrate, pyruvate, lactate and alanine concentrations) at rest and during 60 min of exercise at 56% V ̇O2peak compared with placebo. However, both studies used higher intensities which promotes CHO metabolism, than the COP intensity of 38% V ̇O2peak used in the present protocol. It is likely that active YM thermogenic ingredients work in synergy to promote lipolysis and augment FAO during exercise. The metabolic effects include adrengenic effects and stimulated central nervous system associated with caffeine, anti-lipolytic, and hypocholesterolemic properties in chlorogenic acids (mono- and di-caffeolquinic acids) hydroxycinnamic acids (caffeic acid, quinic acid) and triterpenic saponins, and other minerals and vitamins [5]. Anti-oxidant compounds in YM such as flavonoids and polypheonols are common in other herbal teas and may affect nitric oxide levels, which have been shown to be effective in inducing vasodialatory effects [37] when combined with exercise [27,38]. Anti-oxidant compounds of YM have been recently attributed to accelerating muscle strength recovery 24 h after exercise, suggesting that YM favored the concentration of blood antioxidant compounds [39]. Therefore, YM active ingredients may have played a synergetic role in the metabolic effects found during exercise. However, further research is required to assess active ingredients of YM capsules and analyze their bioavailability following ingestion. Favorable psychomotor effects on mood state and satiety are often expected outcomes of fat and weight-loss supplementation protocols. However, several negative side effects were reported for common thermogenic supplements containing caffeine including jitteriness, mood swings, and headache [40]. It is suggested that these effects can be reduced with YM ingested with other ingredients compared with caffeine [22,40]. The present study found an improvement in almost all measures of POMS and VAS (Table 1). In particular, there was an increased focus (p = 0.022), energy (p = 0.008) and concentration (p = 0.003) in the YM treatment compared with PLC, which was combined with no effects on fatigue scores. Interestingly, the RPE score was not different (Figure 5), suggesting that the positive psychomotor YM effects had no negative effects on the perception of effort and fatigue during exercise, which is important when considering adherence to prescribed exercise for weight loss and sport performance outcomes. There was also a reduction in appetite VAS measures, especially for hunger (p = 0.019) and prospective eating (p = 0.022), (Table 1) following the YM ingestion compared with PLC. These YM appetite control effects are reported in humans for the first time, considering previous positive effects reported in animal models [13,14], and the recent report of appetite suppression following exercise in trained female participants [41]. The reduced VAS appetite scores are also in line with previous results found when YM was combined with other multi-ingredients before and after moderate exercise at Fatmax intensities at 43% V ̇O2peak, which is slightly higher than COP intensities of 38% V ̇O2peak to the present COP intensities [22]. Nonetheless, it is suggested that irrespective of the intensity differences, exercise suppresses appetite hormones (GLP-1, PYY3-36, and acylated ghrelin) and VAS scores in trained women [41]. It has also been reported that exercise combined with satiety-inducing compounds is effective in reducing energy intake in active females [42]. This suggests that YM effects on satiety and mood state may be dependent on augmented metabolism during exercise. Perhaps, exercise combined with YM is most effective in appetite control and improved mood state after performing exercise, which is important for designing lifestyle interventions and weight-loss adherence. HR response is a standard measurement for exercise intensity and training cardiovascular adaptations, but it was not significantly affected by YM ingestion (Figure 5). Cardiovascular benefits for YM consumption have been reported using more sensitive biomarkers such as detecting an increase in vascular endothelium-dependent vasorelaxing activity in rats [43,44]. Such microvascular measurements have been shown to be sensitive to the exercise-dependent effects in human lifestyle interventions [26]. Perhaps HR monitoring is insufficient to detect vascular responses associated with combining YM ingestion with exercise, and future research could determine more sensitive macro- and microvascular health effects associated with YM ingestion during exercise, especially when combined with detecting YM metabolic effects found in the present study. Although there was not significance for all variables, the significant increase in FAO is mathematically accounted for by combined non-significant reductions in glucose oxidation and non-significant elevated TEE for YM (Figure 3). The estimates are approximately 0.35 kcal/min greater FAO, and 0.23 reduced CHO with 0.14 kcal/min greater TEE, and with a corresponding significant YM on the calculated total AUC for FAO, CHO, and TEE (p < 0.001). It is unclear whether there are additional YM effects on glucose and adipose tissue levels not determined in this study [45]. We used indirect calorimetry methodology to estimate metabolic variables, so perhaps future studies could use different techniques to estimate YM effects on different body compartments, tissues, and metabolites by including in vitro and muscle biopsy methods. With respect to the gender-specific effects on metabolism, females' FAO during exercise is known to be higher compared to men [46], possibly due to higher total body fat percentage, fitness level, and exercise modality [47]. The BF% data indicated all females who took part were at the lower BF% percentile, indicating that they were physically active, with higher V ̇O2peak of 38 mL/kg/min, which is higher than 32 mL/kg/min measured for a mixed gender cohort in a previous similarly designed study [18]. The study's participants completed their tests within the same week and reported to be within the same menstrual cycle phase. Some authors suggested that the luteal phase of the menstrual cycle is associated with increased lipid oxidation compared with the follicular phase [48], but no differences were reported by others during prolonged moderate exercise between luteal and follicular phases [46]. All participants within the present study repeated their assessments within the same phase of the cycle, so it is unlikely that menstrual cycle affected the metabolic variables. Nevertheless, gender differences' effects on the combined YM and exercise-induced fat oxidation response needs further investigation. BODY.5. LIMITATIONS: Although the positive effects of YM on fat-loss outcomes during exercise are found only in single trials, future research could test the longitudinal YM effectiveness, especially when combining regular exercise training with YM treatment. This is important for lifestyle disease prevention, given the effectiveness found for YM alone as treatment for human obesity, found in recent randomized controlled trials [10,11]. The present study used COP as an effective and a well-established "fat burning" exercise training intensity [24,32,33], but other established concepts in a similar domain, such as Fatmax intensity, can also be effective as we previously demonstrated [15]. The debate concerning a relevant exercise training intensity to maximize "fat-loss" is beyond the scope of this study, and has been sufficiently discussed elsewhere [23,24,28,32,33]. Most of the studies cited within the present study have attributed their YM ingestion effects to the several bioactive ingredients and only some of those studies have analyzed YM supplement (e.g., total phenolic compounds, caffeine, and saponins) for such ingredients as part of their protocol [39]. However, methodological differences in terms of YM supplement preparations and methods used should be considered for such comparisons. For example, the encapsulated ground green YM leaves used in this study is different from lyophilized YM extracts brewed and used elsewhere [39]. Also, the study did not specifically analyze the polyphenolic content of YM, and only relied on the supplier's certificate of analysis for YM content, which makes it difficult to attribute findings to a single YM ingredient. Nonetheless, it is reasonable to infer synergetic effects of naturally-occurring YM ingredients but, from a biochemical point of view, performing phytochemical analysis on the supplement is important for future research. It would be also interesting to follow up with testing the YM active ingredients' bioavailability post ingestion. Limited research has been conducted in humans prior to this study [10,11,22], which makes it difficult to confirm some parallels made with animal models when explaining YM mechanisms. Nonetheless, our findings in human participants extend previous knowledge and demonstrate novel applications of YM for human metabolic health. BODY.6. CONCLUSIONS: Combining YM ingestion with exercise prescription at COP training intensities improves FAO, measures of satiety and mood state compared with exercise alone. The underlying mechanisms of such effects require further investigation. The combined exercise and YM effects on metabolic, psychomotor, and appetite control outcomes are essential for designing an optimized lifestyle prevention for metabolic health and exercise fat-loss outcomes. Future research should test the longitudinal effectiveness of YM and exercise for metabolic health outcomes.

TITLE: A Randomized Controlled Phase IIb Trial of Antigen-Antibody Immunogenic Complex Therapeutic Vaccine in Chronic Hepatitis B Patients ABSTRACT.BACKGROUND: The safety of the immune complexes composed of yeast-derived hepatitis B surface antigen (HBsAg) and antibodies (abbreviated as YIC) among healthy adults and chronic hepatitis B patients has been proved in phase I and phase IIa trial. A larger number of patients for study of dosage and efficacy are therefore needed. ABSTRACT.METHODS AND PRINCIPAL FINDINGS: Two hundred forty two HBeAg-positive chronic hepatitis B patients were immunized with six injections of either 30 μg YIC, 60 μg of YIC or alum adjuvant as placebo at four-week intervals under code. HBV markers and HBV DNA were monitored during immunization and 24 weeks after the completion of immunization. The primary endpoint was defined as loss of HBeAg, or presence of anti-HBe antibody or suppression of HBV DNA, while the secondary endpoint was both HBeAg seroconversion and suppression of HBV DNA. Statistical significance was not reached in primary endpoints four weeks after the end of treatment among three groups, however, at the end of follow-up, HBeAg sero-conversion rate was 21.8%(17/78) and 9% (7/78) in the 60 μg YIC and placebo groups respectively (p = 0.03), with 95% confidence intervals at 1.5% to 24.1%. Using generalized estimating equations (GEEs) model, a significant difference of group effects was found between 60 μg YIC and the placebo groups in terms of the primary endpoint. Eleven serious adverse events occurred, which were 5.1%, 3.6%, and 5.0% in the placebo, 30 μg YIC and 60 μg YIC groups respectively (p>0.05). ABSTRACT.CONCLUSIONS: Though statistical differences in the preset primary and secondary endpoints among the three groups were not reached, a late and promising HBeAg seroconversion effect was shown in the 60 μg YIC immunized regimen. By increasing the number of patients and injections, the therapeutic efficacy of YIC in chronic hepatitis B patients will be further evaluated. ABSTRACT.TRIAL REGISTRATION: ChiCTR.org ChiCTR-TRC-00000022 BODY.INTRODUCTION: According to the World Health Organization, there are 350 million people worldwide, who are chronically infected with HBV. Prolonged chronic hepatitis B results in the development of liver cirrhosis, liver failure, or hepatocellular carcinoma[1]. The pathogenesis of HBV in chronically infected patients has been well- studied and reviewed. Lack of effective immune responses, notably, defective cell-mediated immune responses (CD4, CD8 and NK cells, cytolytic responses) against HBV, defective dendritic cell (DC) functions and imbalance of cytokine production have been identified as the major mechanisms for virus persistence and initiation of chronic liver disease [2], [3], [4], [5], [6]. Effective host immune responses are crucial to terminate viral persistence. To overcome the defects in immune responses, various therapeutic measures have been designed to boost effective host immune responses [7], [8], [9], [10], [11], [12], [13]. Immune complexes (IC) composed of antigen and antibodies have long been used to induce potent antibody responses against microbial proteins and other proteins in animals [14]. Whether IC can be used for therapeutic treatment of viral hepatitis B patients has been questioned because circulating immune complexes (CIC) have been found in some chronic hepatitis B patients [15]. We hypothesized that the crucial difference between CIC and the immune complexes composed of yeast-derived hepatitis B surface antigen (HBsAg) and antibodies (abbreviated as YIC) used in this study is that, in CIC, the anti-HBs antibodies from the patient are of low affinity, which cannot efficiently bind to HBsAg and clear the protein from the host. In contrast, the anti-HBs used to produce YIC are generated from healthy adults who were immunized multiple times with yeast-derived recombinant HBsAg. Therefore, these are high affinity antibodies that can combine efficiently with HBsAg [16]. When YIC is administered via intramuscular injections, it served as an immunogen to the host, and antigen presenting cells in the immune tolerant host would be forced to uptake the HBsAg complexed to its antibodies via the Fc receptors on antigen presenting cells, and thereby leading to modified antigen processing and presentation in the complex. This hypothesis has been confirmed by our previous experimental studies in animal models and in vitro experiments on human dendritic cells [17], [18]. A recent preliminary study in a small number of chronic hepatitis B patients showed that the therapeutic effect of YIC correlated with both cytolytic and noncytolytic responses [19].Though antiviral drugs are highly effective in inhibiting HBV replication, emergence of drug resistance and rebound of virus replication after withdrawal of drugs are major disadvantages for treatment of persistent viral infections [20], [21]. Conversely, vaccine therapy is an inexpensive and promising approach for the treatment of persistent viral infections [22], [23]. To study the in vivo immunotherapeutic effects of YIC in chronic hepatitis B patients, a double-blind, randomized, placebo-controlled clinical study was conducted, and results are presented. BODY.METHODS: The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. BODY.METHODS.IMMUNE COMPLEXES AND PLACEBO: Both the immune complexes and placebo used in this study were manufactured by Beijing Institute of Vaccine and Biological Products, and the Chinese Good manufacture practice (GMP) regulation was followed. Each dose of 1 mL immune complexes (YIC) consisted of either 30 or 60 μg of HBsAg complexed to human anti-HBs immunoglobulin (HBIG) at an appropriate ratio (described in US patent 6,221,664 B1 and European patent 913157), using alum as the adjuvant, which was a mixture of KAl(SO4)2 and NaOH. The placebo contained 0.1% alum identical to that being used in YIC as the adjuvant. BODY.METHODS.STUDY DESIGN: This double-blind, randomized, and placebo-controlled study was conducted at 12 evaluation centers for the treatment of HBV, which were certificated by the State Food and Drug Administration (SFDA), China. Prior to initiating the clinical trial, the protocol of this study was submitted, registered, licensed and approved by the SFDA, China (license number 2002L0038). The trial was registered at WHO International Clinical Trials Registry Platform. Final approval from the ethics committee at Ditan hospital after ethical evaluating at each participating center was completed in February 2005, and enrollment of patients was initialed in March, 2005. Prior to enrollment, each patient signed a written consent for participating in this trial. The study was designed in a three-arm fashion. Eligible patients were assigned to receive 30 μg YIC, 60 μg YIC or placebo in blocks of 6 (two for the 30 μg YIC vaccine, two for the 60 μg YIC vaccine, and two for the placebo group) through computer generated random numbers on the label of study agent vial in terms of recruiting sequence. An independent biostatistician was in charge of the processing of randomization using SAS program (SAS Institute Inc., Cary, NC, USA). All participants were immunized with six intramuscular injections at 4- week intervals, and followed for 24 weeks after the termination of immunization. Serum samples were collected from each patient at baseline, 12th, 24th, 36th and 44th week after initial injection, and separated into two vials. One vial of serum was used for routine biochemical and hematological tests, such as ALT, AST, and was conducted immediately at each evaluation center. Another vial serum was storied at −70°C at each center and shipped to the reference lab at Beijing Ditan Hospital at the end of study for assays of HBV markers and virus load of all samples from all centers. The study was designed by a chief clinical investigator in Ditan Hospital, and was monitored by TigerMed, China, an independent Contract Research Organization. The principles of good clinical practice and clinical trial-related guidelines issued by SFDA were implemented throughout the study. BODY.METHODS.PATIENTS: Patients with chronic hepatitis B, aged 18–65 years old, who were HBsAg and HBeAg positive for at least 6 months and who were anti-HBe negative with an HBV viral load >100,000 copies/mL and a serum ALT of two to ten times the upper limit of normal value within four weeks before randomization were recruited at each evaluation center. Exclusion criteria were co-infection with hepatitis A, C, D and E virus, or HIV; taking antiviral, hepatotoxic or immunosuppressive drugs or products within the preceding 6 months; other causes of liver disease; serious medical or psychiatric illness; hepatic cirrhosis or AFP >100 ng/mL; abnormal serum creatinine, thrombocyte count, hemaglobin or serum total bilirubin; and pregnancy. BODY.METHODS.ASSAYS: Routine biochemical and hematological tests, such as ALT, AST, were carried out at each evaluation center using automated techniques available at each center. At the end of study, all frozen serum samples from all enrolled patients under code were transferred from all evaluation centers to the reference lab at Beijing Ditan Hospital for assays of HBeAg, anti-HBe and serum HBV DNA levels. Samples at all time points were thawed and assayed using the same lot of reagents. Sequential samples from one patient were tested on the same day. Abbott EIA AxSYM (Abbott, Abbott Park, IL, USA) was employed for HBsAg, HBeAg, and anti-HBe. According to protocols provided by the manufacturer, positive and negative cutoffs were calculated, with the positive and negative controls as required by the diagnostic kits. Serum HBV DNA was quantified by fluorescent PCR assay using the ABI equipment, and reagents were from PiJi, Shenzhen Co, China, with a detection limit of 500 copies/mL. BODY.METHODS.ENDPOINTS: The virologic response was assessed four weeks after the end of treatment (week 24) and 24 weeks after the end of treatment (week 44, the end of follow-up). HBeAg seroconversion was defined by the loss of HBeAg and the presence of anti-HBe antibody. Suppression of HBV DNA was defined as the >2 log10 decrease of viral load. The primary endpoint was defined as loss of HBeAg, or presence of anti-HBe antibody or suppression of HBV DNA. The secondary endpoint was designated as both HBeAg seroconversion and suppression of HBV DNA. BODY.METHODS.SAFETY: All participants were observed for local reactions and systemic symptoms through diary card and follow-up interview. The causality of adverse events was determined by the clinical investigators, and the criteria for severe adverse events were: blood total bilirubin (TB) >3×17.2 μmol/L), prolonged prothrombin time (PTA) <40%), and ALT levels elevated 10 times higher than that of the baseline. The severity of adverse events was classified as mild (easily tolerated; causing minimal discomfort; not interfering normal everyday activities), moderate (Sufficiently discomforting to interfere with normal everyday activities) and severe (Prevents normal everyday activities). Safety analysis was performed on all patients who underwent randomization and received at least one dose of study agent. BODY.METHODS.DATA MANAGEMENT AND STATISTICAL ANALYSIS: The sample size was calculated to ensure an adequate evaluation of the primary endpoint. Based on the literatures and the results of phase IIa trial, a sample size of 78 patients per arm could detect a difference of primary response rate between 60 μg group, 30 μg group and placebo group (response rate in 60 μg YIC group vs placebo group, 35% vs 3%; and response rate in 30 μg YIC group vs placebo group, 20% vs 3%) with a statistical power of 80% at the 0.05 level of significance, allowing for a dropout rate of 20%. All data were double entered into custom-made data entry programs. The data management programs included range and consistency checks. An SAS program (SAS Institute Inc., Cary, NC, USA) was applied for statistical analysis. Analysis was conducted on all eligible patients according to the intent-to-treat principle. HBV DNA was logarithmically transformed for analysis. For binary data, the Chi square test, or Fisher's exact test when data were sparse, were employed. For dichotomous outcomes, ANOVA was used. Repeated measures analysis was performed using a generalized estimating equations (GEEs) method to adjust the dependence among repeated observations made on the same patient while testing the group and time effects [24]. In the model, we included the time effect as a class variable which used three indicator variables. The indicator variable was defined by treating Week 44 as a baseline time. Similarly, the group effect was defined by using two indicator variables, where the placebo group served as baseline group. Since there were only four repeated measurements (week 12, 24, 36, and 44), we applied the unstructured (UN) working covariance matrix which provided robust estimation of covariance to the structure. Since the ALT and HBV DNA are crucial indicators for baseline assessment, adjustments were made for group, baseline ALT and HBV DNA. A p-value <0.05 (two-tailed) was considered statistically significant. BODY.RESULTS.BASELINE CHARACTERISTICS OF ENROLLED PATIENTS: Three hundred and fifty four hepatitis B patients were evaluated for the inclusion criteria. Of these, 242 patients were eligible and assigned randomly to placebo, 30 μg YIC and 60 μg YIC groups in a three-month period. Five patients were found ineligible during the batch assay at the end of treatment (week 24) at the central laboratory, and therefore were excluded from the intent-to-treat analysis. Of 237 patients included in the analysis , 8 receiving placebo, 5 receiving 30ug YIC, and 10 receiving 60ug YIC either did not complete the treatment, did not complete the follow-up or violated the protocol (Figure 1). No significant difference was found in baseline characteristics among three groups (Table 1). 10.1371/journal.pone.0002565.g001Figure 1Summary of participants.Footnote: The numbers of SAE patients are those who discontinued treatment, while other SAE patients are not included in this figure. 10.1371/journal.pone.0002565.t001 Table 1 Characteristics of participants. Characteristics Placebo group (N = 78) 30 µg YIC group (N = 81) 60 µg YIC group (N = 78) Age (yr; SD) 27.9±7.6 28.6±8.6 28.8±8.0 Female sex (no. ; %) 19 (24.4) 22 (27.2) 17 (21.8) Weight (kg; SD) 61.7±11.1 62.3±10.0 63.7±9.3 Course of illness, hepatitis B (yr; SD) 6.3±5.2 6.0±5.8 6.1±5.4 Family history, hepatitis B (no.;%) 46 (59.0) 46 (56.8) 46 (59.0) Alanine aminotransferase (IU/liter; SD) 169.6±80.0 162.5±74.7 171.8±93.4 HBV DNA (log copies/mL; SD) 7.1±0.9 7.1±0.9 7.2±0.8 SD: Standard deviation. BODY.RESULTS.KINETICS OF RESPONSES IN PATIENTS: As shown in Table 2, a delayed response to YIC was observed between 24 weeks and the end of follow-up. For intent-to-treat analysis, at the end of follow-up, 31, 35 and 28 patients achieved the primary endpoint, and 6, 7 and 14 patients reached the secondary endpoint in the placebo, 30 μg YIC and 60 μg YIC groups, respectively. The response rate for secondary endpoint in the 60 μg YIC group was comparatively higher than that of the other groups, though statistical significance had not been reached. However, at the end of follow-up, a significant difference on HBeAg seroconversion was found between 60 μg YIC and placebo groups (p = 0.03, 2-tailed). The 95% confidence interval for this difference was 1.5% to 24.1%. In contrast, a statistical significance had not been reached between 30 μg YIC and placebo groups. 10.1371/journal.pone.0002565.t002 Table 2 Virologic response at the end of treatment and the end of follow-up. End of treatment (week 24) End of follow-up (week 44) Placebo group (N = 78) 30 µg YIC group (N = 81) 60 µg YIC group (N = 78) p value Placebo group (N = 78) 30 µg YIC group (N = 81) 60 µg YIC group (N = 78) p value HBeAg loss (No.;%) 8 (10.3) 9 (11.1) 9 (11.5) 0.99 9 (11.5) 11 (13.6) 18 (23.1) 0.13 Presence of anti-HBe antibody (No.;%) 9 (11.5) 15 (18.5) 15 (19.2) 0.65 10 (12.8) 18 (22.2) 23 (29.5) 0.06 HBeAg seroconversion * (No.;%) 6 (7.7) 8 (9.9) 6 (7.7) 0.97 7 (9.0) 10 (12.3) 17 (21.8) 0.08 HBV DNA 13 (16.7) 21 (25.9) 21 (26.9) 0.46 28 (35.9) 21 (25.9) 29 (37.2) 0.28 >2log decrease (No.;%) Primary endpoint ˆ; (No.;%) 18 (23.1) 22 (27.2) 26 (33.3) 0.63 31 (39.7) 28 (34.6) 35 (44.9) 0.39 Secondary endpoint $ (No.;%) 3 (3.8) 8 (9.9) 6 (7.7) 0.59 6 (7.7) 7 (8.6) 14 (17.9) 0.14 * : 95% confidence interval (2-tailed) for the difference of response rate at week 44 between 60 µg YIC and placebo groups was 1.5% to 24.1%; it was −6.3% to 13.0% between 30 µg YIC and placebo groups. ˆ; : 95% confidence interval (2-tailed) for the difference of response rate at week 44 between 60 µg YIC and placebo groups was −10.4% to 20.6%; it was −20.2% to 9.8% between 30 µg YIC and placebo groups. $ : 95% confidence interval (2-tailed) for the difference of response rate at week 44 between 60 µg YIC and placebo groups was −0.2% to 20.7%; it was −7.6% to 9.5% between 30 µg YIC and placebo groups. Furthermore, when applying the GEEs method to estimate the group and time effects, a significant difference of group effects was found between the 60 μg group and the placebo group in terms of the primary endpoint, and response rate calculated by both primary and secondary endpoints changed significantly over the time in the placebo, 30 μg and 60 μg YIC groups (p<0.05) (Table 3). 10.1371/journal.pone.0002565.t003 Table 3 Repeated measures analysis for time and group effects controlling by baseline ALT and HBV DNA. Group Primary endpoint Secondary endpoint OR (95% confidence interval) p value OR (95% confidence interval) p value 30 µg YIC group vs placebo group 0.9 (0.5–1.4) 0.52 1.3 (0.5–3.6) 0.60 60 µg YIC group vs placebo group 1.7 (1.0–2.7) 0.04 2.2 (0.9–5.4) 0.09 Week 12 vs Week 44 0.3 (0.2–0.5) <0.0001 0.3 (0.2–0.6) 0.0006 Week 24 vs Week 44 0.5 (0.4–0.7) <0.0001 0.6 (0.4–0.9) 0.01 Week 36 vs Week 44 0.7 (0.6–1.0) 0.04 0.6 (0.4–1.0) 0.04 When the baseline serum HBV DNA and levels of HBeAg from all secondary responders at the end of follow-up in the three groups were analyzed, among the 14 patients immunized with 60 μg YIC , five had HBV DNA≥107 copies/ml, eight had HBV DNA ≥106 and <107 copies/ml, only one had HBV DNA 105 copies/ml. In contrast, of the 6 patients immunized with alum, five patients had HBV DNA 105 copies/ml, one had HBV DNA ≥106 and <107 copies/ml, none of them had HBV DNA ≥107 copies/ml. BODY.RESULTS.REVERSION OF HBEAG AND REBOUND OF HBV DNA: The incidence of rebound in virus replication and reversion to serum HBeAg at the end of follow-up were compared among the three groups. For those responders who reached the secondary endpoint, none of the patients from either the 60 μg YIC group or placebo group showed a rebound in virus load nor in reversion to serum HBeAg. In contrast, four responders in the 30 μg YIC group showed a virus load rebound to the baseline level and HBeAg reverted to positive. Interestingly, rebound of viral load and reversion to serum HBeAg were all from responders who only reached the primary endpoint at the end of immunization, while none occurred in those who reached the secondary endpoint, suggesting that patients who achieved the secondary response at the endpoint of treatment were unlikely to develop reversion. BODY.RESULTS.ADVERSE EVENTS AND SEVERE ADVERSE EVENTS: Overall, the most common systematic symptoms were similar for the three groups. However, more local reactions were found in the 60 μg YIC and 30 μg YIC groups. In the placebo group, the most common reaction was pain at the injection site, followed by malaise and fatigue; in the 30 μg and 60 μg YIC group, the most common adverse events were pain at the injection site, pruritus and swelling (Table 4). During study period, eleven patients experienced elevated ALT levels over ten times the normal level, accompanied by high levels of serum bilirubin and thus were hospitalized. 10.1371/journal.pone.0002565.t004 Table 4 Occurrence of most common adverse events during treatment and follow-up. Adverse events No. of patients (%) p value Placebo group (N = 79) 30 µg YIC group (N = 83) 60 µg YIC group (N = 80) Local adverse reaction Erythema 6 (7.6) 15 (18.1) 15 (18.8) 0.08 Swelling 9 (11.4) 19 (22.9) 25 (31.3) 0.009 Pain 32 (40.5) 42 (50.6) 45 (56.3) 0.13 Pruritus 12 (15.2) 22 (26.5) 34 (42.5) 0.0006 Systematic symptom Fever 11 (13.9) 8 (9.6) 11 (13.8) 0.63 Malaise 16 (20.3) 12 (14.5) 12 (15.0) 0.57 Headache 8 (10.1) 9 (10.8) 17 (21.3) 0.09 Dizziness 11 (13.9) 11 (13.3) 18 (22.5) 0.24 Fatigue 14 (17.7) 19 (22.9) 19 (23.8) 0.62 Vomiting 5 (6.3) 5 (6.0) 4 (5.0) 0.94 Nausea 8 (10.1) 9 (10.8) 14 (17.5) 0.35 Abdominal pain 6 (7.6) 6 (7.2) 8 (10.0) 0.84 Diarrhoea 6 (7.6) 6 (7.2) 4 (5.0) 0.81 The occurrence of serious adverse events calculated according to the intent-to-treat principle was 5.1% (4/79), 3.6% (3/83), and 5.0% (4/80) in the placebo, 30 μg YIC and 60 μg YIC groups respectively (p>0.05). Ten of these were males, while one was female. The age of patients with SAE varied between 21 and 41years of age. Four occurred after the first injection (2 in placebo group, 2 in 60 μg YIC group), two occurred after the second injection (1 in placebo group, 1 in 30 μg YIC group), one occurred after 5 injections (in 30 μg YIC group), and the other four appeared after 6 injections (1 in placebo group, 1 in 30 μg YIC group and 2 in 60 μg YIC group). All 11 patients were hospitalized and recovered after treatment without immunomodulating drugs (one patient used antiviral treatment). No deaths were observed during the study period. BODY.DISCUSSION: In this study, statistical significant differences in the preset primary and secondary endpoints among the three groups of patients were not reached at the end of treatment or of follow up. Nevertheless, patients immunized with intramuscular injections of 60 μg YIC showed the highest rates of HBeAg loss (23.1%), HBeAg seroconversion (21.8%) and suppression of HBV DNA (37.2%) at the end of follow-up (44 weeks). When one of the primary endpoints (HBeAg) seroconversion, was compared between 60 μg YIC and placebo groups at the end of follow-up, statistical significance was observed (p = 0.03). Interestingly, these rates in 60 μg YIC group at 44 weeks markedly surpassed those achieved at 24 weeks, namely, HBeAg loss (23.1% vs 11.5%), HBeAg seroconversion (21.8% vs7.7%) and suppression of HBV DNA (37.2% vs26.9%) (Table 2). An important difference between YIC as an active immunotherapeutic vaccine versus passive immunotherapies such as using interferon, thymosin et al for treatment, is that, active immunotherapeutic approach functions through inducing immune responses in the patients; while in passive immunotherapeutic approaches, the immunological modulating products are repeatedly introduced into patients, and thus continuously providing the patients with the necessary immunological modulating products. The former approach needs only few injections at relatively long intervals, while the latter needs repeated injections of products to immunomodulate host immune responses. Therefore, it is not surprising, a late and sustained response versus YIC immunization was observed in a subpopulation of patients. Though the response rates with respect to the secondary endpoints in the 60 μg YIC group were comparatively higher than that of the other groups, due to the unexpected rates of HBeAg seroconversion and suppression of serum HBV DNA in the alum immunized group, statistical significance was not reached. Nevertheless, after adjusting the dependence among repeated observations made on the same patient, a significant change of response rate over the study period was detected in the placebo, 30 μg and 60 μg YIC groups, with respect to either primary or secondary endpoints (p<0.05) (Table 3). It was intriguing that only alum immunization resulted in a decrease of HBV viral load and seroconversion of HBeAg in some patients. As shown in the analysis of patients who reached secondary response at the end of follow-up, the baseline serum HBV DNA in patients who responded to alum alone immunization predominantly were those who had lower levels of serum HBV DNA (105 copies/mL). Whether this phenomenon was due to spontaneous sero-conversion in patients needs to be considered. Ideally, to include a group of patients without injections as additional control in the study may well clarify this issue. However, due to ethical concern, as well as double blinding principle, such study design has not been approved. Using the data from a previous study by Yuen et al as a reference, the one year HBeAg sero-conversion rate in patients treated with IFN-α and untreated patient was 21.1% and 2.2% [25]. In another clinical trial of lamivudine treatment, at the end of one year, the HBeAg sero-conversion rate was 8.3% in the treated group [26]. These studies suggested that the 21.8% of HBeAg seroconversion rate in Chinese patients observed in this study most likely was not due to spontaneous sero-conversion. Besides, by pair-wise comparison, significance of HBeAg seroconversion rate was only found between the 60 μg YIC and the placebo groups, other than between the 30 μg YIC and the placebo groups, which further supported the previous finding was not due to spontaneous seroconversion. As for the 10 patients in the 60 μg YIC immunized group who only reached primary responses at the end of treatment, attained secondary responses during follow-up, could be due to a late response to YIC immunization, however, spontaneous HBeAg sero-conversion should be excluded. Recently, it was reported that immune complex-loaded dendritic cells were superior to soluble immune complexes as an anti-tumor vaccine in animals [27], Furthermore, antigen-antibody immune complexes were reported to empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo [28]. These studies strengthened our confidence in using immune complexes as a therapeutic vaccine for persistent infections. Compared to passive immunotherapy, we consider active immunization by YIC is a simple-to-use, less expensive and promising therapeutic vaccine in a subpopulation of chronic hepatitis B patients. Regarding the effect of alum, it was reported that alum alone can promote B cell activation in mice, which could bypass the priming effect needed for B cell responses [29]. Whether alum also can induce T cell responses is still under discussion [30]. One may speculate that immunization with alum alone activated the B cells in some chronic hepatitis B patients and when these B cells came across the circulating HBsAg in these patients, a low level of immune responses to HBsAg might be triggered and eventually could lead to decrease in virus load, and /or sero-conversion of HBeAg. Patients who developed severe adverse events were distributed almost equally in the three groups. It was surprising that three patients immunized with only alum developed severe adverse events, and two of them even developed severe adverse events after only one injection. The severe adverse events in these two patients were verified by highly elevated ALT levels attaining 937 U/L and 818 U/L with TB 111.6 μmol/L and 54.8 μmol/L respectively. In the forthcoming phase III clinical trial, not only the therapeutic efficacy of YIC should be evaluated, but also severe adverse events will be closely monitored and investigated. BODY.SUPPORTING INFORMATION: Protocol S1Trial Protocol.(0.49 MB DOC)Click here for additional data file. Checklist S1CONSORT Checklist.(0.06 MB DOC)Click here for additional data file.

TITLE: Evaluation of Anesthesia Profile in Pediatric Patients after Inguinal Hernia Repair with Caudal Block or Local Wound Infiltration ABSTRACT.AIM:: The aim of this study is to evaluate anesthesia and recovery profile in pediatric patients after inguinal hernia repair with caudal block or local wound infiltration. ABSTRACT.MATERIAL AND METHODS:: In this prospective interventional clinical study, the anesthesia and recovery profile was assessed in sixty pediatric patients undergoing inguinal hernia repair. Enrolled children were randomly assigned to either Group Caudal or Group Local infiltration. For caudal blocks, Caudal Group received 1 ml/kg of 0.25% bupivacaine; Local Infiltration Group received 0.2 ml/kg 0.25% bupivacaine. Investigator who was blinded to group allocation provided postoperative care and assessments. Postoperative pain was assessed. Motor functions and sedation were assessed as well. ABSTRACT.RESULTS:: The two groups did not differ in terms of patient characteristic data and surgical profiles and there weren't any hemodynamic changes between groups. Regarding the difference between groups for analgesic requirement there were two major points - on one hand it was statistically significant p < 0.05 whereas on the other hand time to first analgesic administration was not statistically significant p = 0.40. There were significant differences in the incidence of adverse effects in caudal and local group including: vomiting, delirium and urinary retention. ABSTRACT.CONCLUSIONS:: Between children undergoing inguinal hernia repair, local wound infiltration insures safety and satisfactory analgesia for surgery. Compared to caudal block it is not overwhelming. Caudal block provides longer analgesia, however complications are rather common. BODY.INTRODUCTION: Prophylactic analgesia with local anesthetics is an attractive concept, especially in pediatric practice, because the evaluation of pain can be very challenging in young children [1]. In contrast to opioids, local anesthetics can be administered safely, and in recent guidelines regional anesthesia is accepted as the cornerstone of post-operative pain relief in the pediatric patients [2]. Although regional anesthesia holds a good safety record overall [3], the global experience with pediatric regional anesthesia is still quite low; even the most commonly performed procedure, caudal block, represents only 2.5% of all central neuraxial blocks performed [4]. Determining the risk-benefit ratio is rather difficult for techniques that are relatively rarely performed. Wound infiltration can produce reliable analgesia for superficial skin surgery. Infiltration itself is extensively used by pediatricians, surgeons and emergency physicians for skin laceration repair or minor superficial surgery [1]. Several studies have compared the local anesthesia so far, including: ilioinguinal and iliohypogastric nerve block plus subcutaneous injection by the surgeon against the caudal anesthesia [5-7]. But, to our knowledge there is still no study comparing the local wound infiltration by itself and caudal anesthesia. The aim of this study was to evaluate anesthesia and recovery profile in pediatric patients after inguinal hernia repair with caudal block (CB) or local wound infiltration (LWI). BODY.METHODS: The study was approved by the Institutional Review Board of and was registered at Clinical Trials.gov (registration number: NCT02620566). This randomized double-blinded study was conducted at a single tertiary medical centre ("Mother Teresa") in Skopje, Republic of Macedonia between September and December 2015. After obtaining written information consent from parents, we enrolled a total of 80 children aged 6 months to 7 years of ASA physical status I or II, undergoing unilateral hernia repair. Exclusion criteria included a history of developmental delay or mental retardation, type I diabetes, known or suspected coagulopathy, known allergy to any local anesthetic, known congenital anomaly of the spine, or signs of spinal anomaly or infection at the sacral or inguinal region. For all the patients included in this trial (n = 80), a standardized anesthetic protocol was used. No premedication was administered. Anesthesia was induced with 2–3 mg/kg of propofol or 8% of sevoflurane in 100% oxygen. Standard monitors including: electrocardiography, noninvasive arterial pressure, pulse oximetry, carbon dioxide, and gas analyzer were applied during induction and maintenance of anesthesia. The airway was established by using a laryngeal mask airway (LMA). Anesthesia was maintained with sevoflurane, and depth of anesthesia was adjusted accordingly with a goal of 80–120% baseline arterial pressure and 4.7–6 kPa end-tidal carbon dioxide (EtCO2). Spontaneous breathing was maintained during surgery. After completion of surgery, the LMA was removed, and the child was sent to a post-anesthetic care unit (PACU) so long as there was no compromise in airway or hemodynamic instability per operatively. According to the analgesic method used, all patients were allocated to two groups. Group C (caudal) (n = 40) and Group L (local wound infiltration) (n = 40). Enrolled children were randomly assigned to either Group C or Group L according to a computer-generated randomization table. For caudal blocks, Group C received 1 ml/kg of 0.25% bupivacaine (maximum volume 20 ml); Group L received 0.2 ml/kg 0.25% bupivacaine (maximum volume 4 ml) applied as local wound infiltration. An investigator who did not participate in the care of the enrolled children prepared all study medications according to group assignment. Another investigator, performed local infiltration or caudal blocks in all patients. After induction of anesthesia, children from Group C were placed in a left lateral decubitus position. After the sacral cornua and hiatus were identified, the location of needle entry site was marked. Then a 5 cm short beveled 22 G block needle was inserted into the sacral epidural space. An aspiration test was conducted to exclude intravascular placement before injection administration. The patients allocated in Group L received local infiltration of the surgical area with 0, 25% bupivacaine 10 min before skin incision. The infiltration technique was standardized as follows: the subcutaneous tissue in the proposed area was infiltrated with 0.25% bupivacaine before incision. A 16-mm 26-gauge needle was inserted in the center of the area, and the medial and lateral parts of the proposed skin incision were infiltrated. With the needle still in central position, a fan-shaped application was administered under the external abdominal fascia. Surgery was initiated ten minutes after performing the caudal block or local infiltration. The caudal block or local infiltration is considered to have failed if the patient moved his or her limbs, had an increased heart rate, had an increase in mean arterial pressure, or both of more than 15% compared with baseline during the surgery. In such instances, the patient is to be withdrawn from the study and treated with 1–2 μg/kg of fentanyl. Another investigator who was blinded to group allocation provided postoperative care and assessments. Postoperative pain was assessed using the Children's Hospital of Eastern Ontario Pain Scale (CHEOPS, 0–10) [8] and the Faces Legs Activity Cry Consolability tool (FLACC, 0– 10) [9] at 15 min, 30 min and 1, 2, and 3 h after operation. A child with a score of more than 4 on both CHEOPS and FLACC received 0.5 μg/kg of fentanyl i.v. for rescue analgesia. Motor function was assessed using the following scale: 0, no motor block; 1, able to move legs; 2, unable to move legs. Assessment of sedation was done with objective score based on eye opening: 0- spontaneously, 1- on verbal stimulation, 2- on physical stimulation. The presence of other adverse events was evaluated as well including: bradycardia, hypotension, respiratory depression, wound infection, fever, wound dehiscence, retching, vomiting, agitation, or urinary catheterization. Hypotension and respiratory depression were defined as 80% of baseline arterial pressure and ≤ 95% of pulse oxygen saturation, respectively. The decision to place a urinary catheter for urinary retention and the evaluation of micturition were made by an urologist. Analgesia on ward was provided with oral acetaminophen (15 mg/kg). The time for first supplemental oral acetaminophen demand (first acetaminophen time) was defined as the time from the end of surgery to the first registration of more than 4 on both CHEOPS and FLACC by the investigator. Twenty-four hours after surgery, reports on delayed side-effects and demands for rescue acetaminophen from the child were gathered. The investigator, who was blinded to the treatment group, documented these data with the medical records. Children were discharged from the hospital after 24 h if they met the following discharge criteria: conscious, hemodynamically stable, tolerating oral intake, voiding, walking in an appropriate manner for age, with the absence of retching, vomiting, and other side-effects [10]. Statistical analysis Statistical analysis was performed by using SPSS 17.0. Data were expressed as mean and ± standard deviation and statistically analyzed using Student's t-test, the Mann–Whitney rank-sum test and the test of Difference. A value less than 0.05 were considered as statistically significant for all tests. BODY.RESULTS: In this trial 80 patients were choose eligible for examination but for several reasons they were excluded (Fig. 1). A total of 60 subjects were enrolled in the study and six in total were excluded. Four subjects (one in Group C and three in Group L) were with inadequate caudal block/local infiltration and required additional analgesia in operation theatre. Figure 1Consort diagram. C: Caudal Group, L: Local Infiltration Group. Two subjects (one in Group C and one in Group L) were excluded because attending anesthesiologists administered propofol to treat agitation that could not be controlled by fentanyl administration. Therefore, these six subjects were all excluded from the study. The two groups did not differ in terms of patient characteristic data and surgical profiles as well as hemodynamic changes between groups (Table 1, 2). Table 1 Patient demographic data and other details (mean ± SD) Group Caudal Group Local (n = 28) (n = 26) Gender (M/F) 23/5 21/5 Age (years) 3.6 ± 1.8 3.4 ± 1.9 Weight (kg) 14.6 ±4.4 16.5 ± 4.6 Duration of Anesthesia (min) 52.7 ± 9.0 53.5 ± 8.1 Duration of surgery (min) 34.9 ± 8.2 36.8 ± 8.1 Fluids (ml) 44.6 ±11.4 49 ± 10.9 Table 2 Hemodynamic changes between groups (mean ± SD) SBP DBP HP SaO 2 Times points Local (n = 26) Caudal (n = 28) Local (n = 26) Caudal (n = 28) Local (n = 26) Caudal (n = 28) Local (n = 26) Caudal (n = 28) T0 102 ± 9.3 103.4 ± 8.3 53.1 ± 7.2 55.6 ± 4.7 138.9 ± 31.5 149.3 ± 14.3 98.3 ± 0.6 98.3 ± 0.7 T1 95 ± 9.5 94.9 ± 8.3 49.6 ± 5.9 49.7 ± 4.4 114.3 ± 22.7 118.3 ± 13.9 99.3 ± 0.4 99.5 ± 0.5 T2 95.2 ± 9.1 92.7 ± 8.3 50.1 ± 6.2 48.3 ± 5.5 114.3 ± 15.6 117.8 ± 14.0 99.4 ± 0.4 99.6 ± 0.4 T3 93.7 ± 8.9 91.6 ± 8.8 47.3 ± 6.3 47.3 ± 6.4 111.8 ± 14.6 113.1 ± 14.1 99.4 ± 0.5 99.5 ± 0.4 T4 98 ± 9.7 94.2 ± 9.2 52.2 ± 6.1 49.4 ± 7.1 119.5 ± 18.8 122.4 ± 14.3 98.8 ± 0.4 99.3 ± 0.4 To – Baseline value; T1 – After induction in anesthesia; T2 – incision; T3 – surgery; T4- end of operation; HR- Heart rate; SBP – Systolic blood pressure; DBP – Diastolic blood pressure; SaO 2 %–Peripheral oxygen saturation. The incidence of rescue fentanyl in the PACU and acetaminophen on ward was significantly lower in children who received caudal block compared to those who received local wound infiltration (Table 4). Two of 28 in the caudal group and nine of 26 in the local group received fentanyl rescue analgesia in PACU. Only one in 28 of the caudal group received acetaminophen on ward and neither one of 26 from the local group received acetaminophen on ward. The difference between groups for analgesic requirement is statistically significant p<0.05. Time to first analgesic administration was not statistically significant p=0.40. Table 3 Postoperative analgesic profile in the groups CaudalGroup Local Group (n = 28) (n = 26) No need of analgesic 25 17 N0 of subject with oral analgesic 3 9 One dose of analgesic 3 9 More than one dose of analgesic 0 0 for 24 hour Rescue fentanyl at PACU 2 9 Acetaminophen on WARD 1 0 Min 15 15 Time to first analgesic Max 840 90 requirement (min) mean ± SD 325 ± 449 55 ± 30 Vomiting 4 0 Agitation 1 0 Complications Urinary 1 0 retention Motor block 1 0 Analgesia Opoids 2 9 Acetaminophen 1 0 Table 4 Post anesthesia assessment in the groups in Post Anesthesia Care Unit Assessments Group Caudal (n = 28) Group Local (n = 26) CHEOPS 2.8 ± 1.6 4.2 ± 1.8 FLACCS 2.6 ± 1.4 3.8 ± 1.9 Post-operative sedation 0.3 ± 0.7 0.4 ± 0.4 Motor function 1.3 ± 0.5 0.0 ± 0.0 (CHEOPS = Children Hospital of Easter Ontario Pain Scale); (FLACCS= Face Legs Activity Cry Consolability scale) Pain scores using CHEOPS and FLACC assessed at the PACU were significantly lower in the caudal group. As for sedation, it was similar in both groups and motor function was better in local group; all subjects had good motor function; in caudal group one of 28 was not able to move the legs and two were able to move their legs and 26 haven't got motor dysfunction (Table 3). There were significant differences in the incidence of adverse effects in caudal and local group including: vomiting (14.29% vs. 0%), delirium (3.5% vs. 0%), and in one subject from caudal group an urologist decided on urinary catheterization (3.5% vs. 0%). Vomiting was well controlled by a single dose of antiemetic. Delirium was controlled with single dose of midazolam. Adverse effects were not noted in the Local group. The time from when the patient entered the recovery room to when they met the discharge criteria did not differ in both groups; all subjects were discharged after 24 hours. BODY.DISCUSSION: To our knowledge, this study is the first to examine the effects of local infiltration alone without ilio-inguinal and ilio-hypogastric nerve block on pain management after pediatric hernia repair surgery. We demonstrated that a single dose of local infiltrated bupivacaine 0.25% 0.2 ml/kg compared with caudal block does not reduce postoperative pain; and compared to caudal block, local wound infiltration is safe in terms of adverse events. In the review article of Martin Jöhe for regional anesthesia in neonates, infants and children, local infiltration can produce reliable analgesia and is widely used by pediatricians, surgeons and emergency physicians [1]. In the same article Jöhe mentions that ultrasound is not essential for performing a CB. On the contrary, it may make a simple procedure complicated and more prone to infection, however it can help in cases of suspected anomalies at palpation and also for teaching purposes [1]. In our study we tried to use the LWI anesthesia alone for hernia repair in children. We did not use ultrasound and failure rate was 3.3% for CB and 10% for LWI. There are studies comparing CB versus LWI but all of them include ilio-inguinal, ilio-hypogastic nerve block and local infiltration [11]. We identified 7 studies in total. Both interventions were performed after surgery in two studies [12-14]; however the other 4 studies performed caudal preoperatively and infiltration postoperatively [5, 13, 15, 16] and only one study performed both techniques preoperatively [17]. All included hernia surgeries only except for Lafferty and colleagues (only orchidopexy) [13]. All used bupivacaine in concentration of 0.25% for CB and 0.25%–0.5% for LWI. The volume ranged from 0.7 to 1.0 ml/kg (CB) and from 0.2 to 0.7 ml/kg (LWI). Only Conroy and colleagues used epinephrine along with bupivacaine [16]. Variations of the infiltration techniques involved infiltration of the wound site through the skin and infiltration of fascia or aponeurosis before closure. No study used image guidance. We performed both interventions preoperatively, CB with 1 ml/kg – 0.25% pure bupivacaine and LWI with 0.2 ml/kg – 0.25% also pure bupivacaine. Since 1992 Ejlersen compared the efficiency of pre-incision and pos-incision wound infiltration with Lidocain 1% on the postoperative pain of adult patients with inguinal herniotomy. The demand for additional analgesics occurred earlier in those who received Lidocaine infiltration after incision. The pre-incisional infiltration group also had fewer patients requiring supplemental analgesic [18]. The findings of our study and the findings of Ejlersen suggest that the inhibition of peripheral sensitization may be of major importance in impeding the development of acute pain and explain why prevention is important in handling operative pain. There are no firmly established dosage schemes for either technique and each has reasonable alternatives. A 1 ml/kg dose of bupivacaine for CB is widely employed, simple and safe [19]. The best method of local anesthesia is unknown. Varieties of techniques have been used and include wound instillation, wound infiltration and local neural blockade of ilio-inguinal, ilio-hypogastric and genitor-femoral nerves [20]. The optimal concentration of bupivacaine is not known; also which combination of local blocks are optimal needs to be determined. For this study we chose a simple, yet effective technique, which we believe to be popular and clinically relevant. There are several limitations to the present study. First we can't close the eyes of the investigator who performed the intervention, which means that potential bias exists. There is also lack of long postoperative follow-up to evaluate whether there are other late-onset complications. Number of investigated subjects is low. In conclusion, between children undergoing inguinal hernia repair, local wound infiltration insures safety and satisfactory analgesia for surgery. Compared to caudal block it is not overwhelming. Caudal block provides longer analgesia, however complications are rather common.

TITLE: Randomized Controlled Study of Metformin and Sitagliptin on Long-term Normoglycemia Remission in African American Patients With Hyperglycemic Crises ABSTRACT.OBJECTIVE: After intensive insulin treatment, many obese African American patients with new-onset diabetic ketoacidosis (DKA) and severe hyperglycemia are able to achieve near-normoglycemia remission. The optimal treatment to prevent hyperglycemic relapses after remission is not known. ABSTRACT.RESEARCH DESIGN AND METHODS: This prospective, 4-year, placebo-controlled study randomly assigned 48 African American subjects with DKA and severe hyperglycemia to metformin 1,000 mg daily (n = 17), sitagliptin 100 mg daily (n = 16), or placebo (n = 15) after normoglycemia remission. Hyperglycemic relapse was defined as fasting glucose >130 mg/dL (7.2 mmol/L) and HbA1c >7.0% (53 mmol/mol). Oral glucose tolerance tests were conducted at randomization and at 3 months and then every 6 months for a median of 331 days. Oral minimal model and incremental area under the curve for insulin (AUCi) were used to calculate insulin sensitivity (Si) and β-cell function, respectively. Disposition index (DI) was calculated as a product of Si and incremental AUCi. ABSTRACT.RESULTS: Relapse-free survival was higher in sitagliptin and metformin (P = 0.015) compared with placebo, and mean time to relapse was significantly prolonged in the metformin and sitagliptin groups compared with the placebo group (480 vs. 305 days, P = 0.004). The probability of relapse was significantly lower for metformin (hazard ratio 0.28 [95% CI 0.10–0.81]) and sitagliptin (0.31 [0.10–0.98]) than for placebo. Subjects who remained in remission had a higher DI (P = 0.02) and incremental AUCi (P < 0.001) than those with hyperglycemia relapse without significant changes in Si. ABSTRACT.CONCLUSIONS: This study shows that near-normoglycemia remission was similarly prolonged by treatment with sitagliptin and metformin. The prolongation of remission was due to improvement in β-cell function. BODY.INTRODUCTION: With intensive insulin treatment, more than half of obese African American patients with new-onset, unprovoked diabetic ketoacidosis (DKA) and severe hyperglycemia achieve near-normoglycemia remission from insulin (1–3). Unlike patients with type 1 diabetes, patients with DKA have a low prevalence of pancreatic autoantibodies (4–8). Near the presentation of DKA and severe hyperglycemia, these patients have defects in insulin secretion and insulin action (1,9–11). After intensive insulin treatment, many patients exhibit improved pancreatic β-cell function and insulin sensitivity (Si) and discontinue insulin therapy (near-normoglycemia remission [HbA1c <7%]) in favor of oral antidiabetic medications (2,10,12). Upon discontinuation of insulin, the period of near-normoglycemia may last for several months to years (10,12,13). Despite significant improvement in insulin secretion and insulin action at the time of remission from insulin (1,14,15), many patients experience recurrence of hyperglycemia if treated with diet alone (10). Few studies have focused on the optimal treatment to prolong the period of near-normoglycemia remission in obese African American patients with DKA and severe hyperglycemia. Sulfonylureas have been shown to maintain remission for ∼16 months compared with diet alone (12,16). However, sulfonylureas increase the risk of hypoglycemia (17), which may be especially detrimental in these patients during the period of near-normoglycemia remission. A small observational study by Low et al. (18) in obese pediatric and adolescent patients with DKA reported that metformin given shortly after presentation improves glycemic control and prevents readmissions for DKA; however, most patients continue to require insulin therapy during follow-up. Sitagliptin is a dipeptidyl peptidase-4 inhibitor recommended as monotherapy for the treatment of type 2 diabetes (17). It prolongs the half-life of incretin hormones GLP-1 and gastric inhibitory polypeptide, which in turn potentiate glucose-mediated insulin release from the β-cell (19). Metformin is recommended as first-line oral therapy for the treatment of type 2 diabetes (17) and has been shown to be effective in preventing the progression to diabetes in high-risk populations (20). However, no prospective randomized controlled studies have investigated whether the use of metformin or sitagliptin is efficacious in prolongation of near-normoglycemia remission in obese African Americans with new-onset DKA and severe hyperglycemia. Therefore, we tested the hypothesis that treatment with metformin and sitagliptin monotherapy compared with placebo allows for a longer period of near-normoglycemia remission after discontinuation of insulin therapy. We also tested whether prolongation of near-normoglycemia is mediated through improvement in insulin secretion and action. BODY.RESEARCH DESIGN AND METHODS.STUDY SUBJECTS: This study was a single-blind randomized placebo controlled trial conducted at Grady Memorial Hospital and Emory University Hospital in Atlanta, Georgia, between September 2009 and December 2013. The study was approved by the institutional review board of Emory University. Patients were included if they were overweight or obese (BMI ≥28 kg/m2), of African American ancestry, between the ages of 18 and 65 years and had new-onset, unprovoked DKA or severe hyperglycemia. Unprovoked DKA was defined as any known lack of precipitant. Patients were excluded if they had contraindications to metformin or sitagliptin; had a history of pancreatitis, moderate or severe congestive heart failure, or significant anemia; were pregnant; or were unable to consent. DKA was defined as blood glucose >250 mg/dL (13.9 mmol/L), pH <7.30, bicarbonate <18 mmol/L, and positive ketonemia defined as a β-hydroxybutyrate >3 mmol/L. Severe hyperglycemia was defined as a blood glucose >400 mg/dL (22.2 mmol/L) with pH ≥7.30 and bicarbonate ≥18 mmol/L and without ketonemia (β-hydroxybutyrate ≤3 mmol/L) (21). BODY.RESEARCH DESIGN AND METHODS.STUDY PROTOCOL: Informed consent was obtained from all subjects between December 2009 and April 2013. Randomization occurred between April 2010 and July 2013, and the study was stopped in December 2013 for all subjects. Eighty-eight subjects were consented and assessed in the Grady Memorial Hospital Clinical Research Unit within 3 days of discharge after resolution of DKA and severe hyperglycemia. Anthropometric measures, family history, glucose, HbA1c, C-peptide, and GAD antibody levels were measured at initial assessment. After discharge, all subjects were treated with intensive subcutaneous insulin to target fasting and premeal blood glucose between 70 and 130 mg/dL (3.9–7.2 mmol/L) during the next 12 weeks. To achieve target blood glucose, insulin was titrated every 2 weeks based on fingerstick glucose levels and through phone calls by the study team. Subjects were considered to be in near-normoglycemia remission if they were able to be off subcutaneous insulin for ≥1 week and have all fasting blood glucose measures <130 mg/dL (7.2 mmol/L) and/or HbA1c measures <7% (53 mmol/mol). In the subjects who achieved near-normoglycemia remission, HbA1c and fasting glucose levels were measured within 1 week after discontinuing insulin. Some subjects were able to achieve and maintain fasting blood glucose levels <130 mg/dL (7.2 mmol/L) and random blood glucose levels <180 mg/dL (10 mmol/L) after discontinuation of insulin for at least 1 week before 3 months from enrollment in the study. These subjects were considered to be in near-normoglycemia remission even if they had an HbA1c >7% (53 mmol/mol) and were randomized. Subjects who did not achieve fasting blood glucose levels <130 mg/dL (7.2 mmol/L) or random blood glucose levels <180 mg/dL (10 mmol/L) or HbA1c <7% (53 mmol/mol) or needed insulin at 12 weeks after diagnosis were considered as failing to wean from insulin and were not randomized. At 12 weeks from enrollment, 19 subjects were unable to discontinue insulin, 18 were lost to follow-up, and 3 withdrew from the study. The 48 subjects who achieved near-normoglycemia remission were randomly assigned to sitagliptin 100 mg daily, metformin 1,000 mg daily, or placebo (one tablet daily) after 1 week of discontinuation of insulin therapy. After randomization (month 0), subjects were initially followed at the Grady Diabetes Clinic every 4 weeks until 3 months and then every 3 months until 27 months or until they experienced hyperglycemia relapse while on oral medications. Hyperglycemia relapse was defined as fasting blood glucose ≥130 mg/dL (7.2 mmol/L), a random blood glucose measure of ≥180 mg/dL (10 mmol/L) for 2 consecutive days, or an HbA1c ≥7% (53 mmol/mol). All randomized subjects underwent a modified oral glucose tolerance test (OGTT) at 0, 3, 9, 15, 21, and 27 months from randomization or if they had a hyperglycemia relapse. During each study visit, subjects also received diet counseling on the plate method for meal planning (22) and encouraged to exercise at least three times a week for a minimum of 30 min/session. BODY.RESEARCH DESIGN AND METHODS.MEDICATIONS: Sitagliptin and placebo were provided by Merck & Co. (Kenilworth, NJ). Metformin was obtained from the research pharmacy at Grady Memorial Hospital. All study medications dispensed to the subjects were labeled as study drug. Merck & Co. had no involvement in the study design, analyses, or writing of the article. BODY.RESEARCH DESIGN AND METHODS.MODIFIED OGTT: After an 8- to 10-h overnight fast, all subjects were admitted to the Grady Memorial Hospital Clinical Research Unit between 8:00 and 10:00 a.m. An antecubital intravenous line was placed. After resting for 30 min, blood was drawn for fasting glucose and insulin levels. A 75-g oral glucose load was administered over 1 min. Blood draws for glucose and insulin levels were performed at 15, 30, 60, 90, and 120 min. Glucose and insulin levels were assessed at additional time points during the OGTT (15, 30, 60, 90 min) for calculations of Si and β-cell function from OGTT-derived measures. BODY.RESEARCH DESIGN AND METHODS.MEASURED OUTCOMES AND CALCULATIONS: Si and β-cell function were calculated by using OGTT-derived measures. Whole-body Si was assessed by the oral minimal model. The original oral minimal model analysis was developed with a 22-point 300-min OGTT (23). A subsequent study validated the 22-point OGTT with a 7-point 120-min OGTT (24). Our modified OGTT contained six time points. The model fit using the 6-point OGTT was similar to the 120-min 7-point OGTT. Therefore, we calculated Si by using glucose and insulin levels from 6-point OGTTs on the basis of the oral minimal model. Pancreatic β-cell function was calculated from the incremental area under the curve for insulin (AUCi). For calculation of incremental AUCi, 15-, 30-, 60-, 90-, and 120-min time point insulin levels were subtracted from fasting insulin levels, and incremental AUCi was calculated with use of the trapezoidal method (25). Disposition index (DI) was calculated as the product of Si from the oral minimal model and incremental AUCi. BODY.RESEARCH DESIGN AND METHODS.ANALYTIC TECHNIQUES: Assays for glucose, insulin, and C-peptide were performed at the Endocrinology/Lipoprotein Laboratory of the University of Tennessee Health Science Center. Glucose levels were analyzed by the hexokinase method (Beckman-Coulter, Los Angeles, CA). Insulin and C-peptide levels were measured by two-site sequential chemiluminescent immunometric assays as previously described (26). HbA1c and GAD antibody tests were measured at the central laboratory at Grady Memorial Hospital. BODY.RESEARCH DESIGN AND METHODS.STATISTICAL ANALYSES: The primary aim of the study was to compare hyperglycemia relapse-free survival while on oral medications between the randomized groups. The secondary aims were to compare the effects of metformin and sitagliptin on β-cell function and Si compared with placebo. We also compared changes in Si and β-cell function between subjects with a hyperglycemia relapse and those who remained in near-normoglycemia remission and between subjects with initial presentation of DKA and severe hyperglycemia. For the subjects who were lost to follow-up or who withdrew from the study, values from the last documented visit were used in analyses. This was an intention-to-treat analysis. Cox proportional hazards and log-rank tests adjusted for age were used to compare rates of hyperglycemia relapse-free survival among the metformin, sitagliptin, and placebo groups. Because of the low number of subjects with hyperglycemia relapse and censoring in the medication groups, we calculated restricted mean survival time to estimate time to hyperglycemia relapse between the combined medication and placebo groups (27,28). On the basis of the normality of the data, continuous variables were compared using ANOVA or Kruskal-Wallis test for three-group comparisons. Student t or Mann-Whitney U tests were used for two-group comparisons. Categorical data were compared using χ2 or Fisher exact test. Repeated-measures ANOVA was used to compare changes in DI, incremental AUCi, and Si over the study period. All data are expressed as mean ± SD unless stated otherwise. Statistical analyses were performed using SAS 9.2 software (SAS Institute, Cary, NC). Based on our previous data, we expected that 70% of obese African American patients with new-onset DKA and/or severe hyperglycemia will achieve near-normoglycemia remission (1,12). At the time of study design, there were no previously published studies in obese African Americans to determine the effect size needed to detect differences in hyperglycemia relapse-free survival. Based on our previous experience, we initially calculated that we would be able to recruit 90 subjects over a 2-year period. Accounting for a 25% attrition rate and 30% failure to wean from insulin, we calculated that we would be able to enroll 48 subjects in the study. We enrolled 88 patients from December 2009 to April 2013. BODY.RESULTS: Forty-eight African American subjects with DKA (n = 22) and severe hyperglycemia (n = 26) were included in the study. Seventeen subjects were randomly assigned to metformin 1,000 mg daily, 16 to sitagliptin 100 mg daily, and 15 to placebo. Four subjects in the metformin group, 6 in the sitagliptin group, and 1 in the placebo group were lost to follow-up. One subject in the sitagliptin group and one subject in the placebo group withdrew from the study. The overall median follow-up after insulin discontinuation was 331 days (interquartile range 102, 612 days) with no differences between randomized groups (Table 1). The subjects lost to follow-up or who withdrew from the study were in near-normoglycemia remission during their last documented study visit. There were no significant differences in baseline characteristics at presentation between subjects who withdrew and those who stayed in the study, except for HbA1c. HbA1c was lower at presentation of DKA/hyperglycemia in subjects who withdrew compared with those who stayed (12.0 ± 2.3% [107 ± 25 mmol/mol] vs. 13.5 ± 2.0% [124 ± 22 mmol/mol], P = 0.03) but was similar at randomization. Table 1 Clinical characteristics of obese African American subjects with DKA and severe hyperglycemia Metformin ( n = 17) Sitagliptin ( n = 16) Placebo ( n = 15) P value Sex ( n ) 0.70  Male 11 11 8  Female 6 5 7 Age (years) 48 ± 9 50 ± 11 46 ± 13 0.73 At diagnosis of diabetes  BMI (kg/m 2 ) 35.0 ± 4.3 37.3 ± 10.0 34.9 ± 5.2 0.96  DKA/severe hyperglycemia ( n ) 7/10 10/6 5/10 0.26  Family history of type 2 diabetes (%) 71 88 80 0.56  FBG 0.49   mmol/L 40.8 ± 13.5 41.7 ± 18.7 43.7 ± 12.2   mg/dL 735 ± 243 750 ± 336 787 ± 219  GAD antibody positivity [ n (%)] 2 (12) 0 (0) * 3 (23) * 0.20  HbA 1c 0.85   % 13.1 ± 2.0 13.1 ± 2.5 13.1 ± 2.3   mmol/mol 120 ± 21 120 ± 27 120 ± 25  Fasting C-peptide (pg/L) 2.8 ± 1.1 3.4 ± 1.2 3.1 ± 1.2 0.43 At randomization  FBG 0.21   mmol/L 6.3 ± 1.0 6.2 ± 0.9 7.0 ± 1.3   mg/dL 114 ± 18 111 ± 16 125 ± 24  HbA 1c 0.28   % 6.2 ± 0.9 6.5 ± 0.7 6.6 ± 0.5   mmol/mol 44 ± 10 48 ± 8 49 ± 6  Insulin dose (units/kg/day) 0.5 ± 0.2 0.6 ± 0.3 0.8 ± 0.4 0.08  Length of insulin use (weeks) 9.5 ± 3.0 7.6 ± 3.2 9.2 ± 2.7 0.14  ΔWeight from enrollment (kg) 1.8 (−1.0, 5.9) −0.1 (−3.5, 5.1) −4.6 (−6.3, 0.5) 0.08 At end of study—all subjects  Duration of treatment † (days) 472 (242, 716) 194 (92, 613) 194 (91, 579) 0.19  ΔWeight from randomization (kg) 0 (−3.6, 3.5) 3.3 (−0.7, 6.0) 1.4 (−2.2, 2.9) 0.10  FBG 0.10   mmol/L 6.7 ± 1.2 6.9 ± 1.5 8.5 ± 3.2   mg/dL 121 ± 22 124 ± 27 153 ± 58  HbA 1c 0.04   % 6.4 ± 1.2 6.6 ± 1.1 7.6 ± 1.6   mmol/mol 46 ± 13 48 ± 12 55 ± 22 Near-normoglycemia remission ( n ) 12 12 4  End-of-study FBG 0.04   mmol/L 6.2 ± 1.1 6.1 ± 0.4 7.4 ± 0.8   mg/dL 112 ± 19 110 ± 7 134 ± 15  End-of-study HbA 1c 0.80   % 5.8 ± 0.7 6.1 ± 0.7 6.1 ± 0.5   mmol/mol 40 ± 8 43 ± 8 43 ± 6 Hyperglycemia relapse ( n ) 5 4 11  End-of-study FBG 0.51   mmol/L 7.9 ± 0.4 9.1 ± 1.3 8.9 ± 3.8   mg/dL 142 ± 7 163 ± 23 160 ± 68  End-of-study HbA 1c 0.94   % 8.0 ± 0.9 8.0 ± 0.9 8.2 ± 1.5   mmol/mol 64 ± 10 64 ± 10 66 ± 16 Data are mean ± SD or median (interquartile range) unless otherwise indicated. FBG, fasting blood glucose. *Missing GAD antibody levels for one subject in the sitagliptin group and two subjects in the placebo group. †Median time of follow-up after randomization and insulin discontinuation. At presentation of DKA and severe hyperglycemia, there were no differences in age and BMI among the metformin, sitagliptin, or placebo groups (Table 1). Although not statistically significant, both the sitagliptin and metformin groups had more men than women, whereas the placebo group had similar proportions of men and women. In the proportion of subjects with DKA or severe hyperglycemia, length and dose of insulin use before randomization (time to near-normoglycemia remission) did not differ between groups. At randomization, there were no significant changes in weight or differences in fasting glucose or HbA1c levels. At the end of the study, there was a significant difference in HbA1c (P = 0.04) between the groups (Table 1). In the patients who remained in near-normoglycemia remission, there was a significant difference in fasting glucose at the end of the study (Table 1). There were no differences at diagnosis of diabetes in the subjects who remained in near-normoglycemia remission compared with those with hyperglycemia relapse at presentation (Table 2). At randomization, fasting glucose levels were higher in subjects who experienced hyperglycemia relapse than in those who remained in remission (Table 2). At the end of the study, fasting glucose and HbA1c levels were higher in the subjects who experienced hyperglycemia relapse (Table 2). Table 2 Clinical characteristics of obese African American patients with DKA and severe hyperglycemia with near-normoglycemia remission compared with those with hyperglycemia relapse Near-normoglycemia remission ( n = 28) Hyperglycemia relapse ( n = 20) P value Sex ( n ) 0.38  Male 19 11  Female 9 9 Age (years) 48 ± 10 49 ± 12 0.73 At diagnosis of diabetes  BMI (kg/m 2 ) 35.2 ± 5.0 36.6 ± 9.0 0.96  DKA/severe hyperglycemia ( n ) 15/13 7/13 0.24  Family history of type 2 diabetes (%) 82 90 0.68  FBG 0.95   mmol/L 42.5 ± 16 41.4 ± 13.2   mg/dL 765 ± 288 745 ± 239  GAD antibody positivity † [ n (%)] 2 (7) 3 (17) 0.64  HbA 1c 0.75   % 13.2 ± 2.2 13.0 ± 2.2   mmol/mol 121 ± 24 119 ± 24  Fasting C-peptide (pg/L) 3.3 ± 1.1 2.8 ± 1.3 0.25 At randomization  FBG 0.007   mmol/L 6.1 ± 0.9 7.0 ± 1.1   mg/dL 110 ± 17 126 ± 20  HbA 1c 0.14   % 6.3 ± 0.8 6.7 ± 0.5   mmol/mol 49 ± 12 60 ± 16  Insulin dose (units/kg/day) 0.6 ± 0.2 0.7 ± 0.4 0.67  Length of insulin use (weeks) 8.6 ± 3.1 9.1 ± 2.9 0.57  ΔWeight from enrollment (kg) −0.4 (−6.9, 1.8) 0.8 (−5.4, 4.5) 0.24 At end of study  ΔWeight from randomization (kg) −0.4 (−2.5, 3.1) 2.1 (−0.4, 5.6) 0.11  FBG <0.0001   mmol/L 6.3 ± 0.9 8.7 ± 2.8   mg/dL 114 ± 17 156 ± 50  HbA 1c <0.0001   % 6.0 ± 0.7 8.1 ± 1.2   mmol/mol 42 ± 8 65 ± 13 Data are mean ± SD or median (interquartile range) unless otherwise indicated. FBG, fasting blood glucose. †Missing GAD antibody levels for one subject in the near-normoglycemia remission group and two subjects in the hyperglycemia relapse group. Hyperglycemia relapse-free survival was significantly higher in the metformin and sitagliptin groups than in the placebo group (P = 0.015) (Fig. 1). The 2-year failure rate was higher in the placebo than in the sitagliptin (77% vs. 44%, P = 0.113) or metformin (77% vs. 34%, P = 0.013) groups. Compared with placebo, patients randomized to metformin (hazard ratio 0.28 [95% CI 0.10–0.814]) and sitagliptin (0.31 [0.10–0.98]) were ∼70% less likely to have a hyperglycemia relapse. However, there was no difference in hyperglycemia relapse-free survival between metformin and sitagliptin (P = 0.75) (Fig. 1). The restricted mean time to hyperglycemia relapse in the combined metformin and sitagliptin groups was significantly higher than placebo (480 vs. 305 days, P = 0.004). Figure 1Cox proportional hazards of failure-free survival among metformin, sitagliptin, and placebo in obese African American patients presenting with DKA and severe hyperglycemia. A significant difference was found among the placebo, metformin, and sitagliptin groups (P = 0.015), but no significant difference was found between the sitagliptin and metformin groups (P = 0.75). We also assessed whether being on medication decreased the severity of hyperglycemia relapse. Because only a small number of subjects experienced relapse in the medication groups (metformin [n = 5], sitagliptin [n = 4]) compared with placebo (n = 11), we combined the metformin and sitagliptin groups. There were no significant differences in HbA1c (8.0 ± 0.8% [64 ± 9 mmol/mol] vs. 8.2 ± 1.5% [66 ± 16 mmol/mol], P = 0.79) or blood glucose (152 ± 18 mg/dL [8.4 ± 1 mmol/L] vs. 160 ± 68 mg/dL [8.9 ± 3.8 mmol/L], P = 0.65) levels at the time of hyperglycemia relapse between the placebo group and the combined metformin and sitagliptin group. In the placebo group, 3 of the 11 subjects who experienced hyperglycemia relapse presented to the emergency department with a glucose level >400 mg/dL (22.2 mmol/mol) or DKA. None of the patients in the metformin and sitagliptin group had a hyperglycemia relapse necessitating a visit to the emergency department or admission to the hospital. The difference in hyperglycemia relapse was explained by improvements in β-cell function. Over the course of the study, DI (P = 0.02) and incremental AUCi (P < 0.001) were significantly higher in subjects who remained in near-normoglycemia remission compared with those who had a hyperglycemia relapse without any differences in Si (P = 0.75). There was a significant interaction between remission status and study visit for both Si (P = 0.01) and DI (P = 0.02), suggesting a different pattern of change in Si and DI over time between subjects with near-normoglycemia remission and hyperglycemia relapse. The difference in DI and incremental AUCi was not present at randomization in the subjects who stayed in near-normoglycemia remission compared with those who experienced a hyperglycemia relapse (Fig. 2A and B). At the last documented follow-up, there were no differences in Si (Fig. 2C); however, DI (P = 0.02) and incremental AUCi (P < 0.001) were significantly higher in subjects who stayed in near-normoglycemia remission than in those who had a hyperglycemia relapse at the end of the study (Fig. 2A and B). Comparison across treatment groups showed no differences at randomization, over the course of the study, or at the last follow-up for DI, incremental AUCi, or Si (data not shown). Similarly, a comparison of subjects with an initial presentation of DKA and severe hyperglycemia showed no differences in Si, incremental AUCi, or DI at randomization, over the course of the study, or at the last follow-up (data not shown). Although there were no significant changes in weight, we performed analyses adjusting for changes in weight throughout the course of the study. No differences were found in Si, incremental AUCi, or DI compared with unadjusted analyses. Figure 2DI, incremental AUCi, and Si at randomization and last follow-up. At randomization OGTT, no significant differences were found in DI, incremental AUCi, or Si in subjects who remained in near-normoglycemia remission at the end of the study compared with those with hyperglycemia relapse. During the last follow-up OGTT, subjects who remained in remission had higher DI and incremental AUCi than those who had a relapse (A and B) without a difference in Si (C). BODY.CONCLUSIONS: To our knowledge, this is the first randomized controlled longitudinal study to determine the efficacy of metformin and sitagliptin in avoiding recurrence of hyperglycemia in African American subjects with DKA and severe hyperglycemia. The study shows that both metformin and sitagliptin significantly prolong near-normoglycemia remission in subjects with both DKA and severe hyperglycemia and that these subjects are ∼70% less likely to experience hyperglycemia relapse than those taking placebo. The prolongation of near-normoglycemia remission was due to improvement in insulin secretion in subjects who remained in remission compared with those who experienced hyperglycemia relapse as shown by the incremental AUCi. Previous studies by our group (1) and others (6,10,13) have shown that subjects with DKA and severe hyperglycemia experience significant improvement in β-cell function after 8–12 weeks of intensive treatment, which allowed for discontinuation of insulin in ∼70% (1). The period of near-normoglycemia remission is variable, with some studies reporting remission lasting between 6 and 120 months (10,13). Despite the initial improvement, most obese African American patients with DKA and severe hyperglycemia (12,16) have a gradual decline in their β-cell function (10) with continued insulin resistance (15) if treated with diet alone. Previous studies with sulfonylureas showed prolongation of near-normoglycemia remission in obese African American patients with an initial presentation of DKA and severe hyperglycemia (12,16), but sulfonylurea treatment may increase the risk for hypoglycemia and weight gain. In the current study, we significantly prolonged near-normoglycemia with metformin and sitagliptin in subjects with an initial presentation of both DKA and severe hyperglycemia without significant changes in weight. In addition, in the subjects who experienced hyperglycemia relapse, those taking metformin and sitagliptin had a milder presentation of relapse than those taking placebo. Prevention of hyperglycemia relapse was through improvement in β-cell function. In this study, subjects who remained in near-normoglycemia remission had a significant improvement in insulin secretion compared with those who experienced hyperglycemia relapse. There were no significant changes in Si between subjects in remission and those with hyperglycemia relapse. This finding is consistent with previous studies in obese African Americans with DKA and severe hyperglycemia (2,10) and in patients with type 2 diabetes from the UK Prospective Diabetes Study, which showed that deteriorating β-cell function rather than insulin resistance is the primary reason for deterioration of glucose control (29). The current findings are also similar to those of reports in subjects with newly diagnosed type 2 diabetes in whom intensive therapy and tight glucose control resulted in significant improvement in β-cell function (30,31). There are several limitations to this study. Subjects lost to follow-up or who withdrew from the study had a lower HbA1c at presentation with DKA and severe hyperglycemia and were in remission at the last documented study follow-up. Most of these subjects were also in the sitagliptin and metformin groups. Therefore, withdrawal of these subjects could have underestimated changes in insulin secretion and Si in the metformin and sitagliptin groups. At the time of study design, no data were available on the effect sizes needed to detect differences in long-term hyperglycemia-free survival. We designated the number needed based on feasibility of recruitment over the study period. Therefore, the study was likely underpowered to detect mechanistic differences contributing to near-normoglycemia remission between treatment groups as well as between subjects with DKA and severe hyperglycemia. Another limitation was that subject follow-up was variable and that the study was stopped in December 2013 irrespective of when subjects were recruited. Therefore, subjects recruited later were followed for a shorter period than those recruited earlier. If all subjects had equivalent follow-up periods, it may have been possible to discern significant differences in Si or insulin secretion between randomized groups. Of note, insulin doses at randomization in the metformin and sitagliptin groups trended lower than those in the placebo group. In addition, fasting glucose levels at randomization were lower in subjects who stayed in near-normoglycemia remission than in those who experienced hyperglycemia relapse. However, there were no differences in Si, incremental AUCi, or DI between the groups at randomization, suggesting that the lower insulin needs in the metformin group and fasting blood glucose levels in the subjects who stayed in near-normoglycemia remission were likely not the reason for the sustained near-normoglycemia remission. In summary, this study showed that near-normoglycemia remission is prolonged by monotherapy with either metformin or sitagliptin in obese African American patients with DKA and severe hyperglycemia. The study also showed that near-normoglycemia can be explained by changes in β-cell function. We did not find any differences in Si or β-cell function between subjects with an initial presentation of DKA and severe hyperglycemia. Because there were no differences in prolongation of near-normoglycemia remission between metformin and sitagliptin, either medication can be used to prolong and maintain near-normoglycemia remission in obese African American patients with DKA and severe hyperglycemia.

TITLE: Effect of interval training program on white blood cell count in the management of hypertension: A randomized controlled study ABSTRACT.OBJECTIVE:: Elevated white blood cell (WBC) count is considered to be prospectively and positively associated with cardiovascular diseases, particularly hypertension. Also, the positive role of exercise in the management of hypertension has been well and long established. However the relationship between WBC count and hypertensive management particularly in the nonpharmacological technique is ambiguous and unclear. Therefore the purpose of the present study was to determine the effect of interval training program on WBC count and cardiovascular parameters in male hypertensive patients. ABSTRACT.MATERIALS AND METHODS:: A total of 245 male patients with mild to moderate (systolic blood pressure (SBP) between 140 mmHg and 179 mmHg and diastolic blood pressure (DBP) between 90 mmHg and 109 mmHg) essential hypertension were age matched and grouped into experimental and control groups. The experimental (n=140; 58.90±7.35 years) group involved in an 8-week interval training (60-79% HR max reserve) program of between 45 minutes to 60 minutes, while the age-matched controls hypertensive (n=105; 58.27±6.24 years) group remain sedentary during this period. Cardiovascular parameters (SBP, DBP, and VO2 max) and WBC count were assessed. Student's t and Pearson correlation tests were used in data analysis. ABSTRACT.RESULTS:: Findings of the study revealed a significant effect of the interval training program on VO2max, SBP, and DBP and WBC count at P<0.05 and VO2max is negatively related to the WBC count (r=–0.339) at P<0.01. ABSTRACT.CONCLUSIONS:: It was concluded that the interval training program is an effective adjunct nonpharmacological management of hypertension and the therapeutic effect of exercise programs may be mediated through suppression of inflammatory (WBC count) reaction. BODY.INTRODUCTION: Several prospective studies have shown a positive and independent association between white blood cell (WBC) count and coronary heart disease, hypertension, and ischemic stroke incidence and mortality.1–6 According to Lee and Associates,3 the relationship is irrespective of sex, ethnic, and smoking status. Chronic low-grade inflammation is believed to be the mechanism behind this association whereby WBC-derived macrophages and other phagocytes contribute to vascular injury, endothelial dysfunction, and atherosclerotic disease progression.7–9 However, inflammation may also contribute to increasing microvascular capillary resistance, initiation of platelet aggregation, increased cathecolamine levels, and there is considerable evidence of a link between inflammation and hypertension.29–12 Another possibility for the relationship between WBC and hypertension might be the close relationship between leucocytes count and activity of corticosteroids in hypertension and other vascular diseases. This has been reported previously in both animal models and humans.1314 The total leucocytes count and blood pressure are increased by cortisol administration in normal human subjects.15 Previous studies have shown that elevated WBC count is associated with a small, but significant increase in the risk of hypertension among white men.1016 Nakanishi and co-workers17 also found this association to be true among Japanese men irrespective of their smoking status. Previous reports18–21 show that moderate to high levels of cardiorespiratory fitness are protective against Cardiovascular disease (CVD) and all-cause mortality, even in individuals with CVD risk factors. Regular exercise induces anti-inflammatory actions. Geffken et al.22 found WBC to be inversely related to physical activity in a large sample (n=5201) composed of men and women from diverse racial backgrounds. Nieto et al.23 reported sport activity index to be inversely related to WBC in whites but not in blacks. Studies that investigated the association between WBC count and hypertension management particularly the nonpharmacological are few and the exact nature of this relationship is ambiguous and unclear.24 Therefore, the purpose of the present study was to investigate the relationship between interval training program and WBC in the noninvasive management of chronic essential hypertension BODY.MATERIALS AND METHODS.RESEARCH DESIGN: In the present study, age-matched randomized independent pretest–posttest–control group design was used to determine the influence of the interval training program on WBC count. BODY.MATERIALS AND METHODS.SUBJECTS: The population for the study was male essential hypertensive subjects attending the hypertensive clinic of Murtala Muhammed Specialist Hospital Kano Nigeria. Subjects were fully informed about the experimental procedures, risk, and protocol, after which they gave their informed consent. BODY.MATERIALS AND METHODS.INCLUSION CRITERIA: Only those who volunteered to participate in the study were recruited. Subjects between the age range of 45-70 years with chronic mild to moderate and stable (>1 year duration) hypertension (systolic blood pressure (SBP) between 140 mmHg and 179 mmHg and diastolic blood pressure (DBP) between 90 mmHg and 109 mmHg) were selected. Only those who had stopped taking antihypertensive drugs or on a single-antihypertensive medication (monotherapy) were recruited.25 They were sedentary and have no history of psychiatry or psychological disorders or abnormalities. BODY.MATERIALS AND METHODS.EXCLUSION CRITERIA: Obese or underweight (body mass index (BMI) between 20 and 30 kg/m2), smokers, alcoholic, diabetic, other cardiac, renal, respiratory disease patients were excluded. Those involved in vigorous physical activities and above averagely physically fit (VO2 max >27 and >33 ml/kg/min for over 60 and 50 years old respectively) were also excluded. A total of 323 chronic and stable, essential mild to moderate male hypertensive patients satisfied the necessary study criteria. Subjects were aged matched and randomly grouped into experimental (162) and control (161) groups. They were fully informed about the experimental procedures, risk, and protocol, after which they gave their informed consent in accordance with the American College of Sports Medicine (ACSM) guidelines, regarding the use of human subjects26 as recommended by the human subject protocol. Ethical approval was granted by the Ethical Committee/Board of Kano State Hospitals Management Board. BODY.MATERIALS AND METHODS.WASH OUT PERIOD: All subjects on antihypertensive drugs were asked to stop all forms of medication and in replaced, were given placebo tablets (consisted of mainly lactose and inert substance) in a single blind method.2728 All subjects including those not on any antihypertensive medications were placed on placebo tablets for 1 week (7 days); this is known as "wash out period." The purpose of the wash out period was to get rid of the effects of previously taken antihypertensive drugs/medications. During the wash out period all subjects were instructed to report to the hypertensive clinic for daily blood pressure monitoring and general observation. The pretest procedure was conducted at the last day of the wash out period, and in the Department of Physiotherapy of Murtala Mohammed Specialist Hospital (MMSH), Kano between 8:00 and 10:00 am. At the end of the 8-week training period, all subjects was asked to stop methyldopa (Aldomet) and subjects were prescribed with placebo tablets in a single-blinded method for 1 week in order to get rid the effect of the methyldopa taken during the training period. BODY.MATERIALS AND METHODS.PHYSIOLOGICAL MEASUREMENT: Subjects resting heart rate (HR), SBP, and DBP were monitored from the right arm as described by Walker et al.29 and Musa et al.30 using an automated digital electronic BP monitor (Omron digital BP monitor, Medel 11 EM 403c; Tokyo, Japan). These measurements were monitored between 8:00 and 10:00 am each test day. BODY.MATERIALS AND METHODS.ANTHROPOMETRIC MEASUREMENT: Subjects' physical characteristics (%body fat, weight [kg] and height [m]) and body composition (body mass index [BMI] (kg/m-2)) assessment were done in accordance with the standardized anthropometric protocol.3132 BODY.MATERIALS AND METHODS.BLOOD SAMPLE COLLECTION (VENIPUNCTURE METHOD): Both pre- and post-treatment venous blood samples were obtained between 8: 00 and 10:00 pm after about 12-hour overnight fast (fasting blood sample). A 5 ml syringe was used for blood sample collection, using the procedure described by Bachorik.33 One milliliter of blood sample was immediately transferred into a special container containing anticoagulant (heparin, 75 U/ml) for WBC count. All samples were stored in a refrigerator at –80°C until analysis.34 BODY.MATERIALS AND METHODS.STRESS TEST: The Young Men Christian Association (YMCA) submaximal cycle ergometry test protocol was used to assess subject's aerobic power.3536 The YMCA protocol uses two to four 3-minute stages of continuous exercise, two HR-power output data points will be needed (steady state HR) of between 110 and 150 beat/min. The two steady state HR were plotted against the respective workload on the YMCA graph sheet. A straight line was drawn through the two points and extended to the subjects predicted maximum HR (220 age). The point at which the diagonal line intersects the horizontal-predicted HR max line represents the maximal working capacity for the subject. A perpendicular line was dropped from this point to the baseline where the maximal physical workload capacity was read in kg/m/min which was used to predict the subjects VO2 max. This procedure was done for both pre- and post-test stress test. BODY.MATERIALS AND METHODS.TEST PROCEDURES: The test procedures were conducted in the Department of Physiotherapy of MMSH, Kano between 8:00 and 10:00 am. BODY.MATERIALS AND METHODS.TRAINING PROGRAM: Following the stress test and prior to the exercise training, all subjects in both control and interval groups were reassessed by the physicians and were prescribed with methyldopa (500 mg-1 g daily in divided doses of 2 to 4 times) based on the subject's responses and tolerance to therapy. Methyldopa was preferred because it does not alter normal hemodynamic responses to exercise,37 and it is a well-tolerated antihypertensive drug in Africa.38–40 In addition, it is the drug prescribed the most in Kano, where the study was conducted and had proved useful in the treatment of mild to moderately severe hypertension either as monontherapy or combination therapy.39–41 Subjects maintained these prescriptions with regular medical consultation and observation throughout the period of exercise training. BODY.MATERIALS AND METHODS.THE INTERVAL GROUP (GROUP 1): Subjects in the interval group exercised on a bicycle ergometer at a low intensity of between 60–79% of their HR max reserve that was estimated from 220 minus the age of a subject as recommended by ACSM.42 The starting workload was 100 kg (17 watts) which was increased at a pedal speed of 50 rpm to obtain a HR max reserve 60% was increased in the first 2 weeks to and level up at 79% HR max reserve throughout the remaining part of the training period at a work/rest ratio of 1:1of 6 minutes each. The initial of exercise session was increased from 45 minutes in the first 2 weeks of training to and leveled up at 60 minutes throughout the remaining part of the training. Exercise session of three times per week was maintained throughout the 8 weeks period of training for the interval group. BODY.MATERIALS AND METHODS.THE CONTROL GROUP (GROUP 2): Subjects in the control group were instructed not to undertake any vigorous physical activity during the 8 weeks period of the study. BODY.MATERIALS AND METHODS.WHITE BLOOD CELL COUNT: The WBC count was analyzed using the Turks method as described by Dacie and Lewis.43 BODY.MATERIALS AND METHODS.BLOOD SAMPLE COLLECTION: Immediately after the post-training wash out period, fasting blood samples were collected as earlier described. Post-training SBP, DBP, WBC count assessment, and stress test were conducted as earlier described in the pretest procedures using standardized protocols, techniques, and methods. All pre- and post-test measurements were recorded on a data sheet. A total of 257 subjects (140 from the interval, and 105 from the control groups) completed the 8 weeks training program. Seventy-eight subjects (22 from interval, and 56 from the control groups) had dropped out because of noncompliance, unfavorable responses to methyldopa, and exercise training or had incomplete data; therefore, the data of 245 subjects were used in the statistical analysis [Figure 1]. Figure 1Study design flow chat BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Following data collection, the measured and derived variables were statistically analyzed. The descriptive statistics (means and standard deviations) of the subject's physical characteristics, estimated VO2 max, WBC count, and cardiovascular parameters were determined. Analysis of covariance (ANCOVA) was used to assess the outcome variables; in the ANCOVA, the post-test values were the outcome variables and the co-covariates were the pretest values, age, baseline BMI, and % body fat. The Pearson product moment correlation test was also computed for the variables of interest; in the correlation test, the difference between subjects post-training and pretraining measurements (changed score) were used as dependent measures. All statistical analysis was performed on a Toshiba compatible microcomputer using the statistical package for the social science (SPSS), (Windows Version 16.0 Chicago, IL, USA). The probability level for all the above tests was set at 0.05 to indicate significance. BODY.RESULTS: The subject's age ranged between 45 and 70 years. The mean age, height, weight, BMI, and % body fat±SD were as follows, respectively: the interval group (58.40±6.91 years, 167.78±7.81 cm, 70.18±11.37 kg, 24.96±3.88 kg/m–2, and 17.69±6.50%) and the control group (58.27±6.24 years, 167.89±5.31 cm, 68.47±17.07 kg, 24.16±4.91 kg/m–2, and 22.27±9.82%). There was no significant difference in age between the groups (t=0.156, P=0.876). Table 1 shows pre- versus (vs.), post-treatment mean BP±SD mmHg, WBC (cells/ml3) count and VO2 max (ml/kg/min) for the exercise (SBP166.05±14.10; DBP, 96.80±3.38; WBC count 7.61±1.17, and VO2 max 23.62±9.15 vs. SBP, 150.00±16.67; DBP, 94.98±5.40; WBC count 7.57±1.26, and VO2 max 37.46±7.42) group and the control (SBP160.87±13.23; DBP, 97.17±1.43; WBC count 7.13±1.30, and VO2 max 21.23±5.76 vs. SBP, 163.47±14.88; DBP, 96.10±2.67; WBC count 8.21±1.60, and VO2 max 22.82±7.44) group. Table 1 Groups mean and standard deviation for pre- and post-test values The Table 2 ANCOVA test results indicated a significant reduction in the exercise groups over control in SBP (P=0.000), DBP (P=0.000), WBC (P=0.001), and VO2 max (P=0.000) at P<0.05. Table 2 Analysis of the covariance test between the interval and control groups The results also showed significant negative correlation Figures 2 and 3 between changes in VO2 max and changes in other parameters such as WBC count (r=–0.339); SBP (r=–0.304); and DBP (r=–0.289) at P<0.01. Changes in the WBC count positively and significantly correlated with both SBP (r=0.200) and DBP (r=0.263) [Figure 4]. Figure 2Correlation between training changes in VO2max and BP Figure 3Correlation between training changes in VO2max and WBC count Figure 4Correlation between training changes in WBC count and BP BODY.DISCUSSION: Findings from the present study revealed a significant decrease in SBP, DBP, and increase in VO2 max in the experimental group over the control group. The favorable changes resulting from aerobic training on both SBP and DBP demonstrated in the present study is consistent with previous studies.44–51 Also, the result of the present study indicated a significant reduction in the WBC count in the experimental group over control. There was a negative significant correlation between changes in VO2 max and WBC count. This finding is in agreement with the report of Kullo et al.,52 they investigated the association between WBC count and VO2 max in men without CHD. In their study, 172 asymptomatic men (age, 51±9.3 years) engaged in a symptomless graded treadmill aerobic exercise. They reported an inverse association between WBC count and VO2 max (r=–0.22, P=0.004). Another study in support of the present study was conducted by Church et al.1 in their study, they investigated 4057 men, after age-adjusted resting levels and risk of having a clinically significant elevation of the WBC count across nine fitness body fatness combinations. They reported that fitness as measured by VO2 max was inversely related to the age-adjusted values of WBC count (P for trend P<0.0001). A contradictory finding was reported by Shankar et al.24 they studied the relationship between WBC count and physical activity in the development of hypertension. They studied 2459 hypertension-free women and men participated after adjusting and stratifying by smoking and several other potential confounding factors. They reported a nonsignificant effect of moderate physical activity of twice per week on the WBC count (P=0.06). Kim et al.53 investigated the relationship between WBC count and cardiorespiratory fitness (VO2 max) after adjusting for several well-known cardiovascular risk factors. Subjects who visited the health promotion center for a medical checkup and treadmill test (n=8241; age: median, 48 years; range, 16–79 years) were classified into three groups based on their WBC counts (group 1, 2200–5300 μl, n=2823; group 2, 5301–6500 μl, n=2709; group 3, 6501–10,000 μl, n=2709). After adjusting for age, body mass index, body fat percentage, smoking history, systolic blood pressure, diastolic blood pressure, serum lipid profile, and fasting plasma glucose, VO2 max still showed a significant association with WBC count (partial r=–0.11, P<0.001). They concluded that the WBC count in the normal concentration range is independently related to cardiorespiratory fitness in Korean men. In cross-sectional analyses, Volpatoe et al.54 found IL-6 levels to be inversely related to exercise tolerance in disabled older women, while Taaffe et al.55 reported an inverse relationship between accumulated moderate and strenuous activity with IL-6 in 880 adults aged 70-79 years. Smith et al.44 found that a 6-month-exercise program reduced Tissue necrosis factor (TNF)- (n=43, average age=49.0 years). Tsukui et al.56 reported exercise training in 29 obese women (average age=56 years) reduced TNF with only modest weight loss. It is generally at large accepted that the physiological mediator of low-grade chronic inflammation and raised WBC count is the TNF-alpha, which has been proven to be downregulated by regular physical activities. Another mechanism is that the postexercise hypotension which is accompanied by a decrease in serum cathecolamines, norepinephrine, dopamine, cortisol, sympathetic nervous system, plasma rennin activity,57–60 thus, suppressing inflammatory reaction and finally downregulating WBC count. Reasons for diversities in findings between the present study and several others might not be unconnected to types of exercise, plasma IL-6 during exercise increase with the intensity and duration of exercise,61 interracial differences that might exist in exercise responses to WBC count.6263 The effect of subjects' condition cannot be ruled out, previous studies utilized normotensive subjects compared to hypertensive patients in the present study. BODY.CONCLUSION: Based on the results of the present study, it was concluded that interval training program is an effective adjunct nonpharmacological management of hypertension. The therapeutic effect of interval training program on blood pressure and VO2 max (aerobic fitness) may be mediated through suppression of inflammatory (WBC count) reaction. BODY.LIMITATIONS: The present study demonstrated a rationale bases for the role of interval exercise training in the down regulation of the blood pressure and WBC count. However, there are limitations of the study, including failure to investigate the effect of severity of hypertension and other markers of inflammation on the outcome variables. It is however unlikely that a single biomarker reflects all health risk.64 These limitations warrant attention in future studies.

TITLE: Patient Education for Endoscopic Sinus Surgery: Preliminary Experience Using 3D-Printed Clinical Imaging Data ABSTRACT: Within the Ear, Nose, and Throat (ENT) medical space, a relatively small fraction of patients follow through with elective surgeries to fix ailments such as a deviated septum or occluded sinus passage. Patient understanding of their diagnosis and treatment plan is integral to compliance, which ultimately yields improved medical outcomes and better quality of life. Here we report the usage of advanced, polyjet 3D printing methods to develop a multimaterial replica of human nasal sinus anatomy, derived from clinical X-ray computed tomography (CT) data, to be used as an educational aid during physician consultation. The final patient education model was developed over several iterations to optimize material properties, anatomical accuracy and overall display. A two-arm, single-center, randomized, prospective study was then performed in which 50 ENT surgical candidates (and an associated control group, n = 50) were given an explanation of their anatomy, disease state, and treatment options using the education model as an aid. Statistically significant improvements in patient ratings of their physician's explanation of their treatment options (p = 0.020), self-rated anatomical understanding (p = 0.043), self-rated understanding of disease state (p = 0.016), and effectiveness of the visualization (p = 0.007) were noted from the population that viewed the 3D education model, indicating it is an effective tool which ENT surgeons may use to educate and interact with patients. BODY.1. INTRODUCTION: Communication with patients has long been regarded as one of the most critical yet challenging parts of a doctor's clinical work [1]. Patient understanding of their diagnosis and treatment regimen is integral to individual compliance and satisfaction with their physician [1,2,3]. Visual aids can be an important clinical tool for educating patients as they tend to understand and retain information more effectively with visual reinforcement [3]. However, classical graphical aids, such as photographs or illustrations, often require patients to view a three-dimensional morphology in a two-dimensional representation. Patient comprehension can be especially difficult with regards to internal anatomic relationships such as the nasal sinus passages, as these are not commonly visualized systems. Poor comprehension can hinder the informed consent process and may result in the patient electing not to move forward with a beneficial and necessary procedure. This is particularly prevalent in the Ear, Nose, and Throat (ENT) medical space, where surgery cancelation rates exceed 16% [4]. Therefore, advanced teaching aids may improve patient understanding and consent, leading to improved clinical outcomes. Three-dimensional (3D) printing has emerged as an invaluable tool for the creation of anatomical models. The intricate complexity that can be replicated with advanced additive technology has enabled the synthesis of patient-specific replica models and vastly increased the wealth of educational and pre-surgical training models currently available [5]. Further driving the innovation in this space are multi-head 3D printers that enable several polymers with varying properties, such as color and hardness, to be mixed in a 3D space within the same model [6]. In particular, the Objet PolyJetTM systems use acrylic polymer substrates with variable side-chain groups to achieve tunable material properties [7]. These materials can be mixed at customizable ratios from each extruder head, at specific spatial coordinates, to yield novel composite models. While the use of 3D printing has been reported in the areas of oncology [8], craniofacial reconstruction [9], orthopedics [10], and organ models [11,12,13], there has been relatively little work reported in the rhinology/otolaryngology space [14]. Here we report the use of PolyJet technology to create an anatomical replica of patient sinus passages, with soft tissue and bone structure represented with malleable and hard polymeric materials, respectively. These customized models can be opened to allow patients to explore and manipulate the nasal sinus anatomy so that they may have a comparable teaching aid to those used in medical school anatomy labs. ENT doctors employed the models as teaching aids to evaluate their ability to improve patients' self-reported understanding of their anatomy, disease state, and proposed surgical treatment versus traditional clinical education tools. BODY.2. RESULTS.2.1. MODEL FABRICATION: A 3D-printed model was generated from X-ray computerized tomography (CT) scans of the nasal sinus anatomy of a patient (Figure 1A). The model was printed using a malleable, translucent polymer to represent soft tissue areas and a stiff, opaque polymer for bone tissue regions. Itwas cut into seven coronally-sliced slabs, which could be disassembled to expose and show the interior sinus and nasal anatomy (Figure 1B). The turbinates and nasal septum, soft nasal tissues, were accessible and easily manipulated by the ENTs to demonstrate physical manifestations of the patient's disease state, altered air flow pathways, and proposed surgical treatments to the patients (Figure 1C). In this fashion, a single model was adaptable to describe the disease states of all individuals, preventing the need to create expensive, patient-specific repicas on a case-by-case basis. BODY.2. RESULTS.2.2. 3D-PRINTED MODEL AS AN EDUCATIONAL TOOL: One hundred adult candidates for sinus or nasal surgery were enrolled in a randomized clinical study to measure the efficacy of anatomically accurate, 3D-printed models in improving patient understanding of their anatomy, disease state, and surgical options. Each patient received an explanation of their condition from their ENT physician, either with a 3D-printed teaching model generated from CT scan data or with standard diagrams and charts. Immediately following the ENT consultation, each patient was given a survey to rate the physician's explanation of the proposed treatment plan, their own understanding of their anatomy and disease state, and how much the educational tools aided the physician's explanation. Patients were also asked if the ENT consultation eased their anxiety and if they intended to go through with the proposed treatment plan, if applicable. Patient ratings for questions 1–5 were averaged and subjected to a non-parametric Mann-Whitney test comparing the 3D model and control groups. Patient responses for questions 6 and 7 were collected and the percentage of "yes" responses was calculated and subjected to a chi-squared test to determine the statistical significance. "N/A" responses in question 6 were removed when calculating the percentage of "yes" responses. The survey questions and averaged results are shown in Table 1. The average score for each survey question was higher for the 3D model group compared to the control group. Patient responses in the 3D model group increased significantly when rating the quality of the physician's explanation, their own understanding of their anatomy, their own understanding of their disease state, and their understanding of the proposed surgical treatment. Averages in both groups approached the ceiling score, which may impact the significance of the results. The largest increase in score was seen in patient understanding of the surgical treatment, which is the most critical information to ensure the patient is making an informed decision. Interestingly, 32 patients said the standard visual aids eased their anxiety, while 39 responded positively to the 3D model. Questions 6 and 7, which were more qualitative in nature, did not result in statistically significant results between the control and 3D model groups, though a higher percentage of patients did respond in the affirmative to each question when presented with the 3D model. Overall, the survey results demonstrate the positive educational benefits of the 3D model as a clinical teaching tool for ENTs. BODY.3. DISCUSSION: The survey data imply that providing patients with a 3D model during their physician consultation is equivalent or superior to the current clinical standard for improving patient self-reported understanding of their anatomy, condition, and treatment options. The results of the survey are notable for a variety of reasons. First, although the scores in the 3D model experimental group were higher across the board, the control group still showed high self-reported scores for each question. On one hand, this suggests that the standard explanation and tools ENTs use provide their patients with a very good overall understanding of their health, anatomy, condition, and treatment options. On the other hand, since these scores are all self-reported, it may convolute the true benefits of improved educational models since so many patients already reported perfect or near-perfect understanding. Additionally, the abundance of high scores clustered close to the maximum value (10) could impact the ability to identify truly significant changes. Future studies with larger cohorts in each group could help in distinguishing which increases are significant, and could also test each patient to objectively measure the understanding of their anatomy. Overall, it was not surprising to see high self-reported survey results as the two ENT physicians, who each saw a randomly distributed subset of patients from each cohort, were highly experienced, with five and 20 years of experience, respectively. A greater impact from use of the model would be hypothesized for less experienced ENT doctors, or perhaps from those in which English is a second language. As mentioned above, the largest difference in score between the control and 3D model groups was in patient understanding of the surgical procedure. The cohort given the 3D model reported a nearly perfect understanding of the surgery, with an average score of 9.82 out of 10. Only one patient reported an understanding level of 8, seven reported 9, and the remaining 42 patients reported a maximum understanding of the surgical procedure. By comparison, the control group had eight reports of scores 8 or below. Given that physicians use educational aids to inform their patients about the details of the proposed surgical treatment, the 3D models appear to be an improved tool for these means. On the whole, the degree of patient teaching varies between doctors. However, a common thread between all doctors is their desire to improve patient outcomes. Physicians may be less likely to explain difficult and complex ideas if they are concerned the information may be misunderstood and may lead patients to opt out of beneficial treatment options due to fear or lack of comprehension. Patient anxiety has been reported as a significant hurdle to opting for a surgical procedure [15]. However, in this study, the use of the 3D models was found to ease patient anxiety, with 97.5% of respondents reporting reduced apprehension, as well as increase self-reported understanding. Understanding of the surgical procedure is critical to patient-centered care models and well-informed individuals are better equipped to make decisions about treatment options that are suitable for them. The results from the patient survey show a trend pointing at the benefits of realistic, interactive teaching models for ENTs. Future work could include additional patient surveys conducted both prior to their physician consultation and at a set period of time after the consultation. This would provide additional insights into the efficacy of the 3D model teaching tools to improve comprehension, understanding, and retention of information regarding the patient condition and treatment options. To get a better measure of patient understanding relative to self-reporting, a quiz could be designed and administered to patients to gather quantitative results, as has been used in other studies to measure patient understanding [16,17]. BODY.4. MATERIALS AND METHODS.4.1. SELECTION OF PATIENTS AND STUDY DESIGN: Prior to recruiting patients, this study was approved by the University of Notre Dame's Institutional Review Board. IRB approval was granted for waiver of written informed consent for this study due to minimal risk and the need to prevent full disclosure that may have induced bias. Each patient received a letter describing the study and agreed to participate. The letter stated: "Dr. (Insert physician name) is participating in a new research study to determine which explanation techniques will help patients to better understand their nasal and sinus anatomy, the disease they have been diagnosed with, and the surgical choices they are being given. You have been asked to participate in this research study because your doctor has recommended nasal or sinus surgery. This study consists of your disease, anatomy and surgical procedure being described to you both verbally and using other forms of education materials. Patients in this study are randomly assigned to one of two groups. The description will be done using different explanation techniques for each group. This will be followed by a seven question survey that you will be asked to fill out. The sponsor of this study is the Notre Dame Biological Imaging Laboratory. The sponsor is paying your surgeon to participate in this study to help cover the activities involved in this study. Participation in this research study is voluntary. You can withdraw from this study at any time. Your participation does not in any way affect your treatment by your physician. Please let your physician know if you would like to participate. Thank you for considering participation in this new research study." The study was a prospective, two arm, single center, randomized study. The randomization of patients was determined using a computational random number generator, with patients entering the study via their surgeon. From February 2015 to April 2015, 100 adult patient candidates for sinus or nasal surgery were included, each aged 18 years or older. Patients were verbally informed about the study design and were given a letter with the highlights. They were randomized into either the study or control group, with 50 patients in each arm. The patient's disease, anatomy, and surgical options were explained in a method consistent with their cohort assignment. The control group had the information about their anatomy, disease, and surgical option(s) explained by their surgeon using the current standard of clinical care, including the use of 2D charts and a verbal explanation. The study group had the same information given to them using an anatomically accurate, 3D printed model of the nasal/sinus region of a standard head to help explain their anatomy, disease, and surgical option(s). The same model was used throughout the study. After this explanation, a seven-question survey was given to each patient to complete. There was no time restriction given to the patient to fill out the survey, and the patient understood that their surgeon would not see the answers to their survey. The survey results for each patient were collected and analyzed in conjunction with the other patients in their respective assigned cohort. For questions 1–5, a median patient score for each cohort was calculated and compared to the other cohort using an upaired, non-parametric Mann-Whitney test. Results were taken to be statistically significant for p < 0.05. For question 6, the number of patients answering "yes," "no," and "not applicable" were totaled. The percentage of patients answering "yes" in each cohort was calculated by summing the number of "yes" responses and dividing by the total number of "yes" and "no" responses; "not applicable" responses were excluded from the calculation. For question 7, the number of patients answering "yes," "no," and "not sure" were totaled. The percentage of patients answering "yes" in each cohort was calculated by summing the number of "yes" responses and dividing by the total number of responses. No patient answered "no" to question 7. For questions 6 and 7 the percentage of "yes" answers were compared between cohorts with a chi-squared test, with p < 0.05 set as the cutoff for determining statistical significance. BODY.4. MATERIALS AND METHODS.4.2. CT DATA PROCESSING AND 3D PRINTING: Pre-existing cone beam CT data acquired with a MiniCAT (Xoran Technologies, Ann Arbor, MI, USA) was used for 3D printed model generation. The CT data was opened in Pmod 3.2 Biological Image Quantification software (Pmod Technologies, Zurich, Switzerland) with the View tool. A volume of interest was drawn around the portion of the scan that included the nose, nasal cavity, and sinuses, but excluded the teeth and other tissue at the edge of the scan. All volumes outside of the volume of interest were masked at a value of −1000 Hounsfield Units (HU). The data was exported in Nifti (.nii) format and opened in 3DSlicer [18]. A 3D surface map for bone was generated using the "Grayscale Modelmaker" tool within 3DSlicer at a threshold of 300 HU. A separate 3D surface map was generated for soft tissue using the same tool and process with a threshold level of −300 HU. Each surface map was exported from 3DSlicer as a .stl file. Soft tissue and bone 3D surface maps were imported separately into MeshLab software (ISTI–CNR) [19]. Within MeshLab, the "Remove Isolated Pieces" algorithm was applied to each surface map with a threshold of 10% to remove unconnected surfaces from the overall 3D surface map. The "Laplacian Smooth" algorithm was applied to each surface map in three steps. Smoothed surface maps were then exported as .stl files. Smoothed bone and soft tissue surface maps were imported into Autodesk Netfabb software (Autodesk, Inc., San Rafael, CA, USA) into the same session [20]. Cuts were made on the coronal plane starting at the plane of the canthi and continuing posteriorly at intervals of 1 cm, until the sphenoid sinus was bisected by at least one cut. A total of 6 cuts were made in the model used for this study, resulting in 7 slices. Cuts were not triangulated, and both parts were aligned to cut at the same time. After the cuts were made, each slab of the model was taken into "Repair" mode in Netfabb, holes were bridged with the "Add Triangles" tool, and the "Automatic Repair" script was applied. Each slab was exported from Netfabb as a separate .stl file. The .stl files were imported into Objet Studio (Stratasys, Ltd., Eden Prairie, MN, USA), aligned, and assigned materials. TangoPlus (Shore A: 27) was used for the soft tissue, and VeroWhite was used for bone (Shore D: 83). Models were printed on an Objet Connex500 3D Printer with a glossy finish. Support material was removed via water jet without disrupting the anatomy prior to delivery to the ENT clinic. BODY.5. CONCLUSIONS: Three-dimensional printing is a powerful tool that is just beginning to find its uses in a variety of areas. New examples of its applications in healthcare are occurring at a rapid pace. Here, we have demonstrated the use of a 3D-printed model of the nasal sinus anatomy as an educational aid for ENTs to teach patients about their anatomy, medical conditions, and surgical treatment options. Across the entire breadth of survey questions given to candidates for nasal surgery, patients reported better understanding and comfort levels when their ENT included a 3D-printed model as part of their consultation compared to being educated with the current standard of 2D diagrams and drawings. The patients' ability to better visualize their condition and potential treatment options led to self-reported greater understanding, improved comprehension, and less anxiety, all of which can contribute to better patient outcomes through informed consent and increased willingness to go through with beneficial procedures.

TITLE: Manipulation of arterial stiffness, wave reflections, and retrograde shear rate in the femoral artery using lower limb external compression ABSTRACT: Exposure of the arterial wall to retrograde shear acutely leads to endothelial dysfunction and chronically contributes to a proatherogenic vascular phenotype. Arterial stiffness and increased pressure from wave reflections are known arbiters of blood flow in the systemic circulation and each related to atherosclerosis. Using distal external compression of the calf to increase upstream retrograde shear in the superficial femoral artery (SFA), we examined the hypothesis that changes in retrograde shear are correlated with changes in SFA stiffness and pressure from wave reflections. For this purpose, a pneumatic cuff was applied to the calf and inflated to 0, 35, and 70 mmHg (5 min compression, randomized order, separated by 5 min) in 16 healthy young men (23 ± 1 years of age). Doppler ultrasound and wave intensity analysis was used to measure SFA retrograde shear rate, reflected pressure wave intensity (negative area [NA]), elastic modulus (Ep), and a single-point pulse wave velocity (PWV) during acute cuff inflation. Cuff inflation resulted in stepwise increases in retrograde shear rate (P < 0.05 for main effect). There were also significant cuff pressure-dependent increases in NA, Ep, and PWV across conditions (P < 0.05 for main effects). Change in NA, but not Ep or PWV, was associated with change in retrograde shear rate across conditions (P < 0.05). In conclusion, external compression of the calf increases retrograde shear, arterial stiffness, and pressure from wave reflection in the upstream SFA in a dose-dependent manner. Wave reflection intensity, but not arterial stiffness, is correlated with changes in peripheral retrograde shear with this hemodynamic manipulation. BODY.INTRODUCTION: The peripheral blood flow waveform in human conduit arteries is triphasic, exhibiting characteristic antegrade (forward) flow during systole and a combination of retrograde (backward) and secondary antegrade flow during early and late diastole, respectively (Mahler et al. 1977; Marquis et al. 1984; Hashimoto and Ito 2010). Whereas antegrade shear is associated with an antiatherosclerotic phenotype, increased retrograde shear is associated with endothelial dysfunction and a proatherogenic phenotype (Gnasso et al. 1996; Kornet et al. 1999; Thijssen et al. 2009; Duivenvoorden et al. 2010; Chiu and Chien 2011). Although highly investigated of late (Padilla et al. 2010, 2011; Young et al. 2010; Casey et al. 2012), the vascular and hemodynamic correlates of retrograde shear in peripheral conduit vessels remain poorly understood (Halliwill and Minson 2010). Pressure from wave reflections is a potentially important hemodynamic factor influencing the contour of the flow waveform. With each cardiac contraction, a pressure wave is produced that traverses the systemic arterial tree. Bifurcations and changes in peripheral vascular/arteriolar tone result in reflection of the pressure wave. It is generally acknowledged that pressure from wave reflections augment incident wave pressure but attenuate antegrade flow (Westerhof et al. 1972; O'Rourke and Avolio 1980; Westerhof and O'Rourke 1995). Whether pressure from wave reflections is associated with retrograde shear in vivo has not been as extensively studied. Arterial stiffness may also affect the blood flow profile in the systemic circulation. Transmission of blood into an elastic conduit artery results in radial expansion and recoil of the vessel, converting pulsatile flow into laminar flow. With an increase in stiffness, this buffering capacity is lost resulting in increased flow pulsatility and altered shear patterns (Nichols et al. 2011). Examination of arterial stiffness and wave reflections as prominent correlates of retrograde shear in the peripheral circulation will provide novel insight into the hemodynamic genesis of pro- and antiatherosclerotic shear profiles in vivo. Inflation of a pneumatic compression cuff to subdiastolic pressures has been demonstrated to effectively increase retrograde shear in a dose-dependent manner (Thijssen et al. 2009; Tinken et al. 2009; Carter et al. 2013; Johnson et al. 2013). External compression has also been used to create physical wave reflection sites and manipulate pressure from wave reflections and arterial stiffness in upstream vessels (Latham et al. 1985; Heffernan et al. 2007). Exploitation of this technique may thus provide a novel model to explore the in vivo relation between localized changes in arterial stiffness and pressure from wave reflections with retrograde shear (Klanchar et al. 1990; Wang and Tarbell 1995). The purpose of this study was to explore the association of superficial femoral artery (SFA) stiffness and pressure from wave reflections with SFA retrograde shear rate during manipulation of regional hemodynamics with distal cuff compression. We hypothesized that external cuff compression would increase SFA retrograde shear in a stepwise fashion and this would be accompanied by dose-dependent increases in SFA stiffness and pressure from wave reflections. BODY.METHODS.PARTICIPANTS: Sixteen healthy men from the University community participated in this study (23 ± 1 years, body mass index 26 ± 1 kg/m2). Exclusion criteria included self-reported (from a health history questionnaire) smoking, hypertension, diabetes mellitus, hyperlipidemia, pulmonary disease, renal disease, neurological disease, and peripheral artery disease. Participants were not taking medications of any kind. This study was approved by the Institutional Review Board of Syracuse University, and all subjects provided written informed consent before study initiation. BODY.METHODS.EXPERIMENTAL PROTOCOL: All testing was conducted in a quiet, dimly lit, temperature-controlled laboratory. Participants were instructed to fast for ≥3 h and avoid vigorous exercise and consuming caffeine or alcohol on the day of testing. Upon arrival, participants rested for 10 min in a supine position. The treatment leg was supported by two foam blocks under the upper leg and ankle. The SFA was chosen for imaging given its clinical significance as an atherosclerosis-prone vessel and its location distal to the confounding influence of prominent upstream reflection sites and sources of turbulent flow (aortoiliac, iliofemoral bifurcations) (Greenwald et al. 1990; Pythoud et al. 1996). A pneumatic compression cuff was tightly secured around the calf at the area of greatest circumference. The calf was selected for the following reasons: (1) to remove the potentially confounding influence of regional femoral compression on local vascular properties (Heffernan et al. 2007); and (2) the calf region is a fundamental source of discrete reflection sites, therefore exogenous compression would be expected to amplify endogenous reflection (O'Rourke and Taylor 1966). The cuff was attached to a rapid cuff inflation system (Hokanson, Bellevue, WA). Participants underwent the following three cuff compression conditions in a randomized order: 0, 35, and 70 mmHg. These pressures were selected based on recent studies using a similar compression schema in the brachial artery to manipulate shear profiles (Thijssen et al. 2009). Pilot investigation in our lab (n = 3) demonstrated that a pressure of 70 mmHg was sufficient to alter the SFA flow profile. Each intervention was approximately 5 min in duration and was separated by 5 min of rest between conditions. The treatment leg (left vs. right) was randomized. SFA measurements of diameter, flow velocity, and wave intensity were taken ∼2 min after the start of cuff inflation to ensure steady-state conditions, as acute venous emptying from external compression may alter vascular function (Tschakovsky and Hughson 2000). This was confirmed using a qualitative substudy described below. BODY.METHODS.VASCULAR MEASUREMENTS: Images of the SFA were obtained using Doppler ultrasound (ProSound α7, Aloka, Tokyo, Japan) attached to a 5.0–13.0 MHz linear array probe during cuff compression conditions. The artery was imaged ∼8–10 cm distal to the bifurcation of the common femoral artery. Wave intensity analysis (WIA) combined with eTracking was used to derive measures of forward and reflected wave intensity and arterial stiffness. This method has been described in detail previously (Harada et al. 2002; Niki et al. 2002). Briefly, this technique simultaneously measures SFA distension waveforms (analogous to the pressure waveform) and flow waveforms. The distance from the near-wall to far-wall lumen–intima interface was continuously traced using eTracking software. The echo-tracking system measures diameter changes within 1/16th of an ultrasound wavelength (0.013 mm) (Ohte et al. 2003) creating a distension waveform almost identical to pressure waveforms (Van Bortel et al. 2001). WIA distension waveforms were calibrated using brachial blood pressure (from simultaneous oscillometric recordings) during each condition. Flow waveforms were measured using range-gated color Doppler signals averaged along the Doppler beam. An insonation angle ≤60° was maintained for all measures. Sample volume was adjusted to encompass the entire vessel. At least eight waveforms were ensemble averaged to gain a representative average waveform. Wave intensity was calculated using time derivatives of blood pressure (P) and velocity (U), where wave intensity = (dP/dt × dU/dt); thus, the area under the dP/dt × dU/dt curve represents the energy transfer of the wave (Sugawara et al. 2009). WIA states that if these wavefronts carry a positive rate of pressure change, they are referred to as compression waves. Conversely, if the wavefront carries a negative rate of pressure change, they are referred to as expansion waves. It should be noted that "expansion" in this setting is an expression from fluid dynamics theory referring to "decreasing pressure" and not to be confused with "dilitation (Sugawara et al. 2009)." (1) W1 represents a forward compression wave produced during early systole that accelerates flow and increases pressure; (2) W2 represents a forward expansion wave that decelerates flow and reduces pressure; (3) the negative area (NA) between W1 and W2 is a backward traveling compression wave due to the sum of waves reflected from the periphery (wave reflection intensity) that decelerates flow but increases pressure. The time interval between the R-wave of the ECG and W1 is analogous to the pre-ejection period (Niki et al. 2002) and was used as a crude proxy of peripheral sympathetic activation (Schachinger et al. 2001). Results obtained from WIA are highly reproducible (Liu et al. 2011a,b) and have been shown to be analogous to results obtained using traditional linear wave separation with impedance analysis for the determination of forward and backward traveling waves (Hughes and Parker 2009). Arterial stiffness measures included stiffness index (β), Peterson's pressure-strain elastic modulus (Ep), and a single-point pulse wave velocity (PWV). All variables were automatically calculated by the echo-tracking subsystem using the formulas listed below: where P and D correspond to pressure and diameter, respectively, and Max and Min refer to maximum (systolic) and minimum (diastolic) values during the cardiac cycle. Blood density, ρ, is assumed constant and equals 1050 kg/m3. We additionally computed the reflection coefficient (RC) as RC = NA/W1 + NA as a proxy of distal vascular tone (Liu et al. 2011a). Systolic antegrade, diastolic retrograde, diastolic antegrade, and mean blood velocities (Vm) were measured using Doppler ultrasound as described above and calculated as follows: Vm = ∫ V(t) dt/FT, where ∫ V(t) dt is the velocity–time integral of the velocity waveform and FT is flow time. Shear rate was calculated as 4 × (Vm/Diameter). SFA intima-media thickness (IMT) was assessed using a longitudinal view of the artery with both near-wall and far-wall lumen–IMT boundaries clearly visible. Once a satisfactory image was obtained, wall thickness was measured separately during systole and again during diastole (determined from simultaneous ECG gating) across a continuous 5-mm region of interest via semiautomated digital calipers. The distance from the lumen–intima interface to the media–adventitia interface was taken as the IMT. BODY.METHODS.SYSTEMIC HEMODYNAMIC MEASURES: Beat-to-beat systolic and diastolic blood pressure (SBP and DBP, respectively) were monitored continuously using digital photoplethysmography (Finapres Medical Systems, Amsterdam, The Netherlands) to ensure that the intervention did not alter systemic hemodynamics (Guelen et al. 2008). Arterial pressure in the finger was measured via the volume-clamp method, which is based on the development of the dynamic pulsatile unloading of the finger arterial walls (Guelen et al. 2003). Beat-to-beat blood pressure measurements were calibrated to brachial pressures prior to experimental testing. The Modelflow method was used to derive stroke volume and cardiac output (heart rate × stroke volume) and total peripheral resistance (mean arterial pressure/cardiac output) (Bogert and van Lieshout 2005). Brachial blood pressure was further obtained via a validated automated oscillometric cuff (EW3109, Panasonic Electric Works, Secaucus NJ) at baseline and immediately pre–post femoral imaging for each compression condition (Bonso et al. 2010). BODY.METHODS.ACUTE SUBSTUDY: In order to gain qualitative insight into hemodynamic temporal changes instigated by direct cuff compression, a subset of participants (n = 6) reported to the laboratory on a separate day and underwent trials at 0-mmHg and 70-mmHg compression. This pressure was selected as it was hypothesized to cause the most robust changes in vascular and hemodynamic parameters. Due to technical requirements of WIA, instantaneous data could not be generated. Therefore, a combination of Doppler ultrasound and tonometry (Sphygmocor, Atcor Medical, Australia) was used to simultaneously capture flow velocity waveforms and pressure waveforms in the left and right femoral artery, respectively, during dual lower limb compression (Hokanson, Bellevue, WA). Due to the overall smooth contour of the resting femoral pulse waveform and lack of a clear inflection point, calculation of global wave reflection using standard metrics (i.e., augmentation index) was not feasible. Data images are displayed for visual inspection only. BODY.METHODS.STATISTICAL ANALYSES: All data are reported as mean ± standard error of the mean and statistical significance was established a priori as P < 0.05. A one-way analysis of variance (three conditions; 0, 35, and 70 mmHg) was used to analyze main outcome variables. If a significant main effect was detected, post hoc comparisons were made using the Tukey method. Associations of interest were examined across conditions using Pearson's correlation coefficients. Absolute change scores were computed as (1) 70-mmHg compression values – 0-mmHg compression values and (2) 35-mmHg compression values – 0-mmHg compression values. Change scores across conditions were combined to examine interassociations between vascular and hemodynamic parameters. To explore day-to-day variability in primary vascular and hemodynamic measures, paired samples t-tests were used to compare means. Intraclass correlation coefficients were used to gauge reliability. All statistical analyses were carried out using IBM SPSS version 20 (SPSS Inc., Chicago, IL). BODY.RESULTS: Day-to-day repeatability of hemodynamic measures calculated from a subset of subjects (n = 6) on two separate days was fair (intraclass correlation coefficients of 0.65–0.94 for all) and resting values did not differ between testing days (P > 0.05). There were no differences in brachial SBP or DBP measured immediately before and after each condition (Table 1). Similarly, there were no differences in SBP, DBP, cardiac output, total peripheral resistance, R-W1, or SFA diameters measured across condition (Table 2). Table 1 Brachial oscillometric blood pressure before and after each condition 0 mmHg 35 mmHg 70 mmHg Before After Before After Before After P -value SBP, mmHg 119 ± 1 119 ± 1 118 ± 1 118 ± 1 119 ± 1 120 ± 1 0.94 DBP, mmHg 73 ± 1 73 ± 1 73 ± 1 73 ± 1 74 ± 1 72 ± 1 0.93 Table 2 Systemic hemodynamics measured by digital plethysmography during each condition 0 mmHg 35 mmHg 70 mmHg P -value SBP, mmHg 126 ± 1 126 ± 1 126 ± 1 0.99 DBP, mmHg 73 ± 1 74 ± 1 73 ± 1 0.80 CO, L min −1 6.1 ± 0.3 6.0 ± 0.3 6.1 ± 0.4 0.99 TPR, mmHg L −1 min −1 1331 ± 68 1344 ± 69 1315 ± 82 0.96 CO, cardiac output; TPR, total peripheral resistance. BODY.RESULTS.SHEAR RATE: There were significant differences in retrograde shear rate, diastolic antegrade shear rate, and consequently mean shear rate across conditions (Table 3 and Fig. 1, P < 0.05). Post hoc pair-wise comparisons revealed that retrograde shear was higher during the 70-mmHg compression condition compared with the 0-mmHg compression condition (Fig. 1, P < 0.05), whereas diastolic antegrade and mean shear were lower during the 70-mmHg compression condition compared to the 0-mmHg compression condition (Table 3, P < 0.05). Table 3 SFA vascular and hemodynamic parameters across conditions 0 mmHg 35 mmHg 70 mmHg P -value Systolic diameter, mm 5.83 ± 0.13 5.86 ± 0.12 5.84 ± 0.13 0.98 Diastolic diameter, mm 6.04 ± 0.11 6.00 ± 0.14 5.93 ± 0.13 0.94 Systolic IMT, mm 0.34 ± 0.02 0.35 ± 0.01 0.34 ± 0.02 0.79 Diastolic IMT, mm 0.34 ± 0.02 0.35 ± 0.01 0.34 ± 0.02 0.94 Systolic antegrade shear rate, sec −1 321 ± 23 314 ± 25 340 ± 13 0.63 Diastolic antegrade shear rate, sec −1 76 ± 4.9 62 ± 5 1 16 ± 6 1 , 2 0.001 Mean shear rate, sec −1 102 ± 9 69 ± 7 1 37 ± 8 1 , 2 0.001 β-stiffness, aU 13.8 ± 0.9 15.6 ± 1.1 17.2 ± 1.1 0.056 W 1 , mmHg m sec −3 7.7 ± 0.5 8.4 ± 0.9 8.8 ± 0.7 0.32 W 2 , mmHg m sec −3 1.7 ± 0.2 1.9 ± 0.2 2.3 ± 0.2 1 , 2 0.031 Reflection coefficient, aU 0.36 ± 0.06 0.46 ± 0.05 0.50 ± 0.07 1 0.047 R-W 1 , msec 242 ± 6 241 ± 6 245 ± 5 0.86 Heart rate, bpm 61 ± 2 62 ± 2 61 ± 2 0.92 1 Significantly different than 0 mmHg ( P < 0.05). 2 Significantly different than 35 mmHg ( P < 0.05). Figure 1Retrograde shear, arterial stiffness parameters (PWV, pulse wave velocity; Ep, elastic modulus), and wave reflection intensity (NA, negative area) across conditions. †Significantly different than 0 mmHg (P < 0.05). BODY.RESULTS.ARTERIAL STIFFNESS AND WAVE REFLECTIONS: There were significant differences in PWV, Ep, W2, NA, and RC across conditions (Table 3 and Fig. 1, P < 0.05). Post hoc pair-wise comparisons revealed significantly higher values for these parameters during the 70-mmHg compression condition compared with the 0-mmHg compression condition (P < 0.05). BODY.RESULTS.CORRELATIONS OF SHEAR PATTERNS AND VASCULAR HEMODYNAMICS: As seen in Table 4, across conditions retrograde shear was associated with NA (P < 0.05) and W2 (P < 0.05). Moreover change in retrograde shear was associated with change in NA (r = 0.43, P < 0.05) and change in W2 (r = 0.39, P = 0.06). There were no associations between retrograde shear and stiffness parameters (across conditions or change between conditions; all P > 0.05). Given colinearity between arterial stiffness measures (all three measures are derived from diameter and blood pressure), PWV was selected as a representative measure of arterial stiffness for correlational display purposes. Table 4 Correlation matrix for hemodynamics and shear components across conditions n = 48 PWV W 2 NA Systolic antegrade shear Retrograde shear W 2 0.36 1 Negative area 0.34 1 0.13 Systolic antegrade shear 0.33 1 0.15 0.06 Retrograde shear 0.18 0.24 1 0.40 1 0.37 1 Diastolic antegrade shear −0.27 1 −0.36 1 0.09 0.07 −0.25 1 1 Significant association, P < 0.05. BODY.RESULTS.COMPRESSION SUBSTUDY: Figure 2 displays a sample image of WIA during the 70-mmHg compression condition. As can be seen from Figure 3, there was an instantaneous increase in retrograde Vm (denoted by the arrow) and diminution of diastolic antegrade Vm with cuff inflation to 70 mmHg. Whereas no clear inflection point could be detected in the femoral pulse waveform during the 0-mmHg compression condition, compression to 70 mmHg produced an instantaneous inflection point on the contour of the pressure wave (also denoted by the arrow) suggesting rapid changes in timing and/or magnitude of pressure from wave reflections. These findings were visually confirmed in all six participants. Figure 2Sample wave intensity analysis during 70-mmHg compression condition. The displayed signals from top to bottom are as follows: the distention waveform (analogous to a pressure waveform), the flow waveform, the wave intensity, and the ECG. WI, wave intensity; P, pressure; U, flow. Figure 3Sample images from two participants showing instantaneous changes in pressure and flow waveforms with external compression to 70 mmHg (denoted "inflation"). Note the immediate appearance of an inflection point on the contour of the pressure wave (white panel, black arrows) signifying increased pressure from wave reflection. Also note the increase in retrograde flow and attenuation of antegrade flow in early and late diastole, respectively (black panel, white arrows). BODY.DISCUSSION: Inflation of a blood pressure cuff around the calf successfully induced a stepwise increase in upstream SFA retrograde shear, which confirms previous observations witnessed in the brachial artery (Thijssen et al. 2009; Tinken et al. 2009; Birk et al. 2012). Moreover, we extend this by adding the novel observation that the increase in retrograde shear in the SFA occurs immediately (i.e., within a single cardiac cycle) after cuff inflation. In addition to the stepwise increases in retrograde shear rate, downstream cuff inflation to 35 and 70 mmHg also resulted in concomitant stepwise elevations in wave reflection intensity and arterial stiffness. Changes in pressure from wave reflections also appear to occur rapidly (i.e., within a single cardiac cycle) after cuff inflation. Finally, we demonstrated that changes in SFA retrograde shear are associated with changes in wave reflection intensity but not arterial stiffness. Current hemodynamic dogma dictates that pressure from wave reflections is a significant determinant of the flow profile in the systemic circulation (O'Rourke and Avolio 1980; Nichols et al. 2011). In vitro simulation studies and mathematical models have proposed that wave reflections not only contribute to flow reversal but also determine the amplitude of vascular wall shear rate (Westerhof et al. 1972; O'Rourke and Avolio 1980; Klanchar et al. 1990; Wang and Tarbell 1995). Retrograde flow is reduced distal from bifurcations (i.e., below wave reflection sites) (Mills et al. 1970; Nichols et al. 2011). In the saphenous, there is no retrograde flow and this has been suggested to be due to distance to downstream reflection sites (Nichols et al. 2011). There is no retrograde flow in the splanchnic branches of the abdominal aorta and this too has been attributed to low peripheral vascular resistance and reflection coefficients in this vascular bed (Nichols et al. 2011). During conditions of reactive hyperemia, there is no retrograde flow in peripheral vessels (Mahler et al. 1977). Similarly, there is reduced magnitude of pressure from wave reflections during reactive hyperemia owing to vasodilation of downstream vessels (Nichols et al. 2011). In these settings, pressure and flow waveforms are virtually identical (Nichols et al. 2011). Our experimental findings using cuff compression to manipulate shear patterns support previous in vitro suggestions that wave reflections are an important correlate of retrograde shear in peripheral conduit vessels in vivo (Mills et al. 1970; Busse et al. 1975). External compression of the calf also resulted in commensurate increases in upstream SFA stiffness. However, change in SFA stiffness was not a direct correlate of change in retrograde shear per se. SFA PWV was, however, associated with wave reflection intensity and this is in keeping with the notion that arterial stiffness is an important potential moderator of wave reflections (both timing and magnitude). Increases in SFA stiffness were also associated with the near elimination of diastolic antegrade shear, a potential manifestation of diminished arterial reservoir function. With each cardiac contraction, the forward pressure wave that is generated causes radial expansion of the vessel. A portion of the ejected stroke volume is temporarily stored within the vessel wall, and upon cardiac relaxation (i.e., diastole), the vessel recoils and stored blood is expelled back into the systemic circulation. Under resting conditions, retrograde flow serves as the substrate for diastolic antegrade flow and this is facilitated by the arterial reservoir (Hashimoto and Ito 2010). The capacitance or reservoir function of the arterial tree allows for adequate convective acceleration of flow (i.e., "run-off") to the periphery during diastole ensuring that flow does not fall to zero (Heffernan et al. 2010). Increased arterial stiffness would portend a reduced capacitance function and hence attenuate stored flow discharge into the periphery. This is important as reduced diastolic antegrade shear was associated with increased retrograde shear in this study, and this is evident when viewing Figure 3. Although speculative, arterial stiffness may indirectly affect retrograde shear via its modulatory influence on pressure from wave reflections and diastolic antegrade shear (i.e., the arterial reservoir). An unanticipated and novel finding was the increase in W2 across conditions that were associated with increased retrograde shear and reduced diastolic antegrade shear. W2 is a forward traveling expansion wave created by myocardial shortening rate and inertial force of aortic blood flow (momentum) that causes a rapid fall in left ventricle (LV) pressure (Jones et al. 2002; Sugawara et al. 2009). This creates a suction wave that applies a "braking" action to the column of blood from behind and decelerates flow (Jones et al. 2002; Feng and Khir 2008). It has been suggested that the effect of this expansion wave on peripheral flow is modest owing to the physical distance from the source of the suction (i.e., the heart) coupled with wave damping and dissipation (Liu et al. 2011b). However, it is possible that an increase in arterial stiffness and wave speed (less viscous damping and greater dispersion) causes this expansion wave to travel more distally (Jones et al. 2002; Zambanini et al. 2005). Indeed, there was an association between PWV and W2 across conditions. The mechanism responsible for acute increases in SFA stiffness, wave reflection intensity, and retrograde shear with external compression of the calf has not been specifically explored. Given that we noted no change in systolic shear but significant changes in diastolic shear, our findings are in keeping with recent suggestions that this external compression model mimics a quasi-Starling resistor (Halliwill and Minson 2010). According to this theory, external compression reduces regional transmural pressure distending the vessel wall which causes momentary precapillary arteriolar collapse during diastole (when critical closing pressure is attained) and creates a "back pressure" to flow (Shrier and Magder 1995); identified as wave reflection intensity herein. The result is an attenuated diastolic runoff into the periphery and retrograde discharge of stored reservoir flow volume upon vessel recoil. There may also be a reflexive and conducted increase in regional vascular tone aimed at preventing further pressure drops across the vessel wall (Nielsen 1991; Jiang et al. 2011). Indeed, we noted a stepwise increase in the RC suggesting that increased external cuff compression of the lower limb mimics an increase in distal vascular tone (Liu et al. 2011a). In response to low-shear conditions, peripheral conduit vessel tone increases instantaneously in vivo with peak low-shear–mediated constriction occurring approximately 3 min after initial exposure (Levenson et al. 2001). Increased vascular tone from decreased wall shear directly increases arterial stiffness (i.e., low-flow/shear–mediated stiffening) (Joannides et al. 2001). The amplitude/intensity of reflected pressure waves is also directly dependent on the value of peripheral resistance (Westerhof et al. 1972). Changes in arterial stiffness, wave reflection intensity, and retrograde shear may thus have a common origin residing, at least partly, in downstream vascular tone. The magnitude of change in SFA retrograde shear was somewhat lower than that previously reported in the brachial artery (Thijssen et al. 2009). The reason for this may be related to the amount of tissue compressed, the amount of discrete reflection sites within the particular vascular bed being manipulated, or the physical distance to the effective reflection site (Sugawara et al. 2010). Another possibility resides in potential limb differences between the SFA and brachial artery in the vascular and hemodynamic response to altered transmural pressure (Newcomer et al. 2008). Limb differences have been described regarding the responsiveness to acute altered retrograde shear (Padilla et al. 2009). Future research is needed to explore potential limb differences in vascular and hemodynamic correlates of retrograde shear. BODY.DISCUSSION.LIMITATIONS: Inflation of a pneumatic compression cuff to alter flow and pressure may not be viewed as cuing a true physiologic response. Our primary purpose was to examine correlates of change in retrograde shear in order to gain deeper insight into underlying mechanism. Other commonly used physiologic perturbations such as cold pressor testing or rhythmic handgrip exercise have not consistently been shown to alter retrograde shear in the upstream brachial artery in young adults (Padilla et al. 2010, 2011). Moreover, increases in heart rate and mean pressure that occur concomitant with cold pressor and exercise are known confounders of change in arterial stiffness, pressure from wave reflections, and retrograde shear (Wilkinson et al. 2000, 2002; Casey et al. 2008; Lydakis et al. 2008; Padilla et al. 2010). We wished to use a method that would enable us to generate a dose–response curve while concomitantly characterizing a fairly isolated regional vascular hemodynamic response devoid of other systemic confounders. Indeed, this perturbation was mild enough not to evoke change in heart rate, stroke volume, pre-ejection period (a proxy of sympathetic activation), systemic vascular resistance, or mean pressure. Another potential limitation is that we did not carry out measures in young women or in older subjects. Therefore, we cannot extrapolate findings to other groups. Finally, this study was not designed to determine if wave reflections directly cause retrograde shear. As seen in Figure 3, changes in retrograde Vm and pressure wave reflection from limb compression were instantaneous precluding ability to distinguish a temporal order. On the basis of our findings we propose that retrograde shear in the SFA may be the culmination of: (1) increased resistance "in front" from increased distal vascular tone preventing flow runoff during diastole (Henderson and Johnson 1912); (2) "cessation of push from behind" from an increased expansion wave (suction wave) causing flow deceleration (Henderson and Johnson 1912); and (3) increased wave reflection intensity contributing to flow reversal. In conclusion, experimental manipulation of regional SFA hemodynamics with lower limb external compression results in rapid and dose-dependent increases in upstream SFA retrograde shear and wave reflection intensity. Changes in SFA retrograde shear are associated with changes in wave reflection intensity but not arterial stiffness.

TITLE: Stock Versus CAD/CAM Customized Zirconia Implant Abutments – Clinical and Patient‐Based Outcomes in a Randomized Controlled Clinical Trial ABSTRACT.ABSTRACT.BACKGROUND: Single‐tooth replacement often requires a prefabricated dental implant and a customized crown. The benefits of individualization of the abutment remain unclear. ABSTRACT.ABSTRACT.PURPOSE: This randomized controlled clinical trial aims to study potential benefits of individualization of zirconia implant abutments with respect to preservation of marginal bone level and several clinical and patient‐based outcome measures. ABSTRACT.ABSTRACT.MATERIAL AND METHODS: Fifty participants with a missing premolar were included and randomly assigned to standard (ZirDesign, DentsplySirona Implants, Mölndal, Sweden) or computer aided design/computer aided manufacturing (CAD/CAM) customized (Atlantis, DentsplySirona Implants, Mölndal, Sweden) zirconia abutment therapy. Peri‐implant bone level (primary outcome), Plaque‐index, calculus formation, bleeding on probing, gingiva index, probing pocket depth, recession, appearance of soft tissues and patients' contentment were assessed shortly after placement and one year later. ABSTRACT.ABSTRACT.RESULTS: No implants were lost and no complications related to the abutments were observed. Statistically significant differences between stock and CAD/CAM customized zirconia abutments could not be demonstrated for any of the operationalized variables. ABSTRACT.ABSTRACT.CONCLUSION: The use of a CAD/CAM customized zirconia abutment in single tooth replacement of a premolar is not associated with an improvement in clinical performance or patients' contentment when compared to the use of a stock zirconia abutment. BODY.INTRODUCTION: The design and stability of the implant‐abutment connection as well as the chemical composition and surface properties of the abutment ground material, influence the function of implant‐supported restorations, and the adjacent soft tissue health and soft tissue stability. Platform‐switched implant‐abutment connections maintain better marginal bone levels than matching ones, as is confirmed in several systematic reviews, meta‐analyses and clinical trials.1, 2, 3, 4, 5 Zirconia has aesthetic benefits over titanium in situations presenting with thin overlying mucosa, but biological superiority is generally not observed.6, 7, 8, 9, 10, 11 Besides chemical composition, surface topography and surface‐free‐energy are relevant factors, with surface roughness being the predominant factor with respect to biofilm formation.12 The ideal abutment surface is smooth enough to inhibit biofilm formation, yet rough enough to allow adhesion of fibroblasts. An optimal surface roughness threshold of R(a) 0.2 micron has been proposed.13 Abutment shape could be another influential factor. In general, the stability of labial mucosal margin and fill of the interproximal area are the outcome parameters studied. Stock abutments are cylindrical or divergent at most, which is clearly different from the emergence profile of natural teeth, hence providing compromised support to the proximal and labial peri‐implant soft tissues. Rompen and colleagues experimented with a stock, gingivally converging abutment design, which resulted in the formation of extra soft tissues,14 but his findings could not be confirmed by others.15, 16, 17, 18, 19 Implant abutments can also be produced by means of Computer Aided Design/Computer Aided Manufacturing (CAD/CAM) technology. Variations are infinite, fitting individual and local circumstances, which offers several advantages. The CAD/CAM process optimally controls the geometry of the abutment including the position of the outline in accordance with the neighboring natural roots and the gingival margin, subsequently reducing the risk on cement remnants deep in the sulcus. The finish of the abutment is controlled, preventing sharp edges and the design can compensate for poor implant angulation. In case of a customized abutment, it is the abutment material that supports and interacts with the soft tissues and not so much the ceramic crown. This is of biological advantage.20, 21 However, the industrial production process best guarantees standard quality of the product. It facilitates the use of biocompatible materials in the permucosal area and reduces the risk of corrosive problems from different alloys in casted and milled parts. Finally, it is less time consuming and does not require extra finishing procedures. This raises the question whether there is a difference in performance between stock and individualized abutments. The aim of the present study is to evaluate whether the use of stock (treatment modality (a) and CAD/CAM customized (treatment modality (b) zirconia abutments results in differences regarding peri‐implant bone level alteration (primary objective), clinical performance and fulfilment of patients' expectations. BODY.MATERIALS AND METHODS: A single‐center, randomized controlled clinical trial was designed, for 50 participants, missing a single mandibular or maxillary premolar. Patients were recruited during a 13‐month inclusion period (January 2013–February 2014). In‐ and exclusion criteria are listed in Table 1. Permission from the medical ethics committee of the University Medical Center Groningen, the Netherlands was granted (METc number 2012.388, ABR number NL 42288.042.12) and informed consent was obtained. Primary outcome measure was peri‐implant bone level alteration after 1 year of function; clinical relevance was set at >0.25 mm difference and a 0.3 mm standard deviation was estimated.23 A power calculation was performed using G*Power24 (Version 3.1.9.2) and revealed that 24 patients in each group would be needed (80% power, normal distribution, 2 tailed). Table 1 Inclusion and Exclusion Criteria ‐Inclusion Criteria Missing first or second premolar in the maxilla or mandible Wish to replace the missing premolar with an implant Willing to sign for informed consent Bone height ≥10 mm beneath the maxillary sinus and ≥10 mm above the mandibular nerve and a bone width of at least 6 mm ‐Exclusion criteria Missing teeth mesial or distal from implantation site Orthodontic treatment at the time of impression taking Severe bruxism Acute periodontitis History of implant loss Documented extreme gagging reflex Poor medical condition (ASA * score 3 or higher) Previous therapeutic radiation of the head–neck region Chronic pain in orofacial system Younger than 18 years at time of inclusion Reduced mental capacity *American Society of Anesthesiologists. BODY.MATERIALS AND METHODS.IMPLANT PLACEMENT: One‐hour pre‐operative antibiotic prophylaxis (3 g amoxicillin or, if allergic to penicillin, 600 mg clindamycin, intraorally) was given. Oral disinfection consisted of a 0.2% chlorhexidine mouthwash, twice daily started one day before surgery and ending 10 days later. All surgeries were performed under local anaesthesia. A full‐thickness muco‐periostal flap was raised and the implant site was prepared following the protocol of the manufacturers. The implant was placed (AstraTech OsseoSpeed TX 3.5S in 9, 11, or 13 mm in length and a diameter of 3.5 mm; DentsplySirona Implants, Mölndal, Sweden). Maximum torque used during implant installation was set according to Astra tech Implant System surgical manual and primary implant stability was estimated manually. The corresponding healing abutment was immediately connected onto the implant. The wound was closed with slowly resorbable sutures (Vincryl & Johnson Health Care, Piscataway, NJ, USA). BODY.MATERIALS AND METHODS.RESTORATIVE PROCEDURES: Restorative treatment commenced 3 months later. An analogue impression with a polyether material (Impregum, 3M ESPE, Seefeld, Germany) in an open, semi‐individual impression tray (Border‐Lock, Clan Dental, Maarheeze, the Netherlands) was made by a single, experienced operator (US). BODY.MATERIALS AND METHODS.FABRICATION AND PROVISION OF THE IMPLANT RESTORATION: A screw‐retained implant restoration was provided 3 weeks after impression taking, consisting of a digitally designed and milled Resin Nano Ceramic crown (RNC crown, Lava Ultimate, 3M ESPE, Seefeld, Germany), bonded to either a stock (ZirDesign, DentsplySirona Implants Mölndal, Sweden, n = 25) or a CAD/CAM customized zirconia abutment (Atlantis, DentsplySirona Implants Mölndal, Sweden, n = 25). The abutment type (Figure 1) was randomly allocated to each of the 50 participants (www.sealedenvelope.com) and patients were assigned to the treatment modality accordingly by US, who also took care of the enrolment of the patients. Figure 1RNC crown bonded to (left) Stock (ZirDesign) and (right) CAD/CAM customized (Atlantis) abutment, both manufactured by DentsplySirona Implants, Mölndal, Sweden. The most appropriate color for the RNC crown was chosen from the available Lava Ultimate shades. The RNC crown was luted extra‐orally to the zirconia abutments following the manufacturers' instructions.25 Blinding of the operator was not possible, due to visual differences between the stock and CAD/CAM customized zirconia abutments. Ground material for both abutment types was yttria‐stabilized tetragonal zirconia polycrystal (Y‐TZP). After verification of adequate fit and proximal contact points the abutment fixation screw was tightened, using a wrench at the recommended torque (20 Ncm). The abutment fixation screw was protected by sterile teflon tape and the screw access hole was sealed with a glass ionomer restorative material (Fuji II, GC Europe, Leuven, Belgium). Static and dynamic occlusion were checked meticulously and oral hygiene instruction was given. All patients with complications where seen as soon as possible. If the abutments remained unaffected, patients were not excluded from the study. During reparation, exact copies of the Crown‐Abutment complex were used, so the emerging profile was left unbiased. These were generated form the same CAD file and available for reasons not related to the present study. After 12 months of clinical service, the patients were examined for data acquisition (Figure 2). Figure 2(A) Stock zirconia abutment (ZirDesign, DentsplySirona Implants, Sweden) with resin nanoceramic crown (3M ESPE, Germany) at position 15 after 1 year of function. (B) CAD‐CAM customized zirconia abutment (Atlantis, DentsplySirona, Sweden) with resin nanoceramic crown (3M ESPE, Germany) at position 25 after 1 year of function. BODY.MATERIALS AND METHODS.IMMEDIATE AND FOLLOW UP CLINICAL OUTCOME PARAMETERS: After the intake (T 0) and implant placement, follow‐up appointments were planned at two weeks (T 1) and 12 months (T 12) after delivery of the restoration at which time clinical outcome parameters were assessed around the implant and both neighboring teeth of the implant site using the following parameters: Plaque accumulation was measured with the modified plaque index,26 score range 0–3 on the neighboring teeth and the implant abutment;Absence or presence (0/1) of dental calculus was assessed on the neighboring teeth and the implant;Probing pocket depth (PPD) was quantified with a plastic periodontal probe. 0.25 Newton of calibrated probing force was applied (Click‐probe, KerrHawe, Bioggio, Switzerland) to measure PPD to the nearest mm from the mucosal margin to the clinical pocket. The neighboring teeth as well as the implant site were measured on three locations (i.e., mesiobuccal, mediobuccal and distobuccal);The bleeding tendency of the neighboring teeth and the implant was recorded with the modified sulcus bleeding index,26 score range 0–3);Peri‐implant inflammation was assessed with the gingiva‐index,27 score range 0–3). All clinical parameters were assessed at T 1 and T 12. BODY.MATERIALS AND METHODS.BONE LEVEL, APPROXIMAL GINGIVAL MARGIN AND SOFT TISSUE APPEARANCE AND DEVELOPMENT: Peri‐implant bone level was measured on two radiographs (T 1 and T 12), taken with individually designed x‐ray trays to ensure the same projection of objects on the radiograph (Figure 3). At least, two known vertical reference points on the implant had to be visible on each of the radiographs of a set. If this was not the case, but the quality of the radiographs was still sufficient for regular clinical care, no new radiograph was made for the sole purpose of this study, in accordance with the ALARA principle. These patients' radiographs were excluded from the study. Designated software (DicomWorks, Biomedical Engineering, University Medical Center Groningen, the Netherlands) was used to measure the distance from a reference point to the marginal bone, as described in detail by others.28 Two researchers (US and ES) were simultaneously introduced to the software and analyzed the radiographs independently. To establish interobserver reliability, a two‐way mixed average measures intraclass correlation coefficient (ICC) for consistency was calculated. Bone level alterations and recession were determined by subtracting the (pooled) values T 1 from T 12 (T 12−T 1) with a negative value indicating growth. Figure 3(A) Radiograph of a Stock zirconia abutment (ZirDesign, DentsplySirona Implants, Sweden) with resin nanoceramic crown (3M ESPE, Germany) at baseline (left) and after one year of clinical service (right) at position 45. (B) Radiograph of a CAD‐CAM customized zirconia abutment (Atlantis, DentsplySirona, Sweden) with resin nanoceramic crown (3M ESPE, Germany) at baseline (left) and after one year of clinical service (right) at position 15. Alteration of approximal gingival margin on both neighboring teeth was clinically measured to the nearest millimeter from a fixed reference point to the mucosal margin at T 1 and T 12. The appearance of the soft tissues was determined on randomly presented digital photographs made at T 1 and T 12 by assessing the Pink Esthetic Score (PES), as proposed by Fürhauser and colleagues.29 Seven items were scored: mesial papilla, distal papilla, soft‐tissue level, soft tissue contour, alveolar process deficiency, soft‐tissue color and texture. All items were assessed on a 2‐1‐0 score, with 2 being the best and 0 being the poorest score. A sum‐score was calculated as an impression of soft tissue appearance and development (range 0–14). Marginal bone level, recession and soft tissue appearance were compared both longitudinally (between T 1 and T 12) and between groups at both moments in time. BODY.MATERIALS AND METHODS.PATIENT‐BASED OUTCOME PARAMETERS: The participants responded to 12 statements regarding their expected emotional, functional and aesthetic contentment with the restoration (T 0) and the perceived contentment at 2 weeks (T 1) and 12 months following delivery of the restoration (T 12). A questionnaire using a visual analogue scale (VAS) adapted from Guljé and colleagues, ranging from 0 to 100 (very discontent, major concerns to very content, no concerns at all) was used.28 Expectations at T 0 and the perceived subjective result at T 1 and T 12 were compared (Figure 4). Figure 4Mean VAS‐scores of several expected (t 0) and achieved (t 1 and t 12) aspects of patient satisfaction for zirconia abutments (Stock and customized CAD/CAM abutments grouped) provided with RNC crowns. T 0 (red bar) is assessed prior to treatment (standard deviation between brackets), t 1 at 2 weeks after placement of the implant crown and t 12 after 12 months of function. Values range from 0 (very discontent, major concerns) to 100 (fully content, no concerns at all). Note that positive values at t 1 and t 12 compared to t 0 (dark blue and light blue bars) imply that expectations were exceeded. * Statistically significantly different from T 0 at T 1 p < .05. ** Statistically significantly different from T 0 at T 1 and T 12 p < .05. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Statistical analysis was performed while blinded with respect to the group a patient was allocated to. Marginal bone level alteration and gingival growth at T 12 was compared across abutment types using a Mann‐Whitney U test, because normality was not observed (using a Kolmogorov‐Smirnov test). Plaque accumulation, dental calculus, PPD, bleeding tendency and gingiva‐index of both abutment types were compared at time point T 1 and T 12 with Mann‐Whitney U tests. Alterations over time were assessed with Wilcoxon signed rank test. The change in overall PES score and scores per item were analyzed per group using a Wilcoxon signed rank test. Changes over time in soft tissue appearance were compared across groups using a Mann‐Whitney U test. Patients' expectations (T 0) and the perceived subjective result at T 1 and T 12 were compared across groups using Mann‐Whitney U tests. Differences across time were analyzed using Wilcoxon signed rank tests. p‐values ≤ .05 were considered statistically significant. All computations were performed using a standard statistical program (SPSS, version 23.0 for Windows, SPSS inc., Chicago, USA). BODY.RESULTS: From the 50 eligible participants (Table 2), all participants were examined at T 1 and T 12, but 7 patients had to be excluded from the radiographic analysis (primary outcome), because one of their radiographs made at T 1 or T 12 were deemed unfit for reliable comparison. One pregnant participant refused to have a radiograph taken at T 12 (Figure 5). Figure 5Consort flow diagram for primary outcome variable: peri‐implant bone level alteration. Treatment consisted of RNC crowns extraorally bonded to (A) stock or (B) CAD/CAM customized zirconia abutments. Table 2 Basic Demographic and Clinical Data of the Research Population Stock Customized Total Gender Male 6 11 17 Female 19 14 33 Age * Mean 48.6 48.1 48,3 Min 18 30 Max 79 71 Tooth Upper 1st premolar 9 6 15 Upper 2nd premolar 11 15 26 Lower 1st premolar 1 1 2 Lower 2nd premolar 4 3 7 *At the time of the placement of the restoration. No implants were lost and no complications related to the zirconia abutments (fracture or screw loosening) were noted in either group. Mean values for the clinical and subjective outcome parameters are presented in Tables 3 and 4 and Figure 4. Tissues were generally healthy, patients were satisfied with the result on all aspects of evaluation and expectations regarding the outcome of treatment were met or exceeded. No statistically significant differences could be demonstrated between the two groups, neither related to the implant restoration, nor related to the neighboring teeth. Table 3 Clinical Outcome Measures Two Weeks After Delivery of the Restoration ( T 1 ) and After 12 Months ( T 12 ), Standard Deviations Between Brackets T 1 T 12 Stock Customized Stock Customized Plaque‐index (0–3, median) 0 (0.51) 0 (0.49) 1 (0.57) 1 (0.40) Calculus‐score (0–1, median) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) Probing pocket depth (in mm, mean) 2.11 (0.60) 2.12 (0.79) 2.32 (0.85) 2.44 (0.78) Bleeding on probing (0–3, median) 0 (0.41) 0 (0.41) 0 (0.56) 0 (0.58) Gingiva‐index (0–3, median) 0 (0.20) 0 (0.00) 0 (0.41) 0 (0.54) Gingival margin apposition at the adjacent teeth (in mm, mean) 0.24 (0.77) 0.40 (0.99) Marginal bone level apposition (in mm, mean) 0.06 (0.23) 0.11 (0.20) Table 4 Pink Esthetic Score (PES) Based on Photographs Taken Two Weeks After Delivery of the Restoration ( T 1 ) and After 12 Months ( T 12 ), Standard Deviations Between Brackets T 1 T 12 Stock Customized Stock Customized PES (sum‐score, 0–14)* 9.2 (1.8) 9.0 (2.5) 10.9 (1.6) 10.6 (2.1) Papilla mesial* 1.0 (0.6) 1.0 (0.8) 1.6 (0.6) 1.7 (0.5) Papilla distal* 1.0 (0.7) 1.0 (0.8) 1.3 (0.8) 1.3 (0.7) Level of the soft tissue margin 1.4 (0.5) 1.5 (0.6) 1.6 (0.5) 1.6 (0.7) Soft tissue contour** 1.2 (0.4) 1.2 (0.4) 1.5 (0.5) 1.5 (0.6) Alveolar process deficiency 1.3 (0.5) 1.4 (0.7) 1.3 (0.5) 1.4 (0.6) Soft tissue color 1.8 (0.4) 1.5 (0.5) 1.8 (0.4) 1.6 (0.5) Soft tissue texture** 1.5 (0.5) 1.4 (0.5) 1.8 (0.4) 1.5 (0.6) Statistical significant improvement between T 1 and T 12 ( p  < .001)* ( p  < .01)**, but not between the groups. For the alterations of the radiographically determined marginal bone levels, the measured ICC was 0.910 indicating excellent reliability between the observers. On average, some marginal bone apposition was observed for both groups between T 1 and T 12 (Table 3). The difference in bone apposition between the 2 groups (stock: 0.06 mm, 95% CI [−0.05 mm; 0.16 mm], standard deviation 0.23mm versus CAD/CAM customized: 0.11 mm, 95% CI [0.02 mm; 0.20 mm]; standard deviation 0.20 mm) was not statistically significant. Clinical examination revealed a significant (T = 24; p < .05) coronal growth of approximal gingival margin (0.32 mm; 95% CI [0,06 mm; 0,58mm]; SD 0.88 mm) on the neighboring teeth after 12 months. There was no statistically significant difference between stock (0.24 mm; 95% CI [−0.09 mm; 0.57 mm]; SD 0.77 mm) and CAD/CAM customized abutments (0.40 mm; 95% CI [−0.01 mm; 0.81 mm] SD 0.99 mm, Table 3). Interobserver reliability was good regarding soft tissue margin and soft tissue color (ICC > 0.6) and excellent for all other PES items (ICC > 0.75). In general, the soft tissue appearance had improved after 12 months (T = 43, p < .001), predominantly because of papilla fill in the mesial and distal proximal areas (p < .001), soft tissue contour and texture (p < .01; Table 4). There were no significant differences between stock and CAD/CAM customized abutments on individual variables of the PES, as well as for the sum‐score at either moment in time. Differences between groups and over time with regard to plaque accumulation, dental calculus, PPD, bleeding tendency and gingiva‐index were generally small and none of them was statistically significant (Table 3). Patients were generally content with the achieved result as reflected by high VAS‐scores at T 1 and T 12 (Figure 4), without a statistically significant difference between the stock and CAD/CAM customized abutments. Expectations were mostly exceeded and general contentment was higher at T1 than patients had expected (T = 29; p < .01) (T 0) but not at T 12 (T = 19; p = .9). A similar pattern for chewing ability, confidence in loading the implant and patients' appreciation of the gum contour was found. Patients experienced significantly less embarrassment and problems with taste, speech, gum color, tooth color and the visibility of the implant at T 1 (p < .01) and T 12 (p < .05) than they had expected at T 0. Self‐confidence was also boosted more at both T 1 and T 12 than patients expected. No differences were found between patients' perceptions and their initial expectations with regard to tooth shape. BODY.DISCUSSION: Studies comparing stock and CAD/CAM customized abutments are rare. Marginal bone level alteration after one year of clinical service was the primary outcome measure of the present study. Some bone apposition was observed (0.06–0.11 mm), whereas according to a recent review, marginal bone resorption ranges on average from 0 to 0.99 mm after one year of function.5 In this review, two of the included studies show very little marginal bone resorption after one year of service (Hürzeler and colleagues30 0.12 mm, SD 0.40 mm, n = 14; Prosper and colleagues31 0.02 mm, SD 0.11 mm, n = 60), so it can be assumed that at least some individuals in those studies showed bone gain instead of bone resorption. A third study by Guljé and colleagues with the same implant type also found a small amount of bone apposition after one year.23 Therefore, measuring "bone level alteration" seems to be a more suitable terminology than measuring "bone resorption." From in vitro studies it was concluded that the specific stock and CAD/CAM customized abutments used, appeared to have a comparable fit for most of the systems evaluated,32, 33 as well as a comparable fracture strength.34 Through individual design, retention and resistance of implant crowns is optimized. In a retrospective clinical study loosening of single crowns cemented with zinc oxide eugenol cement was seen more frequently after 2 years when stock abutments were used compared to customized computer‐milled specimen.35 Stronger cement might have prevented cement failure in clinical practice, but also the design of the abutment and the restoration material used seem play a role.25 Comparing customized zirconia and titanium abutments with metal cast abutments of an undisclosed alloy on the same implant type as used in the present study led Borges and colleagues to conclude that papilla fill was enhanced in the customized abutment group after one year of function.36 However, since the design of a cast‐metal abutment allows the dental technician as much freedom of design as a CAD/CAM abutment, the choice of material or the inferior fit of a cast abutment,37 and not so much the mode of manufacturing may have influenced the result. In a recent multicenter trial, stock and CAD/CAM designed titanium and zirconia abutments were compared with respect to labial recession of the mucosa after 2 years. Titanium CAD/CAM abutments performed better than all other combinations.38 Since at the time of fabrication of the restoration zirconia was selected as abutment material in case of a labial mucosal thickness within 2 mm and titanium was selected as abutment material for situations with a labial mucosal thickness exceeding 2 mm, there was an obvious risk of selection bias. Results from the present study with respect to the position of the labial margin provide similar results when looking at the data for zirconia stock and CAD/CAM customized abutments. No relevant differences were seen for this parameter. For patients, the level of the mucosal margin does not appear to be of particular importance with respect to their appreciation of the aesthetic result in the anterior region anyway, in contrast to the papilla fill, which is considered important.39 In the present study, as also observed by others, papilla fill improved in time.40 However, again no differences were seen between the two abutment types with regard to papilla fill or any of the other clinical, radiographical or patient‐based outcome parameters. Small differences between the two groups might have remained statistically undetected due to the limited number of patients, but given the small differences observed, it is not likely that these will be relevant to clinical practice. Factors that have been reported to be of relevant influence on soft tissue development around implants are vertical implant position and bucco‐palatal angulation, initial soft tissue thickness and soft tissue grafting procedures, as well as the proximal bone level of the neighboring teeth.22 Despite randomization, stock and customized abutments were not evenly distributed among men and women in our study, which is not likely to be of major influence on the results. In general, patients were pleased with the achieved result. Their expectations were met or even exceeded, especially directly after placement of the restorations. We presume that a certain amount of euphoria might have increased the subjective contentment measurement shortly (two weeks) after the placement of the restoration rather than that the satisfaction decreased after one year of clinical service. A further drop in satisfaction might be possible, but does not appear to be very likely. The quality of care from the patients' perspective is largely determined and reflected by the ability of the dental team to meet the patients' expectations. It enhances the reputation of the individual physicians involved, the team as a whole and the field in general and can be accomplished with both, customized or stock abutments. No clinical or satisfaction factors favored one abutment over the other. As a consequence, the choice for a stock or a CAD/CAM customized zirconia implant abutment may just as well be based on secondary factors such as access to software, preference, ease of fabrication or price. BODY.CONCLUSIONS: The use of a CAD/CAM customized zirconia abutment in standard single tooth replacement of a premolar is not associated with a relevant improvement in outcome measures reflecting clinical performance, peri‐implant bone alteration, contentment or the degree to which patients' expectations are met when compared to the use of a stock abutment.

TITLE: Anesthetic Efficacy of Articaine and Ketamine for Inferior Alveolar Nerve Block in Symptomatic Irreversible Pulpitis: A Prospective Randomized Double-Blind Study ABSTRACT.INTRODUCTION:: The aim of this prospective, randomized, double-blind study was to investigate the effect of articaine combined with ketamine on the success rate of inferior alveolar nerve block (IANB) in posterior mandible teeth with symptomatic irreversible pulpitis. ABSTRACT.METHODS AND MATERIALS:: Forty two adult patients with diagnosis of symptomatic irreversible pulpitis of a mandibular posterior tooth were selected. The patients received two cartridges of either containing 3.2 mL 4% articaine with epinephrine 1:200000 and 0.4 mL 50 mg/mL ketamine hydrochloride (A-ketamine group) or 3.2 mL 4% articaine with epinephrine 1:200000 and 0.4 mL normal saline (A-saline group) using conventional IANB injections. Access cavity preparation started 15 min after injection. Lip numbness was required for all the patients. Success was considered as no or mild pain on the basis of Heft-Parker visual analog scale recordings upon access cavity preparation or initial instrumentation. Data were analyzed by independent student t, Mann-Whitney and Chi-square tests. ABSTRACT.RESULTS:: The success rates were 55% and 42.9% for A-ketamine and A-saline group, respectively, with no significant differences between the two groups (P=0.437). ABSTRACT.CONCLUSION:: Adding 0.4 mL 50 mg/mL ketamine hydrochloride to the articaine local anesthetic did not increase the efficacy of IANB for posterior mandibular teeth with symptomatic irreversible pulpitis. BODY.INTRODUCTION: Inferior alveolar nerve block (IANB) is currently considered the most frequent local anesthesia technique for endodontic treatment of mandibular teeth [1, 2]. Clinical research has shown a high failure rate for this technique even with a correct application [3, 4]. On the other hand, achieving adequate anesthesia in teeth with symptomatic irreversible pulpitis has also been proven to be difficult [5-7]. Therefore, anesthetizing posterior mandibular teeth with irreversible pulpitis has been a serious challenge for the dentist, and research to solve the problem would be beneficial [7, 8]. Ketamine hydrochloride is a non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors which has analgesic effects in sub-anesthetic doses. The analgesic effect of ketamine is strong through both the central and peripheral nervous system paths [9]. It has been used alone and in combination with other local anesthetics such as lidocaine, in adeno-tonsillectomy and herniorrhaphy [9, 10]. Combination of a local anesthetic and subanesthetic dose of ketamine during surgical extraction of third molars provides good postoperative analgesia with less swelling and significantly less trismus [11]. It should be noted that providing an effective, safe and painless anesthesia in teeth with symptomatic irreversible pulpitis is critical [7, 12]. Kaviani et al. [13], showed that oral administration of ketamine might be beneficial for enhancing the anesthetic efficacy of IANB in mandibular teeth with symptomatic irreversible pulpitis. Based on ketamine analgesic effects, it may improve the success rate of anesthesia. However, anesthetic efficacy of adding ketamine to anesthetic solution in such teeth has not evaluated yet. Hence, the aim of this prospective, randomized, double-blind study was to investigate the effect of articaine plus ketamine on the success rate of IANB in posterior mandibular teeth with symptomatic irreversible pulpitis. BODY.MATERIALS AND METHODS: Forty two adult patients participated in this prospective, randomized, double-blind study. They were referred to an approved dental clinic of Isfahan University of Medical Sciences for endodontic treatment. The Regional Ethics Committee affiliated to Isfahan University of Medical Sciences approved the study protocol (Grant No.: 388485). Also this study has been registered in the Iranian Registry of Clinical Trial (IRCT138905024440N1). A full description of the study was given to the patients with irreversible pulpitis, and an informed consent was obtained from each of the patients. The participants of this study included the healthy (ASA I or ASA II) patients, aging between 18 to 50 years, who had a vital posterior mandibular tooth (premolar or molar) with pain and a prolonged response (in comparison with control group) to cold testing with Endo-Frost cold spray (Coltene Whaledent, Langenau, Germany) and diagnosed with irreversible pulpitis, had no severe periodontal disease, or any periapical pathological defect seen in the dental radiographs and no contraindications for the drugs and substances used in the study. The exclusion criteria were set in patients showing signs of pulp necrosis upon access cavity opening, patients showing any signs or symptoms of allergy to anesthetic solution, pregnancy and breast-feeding, patients with pain in more than one mandibular tooth, and those who refused to continue at any stage point. Therefore, each patient had a vital mandibular molar tooth with a clinical diagnosis of symptomatic irreversible pulpitis. The patients marked their initial pain on a Heft-Parker Visual Analogue Scale (HP-VAS) [14]. The scale is a 170-mm marked line which is divided into 4 sections with different terms describing the level of pain. No pain, mild pain, moderate pain, and severe pain were indicated by 0 mm, 1-54 mm, 55-113 mm, and 114-170 mm divisions, respectively. Articaine plus ketamine (A-ketamine) local anesthetic solution was prepared as follows: Under sterile conditions, 0.2 mL from a 1.8-mL cartridge of 4% articaine with 1:200000 epinephrine (Artinibsa; Inibsa, Barcelona, Spain) was drawn and replaced with 0.2 mL ketamine (Ketamine Hydrochloride 50 mg/mL, Rotexmedica, Trittau, Germany) using a glass micro liter syringe (Hamilton, Bonaduz, Switzerland). For articaine plus normal saline (A-saline) local anesthetic solution, 0.2 mL from a 1.8 mL cartridge of 4% articaine with 1:200000 epinephrine was drawn and replaced with 0.2 mL of normal saline. A trained dental assistant prepared the two local anesthetic formulations and coded them in a random manner. Patients were assigned equally into two groups of A-ketamine or A-saline. One operator administered two cartridges of either A-ketamine or A-saline local anesthetic solution using IANB technique for each patient in a double-blind trial. Therefore, none of the participants and operators was aware of the group assignment. All the injections were performed using a 27-gauge, 1.5-inch needle (CK ject; CK Dental, Kor-Kyungji-do, Korea) attached to a standard aspirating dental injection syringe. Lip numbness was set as a criterion to IANB achievement. If lip numbness was not profound 15 min after the injection, the patient was excluded from the study. In the present study, no participant was excluded from the study as a result of a lack of lip numbness. Then, the teeth were isolated with a rubber dam, and access cavities were prepared. The patients were asked to rate the pain during the preparation of access cavity or initial file placement. If the patient felt pain, the treatment was stopped, and the patient marked their pain by using HP-VAS. The IANB injection was considered successful if patient felt no pain or mild pain (HP-VAS score≤54). Data were statistically analyzed using SPSS software (SPSS version 16.0, SPSS, Chicago, IL, USA) software. Comparisons between A-ketamine and A-saline groups for the success of the IANB, gender, and tooth type differences were analyzed by Chi square test; age was analyzed by independent t-test and initial pain was analyzed using the Mann-Whitney test. The comparison was considered significant at P<0.05. BODY.RESULTS: Out of 42 adult patients, 18 were women and 24 were men. The age range was 19-56 years, with mean±SD of 30±9.2 and 33±9.4 years in the A-ketamine and A-saline groups, respectively. The age, gender, and initial pain ratings for the A-ketamine and A-saline groups are presented in table 1. There were no differences in age, gender and initial pain between the two groups (P>0.05). Distribution of teeth for A-ketamine and A-saline groups is presented in table 2. There was no difference in tooth type between the two groups (P=0.68). The success rate of IANB was 55.0% for A-ketamine and 42.9% for A-saline group. There was no statistically significant difference in success rates between the two groups (P=0.437). BODY.DISCUSSION: This study was carried out to investigate the effect of articaine plus ketamine on the success rate of IANB in posterior mandible teeth with symptomatic irreversible pulpitis. The results showed that the success rate of IANB in A-ketamine group was a little more than A-saline group. However, the difference was not statistically significant. The participants' age, sex, tooth types and initial pain records were not different between the two experimental groups; therefore, any possible effect from these factors was minimized. Lip numbness indicates successful IANB but does not guarantee pulpal analgesia or anesthesia in all patients [15-18]. Moreover, electric pulp test also seems to overestimate the rate of success when compared to the pain felt by the patients during treatment [17, 18] and cannot ensure pulpal analgesia either [19, 20]. This is attributed to the difference between pulpal anesthesia which is determined by the electric pulp test and pulpal analgesia which is determined through successful clinical pain control of the patient [18]. Therefore, the success of IANB was evaluated by measuring the pain level during endodontic access and initial instrumentation using HP-VAS, and further tests with an electric pulp tester were eliminated in this study. The success rates of anesthesia in the case of pulpitis have been reported to be as low as 20% [15, 21] to as high as 65-70% [18, 22]. The wide range of results could be partly due to differences in the length of time when anesthesia was tested, means of testing pulpal anesthesia or analgesia (e.g. thermal stimulus, electrical stimulus, or clinical treatment), and population differences [16]. Combining anesthetic solution with some medications has been evaluated to overcome the local anesthetic failure of mandibular molars with irreversible pulpitis [23]. Evidence showed that mixing anesthetic solution with mannitol improved the anesthetic efficacy of IANB [24]. However, mixing anesthetic solution with some other medications such as tramadol [25], hyaluronidase [26], meperidine [15], diphenhydramine [27], sodium bicarbonate [28], carbonate [29] did not increase the anesthetic efficacy of IANB in patients with irreversible pulpitis. The results of this study showed that mixing the articaine anesthetic solution with ketamine could not increase the success rate of IANB in patients with irreversible pulpitis. Ketamine interferes with sodium ion channels as a local anesthetic and shares with a binding site of commonly used local anesthetics, increasing their anesthetic effect [30]. Tverskoy et al. [31] showed that after herniorrhaphy, ulcers were infiltrated with a solution of 0.5% bupivacaine and 0.3% ketamine, and an increased level of anesthesia was demonstrated. Furthermore, they found that subcutaneous infiltration with 0.3% ketamine produced a local anesthetic effect. They claimed that ketamine acts via a peripheral mechanism and increases the anesthetic and analgesic actions of local anesthetics completely [31]. In addition, ketamine, as an NMDA receptor antagonist, can block the action potential of nerve fibers by affecting the sodium and potassium ion channels in their membranes. Moreover, its high lipid solubility and fast absorption in the surrounding tissue leads to slight sedative effects and also ketamine was administered as a local anesthetic in order to make use of its analgesic effects [32]. Accordingly, it was theorized that success of IANB is better in A-ketamine group than the A-saline group. Although the results of the present study showed that IANB success rate for the A-ketamine group was a little more than that for the A-saline group, although the difference was not statistically significant. The insignificant effect of adding ketamine to articaine might be partly because of their different onset of action. The effects of ketamine following an intramuscular injection starts in approximately 30 min and lasts for about 15-30 min [32]. Table 1 Mean (SD) of preoperative values for the A-ketamine and A-saline groups Value A-ketamine A-saline P- value * Age (y) 30 (9.2) 33 (9.4) 0.473 Gender Women 9 9 0.451 Men 11 13 Initial Pain ** 96 (30.2) 101 (30.0) 0.571 * There were no significant differences between the two groups (P>0.05) Table 2 Distribution of teeth for A-ketamine and A-saline groups Tooth A-ketamine A-saline First premolar 3 (15%) 1 (5%) Second premolar 5 (24%) 5 (24%) First molar 7 (35%) 10 (48%) Second molar 6 (30%) 5 (24%) Intravenous injection of ketamine may results in the complete emergence of its systemic side effects such as fidgetiness, dizziness, and pulmonary distress [32]. However, in the present study a few patients in the A-ketamine group appeared to have signs of dizziness. The major limitation of the present study was the sample size. Multicenter, prospective, randomized, double-blind trials with more sample size are suggested to achieve more comprehensive results. BODY.CONCLUSION: Under the limited conditions of this study, adding 0.4 mL 50 mg/mL ketamine hydrochloride to the articaine local anesthetic did not improve the success of the IANB for posterior mandibular teeth in patients with symptomatic irreversible pulpitis.

TITLE: Active Experiencing Training Improves Episodic Memory Recall in Older Adults ABSTRACT: Active experiencing (AE) is an intervention aimed at attenuating cognitive declines with mindfulness training via an immersive acting program, and has produced promising results in older adults with limited formal education. Yet, the cognitive mechanism(s) of intervention benefits and generalizability of gains across cognitive domains in the course of healthy aging is unclear. We addressed these issues in an intervention trial of older adults (N = 179; mean age = 69.46 years at enrollment; mean education = 16.80 years) assigned to an AE condition (n = 86) or an active control group (i.e., theatre history; n = 93) for 4 weeks. A cognitive battery was administered before and after intervention, and again at a 4-month follow-up. Group differences in change in cognition were tested in latent change score models (LCSM). In the total sample, several cognitive abilities demonstrated significant repeated-testing gains. AE produced greater gains relative to the active control only in episodic recall, with gains still evident up to 4 months after intervention. Intervention conditions were similar in the magnitude of gains in working memory, executive function and processing speed. Episodic memory is vulnerable to declines in aging and related neurodegenerative disease, and AE may be an alternative or supplement to traditional cognitive interventions with older adults. BODY.INTRODUCTION: Age-related cognitive declines are associated with the development of dementia (National Institutes of Health, 2011) and impact the ability of older adults to live safely and independently (Blake et al., 1988; Grundstrom et al., 2012; Boelens et al., 2013). The rate and magnitude of decline differs across cognitive functions; whereas crystalized intelligence is relatively stable, for example, episodic memory follows a steep negative trajectory (Horn and Donaldson, 1980; Lindenberger et al., 1993; Park, 2000; Bherer et al., 2013). Further, a multitude of factors appear to shape an individual's aging trajectory (Raz and Kennedy, 2009), suggesting the possibility of intervening upon the process to potentially slow decline and promote successful aging. With this aim, various behavioral interventions have been proposed such as physical activity (Colcombe and Kramer, 2003) and cognitive training (Jaeggi et al., 2008; Karbach and Kray, 2009). AE as a form of mindfulness training has been recently identified as a promising avenue for short-term intervention that produces lasting gains in cognitive function among older adults (Noice et al., 2004; Noice and Noice, 2009). These studies provide a good initial framework for examining AE, though, the evidence of gains is limited; the use of small samples that included individuals with elevated risk factors for cognitive decline and limited assessment of multiple cognitive abilities make the generalizability of this finding across cognitive functions in healthy aging unclear. Here, we report the largest study to date of this promising intervention in healthy older adults, including an extensive test battery of cognitive tests. AE is a mindfulness exercise that encourages the individual to be cognizant of the immediate environment and internal state, and is commonly used as preparation for acting (Noice et al., 2004). AE was designed based on interactions with professional actors (Noice, 1996; Noice and Noice, 1999, 2001; Noice et al., 2000). The AE intervention for cognitive aging was developed by extrapolating acting strategies, including motor cues and mnemonic devices (Noice, 1996; Noice and Noice, 1999, 2006), which are often not explicitly demanded of actors but can contribute to successful script performance. AE interventions use a two-stage process of preliminary examination of the script, and engaging the character in rehearsal and performance (Noice et al., 2004). In an AE intervention, older adults with no prior acting experience are instructed to become their character and work to achieve the character's goals (Noice et al., 2004). They do this by "actively experiencing" the character on cognitive, emotional, and physiological levels. Previous evidence suggests that this activity may promote memory functions. Following a 4-week intervention, older adults participating in AE performed better than a no-contact control group on standardized tests of episodic memory and working memory span, but only problem solving ability was significantly improved relative to an active control that completed a visual arts course (Noice et al., 2004). Intriguingly, the AE intervention group maintained higher performance levels in these domains, with potential continued gains in episodic memory (Noice et al., 2004; Noice and Noice, 2009). Thus, AE may be an example of a brief, inexpensive, and enjoyable intervention that can have a sustainable impact on cognitive functions that typically decline during aging. Yet, in part due to the limited scope of cognitive batteries used in these prior reports, the potential mechanism(s) and specificity of AE benefits to memory function over other cognitive abilities are unclear. Unlike cognitive training regimens, AE interventions do not train participants on any specific cognitive tasks or provide any pertinent strategies that are specific to assessment. Instead, older adults indirectly memorize a script by actively engaging in their character and in response to their acting partner (Noice et al., 2015). The intent of this exercise is to use the dialog to achieve the character's motivation, before continuing to the next line of dialog. For example, if the script indicates a character flatter another character, then the first actor attempts to sincerely flatter the second actor using the exact wording in the script. Naturally, with repetition, this type of rehearsal leads to memorization of a short script (e.g., one-three pages), although it is not the explicit goal. In this manner, the AE intervention is a type of mindfulness exercise with the goal of verbatim memorization and recall of complex information, but deliberate memorization independent of the mindfulness exercise is discouraged. Based upon the intervention design, there are two plausible routes of cognitive gains: one, promoting executive control function that is expected to have more general benefits to cognition; another, bolstering mnemonic encoding and recall that would produce more specific gains in memory and problem solving ability. For example, AE as a form of mindfulness may be similar to meditation that is hypothesized to improve attentional control in executive function (Tang et al., 2015) to confer gains in memory and reasoning abilities (Zeidan et al., 2010; Tang et al., 2012), much like the prior reports of AE. However, unlike mindfulness meditation, participants in AE have an explicit task to engage and sincerely act out a script during every rehearsal. A second hypothesized mechanism is specific to memory function. The evaluation of a character's motivation based upon the written script and subsequent performance, and the requirement to respond in character to a dynamic scene, can be conceived as forms of memory training that encourage working memory function and episodic encoding and retrieval. The AE intervention does not explicitly train mnemonic devices, but it is plausible that the acting exercise itself promotes the use and practice of associative memory strategies that aid performance on laboratory memory tasks in older adults (Shing et al., 2008). Yet, for the lack of comprehensive assessment in previous reports, the mechanism(s) and specificity of benefits from AE to cognitive ability in older age is uncertain. We examined these hypothesized cognitive mechanisms of AE benefits by testing intervention-related changes in several cognitive functions—executive function, episodic and working memory, and processing speed—as well as the interaction between changes in different cognitive domains. These aims are aided by a sizable sample of older adults that is the largest study of AE to date. A substantial portion of the extant evidence has come exclusively from samples drawn from the same geographic region, of older age, low-middle socioeconomic status, some living in government-subsidized retirement communities, and on average achieving a high school level education (Noice et al., 2004; Noice and Noice, 2009). Each of these factors can impact cognitive function (Jefferson et al., 2011) and it is logical that carriage of greater risk may produce larger intervention effects. The present study addresses this limitation by implementing a 4-week, randomized control trial of AE intervention among community-dwelling older adults who, on average, obtained a university degree. This sample of older adults is also double the group size of those used in previous AE studies, providing greater power to detect effects and broader representation of the general population. Further, previous reports employed an ANCOVA approach to test intervention gains, which cannot evaluate individual differences in change or the relationship between concurrent and future changes in cognition (see McArdle, 2009). Here, we use latent change models for intent-to-treat analyses, which is the gold standard (McArdle, 2009) to test changes in cognition following intervention and up to 4 months later. Within this framework, we expect the AE group to experience greater gains in cognitive function as compared to the active control group. Additionally, evidence of correlated gains in executive function, working memory and episodic memory may lend insight into the underlying mechanism of AE. BODY.MATERIALS AND METHODS.PARTICIPANTS: The participants in this study were 179 community-dwelling adults aged 60–89 years (M = 69.46, SD = 6.59; 62% female), who on average had a college education (M = 16.80 years, SD = 3.48). For study enrollment, participants were right-handed, scored at least 23 on the MMSE (M = 28.69, SD = 1.39; Folstein et al., 1975), had no contraindication to MRI (not reported in the present article), and provided written consent for study participation. This study was carried out in accordance with the recommendations of the Institutional Review Board at the University of Illinois at Urbana-Champaign with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Review Board. Based on enrollment criteria and initial phone screening, 235 persons were enrolled in the study: 179 completed the intervention and were included in analysis; another 56 individuals were removed from the study due to violations of enrollment criteria that were identified retrospectively (Figure 1). Upon successful entry into the study, participants were assigned to either the active control (n = 86) or the AE condition (n = 93). Group assignment was pseudo random depending on the participant's time of enrollment. Attendance was monitored and participants were required to attend 75% of class sessions. The two groups were similar in age (t(176) = −0.79, p = 0.43), MMSE (t(176) = 0.22, p = 0.82) and years of education (t(176) = 0.86, p = 0.39), as well as representation of sex (χ(1)2 ≤ 0.15, p ≥ 0.70). After completing the 4-week intervention, participants returned for a post-intervention assessment (delay from the first assessment M = 51.48 days, SD = 14.78) and a follow-up 4 months later (delay from post-intervention assessment M = 127.89 days, SD = 10.35). The AE condition experienced a longer delay at post-intervention than the control (t(169) = −3.53, p < 0.001) but groups were similar in follow-up delay (t(143) = 1.78, p = 0.08) and delays between assessments were included as covariates in the model to account for this. Of the 179 individuals included in analyses, 33 did not return for the follow-up assessment, but were similar in demographic characteristics (all t < 1.55, p > 0.12) as those who did return and attrition was similar between the conditions (χ(1)2 = 0.76, p = 0.38). Thus, intent-to-treat analyses were conducted on the total sample and missing data were handled via full information maximum likelihood (FIML) estimation with the assumption of missing at random—a non-imputation approach that leverages all available data during latent model estimation (Muthén and Khoo, 1998). Figure 1CONSORT Flow diagram of participant recruitment, enrollment and attrition over the course of the intervention trial. BODY.MATERIALS AND METHODS.INTERVENTION: Participants were assigned to one of two groups: the Active Control that attended an Understanding the Art of Acting class or the intervention AE class. The control and intervention sessions occurred over a period of 4 weeks with both groups meeting two times a week for 75-min sessions, including a 15-min coffee break to encourage additional social interaction among the participants. Researchers with experience administering AE interventions chose the content of the classes and trained all outside instructors (TN and HN). The Active Control was designed as a theater appreciation class including talks, demonstrations and video clips of stage and film performances. The course topics included the styles of acting, in addition to the history of theater. The Active Control condition was designed to rule out the possibility that learning about a popular art form like acting, along with the social interaction of being engaged in a class, are sufficient to produce the significant improvement in cognitive functioning observed in the previous theatre interventions. The active experience training in the AE group was designed to be internally rewarding and non-competitive (Noice and Noice, 2009). The intervention has been described in detail in previous reports (Noice and Noice, 2009; Noice et al., 2015). Participants performed short scenes with a partner and scripts were 1–3 pages (large print) in length. The activities conducted in the class integrated four key concepts to teach the AE construct. First, participants were discouraged from "pretending" during sessions and instead instructed to perform every action as if it were real life. Second, participants were required to use their imaginations to mentally create the scenario in which they are asked to act. Third, participants were taught to be goal-driven by the scripted scenario and work through difficulties to achieve the character's goal. During the first 2 weeks of AE classes, participants were not required to memorize the scripts but were encouraged to spontaneously pursue the script, by responding to interactions or alterations immediately and naturally. During the training, participants were encouraged to access each of these concepts cognitively, emotionally, and physically. These main constructs were implemented in a range of lessons throughout the intervention period. Finally, during the last 2 weeks of class, participants were expected to perform their assigned scene verbatim from memory. All participants were in the same room during classes, and the training directors circulated to provide active feedback to the acting partners. In previous studies, participants learned very short scenes in less than 1 h and longer scenes in two or three 1-h sessions (Noice and Noice, 2009; Noice et al., 2015). BODY.MATERIALS AND METHODS.COGNITIVE MEASURES: Tasks were selected to address a range of cognitive domains including episodic memory, working memory, semantic knowledge, executive function and processing speed. All computer-based tasks were designed in E-prime version 1.1 (Psychology Software Tools, Pittsburgh, PA, USA) and administered on computers with 17 inch cathode ray tube (CRT) monitors. The same tests were repeated at pre- and post-intervention, and at the follow-up after 4 months. All assessments were administered and scored by research assistants who were blinded to group assignment. BODY.MATERIALS AND METHODS.COGNITIVE MEASURES.EPISODIC MEMORY: The Logical Memory task was taken from the Virginia Cognitive Aging Project (Salthouse and Ferrer-Caja, 2003; Salthouse, 2004, 2005, 2010). Two recorded stories were played for the participant. Each story contained 25 specific story details and eight thematic details. Immediately following the story, participants were asked to recall the story in as much detail as possible. Following the second story, participants were asked to recall as much detail as possible from the first story without having it played back, providing a measure of delayed recall. Number of details recalled for the specific story and thematic categories were used as separate measures of episodic memory. Latent constructs for story and thematic recall were each defined by immediate and delayed recall. BODY.MATERIALS AND METHODS.COGNITIVE MEASURES.SPATIAL WORKING MEMORY: The computerized spatial working memory (SWM) task was administered at variable difficulty memory loads of two, three and four target dots, randomly arranged on the computer screen. Participants completed 12 practice trials followed by 120 test trials. The dots were visible for 500 ms or 1000 ms, replaced with a fixation cross for 3000 ms. Following the fixation, a red dot either appeared in the same location where one of the black dots was or in an altered location. Participants indicated whether the dots were in the original or altered positions. Accuracy of responses for each of the three conditions was used as measures to identify the latent construct. BODY.MATERIALS AND METHODS.COGNITIVE MEASURES.VERBAL WORKING MEMORY: In the computerized N-back test, participants viewed a series of letters presented sequentially for 500 ms with an inter-trial interval of 2000 ms. Participants performed both a 1-back and a 2-back condition, with five runs of 20 letters presented for each condition. In the 1-back condition, participants were instructed to respond by pressing a button when the currently presented letter was the same as the previously presented letter (match trial), but to press a different button when the current letter did not match the previously presented letter. Instructions were similar for the 2-back condition that required participants to indicate if the currently presented letter was the same or different to the letter presented 2 trials previously. For both 1- and 2-back conditions, 50% of trials met the match rule. Response accuracy on each the 1- and 2-back conditions were used as measures to identify the latent construct. BODY.MATERIALS AND METHODS.COGNITIVE MEASURES.SEMANTIC MEMORY: Category fluency of animals and fruits/vegetables was administered to measure semantic memory. Participants were prompted with a category and orally recalled as many items as possible in that category within 1 min. The total number of correct responses per category was used as indicators of category fluency. BODY.MATERIALS AND METHODS.COGNITIVE MEASURES.EXECUTIVE FUNCTION: The computerized Task Switching test consisted of two individual tasks, each presenting a single digit (1–9, excluding 5) for 2500 ms. Participants were required to make a judgment about the presented digit based upon the background color. When presented on a pink background, participants were instructed to indicate if the digit was more or less than 5. When presented on a blue background, participants indicated if the digit was odd or even. Participants completed one block of 40 trials of each task rule individually followed by a block of 160 trials that required switching between response rules, designed to present randomly. The latent construct was defined by accuracy on the switch block and average accuracy of the non-switch blocks. BODY.MATERIALS AND METHODS.COGNITIVE MEASURES.PROCESSING SPEED: The latent processing speed construct was defined by two measures: Part A of the Trail Making Test and Digit Symbol Substitution Task. In Part A of the Trail Making Test, the participant was required to connect a total of 25 numbers (1–25) in ascending and alphabetical order, as quickly as possible without removing the pencil from the page. Longer completion time indicates slower processing speed. In the Digit Symbol Substitution Task participants were provided nine pairs of numbers and symbols, and then were required to fill in the matching symbol for a provided number using the key as a reference. There were 133 possible items and participants were given 2 min to complete as many items as possible working from left to right without leaving any blank. Greater number of correct responses was an index of faster processing speed. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSES: All analyses were completed in a latent modeling framework, estimated in MPlus software (v. 7; Muthen and Muthen). Changes in cognitive ability from pre- to post-intervention, and from post-intervention to follow-up after 4 months, were estimated in latent change score models (LCSM). A LCSM is similar to a difference score, but because it is determined by latent constructs, estimates of change and individual differences therein are free of measurement error (McArdle, 2009). In this model construction, sequential changes between measurement occasions were correlated, and pre-intervention performance was allowed to correlate with subsequent change. To construct the LCSM, several measurement features were imposed. Latent constructs were each determined by multiple measures, fixing one measure factor loading to 1, the other loadings were freely estimated, and all measurement residuals were freely estimated. The following latent constructs were estimated (italics indicates factor loading fixed to 1 for latent identification): story and thematic recall were each defined by immediate and delayed recall; category fluency by accuracy of recall for animal and vegetable/fruit categories; SWM by accuracy on the one, two and three dot conditions; verbal working memory by accuracy on 2-back and 1-back; task switching by accuracy on the switch and non-switch blocks; and processing speed by performance on digit symbol and Trails Part A (see Table 1). To ensure measurement invariance longitudinally, several constraints were added to the LCSM: estimated factor loadings were constrained to be equal over time, measure intercepts and variances were equal at each time point, and repeated measures were allowed to correlate but the magnitude of the correlation was constrained to be equal between occasions. There were a few exceptions: measurement variance of the 3-dot condition of the SWM task was freely estimated and measure intercepts of the task switching block were freely estimated. Table 1 Mean latent change at post-intervention and the 4-month follow-up in the total sample . Post-intervention 4 Month follow-up Construct Pre-intervention variance Mean change [BS 95% CI] Variance in change d Mean change [BS 95% CI] Variance in change d Story recall 0.73* 0.32* [0.21/0.42] 0.49* 0.37 0.12 [0.02/0.25] 0.50* 0.14 Thematic recall 0.73* 0.18 † [0.04/0.32] 0.87* 0.21 0.12 [0.01/0.26] 0.60* 0.14 Category fluency 0.61* 0.07 [−0.10/0.23] 1.00* 0.09 0.21* [0.09/0.36] 0.59* 0.27 Spatial WM 0.89* −0.04 [−0.20/0.12] 1.04* −0.04 0.12 [−0.02/0.26] 1.00* 0.13 Verbal WM 0.25* 0.09 [−0.03/0.19] 0.27* 0.18 0.06 [−0.01/0.17] 0.17* 0.12 Task switching 1.00* 0.14 † [0.02/0.26] 0.70* 0.14 0.16 † [0.03/0.30] 0.77* 0.16 Processing speed 0.85* 0.21* [0.13/0.28] 0.07 0.23 0.07 [−0.01/0.14] 0.08 0.08 Note: Unstandardized change scores reported for measures normed to the mean and standard deviation of the total sample at pre-intervention. All constructs are latent composites of multiple measures.*p < 0.01, † p < 0.05, α’ = 0.01. d is a standardized coefficient of change: d = Mean Change/√(Pre-intervention Variance). BS 95% CI, bias-corrected bootstrapped 95% confidence intervals; WM, working memory . Prior to model construction, all measures were normed to pre-intervention scores in the total sample. Thus, change scores can be interpreted as standardized change from pre-intervention. All reported effects are unstandardized coefficients. A standardized effect size of mean latent change was calculated: d = (Mean Latent Change)/√(Pre-intervention Latent Variance). Cognitive constructs that evidenced significant individual differences in change were further tested for covariates, including age, sex, delay between occasions, as well as group differences. Intervention group differences were tested in a grouped LCSM that included constraints for measurement invariance also between groups. Only the means and variances tested for group differences were freely estimated. To test whether changes in one cognitive ability predict change in another following intervention, parallel change score models were constructed. These included correlated change in cognitive constructs at each assessment occasion, as well as change from pre- to post-intervention in one cognitive ability predicting future change from post-intervention to follow-up in another ability. Model fit was determined by several accepted indices (Hu and Bentler, 1999): non-significant normal theory weighted chi-square (χ2), comparative fit index (CFI > 0.90), root mean square error of approximation (RMSEA < 0.05), and standardized root mean residual (SRMR < 0.08). Model fit was determined for the total sample and with grouped modeling procedures. Analyses were completed with the assumption of intent-to-treat and included the total sample. Missing data were handled via FIML—a non-imputation approach that leverages all available data during effect estimation (Muthén and Khoo, 1998; Larsen, 2011), and the current recommended practice for longitudinal studies with attrition (Little and Card, 2013). To avoid spurious results due to a smaller sample size, all LCSM were bootstrapped with bias-correction (5000 draws; Hayes and Scharkow, 2013) to produce 95% confidence intervals (BS 95% CI) of unstandardized effects. Due to the limitation on the number of parameters that can be reasonably estimated in proportion to the sample size, each cognitive construct was evaluated in a separate model. A Bonferroni correction was made for multiple comparisons (α' = 0.01). BODY.RESULTS.LONGITUDINAL CONSISTENCY IN MEASURES: Prior to testing longitudinal change, longitudinal consistency of the measures was evaluated with Pearson correlations between pre-intervention measures and subsequent testings. Performance on all tasks had acceptable longitudinal consistency: the highest consistency in performance on digit symbol (r = 0.85 and 0.83), and the lowest on n-back task 1-back (r = 0.30 and 0.59) and 2-back conditions (r = 0.55 and 0.49). BODY.RESULTS.LATENT LONGITUDINAL CHANGE IN COGNITIVE ABILITY: Within the entire sample, mean changes from pre- to post-intervention and from post-intervention to the follow-up 4 months later were tested in sequential LCSM. All models had excellent fit: χ(7–31)2 = 8.73–40.49, p = 0.05–0.79; CFI = 0.97–1.00; RMSEA = 0.00–0.07; SRMR = 0.02–0.05. Story recall improved at post-intervention (mean = 0.32, p < 0.001, α' = 0.01; BS 95% CI: 0.21/0.42), as did thematic recall (mean = 0.18, p = 0.03, α' = 0.01; BS 95% CI: 0.04/0.32), although the effect did not survive correction for multiple comparisons. Both tasks evidenced additional gains at follow-up 4 months later, supported by BS 95% CI that do not overlap with zero, but neither effect reached statistical significance (Table 2). Task switching also showed nominal gains at post-intervention (mean = 0.14, p = 0.04, α' = 0.01) and follow-up (mean = 0.16, p = 0.04, α' = 0.01), whereas category fluency only improved at follow-up (0.21, p = 0.005, α' = 0.01; BS 95% CI: 0.09/0.36). Performance on all other tasks was stable over the course of study (Table 2). However, individuals varied in the magnitude of gains in performance on all constructs, except processing speed, and we went on to test possible intervention group differences in performance changes. In addition to intervention group differences, several covariates were tested to explain individual differences in change: pre-intervention performance, age, sex and the delay between assessments as control variables, as well as correlated changes at post-intervention and follow-up. See Table 2 for a summary of all covariates to change. Better performance at pre-intervention was associated with lesser gain at post-intervention in all constructs (r = −0.48 to −0.10, p < 0.001); the same pattern was not consistently observed at follow-up, but individuals who showed greater gains at post-intervention experienced lesser gain at follow-up (r = −0.34 to −0.23, p < 0.001). Final models of covariates had good fit: χ(28–55)2 = 31.11–59.45, p = 0.01–0.33; CFI > 0.95; RMSEA < 0.06; SRMR < 0.06. Possible intervention effects were tested as group differences in the magnitude of change in each cognitive domain, accounting for pre-intervention performance, age and delay between assessments as covariates. Table 2 Covariates of latent change . Construct Change score Pre-intervention performance Age Sex Delay Story recall Post-intervention −0.23* −0.08 0.18 −0.02 Follow-up −0.10 0.14 † −0.03 −0.08 Thematic recall Post-intervention −0.59* 0.06 0.23 −0.10 Follow-up −0.20 −0.01 −0.24 0.01 Category fluency Post-intervention −0.56* 0.33* −0.08 0.00 Follow-up 0.44* −0.39* 0.21 0.01 Spatial WM Post-intervention −0.30* −0.05 0.08 −0.06 Follow-up 0.06 −0.10 0.00 −0.05 Verbal WM Post-intervention −0.10* −0.11 † 0.12 0.00 Follow-up 0.01 0.10 † −0.13 0.02 Task switching Post-intervention −0.33* −0.04 0.22 −0.08 Follow-up −0.24* 0.07 −0.16 −0.01 Note: All covariate effects estimated in latent change score models. Unstandardized coefficients reported for measures normed to pre-intervention means and standard deviations in the total sample. *p < 0.01, † p < 0.05, α’ = 0.01. Constructs are latent composites of multiple measures. WM, working memory . BODY.RESULTS.INTERVENTION GROUP DIFFERENCES: Prior to evaluating group differences in latent change, groups were confirmed to be statistically similar in performance on cognitive measures at pre-intervention (all t(176) = −1.28 to 1.90, p > 0.06), except the AE condition performed worse on immediate story recall (t(176) = 2.09, p < 0.04) and immediate (t(176) = 2.39, p = 0.02) and delayed (t(176) = 3.18, p = 0.002) thematic recall. In latent models testing group differences in change, we constrained pre-intervention latent episodic memory scores to be equal between groups to confirm that this was not a bias in the analysis. The AE intervention produced greater gains in episodic memory than Active Control. Grouped models of episodic memory had excellent fit (story and thematic recall, respectively): χ(62)2 = 78.51 and 65.78, p = 0.08 and 0.35 (AE = 39.49 and 43.23; Active Control = 39.02 and 22.55); CFI = 0.97 and 0.99; RMSEA = 0.06 and 0.03; SRMR = 0.06. The AE condition experienced significant improvement in story recall at post-intervention (0.44, p < 0.001; BS 95% CI: 0.31/0.59) and a trend for the same at follow-up (0.20, p = 0.05, BS 95% CI: 0.04/0.37) whereas the Active Control group showed no significant change in performance over the study (Table 3). Indeed, the intervention produced significantly greater gains at post-intervention as compared to Active Control (difference = 0.26, p = 0.04, BS 95% CI: 0.07/0.46), but additional gains at follow-up were similar between groups (difference = 0.16, p = 0.25, BS 95% CI: −0.06/0.38; Figure 2). A similar pattern was observed for thematic recall—the AE intervention produced greater gains at post-intervention (difference = 0.49, p = 0.002, BS 95% CI: 0.23/0.75) but not at follow-up (difference = 0.04, p = 0.78, BS 95% CI: −0.19/0.29). Although the magnitude of change did not significantly differ between groups at follow-up, the intervention produced a different pattern of gains in episodic memory over the course of study—suggesting better maintenance (and potentially continued gains) of recall ability up to 4 months after the intervention. To ensure that this intervention-related effect was not an artifact of group differences in pre-intervention performance level, we imposed additional model constraints that held groups to be equal at pre-intervention, and the AE condition still demonstrated greater gains in thematic recall at post-intervention (difference = 0.40, p = 0.01; BS 95% CI: 0.14/0.66) and the test of the effect in story recall (difference = 0.19, p = 0.10; BS 95% CI: 0.00/0.38) was likely underpowered based upon the BS 95% CI that slightly overlapped with zero. Therefore, the evidence of group differences in repeated-testing gains in episodic memory is likely an intervention effect and not a bias from a possible ceiling effect in performance. Table 3 Intervention group differences in change at post-intervention and 4-month follow-up . Change at Post-Intervention Change at Follow-up Construct AE [BS 95% CI] Control [BS 95% CI] Difference [BS 95% CI] AE [BS 95% CI] Control [BS 95% CI] Difference [BS 95% CI] Story recall 0.44* [0.31/0.59] 0.18 [0.02/0.33] 0.26 † [0.07/0.46] 0.20 † [0.04/0.37] 0.04 [−0.10/0.19] 0.16 [−0.06/0.38] Thematic recall 0.41* [0.22/0.64] −0.07 [−0.21/0.09] 0.49* [0.23/0.75] 0.13 [−0.05/0.33] 0.09 [−0.06/0.24] 0.04 [−0.19/0.29] Category fluency 0.06 [−0.15/0.28] 0.09 [−0.17/0.33] −0.03 [−0.35/0.31] 0.15 [−0.01/0.32] 0.26 † [0.07/0.47] −0.10 [−0.39/0.13] Spatial WM −0.07 [−0.28/0.13] −0.06 [−0.27/0.16] −0.01 [−0.29/0.27] 0.11 [−0.06/0.31] 0.12 [−0.12/0.36] −0.01 [−0.31/0.29] Verbal WM 0.11 [−0.05/0.26] 0.21* [0.07/0.36] −0.10 [−0.32/0.09] 0.05 [−0.05/0.19] −0.03 [−0.10/0.09] 0.07 [−0.07/0.22] Task switching 0.10 [−0.14/0.27] 0.18 † [0.04/0.32] −0.08 [−0.34/0.15] 0.20 [0.03/0.41] 0.09 [−0.10/0.32] 0.11 [−0.18/0.38] Note: Intervention group comparisons were made in a grouped latent change score model, including covariates and constraints to ensure measurement invariance longitudinally and between groups. Unstandardized coefficients are reported for measures normed to pre-intervention means and standard deviations of the total sample.*p < 0.01; † p < 0.05; α’ = 0.01. AE, active experiencing intervention condition; Control, active control condition; BS 95% CI, bias-corrected bootstrapped 95% confidence intervals; WM, working memory . Figure 2Intervention group differences in latent change in cognitive ability at post-intervention and at follow-up, 4 months later. The Active experiencing (AE) condition produced significantly greater gains in story and thematic recall in episodic memory as compared to the control group at post-intervention, but not at follow-up. Groups were similar in the magnitude of change in all other cognitive constructs. Intervention group comparisons were made in a grouped latent change score model, including covariates and constraints to ensure measurement invariance longitudinally and between groups. Unstandardized coefficients are reported for measures normed to pre-intervention means and standard deviations of the total sample. Error bars represent bias-corrected bootstrapped 95% confidence intervals; *p < 0.01; †p < 0.05; α' = 0.01. AE, AE intervention condition; Control, active control condition. Group differences were only detected in episodic memory ability and groups were equivalent in changes in all other constructs (Table 3). All other group models of cognitive ability had excellent fit: χ(46–117)2 = 46.65–131.46, p = 0.17–0.45 (AE = 25.17–54.55, Active Control = 21.48–76.91); CFI > 0.98; RMSEA < 0.04; SRMR < 0.07. Except the model of task switching that had less-than-optimal fit due to violations to the assumption of measurement invariance between groups: χ(55)2 = 96.33, p < 0.001; CFI = 0.96; RMSEA = 0.09; SRMR = 0.09. BODY.RESULTS.CORRELATED CHANGES IN COGNITION: Although groups did not differ in the magnitude of change in any domain besides episodic memory, individuals varied in changes in several cognitive domains and the pattern of correlated changes across domains may lend insight into the mechanism of AE intervention benefits. In a parallel latent change score model, we evaluated correlations between concurrent changes in cognitive constructs, including story and thematic recall, executive function and working memory. This was first examined in the total sample. As expected, individuals who experienced greater gains in story recall also showed gains in thematic recall immediately following the intervention (r = 0.25, p < 0.001) and at follow-up (r = 0.19, p < 0.001). Greater gains in executive function from post-intervention to follow-up also correlated with concurrent gains in thematic recall (r = 0.11, p = 0.03) but not in story recall at that time (r = 0.07, p = 0.17), and changes from pre- to post-intervention were not correlated with concurrent changes in thematic (r = 0.001, p = 0.99) or story recall (r = 0.04, p = 0.44). Changes in working memory were unrelated to concurrent change in episodic recall at post-intervention (r = −0.06 and r = 0.02, p > 0.09, story and thematic recall, respectively) or at follow-up (r = 0.01 and r = 0.00, p > 0.77, respectively). Thus, while performance on story and thematic recall was correlated, it did not consistently associate with concurrent changes in executive function or working memory. To further evaluate a possible cognitive mechanism of AE intervention gains in episodic memory, the parallel change score model included a test of change in executive function and working memory from pre- to post-intervention predicting future change in episodic recall from post-intervention to follow-up. However, there was no evidence of change in one cognitive domain predicting future change in another. Change in executive function immediately after the intervention did not predict future change in episodic recall assessed 4 months later (b = −0.11 and b = −0.06, p > 0.29, story and thematic recall, respectively), nor did change in working memory (b = −0.17 and b = −0.12, p > 0.46, story and thematic recall, respectively). Due to the lack of evidence for these effects in the total sample, intervention group differences were not further tested. Taken together, group intervention effects were limited to episodic recall and gains in this cognitive domain were unrelated to changes in executive function and working memory. BODY.DISCUSSION: Typical aging is characterized by cumulative and progressive declines in cognition (Horn and Donaldson, 1980; Lindenberger et al., 1993; Park, 2000; Bherer et al., 2013) and the prospect of interventions to slow this decline is intriguing. AE, a form of mindfulness, is a promising intervention that has not been widely explored. Here, we find that a group of older, community-dwelling adults who completed a 4-week AE intervention experienced greater repeated-testing gains in episodic memory recall than the Active Control group. These gains in function were maintained up to 4 months later, although the intervention groups did not significantly differ in performance at follow-up. This is the largest study of AE to date and included a broad range of cognitive assessments. Yet, there was no evidence of intervention benefits to other cognitive abilities, suggesting that AE may specifically bolster episodic memory in healthy aging. Thus, the mechanism of the AE intervention is likely closely related to mnemonic encoding and recall to confer gains within this domain, albeit without additional global cognitive benefits. The pace and magnitude of declines during aging vary across cognitive domains and episodic memory appears to be particularly sensitive (Horn and Donaldson, 1980; Lindenberger et al., 1993; Park, 2000; Bherer et al., 2013). Thus, short-term interventions that bolster this function with sustained benefits could have major implications for public health. Several intervention approaches, including aerobic exercise (Colcombe and Kramer, 2003; Smith et al., 2010; Roig et al., 2013), cognitive training (Melby-Lervåg and Hulme, 2013), and mindfulness meditation (Tang et al., 2015) have been tested and produce mixed results in improving memory function in older adults. Here, we partially replicate previous reports of AE improving memory function relative to active control groups (Noice et al., 2004; Noice and Noice, 2009). Importantly, the present study reports an extensive range of cognitive assessments never before administered in this type of intervention, of which only episodic memory showed improvements. This result indicates a potential selective intervention benefit via a mechanism specific to mnemonic function, and not global improvements in executive function or working memory, for example. Although we find evidence for a selective effect, we can only speculate on the precise mechanism by which AE bolsters memory ability, and it is plausible that it relates to improved use of mnemonic strategies for encoding and retrieval. The AE training required the participant to evaluate a character's motivation and affect in the course of performing a script. Although participants were not instructed to deliberately memorize the script, the repetition and creative development of the character in relation to the scene performance is conceptually similar to elaborative mnemonic strategies that improve subsequent recall in the laboratory (Hertzog and Dunlosky, 2004; Preston and Eichenbaum, 2013). Older adults appear to spontaneously use such strategies less frequently and less effectively than their younger counterparts do, partially explaining worse recall accuracy in later life (Kausler, 1994; Verhaeghen and Marcoen, 1994). Moreover, age-related deficits in episodic recall can be mitigated by supplying deep encoding strategies (Shing et al., 2008) that are similar to those derived from AE training. AE does not explicitly train mnemonic strategies or specific cognitive functions, yet rehearsal over 4 weeks may have encouraged older adults to spontaneously use deep encoding strategies more frequency and effectively when performing tasks other than acting. In this regard, AE training may have implicit benefits to episodic memory function. Moreover, these benefits were sustained up to 4 months later, similar to the long-term benefits of mnemonic strategy training evidenced years after intervention (Gross and Rebok, 2011). However, without independent reports of strategy use, we can only speculate on the source of gains in episodic recall. An alternative hypothesized mechanism of AE benefits to memory and problem solving that have been reported previously (Noice et al., 2004; Noice and Noice, 2009) is a boost in executive control functions that, in turn, promotes cognitive ability, similar to the putative mechanism of benefits following mindfulness meditation (Tang et al., 2012, 2015). However, we found no evidence of intervention gains in other cognitive abilities, including executive function and working memory, and individual differences in the magnitude of change in these domains did not predict subsequent change in episodic recall. Thus, the AE intervention does not appear to directly target executive function per se. The present results are not completely consistent with previous reports of AE (Noice et al., 2004; Noice and Noice, 2009). This may in part be due to differences in sample characteristics. Although the health of previously reported samples was not thoroughly documented (Noice et al., 2004; Noice and Noice, 2009), previous reports were on samples that were older, of lower SES and education level than participants in the current study. Each of these demographics are proxies for multifarious processes in aging that have been shown to predict steeper cognitive declines (Jefferson et al., 2011). Because greater risk can moderate the magnitude of intervention effects (e.g., Colcombe and Kramer, 2003; Smith et al., 2010; Danielsson et al., 2015), the generalizability of AE benefits in cognition to samples with lesser concomitant risk can be questioned. Here, we partially replicate the previous reports in a college-educated and healthy aging sample, and failure to find effects outside of episodic memory may reflect the sample selection. Future studies should consider additional health factors that may account for individual differences in responsiveness to the intervention. Moreover, individual differences in brain structure and function may interact with health factors and demonstrate change in response to the intervention to further explain cognitive function, as has been documented with mindfulness meditation (Tang et al., 2015). We aim to address these hypotheses in future reports and intentionally limited this initial report to the analysis of primary cognitive outcomes. Nonetheless, the moderate effect sizes within episodic memory function measured in this sample and the sustained effect 4 months later demonstrate the promise of the AE intervention in the course of normal cognitive aging. The current report replicates and expands the extant literature on AE and employs a robust analytic approach. Yet, the evidence should be interpreted with consideration of several limitations. In addition to the possible bias introduced by strict sample selection from the Champaign-Urbana, IL metro area, the sample was compromised by some attrition. However, this is the largest sample to date testing the effects of AE. Further, intent-to-treat analyses were completed on the entire sample and we handled missing data via FIML—a non-imputation approach that leverages all covariance information available during model estimation. To avoid spurious results from the smaller sample size, estimated effects were bootstrapped with bias-correction to produce 95% confidence intervals. Yet, we cannot completely eliminate possible bias related to sampling characteristics. A second limitation of the study is pseudo randomization of group assignments, which may be reflected in group differences in episodic memory performance at pre-intervention. The latent models that assessed intervention group differences included constraints to account for this, but we cannot completely account for this possible source of bias. Future studies should consider a true randomization scheme. A third limitation is our assessment of sustained benefits only 4 months after intervention. Longer delays with multiple measurement occasions are necessary to evaluate this further. Despite these limitations, we offer promising evidence of AE intervention benefits to episodic memory function in healthy aging and identify propitious avenues of future study. BODY.CONCLUSION: Previous reports of AE identified it as a promising intervention to promote cognitive function into older age, yet the mechanism of intervention benefits as well as the generalizability of gains across cognitive domains in the course of healthy aging had not yet been examined. Here, we identified specific gains in episodic recall from AE relative to the Active Control, but no other evidence of intervention gains in cognition. The cognitive mechanism of AE intervention benefits appears to be specific to mnemonic encoding and retrieval, as individual differences in executive function and working memory were unrelated to subsequent change in episodic recall. Episodic memory is particularly vulnerable to decline in aging and here we find promising evidence of intervention benefits in a healthy aging sample that is larger than any previously reported in an AE intervention. Because sustainable benefits were seen after a relatively brief intervention, AE may be a promising activity to slow episodic memory declines that are typical in aging. However, despite the extensive neuropsychological battery, no other cognitive domains exhibited benefits from the AE intervention, in contrast to expectations and previous reports. Future studies that include additional measures of brain structure and function, and other health factors, may substantiate AE as an effective intervention to promote successful aging. BODY.AUTHOR CONTRIBUTIONS: SEB, AMD, JRB, GEC, MV, TN, HN and AFK: manuscript preparation and editing; SEB, AMD, JRB, GEC and MV: data analysis and interpretation; GEC, MV, TN, HN and AFK: study design; AFK, TN and HN: study funding. BODY.CONFLICT OF INTEREST STATEMENT: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

TITLE: Treatment of Lumbar Tuberculosis by Mini-Open Anterior Approach Focal Cleaning Combined with Posterior Internal Fixation ABSTRACT.BACKGROUND: The aim of this study was to evaluate the efficiency and clinical outcomes of mini-open anterior approach focal cleaning combined with posterior internal fixation compared to conventional anterior extraperitoneal approach focal cleaning combined with posterior internal fixation in the treatment of lumbar tuberculosis (TB). ABSTRACT.MATERIAL/METHODS: Medical records from 124 patients were collected from February 2010 to April 2015; patients were divided into two groups: group A (mini-open anterior approach focal cleaning combined with posterior internal fixation) and group B (conventional anterior extraperitoneal approach focal cleaning combined with posterior internal fixation in period I). The data on postoperative mechanical ventilation time, preoperative, postoperative, and last follow-up Cobb angle, visual analog scale (VAS), erythrocyte sedimentation rate (ESR), and Frankel classification were collected and analyzed. Operative complications, internal stability, and bone graft fusion were also observed. ABSTRACT.RESULTS: All patients were followed-up for 12 to 36 months (average 22.5 months). Seven cases (five in group A and two in group B) had side psoas abscess and were cured after secondary drainage surgery. The rest of the cases were all cured after primary surgery, with no formation of sinus, incisional hernia, cerebrospinal fluid leakage, or recurrence of spinal TB, with no TB symptoms. Bone graft fusion ranged from 3 to 8 months (average 4.7 months). Compared to group B, group A, which had less time on postoperative mechanical ventilation, had a higher VAS score. Both groups had distinct improvements in Cobb angle, ESR, and Frankel classification after surgery. ABSTRACT.CONCLUSIONS: Treating lumbar TB by mini-open anterior approach focal cleaning combined with posterior internal fixation was safe and effective. BODY.BACKGROUND: Lumbar tuberculosis (TB) still occurs in both developing and developed countries including China and India; lumbar TB may cause kyphosis deformity, neurological deficits and complications, and paraplegia [1–3]. It can be difficult to diagnosis as it often occurs at an advanced stage, which causes a delay in establishing early diagnosis and good curative effect [4,5]. And at the same time, because there are no clear management strategies and the guidelines for lumbar TB, its treatment remains a challenge. Traditional treatment options [5–8] include anti-TB therapy, focal cleaning, and internal fixation at an earlier stage which may reconstruct the stability of spine and shorten the healing period. The best choice for internal fixation material of lumbar TB is a plate for the anterior and a pedicle screw for the posterior [9]. Furthermore, the method of pedicle screw fixation should be conducive to the patients' activities, and is more commonly used in thoracolumbar spinal TB as it is considered better and much more robust than plate fixation [10]. However, more segments are needed for posterior spinal fixation than for anterior fixation, which may impact the normal vertebral body. There is controversy over how to balance the use of these approaches in lumbar TB surgeries [11–13]. The objective of our study was to evaluate the efficiency and clinical outcomes of mini-open anterior approach focal cleaning combined with posterior internal fixation compared to conventional anterior extraperitoneal approach focal cleaning combined with posterior internal fixation. BODY.MATERIAL AND METHODS.CLINICAL INFORMATION: One hundred and twenty-four patients with lumbar TB, which was diagnosed by postoperative pathological examination and bacteriological culture, were analyzed in this study, including 76 male and 48 female aged from 20 to 85 years (average age 50.89 years). All of the patients had a paraspinal abscess initially, and were hospitalized at Integrated Chinese and Western Medicine Hospital of Zhejiang Province from February 2010 to April 2015. This work was approved by the Ethics Committee of Integrated Chinese and Western Medicine Hospital of Zhejiang Province. Written informed consent was obtained from all patients. The duration of follow-up was 5 to 22 month (average period 9.5). Patients had varying degrees of back pain, rigidity, and limitation in flexion and rotation. The patients were divided into two groups. Seventy-six patients were in group A, while 48 patients were in group B. The mini-open anterior approach focal cleaning combined with posterior internal fixation in period I was used in group A, and conventional anterior extraperitoneal approach focal cleaning combined with posterior internal fixation in period I was used in group B. Thirty-seven patients (22 patients in group A and 15 patients in group B) had nerve symptoms, such as lower limb numbness, asthenia, and sphincter disturbance. Nineteen patients (12 patients in group A and 7 patients in group B) had typical TB symptoms, such as low fever, night sweat, and hyperdynamic. There were no significant differences in sex, age, course of the disease, and segmental lesions between group A and group B (p>0.05) (Table 1). Imaging examination showed that vertebral body and intervertebral disc were damaged and paravertebral abscess were observed. T2W1 scanning showed vertebral body compressed and distorted with the heterogeneous high signal. Stream high signal was seen in soft tissue space. The imaging diagnosis was in accordance with the manifestation of lumbar TB. There were 96 cases involving two vertebral bodies and 28 cases involving three vertebral bodies (shown in Table 1). Eleven cases (six in group A and five in group B) had sinus involvement. ERS, Cobb angle, Frankel classification [14] and VAS were evaluated on admission. BODY.MATERIAL AND METHODS.INCLUSION AND EXCLUSION CRITERIA: Inclusion criteria were as follows: spinal instability or kyphosis deformity; neurological impairment and paraspinal abscess; severe pain in the back or lumbar root pain; inefficacious conservative treatment. Exclusion criteria were as follows: the destruction of L1 upper vertebral body and L5 lower vertebral body; thoracic vertebrae TB and sacral TB. BODY.MATERIAL AND METHODS.ANTI-TB THERAPY: All patients had good compliance with TB medications. Isoniazid, rifampicin, pyrazinamide, and ethambutol were used for conventional anti-TB chemotherapy for two to four weeks. Individual anti-TB treatment was used for elderly patients who had abnormal liver functions and allergic reactions. Systemic nutritional support was strengthened. Blood transfusions were used to improve anemia if necessary. TB symptoms and erythrocyte sedimentation rate (ESR) changes were observed. The best operation time was considered to be when symptoms were alleviated, e.g., body temperature ≤37°C, hemoglobin >100 g/L and ESR declined to 60 mm/hour. BODY.MATERIAL AND METHODS.PREPARATION FOR SURGERY: X-ray, CT scan, 2-D reconstruction, and MRI examinations were performed. Focal cleaning approach was chosen based on the range of vertebral destruction and location of the abscess. Individual fixation program was determined by unilateral or bilateral, upward or downward vertebral destruction, and whether there was interference from lesions for placed nail and focal cleaning. BODY.MATERIAL AND METHODS.SURGERY ASSAYS: The blood test results and imaging performance determined the duration of the treatment. Patients were placed in the prone position. Posterior midline approach was done with affected vertebra as a center. Facet joints of both sides were exposed by paraspinal muscle approach. Individual pedicle screws were placed into lesions according to the degree of preoperative vertebral damage. Pedicle screws were 30–45 mm and placed in the upward or downward 1–2 normal vertebral. If there were two vertebral lesions, 2–3 pedicle screws were placed into vertebral, and pedicle screw was placed in the upward or downward 1 normal vertebral. If there were three vertebral lesions, 2–5 pedicle screws were placed into vertebral, and pedicle screw was placed in the upward or downward 1–2 normal vertebral. Distraction reduction and kyphosis correction were done by fixation system (Figure 1A–1G). Patients were in right lateral position in group A. Anterior retroperitoneal approach was used in this study, an approach which is also called inverted "eight" incision. The procedures were as follows: skin incisions for lumbar vertebrae; incision of fibers of external oblique muscle; incision into fibers of internal oblique muscle; exposure of spine before ligation of segmental vessels. The specific method has been described previously [15]. The vertebra with TB was chiseled to show normal bone of the vertebra, with oozing of blood and without caseous material and sequestrum at the surface. Proper iliac bone autograft was cut out for the bone graft. Autologous bone graft was advocated as first-line treatment as it is easier to heal and has good histocompatibility, which can reduce infection. The donor site of the autologous bone graft was usually the ipsilateral ilium. Aseptic technique was used during surgery. When we harvested the bone grafts, disinfection was applied again around the donor site. Gelatin sponge wrapped with streptomycin was applied into the surgical area to kill any remaining mycobacterium TB. Finally, a drainage tube was placed and wounds were sutured. Patients were in right lateral position in group B. Conventional extraperitoneal approach was chosen and the incision was 12–18 cm. After incision with an electrotome, obliquus external abdominis, the internal oblique muscle of abdomen, and the transversus abdominis were separated. The posterior peritoneum was separated, too. The subsequent operations were like group A. BODY.MATERIAL AND METHODS.POSTOPERATIVE TREATMENT: Postoperative drainage tubes were removed 48–72 hours after surgery. Antibiotics were used for 2–3 days. All patients receive braces, and were encouraged to get up and moved around. Most patients could get up and move around after one week post surgery. Few patients had severe preoperative nerve compression. After patients with Frankel grade B or C recovered to grade D or E, they were encouraged to get up and moved around. Systemic nutritional support and anti-TB treatment were continued after surgery (3 HRZE/9-15 HRE). Routine blood tests, ESR, and hepatic and renal function were reviewed every 2–4 weeks during anti-TB treatment. If streptomycin was used, urine was routinely tested. Lumbar CT and MRI were reviewed every 3–6 month after surgery to inform the status of lesion healing, bone graft fusion, and whether there were loosening in the pedicle screw and/or fractures. After TB was controlled and bone graft fusion occurred, the fixation materials were removed (after 1.5–2.5 years). BODY.MATERIAL AND METHODS.OBSERVATION INDEXES: General information (operation time, blood loss, hospitalization days, and anal exsufflation time), clinical symptoms, radiological measurement, laboratory examination (VAS scores, Cobb angle, and ESR one week before surgery, after surgery, and at last follow-up), and Frankel classification was observed. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: SPSS 19.0 was used for the data analysis. The data of sex and the structure of lesions segments were analyzed by the χ2 test. Age, course, operation time, blood loss, postoperative hospital stay, VAS score, Cobb angle, and ESR were analyzed by t-test. Frankel classification was analyzed by Wilcoxon signed-rank test. A p value <0.05 was considered statistically significance. BODY.RESULTS: All patients were followed-up for 12 to 36 months (average 22.5 months). Seven patients (five in group A and two in group B) had side psoas abscess that were healed after secondary drainage surgery. The rest of the cases healed after primary surgery, with no formation of sinus, incisional hernia, cerebrospinal fluid leakage, or recurrence of spinal TB. All TB symptoms disappeared. The time of bone graft fusion was between 3–7 months (average 4.7 months). Five patients had drug-induced liver damage after taking anti-TB drugs. After the adjustment of program and strengthening liver treatment, liver function was improved. In group A, operation time was slightly extended, blood loss was reduced, and hospitalization time was shorter than for group B, but the data were not statistically significant (Table 2). Anal exsufflation time in group A and group B were 1.21±0.48 day and 2.12±0.82 day, respectively, and there were significant differences between the two groups (p<0.05). The VAS scores, Cobb angle, and ESR before surgery, at one week after surgery, and at the time of the last follow-up are shown in Table 3. Compared with preoperative values, the Cobb angle and ESR were significantly lower at one week after surgery and last follow-up in both groups (p<0.01). The VAS scores had significant differences between one week after surgery and before surgery in group A (p<0.05), but the difference was not significant in group B (p>0.05). The VAS scores at the last follow-up were significantly different from that before surgery in both groups A and B (p<0.01). After surgery, the VAS scores in group A were better than that in group B (P<0.05), while the Cobb angle and ESR of the two groups showed no difference. Postoperative nerve compression symptoms were improved (Table 4). There were significant differences in Frankel classification between before surgery and last follow-up in both groups (p<0.01). But there were no significant differences between group A and group B (p>0.05). BODY.DISCUSSION: Lumbar TB is associated with abscess formation, the presence of the neurologic deficit, persistent or local kyphosis, and segmental instability [10]. Generally, the surgical treatment for spinal TB offer relief of severe pain and improved sagittal balance, which completely relieves the lesion, relieves spinal cord compression, rectifies the kyphosis, and reconstructs spinal stability [11]. Many methods for spinal TB have been reported, such as the anterior approach and staged or simultaneous anterior decompression combined with posterior stabilization [16–18]. Among them, one method, which has been recognized by scholars [19], focuses on the focal cleaning in period I and bone graft fusion internal fixation. As we all know, eliminating the TB lesions is the most important aim in the surgical approach, which may help TB recovery, reduce recurrence, and guarantee the internal fixation is robust. From this perspective, the ideal surgery for lumbar TB remains controversial. This study analyzed the clinical effects and advantages of mini-open anterior approach focal cleaning combined with posterior internal fixation compared to the conventional anterior extraperitoneal approach focal cleaning combined with posterior internal fixation. Currently, the main surgeries for lumbar TB are anterior, posterior, and anterior combined with a posterior. The cardinal pathological destruction of spinal TB is in vertebral body and intervertebral space and thus the anterior approach might access the lesions directly, which is convenient for focal cleaning and supporting bone autograft [20–22]. However, sometimes bad effects might occur, such as infection, or the strength of the anterior internal fixation might not be as strong as a posterior approach [23–25]. Meanwhile, the posterior approach could access the anterolateral column, which would not completely remove the lesions in the front of the vertebral body, and reconstruct the anterior spinal column [26]. So some surgeons have advocated for the anterior focal cleaning and strut graft combined with posterior fixation, which might compensate for the defects of simple anterior or posterior approach and be good for the postoperative rehabilitation of patients [27]. Short-segment pedicle fixation has become an important trend in spinal orthopedic surgery [28]. And pedicle screw internal fixation in pathologic vertebrae can reduce internal fixation segment in the normal vertebral body, which could shorten the fixed range, guarantee the reliability of fixation, reduce acceleration and degeneration of adjacent segment caused by long internal fixation segment, reduce spinal activities, and reduce medical cost [29,30]. Zhang et al. achieved a very nice clinical effect in treating multi-segmental spinal TB through posterior internal fixation approach [30]. And individualized operation mode could be done according to the results of CT scan, x-ray, and MRI before surgery, which might show the true condition of the vertebral destruction. Whether the pathologic vertebrae should use pedicle screw internal fixation or not depends on the failure location of the vertebral column. Wang et al. [29] thought that if the normal tissue of anterior and middle vertebral column was >50%, pedicle screw internal fixation could be used in pathologic vertebrae after focal cleaning. According to our study, we concluded that individualized operation was indispensable on the basis of the range of vertebral lesions and the surgical approach. Pedicle screws might be done if one-third of the bone in pathologic vertebrae had no destruction. Pedicle screws were generally located in the contralateral approach of focal cleaning. If it did not impact the focal cleaning and bone graft, pedicle screw placed ipsilateral was acceptable. Whereas, if the three-column structure of pathologic vertebrae is damaged, internal fixation should not be attempted. In our study, there were seven cases that had severe damage in vertebral bodies, which needed no pedicle screw. For the rest of the cases, pedicle screws were placed in vertebral bodies. There were 11 screws exposed in eight cases, but no occurrence of fixation instability occurred in our study. Hence, effective preoperative imaging evaluation was indispensable, and can greatly lessen the incidence of screw exsertion. In our study, group A patients were placed in the right lateral position, and the inverted "eight" incision was chosen as a center, and the length of anterior incision was 6–8 cm; which was much shorter than conventional extraperitoneal approach [31]. As the obliquus external abdominis, the internal oblique muscle of the abdomen and transverse abdominis were separated bluntly; there was potential slight injury in soft tissue and abdominal muscle. During the surgery, only pathologic vertebrae that needed to be cleaned were exposed, which lessened the damage of the vertebrae, surrounding tissues, and segmental vessels. Meanwhile, after surgery, there was a benefit of lighter pain from incision and the recovery was faster. In this study, the degree of pain in group A was lower than that of group B, which was beneficial to the recovery of intestines peristalsis. And patients in group A had shorter anal exsufflation time than people in group B. Although using the small incision decreased visualization to some extent, the operation time and blood loss was not significantly different between the two groups. Blood vessels should be attended to during the surgery. In our study, there were no vascular injuries to injuries to important organs. BODY.CONCLUSIONS: Compared with the conventional anterior extraperitoneal approach of focal cleaning combined with posterior internal fixation in treating lumbar TB, mini-open anterior approach of focal cleaning combined with posterior internal fixation was safer, much more productive, and effective. This approach may present an improvement to clinical treatment programs of spinal TB, especially lumbar TB.

TITLE: An ABSTRACT.OBJECTIVES:: The main goal of this study was to evaluate the fracture resistance of maxillary second premolar teeth with standard and conservative mesio-occluso-distal (MOD) cavity designs. ABSTRACT.METHODS:: Sixty maxillary second premolars were randomly divided into 6 Groups of 10 teeth. G1 consisted of intact teeth. G2 was prepared with separated proximal boxes that were designed to be 1 mm approximately above the cement-enamel junction for the cervical margins. The occlusal outline of the proximal was performed as approximately half of the intercuspal distance buccolingually and one-third of the mesiodistal dimension. The proximal preparation was standardized in all tested groups. G3 was prepared with an occlusal extension that extended approximately one-third of buccolingual width and 2 mm in depth. G4 was prepared with the occlusal extension of 1mm in depth and width. G5: The occlusal extension was 1mm in depth and 2 mm in width. G6: The occlusal extension was 2 mm in depth and 1 mm in width. Samples were restored with composite resin and subjected to load to failure test to evaluate the fracture resistance. ABSTRACT.RESULTS:: G1 showed the highest fracture resistance value (1737.1 N) while G3 had the lowest mean value (522.9 N). Furthermore, the fracture resistance of G4 and G5 was significantly higher than G3 and G6 (P < 0.05), where in both groups, the preparation of the occlusal extension mostly remained in the enamel layer. ABSTRACT.CONCLUSION:: Modified MOD cavity designs with 1 mm depth in the enamel layer have significantly higher fracture resistance than the standard MOD cavity. BODY.INTRODUCTION: Cavity geometry of the tooth preparation for direct and indirect restorations has been reported as a major factor in determining the ability of the tooth resistance to fracture.[1-4] Teeth with large cavity preparations have exhibited greater cuspal deflection than others with small cavity preparations.[3] Greater cuspal deflections potentially enhance dental deformations, and then, restoration failure or cuspal fracture may occur.[5-7] Polymerization shrinkage of composite restorations subjects cusps of teeth with large cavities to high stresses which may generate dental deformation under repeated occlusal load and consequently increase the cuspal deflection.[8,9] This deflection ranged between 6 and 47 μm based on the cavity size, the tooth stiffness, the restoration size, the composite restoration flow, the bonding system, the placement of restoration, and the curing mode and intensity.[8-12] The destructive nature of the tooth preparation has been encountered as the main reason of reduction in the tooth strength. Furthermore, endodontic procedure, occlusal tooth preparation, and mesio-occluso-distal (MOD) cavities are the principal procedures in the tooth weakening and consequently increase the susceptibility of the tooth to fracture.[2,4,13] Reeh et al.[4] reported that 63% reduction of cuspal stiffness happens with MOD cavity preparation. They proposed that this high reduction of tooth stiffness was due to loss of marginal ridge integrity. The fracture resistance of standard MOD cavity has been investigated in the literature. Recently, Firouzmandi et al.[14] evaluated the fracture resistance of standard MOD cavities restored with amalgam restorations, composite restorations, and combination of both restorations. They found that the fracture resistance ranged between 874 N for amalgam restorations and 1287 N for amalgam/composite combination. It has been proposed that the adhesive nature of composite restoration reinforced the remaining tooth structure by stress distribution along the bonding junction that consequently increases the fracture resistance.[14,15] However, composite resin restoration has been indicated for minimal MOD cavity preparations that maintain the remaining tooth structure and then increase the fracture resistance of teeth.[16] The fracture resistance of teeth with standard MOD cavities has been investigated in the literature. However, conservative MOD cavities where the occlusal extension remains in the enamel layer have not been investigated for the cuspal fracture resistance. This study was designed to evaluate the fracture resistance of maxillary second premolar teeth with standard and modified conservative MOD cavity designs where caries lesion does not extend beyond the dentinoenamel junction (DEJ) to mimic the clinical situations. BODY.MATERIALS AND METHODS.STUDY DESIGN AND SAMPLE COLLECTION: Sixty maxillary second premolar teeth were collected after tooth extraction for orthodontic treatment reason and stored in 5% formalin. These teeth were sound and free of cracks, caries lesions, dental restorations, and dental anomalies. The selected teeth were imbedded in auto-polymerized acrylic resin (Trayplast, Vertix Dental, Netherlands) according to the following steps. A measurement of 2 mm below the cement-enamel junction (CEJ) was identified by a periodontal probe UNC-15 (UNC-15, Paterson Dental) for each tooth and marked with a permanent marker to simulate the biologic width of the natural teeth.[9] After that, Wax up line (Modeling wax, BEGO) of 2 mm height was performed on this marker, and it was used as a reference mark during embedment of the teeth in the acrylic resin. A silicon mold was fabricated to mount teeth perpendicular in the resin block with dimensions of 3 mm of resin around the tooth structure, 5 mm below the tooth apex, and 2 mm below the CEJ. All laboratory procedures were performed by the same operator. BODY.MATERIALS AND METHODS.STUDY GROUPS AND SAMPLE PREPARATION: Samples were randomly divided into six experimental groups (n = 10) [Figure 1] where Group #1 (G1) consisted of 10 intact premolar teeth (control group). Group #2 (G2) included 10 samples that were prepared with separated proximal (mesial and distal) boxes that were designed to be 1 mm approximately above the CEJ for the cervical margins. In addition, the occlusal outline of the proximal boxes was performed as approximately half of the intercuspal distance buccolingually and one-third of the mesiodistal dimension for each tooth.[9] Group #3 (G3) consisted of 10 samples where standard MOD cavity design was prepared as proximal boxes as G2 with an occlusal extension that extended approximately one-third of buccolingual width and 2 mm in depth. Group #4 (G4) consisted of 10 samples that were designed for modified MOD cavity (MOD1) as in G3 except for that occlusal extension that was prepared as 1mm in depth and width, and the proximal boxes were kept as G2. Group #5 (G5): 10 samples were prepared for another modified MOD cavity (MOD2) design where the occlusal extension was 1mm in depth and 2 mm in width, and the proximal boxes were kept as G2. Group #6 where 10 samples had the occlusal extension was 2 mm in depth and 1 mm in width, with proximal boxes were kept as G2 (MOD3). Figure 1Images representing study groups; G1: Control group. G2: M and D cavities. G3: Standard MOD cavities. G4: MOD1 cavities. G5: MOD2 cavities. G6: MOD3 cavities BODY.MATERIALS AND METHODS.CAVITY PREPARATION AND ADHESIVE PROCEDURES: Graphite pencil was used to draw the outline of the cavity before preparation. Enamel access preparation was performed with a 330-carbide bur (Brasseler USA Dental, GA, USA) [Figure 2a]. After that, a medium coarse-tapered cylindrical diamond stone with a round end (Brasseler USA Dental, GA, USA) was used to complete the rest of the cavity preparation where a high-speed handpiece was operated with water coolant. These two burs were only used to standardize the cavity preparation for all samples. In addition, the periodontal probe UNC-15 was used to control all measurements. Any used bur was replaced with new one every five tooth preparations.[2] Figure 2Cavity preparations and restoration materials; a: 330 bur. b: Light curing unit. c: Composite restoration After complete cavities' preparation for all the group samples, etching (total etch, Ivoclar Vivadent, Liechtenstein) was applied for each cavity for 15 seconds. After that, the cavity was rinsed thoroughly using water, followed by gently air-drying. Single bond adhesive (Stae, SDI dental limited) was applied to the cavities, then air was blown gently for 2 s with keeping the cavity surface glossy, and then, the light cure was applied for 10 seconds with standard LED lamp with an 8mm diameter light tip, 1200 mW/cm2actual irradiation output, and 440–490 nm [Figure 2b] (Litex 695C Dentamerica, Taiwan) according to the manufacturer instructions. All cavities were restored with A2 radiopaque light curing nanohybrid composite resins using layering technique (Tetric N-Ceram, Ivoclar Vivadent, Liechtenstein) [Figure 2c]. BODY.MATERIALS AND METHODS.THERMAL CYCLING, CYCLIC LOADING, AND LOAD TO FRACTURE TEST: All samples were subjected to artificial aging where thermal cycling and dynamic occlusal loading have been performed. Samples were placed into the thermal cycling machine (Proto-Tech, El Segundo, CA, USA) using mesh bags and subjected to 2000 thermal cycles. Furthermore, the bath temperature ranged between 5°C and 55°C with a dwell time of 30 s for each bath and transfer time of 10 s between different baths [Figure 3a]. Figure 3Samples subjected to artificial aging and fracture resistance test. a: Thermal cycling. b: Chewing simulator. c: Instron machine After that, samples were attached to a custom holder and then undergone to dynamic cyclic loading of 50 N for 10,000 cycles using chewing simulator (CS-4, SD Mechatronic GmbH, Germany) where the environment was wet. The crosshead diameter of the piston, which was contacting the internal inclines of buccal and lingual cusps, was 5 mm with rounded shape [Figure 3b]. To evaluate the fracture resistance of all groups, samples were subjected to load to fracture test under the Universal Testing Machine (Instron 8871 Universal Testing Machine, Instron, Shakopee, MN, USA) [Figure 3c]. A modified steel indenter with a diameter of 3 mm was customized to apply the compression load with a crosshead speed of 0.5 mm/min. The load was being applied vertically until the sample fracture occurred. The load at the sample fracture was recorded and analyzed. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Data analysis was performed through SPSS 20.0 (IBM Product, Chicago, USA). The results were presented into arithmetic mean and standard deviation. Analysis of variance (ANOVA) was applied to compare the mean effect. Post hoc Tukey's test or Wilcoxon Signed rank test was applied to compare pair-wise differences between the means. P ≤ 0.05 was considered statistically significant result. BODY.RESULTS: Fracture resistance values in Newton (N) with mean and SD of all groups were presented in Table 1. G1 showed the highest fracture resistance mean value (1737.1 N) while G3 (standard MODs) had the lowest mean value (522.9 N). Furthermore, the results of this study showed that the fracture resistance of G4 and G5 (MOD1 and MOD2) was significantly higher than G3 (standard MOD)(P < 0.05), where in both groups, the preparation of the occlusal extension mostly remained in the enamel layer [Table 2]. In addition, the fracture resistance of G4 and G5 (MOD1, MOD2) was significantly higher G6 (MOD3) where the preparation of the occlusal extension was 2 mm in the depth and the width (P < 0.05) [Table 2]. Table 1 The fracture resistance values of all the groups Table 2 The statistical analysis of the fracture resistance between the groups However, there was no significant difference between G3 and G6 (MOD3) where both groups had the lowest fracture resistance means' values (522 N and 628 N), respectively, among all the groups (P > 0.05) [Table 2]. BODY.DISCUSSION: This in vitro study was designed to evaluate and compare the fracture resistance of standard and modified conservative MOD cavity designs to mimic the clinical situations where the occlusal caries lesion does not extend beyond the DEJ. The result presented that as the progressive reduction of the tooth structure at the different groups increased as the fracture resistance decreased. Furthermore, the results showed that MOD cavities with occlusal extension in the enamel layer had significantly higher fracture resistance than MOD cavities that extended beyond DEJ. According to Bozkurt et al.,[17] the occlusal enamel thickness of premolar teeth ranged from 1.8 to 1.2 mm even with abrasion patterns after ultrasonic and histologic evaluations. As our methodology was designed to evaluate the effect of violation of DEJ on the fracture resistance of MOD cavities, G4 and G5, where the occlusal cavity preparation did not extend beyond the DEJ (1 mm in depth), presented significantly higher fracture resistance as compared to G3 where the preparation violated the DEJ. In the meantime, G6, where the preparation depth was 2 mm and the width was 1 mm, it showed significantly lower fracture resistance as compared to G4 and G5. Furthermore, there was no significant difference between G6 and G3 (standard MOD) which might be due to the violation of DEJ. In disagreement with this result, Sakaguchi et al.[18] reported in two-dimensional finite element study that the premolar teeth stiffness was not affected by the separation of the enamel layer between the two cusps at the central groove. They suggested that the tooth deformation could be enhanced by dentin elasticity and integrity. This difference could be explained by the different methodology and experimental tests that have been used. In agreement with this study, different studies showed that the standard MOD cavity affected the structural integrity of the tooth and might be considered as a major cause of cupsal fracture. Lopez et al.[9] compared the cuspal flexure between MOD cavity and M and D boxes. They concluded that higher cuspal deflection presented with standard MOD cavities. Mondelli et al.[19] found that MOD cavity had the lowest fracture resistance value as compared to Classes I and II two surfaces, especially when the one-third of the interocclusal distance was prepared. They considered that the isthmus width was the impact factor that affected the fracture resistance of different groups. Several studies have suggested that the composite restoration is the preferred restorative material to restore MOD cavities.[20-22] Liu et al.[20] reported that composite resin significantly provided a higher fracture resistance for MOD cavities with proximal boxes than using ceramic materials. The explanation of this result is that the lower modulus of elasticity of composite resin could cause lower stresses generated around the junction between the tooth structure and the restoration. In addition, the bonding between the surrounding tooth structure and composite resin is better than that with the ceramic materials. In the present study, samples were subjected to 2000 cycles of thermal cycling ranged between 5°C and 55°C to mimic clinical situations where dental restorations affected by the change in the temperature of oral environment. The essential factor for sample aging is the creation of mechanical stresses into the restoration which could affect the fracture resistance of restorations and remaining tooth structure.[23] The selection of temperature range was based on the most tolerated temperature in the oral cavity. Crabtree and Atkinson[24] reported that the temperature of tooth surface during eating hot meal ranged between 43°C and 53°C. The duration of thermal cycling has been varied in the literature to simulate the clinical scenario. Gale and Darvell[25] claimed in their extensive review that 10,000 cycles of thermal cycling test in the laboratory representing a clinical relevant of 1 year in service of dental restorations. Therefore, the number of cycles (2,000 cycles) that was used in this study simulating almost 2-3 months of dental restorations in the oral environment. Limitations of the present study included that although this in vitro investigation was attempted to mimic clinical situations, it was with the limited presentation of actual oral cavity environment where multiple factors may affect the end result such as pH, saliva, oral temperature, and occlusal loading. Moreover, the MOD cavities in the present study were designed with specific dimensions, which in the actual clinical scenario controlled by the extent of caries lesions. Clinical implications could be attributed to MOD cavities with minimum occlusal preparations that may not require full crown coverage because it significantly performed better than standard MOD cavities. BODY.CONCLUSIONS: According to our results, modified MOD cavity designs with 1mm depth in the enamel layer had significantly higher fracture resistance than the standard MOD cavity while MOD cavities with 2 mm depth presented no significant difference as compared to standard MOD cavities.

TITLE: Evaluation of Safety and Efficacy of Qinming8631 DR Implantable Cardiac Pacemaker in Chinese Patients: A Prospective, Multicenter, Randomized Controlled Trial of the First Domestically Developed Pacemaker of China ABSTRACT.BACKGROUND:: High cost of imported pacemakers is a main obstacle for Chinese patients suffering from bradyarrhythmia, and a domestically developed pacemaker will help lower the burden. This study aimed to evaluate the safety and efficacy of Qinming8631 DR (Qinming Medical, Baoji, China), the first domestically developed dual-chamber pacemaker of China, compared with a commercially available pacemaker Talos DR (Biotronik, Berlin, Germany) in Chinese patients. ABSTRACT.METHODS:: A prospective randomized trial was conducted at 14 centers in China. Participants were randomized into trial (Qinming8631 DR) and control (Talos DR) groups. Parameters of the pacing systems were collected immediately after device implantation and during follow-ups. The effective pacing rate at 6-month follow-up was recorded as the primary end point. Electrical properties, magnet response, single- and double-pole polarity conversion, rate response function, and adverse events of the pacing system were analyzed. The Cochran-Mantel-Haenszel Chi-square test, paired t-test, and Wilcoxon signed-rank test were used for measuring primary qualitative outcomes and comparing normally and abnormally distributed measurement data. ABSTRACT.RESULTS:: A total of 225 patients with a diagnosis of bradyarrhythmia and eligible for this study were randomly enrolled into the trial (n = 113) and control (n = 112) groups. They underwent successful pacemaker implantation with acceptable postoperative pacing threshold and sensitivity. Effective pacing rates of trial and control groups were comparable both in the full analysis set and the per protocol set (81.4% vs. 79.5%, P = 0.712 and 95.4% vs. 89.5%, P = 0.143, respectively). In both data sets, noninferiority of the trial group was above the predefined noninferiority limit (−9.5%). ABSTRACT.CONCLUSIONS:: This study established the noninferiority of Qinming8631 DR to Talos DR. The safety and efficacy of Qinming8631 DR pacemaker were comparable to those of Talos DR in treating patients with cardiac bradyarrhythmia. BODY.I: Cardiac arrhythmia is a major clinical issue leading to considerable morbidity and mortality.[1] According to the statistics of American Heart Association, the incidence of bradyarrhythmia was reported to be 4%. It is estimated to affect 5.6–12.0 million people in 2050 and will lead to more than 400,000 annual sudden cardiac deaths in the United States.[2] Implanting a cardiac pacemaker is the most effective way for treating patients with bradyarrhythmia.[3456] In the past decades, cardiac pacemakers has saved millions of patients suffering from cardiac bradyarrhythmia and has improved the quality of life of patients.[7] An estimated 750,000 patients with cardiac bradyarrhythmia undergo cardiac pacemaker implantation every year all over the world.[8] Although the clinical application of pacemakers grew rapidly in China, the implantation cases was 35/1 million in 2012, which was significantly lower than the numbers in Europe (951/1 million in 2010).[9] Although great progression has been made in China at the field of cardiac device implantation,[10111213] we have to recognize that only 3% patients with symptomatic cardiac bradyarrhythmia undergo pacemaker implantation. The high cost of imported pacemakers was found to be the major reason holding Chinese people from pacemaker implantation. A domestically manufactured pacemaker with much lower cost would help more Chinese patients benefit from cardiac pacing.[14] The Qinming8631 DR implantable cardiac pacemaker (Qinming Medical, Baoji, China) is the first domestically developed dual-chamber rate-responsive pacemaker in China. It has been qualified by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. This study aimed to evaluate the safety and efficacy of the Qinming8631 DR implantable cardiac pacemaker. The commercially available Talos DR (Biotronik, Berlin, Germany) pacemaker was used as control, and noninferiority of the Qinming8631 DR to Talos DR pacemaker in Chinese patients was tested. BODY.M.STUDY DESIGN: This prospective, multicenter, randomized controlled, single-blind, noninferiority study was conducted at 14 clinical sites in China between September 2013 and December 2014. Through a central stratified random sampling, enrolled patients were assigned into trial (Qinming8631 DR) or control (Talos DR) groups. Clinical follow-up visits were conducted at 1, 3, and 6 months after pacemaker implantation in all patients. The protocol was conducted in accordance with the Declaration of Helsinki (2013 No. 007) and approved by the Institutional Review Board at the Second Affiliated Hospital of Zhejiang University School of Medicine (Hangzhou, China) and the institutes that participated in the clinical trial. Every patient signed written informed consent. Data were collected at each clinical site by a third-party analyzer who was responsible for data processing and statistical analysis. Bipolar passive and active fixation pacing leads were used in this study. In the trial group, QM7222 (Qinming Medical, Baoji, China) was used as the atrial electrode lead and QM7211 (Qinming Medical, Baoji, China) active lead as the ventricular electrode lead. In the control group, Selox JT (Biotronik, Berlin, Germany) was used as the atrial electrode lead and Setrox S (Biotronik, Berlin, Germany) as the ventricular electrode lead. The effective pacing rate, defined as acceptable pacing capture threshold and sensing amplitude, at the 6-month follow-up was recorded as the primary end point. Electrical properties, magnet response, single- and double-pole polarity conversion, rate response function, and adverse events of the pacing system after implantation were collected and analyzed at the end of the study. All-cause mortality, cardiovascular death, and pacemaker-related adverse events were reported by each center and analyzed at the end of the study. BODY.M.STUDY POPULATION: Inclusion criteria Patients were deemed eligible for the enrollment in the trial if they (1) suffered from bradyarrhythmia due to sick sinus syndrome (SSS) or atrioventricular (AV) block and indicated for dual-chamber pacemaker implantation; (2) were 18–85 years old; (3) signed the informed written consent; and (4) were capable of finishing 6 months of follow-up. Exclusion criteria Patients were excluded from the study if (1) they had tricuspid atresia; (2) the tricuspid valve was replaced with a mechanical valve; (3) auricular appendix was absent; (4) they had persistent atrial fibrillation; (5) they had indications for implantable cardioverter defibrillator; (6) they were contraindicated to 1.0 mg dexamethasone; (7) they were pregnant or lactating; (8) they had a life expectancy of <1 year; (9) they had coagulation dysfunction; (10) they had high-sensitivity carotid sinus syndrome and neurocardiogenic syncope; (11) they had intracardiac mural thrombus or suffered from ventriculotomy or atriotomy in the last 4 weeks; (12) they had heart transplantation, neuromuscular diseases, sleep apnea syndrome, and cardiac sarcoidosis; (13) they had acute or severe infection, malignant tumor, or end-stage diseases; (14) they used other medical devices that might interfere with the pacemaker; or (15) they had any other conditions unsuitable for the trial as considered by researchers. BODY.M.EFFICACY ASSESSMENTS: Primary end points The effective pacing rate at the 6-month follow-up was used to be the primary end point. It mainly comprised the following parameters: Acceptable pacing threshold (acute): With 0.5 ms of the pulse width, the pacing threshold of the right atrium was ≤1.5 V and the threshold of the right ventricle was ≤1.0 V. When the voltage outputs were set to two times the pulse amplitude of the measured pacing threshold, the target atrium or ventricle was continuously captured and observed on a surface electrocardiogram (ECG)Acceptable long-term threshold: When the pulse width was 0.5 ms, the pacing threshold of the right atrium was ≤1.5 V (fluctuated upward ≤20%) and the threshold of the right ventricle was ≤1.0 V (fluctuated upward ≤20%). When the voltage outputs were set to two times the pulse amplitude of the measured pacing threshold, the target atrium or the ventricle was continuously captured and observed on a surface ECG at 1, 3, and 6 months after the procedurePostoperative sensing: The sensitivity threshold of right atrium was ≥2.0 mV and the threshold of the right ventricle was ≥4.0 mV. When the sensitivity was set at 1/3–1/4 of the sensitivity threshold (should not exceed the setting range advised by the manufacturer), an electrocardio signal could be effectively perceivedMagnet response: Magnet response of the pacemaker at 3 months after the procedure was evaluatedPolarity conversion (unipolar and bipolar): Unipolar and Bipolar conversion of the pacemaker at 3 months after the procedure was evaluatedRate response: Rate response of the pacemaker at 6 months after the procedure was evaluated. Secondary end points Failure to pace as detected by the 24-h ambulatory ECG at 6 monthsFailure to sense as detected by the 24-h ambulatory ECG at 6 months. BODY.M.SAFETY ASSESSMENTS: Safety assessments were conducted by recording all-cause death, cardiovascular death, and pacemaker-related adverse events within 6 months after the procedure. Moreover, the clinical symptoms, vital signs (blood pressure and heart rate), and laboratory parameters were monitored. If abnormal changes were noted, their correlation with the pacemaker was analyzed. Laboratory parameters included complete blood count, liver function, renal function, and blood clotting tests. BODY.M.STATISTICAL ANALYSIS: Sample size estimation On the basis of clinical experience, this study hypothesized that the effective pacing rate in the control group was 95% (taking into account the incidence of lead-related complications), and the noninferiority margin was 9.5%. The sample size in each group would have a power of 0.80 with a level of significance of 0.05; if the expected dropout rate was 10%, the final sample size was 100 in each group. Analysis of population The full analysis set (FAS) comprised cases in accordance to the principle of "intention to treat," including all the enrolled patients. For patients without the primary effect assessment, the last observation carried forward (LOCF) was conducted to carry forward the missing data. The per protocol set (PPS) was a subgroup of enrolled patients completing the trial. The safety analysis set (SAS) included cases with at least one result of safety evaluation during the follow-up. Statistical analysis method All continuous values were presented as mean ± standard deviation (SD), and all categorical variables were presented as number of patients (percentages). Group comparisons of categorical data were conducted by Cochran-Mantel-Haenszel Chi-square test or Fisher's exact test (when 25% of the cells have expected frequencies of <5); group comparisons of normally distributed measurement data were analyzed by student t-test; and group comparisons of abnormally distributed measurement data were analyzed by Wilcoxon signed-rank test. Data management was conducted by the EpiData 3.0 software (The EpiData Association, Odense, Denmark). Data analysis was done using the SAS 9.13 software (SAS Institute Inc., Cary, USA). A two-sided P ≤ 0.05 was considered statistically significant. Analysis of efficacy For primary quantitative outcome measurements, group comparisons were conducted by analysis of covariance. After testing the homogeneity of variance of each center, the least mean square (LMS), LMS error between groups, and 95% confidence interval (CI) of dependent variable were calculated. For primary qualitative outcome measurements, the Cochran-Mantel-Haenszel Chi-square test, adjusting the influence of center, was conducted and differences in the event rate between groups and the 95% CI were calculated. To evaluate the proposed noninferiority of the trial instrument, 95% CI of group variances of outcome measurements was compared with the critical value, which was clinically meaningful and specified preliminary in the trial. For other outcome measurements, comparison of the normally distributed measurement data in a group was conducted by the student t-test and that of abnormally distributed measurement data was conducted by the Wilcoxon signed-rank test. Analysis of safety Normal cases and proportions before the treatment and abnormal cases and proportions after the treatment were calculated in the two groups, respectively. Cases and proportions of adverse events were calculated and analyzed by continuous correction U-test or Fisher's exact test. The specific appearance, degree, and relationship with the instrument in each group were recorded. BODY.R.STUDY POPULATION: A total of 225 patients from 14 centers all over China were enrolled in this study and randomized into two groups based on a third-party random assignment: Qinming8631 DR pacemaker was implanted in 113 patients (trial group) and Talos DR pacemaker was implanted in 112 patients (control group). Comparisons of baseline characteristics, such as demographics and comorbidities, are shown in Table 1. No significant difference was found between the two groups, except for the history of syncope, which was higher in the trial group than in the control group. Patients with a diagnosis of SSS, second-degree AV block, and complete AV block were 62 versus 58 (χ2 = 0.215, P = 0.643), 10 versus 11 (χ2 = 0.063, P = 0.802), and 41 versus 43 (χ2 = 0.107, P = 0.744) in the trial versus control group, respectively. No statistical significance of diagnosis was found in these two groups [Table 1]. Study population of FAS and PPS are shown in Table 2. Finally, 87 (77.0%) patients in the trial group and 86 (76.8%) patients in the control group completed the 6-month follow-up (PPS). Reasons for discontinuation are provided in Supplementary Table 1. Table 1 Baseline characteristics of patients with bradyarrhythmia implanted cardiac pacemakers Variables Qinming8631 DR group ( n = 113) Talos DR group ( n = 112) Statistical values P Age (years) 65.6 ± 11.0 65.3 ± 11.6 0.207 0.837 Female 57 (50.4) 50 (44.6) 0.759 0.384 SBP (mmHg) 137.3 ± 22.5 139.1 ± 21.8 −0.613 0.541 DBP (mmHg) 73.9 ± 12.1 74.8 ± 12.2 −0.562 0.575 Heart rate (beats/min) 50 ± 14 53 ± 17 −1.659 0.099 Coronary heart disease 6 (5.3) 7 (6.3) 0.091 0.762 Hypertension 47 (41.6) 53 (47.3) 0.748 0.387 Diabetes 9 (8.0) 13 (11.6) 0.850 0.357 Stroke 3 (2.7) 6 (5.4) Fisher 0.333 Dilated cardiomyopathy 1 (0.9) 0 (0) Fisher 1.000 Valvular disease 1 (0.9) 2 (1.8) Fisher 0.622 MI 0 (0) 2 (1.8) Fisher 0.247 Paroxysmal atrial fibrillation 3 (2.7) 8 (7.1) 2.522 0.112 History of syncope 10 (8.9) 3 (2.7) 4.209 0.040 Diagnosis  Sick sinus syndrome 62 (54.9) 58 (51.8) 0.215 0.643  Second degree AV block 10 (8.8) 11 (9.8) 0.063 0.802  Complete AV block 41 (36.3) 43 (38.4) 0.107 0.744 Fisher: No available statistical value according to Fisher exact test. Data were expressed as n (%) or mean ± SD. SD: Standard deviation; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; AV: Atrioventricular; MI: Myocardial infarction; 1 mmgHg = 0.133 kPa. Table 2 Study deviation of bradyarrhythmia patients underwent pacemaker implantation during 6 months of follow-up Population Qinming8631 DR group ( n = 113) Talos DR group ( n = 112) Cancellation of informed consent, n 0 0 FAS, n 113 112 Deviation from the protocol, n (%)  Absence of primary end points 22 (19.5) 23 (20.5)  Loss of follow-up 4 (3.5) 3 (2.7) PPS, n (%) 87 (77.0) 86 (76.8) FAS = Number of randomized patients − number of patients withdrawing informed consent; Deviation from the protocol = Patients absence of primary end points + patients loss of follow-up; PPS = FAS − deviation from the protocol. FAS: Full analysis set; PPS: Per protocol set. Supplementary Table 1 Patients deviating from the protocol Center Random number Group Gender Age Type Detailed description 01 053 Trial Male 77 Absence of primary end points Death at the 70 th day 01 054 Trial Female 71 Absence of primary end points No assessment of frequency response at 6-month 01 068 Trial Male 57 Absence of primary end points No assessment of frequency response at 6-month 01 069 Control Male 64 Absence of primary end points No assessment of frequency response at 6-month 01 101 Trial Female 64 Absence of primary end points No assessment of perception threshold immediately after operation 01 122 Control Male 55 Absence of primary end points No assessment of perception threshold immediately after operation 01 129 Trial Male 76 Absence of primary end points Death at the 68 th day 02 179 Control Female 74 Absence of primary end points No assessment of perception threshold immediately after operation 02 191 Trial Female 60 Absence of primary end points No assessment of perception threshold immediately after operation 02 192 Control Female 54 Absence of primary end points No assessment of perception threshold immediately after operation 03 004 Control Female 73 Absence of primary end points No assessment of perception threshold immediately after operation 04 002 Trial Male 60 Absence of primary end points No assessment of perception threshold immediately after operation 04 015 Trial Male 71 Absence of primary end points No assessment of perception threshold immediately after operation 04 048 Trial Male 79 Absence of primary end points Death at the 162 nd day 04 049 Control Male 77 Absence of primary end points No assessment of perception threshold immediately after operation 04 056 Trial Male 51 Absence of primary end points No assessment of perception threshold immediately after operation 04 100 Trial Male 75 Absence of primary end points No assessment of perception threshold immediately after operation 04 107 Control Male 66 Absence of primary end points No assessment of perception threshold immediately after operation 04 138 Trial Male 71 Absence of primary end points No assessment of perception threshold immediately after operation 05 021 Trial Male 78 Absence of primary end points No assessment of perception threshold immediately after operation 05 025 Control Male 70 Absence of primary end points No assessment of perception threshold immediately after operation 05 043 Trial Male 74 Absence of primary end points No assessment of perception threshold immediately after operation 05 052 Control Male 81 Absence of primary end points Death at the 62 nd day 05 116 Trial Male 76 Absence of primary end points No assessment of perception threshold immediately after operation 06 203 Trial Female 76 Loss of follow-up 6-month 06 223 Control Female 82 Loss of follow-up 1-month 07 144 Control Male 26 Absence of primary end points No assessment of perception threshold immediately after operation 08 026 Trial Male 26 Absence of primary end points No assessment of perception threshold immediately after operation 08 040 Control Male 81 Absence of primary end points No perception threshold immediately after operation 08 135 Control Male 33 Absence of primary end points No assessment of perception threshold immediately after operation 08 178 Control Male 56 Loss of follow-up 1-, 3-, and 6-month 08 189 Trial Male 80 Loss of follow-up 6-month 09 038 Trial Male 63 Absence of primary end points Death at the 95 th day 09 074 Control Male 78 Absence of primary end points No assessment of perception threshold immediately after operation 09 088 Trial Male 50 Absence of primary end points No assessment of perception threshold immediately after operation 09 098 Control Male 56 Absence of primary end points No assessment of perception threshold immediately after operation 09 175 Trial Female 64 Absence of primary end points No assessment of perception threshold immediately after operation 09 188 Trial Male 84 Absence of primary end points No assessment of perception threshold immediately after operation 09 195 Control Male 69 Absence of primary end points No assessment of perception threshold immediately after operation 09 089 Trial Female 77 Loss of follow-up 3- and 6-month 09 168 Control Female 82 Loss of follow-up 6-month 10 117 Trial Male 43 Absence of primary end points No assessment of perception threshold immediately after operation 10 133 Trial Male 76 Absence of primary end points Death at the 126 th day 10 153 Control Male 76 Absence of primary end points No assessment of perception threshold immediately after operation 13 029 Control Male 53 Absence of primary end points No assessment of perception threshold immediately after operation 13 046 Trial Male 67 Absence of primary end points No perception threshold immediately after operation 13 104 Control Female 52 Absence of primary end points No assessment of perception threshold immediately after operation 13 216 Trial Female 59 Absence of primary end points No assessment of perception threshold immediately after operation 14 091 Trial Female 64 Loss of follow-up 1-month 14 090 Control Male 56 Absence of primary end points No assessment of perception threshold immediately after operation 14 115 Control Male 76 Absence of primary end points Death at the 75 th day 14 197 Control Female 73 Absence of primary end points No pace-making threshold at 6-month after operation; no assessment of frequency response at 6-month Center - 01: The Second Affiliated Hospital of Zhejiang University School of Medicine; 02: The Affiliated Hospital of Qingdao University Medical College; 03: The First People’s Hospital of Hangzhou; 04: The First Affiliated Hospital of Lanzhou University; 05: The First Affiliated Hospital of Xi’an Jiaotong University; 06: Northern Jiangsu People’s Hospital; 07: The First Affiliated Hospital of Suzhou University; 08: The Second Affiliated Hospital of Nanchang University; 09: Bethune International Peace Hospital of the Chinese PLA; 10: The First Affiliated Hospital of Kunming Medical University; 13: Jilin Province People’s Hospital; 14: Tianjin Chest Hospital. BODY.R.PROCEDURE CHARACTERISTICS: All the patients underwent successful pacemaker implantation. More than 75% patients underwent electrode lead implantation through the subclavian vein (around 20% was through the axillary vein) and very few through the cephalic vein, in both groups. About 70% pacemakers were implanted into the left side of the chest, and no difference was found between the two groups (69.9% vs. 70.5%, χ2 = 0.011, P = 0.919). The atrial lead was fixed in the right atrial (RA) appendage in all patients. In 39.8% of the trial group and 40.2% of the control group, the ventricular lead was fixed in the right ventricular (RV) apex, while in others, it was fixed in the ventricular septum. The RA pacing threshold was 0.63 ± 0.28 V vs. 0.65 ± 0.27 V (t = −0.643, P = 0.521) in trail and control groups while RV pacing threshold was 0.67 ± 0.27 V vs. 0.58 ± 0.22 V (t = 2.628, P = 0.009). RA and RV sensing threshold were similar between the two groups (6.09 ± 5.30 mV vs. 5.18 ± 4.36 mV, t = 1.377 P = 0.170; 11.03 ± 5.76 mV vs. 11.62 ± 6.00 mV, t = −0.751 P = 0.453, respectively). BODY.R.EFFICACY ASSESSMENT: Primary end points On the basis of comparability of the two groups, the primary end points in FAS and PPS were evaluated [Table 3]. The results showed that in FAS, the effective pacing rate was 81.4% in the trial group and 79.5% in the control group. The difference was 1.3% (95% CI: −7.0%–10.6%), and thus noninferiority of the trial group compared with the control group with respect to the effective pacing rate was shown with a margin of −7.0%, which was well above the predefined noninferiority limit (−9.5%). In PPS, the effective pacing rate was 95.4% in the trial group and 89.5% in the control group. The difference was 11.8% (95% CI: 0.3%–24.9%), and thus noninferiority of the trial group compared with the control group with respect to the effective pacing rate was shown with a margin of 0.3%, which was also well above the predefined noninferiority limit (−9.5%). The effective pacing rate immediately after the procedure was no significant difference in the two groups (trial: 100% vs. control: 99.1%, P = 1.000). The effective sensitivity rate immediately after the procedure was 94.8% and 93.6% in the trial and control groups, respectively, with no significant difference (P = 0.716). The stability of pacing during follow-up was very high (99.0% in trial and 98.1% in control groups), and the difference was not significant (P = 0.621). Magnet response, single- and double-pole polarity conversion at 3 months, and rate response at 6 months were 100% in both groups. Pacing threshold, sensing threshold, and lead impedance of RA and RV in both groups are shown in Figure 1. Table 3 Primary endpoint after 6 months of follow-up in the full analysis set and per protocol set Variables Qinming8631 DR group ( n = 113) Talos DR group (n = 112) Difference 95% CI P FAS*, n (%)  Valid 92 (81.4) 89 (79.5) 1.3 −7.0–10.6 0.712  Invalid 21 (18.6) 23 (20.5) PPS, n (%)  Valid 83 (95.4) 77 (89.5) 1.8 0.3–24.9 0.143  Invalid 4 (4.6) 9 (10.5) *The first choice for dealing with missing value is LOCF. When there is still missing values after the data was added according to LOCF, the missing value would be regard as non-effective pacing. 95% CI was obtained through CMH Chi-square test adjusting center effect. FAS: Full analysis set; PPS: Per protocol set; CI : Confidence interval; LOCF: Last observation carried forward; CMH: Cochran-Mantel-Haenszel. Figure 1Parameter of RA and RV leads at post-op and during follow-ups. (a and b) Pacing threshold at 0.4 ms pulse width; (c and d) sensing threshold; (e and f) impedance. Post-op: Postoperation; RA: Right atrium; RV: Right ventricle. Secondary end points At 6-month follow-up, the 24-h ambulatory ECG monitoring did not detect any failure of pacing or sensing in both groups. BODY.R.SAFETY ASSESSMENT: Analysis of safety parameters was based on SAS. The rate of adverse event was 3.53% in the trial group and 2.67% in the control group, with no significant difference (P = 1.000). The four cases of adverse events in the trial group (three noncardiovascular and one cardiovascular) and three cases of adverse events in the control group (zero noncardiovascular and three cardiovascular) were severe. However, the adverse events were not associated with the pacing system [Table 4 and Supplementary Table 2]. Table 4 Adverse events in bradyarrhythmia patients underwent pacemaker implantation Adverse events Qinming8631 DR group ( n = 113) Talos DR group ( n = 112) Statistical values P Noncardiac death, n (%)  Nasopharyngeal carcinoma 1 (0.88) 0 Fisher 1.000  Hepatocellular carcinoma 1 (0.88) 0 Fisher 1.000  Intestinal obstruction (postcolectomy) 1 (0.88) 0 Fisher 1.000 Cardiac death, n (%)  MI 0 2 (1.79) Fisher 0.247  HF and acute renal insufficiency 1 (0.88) 0 Fisher 1.000  HF 0 1 (0.89) Fisher 0.498  All-cause death 4 (3.53) 3 (2.67) Fisher 1.000 Fisher: No available statistical value according to Fisher’s exact test. MI: Myocardial infarction; HF: Heart failure. Supplementary Table 2 Description of patients with adverse events Center Random number Group Age Gender Adverse event Day after operation Severity Outcome Instruments treatments Quit the test 01 053 Control 77 Male MI 70 Severe Death No Yes 01 129 Trial 76 Male Intestinal obstruction (postcolon cancer operation) 68 Severe Death No Yes 04 048 Trial 79 Male Hepatocellular carcinoma 162 Severe Death No Yes 05 052 Control 81 Male MI 60 Severe Death No Yes 09 038 Trial 63 Male HF and acute renal insufficiency 95 severe Death No Yes 10 133 Trial 76 Male Nasopharyngeal carcinoma 126 Severe Death No Yes 14 115 Control 76 Male HF 75 Severe Death No Yes Center - 01: The Second Affiliated Hospital of Zhejiang University School of Medicine; 04: The First Affiliated Hospital of Lanzhou University; 05: The First Affiliated Hospital of Xi’an Jiaotong University; 10: The First Affiliated hospital of Kunming Medical University; 14: Tianjin Chest Hospital. MI: Myocardial infarction; HF: Heart failure. BODY.D: This study demonstrated that the first domestically developed dual-chamber rate-responsive pacemaker of China was noninferior with respect to safety or efficacy compared with Talos DR at 6-month follow-up. Qinming8631 DR pacing system showed acceptable pacing and sensing threshold at both acute phase and long-term follow-up and good magnetic response, polarity conversion (unipolar and bipolar), and rate response function. No failure of pacing or sensing was found by 24-h ambulatory ECG. Most importantly, the prevalence of severe adverse events was low and similar in the two groups, and the adverse events were not associated with the pacing system. In China, Qinming2312 implantable cardiac pacemaker, developed by Qinming Corporation in 2008, was the first domestic single-chamber cardiac pacemaker registered by the China Food and Drug Administration. In the past years, Qinming2312 implantable cardiac pacemaker was clinically used in 150 hospitals and spread over 26 provinces in China. The clinical effects obtained from different hospitals demonstrated that Qinming2312 implantable cardiac pacemaker was comparable with imported cardiac pacemaker and was, especially, inexpensive, which could be afforded by the majority of Chinese patients.[1516] However, dual-chamber pacemaker accounted for 60% of all pacemakers in China,[9] which brought an urgent need of domestically developed dual-chamber pacemaker. On the basis of successful clinical application of Qinming2312 single-chamber cardiac pacemaker, Qinming Corporation developed Qinming8631 DR implantable cardiac pacemaker, which would be the first generation of domestically developed dual-chamber cardiac pacemaker of China. This prospective, randomized, controlled, and single-blind study was conducted at 14 clinical centers in China. The Talos DR implantable cardiac pacemaker, which is widely distributed all over the world as the basic model of dual-chamber pacemaker, was selected as the control device. Rigorous inclusion and exclusion criteria were used while enrolling patients. The loss of follow-up rate was 2% at 1 month, 3% at 3 months, and 5% at 6 months. Good pacing performance and reliable safety shown in this study helped improve the confidence when using Qinming8631 DR implantable cardiac pacemaker in clinical practice. BODY.D.CLINICAL SIGNIFICANCE: As a result of the continuous development in economy and improvement in people's living standards, the aging trend has increased in China, leading to a dramatically increased number of patients relying on the treatment with pacemakers. So far, Qinming8631 DR implantable cardiac pacemaker is the first dual-chamber pacemaker developed in China with independent intellectual property, possessing efficacy and safety comparable to those of Talos DR pacemaker, is much cheaper than the imported pacemaker, and has huge potential for alleviating the financial burden of Chinese patients and providing more opportunities to those who are indicated for pacemaker implantation. BODY.D.LIMITATIONS: The follow-up period in this study was relatively short according to the battery expectancy of a dual-chamber pacemaker. A long-term outcome evaluation is warranted for both Qinming8631 DR and Talos DR pacemakers. In conclusion, the present study demonstrated, through a 6-month follow-up trial, that Qinming8631 DR implantable cardiac pacemaker was feasible for treating patients with cardiac bradyarrhythmia with good safety and reliable pacing performance. Supplementary information is linked to the online version of the paper on the Chinese Medical Journal website. BODY.D.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.D.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Efficacy and pharmacokinetic activity of frovatriptan compared to rizatriptan in patients with moderate-to-severe migraine ABSTRACT.BACKGROUND: Migraine is a painful neurological disorder that affects over 10% of the general population. Frovatriptan and rizatriptan are antimigraine agents belonging to the triptan class. Although previous studies have independently compared the efficacy of these agents, contemporaneous data examining both pharmacokinetic (PK) properties and efficacy in parallel have not previously been available. ABSTRACT.MATERIALS AND METHODS: In this single-center double-blind study, 18 subjects (ten female) were treated for a single migraine attack with frovatriptan 2.5 mg or rizatriptan 10 mg. Plasma concentrations were measured predose and at 2, 4, 6, 12, 24, 48, and 72 hours after drug administration. The primary end point of this study was to evaluate the association between PK parameters and efficacy measures and recurrence rate. Secondary end points were pain-free and pain-relief episodes at 2 and 4 hours, recurrent episodes within 48 hours, and cumulative hazard of recurrence within 72 hours. ABSTRACT.RESULTS: At baseline, approximately 17% of patients had mild migraine, while 83% had moderate–severe migraine. Although the time to maximum concentration was similar for both drugs (2.7 versus 2.3 hours), the terminal half-life for frovatriptan was longer than rizatriptan (29.3 versus 3.2 hours, P<0.0001). The proportion of patients who were pain-free at 4 hours without rescue medication was higher in the frovatriptan-treated group, (38.9 versus 5.6%, P=0.045). The cumulative hazard of recurrence over 72 h was reduced by frovatriptan compared to rizatriptan-treated patients (log-rank test, P=0.04). Pain-free and pain-relief episodes for the study period were positively correlated with the concentration:maximum concentration (Cmax) ratio for frovatriptan (r=0.52, P=0.028), but not rizatriptan. Recurrence rate was negatively correlated with the concentration:Cmax ratio for both frovatriptan (r=−0.96, P=0.0024) and rizatriptan (r=−0.98, P=0.0004). Fewer adverse events were observed for frovatriptan compared to rizatriptan (one versus eight, P=0.021). ABSTRACT.CONCLUSION: This pilot study indicates that a similar extent of initial pain relief is afforded by both triptans in migraine treatment. The longer duration of action of frovatriptan parallels and correlates with its PK profile. BODY.INTRODUCTION: Migraine is a common painful neurological disorder that affects millions of individuals worldwide.1 It is frequently underdiagnosed and undertreated, and typically characterized by recurrent attacks of moderate-to-severe headache lasting 4–72 hours.2 Other common symptoms are nausea, vomiting, photo- and/or phonophobia and neurological aura symptoms.2 Although nonpharmacological therapies have been shown to offer symptom relief in the pediatric population,3,4 current guidelines recommend the use of pharmacological therapies as first-line treatment for mild-to-moderate migraine in adults.5 Triptans are a class of selective serotonin 5-hydroxytryptamine (5-HT) 1B/1D agonists with proven efficacy for the treatment of acute migraine.6 The early success of the parent drug of this class, sumatriptan, led to the development of other triptan compounds, with the aim of optimizing pharmacokinetic (PK) properties, efficacy, and safety in migraine treatment.7 Triptans differ markedly in terms of their PK and pharmacodynamic properties. Some triptans, such as sumatriptan, are considered fast-acting while also possessing greater risk of adverse events (AEs). In contrast, other triptans, such as frovatriptan, provide a more prolonged duration of action, with fewer associated side effects.6 Frovatriptan was specifically developed to exhibit a long duration of action combined with reduced potential for side effects and drug interactions.8–11 It is a 5-HT-receptor agonist that binds with high affinity to 5-HT1B and 5-HT1D receptors, but unlike other triptans, frovatriptan has moderate affinity for 5-HT7 receptors.12 A unique PK feature of frovatriptan is its long terminal elimination half-life of approximately 26 hours, unlike other triptans, which range from 3–6 hours.13 Frovatriptan does not inhibit or induce cytochrome P450 isoenzymes, and is only slightly bound to plasma proteins, thus exhibiting a low risk of PK drug interactions.14 Rizatriptan is also a selective serotonin 5-HT1 (1B/1D) agonist.15,16 It is rapidly absorbed, eliminated with a plasma half-life of approximately 2–3 hours, and is reported to induce rapid pain relief.15–17 Since the majority of rizatriptan is metabolized by monoamine oxidase A, cytochrome P450 inhibitors have minimal effects on the PK of rizatriptan.18 While both frovatriptan and rizatriptan have independently proven efficacy and tolerability for the treatment of acute migraine,11,16 only two head-to-head trials have actually compared the efficacy of these two triptans.19,20 Both studies were performed on Italian migraineurs (one of which was in females with migraine that was menstrually related), and both demonstrated similar antimigraine efficacy of the two triptans in terms of pain-free and pain-relief rates. However, the hazard of migraine recurrence after 48 hours was significantly lower with frovatriptan use in both studies.19,20 Migraine headache recurrence is an important clinical problem in migraine management.21 The mechanism of headache recurrence is not well understood, but it is thought to be linked with patient-specific physiological characteristics and PK properties of the drug used.22 A meta-analysis of more than 30 randomized, double-blind studies showed a strong inverse correlation between the frequency of migraine headache recurrence and half-lives of different triptans.22 The triptans with longer terminal elimination half-lives, namely eletriptan, naratriptan, and frovatriptan, had a lower incidence of headache recurrence.22 Although previous studies have independently compared the efficacy of these agents in head-to-head trials, this is the first study to specifically examine both PK properties and efficacy in real time and in parallel. Therefore, the primary objective of this study was to confirm previous data by evaluating the presence of a potential association between recurrence rates and PK parameters for two pharmacokinetically distinct triptans: frovatriptan and rizatriptan. Secondary measures included standard-efficacy parameters of pain-free and pain-relief-rates at 2 and 4 hours in addition to recurrence rates within 48 hours. BODY.MATERIALS AND METHODS.PATIENTS: This pilot study was performed on patients in the Institute for Pharmacokinetic and Analytical Studies Clinic, Ligornetto, Switzerland between July 2008 and March 2010. Patients included both male and female subjects aged between 18 and 55 years, with a current history of migraine with or without aura, according to International Headache Society (IHS) 2004 criteria, and with at least one but no more than six migraine attacks per month in the 6 months prior to entering the study.2 Patients could not be enrolled in the study if they had: 1) uncontrolled hypertension; 2) ischemic heart disease; 3) cardiac arrhythmias or symptomatic Wolff–Parkinson–White syndrome; 4) previous stroke or transient ischemic attack; 5) severe liver or renal impairment; 6) any other severe or disabling medical condition; 7) a history of alcohol, analgesic, or psychotropic drug abuse; 8) known hypersensitivity to study drugs; 9) previously demonstrated inadequate response to at least two triptans; 10) current use of propranolol or ergotamine (and its derivatives) as a prophylactic agent; 11) current use or use in the previous 2 weeks of monoamine oxidase inhibitors; 12) use of either test medication to treat any one of the last three episodes of migraine; and 13) other headaches that had lasted for more than 6 days (at the time of presentation for the study). Patients were permitted to continue with concomitant medications during the trial if prescribed for concomitant illnesses. Ideally, doses were kept stable during the study, but if a change in the concomitant medications and/or doses was considered essential for medical reasons, these measures were recorded in the case-report form and patient diary. Where possible, the dose of any prophylactic medication remained unchanged throughout the study period. During the blood-sampling time, paracetamol was preferred as a concomitant medication for the treatment of headache. Where indicated, emergency medication could be administered at any time during the trial. Pregnant women and breastfeeding mothers were excluded as well, while women with childbearing potential but not practicing an effective method of birth control were to be submitted to a pregnancy test, if clinically indicated. The study was approved by the independent institutional review boards of the study center, was performed in accordance with IHS guidelines, and was in compliance with the Declaration of Helsinki. Written informed consent was obtained from all patients prior to their inclusion into the study. BODY.MATERIALS AND METHODS.STUDY DESIGN: This was a double-blind, single-dose, balanced, two-sequence, two-period, two-treatment, randomized crossover design with at least 14 days of washout between the two treatments. Each patient received 2.5 mg frovatriptan and 10 mg rizatriptan in a balanced computer-generated randomized sequence (1:1), where frovatriptan was followed by rizatriptan or vice versa. Blinding was ensured by preparing identical capsules containing frovatriptan or rizatriptan. The duration of the study varied between subjects, depending on the interval between screening and the first migraine attack and between the two migraine attacks. However, patients were instructed to go to the clinic as soon as a migraine attack occurred (and to take study medication as soon as possible after migraine occurrence). BODY.MATERIALS AND METHODS.SAFETY ASSESSMENTS: The safety of medications administered during the trial was monitored by recording in the patient's diary all AEs occurring during each period of treatment. In addition, vital signs (respiratory rate, blood pressure, and heart rate) were measured and recorded at each visit (before and at end of study). BODY.MATERIALS AND METHODS.EFFICACY ASSESSMENTS: Patients recorded the following information in paper diaries: date and time of onset of migrainedate and time of treatment with study medicationheadache severity on a 4-point scale (0= none, 1= mild, 2= moderate, 3= severe) at the following times: 0 (time of treatment) and 2, 4, 6, 12, 24, 48, and 72 hours after treatmentassociated symptoms (nausea, vomiting, photophobia, phonophobia) on the same 4-point scale and at the same times. BODY.MATERIALS AND METHODS.PHARMACOKINETIC ASSESSMENTS: Plasma was collected for determination of rizatriptan and frovatriptan concentrations at the following times: predose, and at 2, 4, 6, 8, 12, 24, 48, and 72 hours postdose. Deviations in blood-sampling times were taken into account in the calculation of PK parameters. A 10 mL blood sample was collected from an indwelling catheter and allowed to clot at 2°C–8°C without any anticoagulant. Plasma was separated by centrifugation at 4°C and 1,000 g for 15 minutes. Plasma samples were checked and stored in freezers at −80°C±10°C pending the assay. All blood/plasma samples were identified by labels bearing the study code, the subject number, the treatment period, the time of blood collection, and the aliquot identification. Frovatriptan and rizatriptan plasma concentrations were measured by fully validated liquid chromatography–mass spectrometry methods. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The sample-size calculation of 25 subjects was based on a statistical power of 80%, a significance level of 5%, and a variation coefficient of the main efficacy variables (pain-free and pain-relief rates at 2 h) equal to 25%. The primary end point of interest was the correlation between plasma concentration of each triptan (and more specifically the concentration:maximum concentration (Cmax) ratio and the pain-free and pain-relief rates at each time point. Secondary efficacy parameters were: pain-free rates at 2 and 4 hourspain-relief rates at 2 and 4 hoursrecurrence rates at 24, 48, and 72 hoursassociated symptoms (nausea, vomiting, photophobia, phonophobia) at each time pointrescue-medication usedrug preference (frovatriptan, rizatriptan, or no preference).20 Pain-free, pain-relief and pain-recurrence were defined according to IHS criteria.2 All data are presented as means ± standard deviation or percentages. All statistical analysis was performed with the Stata statistical software package, version 10 (StataCorp, College Station, TX, USA) or InStat (GraphPad Software, La Jolla, CA, USA). Nonparametric continuous variables were compared by the Mann–Whitney test. Comparisons between two groups with normally distributed variables were analyzed by unpaired t-tests. Categorical variables were analyzed by χ2 tests. Univariate correlations (eg, between PK parameters and efficacy measures) were assessed by Pearson's correlation coefficient (r). Where comparisons were made, quoted P-values were two-tailed. The PK parameters Cmax and time to Cmax (Tmax) were read directly using WinNonlin software (Pharsight, Mountain View, CA, USA). Terminal half-life (t1⁄2) values were determined by extrapolating the terminal portion of the curve. Area under the plasma concentration–time curve (AUC) values were calculated from individual concentration–time data using WinNonlin software. The AUC from the time of dosing to the last quantifiable concentration was calculated by means of the linear trapezoidal rule. Extrapolation to infinity was obtained by dividing the last quantifiable concentration by the terminal elimination-rate constant and adding this result to AUCt. The concentration: Cmax ratio was calculated by dividing the instantaneous plasma concentration by the observed Cmax. Univariate regression analysis was performed between selected PK parameters (eg, Cmax, AUC, concentration: Cmax ratio) and efficacy parameters (eg, pain-free, pain-relief, combined pain-free and pain-relief rates). Kaplan–Meier curves were used to determine the cumulative hazard of recurrence over 72 hours. A P-value of <0.05 was considered statistically significant. Bonferroni post hoc testing was applied to adjust for potential multiplicity. BODY.RESULTS.BASELINE DEMOGRAPHIC AND CLINICAL CHARACTERISTICS: From a total of 25 screened and randomized subjects, seven were excluded from the analyses as they did not experience a migraine attack within 3 months, as planned in the protocol. These subjects were excluded from the study without having taken any study treatment, and were not replaced. The remaining 18 subjects completed both treatment groups and are included in the PK, safety, and efficacy analyses. Patient disposition is summarized in Figure 1. Baseline clinical characteristics for all 18 patients included in the study are shown in Table 1. The majority of patients (83.3%) had moderate-to-severe migraine at the start of treatment. Seven patients (38.8%) were taking at least one concomitant medication at screening and during both treatment periods. In addition to baseline characteristics, vital parameters were also measured prior to undertaking the study, at the visit between the two study periods (switch after first treatment), and at the end of the study (after second treatment) (Table 1). BODY.RESULTS.PHARMACOKINETIC ANALYSIS: Mean plasma concentrations of frovatriptan and rizatriptan over the study period are shown in Figure 2A, and the observed and calculated PK parameters are shown in Table 2. The concentration:Cmax ratio (%) peaked at the 2-hour time point for both drugs and shifted forward 2 hours (faster absorption rate) for frovatriptan compared to rizatriptan (Figure 2B). Tmax was similar for frovatriptan and rizatriptan (2.7 versus 2.3 hours, respectively), whereas the t1⁄2 for frovatriptan was significantly longer than rizatriptan (29.3 versus 3.2 hours, P<0.0001) (Table 2). Subanalysis of PK parameters by sex was also performed, and this revealed significantly higher Cmax (3.2±1.5 versus 1.49±0.55 ng/mL, P=0.009) and t1⁄2 (32.8±7.8 versus 24.9±7.7 hours, P=0.048) values for frovatriptan in females compared to male subjects. Sex-specific differences in PK parameters were also observed for rizatriptan, but did not attain statistical significance. BODY.RESULTS.PAIN-FREE AND PAIN-RELIEF RATES AND MIGRAINE RECURRENCE: Pain-free, pain-relief, and recurrence rates are summarized in Table 3. Although no differences were observed in pain-free rates at 2 hours, frovatriptan was more effective than rizatriptan at 4 hours (38.9 versus 5.6%, P=0.045). Pain-relief rates were similar between frovatriptan and rizatriptan. In contrast, the number of recurrent episodes was significantly higher for rizatriptan-compared to frovatriptan-treated patients, attaining statistical significance at 48 hours (55.6% versus 27.8%, P=0.046). This finding was confirmed by the observation of a significantly lower cumulative hazard of migraine recurrence (P=0.04) over 72 hours in patients treated with frovatriptan (Figure 3). No difference was observed between male and female subjects for pain-free or pain-relief rates or rate of migraine recurrence. BODY.RESULTS.ASSOCIATION BETWEEN PHARMACOKINETIC PROFILE AND PAIN-FREE OR RECURRENCE RATE: The univariate regression analysis performed between the concentration:Cmax ratio (%) and pain-free/pain-relief parameters (Figure 4) showed a positive correlation between frovatriptan concentration:Cmax ratio (%) and the proportion of patients that were either pain free or experienced pain relief over the entire study period (Figure 4A). No such correlation was seen for rizatriptan (Figure 4B). We next directly correlated cumulative recurrence over the study period with the concentration:Cmax ratio, and observed a significant inverse correlation for both frovatriptan- (r=−0.96, P=0.0028) and rizatriptan-treated (r=−0.98, P=0.0004) groups (Figure 4C). While recurrence rates remained similar at higher concentration:Cmax ratios of 20%–90%, as highlighted in the figure, the curves markedly diverge at a concentration:Cmax ratio of <20%, whereby cumulative recurrence rate nears 60% in patients treated with frovatriptan and reaches 100% in those treated with rizatriptan (Figure 4C). This analysis was extended to other PK parameters, but did not reveal any additional associations. BODY.RESULTS.MIGRAINE-ASSOCIATED SYMPTOMS: Both drugs gradually reduced migraine-related symptoms, with no difference observed between treatments (Figure 5A–D). Although differences were observed at baseline for nausea and photophobia, these differences were not statistically significant. A significant reduction in patients experiencing nausea (P=0.045), photophobia (P<0.001), and phonophobia (P<0.001) was observed at 72 hours for both drugs compared to baseline (Figure 5B–D). BODY.RESULTS.DRUG PREFERENCE: No significant difference was observed in the proportion of patients who preferred frovatriptan versus rizatriptan (five subjects [27.8%] versus six subjects [33.3%]). Seven (38.9%) patients had no preference for either drug. BODY.RESULTS.RESCUE MEDICATION: Ten patients (55.5%) required concomitant medication to treat their migraine when the study drug provided insufficient relief. Five patients received single-medication treatment, whereas the remaining five patients required two or more doses of the same or two or more different medications. In total, 31 different concomitant medications were taken among the patients, the most frequent being paracetamol (n=11), caffeine (n=6), and indomethacin (n=5). Other medications included prochlorperazine (n=4), nimesulide (n=2), meclizine (n=1), pyridoxine (n=1), and propyphenazone (n=1). Twenty different migraine attacks were treated using combinations of rescue medication on top of frovatriptan or rizatriptan. The frequency of rescue medication was lower in patients during frovatriptan treatment (seven of 20) compared to rizatriptan treatment (13 of 20), this difference just failing to reach statistical significance (P=0.058). BODY.RESULTS.SAFETY: A total of nine AEs were reported by nine subjects during the study (Table 4). One event was reported during frovatriptan treatment compared to eight during rizatriptan treatment (relative risk 2.4, confidence interval 1.39–4.1; P=0.021). The majority of AEs (seven events) were of mild or moderate intensity, and no serious AEs were reported during the study. There was a small decrease in respiratory rate in both treatment groups during treatment, but no clinically-relevant change in any of the other vital signs (heart rate, blood pressure, or temperature) in either treatment group. BODY.DISCUSSION: The present head-to-head trial compared the PK and clinical activity of the two triptans frovatriptan and rizatriptan in 18 patients suffering from migraine and treated with these two drugs according to a double-blind, two-way, crossover design, with a 14-day washout period. Although the therapeutic efficacy of frovatriptan has been directly compared to other triptans,19,20,23,24 including rizatriptan,19,20 this was the first study to specifically examine both the PK profile and therapeutic efficacy of two pharmacokinetically distinct triptans in parallel. PK analysis revealed similar kinetics for the concentration:Cmax ratio during the early phase, while both drugs differed substantially over later time points (4–72 hours). A marked difference was observed in the t1⁄2 between frovatriptan (29.3 hours) compared to rizatriptan (3.24 hours). These two distinct PK profiles are already established.8 Frovatriptan has been shown to have a t1⁄2 of approximately 26 hours, which was not affected by sex, dose, or mode of administration.13 Other studies have shown similar t1⁄2 for frovatriptan, which varied slightly due to age and/or sex.25 In contrast, rizatriptan has been shown to have an extremely fast plasma elimination t1⁄2, typically ranging between 2 and 2.5 hours, corroborating findings observed in the present report.15,18 Frovatriptan has distinctive pharmacologic features compared with other triptans, which make it particularly well suited to patients with prolonged migraines and those suffering migraine recurrence.9,10,26 In addition, due to the lack of inhibitory or inducing effects on cytochrome P450 isoenzymes, frovatriptan retains a low risk of drug interactions, and dosage adjustment is unlikely to be warranted. This is particularly relevant, since combination treatment of frovatriptan with analgesic drugs is sometimes required. Although the number of subjects in our study was small, our safety analysis replicated the findings in other studies in which frovatriptan exhibited a favorable tolerability profile that was often better than comparator triptans.11 As expected, both drugs also gradually decreased migraine-related symptoms, as was also previously observed.20 It is worth noting that sex-specific differences were observed for frovatriptan, for some PK parameters examined, but not for rizatriptan. Female subjects had significantly higher Cmax and t1⁄2 values compared to male subjects. These sex-specific effects have also been observed in previous findings in healthy adults and adolescent migraineurs.13,25 This difference has mainly been attributed to greater bioavailability in females compared to male subjects, in addition to a higher volume of distribution and clearance in males.11,13,25 Regardless, these PK sex-specific differences for frovatriptan do not have any clinically significant effect, in terms of efficacy or safety, since frovatriptan has an extremely wide therapeutic window, and thus no dosage adjustment was deemed necessary.13 Supporting this, our findings also demonstrated that the activity of frovatriptan in terms of clinical benefit was similar between males and females. Comparing efficacy of the two drugs in the present study, we did not see any difference between the two agents at 2 hours, but did observe a higher pain-free rate at 4 hours. Other head-to-head trials comparing frovatriptan to another triptan in migraineurs have observed similar extent of benefit afforded by frovatriptan.19,20,23,24 Furthermore, pain-relief rates at 2 hours (ranging between 40% and 50%) were similar for both drugs and also corroborate previous findings.19,20,23,24 These data, collectively, indicate that the extended duration of effect associated with frovatriptan does not come at the expense of a slower onset of action. We also demonstrated that the frequency of pain-free episodes and pain-relief rates were directly correlated with the PK profile of frovatriptan. In addition to providing acute benefit, in terms of pain-free episodes, frovatriptan also significantly reduced recurrent episodes within 48 hours compared to rizatriptan. Recurrent episodes within 48 hours have typically lower incidence, ranging from 20% to 30% for frovatriptan compared to 43% for rizatriptan,20 44% for almotriptan,23 and 50% for zolmitriptan.24 The numerical differences in recurrence rates at 24 and 48 hours also suggest that beneficial effects on recurrence might also have been seen with frovatriptan with a larger sample size. The difference in PK profile as seen by the concentration:Cmax ratio (%) for frovatriptan and rizatriptan from 4 to 72 hours was shown to correlate negatively with recurrence rate. Our findings reinforce the association observed between recurrence rate and PK activity (t1⁄2) of various triptans, previously documented by Géraud et al,22 but a small sample size hampered the strength of our analysis. Although our findings indicated that PK activity may be an important factor in determining frequency of migraine recurrence, this continues to be an extremely complex physiological phenomenon that cannot be explained solely by PK activity.22 While it is important not to overestimate the clinical importance of different PK profiles of triptans,27 previous evidence,22 including the present report, points toward the need for additional trials with greater sample sizes and with well-defined end points. BODY.DISCUSSION.STUDY LIMITATIONS: The main limitation of the present study was the small sample size. This study was slightly underpowered due to the exclusion of seven patients, as they had not experienced a migraine attack within 3 months, as planned in the protocol. A larger sample size would have permitted a multivariate approach to determine more precisely the cause–effect relationship between PK profile and drug effect (ie, antimigraine outcome measures). Regardless, even with the small sample size, secondary efficacy measures (eg, pain-free and pain-relief rates and rate of recurrence) were similar to other previously published studies.19,20,23,24 Additional time points for secondary measures and a slightly longer follow-up period would have been desired. Although the predominant analgesic effect was attributed to triptan use, it should also be taken into account that in some subjects treatment with anti-inflammatory drugs was required, as planned in the protocol, which may have contributed in part to pain relief. Regardless, the double-blind, randomized, crossover design controlled for these and other potential confounders. A second potential weakness of the present study was the fact that PK parameters were measured from plasma samples and not whole blood. Since rizatriptan (and subsequent PK parameters) is normally assayed from plasma,15,18 we also specifically assayed frovatriptan in the same manner, since this was a crossover design. However, this could be particularly relevant for frovatriptan, since it is recognized to bind to red blood cells (60% reversibly bound at steady states), thus potentially compromising assays performed exclusively in plasma.28,29 However, values for different PK parameters measured in the present study were well within the normal range for those described using whole blood,13,25 arguing that the plasma versus whole blood difference (particularly for frovatriptan) does not appear to cause an observable effect, at least at the doses used in the present study. Finally, we included a limited PK sampling schedule in our study with the intention of correlating plasma levels and efficacy at selected time points. This meant that we were unable to fully define the PK profiles of both drugs, and particularly for rizatriptan, the shape of our observed curve suggested that Tmax would have occurred earlier than our first sample time (2 hours). While this is a limitation for a pure PK analysis, it does not necessarily undermine the conclusions we have drawn from the PK efficacy correlations. Indeed, an earlier Tmax and higher true Cmax for rizatriptan would result in even lower concentration:Cmax ratios for rizatriptan and so magnify further the differences between the two agents. BODY.CONCLUSION: In conclusion, this is the first study to specifically examine both the PK profile and therapeutic efficacy of two pharmacokinetically distinct triptans in parallel. Findings from the present pilot study confirmed the expected clinical activity of the two drugs on a target population and confirm previously published PK characteristics. Although both frovatriptan and rizatriptan displayed similar PK activity in the early phase (0–4 hours), marked differences in activity were observed in the intermediate period after dosing, particularly 4–24 hours, where the longer t1⁄2 of frovatriptan and the persistence of its active plasma concentrations may explain its better performance at later time points. This time period (4–24 hours) coincided with improved efficacy by frovatriptan in terms of reduction of migraine attacks and reduction of migraine recurrence compared to rizatriptan. PK parameters were also found to be associated with pain-free and pain-relief episodes, in addition to migraine recurrence. Results from the present trial support the use of frovatriptan over rizatriptan, particularly in patients suffering from relapse and/or migraines of longer duration. Patient-reported AEs and drug-related AEs were lower in patients treated with frovatriptan compared to rizatriptan. A further trial with a larger pool of patients will help confirm the results obtained in the present study.

TITLE: Effect of vitamin D on proteinuria in type 2 diabetic patients ABSTRACT.BACKGROUND: Vitamin D (Vit D) deficiency is a common disorder in diabetic patients and may be a risk factor for ischemic heart disease and exacerbation of diabetic nephropathy(DN). ABSTRACT.OBJECTIVES: The aim of this study was to evaluate the effect of Vit D3 therapy on protein uriain type 2 diabetic patients with deficient or insufficient serum Vit D. ABSTRACT.PATIENTS AND METHODS: In a double blind clinical trial, 60 type 2 diabetic patients with proteinuria greater than 150 mg/day who had Vit D deficiency or insufficiency were randomly enrolled in two equal groups. Pearl of Vit D as 50 000 IU/week and placebo (1 tablet per week) were prescribed in patients of case and control groups respectively for 8 weeks. At the beginning and 2 months later, 24 hours urine protein was checked in all patients. ABSTRACT.RESULTS: There is no difference between serums Vit D level in case and control group at the beginning of the study, however at the end of the study serum Vit D level was significantly higher in the case group. There is no difference in proteinuria between case and control group at the beginning and the end of the study, while a significant difference between the changes of proteinuria before and after the study was seen in two groups (P = 0.028). ABSTRACT.CONCLUSIONS: Vit D deficiency may exacerbate protein uric and DN, hence correction of Vit D deficiency may decrease proteinuria in diabetic patients with nephropathy. BODY: Implication for health policy/practice/research/medical education:Nephropathy is a common complication of diabetes, so management of it could lead to decreasing of mortality and morbidity of the patients. In addition of angiotensin converting enzyme inhibitors and angiotensin receptor blockers, other drugs such as spironolactone, statins, and allopurinol were used for management of diabetic nephropathy with acceptable results in some studies. Vitamin D metabolites may have renoprotective and anti-proteinuric effect, decreasing of insulin resistance and blood pressure lowering effect, as well. Pearl of vitamin D is an inexpensive, safe and probably effective drug for patients with diabetic nephropathy and vitamin D deficiency. BODY.1. BACKGROUND: The prevalence of diabetes Mellitus (DM) worldwide was 2.8% and total number of patients was 171 million in 2000. There are continuing increase in the prevalence and incidence of DM, while the incidence of DM was 7.1% in 2012 (1). Diabetic nephropathy (DN) is the most common cause of chronic kidney disease and end stage renal disease, as about 30% to 35% of dialysis patients have diabetes (2). Diabetes is also the most common cause of renal replacement therapy requirement, in the United States (3). One of the common and serious complications of diabetes is nephropathy, defined by the development of proteinuria and classified based on the severity of proteinuria as microalbuminuria and macroalbuminuria. With onset of proteinuria, glomerular filtration rate decreased gradually at the rate of 10-12 mL annually or 1ml monthly (4,5). Lifestyle modifications (such as regular exercise, weight loss in obese patients, limitation of salt and alcohol intake, and restriction of dietary protein intake), blood pressure and serum glucose controlling may have a role in the prevention of DN (6). The basic drug treatment of DN is inhibition of the renin–angiotensin aldosterone system with ACE inhibitors or angiotensin II–receptor blockers. Combination of angiotensin converting inhibitors plus angiotensin receptor blockers (dual system blocking) was shown more effective than single agent therapy in some studies (7). There are a few novel modalities for treatment of DN, for example, aliskiren, a direct renin inhibitor has shown the anti-proteinuric effect in diabetic patients (8). Non-dihydropyridine calcium channel blockers such as diltiazem also have renoprotective effect probably due to decreasing hyperfiltration and intra-glomerular pressure (9), in addition, fenofibrate was shown to be effective in reducing proteinuria in small clinical trials (10). Serum level of uric acid may be greater in DN patients compared to normal population (11), thus allopurinol was shown to be effective in reducing proteinuria in type 2 diabetic patients (12). Spironolactone an aldosterone receptor blocker probably has renoprotective effect due to its anti-inflammatory property (13). In addition, combination of spironolactone and hydrochlorothiazide may be effective in DN treatment (14). Vitamin D (Vit D) deficiency is a common disorder in diabetic patients and may be a risk factor for ischemic heart disease, deterioration of chronic kidney disease and DN (15). Vit D metabolites may have a role in the inhibition of the renin-angiotensin system and renoprotective effect by preventing of glomerulosclerosis and anti-proteinuric effect. Furthermore, prescription of Vit D has shown decrease in insulin resistance and decrease in blood pressure as well (16,17). BODY.2. OBJECTIVES: Recently, some studies were carried out regarding to effect of Vit D supplementation on reducing proteinuria in diabetic patients. However the results of these studies are controversial. Therefore, we aimed to evaluate effect of Vit D in reducing proteinuria in the type 2 diabetic patients with 25 (OH) Vit D deficiency or insufficient. BODY.3. PATIENTS AND METHODS. 3.1. STUDY PATIENTS : In this double-blind randomized clinical trial, 60 type 2 diabetic patients were randomly enrolled in two equal case and control groups. Inclusion criteria were: proteinuria greater than 150 mg/dl, glomerular filtration ratio greater than 50 mL/min or serum creatinine < 2 mg/dL and Vit D deficiency (Vit D <25 nmol/L) or insufficiency (Vit D between 25 and 75 nmol/L). Exclusion criteria including; noncooperation of the patients during the study, Ca × Phosphorus >55mg2/dL2, of Vit D or Ca supplement consumption during 2 recent months and Ca>10 mg/dL. In 30 patients in case group, Vit D 50000 IU was weekly prescribed for 8 weeks. For 30 patients in control groups, placebo was prescribed similar to control group (weekly for 8 weeks). Demographic criteria of the patients were obtained and fasting blood sugar (FBS), glycosylated hemoglobin (HbA1C), Ca, phosphorus (P), Albumin, and 25 (OH) Vit D were checked in the beginning and at the end of the study in all of the patients. Urine protein was measured also in the end of the study for all of the patients. BODY.3. PATIENTS AND METHODS. 3.2. ETHICAL ISSUES : 1) The research followed the tenets of the Declaration of Helsinki; 2) informed consent was obtained, and they were free to leave the study at any time and 3) the research was approved by the ethical committee of Shahrekord University of Medical Sciences (Ethical cod:1128 and IRCT code: IRCT2015081723656N1). BODY.3. PATIENTS AND METHODS. 3.3. STATISTICAL ANALYSIS : At the end of the study, data were entered to SPSS software, (version 21.0, SPSS Inc, Chicago, IL, USA). Then data were analyzed for comparisons of groups using the chi-square test, and independent and paired t test, and P values less than 0.5 were considered significant. All information was remaining confidential, so written consent forms were filled in by all cases. The study was done under permission and support of research deputy of Shahrekord University of Medical Sciences. BODY.4. RESULTS: Fifty-seven patients were participated in this study and 3 patients were left due to non-cooperation and withdrawal of one patient of case group and 2 patients of control group. Twelve patients of case group and 18 patients of control group were female and other were male (P=0.120). Mean age of case and control groups were 62.9±9.3 and 62.4±9 years respectively (P=0.85). At the beginning of study, mean serum level of Vit D in case and control groups were 36.76±19.16 (nmol/L) and 32.19±17.76 (nmol/L), respectively (P=0.33). However at the end of the study mean serum level of Vit D was 89.44±34.35 (nmol/L) and 38.02±23.90 (nmol/L) in the case and control groups, respectively (P=0.0001). There was a significant difference between serum level of Vit D before and after the study in the case group (P=0.001) as well as control group (P=0.02). As shown in Table 1, mean level of proteinuria in the patients of case group and control group were 962.62±885.99 mg/day and 755.71±640.94 mg/day, respectively (P=0.70). While, at the end of the study, it was 892.24±879.40 (mg/day) and 971.60±940.24, respectively (P=0.48). Difference of proteinuria before and after the study in the case and control groups were 70.38±553.71 and 215.89±451.44, respectively (P=0.028), which revealed a significant difference among them. There were no significant difference between two groups of the patients based on FBS, HbA1C, ESR and CRP levels (P>0.05). Table 1 Comparison of variables in two groups of patients before and after the study Variable Case group Control group P Vit D (nmol/L) Before 36.76±19.16 32.19±17.76 0.326 After 89.44±34.35 38.02±23.90 0.0001 P 0.0001 0.02 - Before and after difference 52.68±27.48 5.82±14.08 <0.001 Proteinuria (mg/day) Before 962.62±885.99 775.71±640.94 0.702 After 892.24±879.40 971.60±940.24 0.482 P 0.27 0.025 - Before and after difference 70.38±553.71 215.89±451.44 0.028 HbA 1c (%) Before 7.82±0.86 8.14±1.26 0.318 After 8.02±1.23 8.10±0.96 0.523 P 0.259 0.895 - Before and after difference 0.2±0.99 0.04±1.05 0.406 FBS (mg/dL) Before 146.69±53.03 151.50±52.35 0.571 After 152.72±49.4 154.89±73.26 0.354 P 0.482 0.882 - Before and after difference 6.03±35.54 3.39±60.05 0.544 BODY.5. DISCUSSION: The study showed significant difference between changes of proteinuria between 2 groups of the patients during the study (P=0.028), thus we concluded that prescription of Vit D in type 2 diabetic patients with nephropathy and Vit D deficiency may decreased proteinuria (Figure 1). Figure 1The mean of proteinuria during the study in the case and control group. Prevalence of Vit D deficiency is higher in diabetic patients, in addition association of Vit D deficiency or Vit D insufficiency with the DN was reported in some studies. (15,18). Vit D deficiency may be associated with other microvascular complication of diabetes such as diabetic retinopathy (19). In addition to angiotensin converting enzyme inhibitors and angiotensin receptor blockers, other drugs such as spironolactone (aldosterone receptor blocker)(14), non- dihydropyridine calcium channel blockers (diltiazem), antihyperlipidemic agents (20), allopurinol (12) were used in the treatment of DN. Effect of Vit D prescription in DN was evaluated in some clinical and experimental studies with controversial results. Ahmadi et al, in a clinical trial on 51 diabetic patients with DN and Vit D3 deficiency, it was found that Vit D3 prescription for three months had not any effect on decreasing of proteinuria (21). Kim et al in the study on 63 diabetic patients with nephropathy and low level of serum Vit D during seven months showed that repletion with cholecalciferol could decrease albuminuria. They concluded that dietary Vit D in patients with DN may have a beneficial effect in delaying the progression of disease (22). In a study by Bonakdaran et al, of 119 diabetic patients, 31 cases had Vit D deficiency, any significant reduction in the proteinuria after using of calcitriol for 8 weeks was detected (23). In a study by De Zeeuw et al, on 281 diabetic patients, they could show that 2 mg/day of paricalcitol in addition of rennin-angiotensin-aldosterone blockers, could decrease proteinuria (24). In another rstudy by Huang et al deficiency of serum 25(OH) Vit D3 levels was associated with microalbuminuria, and administration of cholecalciferol significantly decreased albuminuria in the early stages of treatment. They concluded that conventional doses of cholecalciferol may have antiproteinuric effects on Chinese diabetic patients (25). We found no association between using of Vit D and serum Hb A1c, however different results were reported in other studies, for example, in Madar et al. 16-week administration of Vit D3 to healthy individuals with low Vit D status showed no improvement for HbA1c (26). Likewise, Ahmadi et al reported no significant difference based on HbA1C in both case and control groups (21). Otherwise Bonakdaran et al showed a significant reduction in HbA1c in diabetic patients (23). BODY.6. CONCLUSIONS: Administration of Vit D in type 2 diabetic patients with Vit D deficiency or insufficiency leads to normalization of serum Vit D level and decrease proteinuria compared to control group, however we did not show any improvement of glycemic control indices in the patients. Thus, we concluded that, correction of Vit D deficiency may be an effective and safe modality of treatment for DN. BODY.LIMITATIONS OF THE STUDY: Small proportion of the patients was a limitation of our study. BODY.ACKNOWLEDGMENTS: This study is adapted from the internal medicine residency thesis of Mohsen Kabiri. Hereby, we thank the Research and Technology Deputy of Shahrekord University of Medical Sciences. BODY.CONFLICTS OF INTEREST: The authors declared no competing interests. BODY.AUTHORS’ CONTRIBUTION: AM; study design, preparation of manuscript and final revision. MM; consultant of theses. MK; data gathering and data interpretation. SK; statistical consultant. BODY.FUNDING/SUPPORT: This study was a funded thesis at Shahrekord University of Medical Sciences (Grant# 1128).

TITLE: Quality of life during olaparib maintenance therapy in platinum-sensitive relapsed serous ovarian cancer ABSTRACT.BACKGROUND:: Maintenance monotherapy with the poly(ADP-ribose) polymerase inhibitor olaparib significantly prolongs progression-free survival over placebo in patients with platinum-sensitive relapsed serous ovarian cancer, with greatest benefit seen in patients with a BRCA1/2 mutation (BRCAm). Preservation of health-related quality of life (HRQoL) is important during maintenance therapy; we evaluated the effect of olaparib on HRQoL in this Phase II trial (NCT00753545, Study 19). ABSTRACT.METHODS:: Patients received olaparib 400 mg b.i.d. (capsules) or placebo until progression. Patient-reported HRQoL and disease-related symptoms were evaluated using the FACT-Ovarian (FACT-O) questionnaire (completed at baseline and every 28 days until progression), the FACT/NCCN Ovarian Symptom Index (FOSI) and the Trial Outcome Index (TOI). TOI of the FACT-O was the primary measure. ABSTRACT.RESULTS:: Overall, 265 women were randomised to maintenance olaparib (n=136) or placebo (n=129). Compliance for HRQoL assessment was high (∼80% over time). Most patients in both arms reported a best response of 'no change' on TOI (81%) and other HRQoL measures. There were no statistically significant differences in time to worsening or improvement rates of TOI, FOSI and FACT-O scores in the overall, BRCAm and germline BRCAm populations. ABSTRACT.CONCLUSIONS:: Maintenance treatment with olaparib was well tolerated and had no adverse impact on HRQoL in this study of patients with platinum-sensitive relapsed serous ovarian cancer who had responded to their most recent platinum-based therapy (partial or complete response). Interpretation of the HRQoL results in this population may differ from patients who have not responded to their most recent platinum-based therapy. BODY: Health-related quality of life (HRQoL) is a multidimensional concept encompassing: physical, cognitive and emotional wellbeing; social functioning domains; disease-related symptoms; therapy-induced side effects; and potential financial and family burden. These HRQoL measures are a particularly important consideration in the maintenance setting after response to chemotherapy, when the majority of patients do not have any symptoms related to recurrent cancer, as the aim of maintenance treatment is to prolong the time to progression and to delay the need for further chemotherapy without compromising the quality of life of the patients on treatment (Friedlander and King, 2013). Olaparib (Lynparza) is an oral poly(ADP-ribose) polymerase (PARP) inhibitor that blocks base-excision repair by trapping PARP at sites of DNA damage, leading to synthetic lethality in tumour cells with deficiencies in homologous recombination repair, such as those with BRCA1/2 mutations (BRCAm) (Evers et al, 2008; Rottenberg et al, 2008). Olaparib has been extensively studied and in several Phase II trials, olaparib monotherapy exhibited antitumour activity in patients with breast and ovarian cancer, particularly in those with BRCAm (Audeh et al, 2010; Tutt et al, 2010; Gelmon et al, 2011). In December 2014, olaparib obtained regulatory approval in the EU as maintenance monotherapy for adult patients with platinum-sensitive recurrent (PSR) BRCAm (germline and/or somatic) high-grade serous ovarian cancer (SOC), fallopian tube or primary peritoneal cancer, who are in complete or partial response to platinum-based chemotherapy. This EU approval was based on the results of a randomised, double-blind, placebo-controlled, Phase II study (NCT00753545, D0810C00019, Study 19), in which maintenance monotherapy with olaparib (capsules) significantly prolonged progression-free survival (PFS) vs placebo in patients with PSR SOC and patients with a BRCAm were most likely to benefit from treatment (Ledermann et al, 2012, 2014). The toxicity profile from this study demonstrated that adverse events (AEs) were manageable in most patients (Ledermann et al, 2012, 2014). Dose reductions were performed as a result of AEs in 24% and 4% of patients in the olaparib and placebo arms, respectively. The common AEs of nausea, vomiting, fatigue and anaemia led to dose reduction in 4%, 3%, 4% and 4% of olaparib patients (0%, 1%, 1% and 1% of placebo patients), respectively. The discontinuation rate as a result of AEs was 4.4% for olaparib patients and 1.6% for placebo patients. HRQoL was assessed as a secondary objective in this study and we report the impact of olaparib on HRQoL and disease-related symptoms. BODY.MATERIALS AND METHODS.STUDY DESIGN AND PATIENTS: In this Phase II trial (NCT00753545), adult patients with platinum-sensitive, relapsed, high-grade SOC who had received at least two platinum-based regimens and had a partial or complete response to their most recent platinum-based regimen were enrolled (Ledermann et al, 2012). Patients were randomised to receive olaparib 400 mg capsules or placebo twice daily within 8 weeks of completing platinum-based chemotherapy. Assessment of BRCAm status was not required at enrolment and was either reported on case report forms after local testing or established retrospectively using blood samples (germline BRCAm (gBRCAm)) and/or archival tumour samples (tumour BRCAm) (Ledermann et al, 2014). The trial design, including inclusion/exclusion criteria, and a planned retrospective analysis of outcomes by BRCA status have been published previously (Ledermann et al, 2012, 2014). The secondary endpoints reported here had the same data cut-off as the primary analysis (30 June 2010). All patients provided written informed consent. The institutional review boards or independent ethics committees of all investigational sites approved the protocol. The study was performed in accordance with the Declaration of Helsinki, Good Clinical Practice and the AstraZeneca Policy on Bioethics (AstraZeneca, 2015). BODY.MATERIALS AND METHODS.HRQOL TOOL SELECTION: HRQoL analysis assessed the impact of maintenance therapy with olaparib, relative to placebo, on HRQoL and disease-related symptoms in patients with PSR SOC. HRQoL was assessed using the Functional Assessment of Cancer Therapy Ovarian (FACT-O) questionnaire, which is a multidimensional questionnaire developed and validated for ovarian cancer patients. The FACT-O questionnaire was linguistically validated in multiple languages; however, no Ukrainian translation was available. BODY.MATERIALS AND METHODS.ASSESSMENTS: The FACT-O questionnaire comprises questions regarding physical, social, emotional and functional wellbeing and additional concerns (n=7, 7, 6, 7 and 12 items, respectively; Supplementary Material and Supplementary Figure S1). The Trial Outcome Index (TOI), a subset analysis of the FACT-O, was the primary HRQoL measure. Whereas the FACT-O score derives from 39 items (score range: 0−152; higher score indicates better health state), the TOI score derives from 26 physical and functional wellbeing items and ovarian cancer concerns subscales (score range: 0−104). The Functional Assessment of Cancer Therapy/National Comprehensive Cancer Network Ovarian Symptom Index (FOSI) assessment derives from eight symptom-related FACT-O items (score range 0−32). Individual concepts of nausea, vomiting and fatigue were assessed from specific FACT-O questions ('I have nausea', 'I have been vomiting', 'I have a lack of energy') that were also captured in FOSI scores (Supplementary Material). There is currently no standard HRQoL assessment for use in oncology maintenance clinical trials; the TOI of the FACT-O was chosen as the primary HRQoL assessment in this study because it is a validated tool and contained the most concepts of importance to ovarian cancer patients and those that a pharmacological product would be expected to impact (physical well-being, functional well-being and specific concerns for ovarian cancer patients). The FACT-O is a multidimensional questionnaire developed and validated for use by ovarian cancer patients; it includes the 27-item FACT-General (FACT-G) targeted to general cancer patients and 12 questions specific to issues faced by ovarian cancer patients (FACT-O subscale). The FACT-G questionnaire includes the following four subscales: physical well-being (PWB; seven items), social well-being (seven items), emotional well-being (six items) and functional well-being (FWB; seven items). These subscales can be analyzed separately or aggregated to produce a total HRQoL score. The FACT-G has demonstrated reliability, validity and responsiveness to change over time (Cella et al, 1993). Two of the FACT-G subscales (PWB and FWB) plus the FACT-O subscale are summed to represent the TOI. BODY.MATERIALS AND METHODS.ANALYSES: Patients completed the FACT-O questionnaire at baseline and monthly until progression. If patients discontinued for reasons other than progression (assessed by Response Evaluation Criteria in Solid Tumours (RECIST)), HRQoL assessments continued until progression was confirmed. Individual symptom severity over the previous 7 days was measured using the five-item Likert scale (not at all (0), a little bit (1), somewhat (2), quite a bit (3) and very much (4)). Better wellbeing was generally indicated by higher scores; where appropriate, raw scores were reversed (Supplementary Material). Improvement and worsening rates in HRQoL values were evaluated against prospectively determined minimally important differences relevant to each endpoint (Supplementary Material) (Osoba et al, 2005). A retrospective exploratory analysis using linear mixed-model repeated-measures (MMRM) modelling, adjusting for score at baseline, time and treatment-by-time interaction, estimated the mean effect over time for HRQoL (Stockler et al, 2014). Estimates of the least-squares means for treatment effects within and between treatment groups were reported with corresponding 95% confidence intervals (CIs). Mixed-model repeated-measures analyses were performed on the overall population, as well as the BRCAm and gBRCAm subgroups. BODY.RESULTS.PATIENTS: Baseline demographics have been reported for the overall population and BRCAm subgroups (Ledermann et al, 2012, 2014) and are summarised, alongside data for gBRCAm patients, in Table 1. No significant differences in patient demographics were observed between treatment groups. Following retrospective gBRCAm and somatic BRCAm testing, BRCAm status data were available for 254 of 265 patients (96%), of whom 136 of 254 (54%) had a known/suspected deleterious gBRCAm and/or somatic BRCAm (Ledermann et al, 2014). Ninety-six patients (36%) had a gBRCAm (Ledermann et al, 2014). For the 265 randomised patients (n=136 olaparib, n=129 placebo), compliance with study treatment was good (mean 97% (standard deviation (s.d.) 9%) olaparib, mean 99% (s.d. 3%) placebo). Compliance rates for TOI, FOSI and FACT-O assessment were high at baseline and similar in each arm (85%, 86% and 84% for olaparib-treated patients vs 86%, 89% and 86% for placebo-treated patients, respectively). Overall compliance rates over 16 months were also high (69%, 70% and 69% for olaparib-treated patients and 69%, 70% and 69% for placebo-treated patients, respectively). Compliance rates over time for the first 6 months of treatment for TOI, FACT-O and FOSI are detailed in Supplementary Table S1. A total of 41 patients were excluded from HRQoL analyses (15% n=23 olaparib, n=18 placebo); 33 (12% n=19 olaparib, n=14 placebo) did not complete the FACT-O questionnaire at baseline or were not evaluable for FACT-O/TOI and were excluded from HRQoL analyses. Of these patients, 14 required a Ukrainian translation (n=9 olaparib, n=5 placebo), which was unavailable, 20 did not receive the questionnaire because of administrative failure (n=11 olaparib, n=9 placebo), two patients (olaparib) were not evaluable for FACT-O and five patients were not evaluable for FACT-O and TOI (n=1 olaparib, n=4 placebo). Patient-level HRQoL data were collected until either progression or the primary analysis. As the median PFS from randomisation after the end of chemotherapy was 4.8 months in the placebo arm, the majority of placebo patients (68%) did not contribute HRQoL data beyond 6 months. Therefore, no data are presented beyond 6 months. BODY.RESULTS.HRQOL: At baseline mean (standard deviation) scores were balanced between olaparib- and placebo-treated patients for all assessments (TOI, FOSI and FACT-O; Table 2). Across the overall, BRCAm and gBRCAm groups, most patients achieved best HRQoL responses of 'no change' (Table 2). Results from MMRM analyses involving BRCAm or gBRCAm patients were consistent with results from patients irrespective of BRCAm status. BODY.RESULTS.TOI: Most patients reported a best response of 'no change' (Table 2); statistical analysis of improvement rates showed no statistically significant difference between treatment groups in the overall population (odds ratio (OR) 1.14; 95% CI 0.58, 2.24; P=0.7). For BRCAm and gBRCAm patients, there was no statistically significant difference in improvement rates, although numerically more patients receiving olaparib had a best response of 'improved' vs placebo (BRCAm: 25% vs 19%, respectively; OR 1.37; 95% CI 0.56, 3.46; P=0.5; gBRCAm: 27% vs 8%, respectively; OR 4.08; 95% CI 1.11, 19.9; P=0.03), indicating no detriment to HRQoL. Supplementary Table S2 provides further details for the time-to-worsening analysis using Cox's proportional hazards model for TOI. Patients had high baseline TOI scores (mean±s.d., 81.7±11.8 with olaparib and 81.5±11.6 with placebo in the overall population; 79.9±12.1 and 79.5±12.1, respectively, for BRCAm patients; 79.5±12.3 and 81.0±11.0, respectively, for gBRCAm patients), with TOI scores remaining consistent over time and similar between groups (Figure 1a–c and Supplementary Figure S2a and b). Details of further subscales are provided in Supplementary Figure S3. The greatest decrease was observed at month 1 for olaparib and month 4 for placebo (overall population, BRCAm and gBRCAm). In the overall population, there was no statistically significant difference in median time to TOI worsening with olaparib vs placebo, although the median time to worsening was numerically shorter with olaparib (3.8 vs 4.6 months, respectively; hazard ratio (HR) 1.08; 95% CI 0.75, 1.55; P=0.7). Median time to TOI worsening was numerically longer with olaparib vs placebo for BRCAm patients (5.7 vs 3.7 months, respectively; HR 0.8; 95% CI 0.48, 1.34; P=0.4) and gBRCAm patients (7.4 vs 3.6 months, respectively; HR 0.54 95% CI 0.30, 0.99; P=0.048). There was no HRQoL detriment in the overall, BRCAm and gBRCAm populations. BODY.RESULTS.FOSI: In the overall population, the percentage of patients with a best response of 'improved' in FOSI was similar between treatment groups (17% vs 15% for olaparib vs placebo; Table 2), with no significant differences (OR 1.22; 95% CI 0.60, 2.51; P=0.59). These values were 21% vs 16%, respectively, in BRCAm patients (OR 1.41; 95% CI 0.56, 3.70; P=0.47) and 26% vs 13%, respectively, in gBRCAm patients (OR 2.31; 95% CI 0.75, 8.10; P=0.15). Supplementary Table S2 provides further details for the time-to-worsening analysis using Cox's proportional hazards model for FOSI. Patient experience of important symptoms on FOSI remained consistent with baseline and comparable between treatment groups and over time (Figure 2a–c and Supplementary Figure S2c–d). Baseline mean FOSI scores±s.d. were 26.1±3.4 with olaparib and 25.4±3.8 with placebo for the overall population; 25.9±3.4 and 24.8±4.1, respectively, for BRCAm patients; and 25.8±3.3 and 25.1±4.1, respectively, for gBRCAm patients. In the overall population, median time to FOSI worsening was 2.8 vs 3.7 months for olaparib and placebo, respectively; there was no significant difference (HR 1.22; 95% CI 0.88, 1.71; P=0.228). Median time to FOSI worsening was 2.8 vs 3.7 months, respectively, in BRCAm patients (HR 1.15; 95% CI 0.74, 1.81; P=0.53) and 3.7 vs 3.3 months, respectively, in gBRCAm patients (HR 0.71; 95% CI 0.42, 1.22; P=0.212). BODY.RESULTS.FACT-O: No statistically significant differences were observed between treatment groups (21% vs 19% for olaparib vs placebo) for improvement in the FACT-O score in the overall population (OR 1.17; 95% CI 0.60, 2.27; P=0.65). In BRCAm patients, the percentage of patients with a best response of 'improved' was 27% vs 21% for olaparib vs placebo, respectively (OR 1.38; 95% CI 0.58, 3.39; P=0.47). These values were 29% vs 11%, respectively, for gBRCAm patients (OR 3.26; 95% CI 1.00, 12.9; P=0.05). Supplementary Table S2 provides further details for the time-to-worsening analysis using Cox's proportional hazards model for FACT-O. The FACT-O total score remained consistent with baseline and comparable between groups and over time (Figure 3a–c and Supplementary Figure S2e–f). Baseline mean FACT-O scores±s.d. were 121.9±17.3 with olaparib and 119.7±17.4 with placebo for the overall population; 118.9±18.1 and 115.9±18.9, respectively, for BRCAm patients; and 119.5±18.5 and 118.6±17.2, respectively, for gBRCAm patients. Time to FACT-O score worsening in the overall population was numerically shorter with olaparib vs placebo (2.8 vs 4.6 months, respectively; HR 1.16; 95% CI 0.83, 1.64; P=0.39). There were no clinically relevant or statistically significant differences in FACT-O score time to worsening for BRCAm patients (3.2 vs 4.4 months, respectively; HR 1.04; 95% CI 0.65, 1.69; P=0.87) or gBRCAm patients (3.2 vs 3.7 months, respectively; HR 0.84; 95% CI 0.48, 1.48; P=0.55). BODY.RESULTS.NAUSEA, VOMITING AND FATIGUE: Minimal changes were observed during the course of treatment for nausea, vomiting and fatigue in the overall population (Supplementary Figure S4). Most patients did not report nausea at baseline or discontinuation. At baseline, the mean nausea score was 3.77 with olaparib and 3.71 with placebo in the overall population, with a shorter median time to nausea worsening with olaparib (1.1 months) than placebo (6.5 months). Although olaparib patients experienced more nausea during the first months of treatment and a higher proportion had a best response of 'worsening' vs placebo (32.2% vs 16.8%, respectively), scores became similar with increasing time on olaparib. In gBRCAm patients, decreases in nausea scores were transient and only observed at month 1. The percentage of patients with a score of 3 or 4 (symptoms: 'a little bit' or 'not at all') for nausea was consistently above 65% (olaparib) and 75% (placebo) in the overall population; these values were 60 and 65%, respectively, for BRCAm patients and 60% and 75%, respectively, for gBRCAm patients. There were no differences in vomiting or fatigue scores between the olaparib and placebo groups in the overall, BRCAm or gBRCAm populations (Supplementary Figure S4b and c). BODY.DISCUSSION: Stability of HRQoL with acceptable side effects of treatment is an important consideration for patients with recurrent ovarian cancer receiving maintenance therapy after response to chemotherapy. In this study, there were no statistically significant or clinically relevant differences in HRQoL between treatment arms on TOI, total FACT-O and FOSI assessments among all patients and in the BRCAm or gBRCAm subgroups. These data demonstrate that maintenance treatment with olaparib had no apparent adverse impact on HRQoL in patients with PSR SOC, which is also supported by the high compliance with treatment and very low discontinuation rates because of AEs. Evidence of maintained HRQoL complements and provides additional support of the primary study results of a significantly prolonged PFS with olaparib vs placebo (difference in median PFS 3.6 months, P<0.001), with BRCAm patients most likely to benefit (difference in median PFS 6.9 months, P<0.0001). In addition, AEs in this study were generally manageable (Ledermann et al, 2012, 2014). Olaparib did not appear to affect patients' experience of vomiting, with similar scores between olaparib- and placebo-treated patients across all time points. Olaparib patients experienced more early nausea and, to a lesser extent, fatigue, but this is consistent with known AE profiles and, with increasing time on therapy, scores became similar to those for placebo. This is reflected by the initial worsening of physical wellbeing scores in olaparib-treated patients, however, after 4 months of treatment with olaparib the HRQoL scores were similar to those in patients treated with placebo. The patient population evaluated in the current study included patients with complete or partial responses to chemotherapy. As expected, baseline HRQoL scores in this group were relatively high compared with scores from other cancer cohorts. These high baseline scores are a consideration when interpreting time-to-worsening measures, particularly those for vomiting, which was not a prominent symptom impacting patients in either arm of the study at entry. A trend towards a higher proportion of patients in the overall population reporting improvements in TOI, FOSI and total FACT-O following treatment with olaparib vs placebo was seen across the endpoints evaluated. It is possible that any improvement during the maintenance treatment phase reflects recovery from the side effects of prior treatment, rather than from amelioration of cancer-related symptoms, as these would be expected to be minimal at study entry for patients whose disease is in response. The HRQoL finding in gBRCAm patients is worth emphasising. Although the least number of patients was included in this subgroup analysis, patient numbers were not much smaller than those in the BRCAm subset. As the study design did not include a spending strategy to control type I error rates among the patient-reported outcome (PRO) endpoints, all analyses should be considered exploratory and the P-values nominal. Of the 136 BRCAm patients in this Phase II study, 96 were known to have a gBRCAm (Ledermann et al, 2014). It is unknown whether these findings originate from a biological basis. Limitations of this study include HRQoL data not being collected beyond disease progression. Patients with progressive disease usually receive further courses of chemotherapy, an intervention associated with reductions in HRQoL. Olaparib significantly delays progression (Ledermann et al, 2012); therefore, post-progression patient outcome data may have indicated a benefit with olaparib. Delays in time to first subsequent therapy or death (TFST) may also delay detriments in HRQoL; it is of interest that future studies collect HRQoL data during PFS, TFST and beyond. Additionally, this analysis is limited in that primary and secondary PRO hypotheses were not defined prior to the study. At the time of the development of this study, there were limited studies on the QoL of cancer patients receiving maintenance treatment and there were no specifically agreed PRO or QoL assessments for use in this setting in oncology trials. Studies in other cancer types, such as non-small-cell lung cancer and metastatic breast cancer, where maintenance treatment is more common, have used a range of QoL assessments, including cancer-specific variations of the EORTC QLQ-C30, EuroQol 5-dimensional questionnaire (EQ-5D) and FACT tools (Gridelli et al, 2012; Park et al, 2015; Spigel et al, 2015). More recently, the importance of QoL assessments during clinical trials has been noted (Friedlander and King, 2013) and specific guidance such as the CONSORT-PRO recommendations should be followed in future trials (Calvert et al, 2013). The Phase III study of olaparib maintenance treatment in patients with BRCAm PSR ovarian cancer (SOLO2; NCT01874353) has included several predefined QoL endpoints to better assess the impact of this treatment on QoL. Finally, although the compliance rate of approximately 80% is favourable, it is incomplete. Reasons for reduced HRQoL assessment completion included no validated Ukrainian translation of the FACT-O questionnaire and administrative failure in questionnaire distribution. In conclusion, olaparib maintenance therapy demonstrated no detrimental impact on HRQoL outcomes compared with placebo. Phase III trials of olaparib in patients with BRCA-mutated ovarian cancer are ongoing (NCT01844986, NCT01874353 and NCT02282020). These studies will assess HRQoL using the FACT-O and EQ-5D-5L (five-level EuroQol) assessments and will collect HRQoL data over a longer period, beyond disease progression.

TITLE: Adjunctive daily supplementation with encapsulated fruit, vegetable and berry juice powder concentrates and clinical periodontal outcomes: a double-blind RCT ABSTRACT.AIM: A double-blind randomized controlled trial to determine whether dietary supplementation with fruit/vegetable/berry juice powder concentrates, simultaneously with non-surgical periodontal therapy, improved 2-month treatment outcomes. ABSTRACT.METHODS: Volunteers with chronic periodontitis were randomly assigned to one of three groups: fruit/vegetable (FV), fruit/vegetable/berry (FVB) or placebo. Supplements were taken daily during non-surgical debridement and maintenance and outcomes assessed at 2, 5 and 8 months after completion. Primary outcomes were mean probing pocket depth (PPD), clinical attachment gain, % sites bleeding on probing (% BOP) at 2 months. Adherence and plasma β-carotene were determined. ABSTRACT.RESULTS: Sixty-one nutritionally replete (by serum biochemistry) volunteers enrolled and 60 (n = 20 per arm) completed the 2-month review. Clinical outcomes improved in all groups at 2 months, with additional improvement in PPD versus placebo for FV (p < 0.03). Gingival crevicular fluid volumes diminished more in supplement groups than placebo (FVB; p < 0.05) at 2 months, but not at later times. The % BOP (5 months) and cumulative plaque scores (8 months) were lowered more in the FV group (p < 0.05). ABSTRACT.CONCLUSIONS: Adjunctive juice powder concentrates appear to improve initial pocket depth reductions in nutritionally replete patients, where plasma micronutrient bioavailability is attainable. Definitive multicentre studies in untreated and treated patients are required to ascertain the clinical significance of such changes. BODY: Periodontitis is a complex chronic inflammatory disease requiring the emergence of a pathogenic biofilm whose expression is governed by a number of host-dependent component causes. The qualitative and quantitative contributions of the various component causes to disease initiation and/or progression varies from patient to patient, resulting in a widely heterogenic clinical phenotype. Disease severity and extent also appear to be influenced by the same exposures, in conjunction with local anatomical features. Tissue destruction arises largely from a dysregulation of chronic inflammatory processes that the body's homoeostatic maintenance systems fail to resolve, resulting in a failure to eliminate pathogenic components of the subgingival biofilm and a persistence of chronic non-resolving inflammation (Van Dyke 2008). Exposure categories believed to contribute component causes to periodontal inflammation include: genetic (Michalowicz et al. 1991); environmental (e.g. biofilm, stress); lifestyle/behavioural (e.g. smoking, diet) and pharmacological (e.g. corticosteroids); and "nutrition", which is influenced by lifestyle, environmental and genetic exposure categories (Chapple 2009). Evidence is emerging for associations between such lifestyle factors and periodontal inflammation (Bawadi et al. 2011). Epidemiological studies demonstrate that a higher intake of fruit and vegetables (FV) is associated with a lowered risk of atherogenic cardiovascular disease (Joshipura et al. 1999), ischaemic stroke (Joshipura et al. 2001) and mortality (Knekt et al. 1996, Sauvaget et al. 2003). Diets rich in vegetables and Vitamin C also appear to associate positively with better periodontal health and negatively with periodontal disease progressing more rapidly in undernourished populations (Enwonwu et al. 2002). Given the high prevalence of periodontitis in the population (moderate disease prevalence 20–50%; König et al. 2010) and its impact upon elevating plasma biomarkers of systemic inflammation (D'Aiuto et al. 2004a, b), it appears that periodontal inflammation makes a significant contribution to the systemic inflammatory burden. In recent years, strong evidence has emerged that diets rich in refined carbohydrates and saturated fats are pro-inflammatory, whereas those rich in polyunsaturated fats (fish oils), antioxidant micronutrients (fruits, berries and vegetables) and certain nuts (cashews) are anti-inflammatory (O'Keefe et al. 2008). Pro-inflammatory diets drive oxidative stress within cells and tissues through metabolic (mitochondrial) and receptor-mediated pathways (reviewed by Chapple 2009) and such post-prandial oxidative stress (Sies et al. 2005), termed "meal induced inflammation" (O'Keefe & Bell 2007) positively correlates with the magnitude (Esposito et al. 2008) and frequency (Ceriello et al. 2008) of post-meal surges in glucose and triglycerides. The latter, a consequence of the rapid absorption of glucose and lipids into the blood stream following intake of dietary refined carbohydrate and saturated fats, gives rise to elevations in plasma levels of CRP and pro-inflammatory cytokines (Monnier et al. 2006) and oxidative stress (Chapple 2009). Indeed, recent evidence has demonstrated a significant role for oxidative stress in promoting bone resorption via activation of certain transcription factors (FoxOs, which decreases wnt signalling), modulated by insulin resistance and increasing age (reviewed by Galli et al. 2011). Antioxidant micronutrients combat such pro-inflammatory cascades through modulation of oxidative stress by directly scavenging reactive oxygen species (ROS) and also by down-regulation of some redox-sensitive pro-inflammatory gene transcription factors such as nuclear factor-kappa B and activator protein-1 while up regulating anti-inflammatory gene transcription factors such as nuclear factor (erythroid-derived 2)-like 2 (Nrf2: reviewed by Chapple & Matthews 2007). Indeed, adjunctive phytonutrient supplementation using juice powder concentrates of fruit, vegetables and berries (FVB) has been shown to reduce the immediate impact of a high fat test meal upon inflammatory biomarkers and to improve flow mediated dilatation of the brachial artery (Plotnick et al. 2003). Oxidative stress, defined as an imbalance between oxidants and antioxidants in favour of the oxidants, leading to a disruption of redox signalling and control and/or molecular damage (Sies & Jones 2007) is a key pathological event underpinning periodontal tissue destruction (Chapple & Matthews 2007, Bullon et al. 2009, Galli et al. 2011). Since the first indirect demonstration of oxidative stress within the periodontal tissues (Chapple et al. 1997), a body of evidence has accumulated demonstrating elevated biomarkers of oxidative stress (Sugano et al. 2000, Takane et al. 2002, Wei et al. 2004, Panjamurthy et al. 2005, D'Aiuto et al. 2010) and antioxidant compromise (Chapple et al. 1997, 2002, Brock et al. 2004, Panjamurthy et al. 2005, D'Aiuto et al. 2010) in periodontitis patients. Recent data from our own laboratory demonstrated that the antioxidant depletion manifest locally within the periodontal tissues appears to be an effect rather than a cause of the oxidative stress, which develops during periodontal inflammation (Chapple et al. 2007, Grant et al. 2010). Nevertheless, it is biologically plausible that boosting the antioxidant micronutritional status of patients may have preventive and/or adjunctive therapeutic benefit, in particular in those patients who show micronutrient deficiency (Tonetti & Chapple 2011). The potential impact of diet upon periodontal inflammatory status was recently illustrated in a Swiss study, which found that when 10 adults were placed in a "stone-age" environment for 4 weeks, with negligible oral hygiene and diets were "stone-age" in nature (id est. low in simple sugars and high in antioxidant micronutrients, fish oils and fibre), they remarkably demonstrated significant decreases in gingival bleeding and probing depths, despite significant increases in plaque accumulation (Baumgartner et al. 2009). Recent case–control studies have demonstrated that periodontitis patients have significantly lower serum antioxidant micronutrient levels than unaffected controls (Brock et al. 2004, Panjamurthy et al. 2005, Konopka et al. 2007), and also compromised gingival crevicular fluid (GCF) antioxidant status (Chapple et al. 2002, 2007b, Brock et al. 2004). Non-surgical periodontal therapy has been shown to restore certain antioxidant components in a process attributed to reducing levels of oxidative stress secondary to the resolution of periodontal inflammation (Chapple et al. 2007). However, non-surgical therapy did not restore levels of the important small molecule antioxidant species, glutathione (GSH), although the ratio of GSH to its oxidized counterpart was restored, implying at least a restoration of the redox balance within periodontal cells and tissues following successful periodontal therapy (Grant et al. 2010). Large scale epidemiological studies also consistently support a strong inverse association between serum antioxidant micronutrient concentrations and periodontitis prevalence and severity, in several different populations (Nishada et al. 2000, Amarasena et al. 2005, Amaliya et al. 2007, Chapple et al. 2007, Linden et al. 2009). Jenzsch et al. (2009) demonstrated that diets rich in vegetables, fruits, legumes and dairy products, when employed as the only intervention, significantly improved pocket depth and gingival inflammation in periodontitis patients with metabolic syndrome. The reported data point towards the biological plausibility of beneficial periodontal outcomes being derived from phytonutritional interventions of antioxidant micronutrients, acting by direct scavenging of ROS, and also by modulation of redox-sensitive pro-inflammatory gene transcription factors. There remains a paucity of data from prospective intervention studies on the role of nutrition in the pathogenesis of periodontitis (van der Velden et al. 2011). Specifically, there are no reports from placebo-controlled randomized trials on the efficacy of nutritional supplements upon periodontal outcomes. Therefore, we hypothesized that: Daily supplementation with a primarily antioxidant juice powder phytonutrient (Juice Plus+®-FV) would significantly improve treatment outcomes over placebo supplementation, at 3 months post-therapy commencement (2 months post-therapy completion), when used as an adjunct to conventional non-surgical periodontal therapy.Triple therapy with the above supplement (Juice Plus+®-FVB) would produce additional treatment benefit over dual therapy (FV) and over placebo.Improved treatment outcomes with the supplement will be maintained at 9 months post-therapy commencement. The aim of this preliminary investigator-led study was to ascertain whether or not daily dietary supplementation with capsules containing primarily FVB juice powder concentrates (Juice Plus+®; NSA LLC, Collierville, TN, USA), taken during standard non-surgical periodontal therapy (no pre-dosing), improved clinical outcomes of periodontal treatment 2 months following completion of non-surgical therapy (simultaneous supplementation). BODY.MATERIAL AND METHODS.STUDY ETHICS AND GOVERNANCE: This was an investigator-led, three-arm, placebo-controlled, double-blind randomized intervention study (NCT00952536) and was approved by South Birmingham Local Research Ethics Committee (05/Q2707/252), and research governance was overseen by South Birmingham Primary Care Trust Research Management and Governance (Project Number – SouthDent116/742). The study was conducted in accordance with international Good Clinical Practice standards. BODY.MATERIAL AND METHODS.STUDY DESIGN AND VOLUNTEERS: Volunteers (n = 61) were recruited from new patient consultation clinics by one study team member (P. W.), and were both non-smokers and medically healthy by medical history questionnaire. A rolling recruitment protocol was adopted and non-smokers selected, as smoking impacts upon oxidative stress (Chapple & Matthews 2007) and was deemed to be a likely confounder of study outcomes. Volunteers were aged between 30 and 60 years, either men or women, and had chronic periodontitis as defined by a minimum of two sites per quadrant with pocketing or interproximal attachment loss of >6 mm and one-third radiographic bone loss. The following exclusion criteria were applied: patients with aggressive disease, patients with physical or mental disability, pregnant women, patients whose medical history may place them at risk of complications from periodontal therapy (e.g. need for antibiotic prophylaxis, Warfarinised patients), patients taking long-term antimicrobial or anti-inflammatory drugs, patients unable to swallow capsules, patients unable to provide informed consent, current smokers (or within 5 years), patients taking regular vitamin supplementation. The study was designed to exit 60 volunteers at 9 months following recruitment (8 months following completion of non-surgical treatment) and was conducted over a 4.5-year period (2005–2010). Volunteers were provided with a detailed information sheet and allowed 2 weeks to consider their participation, prior to obtaining their informed consent and enrolment. As volunteers were enrolled (by M. M.) they were allocated sequential study numbers (by K. A.) and provided with their capsules, sufficient for 3 months. Randomization was performed by the study statistician (G. D.), who was independent of the clinical study team and site, using a computer-generated program http://www.randomization.com (volunteer numbers generated from 001 to 070 to allow for loss of up to 10 participants). The capsules were supplied in pre-labelled containers. Baseline measures were also collected at the enrolment visit, prior to which volunteers were asked to fast overnight, not to brush and not to chew gum or drink within 2 h of their appointment. Protocol adherence was checked prior to sample collection, and patients were re-appointed if necessary. Biological samples were sequentially collected. The GCF from three deep sites (≥6 mm) and three shallow sites (≤3 mm) was collected as previously reported (Chapple et al. 2007) and deep sites were pooled, on a volunteer and visit basis, as were samples from the three shallow sites. The GCF sampling was followed by a venous blood sample, probing pocket depth (PPD) and recession measures, bleeding scores, gingival colour index [Modified Gingival Index (MGI) – Lobene et al. 1986] and plaque indices (Fig. 1). Patients then had scaling and root surface debridement performed on a quadrant by quadrant basis within 1 month and over four visits. At each visit, adherence was re-checked and recorded. Capsule re-supply occurred at recall visits and patients were recalled at 2, 5 and 8 months following completion of the last session of instrumentation. At each recall visit, clinical samples and measurements were repeated and volunteers exited the study at the 8-month recall appointment, when capsule containers were returned and counted. Volunteers were also asked to maintain a supplement diary recording of how many capsules had been missed. If further therapy was required at this stage, as judged by clinical examination, it was performed by the same therapist (N. L.-M.), but outside of the study. Fig. 1Study flow chart. All volunteers completed standard non-surgical periodontal therapy, which was performed by a single operator (N. L.-M.; 15 years experience and MPhil degree in clinical periodontal trials) within 4 weeks of commencing treatment and all indices were recorded by a single GCP-trained examiner (M. M.). Scaling and debridement was performed using a traditional quadrant by quadrant protocol, using a single ultrasonic (Densply CavitronPlus SPS; Densply UK) device and FSI-100 inserts, and Gracey curettes (LM-Dental UK). Treatment was performed systematically until the root surfaces were considered sufficiently clean, each quadrant taking 30–45 min. Patients commenced their assigned capsule supplement at their study enrolment study visit, within 1 week of physical instrumentation commencing (Fig. 1) and continued supplementation during treatment (1 month) and also for 8 months of maintenance (total 9 months of supplementation). Compliance was checked by capsule count and also by analysing serum β-carotene levels. There were three study arms: Test 1 – Six capsules daily containing the total equivalent of four FV capsules, and two placebo capsules during periodontal therapy and for 8 months thereafter.Test 2 – Six FVB capsules daily during periodontal therapy and for 8 months thereafter.Control – Six placebo capsules daily during periodontal therapy and for 8 months thereafter. In all three study groups, capsules were taken with food twice daily (three in the morning, three in the evening). BODY.MATERIAL AND METHODS.OUTCOME MEASURES: Primary outcome measures were (1) reductions in PPDs (mean per patient reduction in PPD), (2) reduction in percentage sites bleeding on probing (% BOP) from the marginal tissues (as a primary outcome measure of tissue inflammation), and (3) mean increase in clinical attachment level (CAL) at the 2-month post-therapy recall visit (3 months post-therapy commencement). Secondary outcome measures were reductions in GCF volume from both deep and shallow sites, gingival redness (MGI – Lobene et al. 1986), cumulative plaque index (Lobene et al. 1982) and recession. Although not part of the original protocol, a decision was made to additionally express (4) pocket depth reductions as "number of residual sites >4 mm", consistent with contemporary studies published subsequent to protocol preparation (Wennström et al. 2005). Probing measures were performed in duplicate at six sites per tooth (two marginal and four proximal) with a constant force (UNC CP-15 markings – 0.2 N force) probe and where differences between duplicate measures of greater than 1 mm arose, a third measure was taken. The mean of the two closest measures was used. Mean probing attachment (derived from pocket depth + recession), and PPD measures per subject were calculated at each examination point (baseline, 2, 5 and 8 months post-therapy). Marginal bleeding was recorded dichotomously (four sites per tooth, mesial, distal, mid-facial, mid-lingual) and expressed as mean percentage sites bleeding on probing (% BOP) per subject at each examination point. Plaque levels were quantified using a modification of the Quigley-Hein index (Lobene et al. 1982) and expressed as a whole mouth total (cumulative) score per volunteer at each examination point. The study visits and procedures are summarized in Fig. 1. Outcomes at the 5- and 8-month recall visits were considered secondary outcomes, as pathogenic biofilm changes are evident at this stage (Quirynen et al. 2005) and may lead to early signs of recalcitrant disease. BODY.MATERIAL AND METHODS.BIOLOGICAL SAMPLES COLLECTED: Gingival crevicular fluid samples were collected over 30 s using PeriopaperTM (Oraflow Inc., Smithtown, NY, USA) strips from the mesio-buccal aspects of the three deep and three shallow molar sites per subject as previously described (Brock et al. 2004). Volumes were read on a pre-calibrated Periotron 8000TM according to standard methodologies (Chapple et al. 1999). Blood was collected into VacutainerTM (NHS supplies, Alfreton, Derbyshire, UK) tubes for serum and plasma preparation (vide infra). Platelet depleted plasma was prepared by centrifugation at 1000 g for 30 min. (4°C). Serum was aliquoted into cryogenic vials, snap frozen and stored at −80°C for subsequent analysis of β-carotene and vitamin E. For vitamin C assay, 0.75 ml metaphosphoric acid (100 g/l) was added to 0.75 ml plasma to precipitate proteins, prior to storage at −80°C. All samples were stored in the dark and kept free from direct sunlight at all stages of handling. BODY.MATERIAL AND METHODS.TEST PRODUCTS: The verum test capsules are marketed commercially as Juice Plus+® and contain a fine, granular powder, encapsulated in a size 00 gelatin capsule. The placebo test capsules were of identical appearance and contained primarily microcrystalline cellulose. The FV capsule contents consisted primarily of a blended FV pulp and juice powder concentrate derived from Acerola cherry, apple, beet, beetroot, broccoli, cabbage, carrot, cranberry, dates, garlic, kale, orange, peach, papaya, parsley, pineapple, prune, spinach, sugar beet, tomato, with Spirulina pacifica, Lactobacillus acidophilus, rice bran, oat bran and Dunaliella salina. These active ingredients were supplemented to provide declared totals (per daily dose) of β-carotene (7.5 mg), vitamin E (46 mg), vitamin C (200 mg) and folic acid (400 μg). Although the phytonutrient capsules are known to contain polyphenolic antioxidant micronutrients, these vary according to growing and harvest conditions and absolute levels were not analysed. The FVB capsule contents consisted primarily of a blended fruit, berry and vegetable pulp and juice powder concentrate. In addition to all the components within the FV capsules, FVB capsules also contained material derived from blackberry, black currant, blueberry, bilberry, concord grape, elderberry, raspberry, red currant, green tea, ginger root, artichoke leaf, grape seed extract in addition to arginine, carnitine and co-Enzyme Q10. These active ingredients were supplemented to provide declared totals (per daily dose) of β-carotene (7.5 mg), vitamin E (66 mg), vitamin C (222 mg) and folic acid (640 μg). Small quantities of anti-caking agents (calcium carbonate, magnesium oxide, magnesium stearate, silicon dioxide, cellulose) and thickeners (citrus pectin, guar gum) are added to assist the manufacture of capsules. BODY.MATERIAL AND METHODS.Β-CAROTENE ANALYSIS (FOR ADHERENCE): Adherence to supplement usage was assessed by residual capsule counts and a supplement adherence diary at recall visits and also biochemically by analysis of serum β-carotene levels. β-carotene was determined by HPLC using isocratic elution, a reversed phase column (pKb-100, 250 × 4.6 mm2; Supelco, Bellefonte, PA, USA), protected by a guard column (4.6 × 4.6 mm2) with the same stationary phase and a Merck-Hitachi L-7100 pump connected with a Merck-Hitachi UV/Vis detector (Merck-Hitachi, Darmstadt, Germany). β-carotene was detected at 450 nm and the concentration calculated from external calibration curves generated with original standard compounds and internal standards as previously reported (Stahl et al. 1993). BODY.MATERIAL AND METHODS.PLASMA VITAMIN E AND VITAMIN C DETERMINATION AT BASELINE: These analyses at baseline were performed to assess whether or not any of the volunteers were vitamin deficient. Alpha-tocopherol was extracted and detected simultaneously with the UV/VIS detector set at 292 nm (Polidori et al. 2001). Vitamin C was determined by HPLC using a Merck- Hitachi L-6200, a pump connected with a Merck- Hitachi UV/Vis detector (Merck-Hitachi) and using a commercially available analytical kit (Chromsystems Instruments & Chemicals GmbH, Munchen, Germany), run according to manufacturer's instructions. BODY.MATERIAL AND METHODS.POWER CALCULATION AND STATISTICAL ANALYSIS: The reported study was the first of its type to the authors' knowledge and therefore a power calculation based upon mean (±SD) outcomes from pilot work was not possible for antioxidant outcomes. However, using the primary outcome measure of reduction in PPD and assuming a mean additional PPD reduction in the supplement groups of 0.4 mm (as this has been demonstrated in pharmacological interventions) over non-surgical therapy alone, 17 volunteers per treatment group were needed to complete the study for a two-sided test of equality of means at the 0.05 level of significance with 80% power. This was based on accepted mean PPD reductions for mild to moderate periodontitis of 1 mm (moderate) to 2 mm (deep sites) with non-surgical therapy, and also upon data from a previous study that demonstrated mean PPD reductions of 1.1 ± 0.4 mm (Chapple et al. 2007). The study was powered to detect differences between test and placebo groups only. A total of 61 volunteers were sequentially recruited. Statistical analysis was performed by the study statistician (G. D.) and utilized analysis of covariance, with baseline measures employed as the covariates. Tukey's Honestly Significant Difference was used to compare the three groups. Analyses were performed using SAS for Windows, version 9.2 (SAS Institute Inc., Cary, NC, USA). All tests were two-sided and a result was judged statistically significant if its observed significance level (p-value) was less than 0.05. An intention-to-treat (ITT) analysis was performed, employing data from all subjects regardless of adherence to treatment. Serum β-carotene concentrations were also used as a biochemical measure of adherence and/or an indirect measure of carotenoid bioavailability. Missing data: The primary outcome measures were taken as part of standard care during the subjects' 8-month review. There was no reason for missing data to be related to treatment or outcome, but missing data were checked for any relationship to treatment assignment. Otherwise missing values were ignored in the analyses because they were deemed to be missing completely at random. Code breaking: The code was retained by the statistician and not broken until the last patient had completed their 8-month recall visit, and all data analysis had been completed. BODY.RESULTS.VOLUNTEER FLOW THROUGH STUDY: The flow of patients through the study stages is illustrated in Fig. 1. Sixty-one patients were enrolled and sixty completed the 2-month post-therapy visit, providing primary outcomes for 20 volunteers in each study group (FV, FVB and placebo). Six patients withdrew at the 2-month post-therapy visit. The reasons for withdrawal were: capsules too large to swallow, moved away from area, difficulty attending appointments, unrelated medical problems, lost to follow-up for unknown reason. One patient was withdrawn (placebo group) as they telephoned to inform that they were experiencing difficulty swallowing, and although this could not be attributed to the supplements as opposed to a respiratory tract infection, the principal investigator (I. L. C.) decided to withdraw the volunteer by telephone and to complete treatment outside of the study. This last volunteer was replaced with the 61st enrolee. To ensure groups remained balanced, whilst at the same time maintaining the blinding of the study, the statistician provided a replacement study number. BODY.RESULTS.DEMOGRAPHICAL AND BASELINE NUTRITIONAL DATA: The volunteer demographics are recorded in Table 1, and although more women were enrolled than men, the groups were similar with regard to age and gender. There were no baseline differences between groups in peripheral blood levels of vitamin C (range = 48–58 μmol/l) and vitamin E (range = 17–19 μmol/l), which were within SI reference ranges (Table 1). Baseline β-carotene concentrations (range = 0.5–0.7 μmol/l) were below the low end of one reference range (Young 1987), but within another commonly used range (Iverson et al. 2007); however, there were no differences between the study groups. Demographical and baseline micronutrient data [mean (μmol/l) ± SD] of the test groups Test group No. Men Women Age (years) β-carotene * (0.9–4.6) † (0.2–1.6) ‡ Vitamin C § (30–110) † (23–85) ‡ Vitamin E * (18–29) † (12–42) ‡ Mean ± SD Range Fruit and vegetable 20 6 (30%) 14 (70%) 48.3 ± 8.4 35–69 0.7 ± 0.45 48.1 ± 16.2 18.3 ± 4.4 Fruit, vegetable and berry 20 8 (40%) 12 (60%) 48.1 ± 7.4 33–58 0.6 ± 0.3 57.4 ± 17.6 18.6 ± 5.9 Placebo 20 7 (35%) 13 (65%) 47.9 ± 6.6 38–60 0.5 ± 0.25 57.9 ± 22.0 17.2 ± 3.2 * serum. § plasma. † SI reference ranges (μmol/l) ( Young 1987 ). ‡ SI ranges from Iversen et al. AMA Manual of Style: a guide for authors and editors. 10th Edition, New York, Oxford University Press, 2007. BODY.RESULTS.ADHERENCE/BIOAVAILABILITY OF Β-CAROTENE: The supplement diaries and capsule counts indicated that protocol adherence had been equivalent in all groups. Serum β-carotene concentrations increased significantly at 2 months in both FV and FVB groups (FV by 5.4 μmol/l, p < 0.0001; FVB by 3.5 μmol/l, p < 0.0001), but there was no change in the placebo group. The differences between the supplement and placebo groups post-supplementation were significant (p < 0.001) and the increase in serum β-carotene concentrations was highest in the FV group (Fig. 2) indicating greater micronutrient bioavailability in this group compared with the FVB group, which does not appear to result from between group differences in adherence, but might have impacted upon clinical outcomes. Fig. 2Percentage change in plasma beta-carotene concentrations from baseline. BODY.RESULTS.CHANGES IN PRIMARY OUTCOMES: The improvement in clinical outcomes in the placebo group was consistent with the literature (Cobb 2002) and statistically significant. This is evident from the statistically significant reduction of sites >4 mm at 2, 5 and 8 months post-therapy completion (Fig. 3a; p < 0.0001). Thus, after a single phase of non-surgical therapy, "closed pockets" (Wennström et al. 2005) increased in number from an average of 70% of sites in all three groups at baseline to 98% (FV group) and 91% (FVB and placebo groups). Fig. 3Changes in primary outcomes during the study period (mean ± SD). (a) Sites >4 mm deep; (b) PPD (mm); (c) CAL (mm); (d) %BOP. Bars represent comparisons with baseline. Symbols alone represent comparisons with placebo. *p < 0.0001; ▪p = 0.002; •p < 0.01; +p < 0.03; ♦p < 0.05. Other primary outcome measures, pre- and post-completion of non-surgical scaling and root surface therapy, are illustrated in Fig. 3b–d (and mean values documented in Table S1). At 2, 5 and 8 months post-treatment completion there were significant reductions in mean PPD compared with baseline, consistent with literature reports for moderate periodontitis (Cobb CM 2002; van der Weijden & Timmermann 2002) for all three groups (p < 0.0001). At 2 months, reductions in PPD were statistically significantly greater in the FV supplement group relative to the placebo group (p < 0.03; Fig. 3b). This was not the case for the FVB group (p = 0.7; Fig. 3b). Although the PPD levels in the FV group remained lower than the placebo and FVB groups at 5- and 8-month recalls, differences were no longer statistically significant at these secondary time points. Significant post-treatment CAL gains were detected at all time points in both FV (p < 0.05) and FVB (p < 0.02) supplement groups (Fig. 3c). In contrast, CAL gains in the placebo groups were only significant over baseline at 5 months. Despite this, CAL gains within supplement groups over those measured in the placebo group did not reach statistical significance (p > 0.09). At all post-treatment reviews, the percentage of sites bleeding on probing (% BOP; Fig. 3d) was significantly reduced in all three study groups (p < 0.002). Although the reduction in % BOP was greatest at all time points for the supplement groups, the additional improvement relative to placebo was only statistically significant at the 5-month secondary time point for the FV group (p < 0.05). BODY.RESULTS.CHANGES IN SECONDARY OUTCOMES: Secondary outcomes, pre- and post-treatment completion, are illustrated in Fig. 4 (and mean values documented in Table S2). Fig. 4Changes in secondary outcomes during the study period (mean ± SD). (a) Gingival crevicular fluid (GCF) volumes (μl) – shallow sites; (b) GCF volume (μl) – deep sites; (c) cumulative plaque index; (d) recession (mm); (e) Gingival redness (MGI). Bars represent comparisons with baseline. Symbols alone represent comparisons with placebo. *p < 0.0001; ‡p < 0.007; ▪p < 0.005; ≠p < 0.002; •p < 0.01; □ p < 0.02; +p < 0.03; ♦p < 0.05; $p < 0.001. Gingival crevicular fluid volumes at both shallow and deep sites reduced with time post-therapy (Fig. 4a and b). Reductions in volume at shallow sites were small and changes only reached statistical significance at 8 months in the placebo (p = 0.007) and FVB (p < 0.02) groups, with supplementation having no effect different to that shown by the placebo group. In contrast, GCF volumes at deep sites were significantly reduced at all time points in all groups post-therapy, with reductions being consistently greater in supplementation groups (p < 0.002; Fig. 4b). However, the greater effect of supplementation on reducing GCF volume at deep sites was only significant at 2 months for the FVB group (p < 0.05). Significant reductions in cumulative plaque scores were detected at all time points post-therapy in all groups (Fig. 4c; p < 0.03). No differences were evident between groups until month 8, when the reduction in plaque scores relative to baseline were significantly higher in the FV group (p < 0.05) compared with the placebo group. There were progressive improvements in recession and MGI post-therapy in all three study groups (Fig. 4d and e; p < 0.001), but no significant differences in the magnitude of improvement between groups at any time point. BODY.DISCUSSION: The current study is the first to report the impact of providing periodontitis patients with an adjunctive phytonutrient supplement during standard mechanical non-surgical periodontal therapy. We hypothesized that additional clinical benefit would result from supplementation with a juice powder concentrate (FV) and that the triple therapy (FVB) would provide enhanced outcomes over the dual supplement (FV) due to additional polyphenolic antioxidants. The rationale for such hypotheses was based upon the substantial literature base concerning the efficacy of antioxidant micronutrients in reducing extracellular oxidative stress, and their intracellular role in the down-regulation of redox-regulated pro-inflammatory gene transcription factors (reviewed by Chapple & Matthews 2007, Chapple 2009). Statistically significant additional reductions in PPD were seen at the 2-month post-therapy recall visit for FV verses placebo, providing partial fulfilment of the first hypothesis. Therapy employing the FVB supplement did not, however, provide significantly improved primary outcomes relative to the placebo group, despite the presence of additional polyphenolic micronutrients within. Hypothesis two was therefore not fulfilled and nor was hypothesis three, as the initially greater pocket depth reduction in the FV group was not sustained at 8 months post-therapy. It was surprising not to see similar additional improvements for the FVB as the FV group, but this may be explained by the reduced bioavailability of micronutrients in this group as demonstrated by the serum β-carotene levels (Fig. 2). It may also be explained by nutrigenetic differences between groups, as polymorphisms in the BCMO1 gene (β-carotene 15,15′-monoxygenase) have been identified (Leung et al. 2009) which appear to explain, at least in part, the low conversion rates of β-carotene to its bioactive form in many individuals. The attenuation in serum β-carotene concentrations at 5 and 8 months could also have arisen due to reduced adherence to supplementation in the FVB group; however, this thesis was not supported by the supplement diary and capsule count analysis. An alternative explanation is that micronutrient absorption across the gastrointestinal tract wall may have been lower in the FVB than the FV group, or that the additional polyphenolic compounds within the FVB supplement antagonized beneficial activities of other components. Whatever the explanation, the plasma β-carotene data demonstrate reduced bioavailability of some micronutrients in the FVB group relative to FV. Significant additional reductions in percentage sites bleeding on probing arose for the FV group at 5 months (following the increased 2-month pocket depth reductions). The cumulative plaque index data recorded in the supplementation group are also interesting, as significant additional decreases were observed at the 8-month follow-up visit for FV verses placebo, 3 months after the additional improvements in percentage sites bleeding recorded in the same group (FV). Although it could be argued that the improved plaque index in the supplement group arose due to better home care/compliance, this seems unlikely, as there were no differences between groups in plaque indices at 2- and 5-month recalls, where the most substantial reductions in plaque took place. An alternative and intriguing explanation is that additional reductions in inflammation evident at 5 months were reflected in the composition of the GCF, which in turn led to a reduced biofilm accumulation. Although this is consistent with current evidence that the inflammatory status of the gingival tissues influences plaque biofilm accumulation (Hillam & Hull 1977, Baumgartner et al. 2009) and with current consensus views on host response/biofilm inter-relationships (Tonetti & Chapple 2011), plaque re-growth studies and those analysing GCF composition would be necessary to substantiate such a thesis. A prebiotic effect upon biofilm accumulation is unlikely as capsules were swallowed and nutrients not applied locally. Any effect therefore would appear likely to be due to the influence of an altered systemic (and downstream local) host response upon the periodontal biofilm (Marsh & Devine 2011). The healing and resolution of inflammation arising from the non-surgical mechanical therapy alone (placebo group) was substantial and consistent with the literature (van der Weijden & Timmermann 2002), with clinically significant increases in the number of closed pockets apparent at 2 months and at subsequent recalls. Moreover, there were no baseline differences between groups in circulating concentrations of vitamin E, vitamin C or β-carotene, all of which were within or close to the low end of published SI reference ranges (Young 1987). Taken together, it is perhaps surprising that any additional clinical benefit was achieved in patients who were effectively nutritionally replete and in whom non-surgical management alone was highly successful. Some of those benefits were not retained after 8 months, a finding that does not fulfil our third hypothesis and which may be explained by a number of factors, including a tendency for recalcitrant disease to develop at this stage (Quirynen et al. 2005). However, given that there is a more limited rationale to the expectation of improved therapeutic outcomes in nutritionally replete verses deplete patients (Schifferle 2009) and that non-surgical therapy was successful without adjunctive nutritional support, it is possible that more substantial clinical benefits may be realized from such dietary augmentation in nutritionally depleted periodontitis patients or in those who do not have access to dental services for periodontal therapy. The public health benefits of such an approach, in a society where obesity trends are increasing and diets are decreasing in quality (Jenzsch et al. 2009) may be substantial if proven. Indeed, there is evidence from one intervention study in patients with metabolic syndrome that inflammatory periodontal outcome measures improved following a nutritional intervention (Jenzsch et al. 2009). Such findings when allied to the impact of periodontal inflammation upon systemic inflammatory status may further impact upon chronic inflammation in the body in general (Kawashima et al. 2007, Jin et al. 2010). There are limitations to the current study. Firstly, the study may have lacked the power to detect the beneficial effects of FV and FVB because we had no prior data upon which to base sample size estimates. Secondly, assessing protocol adherence was difficult, preventing a "per protocol" analysis. The analysis presented is an "ITT" analysis, which assumes that non-adherence will be random and thus equitable across the three groups. We did not attempt to ascertain nutrigenetic parameters which are likely to dictate "high responders" and "low responders", largely because this science is in its infancy and the most appropriate outcome measures are unknown, with the exception of recent discoveries in relation to β-carotene converter status and polymorphisms in the BCMO1 gene (Leung et al. 2009). Thirdly, the high improvements recorded in the placebo group from mechanical debridement alone are likely to have created a ceiling effect, whereby further improvement from adjunctive therapy is less likely. We conclude that adjunctive daily supplementation with an encapsulated FV juice powder concentrate, during non-surgical periodontal therapy, appears to offer additional initial pocket depth reductions, and subsequent additional improvements in bleeding on probing and plaque scores even in high responders to conventional therapy and in those who are not nutritionally compromised. However, outcomes seem to depend upon the serum/plasma micronutrient concentrations achieved, and this requires patient adherence and likely favourable absorption across the gastrointestinal tract wall. Further studies are necessary to assess such approaches in larger and more diverse populations, employing different supplement and therapeutic regimes. In particular, monotherapy studies (phytonutrient supplementation alone) and studies in nutritionally compromised patients are necessary to help build a clearer picture of the clinical impact of antioxidant phytonutrient supplementation upon periodontal and systemic inflammatory status.

TITLE: Influence of a prudent diet on circulating cathepsin S in humans ABSTRACT.BACKGROUND: Increased circulating cathepsin S levels have been linked to increased risk of cardiometabolic diseases and cancer. However, whether cathepsin S is a modifiable risk factor is unclear. We aimed to investigate the effects of a prudent diet on plasma cathepsin S levels in healthy individuals. ABSTRACT.FINDINGS: Explorative analyses of a randomized study were performed in 88 normal to slightly overweight and hyperlipidemic men and women (aged 25 to 65) that were randomly assigned to ad libitum prudent diet, i.e. healthy Nordic diet (ND) or a control group (habitual Western diet) for 6 weeks. Whereas all foods in the ND were provided, the control group was advised to consume their habitual diet throughout the study. The ND was in line with dietary recommendations, e.g. low in saturated fats, sugars and salt, but high in plant-based foods rich in fibre and unsaturated fats. The ND significantly decreased cathepsin S levels (from 20.1 (+/-4.0 SD) to 19.7 μg/L (+/-4.3 SD)) compared with control group (from 18.2 (+/-2.9 SD) to 19.1 μg/L (+/-3.8 SD)). This difference remained after adjusting for sex and change in insulin sensitivity (P = 0.03), and near significant after adjusting for baseline cathepsin S levels (P = 0.06), but not for change in weight or LDL-C. Changes in cathepsin S levels were directly correlated with change in LDL-C. ABSTRACT.CONCLUSIONS: Compared with a habitual control diet, a provided ad libitum healthy Nordic diet decreased cathepsin S levels in healthy individuals, possibly mediated by weight loss or lowered LDL-C. These differences between groups in cathepsin S were however not robust and therefore need further investigation. BODY.FINDINGS.INTRODUCTION: Cathepsin S is highly expressed in antigen presenting cells [1–3] and has important functions in the major histocompatibility complex (MHC) class II antigen presentation [4]. Furthermore it degrades extracellular matrix [5]. Elevated circulating cathepsin S concentrations predict mortality in elderly men [6] and it has been linked to cardiovascular disease (CVD), type 1 and type 2 diabetes, cancer [7–10] and inflammation [11, 12]. Cathepsin S is also associated with LDL-C and HDL-C [13–15]. Furthermore it is increased in adipose tissue and serum of obese persons [16, 17], whereas studies in obese women have indicated that weight loss by obesity surgery or energy-restricted diet decrease serum cathepsin S [17, 18]. As cathepsin S appears to be a novel promising risk marker of both cardiometabolic and malignant diseases, it is important to examine the effect of diet on cathepsin S concentrations. We hypothesized that a healthy nordic diet (ND) could reduce plasma levels of cathepsin S. The aim was to investigate the effects of a ND, eaten ad libitum, on plasma levels of cathepsin S in the NORDIET-trial. A secondary aim was to investigate the relationships between changes in cathepsin S concentrations and changes in cardiometabolic risk factors. BODY.FINDINGS.SUBJECTS AND METHODS.SUBJECTS: During December 2007, subjects living in Bollnäs, Sweden, were recruited by advertisements in the local newspaper. The inclusion criteria were healthy (as assessed by a physician), men and women between 25 and 65 years, plasma LDL-C ≥3.5 mmol/L ̄1, body mass index (BMI) ≥20 and ≤ 31 kg m2, for women and men respectively [19]. Subjects with hypertension, CVD, diabetes and other chronic disease and those on lipid-lowering drugs were excluded. BODY.FINDINGS.SUBJECTS AND METHODS.STUDY DESIGN: The study was conducted between February and May, 2008. Eighty-eight subjects were randomly assigned to one of two groups: a ND or a control group following their usual diet. Clinical and laboratory assessments were performed at baseline and after 6 weeks. The trial was conducted in accordance with the CONSORT statement and registered in the Current Controlled Trials database (http://www.controlled-trials.com); International Standard Randomized Controlled Trial Number (ISRTCTN): 77759305. Written informed consent was given by all subjects. The study was approved by the regional ethical committee in Uppsala. BODY.FINDINGS.SUBJECTS AND METHODS.OUTCOME MEASURES: In the present post-hoc study of the NORDIET-trial we aimed to investigate changes in plasma cathepsin S levels during ND as compared with a control group. We also investigated possible relationships between change in plasma cathepsin S and changes in weight, insulin sensitivity, triglycerides (TG), LDL-C, HDL-C, systolic blood pressure (SBP) and diastolic blood pressure (DBP). BODY.FINDINGS.SUBJECTS AND METHODS.INTERVENTION: All main meals were provided to the subjects in the ND group. The assessment of diet and the change in diet during the study has been described previously [19]. BODY.FINDINGS.SUBJECTS AND METHODS.PRUDENT DIET: The ND was based on the Nordic nutrition recommendations (NNR) [20] and contained characteristic foods used in Nordic countries including fruits (e.g. apples) and berries (e.g. blueberries), legumes, vegetables, low-fat dairy products and fatty fish (e.g. salmon). The ND also included LDL-C lowering foods (e.g. oats; barley, almonds and psyllium seeds) [21, 22]. The ND was provided ad libitum. BODY.FINDINGS.SUBJECTS AND METHODS.CONTROL GROUP: The subjects in the control group were instructed to follow their habitual diet, eaten ad libitum, and continue their usual physical activity. BODY.FINDINGS.SUBJECTS AND METHODS.CLINICAL ASSESSMENT: Bodyweight was measured (kg) in light clothing without shoes, on a digital scale. Blood pressure was measured manually in a sitting position after 5 minutes rest. Two measurements were performed with a 2 minutes interval, and the average value was calculated. BODY.FINDINGS.SUBJECTS AND METHODS.BIOCHEMICAL ANALYSIS: Venous blood was collected after a 12 h fast and plasma separated and frozen in -70°C before analyses. Plasma cathepin S was measured by ELISA (human cathepsin S (Total), DY1183, R&D Systems). The intraassay CV was 7%. Glucose, total cholesterol, TG and HDL-C plasma concentrations were measured using a Roche Diagnostics Cobas® 6000. Plasma LDL-C was calculated by Friedewalds formula [23]. Plasma insulin was measured by an enzyme-linked immunoassay kit (Mercodia AB, Uppsala, Sweden). Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated [24]. BODY.FINDINGS.SUBJECTS AND METHODS.STATISTICAL ANALYSIS: Data are presented as mean ± SD. Per protocol analysis was used to assess effects of diet on outcome measures. Unpaired T-test was used to assess differences in plasma levels of cathepsin S during follow up, between the two groups, and as a second step, we used ANCOVA to adjust for sex, weight and cardiometabolic risk factors. To assess associations between change in cathepsin S and change in cardiometabolic risk factors linear regression and Pearson's correlation was used. A two-tailed P-value of 0.05 was regarded as significant. STATA, version 11 was used for statistical analysis. BODY.RESULTS: Only two subjects (one in each group) dropped out [19], leaving 86 subjects with data on cathepsin S. After randomization, the two groups were almost identical with regards to the baseline characteristics, with the exception of a significant difference in cathepsins S levels between the groups (Table 1).Table 1 Baseline characteristics after randomization CharacteristicsControl groupHealthy Nordic dietP-value Subjects, n 4244 Age (year) 53.4 ± 8.152.6 ± 7.80.63 Men/women 15/2717/270.83 Body weight (kg) 78.0 ± 13.376.0 ± 10.50.44 Body mass index (kg m ̄ 2 ) 26.5 ± 3.326.3 ± 3.20.79 SBP (mmHg) 123 ± 14128 ± 120.50 DBP (mmHg) 83 ± 981 ± 70.16 Plasma TG (mmol/L ̄ 1 ) 1.4 ± 0.81.6 ± 0.80.32 Plasma cholesterol (mmol/L ̄ 1 ) 6.4 ± 0.76.2 ± 0.80.36 Plasma LDL cholesterol (mmol/L ̄ 1 ) 4.2 ± 1.04.0 ± 0.60.33 Plasma HDL cholesterol (mmol/L ̄ 1 ) 1.5 ± 0.51.5 ± 0.40.28 LDL/HDL ratio 2.8 ± 1.02.9 ± 0.80.80 Plasma glucose (mmol/L ̄ 1 ) 4.9 ± 0.64.9 ± 0.50.54 Insulin resistance (HOMA-IR) 1.3 ± 0.61.2 ± 0.60.47 Cathepsin S μg/L 18.3 ± 2.9920.1 ± 4.10.02Data are means ± SD. HDL: High-density lipoprotein; LDL: Low-density lipoprotein; HOMA-IR: Homeostasis model assessment-insulin resistance; SBP: systolic blood pressure; DBP: diastolic blood pressure; TG: triglycerides. Differences between the Nordic Diet and Control groups were assessed using unpaired two-tailed t-tests. BODY.RESULTS.EFFECT OF DIET ON CATHEPSIN S: As previously reported [19], ND reduced body weight (mean 3 kg) as compared with the control group. Here we report a significant difference between ND and control group in the change of cathepsin S concentrations (P = 0.03 for between group difference, Figure 1).Figure 1 Differences in serum cathepsin S levels between the control diet and healthy Nordic diet (ND) from baseline to 6 weeks. Serum levels of cathepsin S were decreased compared with the control diet (P = 0.03). The ND decreased plasma cathepsin S levels, from 20.1 (+/-4.05 SD) to 19.7 μg/L (+/-4.3 SD) whereas the control group changed from 18.2 (+/-2.9 SD) to 19.1 μg/L (+/-3.8 SD) (Figure 1). Mean difference in change between groups were 12.0 μg/L (+/-25.2 SD). These differences remained after adjusting for sex (p = 0.03) and insulin sensitivity (P = 0.03), whereas adjusting for weight change (P = 0.22), LDL-C (P = 0.36) and total cholesterol (P = 0.48) abolished the significant difference between groups. However, adjusting for baseline cathepsin S levels attenuated the difference between groups (p = 0.06), as well as adjustments for differences in systolic and diastolic blood pressure (p = 0.06). As described previously [19], dietary compliance was overall excellent and the dietary goals were achieved for most nutrients". E.g. total fat was 27%E (goal 25-35%E), carbohydrate 52%E (45-60%E), protein 19%E (10-20%E), fibre 54%E (25–35) and saturated fat 5%E (<8%E). BODY.RESULTS.CORRELATIONS BETWEEN CHANGES IN CATHEPSIN S AND CARDIOMETABOLIC RISK FACTORS: Changes in cathepsin S tended to be correlated with changes in weight (P = 0.05), which remained after adjusting for sex (P = 0.04). Furthermore, change in cathepsin S were correlated with change in LDL-C (P = 0.03) and total cholesterol (P = 0.01). These associations remained significant after adjusting for sex (P = 0.03 and 0.01 respectively). Change in cathepsin S did not correlate with changes in insulin sensitivity, TG, HDL-C, SBP or DBP (Table 2).Table 2 Correlations between change in serum cathepsin S concentrations and changes in weight and cardiometabolic risk factors during 6 weeks in the whole sample (n = 86) CharacteristicsrP-valueβ-coefficient (95% CI)P-valueWeight0.220.04241.60 (3.37-479.81)0.05HOMA-IR0.040.72166.02 (-734.40-1066.44)0.72LDL-C0.240.03794.11 (84.86-1503.36)0.03HDL-C0.180.121914.54 (-505.34-4334.43)0.12TG0.020.84114.98 (-1007.58-1237.56)0.84Cholesterol0.270.01755.29 (163.79-1346.90)0.01SBP0.150.1829.43 (-13.37-72.24)0.18DBP0.170.1246.16 (-12.39-104.71)0.12Data are correlation coefficients and regression coefficients, with 95% CI. All models are adjusted for sex. HOMA -IR: Homeostasis model assessment-insulin resistance, HDL-C:High-density lipoprotein cholesterol; LDL -C: Low-density lipoprotein cholesterol, TG: Triglycerides, SB P: Systolic blood pressure, DBP: Diastolic blood pressure; Pearson's correlation and Linear regression has been used. BODY.DISCUSSION: Adherence to an ad libitum ND for 6 weeks slightly decreased levels of plasma cathepsin S in normal or slightly overweight individuals, compared with the control group. Change in circulating cathepsin S concentrations were correlated with changes in body weight, LDL-C and total cholesterol suggesting that these factors may mediate the effect on cathepsin S levels. To our knowledge, there are no studies investigating the effects of a prudent diet on cathepsin S concentrations. In accordance with our results, studies in obese women, showed that energy restriction and weight loss reduced cathepsin S mRNA and cathepsin S release in adipose tissue as well as serum levels [17, 18]. Body weight decreased by on average 3 kg during ND [19], and there was a near significant correlation between the change in body weight and change in cathepsin S. This study suggests that an ad libitum diet reduces weight and cathepsin S levels also in non-obese subjects, including men. The decreased cathepsin S levels did not remain significant after adjusting for weight change, suggesting that weight reduction mediated some of the dietary effect on cathepsin S. Adjusting for baseline levels of cathepsin S resulted in a P-value of 0.06 which may indicate lack of statistical power, rather than a lack of effect on cathepsin S. However, regression-towards-the mean effect cannot be completely excluded. Cathepsin S is strongly associated with cardiovascular risk factors, such as elevated triglycerides [17] and LDL-C [13]. Subjects on ND for 6 weeks markedly improved their cardiovascular risk profile, including lowering of LDL-C, insulin resistance, and blood pressure [19] and it is possible that this improvement affected the levels of cathepsin S. Our study supports such findings since adjustment for change in LDL-C and total cholesterol as well as change in body weight abolished the significant difference between the groups. Higher levels of circulating cathepsin S are associated with insulin sensitivity [10]. In the current study we could not find a correlation between changes in serum cathepsin S and changes in HOMA-IR. However, the change in insulin sensitivity induced by the 6-week ND was moderate, albeit statistically significant [19]. Perhaps the discrepancy also could be explained by insulin sensitivity being measured with euglycemic clamp in the observational study, whereas it was estimated by HOMA-IR in the present study. The strengths of this study include the randomized controlled design, and also all foods were provided to the ND group ensuring high compliance and low drop-out rates. It should however be noted that some of the observed effect on cathepsin S levels was likely to be caused by the fact that all foods was provided to the ND, but not the control group. Also, these results cannot be directly translated to clinical settings were dietary advice alone is given. Further studies are needed where all foods are provided to both ND and control groups, or dietary advice is given to both groups. Although all subjects in both groups were instructed to maintain their physical activity level during the study, possible differences between groups in physical activity level and smoking were not assessed and may thus have introduced some bias. Since only Caucasian Swedish subjects were included, generalizability to other ethnic groups is unclear. Further, it should be noted that this is a post-hoc study of the NORDIET trial, and thus there was no power calculation done on cathepsin S when designing this trial. BODY.CONCLUSION: These results suggest that a prudent diet comprising healthy Nordic foods may moderately reduce cathepsin S levels in non-obese men and women. This association seems to be partly, mediated by diet-induced weight loss and/or reduced LDL-C concentrations. Given the close link between cathepsin S and various obesity-linked diseases and increased mortality risk, the present results warrant further investigation in further studies.

TITLE: An ABSTRACT: Cervical degenerative disease is one of the most common spinal disorders worldwide, especially in older people. Anterior cervical corpectomy and fusion (ACCF) is a useful method for the surgical treatment of multi-level cervical degenerative disease. Anterior cervical disc replacement (ACDR) is considered as an alternative surgical method. However, both methods have drawbacks, particularly the neck motion decrease observed after arthrodesis, and arthroplasty should only be performed on patients presenting with cervical disc disease but without any vertebral body disease. Therefore, we designed a non-fusion cervical joint system, namely an artificial cervical vertebra and intervertebral complex (ACVC), to provide a novel treatment for multi-level cervical degenerative disease. To enhance the long-term stability of ACVC, we applied a hydroxyapatite (HA) biocoating on the surface of the artificial joint. Thirty-two goats were randomly divided into four groups: a sham control group, an ACVC group, an ACVC-HA group, and an ACCF group (titanium and plate fixation group). We performed the prosthesis implantation in our previously established goat model. We compared the clinical, radiological, biomechanical, and histological outcomes among these four different groups for 24 weeks post surgery. The goats successfully tolerated the entire experimental procedure. The kinematics data for the ACVC and ACVC-HA groups were similar. The range of motion (ROM) in adjacent level increased after ACCF but was not altered after ACVC or ACVC-HA implantation. Compared with the control group, no significant difference was found in ROM and neutral zone (NZ) in flexion-extension or lateral bending for the ACVC and ACVC-HA groups, whereas the ROM and NZ in rotation were significantly greater. Compared with the ACCF group, the ROM and NZ significantly increased in all directions. Overall, stiffness was significantly decreased in the ACVC and ACVC-HA groups compared with the control group and the ACCF group. Similar results were found after a fatigue test of 5,000 repetitions of axial rotation. The histological results showed more new bone formation and better bone implant contact in the ACVC-HA group than the ACVC group. Goat is an excellent animal model for cervical spine biomechanical study. Compared with the intact state and the ACCF group, ACVC could provide immediate stability and preserve segmental movement after discectomy and corpectomy. Besides, HA biocoating provide a better bone ingrowth, which is essential for long-term stability. In conclusion, ACVC-HA brings new insight to treat cervical degenerative disease. BODY.INTRODUCTION: Cervical degenerative disease is a prevalent problem in our aging population. It can be in the form of herniated discs and spondylosis in the cervical spine associated with axial neck and/or radicular arm pain and neurological symptoms. Surgical and nonsurgical treatments have been used to halt or reverse the disease process. Posterior approachesas well as anterior techniques, such as anterior cervical discectomy and fusion (ACDF) and anterior cervical corpectomy and fusion (ACCF), are the most commonly used approaches. Because of its high rate of clinical success (relief of symptoms and favorable outcomes),anterior cervical discectomy and fusionhas been considered by many orthopedist and neurosurgeons as the standard surgical treatment for cervical myelopathy or radiculopathy, secondary to cervical degenerative disease and refractory to a conservative treatment[1]. The anterior cervical discectomy or subtotal corpectomy directly remove the compression induced, for example, by the protrude disc, and also permanently eliminate the pathogenic factor by fusing the degenerative segments[2, 3]. However, ACCF has some limitations that include graft donor site morbidity, subsidence, debris, pseudarthrosis, and adjacent segment disease. Adjacent segment cervical disease occurs in approximately 3% of patients per year, with an expected incidence of 25% within the first 10 years following fusion[4]. Goffinet al.[5] found that after anterior cervical fusion, 92% of patients displayed additional radiological adjacent segmental degeneration, from which 25% presented with clinical adjacent segmental disease in a 10-year follow-up. Matsumotoet al.[6] performed a prospective 10-year follow-up magnetic resonance imaging (MRI) study on 64 patients who underwent anterior cervical discectomy and fusion and 201 asymptomatic healthy control subjects. They demonstrated that although both groups showed a progression towards disc degeneration during the 10-year period, patients treated with anterior cervical fusion had a significantly higher incidence of progression towards disc degeneration at adjacent segments than controls, even if this was not always related to the development of clinical symptoms. Besides, although the relationship between adjacent segmental disease and anterior cervical fusion remains unanswered, the motion loss following ACCF results in an increased range of motion and a higher intradiscal pressure in the facet joint adjacent to the fusion level, which is considered to be related to the adjacent segmental disease [7]. This concern has led to the development of motion-preserving devices such as artificial cervical discs. Artificial cervical disc replacement (ACDR) has developed and is now increasingly used as an alternative to ACCF. The first clinical trial involving spinal arthroplasty occurred between 1962 and 1970 and implied the implantation of Fernstrom ball in 207 cases. Nowadays, some clinical trials are currently testing various artificial cervical disc devices while some have been approved by the United States Food and Drug Administration. Arthroplasty seems to be as safe as ACCF and is becoming a substitute for fusion. Cepoiu-Martin et al.[8] performed a systematic review and reported that ACDR appeared to be at least as effective as ACDF in the short-term follow-up (up to 2 years). The theoretical advantages of ACDR are the restoration of the intervertebral disc and the prevention of nerve root compression recurrence. The preservation of physiological spine mobility, rather than the fusion of the degenerative spine, may limit the progression of adjacent segmental degeneration. However, ACDR has some drawbacks, such as more strict criteria about the intervertebral disc and the vertebral body than ACCF. For multi-level cervical degeneration disease, there is currently no method to rebuild cervical stability and preserve the motion function after decompression. There is no role for the indiscriminate use of ACDR. ACDR should simply not be considered as superior or as a replacement of fusion. Besides, if there is a vertebral body disease, such as a primary tumor, metastatic cancer, tuberculosis, vertebral body deformity, fusion is still the most commonly used surgical technique. To address these issues, we developed a novel artificial cervical vertebra and intervertebral complex (ACVC) that can provide immediate stability after anterior cervical corpectomy and maintain the motion of the surgery segment. Theoretically, this motion-preserving joint system may prevent adjacent segment degeneration. Moreover, long-term stability of prostheses is necessary for orthopedic implants. For uncemented prostheses, many parameters could affect long-term stability, including the implant design, surface texture, material biocompatibility, the presence of a bioactive coating, and the biomechanical properties of the surrounding bone. Various biocoatings have been proposed as effective methods for improving the bone-implant interface stability. In recent decades, hydroxyapatite ((Ca10(PO4)6(OH)2; HA) has been reported to have a high biocompatibility, a low degradation rate, and the ability to increase the efficiency of bone-implant integration in order to improve osteointegration of implants because of its similar composition to bone tissue and its ability tocreate chemical bonding withbones[9]. Xie and Luan[10] reported that the precoated HA could further improve the stability of the Ti6Al4V alloy with a composite layer of TiO2 and HA. This suggests that artificial joints pretreated with an HA coating may induce strong bonding with the bone via the natural growth of new HA. For this reason, we applied HA biocoatings to the surface of the ACVCs to determine whether the HA biocoating would increase the implant's interaction with bone and improve its long-term stability. BODY.MATERIALS AND METHODS.STUDY DESIGN: In our previous study [11], we found that goat was an appropriate animal model for the biomechanical study of the cervical spine. Specifically, we showed that the C2-C4 vertebrae were the best segments for obtaining biomechanical data comparable to the human cervical spine. We successfully designed the ACVC and implemented an anterior cervical corpectomy and ACVC replacement in goat model. However, some concerns remained, notably regarding the potential advantages of using ACVC over ACCF. Does ACVC result in less kinematic changes compared with ACCF, which is the traditional surgery for cervical degeneration disease? Moreover, how is the long-term stability of ACVC compared with ACCF? To solve these issues, we developed a titanium mesh cage and an anterior cervical plate system for the goat to implement the ACCF animal model, and the ACVC-HA(ACVC with an HA biocoating). In the present study, we compared the clinical features and the biomechanical properties of the ACVC and ACVC-HA with those of the intact state and of the titanium cage and the anterior plate fusion state in goats. Thirty-two goats were randomly divided into four groups and were followed-up for 24 weeks after surgery. Clinical evaluation, radiological observation, biomechanical testing, and histology staining were performed. 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 Xi'an Jiaotong University Second Affiliated Hospital approved protocol (Permit Number: 2013–16). All surgeries were performed under anesthesia, and all efforts were made to minimize suffering during the surgical procedures and post-surgery recovery. BODY.MATERIALS AND METHODS.INTERNAL INSTRUMENTS AND ANIMAL GROUPING: Thirty-two adult Chinease White goats (age: 23±5 months; average body weight: 57.4±6.7 kg; equal numbers of female and male goats) from the Animal center of Xi'an Jiaotong University Second Affiliated Hospital were randomly divided into 4groups. This included: a control group (Group A), an ACVC group (Group B), an ACVC-HA group (Group C), and an ACCF group (Group D). In the control group, each goat received a sham anterior cervical operation that fully exposed the anterior vertebral surface without disturbing the bony, disc and ligamentous structures. In the ACVC and ACVC-HA groups, ACVC and ACVC-HA were separately implanted in the goats after decompression. In the ACCF group (decompression and fusion group), each animal received an anterior cervical decompression and fusion, using a titanium mesh cage and an anterior plate. To exclude cervical spine abnormalities, radiological images were obtained using a C-arm fluoroscopic imager before surgery. All the internal instruments were made by professional engineers in Northwest Nonferrous Metal Research Institute (China) and were composed of titanium alloy (Ti6Al4V). The ACVC (Patent NO.: US9452062B2; Fig 1B) consisted of two endplate components, two vertebral components, one length-locking screw, and four self-drilling trapping screws. The dimensions of this artificial joint were based on the anatomical and radiological data from the goats. The ACVC is fixed using four self-drilling trapping screws. To create the ACVC-HA (Fig 1C), an HA biocoating with a 30-μm thickness was applied to the surface of the vertebral components, the upper surface of the upper endplate component, the lower surface of the lower endplate component, and the surface of the self-drilling trapping screws, using microarc oxidation (MAO) technology. The biocoating may induce more bone ingrowth, which would facilitate long-term stability. 10.1371/journal.pone.0178775.g001Fig 1Scheme diagram and photos of the three different artificial implants.A, Scheme diagram of the ACVC; B, actual photo of the ACVC; C, actual photo of the ACVC-HA; D, actual photo of the titanium cage, anterior plate, and fixing screws. The design of the titanium cage and the anterior plate (Fig 1D) used in the present study were based on the anatomical data of goat cervical spines and were inspired by the titanium cage and the anterior plate used in human ACCF. Autologous bone could be placed within the hollow structure of the titanium cage. Four to eight self-drilling trapping screws could fix the plate into the adjacent vertebral bodies. BODY.MATERIALS AND METHODS.ARTIFICIAL CERVICAL JOINT IMPLANTATIONS AND POSTOPERATIVE OBSERVATIONS: The goats were allowed one-week acclimatisation before the surgery and were housed individually in a thermo-hygrostat cage (23°C, 45%–50% humidity) with 12-hours dark-light cycle. Each goat was given access to food and water ad libitum. The surgical procedures and postoperative animal care have been well documented in our previous study[11]. Briefly, all the animals were given 0.04 mg/kg of atropine subcutaneously, followed by 10 mg/kg of ketamine and 0.3 mg/kg of xylazine intramuscularly. 6 mg/kg of 2.5% thiamylal sodium solution was administered intravenously after 10 minutes. General anesthesia was maintained with approximately 1% halothane in O2 through 8-mm diameter endotracheal tube. Then the surgeries were performed in supine position by a transverse skin incision at the C3 level from the right side[12]. The anterior surface of C2-C4, between the carotid sheath and tracheal esophageal sheath, was exposed. At this point, the goats of the control group were sutured. All the other goats underwent anterior C2/C3 and C3/C4 discectomies, and a C3 subtotal corpectomy. Great care was taken to remove the cartilaginous tissue, while preserving intact endplates. After removal of the interbody discs and partial C3 vertebral body, preparation of the endplates with arongeur and a curette, the space left free by the intervertebral discs and the C3 vertebral body was filled with the corresponding implant under anterior distraction. Thus, the goats underwent either ACVC implantation, ACVC-HA implantation or titanium mesh cage and anterior plate implantation according to their group assignments. Final X-ray films were taken to verify the placement of implants before the animals were sutured in layers. Then, the goats were placed in plaster neck casts for 4 weeks to restrict neck activity and facilitate fixation of the implant. Surgery time, blood loss, recovery time, and time before eating were recorded. Surgery time was the period from skin incision to skin suture. Blood loss was estimated using a vacuum extractor and blood gauze. Recovery time was the period from the end of surgery until the goat could stand by itself. Time before eating was the period from the completion of surgery until the goat began to eat by itself. Food and water intake was limited during the first 24 hours after surgery to reduce the risk of intestinal tympanites. A normal diet was established two days after surgery. Cefazolin (25 mg/kg) was administered twice a day for two days after surgery. Goat care was ensured by trained animal care staff. Eating habits, ambulatory activities, health status, and neurological functions were monitored daily for the first 4 weeks, twice weekly for Weeks 5 to 12, and weekly from the 12th to 24th week. Post-surgery pain was the major suffering during the post-surgery recovery. Analgesia or anesthesia was used to relieve the post-surgery pain. NSAIDS (non-steroidal anti-inflammatory drugs, such as ibuprofen) and opioids (such as meperidine and morphine) were used to relieve the post-surgery pain if necessary. Soft food (such as cabbages) was provided within the first week after surgery. Anteroposterior and lateral radiographic films were taken under general anesthesia at weeks 1, 6, 12, and 24 after surgery. Computed tomography (CT) analyses were performed to determine the extent of bony fusion and the position of the prosthesis at weeks 6, 12 and 24 post-surgery. All animals were observed for 24 weeks before euthanasia with an overdose of pentobarbital (200 mg/kg). Criteria for early euthanasia during the experiment for humane endpoints: weight loss of 20–25%, inappetance up to 5–7 days, severely weakness, moribund state, infection fails to respond to antibiotic therapy, and severe organ system dysfunction non-responsive to treatment. BODY.MATERIALS AND METHODS.BIOMECHANICAL TESTS: The biomechanical test protocol has been described previously [11]. To investigate the adjacent level movement, the C1-C7 segments were dissected from the harvested cervical spines immediately after sacrifice and cleaned of residual soft tissue, with care taken to not disturb the spinal bony and ligamentous attachments. The C1-C7 vertebrae segments were embedded in a special metal mould containing polymethyl-methacrylate to keep the specimens in a set position with the C3 vertebra parallel to the horizontal plan. Three-dimensional flexibility tests were conducted on each of the specimens according to the protocol established by Zhu et al.[13]. All data were recorded using a servohydraulic material testing machine (MTS 858 Bionix machine, MTS System Inc., Minneapolis, MN, USA). A pure moment of 2.5Nm was applied to the top vertebra (C1), while the specimen was allowed to move in an unconstrained 3-D manner. This continuous moment was applied at a rate of approximately 0.5°/s in all three primary directions of loading, namely flexion-extension, lateral bending, and axial rotation. The load was applied for five complete loading cycles. The first four cycles were used to precondition the specimen and minimize viscoelastic effects, and the fifth cycle was used for data analysis. The total angular range of motion was calculated for the last cycle. Throughout the fifth load cycle, inter segmental motions were collected via five infrared light-emitting diodes(LEDs)markers rigidly affixed to each of the C2 to C6 vertebrae, this serving as the definable points for three-dimensional motion. A marker carrier with four non-collinear LEDs on the base of the spine machine defined a general anatomical specimen coordinate system. A 3D laseroptoelectronic camera system (Optotrak 3020; Northern Digital, Waterloo, Canada; frame rate of 20 Hz) was used to measure the movement of the whole spine and of each segment. The kinematic behavior of each specimen was compared by examining the range of motion (ROM) of the C2-C4, C4-C5, C5-C6, and C2-C6 segments of intact and instrumented goat spines from the fifth loading cycle. To determine the stability of the specimens in the different groups, the C6 and C7 segments were removed. A multidirectional flexibility test was used in a non-destructive manner for the C1-C5 segments. The end vertebrae of the specimens (C1 and C2, C4 and C5) were transfixed with perpendicular pins, to enhance the fixation with mounting jigs. The C1 to C5 vertebrae were then mounted in fast-drying epoxy resin (Huntsman Advanced Materials (HK) Limited, HK). A fatigue test comprised of 5,000 repetitions of axial rotation (fatigue load:1.0 Nm; frequency:0.25 Hz) was used during the kinematic test. A similar biomechanical test was performed. The kinematic behavior of each specimen was compared by examining the range of motion (ROM) and the neutral zone (NZ) of the C2-C4 segments of the intact and instrumented goat spines from the fifth loading cycle before and after the fatigue test. Throughout the biomechanical testing, special care was taken throughout the tests to keep the specimens moist using a 0.9% saline solution. A stability index ROM (SI ROM) and a stability index NZ (SI NZ) were introduced to quantify the stability provided by the implantation to the spine. These indices were similar to those described by Zhang et al. [14]. The SI ROM and SI NZ were defined using the following equations: SI ROM = (ROMintact − ROMinstrumented)/ROMintact, and SI NZ = (NZintact − NZinstrumented)/NZintact. BODY.MATERIALS AND METHODS.HISTOLOGICAL EVALUATION: After the biomechanical testing, each sample was fixed in cold Karnovsky fixative containing 4% paraformaldehyde and 5% glutaraldehyde for two weeks. The specimens were dehydrated in an alcohol series and embedded in polymethyl-methacrylate. Transverse sections of 100 μm thickness were carried out with a Leica SP1600 sawing microtome (Leica, Nussloch, Germany). The specimens were then ground to the desired thickness. Masson's trichrome staining and Van Gieson staining were used for transmitted light microscopy. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: All results were analyzed using SPSS Version 13.0 (SPSS Inc., Chicago, IL, USA). The data regarding surgery characteristics, ROM, NZ, and stiffness were analyzed using are repeated-measure analysis of variance (ANOVA). Post hoc tests were performed using Bonferroni correction for multiple comparisons. The level of statistical significance was set at P<0.05. BODY.RESULTS.DESIGN OF DIFFERENT ARTIFICIAL CERVICAL JOINTS: Fig 1 shows photographs of the ACVC (Fig 1B), the ACVC-HA (Fig 1C), and the titanium cage and the anterior plate (Fig 1D) implants. The ball-in-trough structure (Fig 1A-7 and 1A-8) is the most important part of this unconstrained metal-on-metal ACVC joint. In theory, the ball-in-trough articulation allows a 20° ROM in flexion-extension, a 12° ROM in lateral bending, a 360° ROM in rotation, and a 1.5-mm anterior-posterior slide horizontally. The ACVC-HA is an ACVC treated with a 30-μm hydroxyapatite biocoating via microarc oxidation technology. The titanium cage and the anterior plate were used for goat anterior cervical discectomy and fusion. The titanium cage was 8 mm wide and could be cut to a suitable length. Because the vertebral bodies of goats are much longer than those of humans, four to eight screws could be used to fix the plate and the cage. The vertebral components were hollow-structured, which allowed the surgeons to implant the autologous bone in the hollow space. Thus, the surrounding bone could grow into the vertebral components through the holes, this providing long-term stability. BODY.RESULTS.CLINICAL AND RADIOLOGICAL ASSESSMENTS OF THE DIFFERENT PROSTHESIS IMPLANTATIONS IN GOATS: All surgeries were successful. Surgery time, blood loss, recovery time, and time before eating are shown in Table 1. No significant differences were found in surgery time, blood loss, recovery time, and time before eating among ACVC, ACVC-HA, and ACCF groups. These findings indicate that ACVC or ACVC-HA implantation did not increase surgery time, blood loss, recovery time, and time before eating compared with the ACCF group. 10.1371/journal.pone.0178775.t001 Table 1 Surgery characteristics of the four different groups. control group ACVC group ACVC-HA group ACCF group Surgery time (min) 12±6 68±11 67±16 65±13 Blood loss (ml) 10±6 55±18 56±19 51±20 Recovery time (hours) 1.1±0.6 3.6±1.7 4.1±2.0 3.5±1.4 Eating time (hours) 1.7±0.5 4.5±1.8 4.5±2.2 3.9±2.3 No significant difference was found among ACVC, ACVC-HA, and ACCF groups. The goats tolerated the artificial joints very well during the experiment. All goats survived until 24 weeks after surgery. No abnormal neurological reflexes or neurological complications were found. One goat in the ACVC group and two goats in the ACCF group developed wound infections 8 to 12 days after surgery. These wound infections were cured by applying antibiotics and changing bandages daily. One goat in the ACVC-HA group and another goat in the ACCF group suffered respiratory infections and recovered by using antibiotics. No bone fracture, joint dislocation, or screw loosening were found during the experimental period. Fig 2 shows the CT images of goats in the three different groups 24 weeks after surgery. In the ACVC group, the implant was properly located in the C3 vertebral body (Fig 2B). The vertebral body component of the ACVC was located in the middle of the C3 vertebral body (Fig 2E). The two lower screws were fixed to the C4 vertebral body (Fig 2H). The titanium cage was located between C2 and C4. The vertebral bodies from C2 to C4 were fixed by the plate (Fig 2C). The titanium cage was located in the trough of the C3 vertebral body (Fig 2F). The anterior plate was fixed by 3 screws to the C2 vertebral body and by 3 additional screws to the C4 vertebral body (Fig 2I). 10.1371/journal.pone.0178775.g002Fig 2CT images of goats in control group, ACVC group, ACCF group 24 weeks after the surgery (ACVC and ACVC-HA appears similar).A, B, C, Lateral views of C2 to C4 for the three groups; D, G, transverse views of C3 and C4 vertebral bodies in control group; E, H, transverse views of C3 and C4 vertebral bodies in ACVC group; F, I, transverse views of C3 and C4 vertebral bodies in ACCF group. BODY.RESULTS.BIOMECHANICAL RESULTS OF THE DIFFERENT PROSTHESIS IMPLANTATIONS IN GOATS: To investigate the cervical spine ROM separately, we used the three-dimensional laser optoelectronic camera system (Table 2). There were no significant differences among the control, ACVC, and ACVC-HA groups inflexion-extension and lateral bending for the C2-C6, C2-C4, C4-C5, and C5-C6 segments. The rotation ROM of the ACVC and ACVC-HA groups were greater than the ROM of the control group for the C2-C6 and C2-C4 segments. Accordingly, the ROM in rotation decreased in the ACVC and ACVC-HA groups for the C4-C5 and C5-C6 segments. For the C2-C6 and C2-C4 segments, the ROM was less for the ACCF group than for the control group in all three directions. Accordingly, the ROM of the C4-C5 segment in all three directions and the ROM of the C5-C6 segment in rotation increased in the ACCF group. 10.1371/journal.pone.0178775.t002 Table 2 Ranges of motion profiles of the cervical segments of the four groups before the fatigue test in all directions. Group A (Control group) Group B (ACVC group) Group C (ACVC-HA group) Group D (ACCF group) C2-4 (in Degrees) Flexion-Extension 4.0±0.6 3.9±0.6 4.0±0.3 0.4±0.1 a , b Lateral bending 5.9±0.7 5.6±0.8 5.8±0.5 1.3±0.3 a , b Rotation 7.2±0.6 11.2±0.9 a 12.3±0.7 a 5.6±0.5 a , b C4-5 (in Degrees) Flexion-Extension 1.3±0.2 1.5±0.3 1.4±0.3 2.2±0.3 a , b Lateral bending 1.8±0.2 2.0±0.3 2.0±0.4 2.9±0.3 a , b Rotation 2.2±0.3 1.2±0.3 a 1.4±0.2 a 3.4±0.2 a , b C5-6 (in Degrees) Flexion-Extension 1.4±0.2 1.3±0.2 1.4±0.1 1.6±0.2 Lateral bending 2.0±0.3 1.9±0.3 2.2±0.4 2.2±0.4 Rotation 2.4±0.2 1.8±0.2 a 1.9±0.3 a 1.6±0.1 a , b C2-6 (in Degrees) Flexion-Extension 6.5±0.9 6.2±0.7 6.3±1.1 4.2±0.4 a , b Lateral bending 9.6±1.1 9.1±1.5 9.2±0.9 6.9±0.4 a , b Rotation 11.2±0.8 14.5±1.3 a 15.2±1.8 a 9.8±0.8 a , b no significant differences between ACVC and ACVC-HA group a P<0.05 versus the Group A (control) b P<0.05 versus the Group B (ACVC group) or Group C (ACVC-HA group) The biomechanical data of the C2-C4 segment recorded before the fatigue test (Table 3, Figs 3 and 4) confirmed that the ROM of ACVC and ACVC-HA groups in flexion-extension and lateral bending were similar to those of the control group. It also indicated that the ROM of ACVC and ACVC-HA groups in rotation increased compared with that of the control group. Also, compared with the control group, ROM in all directions was decreased in ACCF group. 10.1371/journal.pone.0178775.t003 Table 3 Ranges of motion, neutral zones, and stiffness profiles of the C2-4 segments of the four groups before the fatigue test in all directions. Motion Group A (Control group) Group B (ACVC group) Group C (ACVC-HA group) Group D (ACCF group) Range of Motion (in Degrees) Flexion-Extension 6.7±1.1 6.5±0.8 6.6±0.9 0.6±0.3 a , b Lateral bending 10.1±1.2 9.7±1.4 9.8±1.2 2.3±0.6 a , b Rotation 10.4±0.9 18.5±1.4 a 17.3±1.3 a 7.4±0.6 a , b Neural Zone (in Degrees) Flexion-Extension 1.2±0.6 1.4±0.7 1.5±0.5 0.3±0.1 a , b Lateral bending 1.1±0.5 1.6±0.8 1.6±0.7 0.3±0.1 a , b Rotation 1.2±0.2 3.5±0.5 a 3.2±0.4 a 0.5±0.2 a , b Stiffness (in Nm/Degree) Flexion-Extension 0.63±0.10 0.30±0.07 a 0.33±0.11 a 7.20±1.33 a , b Lateral bending 0.13±0.03 0.05±0.02 a 0.05±0.01 a 2.53±0.67 a , b Rotation 0.21±0.02 0.02±0.00 a 0.02±0.00 a 1.53±0.42 a , b no significant differences between ACVC and ACVC-HA group a P<0.05 versus the Group A (control) b P<0.05 versus the Group B (ACVC group) or Group C (ACVC-HA group) 10.1371/journal.pone.0178775.g003Fig 3Typical hysteresis curves of C2-C4 level of the four groups before fatigue test in the directions of flexion-extension (A), lateral bending (B), and rotation (C). 10.1371/journal.pone.0178775.g004Fig 4Average unidirectional ROM (A), NZ (B), and stiffness (C) of the four different groups at 24 weeks after surgery in all directions before the fatigue test. There was no significant NZ difference in flexion-extension and lateral bending among control, ACVC and ACVC-HA groups. In rotation, NZ was significantly greater for ACVC and ACVC-HA groups than for the control group. For the ACCF group, NZ was significantly decreased in all directions. The stiffness was significantly decreased in all directions for the ACVC and ACVC-HA groups, while it was increased for the ACCF group. We found similar results for the ROM, NZ, and stiffness in the ACVC and ACVC-HA groups. Similar C2-C4 biomechanical data were found after the 5,000repetitions of the axial rotation fatigue test (Table 4, Figs 5 and 6). 10.1371/journal.pone.0178775.t004 Table 4 Ranges of motion, neutral zones, and stiffness profiles of the C2 to C4 segments of the four groups after the fatigue test in all directions. Motion Group A (Control group) Group B (ACVC group) Group C (ACVC-HA group) Group D (ACCF group) Range of Motion (in Degrees) Flexion-Extension 6.5±0.8 6.2±0.9 6.3±1.1 0.7±0.4 a , b Lateral bending 10.5±1.5 10.1±1.5 10.2±1.3 2.5±0.5 a , b Rotation 10.6±1.2 18.3±1.7 a 18.5±1.9 a 7.0±0.3 a , b Neural Zone (in Degrees) Flexion-Extension 1.1±0.5 1.3±0.5 1.3±0.6 0.2±0.1 a , b Lateral bending 1.2±0.7 1.5±0.7 1.4±0.6 0.4±0.2 a , b Rotation 1.2±0.3 3.6±0.5 a 3.4±0.5 a 0.6±0.2 a , b Stiffness (in Nm/Degree) Flexion-Extension 0.69±0.20 0.35±0.10 a 0.34±0.12 a 7.47±1.45 a , b Lateral bending 0.15±0.04 0.04±0.01 a 0.05±0.02 a 2.67±0.68 a , b Rotation 0.26±0.04 0.02±0.00 a 0.02±0.00 a 1.62±0.47 a , b no significant differences between ACVC and ACVC-HA group a P<0.05 versus the Group A (control) b P<0.05 versus the Group B (ACVC group) or Group C (ACVC-HA group) 10.1371/journal.pone.0178775.g005Fig 5Typical hysteresis curves of C2-C4 level of the four groups after fatigue test in the directions of flexion-extension (A), lateral bending (B), and rotation (C). 10.1371/journal.pone.0178775.g006Fig 6Average unidirectional ROM (A), NZ (B), and stiffness (C) of the four different groups at 24 weeks after surgery in all directions after the fatigue test. The SI ROM and SI NZ are reported in Table 5 and Fig 7. The SI ROM of the ACVC and ACVC-HA groups were positive in flexion-extension and lateral bending but negative in rotation. The SI NZ of the ACVC and ACVC-HA group were negative in all directions. The SI ROM and SI NZ in the ACCF group were positive in all directions. 10.1371/journal.pone.0178775.t005 Table 5 Stability index of the flexibility test before the fatigue test. Motion Group B (ACVC group) Group C (ACVC-HA group) Group D (ACCF group) Stability Index Range of Motion (in Degrees, SI ROM) Flexion-Extension 0.030±0.016 0.030±0.012 0.090±0.042 * Lateral bending 0.040±0.019 0.030±0.015 0.228±0.079 * Rotation -0.779±0.156 -0.663±0.192 0.712±0.281 * Stability Index Neural Zone (in Degrees, SI NZ) Flexion-Extension -0.167±0.198 -0.250±0.122 0.250±0.076 * Lateral bending -0.4555±0.362 -0.455±0.321 0.273±0.122 * Rotation -1.917±0.876 -1.667±0.911 0.417±0.182 * no significant differences between ACVC and ACVC-HA group * P<0.05 versus the Group B (ACVC group) or Group C (ACVC-HA group) 10.1371/journal.pone.0178775.g007Fig 7Average changes in the stability index ROMs and stability index NZs from ACVC group, ACVC-HA group and ACCF group. BODY.RESULTS.HISTOLOGY: Histology sections of the goat cervical spines showed no inflammatory response 24 weeks after surgery, as well as no significant metallic wear debris at the implant-bone interface. Newly formed bone was found around the vertebral body component of the ACVC and the self-drilling fixation screws. In the sections located at the ACVC vertebral body component level (Fig 8A and 8C), we found less new bone formation and more fibrous tissue around the implant for the ACVC group (Fig 8A) than for the ACVC-HA group (Fig 8C). In the sections located at the self-drilling fixation screw level (Fig 8E and 8G), we found significantly more new bone formation around the screws for the ACVC-HA group (Fig 8G) than for the ACVC group (Fig 8E). Moreover, the ACVC group had much more fibrous tissue around the screws than the ACVC-HA group. These findings indicate that, compared with the ACVC group, the ACVC-HA group had a significantly higher bone-implant contact (BIC) in both sections respectively located at the vertebral body component and screw level (Fig 8I), which is an appropriate index for measuring bone ingrowth into the implant. 10.1371/journal.pone.0178775.g008Fig 8Comparison histological images of ACVC and ACVC-HA.A and B, 25X- and 100X- magnified images at the level of ACVC vertebral body component; C and D, 25X- and 100X- magnified images at the level of ACVC-HA vertebral body component; E and F, 25X- and 100X- magnified images at the level of fixation screws of ACVC; G and H, 25X- and 100X- magnified images at the level of fixation screws of ACVC-HA; I, Bone implant contact (BIC) of ACVC and ACVC-HA at vertebral body and screw level. The magnified images showed that bone formation around the implant surfaces was fragmentary and that a large portion of the bone tissue was separated from the implant surface by soft tissue in the ACVC group (Fig 8B and 8F). In contrast, in the ACVC-HA group, a large amount of the implant surface area was surrounded by a thin layer of cartilage on the vertebral body component of the ACVC (Fig 8D) and by extensive and continuous newly formed bone at the level of the fixation screws (Fig 8H). BODY.DISCUSSION: ACCF and ACDF have been considered to represent the standard surgical treatments of cervical myelopathy or radiculopathy with degenerative disc disease that are refractory to conservative treatment. Because of its high rate of clinical success in relieving symptoms, ACCF is usually chosen to treat multi-level cervical degeneration disease[15]. The surgical treatments of multi-level cervical spine degeneration diseases by ACCF and ACDF seem to provide similar results. However, ACCF appears to provide a higher fusion rate than multi-segmental ACDF[16]. Although ACCF was considered to result in a higher fusion rate and a full exposure to complete decompression, the decreased number of fusion surfaces in multilevel ACDF leads to a lower risk of pseudarthrosis[17–19]. However, there is currently no powerful evidence for the superiority of one method over the other. Weber et al.[20] performed a retrospective cohort study to assess the subsidence and revision rates associated with different interbody cages following anterior cervical corpectomy and reconstruction. They found that for the fibula allograft, the average graft subsidence was 3 mm with are vision rate of 25% while these were 2.9 mm and 11.1%, and 2.9 mm and 18.8% respectively for the titanium mesh cages and the titanium expandable cages, indicating that the titanium cage did not increase the subsidence after cervical fusion. Therefore, we chose ACCF using titanium cages and anterior cervical plates as the gold standard to compare the clinical and biomechanical properties of the ACVC and ACVC-HA. BODY.DISCUSSION.MOBILITY AND LONG-TERM STABILITY ARE ESSENTIAL FOR ACVC: The two main advantages of this metal-on-metal unconstrained artificial cervical joint system are its ability to allow physiological motions and its potential to reduce stress at the bone-implant interface. This articulation allows anterior-posterior translation, constrained flexion-extension, and unconstrained rotation, which theoretically mimics the normal cervical physiological motion. To eliminate the risk of dislocation, it is important to have the articulation closed and the posterior longitudinal ligament and surrounding soft tissues aid in providing stability. Besides, we designed several structures to enhance the long-term stability, such as the HA biocoating, the rough and grit-blasted endplate component, and the hollow vertebral body component. Autologous grafting is the gold criteria for fusion in various types of ACCF techniques because it has a high fusion rate compared with discectomy alone or implanted cages. However, the main shortcoming of autologous bone graft is the harvesting of bone, primarily from the iliac crest, which may results in short- and long-term morbidities. These include increased blood loss, infection, donor site pain, hematoma, and peripheral nerve injury[21]. Compared with autografts, allografts have a lower fusion rate, as well as increased infection and immune issues. In contrast, interbody cages provide initial stability and require much less structural bone graft. The anterior plate can provide immediate stability and maintain the cervical spine alignment. This is the reason why we designed a hollow structure to hold autologous bone from the corpectomy, in order to exclude the donor site pain and promote bone ingrowth. The selection of biomaterials for the artificial joint system depend on the good mechanical properties (high strength and low modulus closer to bone), biocompatibility, superior corrosion and wear resistance, and osseointegration. The reasons we chose Ti6Al4V were because of its adequate mechanical properties, good biocompatibility, valuable imaging characteristics, and low elasticity modulus. Besides, there was no acute neural orsystemic histopathologic response to the Ti6Al4V included in a 6-month in vivo animal model[22]. But long term performance of these alloys has raised some concerns of release of aluminum and vanadium ions, poor shear strength, severe wear, which may result in inflammatory reaction leading to loosening of implants due to osteolysis. BODY.DISCUSSION.ACVC AND ACVC-HA PRESERVE CERVICAL MOTION COMPARED WITH ACCF: We designed this ACVC implant to preserve motion in the degenerative segment of the cervical spine after the anterior cervical discectomy and subtotal corpectomy. To test the ACVC biomechanical properties, we compared its kinematic data with those of the intact and ACCF states. First of all, we investigated the adjacent segmental movement. Our data confirmed that once the cervical vertebrae (C2-C4) were fused, the adjacent segment (C4-C5) would compensate for the decreased ROM. Finally, the total motion of the cervical spine (C2-C6) was still less than that of the control group. In contrast, by using this novel motion-preserving cervical joint system, namely ACVC or ACVC-HA, the movement of C2-C4 could be preserved and C4-C5 would not compensate for the altered ROM in flexion-extension and lateral bending. Moreover, rotation increased at the C2-C4 level after ACVC and ACVC-HA implantations, which resulted in a decreased ROM for the C4-C5 and C5-C6 segments. Park et al.[23] reported that the segmental rotation, translation, center of rotation, disc height, and disc angle were increased after arthroplasty, while they were unaltered after fusion in an in vivo kinematic study. Prasarn et al.[24] also showed that the biomechanics was changed at the levels adjacent to a cervical spine fusion, notably the adjacent segmental motion which was increased. Schwab et al. [25]reported that motion compensation increases at segments immediately adjacent to a single-level fusion. Our data showed that the closest segment played an important role in compensating for the loss of cervical mobility, which indicated that the ACVC maintained a physiological distribution of ROM throughout the cervical spine. Compared with the intact state (control group), no difference was observed for both ACVC and ACVC-HA groups in the ROM and NZ during flexion-extension and lateral bending. During rotation, the ROM and NZ were increased. The stiffness was decreased in all directions. The fold change of stiffness decrease in rotation (0.10) was much greater than that in flexion-extension (0.48) and lateral bending (0.38). Compared with the ACCF group, the ROM and NZ were significantly increased and the stiffness decreased in all directions. These biomechanical results indicated that thanks to the ball-in-trough articulation structure design, the ACVC could preserve the three-dimensional motion of cervical spine. Compared with the intact state, the fold change of ROM decrease in the ACCF group was much greater in flexion-extension (0.09) than in lateral bending (0.23) and rotation (0.71). The fold change of NZ decrease was also much greater in flexion-extension (0.25) than in lateral bending (0.27) and rotation (0.42). The fold change of increase in stiffness was much greater in lateral bending (×19.46) than in flexion-extension (×11.43) and rotation (×7.29). The cylinder titanium cage combined with the anterior plate successfully provided strong stability in all directions. A positive, null, or negative stability index value indicates that the spinal construct was respectively, more stable than, equally as stable as, or less stable than the intact spine. Therefore, a higher stability index value indicates a greater immediate stability of the spinal construct [14]. In the present study, the SI ROM and negative SI NZ values observed in the ACVC group were expected because of the ball-in-trough and anterior-posterior slide design of the articulation. The SI ROM and SI NZ values observed in the ACCF group confirmed that this method can provide excellent stability after discectomy and corpectomy. All of the above results indicate that, compared with the intact state and the ACCF group, the ACVC (or ACVC-HA) successfully restored the flexion-extension and lateral bending abilities after discectomy and subtotal corpectomy. The absence of any biomechanical difference before and after the fatigue test for every group indicate that the fixation screw could provide primary stability. BODY.DISCUSSION.THE HA COATING COULD ENHANCE THE LONG-TERM STABILITY BY IMPROVED BIC: The histological results indicated that the amount of new bone formed was much greater in the ACVC-HA than the ACVC group. Furthermore, the bone-implant contact was much higher in the ACVC-HA than the ACVC group. This higher bone-implant contact indicate that the HA biocoating offered great osteoconductivity. In the sections located at the screw level, we found fibrous tissue around the screws in the ACVC group and newly formed bone in the ACVC-HA group. This may have occurred because there were less micro-motion and osteolysis after implantation in the ACVC-HA than the ACVC group. The HA coating formed a strong interface between the bone and screws, providing strong initial stability in the early stage after implantation. The MAO-treated, HA-coated screws significantly improved the bone reaction to implant and increased the fixation stability. Thus, they served as an effective method to decrease screw-loosening by providing both mechanical interlocking and biochemical support at the bone-implant interface. The histological results suggest that the HA coating may encourage bony ingrowth to provide a morphological fixation of the implant to the bone, which may improve the interlocking between the implant and the bone. The bony ingrowth into the implant is accelerated by the bioactivity and the osteoconductive property of the HA coating. The HA coating on the titanium alloy implant increased the osteoconductivity and inhibited soft tissue infiltration, thus offering a mechanical interlocking attachment for short- and long-term fixation. Furthermore, the HA coating immobilizes osteoblastic cells and facilitate vascularization in bone tissue at the early stage after implantation. During the later stage, peri-implant bone formation was stimulated by the calcium ions degraded by the HA coating [26]. Our histological results are in agreement with the findings of other studies that have used calcium-phosphate ceramics for various applications. Lim et al.[27] performed an in vitro pilot study and found that microarc oxidation could be a reasonable option for treating titanium alloy surfaces in order to facilitate a better osseointegration. Huang et al.[28] reported that the apatite layer on the titanium implant processed with microarc oxidation is expected to enhance both the bony ingrowth into the implant and the interlocking between the implant and bone. Quaranta et al. [29]reported that hydroxyapatite coatings have an obvious advantage regarding osteogenesis facilitation and play a critical role in the stability of prostheses. Our results are consistent with those of other studies reporting that HA has a stimulatory effect on bone osseointegration and osteogenesis. BODY.DISCUSSION.RESEARCH LIMITATIONS: Several limitations are inherent to this study. First of all, this is an in vivo animal study. The length and size of cervical vertebrae in goat is different from those of humans. Besides, in humans, the most common symptomatic levels are C5/6 and C6/7. Agenetic and environmental etiology may likely explain this difference. In our study, we choose the C2-C4 level as the surgery level, because of the biomechanical properties of C2-C4 in goat, which is similar to those of humans. Secondly, the follow-up duration is limited. In animals, it may be appropriate to follow an in vivo study up to only 24 weeks. However, it is difficult to comment on the function and longevity of a device designed to remain intact and preserve motion after only 24 weeks in humans. That is why we included a fatigue protocol in the biomechanical test, though the number of fatigue cycles was limited as well. Thirdly, we are planning to conduct further experiment aiming at improving the durability and stabilization capacity of our ACVC for clinical application, such as applying biocoating on the surface of ACVC to improve the bone-implant integration for long-term stability. Fourthly, the low wear resistance of Ti6Al4V was the main shortcoming of this joint system, which may result in implant loosening and wear debris. Thus we are planning to develop a metal-on-UHMWPE articulation to solve this problem. BODY.CONCLUSIONS: We have successfully performed the in vivo implantation of ACVC, ACVC-HA and titanium cage in goats. Goat has been proven to been excellent animal model for cervical spine biomechanical studies, especially for the artificial joints and biomedical materials. The biomechanical results indicate that, compared with intact state and ACCF, ACVC and ACVC-HA provide stability and preserve segmental movement for at least 6 months after discetcomy and corpectomy. The histological results demonstrate that hydroxyapatite biocoating provide better bone ingrowth, which is essential for long-term stability. Also, ACVC-HA brings a new insight to treat cervical degenerative disease.

TITLE: Usefulness of Sputum Induction with Hypertonic Saline in a Real Clinical Practice for Bacteriological Yields of Active Pulmonary Tuberculosis ABSTRACT.BACKGROUND: Mycobacterial identification in active pulmonary tuberculosis (APTB) is confirmative, even though successful rates using self-expectorated sputum are limited. Sputum specimens collected by hypertonic saline nebulization showed higher bacteriologic diagnostic sensitivities over those of self-expectoration, mostly studied in smear-negative or sputum-scarce patients. The efficacy of induced sputum was rarely assessed in real clinical settings. ABSTRACT.METHODS: A prospective randomized case-control study was performed in one hospital. The subjects highly suspicious of APTB were asked to provide 3 pairs of sputum specimens in 3 consecutive days. The first pairs of the specimens were obtained either by self-expectoration (ES) from the next day of the visit or sputum induction with 7% saline nebulization in clinic (SI), and the other specimens were collected in the same way. The samples were tested in microscopy, culture, and polymerase chain reaction (PCR). The outcomes of the bacteriological diagnosis were compared. ABSTRACT.RESULTS: Seventy six patients were assigned to either ES (38 subjects, median age of 51, 65.8% male) or SI (38 subjects, median age of 55, 52.6% male). APTB was clinically confirmed in 51 patients (70.8%), 27 in ES and 24 in SI. Among the APTB, more adequate specimens were collected from SI (41/65, 63.1%) than ES (34/80, 42.5%) (p=0.01). Bacteriological confirmation was achieved in 14 (58.3%) patients in SI, and 13 (48.1%) in ES (p=0.46). In the same-day bacteriological diagnosis with microscopy and PCR, there were positive results for 9 patients (37.5%) in SI and 7 patients (25.9%) in ES (p=0.37). ABSTRACT.CONCLUSION: Sputum induction improves sputum specimen adequacy. It may be useful for the same-day bacteriological diagnosis with microscopic examination and PCR. BODY.INTRODUCTION: The global prevalence of active Mycobacterium tuberculosis infection is unacceptably high. Despite efforts to counteract the disease, millions of new cases of active tuberculosis per year are being identified worldwide, more than one-third of which are in low-income countries1. People with active pulmonary tuberculosis (APTB) are the main community source of the mycobacteria. Rapid and accurate diagnosis and immediate appropriate chemotherapy of APTB are the key concepts of tuberculosis control, especially in high-burden regions. Microscopic examination of sputum samples for acid-fast bacilli (AFB) and their culture with subsequent drug-susceptibility testing for antituberculosis drugs is the standard diagnostic method for the diagnosis of APTB, although it has insufficient sensitivity and specificity. As a supplement to improve the rapidity and accuracy of diagnosis, nucleic acid amplification, imaging, and histopathologic examination are useful, but are rarely available in low- and middle-income countries2,3. Mycobacterial identification in respiratory specimens is confirmative for the diagnosis of APTB. The success rate of the identification depends on the quality of the collection technique, and the quality of specimens collected by patients' self-expectoration (ES) is frequently unsatisfactory. To overcome the relatively low sensitivity of the standard methods in patients in whom active disease is suspected, two or three consecutive daily sputum samples, preferably expectorated early in the morning, are requested and tested in the laboratory by AFB smear and mycobacterial culture4. Nebulization with hypertonic saline facilitates sputum expectoration even in patients who usually do not expectorate. This method has been applied in patients with cystic fibrosis to enhance mucus clearance and for identification of infectious agents, and for cytological examination in inflammatory airway disorders5,6,7. When applied to the diagnosis of APTB, sputum induction (SI) by hypertonic saline increases the diagnostic sensitivity, especially in sputumless patients. In most previous studies, SI was applied to patients who had scanty sputum at presentation or who were suspected of having APTB but had a negative AFB smear8,9,10,11. Its diagnostic sensitivity is higher than that of self-expectorated sputum, nasopharyngeal aspiration, or gastric lavage, and is equal to that of invasive techniques such as bronchoscopic lavage12,13,14. Despite the benefits reported in the literature, SI is not recommended as a supplemental standard method even in patients who spontaneously produce scanty sputum. In a real clinical practice, we prospectively applied SI with hypertonic saline on the day of the clinic visit for patients suspected of having APTB, and investigated its usefulness compared with the standard method of sputum sample collection for bacteriological confirmation of APTB. BODY.MATERIALS AND METHODS: This prospective, randomized, case-control comparative study was planned in a clinic in a regional referral hospital in a high-income, low-human immunodeficiency virus (HIV)-prevalence country, and the study protocol was approved by the Institutional Review Board of the Jeju National University Hospital in Jeju, Korea (IRB no. 2011-58). Patients over 18 years old who were clinically suspected of having APTB were enrolled from January 2012 to August 2012. Patients exposed to antituberculosis agents within the previous 3 months were excluded. Clinically suspicious cases was subjectively identified by one of 3 experienced specialists based on the patients' symptoms, physical examination, chest imaging and/or laboratory tests. With informed consent, each patient, regardless of his or her subjective amount of sputum at presentation, was randomized to either SI or ES. Patients were briefed on sputum collection techniques and their further utilization. Patients who were randomized to SI were, on the day of the clinic or emergency toom (ER) visit (SI D1), required to breathe under nebulization (PARI Company, Starnberg, Germany) with 5 mL of 7% NaCl solution for at least 5 minutes, and thereafter to produce 3 sputum samples into aseptic sputum jars for AFB smear, mycobacterial culture, and real-time polymerase chain reaction (PCR) for M. tuberculosis and nontuberculous mycobacterium (MTB & NTM kit; BioSewoom Inc., Seoul, Korea). On each of the following two days (SI D2, D3), the SI patients were asked to submit 2 sputum specimens for AFB smear and culture, which were collected early in the morning at home or on the ward. Patients randomized to ES were asked to submit 3 sputum samples for AFB smear, culture, and PCR collected early in the morning on the day after the clinic or ER visit (ES D2). Other samples were collected on day 3 (ES D3) and day 4 (ES D4) by the same method as those used for SI D2 and SI D3. Patients who for any reason did not submit more than 2 specimens were excluded from the outcome analyses (Figure 1). The adequacy of the specimens was measured according to microscopic examination. An adequate specimen was defined as sputum that contained >25 neutrophils/low-power field (LPF) and <25 squamous epithelial cells/LPF (grades 4 and 5)15. Sputum smear microscopy was done in a two-step procedure, where samples were screened by auramine-rhodamine stain and positives were confirmed by classical Ziehl-Neelsen staining. An AFB smear was defined as positive if 1+ (10 to 99 AFB per 100 LPF) or more bacilli were detected. Positive mycobacterial culture was defined as identification of either or both M. tuberculosis and nontuberculous mycobacterium in an automated liquid or solid culture system requested in the Korea Institute of Tuberculosis (http://www.kit.or.kr). PCR positive was defined as per the manufacturer's instructions. Radiological suspicion of APTB was identified by an experienced radiologist blinded to the experienced clinical specialist's opinion. Bacteriological confirmation of APTB in a patient was defined as when at least one of the AFB smear, culture, or PCR from the patient was positive. A patient who had bacteriological confirmation of APTB or who showed clinical and radiological improvement recognized by one of the specialists after having maintained antituberculosis chemotherapy for longer than 3 months was considered as "a confirmed APTB case." BODY.MATERIALS AND METHODS.1. STATISTICAL ANALYSIS: All statistical analysis was performed using PASW Statistics ver. 14 (SPSS Inc., Chicago, IL, USA). An α level less than 0.05 was considered to be statistically significant. The χ2 test was used to compare categorical variables. Continuous variables were analyzed using Student's t-test or the Mann-Whitney U test. BODY.RESULTS.1. DEMOGRAPHIC CHARACTERISTICS: Seventy six patients (median age, 53 years; interquartile range [IQR], 39-68 years; male, 57.7%) were randomized to either SI (median age, 55 years; IQR, 34-68 years; male, 52.6%) or ES (median age, 51 years; IQR, 42-67 years; male, 65.8%) (p=0.54 and p=0.24, respectively). Productive cough was reported in 18 patients (47.3%) of the SI group and 17 (44.7%) of the ES group (p=0.82). Four patients (5.3%) did not submit sputum specimens, 1 in the SI group and 3 in the ES group. No HIV-positive patient was included. Of 209 specimens collected for microscopic examination, 106 (50.7%) adequate sputum specimens were obtained, 48 (45.3%) of 106 specimens from the SI group and 58 (56.3%) of 103 specimens from the ES group (p=0.11). APTB was suspected by the radiologist in 58 patients (76.3%), 26 (68.4%) from the SI group and 32 (84.2%) from the ES group (p=0.11) (Table 1). BODY.RESULTS.2. COMPARISONS OF BACTERIOLOGICAL YIELDS IN PATIENTS WITH CONFIRMED APTB: Among the 72 patients who submitted more than 2 specimens, APTB was clinically confirmed in 51 patients (70.8%), 24 of 37 (64.7%) patients in the SI group, and 27 of 35 (77.1%) in the ES group (p=0.25). In the SI group, of 65 samples that underwent microscopic examination, 41 specimens (63.1%) were noted as adequate sputum specimens, whereas in the ES group only 34 (42.5%) of 80 specimens were adequate (p=0.01). In the SI group, 39 individual sputum specimens (25.3%) of 154 samples were positive for M. tuberculosis or nontuberculous mycobacteria, whereas in the ES group, 35 (18.6%) of 188 samples were positive (p=0.13). Among the 51 confirmed APTB cases, M. tuberculosis or nontuberculous mycobacteria was identified in one or more samples from 27 (52.9%) patients, 14 (58.3%) from the SI group and 13 (48.1%) from the ES group (p=0.46). The first samples from each group (SI D1, ES D2) included positive samples from 22 (43.1%) patients, 10 (41.7%) in the SI group and 12 (44.4%) in the ES group. Of the second samples from each group (SI D2, ES D3), 4 additional patients (7.8%) showed positive results, 3 (12.5%) in the SI group and 1 (3.7%) in the ES group. Of the third day samples, only one additional patient (2.0%) from the SI group was newly positive (Table 2, Figure 2). BODY.RESULTS.3. COMPARISONS OF THE BACTERIOLOGICAL YIELDS AMONG THE FIRST-DAY'S SPUTUM SAMPLES FROM APTB-CONFIRMED CASES: The first-day sampling methods were the only difference between the two groups (SI D1 vs. ES D2). Among the confirmed APTB cases, mycobacteria were identified in 30 first-day samples (15 samples from each group). In the first-day samples from the SI group (SI D1), 3 were microscopy positive, 6 culture positive, and 6 PCR positive (15/24, 62.5%). In the first-day samples from the ES group (ES D2), 3 were microscopy positive, 8 culture positive and 4 PCR positive (15/27, 55.6%). The AFB smear and PCR techniques give prompt bacteriological diagnosis, referred to as same-day diagnostics. In 16 patients (31.4%), M. tuberculosis or nontuberculous mycobacteria was detected in at least one of microscopy or PCR, 9 patients from the SI group and 7 from the ES group (37.5% and 25.9%, respectively; p=0.37) (Figure 3). BODY.RESULTS.4. ADVERSE EVENTS: There were no patients who did not tolerate the procedure of 5 minutes' respiratory exposure to a mist of 7% saline. As expected, mild adverse events including cough or salty taste in the mouth and the nose were noted in most patients. There were no serious adverse events including shortness of breath experienced by any of the 38 patients. SI by 7% hypertonic saline was found to be tolerable and safe. BODY.DISCUSSION: Human sputum consists mainly of water and mucin proteins that are highly glycosylated and condensed in the mucin-secreting cells. Once secreted, the physical properties of mucins, including their viscosity, are determined by the content of water and bicarbonate in the airway lumen. The water content in the airway originates from the blood flow surrounding the airway, and diffuses into the lumen through an osmotic pressure that is mainly determined by sodium and chloride ion concentration16,17. Salt delivered by nebulization increases the osmotic pressure in the airways and draws more water into the lumen. As a consequence, mucins in the airways are diluted, facilitating sputum expectoration. In addition to its dilutional effects, hypertonic saline also has a protussive effect by stimulating cough reflex, which is believed to facilitate sputum expectoration18. Although there is no standardized method noted in the literature, nebulization with 20 mL 3% saline, which takes 40-60 minutes, is most frequently used11,19,20. This time-consuming method may be unpleasant for both the patient and the observer of the procedure. Nausea, vomiting, and, rarely, bronchospasm related to this procedure have been observed20. Because the observers are exposed to the risk of airborne infection from patients, a facility with a ventilated booth or at least performance of the procedure in the open air is required. Peter et al.21 suggested that sputum collection facilitated by instructions from a health-care worker may be more feasible than SI in circumstances where facilities are limited because of low income. Nonetheless, SI by hypertonic saline is considered to be a safe, cheap, and well-tolerated procedure. If applied in clinical practice, it should result in more compliant sputum collection than collection of early morning specimens at home. Same-day diagnosis may be possible, allowing immediate initiation of treatment. In this study, nebulization with 5 mL of 7% saline for 5 minutes was applied. A higher concentration of saline can shorten the duration of the procedure and achieve a similar sputum-inducing effect. No serious adverse events were noted in our study. In a low-income, high-burden country, Atiq-ur-Rehman et al.19 compared 164 paired collections of self-expectorated sputum and sputum induced with 3% saline for 20 minutes. Although adequate specimens were successfully obtained by induction in two-thirds of sputumless patients, the study failed to demonstrate any statistical benefits of SI over ES in bacteriological confirmation of APTB19. In a recent study in which the efficacy of induced sputum collection was compared with that of health-care worker-instructed collection in sputumless patients, sputum adequacy and culture positivity were significantly higher with induction. However, the rates of same-day diagnosis by microscopic examination and molecular diagnostics were similar in the two groups21. In a systemic review of 23 studies, the overall success rates of sputum collection by saline nebulization were generally high, from 76% to 100%, with few adverse events. In only 8 studies, the results of microscopic examination were compared with those of mycobacterial culture. Higher yields in induced sputum were generally shown, but the sensitivities ranged widely from 0% to 100%12. In the present study, the usefulness of hypertonic saline SI for improving bacteriological yield was investigated exclusively in a real clinical practice. Although the results consistently supported the benefits of induced sputum for bacteriological yield, especially in the same-day samples obtained at the clinic visit, this study failed to demonstrate any significant differences, except in the rate of collection of adequate sputum specimens. This is mainly because of the size of the study, low participant numbers and because of a relatively lower rate of culture positivity in patients undergoing SI. We cannot exclude the possibility that airway exposure to high-concentration saline might influence the viability of mycobacteria in respiratory specimens. Mycobacterial culture is important for discriminating nontuberculous mycobacterial infection and for drug-susceptibility tests, so patients with nontuberculous mycobacteria were included in this study. The incidence and proportion of nontuberculous mycobacteria in respiratory samples is consistently increasing in Korea22. Needless to say, treatment strategies for nontuberculous mycobacteria are different from those for APTB, but in most cases nontuberculous mycobacterial infection can barely be discriminated clinically or radiologically from APTB. In this study, the third-day samples for AFB smear and culture provided little additional bacteriological yield, with only a 2% increase in confirmed APTB cases. Sampling on two separate days might be sufficient. In conclusion, to increase the diagnostic yield in a patient with suspected APTB, it is suggested that in a real clinical practice 1) at the clinic visit, SI with hypertonic saline is applied for same-day diagnosis with microscopic examination and PCR and 2) for the next 2 days, morning self-expectorated specimens are collected for mycobacterial culture. A follow-up larger-scale randomized trial should be performed.

TITLE: Effects of oral versus transdermal menopausal hormone treatments on self-reported sleep domains and their association with vasomotor symptoms in recently menopausal women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) ABSTRACT.ABSTRACT.OBJECTIVE:: This study determined whether two different formulations of hormone therapy (HT): oral conjugated equine estrogens (o-CEE; 0.45 mg/d, n = 209), transdermal 17β-estradiol (t-E2; 50 μg/d, n = 201) plus cyclic progesterone (Prometrium, 200 mg) or placebo (PBO, n = 243) affected sleep domains in participants of the Kronos Early Estrogen Prevention Study. ABSTRACT.ABSTRACT.METHODS:: Participants completed the Pittsburgh Sleep Quality Index at baseline and during the intervention at 6, 18, 36, and 48 months. Global sleep quality and individual sleep domain scores were compared between treatments using analysis of covariance, and correlated with vasomotor symptom (VMS) scores using Spearman correlation coefficients. ABSTRACT.ABSTRACT.RESULTS:: Global Pittsburgh Sleep Quality Index scores (mean 6.3; 24% with score >8) were similar across groups at baseline and were reduced (improved sleep quality) by both HT (average change −1.27 [o-CEE] and −1.32 [t-E2]) when compared with PBO (−0.60; P = 0.001 [o-CEE vs PBO] and P = 0.002 [t-E2 vs PBO]). Domain scores for sleep satisfaction and latency improved with both HT. The domain score for sleep disturbances improved more with t-E2 than o-CEE or PBO. Global sleep scores significantly correlated with VMS severity (rs = 0.170, P < 0.001 for hot flashes; rs = 0.177, P < 0.001 for night sweats). Change in scores for all domains except sleep latency and sleep efficiency correlated with change in severity of VMS. ABSTRACT.ABSTRACT.CONCLUSIONS:: Poor sleep quality is common in recently menopausal women. Sleep quality improved with both HT formulations. The relationship of VMS with domains of sleep suggests that assessing severity of symptoms and domains of sleep may help direct therapy to improve sleep for postmenopausal women. BODY: Chronic sleep deprivation is associated with both short and long-term health consequences including fatigue, impaired memory, and increased risk for cardiovascular disease and diabetes.1 Forty to sixty per cent of women report problems sleeping during the perimenopause and early menopause.2 Data from The North American Menopause Society posits that about 73% of postmenopausal women report vasomotor symptoms (VMS) (ie, hot flashes and night sweats). VMS may also be a risk factor for future cardiovascular disease.3,4 Hormone therapy (HT) is an effective treatment for common VMS, and also for alleviation of some sleep problems during menopause.5-7 Many studies have evaluated the relationships among HT, sleep, and VMS in perimenopausal women.8-10 It is difficult overall to disentangle age-related worsening of sleep from menopause-driven change in sleep. In one 14-year longitudinal examination of the menopausal transition, no strong relationship between menopause and worsening sleep was observed. However, a small group of "at-risk" women were identified who had poor sleep at baseline, and who reported VMS.11 This group of women was most likely to have acute worsening of their sleep as they traversed the menopause. In agreement with this body of research, a systematic appraisal of the literature suggests that only a subgroup of women with concomitant VMS may derive a modest improvement in sleep from HT.12 Therefore, questions remain regarding the association of domains of sleep (sleep disturbances compared with overall sleep quality) in relationship to VMS in menopause and HT.13,14 There are three major difficulties in assessing the relationships between VMS, HT, and sleep. First, a variety of HT formulations, doses, and routes of administration are used to treat menopausal symptoms, making comparisons among studies difficult. No studies have compared the effectiveness of two types of HT on their relationship to alleviation of VMS and domains of sleep. The second difficulty emanates from a heterogeneity in the tools used to evaluate and characterize sleep.15 Clinically, poor sleep quality is defined as perceived sleep problems that are bothersome, but do not meet criteria for a clinical sleep disorder.16,17 However, poor sleep quality may be comprised of different factors, or domains, for different individuals; therefore, a standard tool that measures sleep through multiple domains allows for optimal characterization of sleep quality15 and may provide a better insight into the associations between alleviation of VMS and sleep in menopausal women. A third difficulty is that self-reported changes in VMS may not accurately reflect their frequency, duration, and severity.18-20 The purpose of this study was to evaluate the effects of two formulations of HT on self-reported sleep quality and sleep domains using the broadly accepted Pittsburgh Sleep Quality Index (PSQI); and to assess the relationship between the changes in global sleep quality, and the domains of sleep with VMS for each HT formulation compared with placebo (PBO) in recently menopausal women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS). BODY.METHODS.PARTICIPANTS: The KEEPS (NCT00154180)—a randomized, double-blind, PBO-controlled multisite trial—enrolled women from the communities surrounding each recruitment site who were between the ages of 42 and 58 years, between 6 and 36 months since their last menses, and had serum follicle-stimulating hormone level ≥35 mIU/mL and/or estradiol level <40 pg/mL. Women were excluded if they had a coronary artery calcium score of >50 Agatston Units, history of cardiovascular disease, body mass index (BMI) >35 kg/m2, uncontrolled hypertension (systolic blood pressure >150 mm Hg and/or diastolic blood pressure >95 mm Hg), low-density lipoprotein cholesterol (LDL) >190 mg/dL, triglycerides (Tg) >400 mg/dL, fasting blood glucose >126 mg/dL (or history of diabetes), current or recent (6 months) use of cholesterol-lowering medications (statins, fibrate, or >500 mg/d niacin), if they reported smoking more than 10 cigarettes per day, or had a diagnosis of clinical depression. The study was approved by Institutional Review Boards at each participating institution. All participants were recruited and all gave written informed consent. The study design and methods have been described in detail elsewhere.21 BODY.METHODS.STUDY DESIGN: Participants (n = 727) were randomized (4:4:5 ratio) to either oral conjugated equine estrogens (o-CEE; Premarin, 0.45 mg/d) plus a PBO transdermal patch (n = 230), transdermal 17β-estradiol (t-E2; Climara 50 μg/d) plus a PBO pill (n = 222), or PBO pills and patch (n = 275). Women in the active treatment groups also received oral micronized progesterone (Prometrium, 200 mg) for the first 12 days of each month, whereas women in the PBO group received a PBO capsule for the first 12 days of each month. The randomization sequence generation was done using a random number table. Study drugs were supplied to the clinical sites identified only by the participant's ID number, with both research participants and investigators blinded to treatment. Treatment was given for 4 years. Participants at all sites were invited to complete the PSQI before randomization (baseline) and then at 6, 18, 36, and 48-month study visits, which coincided with cognitive testing (NCT00623311).22,23 Changes in the domains of sleep were not prespecified outcomes from the main KEEPS protocol, but including the PSQI questionnaire to the study visits was approved as an ancillary study to the main protocol. BODY.METHODS.OUTCOMES.PSQI FOR SLEEP PARAMETERS: The PSQI is a brief self-report, nine multi-item questionnaire, assessing seven domains of sleep quality: sleep satisfaction, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medications, and staying awake during daytime activities (daytime dysfunction).24 The PSQI has strong reliability and validity, in both clinical and nonclinical samples.25 Each of the seven domains of sleep is clinically relevant for evaluating sleep and is scored from a range of 0 to 3 points: 0 = no difficulty, 3 = severe difficulty.24 The global sleep score represents a summation of the individual sleep domain scores 0 to 21. Poor sleep quality was defined by a PSQI global score >8, which is the clinical threshold for requiring complete somnological evaluation.26 Analyses were performed on PSQI global scores and on six out of seven individual domains: sleep satisfaction, sleep latency, habitual sleep efficiency, sleep disturbances, sleep duration, and daytime dysfunction. Use of medication was not included in the present analysis, because the mean baseline score for this domain was 0.61, with 69% of the participants (71% in PBO, 67% in t-E2, and 68% in o-CEE) reporting not taking medications for sleep in the previous month. Caffeine intake was not recorded. BODY.METHODS.OUTCOMES.VASOMOTOR SYMPTOM SCALE: To assess the burden of hot flashes and night sweats over time, participants completed a questionnaire reporting symptoms of hot flashes and night sweats over the past 3 months, ranking symptoms on a 4-point Likert scale as none (0), mild (1), moderate (2), or severe (3). As reported previously,27 these symptoms were evaluated retrospectively at baseline and annually during the study period, with an average score calculated and compared with baseline for each symptom. BODY.METHODS.STATISTICAL ANALYSIS: Descriptive statistics on baseline characteristics are reported as number and percentage for categorical variables (which were dichotomized), and as mean and standard deviation (SD) for continuous variables. Baseline comparisons between randomized treatment groups were performed using Pearson's chi-square test or analysis of variance, as appropriate. The study outcomes of interest included the PSQI global score (0-21) and six preselected domain scores (0-3), and also two menopausal symptom severity scores (0-3). To describe the treatment response for each outcome treated as a continuous variable, repeated measurements were reduced into a single measure of response by computing the average score per participant during the treatment period and subtracting their baseline value. Since changes in the outcome variables were generally observed early at 6-month assessment and sustained through the study visits at 48 months, it was reasoned that the average change provides an adequate summary of the response and makes better use of available information than the change computed at a single follow-up time. To augment the description of PSQI global scores in this study, the measured continuous scale was also dichotomized at a clinically relevant threshold score of >8 to indicate poor sleep quality.28 Formal comparison of the change in response between treatments was performed via analysis of covariance (ANCOVA) using all available follow-up measurements, with use of the generalized estimating equation (GEE) method to account for correlation among the repeated measures on participants. One ANCOVA model was fit to each outcome variable, with the follow-up score included as the dependent variable, treatment group as the primary independent variable, and baseline score as the covariate. Study site was included in the models as an additional adjusting covariate. Race was not included as a covariate as the majority of participants were white by self-report and ancestry genotype.29,30 To avoid deletion of a small subset of individuals lacking a baseline PSQI measurement (n = 38), the mean baseline value of the combined sample was imputed for these individuals. Each ANCOVA model yielded a least squares mean estimate of the baseline-adjusted change within treatment group, presented as mean and 95% confidence interval (CI). Treatment group differences in these adjusted changes were assessed by testing the group term in the model for significance, using an omnibus 2 degree of freedom test; individual pair-wise comparisons were carried out only if the overall test revealed evidence of any statistically significant difference (if P ≤ 0.05). An analogous model based on the GEE formulation of logistic regression was used for the binary PSQI outcome to test whether the change in rate of poor sleep quality differed between treatment groups after adjustment for baseline rate. Secondary analyses were performed to assess the relation between changes in PSQI scores (global and individual domain scores) and changes in the menopausal symptom scores based on the partial Spearman's rank correlation coefficient (rs), which were adjusted for treatment group. The same method was used to perform a post hoc subgroup analysis to assess the correlation between changes in sleep quality and changes in menopausal symptoms in women who reported moderate to severe hot flashes and night sweats (score 2 or 3 on VMS scale) at baseline. All analyses were carried out in SAS software version 9.4 (SAS Institute Inc., Cary, NC). BODY.RESULTS: In all, 727 women were enrolled in the KEEPS, with 275 randomly assigned to PBO, 230 to oral (o-CEE), and 222 to transdermal (t-E2) treatments. Baseline clinical parameters and menopausal symptoms (Table 1), and also education status, incomes, marital status, and number of term pregnancies27,31 were similar among treatment assignments and were not different from women excluded from the main KEEPS protocol.29 For the primary analysis, the effects of treatment on aspects of sleep were assessed on 653 (90%) participants who completed the PSQI at one or more study visits during the 4-year intervention period (Fig. 1). To examine the potential for selection bias, the 653 PSQI respondents were compared with the remaining 74 KEEPS participants who did not complete the PSQI, and no significant differences were found in baseline clinical parameters (results not shown). TABLE 1 Comparisons of baseline characteristics among treatment groups for total KEEPS Variable n No. (%) missing PBO (n = 275) t-E2 (n = 222) o-CEE (n = 230) P Age, y 726 1 (<1%) 52.5 ± 2.5 52.7 ± 2.6 52.8 ± 2.6 0.394 White, by self-report 690 37 (5%) 210 (80%) 169 (82%) 176 (80%) 0.863 Weight, kg 727 0 71.1 ± 12.2 69.9 ± 12.0 69.7 ± 11.9 0.322 Height, cm 727 0 164.1 ± 6.2 163.9 ± 6.1 163.6 ± 6.3 0.748 Body mass index, kg/m 2 727 0 26.4 ± 4.3 26.0 ± 4.4 26.0 ± 4.3 0.502 Waist circumference, cm 712 15 (2%) 84.9 ± 12.0 83.8 ± 11.8 84.2 ± 11.3 0.566 Systolic blood pressure, mm Hg 727 0 119.8 ± 14.4 117.3 ± 15.8 118.9 ± 14.5 0.158 Diastolic blood pressure, mm Hg 727 0 75.3 ± 9.5 74.0 ± 9.7 75.2 ± 8.3 0.237 Total cholesterol, mg/dL 727 0 216.7 ± 30.4 215.2 ± 33.6 214.7 ± 32.1 0.759 Low-density cholesterol level, mg/dL 727 0 130.5 ± 29.3 127.8 ± 31.1 127.5 ± 27.9 0.459 High-density cholesterol level, mg/dL 727 0 63.8 ± 16.2 66.2 ± 18.5 66.4 ± 18.0 0.176 Triglyceride level, mg/dL a 727 0 92.7 ± 51.5 89.0 ± 45.6 89.3 ± 52.0 0.658 Menopausal symptoms Hot flash symptoms by history b 727 0 126 (46%) 92 (41%) 100 (43%) 0.617 Night sweats by history b 727 0 99 (36%) 72 (32%) 83 (36%) 0.643 Mood swings b 727 0 43 (16%) 38 (17%) 34 (15%) 0.789 Depression b 727 0 21 (8%) 19 (9%) 23 (10%) 0.641 Trouble sleeping b 727 0 93 (34%) 78 (35%) 66 (29%) 0.297 Irritability b 727 0 42 (15%) 42 (19%) 39 (17%) 0.559 KEEPS, the Kronos Early Estrogen Prevention Study; o-CEE, oral conjugated equine estrogen; PBO, placebo; t-E2, transdermal estradiol. a Variable was transformed to the natural logarithmic scale before performing the statistical testing so that the test assumptions were not violated. b Severity scales for menopausal symptoms were dichotomized as binary to indicate a response of either “moderate” or “severe.” FIG. 1Flow diagram of the Kronos Early Estrogen Prevention Study (KEEPS) participants included in study analysis. HF, hot flashes; NS, night sweats; o-CEE, oral conjugated equine estrogen; PBO, placebo; PSQI, Pittsburgh Sleep Quality Index; t-E2, transdermal estradiol; VMS, moderate to severe vasomotor symptoms at baseline subgroup. It should be noted that the numbers for women with hot flashes and night sweats are not mutually exclusive. BODY.RESULTS.PSQI GLOBAL SCORE: At baseline, PSQI global score did not differ significantly among treatment groups (Table 2). Overall, 24% of participants (22% PBO, 21% t-E2, and 28% o-CEE) had poor sleep quality (global scores >8) at study entry. Compared with baseline values, each of the two HT groups and the PBO group showed a reduction in average PSQI global scores over 4 years of treatment (each P < 0.001). The average reduction in the global score was similar between the two hormone-treated groups (average change of −1.27 [o-CEE] and −1.32 [t-E2] points), and both were significantly greater than that in the PBO group (−0.60 points; P = 0.001 [o-CEE vs PBO] and P = 0.002 [t-E2 vs PBO]; Table 2). Similarly, the percentage of women with poor sleep quality (PSQI global score >8) decreased with t-E2 (from 21% to 9%; P < 0.001) and o-CEE (from 28% to 16%; P < 0.001), with a smaller decline with PBO (from 22% to 17%; P = 0.06). When compared with PBO, the reduction in the percentage of women with poor sleep quality (ie, the number of women with improved sleep quality) was significantly greater in the t-E2 group (P = 0.003) and modestly but not significantly greater in the o-CEE group (P = 0.07). TABLE 2 PSQI global and individual domain scores Measurement Placebo (PBO) Transdermal (t-E2) Oral (o-CEE) Overall P PBO/t-E2 Contrasts PBO/o-CEE t-E2/o-CEE PSQI score and subscales (n = 653)  Global score   Baseline score a 6.17 ± 3.06 6.10 ± 3.12 6.57 ± 3.36   Mean follow-up score 5.69 ± 2.95 4.92 ± 2.63 5.23 ± 2.71   Mean change (95% CI) b −0.60 (−0.89, −0.31) c −1.32 (−1.64, −1.00) c −1.27 (−1.58, −0.95) c 0.001 0.002 0.001 0.975  Satisfaction/quality   Baseline score a 1.12 ± 0.66 1.17 ± 0.75 1.20 ± 0.72   Mean follow-up score 0.97 ± 0.55 0.85 ± 0.57 0.84 ± 0.55   Mean change (95% CI) b −0.19 (−0.25, −0.12) c −0.32 (−0.39, −0.25) c −0.34 (−0.41, −0.27) c 0.001 0.008 <0.001 0.449  Sleep latency   Baseline score a 1.02 ± 0.92 0.95 ± 0.88 1.06 ± 0.93   Mean follow-up score 0.97 ± 0.79 0.74 ± 0.70 0.79 ± 0.73   Mean change (95% CI) b −0.08 (−0.15, 0.00) −0.27 (−0.36, −0.19) c −0.28 (−0.36, −0.19) c <0.001 0.002 <0.001 0.822  Sleep efficiency   Baseline score a 0.46 ± 0.78 0.45 ± 0.72 0.61 ± 0.91   Mean follow-up score 0.40 ± 0.64 0.35 ± 0.47 0.39 ± 0.64   Mean change (95% CI) b −0.10 (−0.16, −0.03) c −0.15 (−0.23, −0.08) c −0.16 (−0.24, −0.09) c 0.451  Sleep disturbances   Baseline score a 1.52 ± 0.54 1.44 ± 0.61 1.52 ± 0.60   Mean follow-up score 1.43 ± 0.47 1.26 ± 0.41 1.39 ± 0.47   Mean change (95% CI) b −0.08 (−0.13, −0.03) c −0.22 (−0.27, −0.16) c −0.12 (−0.18, −0.07) c 0.001 <0.001 0.166 0.029  Sleep duration   Baseline score a 0.54 ± 0.68 0.51 ± 0.70 0.57 ± 0.77   Mean follow-up score 0.47 ± 0.64 0.43 ± 0.60 0.41 ± 0.58   Mean change (95% CI) b −0.08 (−0.14, −0.01) c −0.10 (−0.18, −0.03) c −0.15 (−0.22, −0.07) c 0.377  Daytime dysfunction   Baseline score a 0.91 ± 0.75 0.89 ± 0.70 0.84 ± 0.68   Mean follow-up score 0.79 ± 0.59 0.71 ± 0.58 0.73 ± 0.55   Mean change (95% CI) b −0.11 (−0.17, −0.05) c −0.19 (−0.26, −0.12) c −0.14 (−0.21, −0.08) c 0.258 Vasomotor symptom scores (n = 662)  Hot flashes severity   Baseline score a 1.39 ± 0.79 1.38 ± 0.82 1.37 ± 0.88   Mean follow-up score 0.89 ± 0.68 0.44 ± 0.60 0.39 ± 0.46   Mean change (95% CI) b −0.48 (−0.55, −0.42) c −0.93 (−1.00, −0.85) c −0.98 (−1.05, −0.90) c <0.001 <0.001 <0.001 0.343  Night sweats severity   Baseline score a 1.08 ± 0.92 1.12 ± 0.95 1.08 ± 0.98   Mean follow-up score 0.62 ± 0.67 0.33 ± 0.54 0.33 ± 0.46   Mean change (95% CI) b −0.47 (−0.54, −0.41) c −0.76 (−0.83, −0.69) c −0.76 (−0.83, −0.69) c <0.001 <0.001 <0.001 0.919 PSQI results are reported on a total of 653 respondents (n = 243 [PBO], 201 [t-E2], 209 [o-CEE]), whereas vasomotor symptom results are presented on 662 respondents (n = 243 [PBO], 205 [t-E2], 214 [o-CEE]); to describe the treatment responses, repeated measurements were summarized by the average score per woman during the treatment period, whereas comparisons between treatments utilized all serial data points available via repeated measures analysis (see “Methods” section for details). Treatment groups were tested for a difference in response in ANCOVA models that included site and baseline score as covariates, with the GEE method used to account for within-participant correlation from repeated measurements. From each ANCOVA, the three-level treatment effect was screened for significance and only when such evidence was revealed did we proceed with post hoc testing of pair-wise group differences. CI, confidence interval; o-CEE, oral conjugated equine estrogen; PBO, placebo; PSQI, Pittsburgh Sleep Quality Index; t-E2, transdermal estradiol. a There were no significant differences in baseline scores between treatments ( P  > 0.05 from analysis of variance [ANOVA]). To avoid case-wise deletion in analyses of treatment responses, the mean baseline estimate of each PSQI scale was imputed on n = 38 women who lacked a baseline measurement. b Baseline-adjusted mean change over follow-up assessments was estimated via analysis of covariance [ANCOVA] modeling, and is reported as mean and 95% confidence interval. c Indicates that the baseline-adjusted change within corresponding group was statistically significant ( P  < 0.05). BODY.RESULTS.PSQI INDIVIDUAL DOMAINS SCORE: Compared with baseline scores, significant improvements in all six domain scores were observed during treatment within each group, with the exception of sleep latency in the PBO group (P = 0.06; Table 2). The average improvement in score differed between treatments for three out of the six domains (sleep satisfaction, sleep latency, and sleep disturbances), with each of these three domains showing significantly greater improvement in one or both of the HT groups when compared with PBO (Fig. 2). Among the three domains, only sleep disturbances showed a statistically significant difference between the two HT formulations (P = 0.029), with t-E2 improving more during follow-up than o-CEE (Table 2). There were no significant differences between groups for the changes in domain scores pertaining to sleep efficiency (P = 0.45), sleep duration (P = 0.38), or daytime dysfunction (P = 0.26) across follow-up. FIG. 2Longitudinal analysis for overall Pittsburgh Sleep Quality Index (PSQI) score and three domains of sleep. Line graphs show mean and 95% confidence intervals at each study visit to illustrate trends by treatment for the pooled set of participants These values are based on a total of 653 respondents (n = 243 [PBO], 201 [t-E2], and 209 [o-CEE]) with at least one survey during study follow-up, of whom 85% (86% [PBO], 87% [t-E2], and 81% [o-CEE]) contributed a score at the end-of-study 48-month visit. These graphs demonstrated significantly greater improvement (decrease in scores) in each of the two hormone-treated groups relative to those treated with PBO for global PSQI score, domains for sleep latency and sleep satisfaction. For the domain of sleep disturbance, only t-E2 showed a significantly greater improvement (decrease in score) than PBO, and was also the only scale to show a significant difference between the hormone treatment groups (greater improvement in those randomized to t-E2 than to o-CEE). o-CEE, oral conjugated equine estrogen; PBO, placebo; t-E2, transdermal estradiol. BODY.RESULTS.ASSOCIATION WITH VMS: A total of 662 women providing responses of VMS severity during the course of treatment were retained in the corresponding treatment comparisons. As reported previously for the entire KEEPS cohort,27 the average severity scores of hot flashes and night sweats were significantly reduced by both HT formulations when compared with PBO (P < 0.001 for both), with no difference between the o-CEE and t-E2 groups (P = 0.343 and P = 0.919, respectively; Table 2). For the 646 participants for whom both PSQI and VMS data were available, there were positive associations between the average change in global PSQI score and the average changes in severity of hot flashes (rs = 0.170, P < 0.001) and night sweats (rs = 0.177, P < 0.001; Table 3). Among the correlations assessed between changes in the individual sleep domain scores and changes in scores for hot flashes, all domains except sleep latency and sleep efficiency correlated positively with change in severity. In addition to the domains of sleep latency and sleep efficiency, the domain of sleep duration also did not correlate with changes in night sweats. TABLE 3 Correlation ( r s ) of average change in PSQI scores with average change in VMS scores Overall analysis Subgroup analysis women with moderate/severe symptoms at baseline PSQI measure Hot flashes (n = 646) Night sweats (n = 646) Hot flashes (n = 279) Night sweats (n = 226) Global score 0.170 ( P  < 0.001) 0.177 ( P  < 0.001) 0.181 ( P  = 0.002) 0.207 ( P  = 0.002) Satisfaction/quality 0.183 ( P  < 0.001) 0.222 ( P  < 0.001) 0.179 ( P  = 0.003) 0.233 ( P  < 0.001) Sleep latency 0.072 ( P  = 0.069) 0.061 ( P  = 0.125) 0.137 ( P  = 0.022) 0.147 ( P  = 0.028) Sleep efficiency 0.068 ( P  = 0.086) 0.053 ( P  = 0.182) 0.085 ( P  = 0.160) 0.073 ( P  = 0.277) Sleep disturbances 0.135 ( P  < 0.001) 0.148 ( P  < 0.001) 0.091 ( P  = 0.129) 0.118 ( P  = 0.078) Daytime dysfunction 0.079 ( P  = 0.044) 0.108 ( P  = 0.006) 0.039 ( P  = 0.518) 0.075 ( P  = 0.263) Sleep duration 0.105 ( P  = 0.008) 0.067 ( P  = 0.089) 0.239 ( P  < 0.001) 0.169 ( P  = 0.011) PSQI, Pittsburgh Sleep Quality Index; VMS, vasomotor symptom. In multivariable analysis to examine the effect of treatment on change in sleep quality while controlling for changes in VMS, the difference in average improvement in PSQI global score between treatments, though attenuated, remained significant after adjustment for average improvement in each symptom (P = 0.020 adjusting for changes in hot flashes; P = 0.004 adjusting for changes in night sweats). Controlling for the effects of treatment, the associations between the change in each VMS and change in sleep remained significant as well (P = 0.002 for hot flashes and P = 0.029 for night sweats). BODY.RESULTS.POST HOC VMS SUBGROUP ANALYSIS: At baseline, 226 women reported having moderate to severe night sweats, and 279 women (some overlap with those having moderate to severe night sweats) reported having moderate to severe hot flashes. In these respective subgroups of women, there were significant and positive correlations of changes in the global PSQI score, with changes in severity of night sweats (rs = 0.181, P = 0.002) and with changes in severity of hot flashes (rs = 0.207, P = 0.002; Table 3). In contrast to the results obtained on the overall set of participants, improvement in sleep latency in these women with moderate/severe symptoms correlated significantly with reduced severity of hot flashes (rs = 0.137, P = 0.022) and night sweats (rs = 0.147, P = 0.028); in addition, sleep duration correlated more strongly with reduced severity of hot flashes (rs = 0.239, P < 0.001) and night sweats (rs = 0.169, P = 0.011). In models adjusting for HT, the association between average changes in VMS and the average change in PSQI global score remained significant in both VMS subgroups (both P < 0.001). In contrast, the association of treatment with average change in PSQI global score was attenuated and no longer significant in both subgroups after adjustment for the average change in the corresponding VMS (P = 0.525 and 0.128 from partial tests of treatment effects in the subgroup with baseline hot flashes and with baseline night flashes, respectively). BODY.DISCUSSION: In a population of a majority of white recently menopausal women, improvements in sleep quality were observed with the use of low-dose HT (oral and transdermal) over a 4-year period. The global sleep score indicative of sleep quality in women at baseline is consistent with what has been reported for population-based studies.2 The average change in the global sleep score during treatment was a reduction of about 1.3 points in both groups randomized to HT. The magnitude of change in sleep score with HT was about twice as great as that reported by the PBO group (0.06), which is consistent with effects of HT on sleep reported in other studies.12 A second finding of the present study is that changes in sleep quality correlated with changes in VMS (hot flashes and night sweats)—a finding that persisted after controlling for treatment assignment to HT and consistent with other studies.10 However, a multivariable analysis on the overall set of women demonstrated significant partial effects of both treatment and change in VMS, indicating the alleviation of VMS does not fully account for the improved sleep outcomes among those assigned to HT and suggesting that HT affects other mechanisms associated with sleep. A causal relationship between these factors is hard to establish, as there is a bidirectional relationship of perceived sleep quality and VMS, in that poor sleep quality is both a consequence of VMS and also an influence on the extent to which VMS are perceived as bothersome. Some insight into why HT may affect sleep through mechanisms other than alleviation of VMS is provided by the subanalysis of the relationship between symptom relief and sleep domains in women reporting moderate/severe VMS at baseline. In these women, unlike the entire set of women, the association between the change in symptom severity and the change in PSQI score was attenuated and no longer significant after adjustment for treatment. These results suggest that in women with moderate to severe symptoms, but not in those with none to mild, the effects of HT on sleep are mediated through symptom relief—a finding consistent with conclusions of the recent meta-analysis of other studies of sleep, VMS, and HT.12 A third finding of the present study is the direct comparison between two formulations and doses of HT, which are commonly used in clinical practice, on sleep domains. The majority of previous sleep studies evaluated o-CEE at 0.625 mg/d.12 In the present study, following clinical guidelines that followed the cessation of the Women's Health Initiative in 2002 for use of lower doses of HT,32 0.425 mg/d of o-CEE was used in KEEPS when it was designed in 2004. Although overall sleep quality was improved with HT, not all domains of sleep showed significant change averaged across the treatment period. The finding that t-E2 was more efficacious than o-CEE in alleviating sleep disturbances may be related to the pharmacokinetics of these two formulations. Transdermal E2 is likely to provide more consistent 24-hour estradiol dosing, whereas o-CEE may engender daytime peaks and night-time troughs if women took their o-CEE in the morning, thus, leading to less relief of sleep disturbances. In the entire group, sleep efficiency and sleep duration were not affected by either HT, reflecting, perhaps, that other factors such as life circumstances may impact these domains, especially in women who do not report moderate to severe symptoms. Unlike what has been reported in other studies,12 daytime dysfunction was not improved by HT in the KEEPS cohort. This difference may be explained, in part, by a lower prevalence of women with poor sleep quality (global score >8) in the present study sample (24% at baseline) compared with other studies.33,34 In addition, the majority of participants in the KEEPS were white and recently menopausal as confirmed by strict criteria, whereas in other studies, women were more often of mixed ethnicity and were perimenopausal and also postmenopausal. The possibility that doses or formulations of HT directly influence specific domains of sleep apart from changes in VMS in women of different ethnicities and ages will require additional study. There are difficulties in comparing self-reported sleep outcomes among clinical trials due to heterogeneity among the available questionnaires and the absence of objective testing.12 For example, self-reported sleep tools may include validated and nonvalidated scales, single-item or multiple-items measures, visual analog scales, and diaries such that clinical applicability of results in various reports to clinical management is uncertain.35 Yet, the use of sleep architecture measurements (polysomnography, wrist actigraphy) does not always correlate with perceived sleep quality,36,37 and due to costs and accessibility, these tests have limited applicability in large population settings. Therefore, self-report instruments remain crucial in the clinical assessment of outcomes after interventions to improve sleep. In studies of menopause, self-report instruments may allow clinicians to evaluate who might benefit from HT for sleep and who might benefit from additional clinical assessment, such as testing for sleep apnea. Although the PSQI contains questions regarding symptoms of snoring and stopping breathing during the night, the responses to these questions are included in an overall score for sleep disturbances and may not provide accurate information regarding sleep apnea for those individuals who sleep alone. However, the answers to these questions may provide information for the individual physician who examines the questionnaire for their individual patient. This present study has a number of strengths. Evaluating the recently menopausal women enrolled in KEEPS allowed many of the deficiencies of the prior literature to be addressed. First, KEEPS was a randomized clinical trial enrolling a large number of well-characterized and otherwise healthy, recently menopausal women meeting the standard and stringent clinical and biochemical criteria for menopause. In addition, the design allowed for a direct comparison between the two modalities of menopausal HT commonly used in current clinical practice—o-CEE and t-E2. The differences in pharmacokinetics and dynamics between the products may help to direct clinical decisions related to which formulation best meets the women's needs. A second strength of the study is the use of a validated tool for self-reporting global sleep quality and domains of sleep. The PQSI provided important qualitative information on the domains of sleep that cannot be obtained by laboratory analysis with important implications for women whose major sleep complaints relate to their ability to fall asleep, sleep disturbances, and overall sleep satisfaction. Because data regarding VMS was by self-report, responses of symptom severity are susceptible to misclassification bias and may under or over-represent the symptoms. However, the associations between sleep and symptom severity are, to our knowledge, the first reported with use of low doses and two different routes of two different estrogen products in the same study. A limitation of the present study is that other stressors related to sleep quality such as marital, employment and socioeconomic status, allergies, caffeine intake, and numbers of children in the home were not considered. Although the study could be criticized for eliminating the domain of sleep medication, the use of such medications was low at baseline, limiting statistical power, and their use is not well-established with health outcomes.15 Additionally, no clinical assessments were obtained regarding other potential confounders of sleep such as emotional stress, obstructive sleep apnea, or restless leg syndrome. As with other studies, it is important not to generalize the results of this study to other groups not defined by the inclusion and exclusion criteria. BODY.CONCLUSIONS: In recently menopausal women, the overall sleep quality was improved by both HT regimens compared with PBO, with the transdermal estrogen formulation performing modestly better than the oral formulation. Of the studied sleep domains, sleep satisfaction, disturbances, and duration were improved, with sleep disturbances more improved by t-E2 than by o-CEE. Domains of sleep latency, efficiency, and daytime dysfunction were not affected by the HT regimes used in this study. Alleviation of VMS was associated with improvements in overall sleep quality. This result in not an unexpected outcome as it is common to use amelioration of VMS as a clinical guide to treatment. In a subset of women reporting moderate to severe VSM at baseline, reduced symptoms also correlated with improvement in the domain of sleep latency. These findings suggest that at least one way to approach the use of HT for sleep complaints is to assess the severity of VMS, and perhaps explore additional underlying problems affecting sleep, for example, obstructive sleep apnea. Sleep disorders in midlife women warrant evaluation because treatment can lead to substantial improvements in quality of life and health outcomes.1,17 These results from a sufficiently powered, randomized clinical trial suggest that fostering a conversation about sleep quality, and sleep domains during clinical encounters may be a more appropriate guide for a patient-centered approach for achieving optimal sleep health.

TITLE: Everolimus for treatment of tuberous sclerosis complex‐associated neuropsychiatric disorders ABSTRACT.ABSTRACT.OBJECTIVE: To evaluate if short‐term treatment with everolimus was safe and could improve neurocognition and behavior in children with TSC. ABSTRACT.ABSTRACT.METHODS: This was a prospective, double‐blind randomized, placebo‐controlled two‐center phase II study. Participants diagnosed with TSC and age 6–21 years were treated with 4.5 mg/m2 per day of oral everolimus (n = 32) or matching placebo (n = 15) taken once daily for 6 months. For efficacy, a comprehensive neurocognitive and behavioral evaluation battery was performed at baseline, 3 months, and 6 months. For safety, adverse events recorded continuously via patient diary were categorized and graded per NCI Common Toxicity Criteria for Adverse Events, version 3.0 (CTCAE 3.0). Analyses were performed on the intention‐to‐treat population (n = 47). ABSTRACT.ABSTRACT.RESULTS: Nearly all assessment measures failed to demonstrate significant differences between the two groups at the end of 6 months. Only one measure each of executive function (Cambridge Neuropsychological Test Automated Battery Stockings of Cambridge) favoring placebo (P = 0.025) and social cognition (Social Responsiveness Scale Social Cognition Subscale) favoring everolimus (P = 0.011) was observed. A total of 473 adverse events (AE) were reported. The average number of total AE per subject was similar for both placebo and everolimus. Most were mild or moderate in severity and serious AE were rare. ABSTRACT.ABSTRACT.INTERPRETATION: While safe, oral everolimus administered once daily for 6 months did not significantly improve neurocognitive functioning or behavior in children with TSC. BODY.INTRODUCTION: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs due to mutations in either TSC1 or TSC2 leading to hyper‐activation of the mechanistic target of rapamycin (mTOR) pathway.1, 2 TSC affects all organ systems, but involvement of the central nervous system presents early and is associated with significant morbidity including subependymal giant cell astrocytoma (SEGA), epilepsy, and TSC‐associated neuropsychiatric disorders (TAND). TAND spectrum is broad consisting of cognitive, behavioral and psychiatric conditions such as autism, intellectual disability, mood disorders, and specific neuropsychological deficits.3, 4, 5 These neuropsychiatric disorders result in the greatest burden of care and treatment in TSC.5, 6 In recent years, mTOR inhibitors have been approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for treatment of SEGA, renal angiomyolipomas, and lymphangioleiomyomatosis (LAM). Most recently, everolimus has been shown effective as adjunctive treatment of focal seizures in TSC.7, 8 Studies have shown that over‐activation of the mTOR pathway in neurons result in aberrant axonal and dendritic connectivity, enlarged cell size, increased cellular stress, reduced myelination and synaptic dysfunction.9, 10, 11, 12, 13 In animal models, mTOR inhibitors such as sirolimus (rapamycin) and everolimus improved synaptic function, myelination, and behavioral deficits including learning, memory, and autistic‐like features.14, 15, 16, 17, 18, 19 For example, just 5 days of sirolimus treatment improved long‐term potentiation and hippocampal learning in a TSC2+/− mouse model.19 A conditional knockout of Tsc1 in cerebellar Purkinje cells alone led to social interaction and reversal learning deficits, which also improved with sirolimus treatment.15 However, concerns have also arisen about the potential for mTOR inhibitors to interfere with competing processes that are essential to normal human growth and development in younger individuals, despite the observed neurocognitive gains in preclinical models.20, 21, 22, 23 An early‐phase clinical trial of sirolimus in individuals with angiomyolipomas associated with TSC and/or sporadic LAM examined memory and executive skills in adults with TSC.24 Immediate recall memory and executive function improved with treatment, whereas other neuropsychological measures showed reduction in some participants. No control group was included and to date, no placebo‐controlled human clinical trials have directly and prospectively evaluated neurocognitive effects of mTOR inhibitors. Here, we asked firstly, whether the mTOR inhibitor everolimus would be safe in children with TSC in terms of neurocognition, and secondly, whether we could identify any specific components of TAND that showed improvement on everolimus. We selected neurocognitive measures that were quantifiable, using standardized instruments, including computer‐based testing. We were deliberately signal‐seeking and therefore included measures across a broad range of TAND domains that in prior preclinical15, 19 and clinical studies7, 24 of TSC have suggested treatment with mTOR inhibitors to be beneficial. BODY.PATIENTS AND METHODS.PARTICIPANTS: Participants were enrolled at Boston Children's Hospital (BCH) and Cincinnati Children's Hospital Medical Center (CCHMC) between 2011 and 2014. Participants had to have an established diagnosis of TSC,25 and be aged 6–21 years, and medically stable. Previous treatment with an mTOR inhibitor was not allowed, and no changes in antiepileptic medications except dose adjustments within the previous 6 months of enrollment were permitted. To complete as much of the assessment battery as possible and avoid assessment limitations of previous studies,26 in addition to minimum of being 6 years of age, participants were required to have English as their primary language and a baseline Verbal, Performance or Overall IQ ≥ 60. The study protocol was approved by the Internal Review Boards at both institutions and listed on clinicaltrials.gov (NCT01289912). Individual informed consent/assent was obtained before enrollment. BODY.PATIENTS AND METHODS.STUDY DESIGN AND RANDOMIZATION: This was a prospective, double‐blind randomized, placebo‐controlled two‐center phase II study. Participants were treated with 4.5 mg/m2 per day of oral everolimus or matching placebo taken once daily for 6 months. Randomization was 2:1 everolimus versus matching placebo, using the SciRan (Scientific Randomization) program developed at BCH. Randomization was stratified by age and IQ with two levels for each factor. All study staff were blinded to randomization assignment, except for one physician at BCH to whom treatment assignment and serum trough levels were available to make protocol‐defined dose‐adjustment recommendations aimed at achieving serum trough levels between 5 and 15 mcg/mL. The unblinded physician otherwise had no direct access to participants, clinical data, or assessment results. BODY.PATIENTS AND METHODS.STUDY PROCEDURES AND OUTCOME MEASURES: Nine study visits occurred during the 6‐month period starting with screening and baseline visit. Neuropsychological testing took place at baseline, 3 months and 6 months. An additional telephone follow‐up was scheduled 28 days after the last dose to assess for unresolved or new adverse events (AEs). A Data and Safety Monitoring Board (DSMB) monitored the safety of participants. For safety, adverse events were categorized and graded per National Cancer Institute Common Toxicity Criteria for Adverse Events (CTCAE), version 3.0. Relationship to study drug, duration, and clinician action taken also were captured. For impact on neurocognition including safety and efficacy, TAND domains were measured by well‐validated, standardized, assessment tools that used direct testing and parent/caregiver report rating scales. Global intellectual ability was assessed using the Wechsler Abbreviated Scale of Intelligence (WASI);27 language using the Expressive Vocabulary Test 2 (EVT2)28 and Peabody Picture Vocabulary Test 4 (PPVT4);29 learning and memory using Cambridge Neuropsychological Test Automated Battery (CANTAB)30 and Wide Range Assessment of Memory and Learning 2 (WRAML2);31 attention using CANTAB and WRAML2; executive function using CANTAB; and processing speed using CANTAB and grooved pegboard.32 Parent rating scales included the Vineland Adaptive Behavioral Scales‐II (VABS2);33 Behavior Rating Inventory of Executive Function (BRIEF),34 Behavior Assessment System for Children 2 (BASC2),35 Strengths and Difficulties Questionnaire (SDQ),36 and Social Responsiveness Scale (SRS).37 Academic skills were measured by Reading and Math subtests of the Wide Range Achievement Test‐4 (WRAT4).38 To ensure intersite reliability on neuropsychological and other psychometric assessments, a baseline face‐to‐face meeting was held with all study neuropsychologists, psychometrists, and neuropsychiatrist, and followed up with regular teleconferences throughout the trial. BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: The study was designed to detect an effect size of at least 0.87SD with 80% power while maintaining Type I error rate of 5% using 2:1 allocation, comparing everolimus in individuals with TSC to placebo. The treatment effect size of 0.87SD was based on observed changes in multiple TAND‐associated neurocognitive and behavioral domains following everolimus treatment in a similarly aged pediatric TSC population with epilepsy.7 Assuming a dropout rate of 10%, the target enrollment was 55 patients. Actual enrollment was less (52 subjects) at time of study conclusion. Multiple reasons that were not mutually exclusive account for the smaller population size, including prior or current treatment with mTOR inhibitors as part of previous or ongoing clinical trials targeting epilepsy and SEGA in TSC,7, 8, 39, 40 FDA approval of everolimus to treat SEGA in 2010 made mTOR inhibitors commercially available to TSC patients, and multiple potential enrollees failed to meet minimum IQ inclusion criteria. Demographic and baseline characteristics of the randomized study groups were compared, using chi‐square tests for categorical variables and t‐tests for continuous variables. Outcome measures were analyzed using a linear mixed‐effects model that evaluated between‐subject variability and within‐subject correlations. The slope parameter corresponding to interaction of group indicator and time summarized the difference in rate of change per 3‐month interval. For multiple comparisons, we used the Benjamini–Hochberg false discovery rate procedure.41 Missing observations were assumed obsolete and missing at random,42, 43, 44 as maximum likelihood estimation for the linear mixed‐effects model provides unbiased estimates. Final model selection was performed using Akaike Information criterion (AIC). Variance covariance parameters of random‐effects were chosen using the Likelihood ratio tests (LRTs) in the nested models. Group comparisons were examined using age (≤10,>10 years) and IQ (≤80, >80) stratification cut‐offs to determine an equal number of sample sizes in placebo group within each stratum. Analyses were conducted using SAS Version 9.4 (SAS Institute, Cary, NC, USA). BODY.RESULTS.BASELINE CHARACTERISTICS: Of the 52 participants enrolled (Fig. 1), 47 were randomized to receive everolimus (n = 32) or placebo (n = 15). Three participants did not meet baseline IQ inclusion criteria, one was unable to complete baseline neurocognitive testing, and one was newly diagnosed with SEGA and started clinically indicated treatment with everolimus. Forty‐two of the 47 randomized (89%) completed the study. Demographic and baseline TSC characteristics differed only in hypomelanotic macules (Table 1). Detailed neurological and physical examinations identified two subjects in the placebo group with reported abnormal genitourinary examinations. Comprehensive laboratory assessment found a few minor statistically significant, but clinically insignificant, differences in hematocrit (∆2.1%, P = 0.03), eosinophil count (∆ 1.8%, P = 0.04), and basophil count (∆ 0.3%, P = 0.01). Figure 1Patient flow diagram. Table 1 Baseline patient demographics and clinical characteristics   PLACEBO ( n  = 15) EVEROLIMUS ( n  = 32) TOTAL ( n  = 47) P ‐value Age, years (SD) 11.47 (5.30) 13.25 (5.06) 12.68 (5.15) 0.27 Gender 0.83 Male 8 (53.3%) 16 (50.0%) 24 (51.1%) Female 7 (46.7%) 16 (50.0%) 23 (48.9%) Race 0.34 White 12 (80.0%) 26 (83.9%) 38 (82.6%) Black/African American 0 (0.0%) 3 (9.7%) 3 (6.5%) Asian 2 (13.3%) 1 (3.2%) 3 (6.5%) Other 1 (6.7%) 1 (3.2%) 2 (4.3%) Clinical Features Autism Spectrum Disorder (ASD) 3 (20.0%) 13 (40.6%) 16 (34.0%) 0.50 Epilepsy 10 (66.7%) 25 (78.1%) 35 (74.5%) 0.40 Subependymal Nodules (SEN) 14 (93.3%) 31 (100%) 45 (97.8%) 0.15 Subependymal Giant Cell Astrocytoma (SEGA) 3 (20.0%) 5 (16.1%) 8 (17.4%) 0.75 Hypomelanotic Macules a 13 (86.7%) 32 (100%) 45 (95.7%) 0.04 Shagreen Patch 4 (26.7%) 17 (53.1%) 21 (44.7%) 0.09 Facial Angiofibromas 10 (66.7%) 26 (83.1%) 36 (76.6%) 0.27 Retinal Nodular Hamartomas 2 (13.3%) 10 (31.3%) 12 (25.5%) 0.19 Renal Angiomyolipoma 8 (53.3%) 16 (51.6%) 24 (52.2%) 0.91 Vitals Weight, kg (SD) 39.01 (24.38) 48.62 (25.68) 45.55 (25.41) 0.23 Height, cm (SD) 138.21 (21.12) 146.74 (19.73) 144.01 (20.35) 0.18 Systolic BP, mmHg (SD) 107.73 (8.11) 110.69 (10.97) 109.74 (10.15) 0.36 Diastolic BP. mmHg (SD) 63.93 (11.08) 64.34 (8.50) 64.21 (9.28) 0.89 Body Surface Area, m^2 (SD) 1.19 (0.44) 1.37 (0.42) 1.32 (0.43) 0.19 Continuous variables rows include sample mean (standard deviation), P ‐value is based on 2‐sample t ‐test. Categorical variables include frequency (percentage across categories), P ‐value is based on chi‐square test. a Indicates significant difference ( P  < 0.05) between placebo and everolimus groups at baseline. John Wiley & Sons, Ltd Epilepsy is common in TSC and closely associated with TAND outcomes.45, 46 As expected, a history of epilepsy was common for both groups at baseline (Table 1). The decision to exclude participants with baseline IQ <60 enabled comprehensive testing and resulted in fewer with uncontrolled epilepsy and breakthrough seizures. Seventy‐nine percent reported no seizures and only 10% (n = 5; 1 placebo, 4 everolimus) reported > 2 seizures over the 6 month treatment period. No participant developed new seizures during the study. Global intellectual ability (IQ) was assessed at baseline only, as part of the screening process to ensure subject eligibility. Full Scale IQ showed a wide range (55–122) and without detectable difference between placebo and treatment groups (P = 0.80) (Table 2). Most participants were in the Low Average range (average: 80.1, IQR: 67.3–90.3). No significant baseline differences were identified in language, learning and memory, attention and executive function, socialization and behavior, motor skills, or academic skills. Table 2 Neurocognitive and behavioral assessment results at baseline and after treatment with everolimus for 6 months   Baseline 6 months P ‐value Placebo ( n  = 15) Everolimus ( n  = 32) Placebo ( n  = 13) Everolimus ( n  = 29) Intellectual function Full scale IQ (WASI) 80.93 (11.35) 76.69 (17.09) – – n.a. Verbal IQ (WASI) 80.80 (11.07) 83.13 (16.52) – – n.a. Performance IQ (WASI) 84.47 (12.64) 79.56 (16.98) – – n.a. Language Receptive (PPTV4) 86.00 (17.78) 81.06 (23.96) 88.54 (17.21) 82.50 (22.32) 0.609 Expressive (EVT2) 85.47 (12.22) 90.00 (21.15) 89.15 (16.08) 82.55 (21.22) 0.870 Learning and memory Verbal Learning (WRAML2) 8.00 (2.00) 7.14 (3.16) 8.46 (2.44) 7.85 (2.92) 0.990 Verbal Recall (WRAML2) 7.07 (2.94) 7.52 (2.53) 8.23 (3.49) 8.08 (2.77) 0.674 Pattern Recognition Memory (CANTAB) −1.48 (1.90) −1.23 (2.11) −0.91 (1.60) −1.09 (1.79) 0.901 Spatial Recognition Memory (CANTAB) −2.33 (1.40) −2.24 (1.33) −2.62 (1.58) −2.19 (1.56) 0.402 Spatial Span (CANTAB) −1.32 (1.18) −1.02 (1.20) −1.29 (1.41) −1.32 (1.37) 0.585 Spatial Working Memory (CANTAB) −0.66 (0.79) −0.67 (1.57) −0.73 (0.97) −0.27 (2.87) 0.582 Attention and executive function Reaction Time (CANTAB) −2.24 (3.63) −1.15 (2.22) −0.88 (1.38) −2.91 (3.84) 0.385 Rapid Visual Processing (CANTAB) −0.96 (2.59) −1.41 (2.80) −0.97 (2.71) −1.20 (2.15) 0.963 Stockings of Cambridge (CANTAB) a −1.38 (1.32) −1.89 (1.47) −0.58 (1.11) −1.84 (1.32) 0.025 Intraextra Dimensional Set Shift (CANTAB) −1.02 (1.65) −1.48 (2.05) −1.05 (1.64) −2.24 (3.32) 0.951 Global Executive Composite (BRIEF) 70.50 (15.13) 69.48 (12.98) 62.25 (12.53) 64.68 (10.88) 0.370 Behavioral Regulation Index (BRIEF) 70.93 (16.89) 66.53 (14.50) 60.83 (12.41) 62.57 (12.00) 0.180 Metacognition Index (BRIEF) 68.07 (14.52) 68.93 (12.12) 61.83 (12.97) 64.57 (10.65) 0.508 Socialization and behavior Total Social Responsiveness (SRS) 77.14 (17.50) 76.43 (18.62) 80.62 (22.83) 75.69 (19.27) 0.160 Social Awareness (SRS) 67.40 (14.69) 62.90 (15.32) 67.38 (15.51) 64.86 (17.19) 0.773 Social Cognition (SRS) a 76.43 (19.59) 75.70 (18.77) 81.15 (18.86) 76.17 (18.98) 0.011 Social Communication (SRS) 74.36 (16.75) 72.23 (17.18) 76.85 (21.55) 71.69 (17.98) 0.106 Social Motivation (SRS) 69.80 (16.60) 69.60 (14.76) 72.77 (20.77) 67.24 (16.37) 0.386 Autism Mannerisms (SRS) 76.71 (17.32) 80.03 (20.24) 79.77 (24.50) 78.66 (20.16) 0.812 Externalizing Problems (BASC2) 57.36 (15.58) 53.24 (9.85) 52.62 (9.26) 52.34 (8.59) 0.920 Internalizing Problems (BASC2) 58.43 (18.34) 51.55 (12.09) 56.00 (19.37) 51.45 (14.42) 0.569 Behavioral Symptoms Index (BASC2) 62.29 (12.61) 58.97 (10.87) 57.54 (10.02) 55.41 (11.26) 0.836 Adaptive Skills (BASC2) 38.79 (13.16) 36.72 (10.22) 42.08 (13.27) 40.00 (12.02) 0.744 Adaptive Behavior Composite (VABS2) 78.79 (19.69) 72.06 (13.90) 79.00 (15.26) 74.72 (14.57) 0.685 Communication (VABS2) 81.27 (21.87) 73.68 (15.45) 81.77 (16.66) 76.79 (14.76) 0.930 Socialization (VABS2) 79.07 (19.67) 74.10 (15.46) 80.38 (17.78) 76.86 (16.40) 0.931 Daily Living Skills (VABS2) 78.80 (19.50) 74.68 (15.69) 81.69 (14.74) 76.41 (17.37) 0.572 Motor Skills (VABS2) 89.87 (18.26) 78.83 (21.68) 92.69 (18.65) 87.79 (18.12) 0.899 Total Difficulties Scale (SDQ) 16.00 (6.93) 15.46 (5.61) 14.42 (5.09) 13.20 (5.90) 0.336 Motor skills Dominant Hand Speed (Pegboard) 101.33 (54.27) 103.06 (41.18) 89.83 (24.99) 97.25 (39.02) 0.284 Nondominant Hand Speed (Pegboard) 105.27 (37.10) 119.30 (44.19) 92.15 (34.26) 112.78 (50.89) 0.203 Academic skills Word Reading (WRAT4) 86.80 (16.79) 84.28 (20.07) 91.15 (20.76) 84.45 (20.45) 0.317 Math Computation (WRAT4) 76.00 (15.64) 74.60 (17.99) 77.31 (16.93) 73.81 (16.01) 0.444 Shown is mean ± standard deviation (S.D.) for each group at each time point. P ‐values are based on Wald test statistic, using mixed‐effect models. a Indicates significant difference ( P  < 0.05) between placebo and everolimus groups after 6 months treatment. John Wiley & Sons, Ltd BODY.RESULTS.EVEROLIMUS EFFECT ON TAND FEATURES: In examining change scores from baseline for multiple neurocognitive and behavioral domains, nearly all assessment measures failed to demonstrate significant differences between the two groups at the end of 6 months (Table 2). When comparing treatment to placebo, only the CANTAB Stockings of Cambridge (SOC) (objective performance measure of executive function) and SRS Social Cognition (parent rating of social behavior) were significant. Placebo was associated with more favorable outcome on SOC than everolimus. Analysis of individual responses revealed that most individuals in the everolimus group and all in the placebo group improved compared to baseline on the SOC. However, individuals who increased the average number of errors compared to baseline were all in the everolimus group (Fig. 2A). For the SRS, those in the everolimus group were more likely to be reported by caregivers to improve social cognition versus the placebo group (Fig. 2B). In addition, the social communication domain of the SRS demonstrated a quadratic trend at 3 months but this was not maintained at 6 months. Figure 2Individual Treatment Response for CANTAB Stockings of Cambridge (A) and SRS Social Cognition (B). Waterfall plots show percentage change in average number of errors on the SOC (A) and SRS T‐score (B) after 6 months treatment with everolimus (blue) or placebo (red), compared to baseline, for each participant completing both assessments. Given the wide range of intellectual ability seen at baseline, we explored if baseline intellectual ability predicted treatment response. To separate the groups, we divided the sample by baseline IQ (≤ or >80). Most domains were not significantly different (Tables S1–S2). In the more severely affected group (IQ ≤ 80), placebo performed better than everolimus on BRIEF Behavioral Regulation and Metacognition subdomains (executive function), WRAT4 Word Reading (academic performance), BASC Externalizing Problems. Conversely, the VABS2 Daily Living Skills subdomain (socialization and behavior), the everolimus group performed better than the placebo group. In those more mildly affected (IQ > 80), we observed no changes in executive function or academic performance although multiple subdomains of socialization and behavior showed change in relation to everolimus treatment, including the SRS Social Cognition, BASC2 Externalizing Problems, and SDQ Total Difficulties scores. Quadratic analysis again identified a trend for improvements at 3 months that worsened by 6 months for SRS Social Cognition and Social Communication. We also evaluated age as an independent factor to determine if younger children (age <10 years), for whom neuroplasticity might be assumed to be more prominent and differentially impact treatment response. Most subdomains did not show any age‐dependent treatment‐related differences (Tables S3–S4). SRS Social Cognition was significant in the younger cohort but favored placebo rather than everolimus, as did SRS Social Awareness and BRIEF Behavioral Regulation in the older subgroup. The SRS Total Social Responsiveness Scale showed quadratic trend improvements at 3 months but not 6 months. BODY.RESULTS.EVEROLIMUS SAFETY: Over 6 months of treatment and 1 month of posttreatment follow‐up, a total of 473 AEs were reported (Table 3). The average number of AE per subject in each group was nearly identical (9.8 placebo vs. 10.2 everolimus). The majority of AE were mild or moderate (CTCAE3.0 Grade 1 or 2), accounting for 97% of all AE. More severe grade 3 or 4 AE were more likely to occur in the everolimus group, reported by 10 of 32 subjects vs. 1 of 15 subjects in the placebo group (P = 0.02). Treatment‐related grade 3/4 AE included fever/infections (n = 8), aphthous ulcers (n = 3), and seizure (n = 1). Grade 3/4 AE unrelated to treatment were behavior changes (n = 4) and syncope (n = 2). Table 3 Summary of adverse events Placebo ( n  = 15) Everolimus ( n  = 32) Total AE 147 326 Serious AE 0 (0%) 7 (2%) Nonserious AE 147 (100%) 319 (98%) AE Severity Grade 1 131 (89%) 250 (77%) Grade 2 15 (10%) 59 (18%) Grade 3 1 (1%) 15 (5%) Grade 4 0 (0%) 2 (1%) Relationship to study drug Suspected 62 (42%) 161 (49%) Not Suspected 85 (58%) 165 (51%) Action Taken None 97 (66%) 162 (50%) Dose Held or Adjusted 4 (3%) 22 (7%) Discontinued Treatment 0 (0%) 1 (0%) Concomitant Medication Given 41 (28%) 135 (41%) Nondrug Therapy Given 4 (3%) 1 (0%) Hospitalization 0 (0%) 5 (2%) Not Specified 1 (1%) 0 (0%) Shown is frequency (percentage across categories), separated by treatment arm. John Wiley & Sons, Ltd Regardless of treatment arm or AE grade, AE were just as likely to be attributed to study medication as not and in most instances, and required no action by managing clinicians. Gastrointestinal complaints, primarily stomatitis/aphthous ulcers, were the most common AE (28% overall) (Table 4), although the frequency between treatment and placebo groups was not significant for gastrointestinal complaints in general (P = 0.80) nor stomatitis/aphthous ulcers (P = 0.25). Infections (primarily URI) and neurological complaints (headache) were the only other AE accounting for more than 10% of all reported AE. The only category to demonstrate significant difference between groups was respiratory AE (cough), which was significantly higher in the placebo group compared to everolimus group (P < 0.001). Table 4 Adverse event categories   Placebo ( n  = 15) (%) Everolimus ( n  = 32) (%) AE Category Cardiac 0 (0) 1 (0) Dermatologic 12 (8) 21 (6) Gastrointestinal 40 (27) 92 (28) General 6 (4) 18 (6) Genitourinary 2 (1) 6 (2) Hematologic 7 (5) 11 (3) Infectious 28 (19) 73 (22) Metabolic 1 (1) 1 (0) Musculoskeletal 6 (4) 13 (4) Neurologic 29 (20) 70 (21) Psychiatric 3 (2) 12 (4) Respiratory 13 (9) 7 (2) Unspecified 0 (0) 1 (0) Shown is frequency (percentage across categories), separated by treatment arm. John Wiley & Sons, Ltd Serious AE (SAE) were infrequent (Table 3), consisting of 7 hospitalizations in 3 subjects from the everolimus treatment group only. The first SAE was due to pneumonia that resolved with antibiotic treatment. However, the participant subsequently elected to discontinue treatment. The other SAE consisted of hospitalizations for pyelonephritis and behavioral/personality changes, and in each case treatment with study drug was resumed after resolution of the SAE. Of note, the behavior/personality changes were not suspected to be treatment‐related as the participant had prior history of psychiatric illness with psychiatric hospitalizations that preceded study participation. BODY.DISCUSSION: We performed the first placebo‐controlled clinical trial that directly and prospectively evaluated the impact of mTOR inhibitors on TAND in children with TSC. The major finding of this trial is that treatment with mTOR inhibitors does not interfere with normal behavior and development in this population. Given that everolimus is FDA‐approved for SEGA and renal angiomyolipomas, it is reassuring that there are no suggestions of deleterious effects in neuropsychological, academic skills and adaptive behavior over the short term. Safety in this regard over the long term (>6 months treatment duration) still needs to be verified. Everolimus generally was well‐tolerated with only one subject discontinuing treatment due to safety concerns of the parent. Overall frequency of AE was identical in the placebo and everolimus groups, and the types of AE encountered were consistent with those previously reported in human everolimus clinical trials for SEGA, epilepsy, and angiomyolipoma in individuals with TSC.8, 47, 48 We did find that grade 3/4 AE and SAE were more common in the everolimus group compared to placebo. While many of these were judged to be disease‐related rather than treatment‐related, this observation highlights continued need to demonstrate clear benefit for everolimus treatment of TAND that justifies potential risks. We were disappointed to find no significant difference in most neuropsychological measures (memory, attention, executive function, behavior) in individuals with TSC treated with everolimus versus placebo. This is the first instance in which mTOR inhibition has not clearly improved clinical manifestations of TSC despite convincing preclinical evidence in TSC animal models that supports the use of mTOR inhibitors for neurocognitive improvement,14, 15, 16, 17, 18, 19 There were some trends suggesting improvement in children in the everolimus group compared to placebo in standardized parental ratings of behavior versus direct measurements of neuropsychological skills. Older children and adolescents in the treatment group demonstrated some reported improvement in social skills over the 6‐month trial whereas younger children showed decreased symptoms of depressed mood, but these effects were not statistically significant. We suspect age may prove key for mTOR‐targeted treatment to change TAND outcomes in TSC, given that TAND symptoms and features initially present within the first 12–24 months of life.45, 49, 50 Onset of epilepsy occurs during the same time period and has been closely correlated with TSC neurocognitive and neurodevelopmental outcomes.45, 46 Aggressive even presymptomatic treatment of epilepsy in TSC infants and toddlers has been reported to improve long‐term neuropsychological outcomes.51 While evaluating everolimus effect on TAND features in this younger age group would have been ideal, our study limited enrollment to 6 years and older, as TAND assessment tools for younger children lack needed precision compared to standardized and validated instruments available to assess and characterize older children. Furthermore, the safety of everolimus in TSC under the age of 2 years has yet to be established. Overcoming these obstacles to assess everolimus in this younger age group and in individuals with coexistent epilepsy might reveal more profound effects on TAND than we were able to observe in this study. Our trial highlights limitations in obtaining and interpreting accurate assessments of mTOR‐associated changes in neuropsychological and behavior domains of TAND for children with TSC. Detailed TAND characterization in TSC children has been reported only in small cohorts, using a wide variety of assessment tools, age ranges, and populations.49, 52, 53, 54, 55, 56 Prior attempts to measure TAND‐related outcomes in human TSC clinical trials with mTOR inhibitors have taken different approaches to overcome these obstacles. Krueger et al. (2010) treated 22 children with TSC diagnosed with SEGA with everolimus for 6 months.26 Four participants were cognitively and behaviorally impaired to an extent that standardized assessment was not possible. Few participants in the study could complete the comprehensive testing battery and no changes were found. Davies et al. (2011) took a similar approach in 8 adults with TSC with angiomyolipoma or LAM treated with sirolimus, all of whom completed testing.24 Results were mixed, immediate recall memory and executive function were improved though immediate recognition memory worsened. In a more recent study using everolimus to treat individuals with TSC and refractory epilepsy, a simpler approach relying exclusively on broad‐based parental surveys and questionnaires was used to ensure all participants would be able to complete all measures.7 Parents reported subjective improvement in attention, adaptive social behavior, conduct problems, insecurity/anxiety, and quality of life following treatment after 3 months. The lack of uniform improvement in cognition across our study participants is likely multifactorial. Despite our attempts to minimize heterogeneity, TSC symptoms are further compounded by comorbid medical conditions and medications.4 For example, poorer neurocognitive function and neurodevelopmental outcomes are closely associated with epilepsy in TSC, especially when seizures are not fully controlled.45, 46, 57 Although the prevalence of epilepsy in our cohort was representative of that reported for the overall TSC population,46 the inclusion criteria limiting to participants with a minimum IQ selected for individuals whose seizures generally were well‐controlled. Thus, any secondary neurocognitive and behavioral improvements as a result of improved seizure control could not be assessed. Concurrent antiepileptic drugs (AEDs) could also negatively impact neurocognitive function independent of underlying disease or mTOR‐mediated effects. AEDs can affect everolimus through enzyme induction of the P450 system in the liver, although this was mitigated by dosing according to serum trough levels versus a standardized dosing scheme based on age or body size. The constellation and severity of other TAND features may have additionally obscured group differences in such a small sample. Using statistical means to address these limitations by stratifying age and IQ, we found domains with differences between placebo and treatment groups. Future studies will be needed to target‐specific behavior and neuropsychological domains in these restricted populations based on a narrower age range, specific TAND phenotypes, symptom severity, or concurrent medications. Additionally, a significant placebo effect was evident in our study, which is common in similar clinical trials for other neurodevelopmental disorders and obscures smaller but genuine treatment‐related changes. These limitations can be overcome not only with improved outcome measures that emphasize objective assessment over subjective reporting, but also development and inclusion of well‐validated biomarkers and larger sample sizes. We followed everolimus dosing and treatment guidelines reflected in clinical trials using current FDA‐approved treatment of SEGA in children with TSC.26, 39 Earlier trials suggested benefit might be achievable in this dosing range and duration of treatment.7, 26 However, optimal dosing and treatment duration have yet to be established for neurocognitive and behavioral change, and we may have not allowed sufficient time or achieved adequate dosing levels for neurocognitive benefit to occur. It also is possible that everolimus treatment is not sufficient to improve neurocognitive skills unless it is combined with a behavioral intervention. Such combination studies are being conducted in other neurodevelopmental disorders and may also be a model for children with TSC. BODY.AUTHOR CONTRIBUTIONS: Darcy A. Krueger, MD, PhD: study concept and design, acquisition of data, analysis and interpretation of data, and drafting and revision of manuscript for intellectual content. Anjali Sadhwani, PhD: acquisition of data, analysis and interpretation of data, and drafting and revision of manuscript for intellectual content. Anna W. Byars, PhD: acquisition of data, analysis and interpretation of data, and drafting and revision of manuscript for intellectual content. Petrus J de Vries, MBChB, MRC, Psych, PhD: study concept and design, analysis and interpretation of data, and revision of manuscript for intellectual content. David Neal Franz, MD: study concept and design, acquisition of data, revision of manuscript for intellectual content. Vicky H. Whittemore, PhD: study concept and design, revision of manuscript for intellectual content. Rajna Filip‐Dhima: analysis and interpretation of data, revision of manuscript for intellectual content. Donna Murray, PhD, CCC‐SLP: study concept and design, analysis and interpretation of data, and revision of manuscript for intellectual content. Kush Kapur, PhD: analysis and interpretation of data, and revision of manuscript for intellectual content. Mustafa Sahin, MD, PhD: study concept and design, acquisition of data, analysis and interpretation of data, and drafting and revision of manuscript for intellectual content. BODY.CONFLICT OF INTEREST: Darcy A. Krueger, MD, PhD: Dr. Krueger has received consulting and speaking fees and travel expenses from Novartis and additional research support from the National Institute of Neurological Disorders and Stroke of the NIH (U01‐NS082320, U54‐NS092090, P20‐NS080199), the Tuberous Sclerosis Alliance, the Van Andel Research Institute, Novartis, and Upsher‐Smith Pharmaceuticals. In addition, he serves on the professional advisory board and international relations committee for the Tuberous Sclerosis Alliance and the editorial board of Pediatric Neurology. Anjali Sadhwani, PhD: Dr. Sadhwani reports no disclosures. Anna W. Byars, PhD: Dr. Byars has received additional research support from the National Institute of Neurological Disordders and Stroke of the NIH (U01‐NS082320) and serves on the Professional Advisory Board of the Tuberous Sclerosis Alliance. Petrus J de Vries, MBChB, MRC, Psych, PhD: Dr. de Vries has received consulting and speaking fees and travel expenses from Novartis and additional research support from the University of Cape Town, National Research Foundation, and the Struengmann Fund. Vicky H. Whittemore, PhD: Dr. Whittemore is an employee of the National Institute for Neurological Disorders and Stroke of the National Institutes of Health.Donna Murray, PhD, CCC‐SLP: Dr. Murray is an employee of the Autism Speaks Foundation. Kush Kapur, PhD: Dr. Kapur reports no disclosures. Mustafa Sahin, MD, PhD: Dr. Sahin is supported by the NIH (U01 NS082320) and the Developmental Synaptopathies Consortium (U54 NS092090), which is part of the NCATS Rare Diseases Clinical Research Network (RDCRN). His lab receives research funding from Roche, Pfizer, Novartis and LAM Therapeutics, and he has served on the Scientific Advisory Board of Sage Therapeutics. In addition, he serves on the Professional Advisory Board of the Tuberous Sclerosis Alliance and is an Associate Editor of Pediatric Neurology. BODY.CLINICAL TRIALS REGISTRY: Clinicaltrials.Gov Identifier: NCT01289912 BODY.SUPPORTING INFORMATION: Table S1. IQ Stratification (Full Scale IQ ≤ 80) subgroup analysis of neurocognitive and behavioral assessment results at baseline and after treatment with everolimus for 6 months. Table S2. IQ Stratification (Full Scale IQ > 80) subgroup analysis of neurocognitive and behavioral assessment results at baseline and after treatment with everolimus for 6 months. Table S3. Age stratification (Age ≤ 10 Years) subgroup analysis of neurocognitive and behavioral assessment results at baseline and after treatment with everolimus for 6 months. Table S4. Age stratification (Age > 10 Years) subgroup analysis of neurocognitive and behavioral assessment results at baseline and after treatment with everolimus for 6 months.Click here for additional data file.

TITLE: Mozart K.448 listening decreased seizure recurrence and epileptiform discharges in children with first unprovoked seizures: a randomized controlled study ABSTRACT.BACKGROUND: Increasing numbers of reports show the beneficial effects of listening to Mozart music in decreasing epileptiform discharges as well as seizure frequency in epileptic children. There has been no effective method to reduce seizure recurrence after the first unprovoked seizure until now. In this study, we investigated the effect of listening to Mozart K.448 in reducing the seizure recurrence rate in children with first unprovoked seizures. ABSTRACT.METHODS: Forty-eight children who experienced their first unprovoked seizure with epileptiform discharges were included in the study. They were randomly placed into treatment (n = 24) and control (n = 24) groups. Children in the treatment group listened to Mozart K.448 daily before bedtime for at least six months. Two patients in the treatment group were excluded from analysis due to discontinuation intervention. Finally, forty-six patients were analyzed. Most of these patients (89.1%) were idiopathic in etiology. Seizure recurrence rates and reduction of epileptiform discharges were compared. ABSTRACT.RESULTS: The average follow-up durations in the treatment and control groups were 18.6 ± 6.6 and 20.1 ± 5.1 months, respectively. The seizure recurrence rate was estimated to be significantly lower in the treatment group than the control group over 24 months (37.2% vs. 76.8%, p = 0.0109). Significant decreases in epileptiform discharges were also observed after 1, 2, and 6 months of listening to Mozart K.448 when compared with EEGs before listening to music. There were no significant differences in gender, mentality, seizure type, and etiology between the recurrence and non-recurrence groups. ABSTRACT.CONCLUSIONS: Although the case number was limited and control music was not performed in this study, the study revealed that listening to Mozart K.448 reduced the seizure recurrence rate and epileptiform discharges in children with first unprovoked seizures, especially of idiopathic etiology. We believe that Mozart K.448 could be a promising alternative treatment in patients with first unprovoked seizures and abnormal EEGs. Further large-scaled study should be conducted to confirm the effect. ABSTRACT.TRIAL REGISTRATION: NCT01892605, date: June-19-2013 BODY.BACKGROUND: Music has been used for healing mental and physical diseases. Rauscher et al. report that brief exposure to Mozart's Sonata for Two Pianos in D major, K.448 (Mozart K.448), produces a temporal increase in spatial reasoning scores [1], the so-called "Mozart Effect." In addition to improvement of cognitive function, subsequent studies reveal positive effects from listening to music for many medical diseases, including hypertension, anxiety, and dementia [2]–[4]. For example, Särkämö et al. investigated patients with strokes who listened to their favorite music for two months. Their results show that recovery in the domains of verbal memory and focus attention improved significantly more in the music group than in the control group, even six months after the stroke [5]. In patients with Parkinson disease, after listening to self-selected music, motor coordination with "Vienna Test System" shows improvement in aiming and line tracking [6]. This study provides evidence that specific music can improve the precision of arm and finger movement. Relaxing classical music was also used in sleep disorders. 94 students with sleep complaints participated a music study. They listened to relaxing classical music, including some popular pieces from Baroque to Romantic, for 45 minutes every night at bedtime for 3 consecutive weeks, or audiobooks-a CD containing 11 hours of short stories by Hungarian writers such as Frigyes Karinthy, Gyula Krúdy, Géza Gárdonyi, Zsigmond Móricz and Mihály Babits for 45 minutes every night at bedtime for 3 consecutive weeks, or no intervention for 3 weeks. The results show music significantly improves sleep quality according to the Pittsburg sleep quality index and depressive symptoms. However, sleep quality and depressive symptoms did not improve in the audiobook and control group [7]. Regarding epilepsy, Hughes et al. and our previous study show that epileptiform discharges decrease when listening to Mozart K.448 in patients with epilepsy [8,9]. Furthermore, our study also reveals that listening to Mozart K.448 reduces seizure frequencies in children with intractable seizures [10]. The seizure recurrence rate after the first unprovoked seizure in pediatric patients is highly variable, depending on the follow-up period and patient selection. Age at onset of epilepsy under 1 year, remote symptomatic etiology, developmental delay/mental retardation, abnormal EEG background, frequent epileptiform discharges, and abnormal neuroimaging are all significant predictors of a higher risk for recurrence of epilepsy [11,12]. Children with a recurrence have a similar epileptic outcome when compared to children presenting with multiple seizures, regardless whether they were treated after the first unprovoked seizure or not [13]. This argues in favor of withholding anti-epileptic drug treatment at least until a second seizure has occurred to reduce the potential for adverse effects of drugs, including unpleasant physical effects, adverse cognitive and behavioral changes, and potential teratogenicity [13,14]. Until now, there has been no effective method to reduce seizure recurrence after the first unprovoked seizure. In this study, we investigated the effect of Mozart K.448 on the seizure recurrence in children with their first unprovoked seizure who have epileptiform discharges. BODY.METHODS.SUBJECTS.INCLUSION CRITERIA AND EXCLUSION CRITERIA: Children less than 18 years of age with first unprovoked seizure were investigated in the out-patient department of Kaohsiung Medical University Hospital. Patients with epileptiform EEGs were included in this study. Patients who (1) had neurodegenerative diseases, or (2) did not have epileptiform discharges were excluded. Participants in the study received EEG examinations after their first seizure occurred. Each EEG was recorded digitally (Harmonie DVN V5.1, Montreal, Canada). Electrodes were placed according to the International 10–20 System and two neurologists reviewed the EEG recordings. Informed consent was given by a family member or legal guardian in each case. This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH-IRB-970391). BODY.METHODS.SUBJECTS.MUSIC LISTENING AND EVALUATING SEIZURE RECURRENCE: This randomized controlled study was conducted in Kaohsiung Medical University Hospital. The patients in this study were randomly placed in the control or treatment group by the chief investigator in a 1:1 ratio, by randomly generating even (for treatment) and uneven (for control) numbers using a computer program (http://www.random.org). No stratification for age, sex, or seizure type was performed. The patients in the study did not receive AED after their first unprovoked seizure. The treatment group listened to the first movement of Mozart K.448 for eight minutes once daily before bedtime for at least six months. None of the participants had reported listening to Mozart K.448 music in the past. The control group did not receive any music. All of the patients received follow-up telephone calls monthly. Patients who experienced seizure recurrence were advised to begin anti-epileptic drug treatment. All the data were collected at the Kaohsiung Medical University Hospital from April 2009 to December 2011. BODY.METHODS.SUBJECTS.FOLLOW UP EEG: Patients who listened to Mozart K.448 without seizure recurrence received EEG examinations before, and after 1, 2, and 6 months of listening. Patients in the control group received EEG examinations at the beginning of study and before anti-epileptic drug treatment. The epileptiform discharges were defined as distinct waves or complexes, distinguished from background activity. Two pediatric neurologists analyzed these EEGs and calculated the frequency of epileptiform discharges by blind. The total number of epileptiform discharges during each section (before, and after 1, 2, and 6 months of listening) were divided by the duration (in minutes) of the section and compared. Percentage changes in epileptiform discharge were calculated as ((the discharge before music listening - discharge after music listening)/discharge before music listening) × 100. All recordings were performed during the daytime. To decrease the factors influencing epileptiform discharges, each patient maintained the same state of wakefulness during each recording period. BODY.METHODS.SUBJECTS.STATISTICAL ANALYSIS: Seizure recurrence rates were determined by Kaplan-Meier estimates, defining an event as the recurrence of a second seizure. Sample size for Kaplan-Meier approach was estimated by the survival power calculator (http://www.statstodo.com/SSizSurvival_Pgm.php). The follow-up time for patients with seizure recurrence was from the beginning of music treatment to the last clinical visit. Differences in the distribution of treatment and control groups were calculated using the Chi-square test. The paired t-test and ANOVA were used to compare the percentage of epileptiform discharge reduction at the 1st, 2nd, and 6th month of music listening. A p value less than 0.05 was considered significant. BODY.RESULTS: 153 patients with their first unprovoked seizure were investigated for inclusion in this study and 107 patients were excluded from analysis. No epileptiform discharges were found in 105 patients. Forty-eight patients with epileptiform discharges were enrolled. Twenty-four patients were placed in the treatment group and 24 patients in the control group (Figure 1). Two patients who listened to music less than six months in the treatment group were excluded. Finally, forty-six patients were analyzed. The mean age of the treatment group was 9 years 6 months ± 3 years 10 months, and the control group was 8 years 7 months ± 3 years 10 months. Forty-three patients demonstrated normal intelligence, two patients had a reduced IQ, and one patient had an undetermined IQ level. The majority of patients (n = 41) were idiopathic in etiology, and five patients were symptomatic (Table 1). There were no significant differences in gender, mentality, seizure type, and etiology between the treatment and control groups. None of the patients suffered from musicogenic epilepsy at the time of study. Figure 1The patient recruitment flowchart. Table 1 Profile comparison between treatment with Mozart K. 448 and control group   Treatment n (%) Control n (%) p-value Sex         Male 13 (59.1) 12 (50) 0.536   Female 9 (40.9) 12 (50)   Age 9 y 6 m ± 3 y 10 m 8 y 7 m ± 3 y 10 m 0.413 Mentality         IQ≥70 20 (90.9) 23 (95.8) 0.209   IQ<70 2 (9.1) 0 (0)     undetermined 0 (0) 1 (4.2)   Seizure type         Generalized 4 (18.2) 7 (29.2) 0.383   Focal 18 (81.2) 17 (65)   Etiology         Idiopathic 19 (83.4) 22 (89.1) 0.564   Symptomatic 3 (16.6) 2 (10.9)   BODY.RESULTS.SEIZURE RECURRENCE AFTER MUSIC LISTENING: The average follow-up durations in the treatment group and control group were 18.6 ± 6.6 (range from 6 to 24 months) and 20.1 ± 5.1 (range from 10 to 24 months) months, respectively. During the follow-up period, 8 of 22 patients in the treatment group had seizure recurrence, while 18 of 24 patients in the control group had seizure recurrence. After at least six months of Mozart K.448 listening, the seizure recurrence rate in the treatment group at the 6 months follow-up was 22.7% and was estimated to be 37.2% by 12 and 24 months. In the control group, at the 6 months follow-up, the seizure recurrence rate was 58.3% and was estimated to be 76.8% by 12 and 24 months. A significantly higher recurrence rate was noted in the control group than the treatment group (p = 0.0109) (Figure 2). There were no significant differences in gender, mentality, seizure type, and etiology between the recurrence and non-recurrence subjects in either treatment or control group (Table 2). Figure 2The seizure recurrence rates after the first unprovoked seizure between treatment and control groups. The seizure recurrence rate was estimated to be significantly lower in the treatment group than the control group by 24 months (p = 0.0109). Table 2 Profile comparison between recurrence and non - recurrence group   Treatment Control   Recurrence Non-recurrence p - value Recurrence Non-recurrence p - value   n (%) n (%) n (%) n (%) Sex               Male 3 (37.5) 10 (71.4) 0.119 10 (55.6) 2 (33.3) 0.346   Female 5 (62.5) 4 (28.6)   8 (44.4) 4 (66.7)   Mentality               IQ≥70 8 (100) 12 (85.7) 0.262 17 (94.4) 6 (100) 0.555   IQ<70 0 (0) 2 (14.3)   0 (0) 0 (0)     undetermined 0 (0) 0 (0)   1 (5.6) 0 (0)   Seizure type               Generalized 2 (25) 2 (14.3) 0.531 4 (22.2) 3 (50) 0.195   Focal 6 (75) 12 (85.7)   14 (77.8) 3 (50)   Etiology               Idiopathic 7 (87.5) 12 (85.7) 0.906 16 (88.9) 6 (100) 0.394   Symptomatic 1 (12.5) 2 (14.3)   2 (11.1) 0 (0)   BODY.RESULTS.CHANGES IN FREQUENCY OF EPILEPTIFORM DISCHARGES IN THE TREATMENT AND CONTROL GROUPS: There was no significant difference between baseline and follow-up (the interval between baseline and follow-up EEGs ranged from two to seven months) epileptiform discharge frequencies in the control group (3A). Fourteen patients did not experience seizure recurrence during any of the follow-up periods in the treatment group. Among them, eleven patients received EEG follow-ups after1 month, ten patients after 2 months, and eight patients after 6 months of listening to Mozart K.448. The absolute frequency of epileptiform discharges demonstrated a trend of decrease after music exposure (Figure 3B). Since the average epileptiform discharge frequency in each patient of treatment group before Mozart K.448 listening was highly variable, ranging from 0.3/min to 30.2/min, the change in epileptiform discharges was expressed by percentage of reduction. Significant decreases in epileptiform discharges were found after 1, 2, and 6 months of listening to Mozart K.448 when compared with EEGs before listening to music (decreased by 79.4 ± 20.0%, 71.2 ± 40.3%, and 82.1 ± 30.6% respectively, p < 0.001). However, the decreases of epileptiform discharges did not show a significant difference between 1, 2, and 6 months of listening to Mozart K.448 (Figure 3B). On the contrary, during the average period of 3.3 months (ranged from 2 to 7 months), ten patients in the control group received EEG follow-ups, and there was no significant change in epileptiform discharges between EEGs at the beginning of study and before anti-epileptic drug treatment (increased by 0.3 ± 36.4%, p =0.310) (Figure 3A). Figure 3The comparison of reduction of epileptiform discharges in absolute frequency and percentage of reduction between treatment and control groups. There was no significant difference between baseline and follow-up (the interval between baseline and follow-up EEGs ranged from two to seven months) epileptiform discharge frequencies in the control group (A). Significant decreases in epileptiform discharges were observed after 1, 2, and 6 months of listening to Mozart K.448 when compared with EEGs before listening to music (B). * p < 0.05. BODY.DISCUSSION: Mozart K.448 listening is reported to diminish the intensity of tinnitus, improve the paper-folding and cutting tests in patients with mild cognitive impairment, and increase weight gain in preterm infants by reducing resting energy expenditure [15]–[17]. Regarding epilepsy, our previous works show that interictal discharges were reduced in most patients with epilepsy when they listened to Mozart K.448 [9]. In addition, 72.7% of the patients with refractory epilepsy became seizure free or had a very good response by listening to Mozart K.448 [10]. Our previous report also demonstrates that Mozart K.448 is not the only piece of music to have beneficial effects on children with epilepsy, and that listening to Mozart K.545 with similar lower harmonics can decrease epileptiform discharges in epileptic children as well [18]. In this current study, we investigated the effect of Mozart K.448 on seizure recurrence after the first unprovoked seizure. The results showed that listening to Mozart K.448 once daily reduced the seizure recurrence rate and epileptiform discharges. The estimated seizure recurrence rate in the control group was 76.8%, while it was 37.2% in music treated patients. The epileptiform discharges also showed an approximate 70-80% reductions after 1, 2, and 6 months of music listening. Seizure recurrence after the first unprovoked seizure in pediatric patients ranges from 26-71% [19]. Risk factors for seizure recurrence include a remote symptomatic etiology, an abnormal EEG, a seizure occurring while asleep, a history of prior febrile seizures, and Todd's paresis [12]. In a large study, 564 patients, including adults and children who had first unprovoked seizures, have been followed up for 2–4 years. Sixty-seven percent of them had a recurrence within 12 months of the first seizure, and 78% had a recurrence within 36 months [20]. In the under the age of 16 group, the seizure recurrence rate was 83% by 36 months [20]. In our study, the seizure recurrence rate was estimated to be 76.8% by 24 months in the control group. An abnormal EEG and an age less than 16 appeared to be risk factors for a higher recurrence rate in our study. However, the seizure recurrence rate did not demonstrate a significant difference in patients with different gender, mentality, seizure type, and etiology. The results were similar to our previous studies which show that gender, mentality, and etiology of epilepsy do not influence the short-term or long-term music effect on epileptiform discharges [9,21]. The epileptiform discharges were significantly reduced in EEGs performed 1, 2, and 6 months after initiating music listening in patients without seizure recurrence. However, the epileptiform discharges did not decrease in a duration-dependence manner. EEG improvement after one month of listening to Mozart K.448 may serve as an indicator in determining the long term outcome of music intervention. Although it is not possible to predict how long the patients should be treated, the effectiveness of music listening was demonstrable within one month and continued for at least 6 months. We suggest that listening to music daily for 6 months has a beneficial effect in decreasing seizure recurrence in children with first unprovoked seizures. Recently, several theories have been introduced regarding the effects of sound on the brain. Poor health is reported to be associated with lower parasympathetic tone in several medical conditions, including epilepsy [22]. Lotufo et al. report a sympathovagal imbalance in epilepsy, as shown by lower high frequency (HF), the standard deviation of the RR interval (SDNN), and the square root of the mean squared differences of successive RR intervals (RMSSD) values when compared to controls [23]. One study shows that a two-hour music intervention in cancer patients increases their relaxation scores and parasympathetic activities [24]. Another study shows that forty-five minutes of music therapy once a week in patients with cerebrovascular disease enhances parasympathetic activities and decreases congestive heart failure events by reducing plasma cytokine and catecholamine levels [25]. Our previous data also demonstrates that significant increases in HF, RMSSD, the standard deviation of differences between adjacent RR intervals (SDSD), and a decrease in mean beats per minute in heart rate variability analysis occurs while listening to Mozart music in children with epilepsy. At the same time, epileptiform discharges are significantly reduced during and right after listening to Mozart music. The results suggest that Mozart music stimuli induces parasympathetic activation [26]. It is possible that musical enhancement of parasympathetic tone may account for the beneficial effects on epilepsy. Neurotransmitter pathways may also be involved in the effect of Mozart K.448 on epilepsy. Musical exposure is known to increase the expression of dopamine levels in the brain [2]. In recent years, the role of dopamine in the pathophysiology of epilepsy has been well documented. A Positron Emission Tomography study shows that impaired dopamine uptake in the midbrain is hypothesized to contribute to seizures in juvenile myoclonic epilepsy [27]. In a recent animal study, the authors report that pentylenetetrazole induced seizures decrease the dopamine levels in striatal and hippocampal areas, accompanying the induction and propagation of seizures [28]. It is possible that listening to music modifies the dopaminergic pathways contributing to the beneficial effects in epilepsy therapy. There are limitations to this present study. First, the number of participants was somewhat limited and most of the patients were idiopathic in etiology. The statistical power is 0.74, under alpha error 0.05, based on 22 treatment and 24 control subjects with non-recurrence rates 0.63 and 0.23, respectively. Although the power is not sufficient, our findings may provide preliminary evidence that Mozart K.448 listening is beneficial for children with first unprovoked seizures. Second, the lack of control music made it impossible to say that the effects were specific to Mozart K.448 or to a placebo effect. Third, because we did not follow up the EEG in all patients in the control group, it could not be determined whether time duration itself could have caused the decreases in epileptiform discharges, although we had shown that there were significant decreases in epileptiform discharges after listening to Mozart K.448 in treatment group. Fourth, we used per protocol analysis with compliant patients instead of using intention-to-treat analysis, although the result of intention-to-treat is also significant (p = 0.029). BODY.CONCLUSIONS: In conclusion, listening to Mozart K.448 reduced the seizure recurrence rate and epileptiform discharges in children with first unprovoked seizures, especially of idiopathic etiology. Although there were limitations to the case numbers in this study, the results highlight that Mozart K448 listening is a promising alternative treatment in patients with first unprovoked seizures and abnormal EEGs. More investigations should be performed to substantiate the effects of music on first unprovoked seizures. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: LCL carried out the study, participated in the evaluation of data, and helped draft the manuscript. MWL, RCW, and HKM conceived the study and participated in the evaluation of data. RCY participated in the design of the study, evaluation of data, and wrote the final version of the manuscript. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1472-6882/14/17/prepub

TITLE: Effects of hope promoting interventions based on religious beliefs on quality of life of patients with congestive heart failure and their families ABSTRACT.BACKGROUND:: Heart failure is one of the most important and prevalent diseases that may have negative effects on the quality of life (QOL). Today, the promotion of QOL in patients with heart failure is important in nursing care programs. This research aimed to determine the efficacy of hope-promoting interventions based on religious beliefs on the QOL of patients with congestive heart failure (CHF). ABSTRACT.MATERIALS AND METHODS:: In this randomized clinical trial (IRCT2014100619413N1) conducted in Isfahan, Iran, 46 adult patients with CHF were selected and randomly assigned to study and control groups. Ferrans and Powers Quality of Life Index (QLI) was completed by both groups before, immediately after, and 1 month after the intervention. For the study group participants and their families, 60-min sessions of hope-promoting interventions based on religious beliefs were held twice a week for 3 weeks. Independent t, repeated measures analysis of variance (ANOVA), Chi-square, Mann–Whitney, and Fisher's exact tests were adopted for data analysis. ABSTRACT.RESULTS:: The mean (standard deviation) overall QOL score in the area of satisfaction significantly increased in the study group, compared to the controls, immediately [70.7 (8.5) vs. 59.2 (12.5)] and 1 month after the intervention [75.2 (7.4) vs. 59.4 (12.9)] (P < 0.05). There was also a similar difference between the two groups in the area of importance immediately [73.6 (5.8) vs. 65.7 (7.5)] and 1 month after the intervention [76.3 (8.1) vs. 66.8 (8.5)] (P < 0.05). ABSTRACT.CONCLUSIONS:: Hope-promoting intervention based on religious beliefs is a useful method for improving QOL in patients with CHF. BODY.I: Heart disease is a major cause of disability and mortality in different countries of the world. Despite the different preventive approaches developed, it has still increased in prevalence. Among heart diseases, heart failure is one of the most important diseases and has the highest incidence of mortality and disability.[1] Almost 5 million people in America suffer from chronic heart failure.[2] In Iran, there are over 1 million patients with heart failure[3] The trend in percentage of deaths from cardiovascular diseases in Tehran, Iran, is similar to that of developed countries.[1] Heart failure is a global pandemic and effects communities through the financial burden on the healthcare system.[4] Compared to other chronic diseases, heart failure has a more acute impact on the quality of life (QOL).[1] In addition, this disease impairs the functional capacity and QOL of patients.[5] Researchers believe that the QOL in heart failure patients is lower than that in cancer patients.[6] This disease affects all aspects of the patients' life, including physical, mental, and social status; therefore, it is recommended that medical staff, especially nurses, pay attention to the QOL of patients and provide treatment, care, and educational measures.[7] On the other hand, diagnosis and treatment of diseases cause reduction or loss of hope in patients and patients with heart failure are not an exception. The severity of heart failure and the uncertain outcome of the disease among patients highlights the importance of hope in these patients.[8] Hope is defined as an inner force that can enrich life and enable patients to have an outlook beyond their current situation and suffering[9] Berendes et al. found that higher levels of hope were associated with elevated mood, better physical health, and greater ability to cope with illness and endure more pain.[10] In general, lack of hope and purpose in life leads to reduced QOL for the patient and negative beliefs.[9] Supportive interventions associated with hope in patients with heart failure can have an impact on health goals.[8] Thus, giving hope to patients has been considered as an important intervention in medicine, nursing, and mental health, but the practical and predictive value of hope remains unclear.[11] Many studies have been conducted in relation with hope-promoting interventions. In most of these studies, hope treatment has been based on the theory of positive psychology, particularly Snyder's Hope Theory,[1213] and less attention has been paid to religious beliefs to raise hope. Islam and its teachings include all the necessities of human life, and based on its many traditions and verses, all the sciences have been included in the Quran. There is a significant difference between the theory of hope in psychology and in the Holy Quran. The theory of hope in the Quran is based on the purpose of creation, is parallel to the human evolution, and its foundation is faith in God. However, in psychology, this central core has been overlooked. In psychology, the objectives of the theory of hope are defined by the individual and have material aspects. Nevertheless, the Quran has already identified these objectives as the worship of God in the vicegerent of God. Obstacles to achieving goals in the theory of hope in psychology also have material aspects, whereas in the Quran, obstacles are defined as desire and Satan. The models presented in the Quran for strengthening hope are the prophets, and compared to the patterns existing in psychology, they have a grand and glorious position.[14] It can be stated that Islamic teachings contain all that is required for growth, health, and human perfection. Knowing, believing, and applying these teachings in life have a significant role in promoting positive behavior and preventing mental and physical illness.[15] Baljani et al. believe that nursing and care interventions designed according to the religious beliefs of patients will maintain hope.[16] Therefore, in this study, hope-promoting interventions based on religious beliefs, instead of Snyder's Hope Theory, have been used among Muslims. There were only a few researches in the context of a hope-promoting intervention for heart failure patients based on religious beliefs in Iran, despite the country having a Muslim majority. Furthermore, there were no conflicting study results showing the lack of positive effect of hope therapeutic interventions. Therefore, nurses benefit from this holy book and religious beliefs as an important source of hope for their hope-promoting interventions. However, studies show that patients with heart failure and their caregivers experience poor QOL and depressive symptoms, and the burden of caregivers' responsibilities and symptoms of depression are associated with patients' QOL.[17] Thus, when the nurse determines the patient's care needs, he/she must not ignore the needs of the family, especially when the family has the responsibility of caring for the patient.[18] Most studies focus on the patient and the disease, and less attention is given to the family caregivers of patients. Therefore, in this study, the patients' family members were also considered. The aim of this study was to evaluate the impact of hope-promoting interventions based on religious beliefs of patients and their family on the QOL of patients. BODY.M: This study was a randomized clinical trial registered with the code IRCT2014100619413N1. It had two groups and was performed in three stages (before, immediately after, and 1 month after the intervention for follow-up).[19] The impact of hope-promoting interventions based on religious beliefs of the patients and their families was analyzed as the independent variable and patients' QOL as the dependent variable. The study population included patients with heart failure class I and II (with ejection fraction of more than 40%) who referred to hospitals and clinics affiliated to Isfahan University of Medical Sciences, Isfahan, Iran. Primary sampling was performed via convenient sampling method based on confidence interval of (Z1) 95%, meaning 1.96, test power factor (Z2) of 80%, meaning 0.89. The minimum difference in QOL score between the two groups (d), which showed the difference to be significant, was considered to be 0.83. Sample size was calculated as 23 patients in each group. Inclusion criteria included age of 18 years and above, diagnosis of heart failure class I and II by a specialist physician, history of heart failure of at least 1 year,[1] full awareness and knowledge of the time, place, and person, lack of dementia, willingness to participate in the study,[4] belonging to Muslim faith, lack of speech, mental, and hearing disorders confirmed by a specialist, and lack of any other serious and limiting medical illnesses that could impact the QOL and mental health confirmed by a specialist.[12] Patients were randomly assigned to the groups. For the random placement of the samples in the groups, using a table of random numbers, the researcher selected a number with closed eyes with up and right directions. If the number on the right was an even number, the sample was placed in the experimental group and if it was an odd number, the sample was placed in the control group. For the random allocation, each of the numbers was written on a card and placed in sealed envelopes, and then, a code was given to them. Envelopes were placed in a box and were not opened until sampling in the research environment. On the day of sampling, each eligible subject was given an envelope in order of the codes. This action was continued until reaching the desired sample size in all the sampling centers with 20% probability of sample loss. Sampling lasted for 4 months from September to February 2013. The patients were asked not to use other methods of alternative medicine that were associated with spiritual intervention during the execution of this study and to continue with their normal lives. Data were collected through a questionnaire that consisted of two parts. The first part included demographic characteristics (six questions for the patient and four questions for the family members) and questions about the status of the disease (three questions). The second part included the Ferrans and Powers Quality of Life Index (QLI),[20] which was answered by the patients in complete willingness. The QLI was designed in 1999 and the questions were developed in the two aspects of assessing importance of and satisfaction with health and performance, socioeconomic, psychological, spiritual, and familial dimensions. Each section contained 35 questions. In each section, 15 questions were related to health and performance measurement, 8 questions to the assessment of the socioeconomic dimension, 7 questions to psycho-spiritual assessment, and 5 questions to family relations.[20] The respondents determined satisfaction and importance based on a 6-point Likert scale: 0 (very dissatisfied) to 5 (very satisfied), and 0 (extremely unimportant) to 5 (very important). In this questionnaire, the rating scale was set from the lowest score to the highest score, from 0 to 5. The scores of each area were added and multiplied by 100, and then divided by the result of multiplying the number of questions by 5 to make the scores of each area and overall QOL understandable in the range of 0–100. The QLI could be used in any culture without any significant changes due to the lack of specific cultural problems.[21] The validity of the QLI was determined by Shojaei in 2008 through content validity and its reliability was confirmed by Cronbach's alpha of 0.86.[20] Panthee et al. also confirmed its reliability in their study in 2011 with a Cronbach's alpha of 90%.[22] After obtaining informed consents from the subjects, the patients of the experimental group and their family members received hope-promoting intervention as a group. They received the intervention twice a week, each time for an hour, for 3 weeks. This intervention consisted of two stages of creating hope (including two steps of finding hope and strengthening it) and increasing hope (two steps of enrichment and maintenance of hope).[14] Meetings were held by the researcher, a monitoring colleague with a PhD in theology, and a psychiatrist consultant as group discussions and questions and answers sessions. At the end of each session, a booklet containing the content of the meeting and the next session's task was given to the patients to ascertain that they will be remembered by the patients and evaluate the meetings' efficiency. For the control group, two sessions were held on the disease. Contents of intervention sessions based on the Quran and Islamic religious beliefs were as follows: BODY.M.ETHICAL CONSIDERATIONS: The researcher obtained a referral letter from the School of Nursing and Midwifery of Isfahan University of Medical Sciences. Subsequently, the researcher presented the introduction letter and explained the objectives of the study to the hospital management, and after obtaining their consent, performed sampling. This research was scientifically and ethically validated by the ethics committee of Isfahan University of Medical Sciences. BODY.M.FIRST SESSION.AIM: Explaining the objectives and rules, the relationship between despair and disease, and the importance and value of patient care in the Quran and other religious beliefs, requesting the patients to consider several important purposes of life, comparing the signs of disappointment with what has been offered in the Quran and doctrines and religious beliefs, the importance and value of having hope and denouncing despair. BODY.M.FIRST SESSION.TASK: To note the effects of the Quran and other religious beliefs on their perspective of despair, mood, and attitude. BODY.M.SECOND SESSION.AIM: Recalling God's blessings in the lives of individuals, considering the hopeful stories of the Holy Quran and the lives of saints and Imams seeking hope (the first step in creating hope). BODY.M.SECOND SESSION.TASK: Preparing a list of miracles that have occurred in their lives. BODY.M.THIRD SESSION.AIM: Setting Imams as their model, belief in resurrection, and having hope in benefiting from the worldly intercession of saints in order to strengthen hope (the second step of creating hope). BODY.M.THIRD SESSION.TASK: Noting the level of relationship with one of the saints or attempts to communicate specifically and intimately with one of the saints, for example, Imam Hussein, Imam Reza, or Hazrat Abalfzl. BODY.M.FOURTH SESSION.AIM: Explaining the effect of individual strategies, such as prayer, thought of God, enriching hope through familiarity with the Quran (the first step to increase hope). BODY.M.FOURTH SESSION.TASK: The use of at least one of the approaches presented and explaining its effect on the level of hope. BODY.M.FIFTH SESSION.AIM: Explaining the effect of social practices like charity and forgiveness in raising hope (the first step to increase hope). BODY.M.FIFTH SESSION.TASK: The use of at least one of the solutions presented and expressing its effect on one's hope and on others (as a society). BODY.M.SIXTH SESSION.AIM: Identifying evil and carnal desires as enemies of humanity and obstacles to achieving goals (unproductive thoughts resulting from desires and temptations of the devil) in order to preserve and maintain hope (second step to increase hope),[14] and reviewing previous meetings and thanking the participants. BODY.R: The results of demographic characteristics of the subjects including age, gender, marital status, employment status, educational level, and duration of heart disease, hospitalizations, and other chronic diseases are presented in Tables 1 and 2. In general, the results showed that the two groups were similar in terms of demographic characteristics and had no significant differences [Tables 1 and 2]. Independent t-test results showed no significant differences before the intervention between the two groups in terms of the mean QOL score in the performance and health, socioeconomic, psychological, spiritual, and familial dimensions and areas of satisfaction and importance. Immediately after and 1 month after the intervention, a significant difference was observed between the groups in terms of mean QOL score in the four dimensions and areas of satisfaction and importance, and the overall QOL score (P < 0.05) [Tables 3 and 4]. Repeated measures analysis of variance (ANOVA) also showed a significant difference between the three time durations in the control group in terms of mean QOL score in the four dimensions and two areas of satisfaction and importance. However, in the experimental group, there was a significant difference between the three time durations in terms of mean QOL score in the four dimensions and two areas of satisfaction and importance (P < 0.05). Moreover, mean QOL score had increased. According to the findings, it can be concluded that the implementation of hope-promoting interventions based on religious beliefs was effective in improving the QOL of patients of the experimental group. Table 1 The mean age, duration of disease, and frequency of hospitalizations in the experimental and control groups Table 2 Comparison of the distribution frequency of gender, marital status, education level, employment status, and other chronic diseases in the experimental and control groups Table 3 Mean overall score of quality of life in the experimental and control groups in the area of satisfaction at different times Table 4 Mean overall score of quality of life in the experimental and control groups in the area of importance at different times BODY.D: Numerous studies have indicated the poor QOL of patients with heart failure; therefore, therapeutic interventions, care, and training by healthcare workers, particularly nurses, must be performed with the aim of enhancing the QOL of these patients. According to the present study, implementation of hope-promoting programs based on religious beliefs for patients with heart failure and their family members improves the QOL of these patients. The findings showed that, 1 month after the intervention, the mean QOL score of experimental group patients, in the four dimensions and two areas of importance and satisfaction, was higher than that in the control group. This result indicated the endurance of the impact of the hope-promoting intervention in these patients even up to a month after the intervention. Khaledi Sardashti also found, in his study, that the effect of the intervention persisted 1 month after the treatment in patients with diabetes.[19] There are few studies on hope-promoting interventions based solely on religious teachings, and most studies in this field are based on Snyder's hope therapy. Sotodehasl et al. aimed to compare the effect of medication therapy and hope therapy on QOL of patients with primary hypertension.[12] They found that Snyder's hope therapy improved the QOL of patients with primary hypertension more than medication therapy and the mean QOL score immediately after and 1 month after the hope therapy intervention had increased compared to before the intervention.[12] The study by Ghezelseflo and Esbati was also conducted on two groups (experimental and control).[13] The experimental group received eight sessions of 2-h duration of group hope therapy intervention based on Snyder's theory, and no intervention was conducted for the control group.[13] Their results showed that group hope therapy had a positive effect on the QOL of men who were HIV positive.[13] However, Khaledi Sardashti studied the effect of group hope therapy on the level of hope in people with diabetes.[19] He conducted eight 2-h sessions of a combination of Snyder's hope therapy and Quranic verses and sayings and stories such as the story of Joseph story.[19] The results showed a positive impact on the level of hope of people with diabetes in the intervention group.[19] Hope definition, two-staged hope-promoting therapy, using hope generating stories, focusing on past successes to create hope, and understanding the purpose and limitations to maintaining hope are some of the similarities between the intervention of this study and the present study. The main difference between the present study and other studies on hope-promoting therapy is that this study used only the Quranic theory of hope, contrary to the positive psychology theory used in other studies. The hope-promoting intervention used in this study was based on the purpose of human creation, was parallel to human evolution, and its foundation was based on the faith in God. Through this faith, such trust and confidence is achieved in the human psyche that he/she will become patient and resistant against all adversities of life and will not be disappointed. Moreover, the models used in this intervention to strengthen hope are the prophets of God, and have a grand position compared to the models existing in psychology. In general, patients' need for communication with God is an innate need, and the establishment of true peace and the strengthening of real hope are possible only through a relationship with the creator (Raad 28/13). Interventions based on the religious beliefs of these patients can be very effective. Patients need to feel safe and supported, and, more importantly, hopeful during their illness more than at other times. In addition, an Islamic culture exists in Iran, and 98% of Muslims adhere to their religious orders and values and benefiting from the Quran and Islamic teachings has a special place among them. Therefore, religious practices can be used to create, maintain, and enhance hope, and thus, improve the patients' QOL. Jahani et al. studied the relationship between spiritual well-being and QOL in patients with coronary artery disease.[23] They stated that the existence of a relationship between spiritual health and QOL in these patients must be taken into consideration in designing of care program therapies in countries with rich religious and cultural beliefs, such as Iran.[23] Reviewing previous studies showed that most studies have focused on the patient and the disease, and less attention has been paid to the patient's family. Hooley et al. conducted a study in 2005 entitled "The relationship of quality of life, depression, and caregiver burden in outpatients with congestive heart failure" in Canada.[17] This study showed that patients with heart failure and their caregivers experience poor QOL and symptoms of depression, and that burden of care and symptoms of depression were associated with patients' QOL.[17] Ghahremani et al. also believed that family is the most valuable and vulnerable resource for disabled patients, and family members play an important role in supporting patients, and thus, require attention.[24] Therefore, in this study, families of patients with heart failure also attended the hope-promoting interventions with their patients. BODY.C: Hope-promoting interventions based on religious beliefs are effective in improving the QOL of patients with heart failure. Hence, this method is recommended in the care of patients with heart failure. Furthermore, due to the importance of family, its role in patients' QOL, and the profound effect of the physical and mental health of these patients' caregivers on their care, especially in home care, nurses should also attend to patients' family members. BODY.C.FINANCIAL SUPPORT AND SPONSORSHIP: Vice Chancellor for Research of Isfahan University of Medical Sciences, Isfahan, Iran. BODY.C.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Effect of rifampin on the pharmacokinetics of Axitinib (AG-013736) in Japanese and Caucasian healthy volunteers ABSTRACT.PURPOSE: Axitinib, a potent and selective inhibitor of vascular endothelial growth factor receptors 1, 2, 3, is metabolized by cytochrome P450 3A4 and glucuronidation. This study evaluated the effect of rifampin, a potent inducer of drug-metabolizing enzymes, on axitinib plasma pharmacokinetics. Equal numbers of Japanese and Caucasian subjects were enrolled to assess the potential differences in axitinib pharmacokinetics between the two ethnicities. ABSTRACT.METHODS: Forty healthy volunteers were randomized to receive 5 mg axitinib alone and with 600 mg rifampin. ABSTRACT.RESULTS: Rifampin expectedly decreased AUCinf and Cmax of axitinib (geometric mean reduced by 79 and 71%, respectively). However, differences in axitinib pharmacokinetics were not observed between Japanese and Caucasian subjects (geometric mean ratios for axitinib treatment alone for AUCinf and Cmax were 103 and 96%). ABSTRACT.CONCLUSIONS: The results support a common axitinib starting dose in both populations. Potent inducers of drug-metabolizing enzymes reduce axitinib exposure and dose adjustments may be needed for optimal efficacy. BODY.INTRODUCTION: Axitinib (AG-013736) is an oral, potent, and selective inhibitor of vascular endothelial growth factor receptors 1, 2, 3 [1]. Early clinical trial experience in over 500 cancer patients has demonstrated that it has activity and is well-tolerated as a single agent [2–4] and in combination with various chemotherapeutic regimens [5, 6]. Axitinib is currently in phase III development for advanced renal cell carcinoma and in phase II development for various other tumor types. Clinically, axitinib is administered 5 mg twice daily as an oral starting dose and its pharmacokinetics are characterized by rapid oral absorption (with peak plasma concentrations occurring 1–2 h after dosing in the fasted state) and a terminal plasma half-life of 3–5 h. Axitinib has an oral absolute bioavailability of approximately 58% (Pfizer Inc., Data on file 2008) with generally linear pharmacokinetics [7]. Axitinib is primarily metabolized by cytochrome P450 (CYP) 3A4 and to a lesser extent by CYP1A2, CYP2C19 and uridine diphosphate glucuronosyltransferase (UGT) 1A1 [6]. Therefore, axitinib plasma exposure has the potential to be affected by the co-administration of drugs that inhibit or induce CYP3A. This has been previously observed with the co-administration of CYP3A inhibitor ketoconazole, which increased the area under the plasma concentration–time curve from time 0 to 24 h (AUC(0–24)) and maximal plasma concentration (Cmax) of axitinib approximately 2- and 1.5-fold, respectively [8]. Rifampin is a known potent CYP3A4 inducer along with other drug-metabolizing enzymes including CYP1A2 and UGT1A1, and is hence likely to affect the pharmacokinetics of axitinib [9–11]. In addition, subjects who are homozygous for UGT1A1*28 variant gene have the potential for reduced glucuronidation of axitinib. Due to differences in the frequency of UGT1A1*28 genotype between Caucasians (12%, range 9–16%) and Japanese (4%, range 1–6%) [12–22], this may also contribute to differences in the pharmacokinetics between these ethnicities. The primary objective of this phase I study was to investigate potential pharmacokinetic interaction in healthy volunteers when axitinib is administered in combination with rifampin. Secondary objectives included: (1) assessing potential inter-ethnic differences between Caucasian and Japanese subjects, (2) evaluating the effect of UGT1A1 genotype on the pharmacokinetics of axitinib, (3) evaluating the safety and tolerability of axitinib alone and when co-administered with rifampin. BODY.METHODS.SUBJECTS: This study was conducted in compliance with the ethical principles originating in or derived from the Declaration of Helsinki (Revised Edinburgh, 2000) and in accordance with all International Conference on Harmonization and Good Clinical Practice guidelines. The final protocol and informed consent documentation were reviewed and approved by the Institutional Review Board at the study center (Radiant Covance, Hawaii, USA). Key inclusion criteria included: female of non-childbearing potential or male, age 18–55 years; Caucasian or first-generation Japanese (both parents of Japanese descent, born in Japan and resident outside of Asia for ≤5 years); and body mass index 18–28 kg/m2 with total body weight >45 kg (99 lb). Key exclusion criteria included: clinically significant medical or surgical conditions with the potential to interfere with the absorption, distribution, metabolism or excretion of the study drugs; history of alcohol consumption >14 drinks/week for men or 7 drinks/week for women within 6 months of screening; smoker; electrocardiogram (ECG) abnormality; blood pressure >140/90 mmHg on two evaluations at least 10 min apart; and treatment with an investigational drug or any known CYP450 enzyme-inducing/-inhibiting agents or herbal supplements within 30 days prior to first dose of study medication. Subjects were to abstain from using prescription and non-prescription drugs (other than acetaminophen as deemed necessary), vitamins and dietary supplements within 7 days prior to the first dose of study medication and throughout the study. BODY.METHODS.TREATMENTS: This single-center, open-label study involved subjects being assigned to two separate treatment sequences according to a two-way crossover design. The two treatments involved subjects receiving single 5 mg axitinib as an oral dose on day 1 (treatment A), and subjects receiving rifampin (Rifadin®, Aventis Pharmaceuticals Inc., Bridgewater, NJ, USA) 600 mg/day orally for 9 days with a single 5 mg oral dose of axitinib co-administered on day 8 (treatment B). In both treatment arms, axitinib was administered in the morning after an overnight fast of at least 10 h. To standardize conditions, subjects were not allowed to eat or drink (except water) for at least 4 h following dosing. To ensure full compliance, all doses of study medication were administered under medical supervision at the clinic. Subjects received treatments A and B according to a crossover design. Volunteers initially receiving treatment A adhered to a 7-day washout period before commencing treatment B; those initially receiving treatment B adhered to a 21-day washout before treatment A began. Washout was required to allow elimination of axitinib from plasma and for recovery from rifampin-mediated enzyme induction. BODY.METHODS.STUDY ASSESSMENTS.SAMPLE COLLECTION: Blood samples (5 mL) for determination of axitinib plasma concentration were collected pre-dose and at 0.5, 1, 1.5, 2, 4, 6, 8, 12, 16, 24 and 32 h following axitinib administration on both treatment arms. BODY.METHODS.STUDY ASSESSMENTS.BIOANALYTICAL METHODS: Plasma concentrations of axitinib were measured using a validated high-performance liquid chromatography with tandem mass spectrometric detection method (Charles River Discovery and Development Services; Worcester, MA, USA) [7]. The limit of quantification for the axitinib assay in plasma was 0.1 ng/mL, the between-day precision was <15.7% and bias <12.8% for quality control samples evaluated in the concentration range of interest. Pharmacodynamic evaluations consisted of the ratio of urinary 6, β-hydroxycortisol/cortisol concentrations as a marker of CYP3A4 induction [23]. The limit of quantification for the cortisol assay in urine was 2.5 ng/mL, the between-day precison was <12.5% and the bias was <15%. For 6, β-hydroxycortisol in urine, the limit of quantification was 12.5 ng/mL, the between-day precision was <8.7% and bias was <16%. The urinary 6, β-hydroxycortisol/cortisol ratio was calculated for all subjects. During treatment A, urine samples were collected pre-dose (baseline). During treatment B, urine samples were collected pre-dose (baseline) and in the morning on days 8 and 9 following combination treatment with axitinib and rifampin. Urine concentrations of cortisol and 6, β-hydroxycortisol were determined using a validated high-performance liquid chromatography method with tandem mass spectrometric detection (ABC Laboratories; Columbia, MO, USA). BODY.METHODS.STUDY ASSESSMENTS.GENOTYPING: To assess possible differences in metabolism, subjects were genotyped for metabolizing enzymes including UGT1A1. DNA was extracted from whole blood using Qiagen's QIAamp kit. The polymerase chain reaction was used to amplify DNA samples. The Sequenom MassArray was used to perform the genotyping of the UGT1A1*28 (Sequenom Discovery) genotyping assay. The genotypes were determined with MassARRAY RTTM software (SpectroTYPERTM Version 3.4, Sequenom). The UGT1A1*28 genotype was investigated as an exploratory variable to determine the potential correlation with pharmacokinetic variability. Specifically, the variants of UGT1A1 to be assessed included UGT1A1*60, UGT1A1-3156 (−3156 G→A nucleotide change), UGT1A1 promoter TA repeat (*28, *36, *37), UGT1A1*6 and UGT1A1*27. BODY.METHODS.STUDY ASSESSMENTS.SAFETY: Adverse event (AE) monitoring, vital signs (heart rate and blood pressure), 12-lead ECG recordings, and clinical laboratory tests were carried out. AE severity was based on the National Cancer Institute Common Terminology Criteria for Adverse Events criteria; relationship to treatment was determined by the investigator. BODY.METHODS.PHARMACOKINETIC AND STATISTICAL EVALUATIONS: The pharmacokinetic evaluation of axitinib was performed with WinNonlin (Version 4.01, Pharsight, Mountain View, CA) using a non-compartmental approach. Pharmacokinetic parameters estimated for orally administered axitinib included AUC from time zero extrapolated to infinity (AUCinf) and from time zero to the time for the last quantifiable concentration (AUClast), Cmax, time to Cmax (Tmax), terminal half-life, oral clearance and apparent volume of distribution. Pharmacokinetic parameters were summarized by treatment, ethnicity and genotype. For the parameters AUCinf, AUClast and Cmax, a statistical analysis to determine the estimated ratio of adjusted geometric means and the associated 90% confidence interval (CI) was performed. A sample size of 32 subjects was determined to have sufficient power to detect treatment differences in AUCinf and Cmax. An additional eight subjects were enrolled to account for possible dropouts. BODY.RESULTS.SUBJECTS: A total of 40 male subjects (Caucasian, n = 20; Japanese, n = 20) with a mean (±standard deviation) age of 29 (±7.6) years (range 21–52 years) and a mean weight of 72.3 (±9.5) kg (range 54.0–96.7 kg) were enrolled. During the enrollment period of 6 months for this study, no women of non-childbearing potential who otherwise met study entry criteria were found; hence, only male subjects eventually were enrolled in this study. One subject failed to complete both treatment periods for reasons unrelated to treatment (failed drug test) but was included in the evaluable population. Hence, all subjects (n = 40) received at least one axitinib 5 mg oral dose and 39 subjects received axitinib and rifampin combination therapy. BODY.RESULTS.CYP3A4 INDUCTION: The mean urinary 6, β-hydroxycortisol/cortisol ratio was 4.2 ± 3.5 at baseline and increased to 18.6 ± 15.8 after 8 days of rifampin dosing. After cessation of rifampin treatment, the mean 6, β-hydroxycortisol/cortisol ratio returned to near baseline values (4.6 ± 3.4). Similar results were observed in Caucasian and Japanese subjects. In Caucasian subjects, mean 6, β-hydroxycortisol/cortisol ratio was 4.5 ± 2.6 at baseline and increased to 15.3 ± 9.1 after 8 days of rifampin dosing and in Japanese subjects the mean ratio was 4.0 ± 4.3 at baseline and 21.7 ± 20.0 after 8 days of rifampin dosing. BODY.RESULTS.PHARMACOKINETICS: The plasma concentration–time profiles of axitinib in the absence and presence of rifampin are shown in Fig. 1a. Axitinib plasma concentrations were reduced when co-administered with rifampin. Axitinib pharmacokinetic parameters for the entire evaluable subject population are reported in Table 1 and Fig. 1b, c. Geometric means of AUCinf and AUClast for axitinib were decreased by 79 [geometric mean ratio 21% (90% CI 18–24)] and 80% [geometric mean ratio 20% (90% CI 17–23)] respectively, when co-administered with rifampin. The geometric mean for axitinib Cmax was reduced by 71% when co-administered with rifampin [geometric mean ratio 29% (90% CI 24–35)]. The axitinib plasma half-life appeared to be decreased in the presence of rifampin (Table 1); however this should be interpreted with caution since this is likely due to axitinib plasma concentrations falling below the limit of quantification at later time-points in the presence of rifampin which resulted in poorly estimated terminal half-life in many subjects.Fig. 1a Mean (±standard deviation) axitinib plasma concentration profile in subjects administered axitinib alone (open symbols) (n = 40), and co-administered axitinib and rifampin (closed symbols) (n = 39) (Inset same plot with y-axis on log scale). b Comparative box plot of axitinib area under the plasma concentration–time curve from 0 h to infinity (AUCinf; ng h/mL) in all subjects in the absence (n = 40) and presence (n = 39) of rifampin. c Comparative box plot of axitinib maximal plasma concentration (Cmax; ng/mL) in all subjects in the absence (n = 40) and presence (n = 39) of rifampin. Box plot represents 25th and 75th percentiles, whiskers extend to 5th and 95th percentiles. Median is indicated by line within box. Circles represent values for individual subjectsTable 1Mean axitinib plasma pharmacokinetic parametersParameter (units)aAxitinib 5 mg (n = 40)Axitinib 5 mg + rifampin 600 mg/day (n = 39b)Ratio of adjusted meansd, % (90% CI)AUCinf (ng h/mL)190 (152–238)40 (31–53)c (n = 38)21 (18–24)AUClast (ng h/mL)187 (149–235)37 (28–50)20 (17–23)Cmax (ng/mL)50.1 (39.5–63.7)14.5 (10.6–19.8)29 (24–35)Tmax (h)1.5 (0.5–4)1.5 (1–4)t1/2 (h)7.7 (145)2.5 (188)c (n = 38)CL/F (L/h)26.3 (21.0–32.9)123.5 (95.0–160)c (n = 38)Vz/F(L)199 (146–271)296 (211–413)c (n = 38)AUCinf area under the plasma concentration–time curve from 0 h to infinity, AUClast area under the plasma concentration–time curve from 0 h to last quantifiable concentration, CI confidence interval, CL/F apparent oral clearance, Cmax maximal plasma concentration following single-dose administration, t1/2 plasma elimination terminal half-life, Tmax time to maximal plasma concentration, Vz/F apparent volume of distribution during the elimination phaseaGeometric means (95% CI) for AUCinf, AUClast, Cmax, CL/F and Vz/F, arithmetic means (%CV) for t1/2 and median (range) for Tmaxbn = 39, unless otherwise specifiedcn = 38 because parameter could not be estimated for one subject with a non-estimable elimination half-lifedRatio of axitinib in combination with rifampin versus axitinib alone BODY.RESULTS.PHARMACOKINETICS.EFFECT OF ETHNICITY ON AXITINIB PHARMACOKINETICS: Axitinib pharmacokinetics were comparable between Caucasian and Japanese subjects in the presence and absence of rifampin (Table 2; Figs. 2, 3). The geometric mean ratio (Japanese versus Caucasian) for axitinib AUCinf and Cmax was 103 (90% CI 71–151) and 96% (90% CI 64–144) respectively. When axitinib was administered alone, median peak plasma concentrations of axitinib were achieved in 1.5 h (range 1.5–2 h) after dosing in Caucasian volunteers and 1.75 h (range 0.5–2 h) in Japanese subjects as shown in Fig. 2a. In subjects administered axitinib and rifampin in combination, axitinib mean peak plasma concentrations were observed at 1.5 h after dosing in both ethnic groups (Fig. 2b). Figure 3a, b compares the AUCinf and Cmax, respectively, of axitinib in Caucasian and Japanese volunteers in the presence and absence of rifampin and demonstrate no differences. Inter-subject variability for axitinib AUCinf measured by coefficient of variation was similar in Japanese and Caucasian subjects at 65 and 54%, respectively.Table 2Axitinib pharmacokinetic parameters for each ethnic groupParameter (units)Axitinib 5 mg (n = 40), adjusted geometric meanAxitinib 5 mg + rifampin 600 mg/day (n = 39), adjusted geometric meanRatio of adjusted means, %b (90% CI)Caucasian AUCinf (ng h/mL)1873820 (16–25) Cmax (ng/mL)51.114.628.6 (21.6–37.7) t1/2 (h)a9.41.6N/AJapanese AUCinf (ng h/mL)1934121 (17–26) Cmax (ng/mL)49.214.529.4 (22.4–38.7) t1/2 (h)a6.03.3N/AAUCinf area under the plasma concentration–time curve from 0 h to infinity, AUClast area under the plasma concentration–time curve from 0 h to last quantifiable concentration, CI confidence interval, Cmax maximal plasma concentration following single-dose administration, NA not applicableaArithmetic meanbRatio of axitinib in combination with rifampin versus axitinib aloneFig. 2a Mean (±standard deviation) axitinib plasma concentration profile in Japanese (closed symbols) (n = 20) and Caucasian (open symbols) (n = 20) subjects after administration of axtitinib, 5 mg single dose, alone (Inset same plot with y-axis log scale) b Mean (±standard deviation) axitinib plasma concentrations observed over time in Japanese (closed symbols) (n = 20) and Caucasian (open symbols) (n = 19) subjects in the presence of rifampin (Inset log scale)Fig. 3Comparative box plot of axitinib a area under the plasma concentration–time curve from 0 h to infinity (AUCinf; ng h/mL) and b maximal plasma concentration (Cmax; ng/mL) in Japanese (n = 20) and Caucasian (n = 20) subjects in the absence and presence of rifampin. Box plot represents 25th and 75th percentiles, whiskers extend to 5th and 95th percentiles. Median is indicated by line within box. Circles represent values for each subject BODY.RESULTS.PHARMACOKINETICS.EFFECT OF UGT1A1 GENOTYPE ON AXITINIB PHARMACOKINETICS: During the 6-month enrollment period of the study, recruitment was kept open in an effort to enroll at least three Caucasian and three Japanese subjects who were variant for the UGT1A1*28 allele. Of the 40 subjects eventually recruited over the enrollment period, three Caucasian and one Japanese subject were homozygous variant for UGT1A1*28. In both Caucasian and Japanese subjects, pharmacokinetic parameters (AUCinf and Cmax) were similar for subjects with all UGT1A1*28 genotypes as shown in Fig. 4a and b respectively.Fig. 4Comparative box plot of axitinib a area under the plasma concentration–time curve from 0 h to infinity (AUCinf, ng h/mL) b maximal plasma concentration (Cmax, ng/mL) in UGT1A1*28 wild type (6/6, n = 23), heterozygous (6/7, n = 13), and variant (7/7, n = 4) subjects. Data provided in this figure are following administration of axitinib alone (i.e., in the absence of rifampin). Box plot represents 25th and 75th percentiles, whiskers extend to 5th and 95th percentiles. Median is indicated by line within box. Circles represent values for each subject BODY.RESULTS.SAFETY: None of the AEs were treatment-related or resulted in treatment discontinuation, dose reduction, or death during the study. No clinically relevant mean changes in hematology, vital sign or ECG findings were noted in the overall safety population or in either ethnic groups. BODY.DISCUSSION: The results of this study demonstrate three important findings. First, as expected, axitinib exposure is decreased when concomitantly administered with rifampin in healthy volunteers. Second, there is no difference in the plasma pharmacokinetics of axitinib in healthy Caucasian and Japanese subjects. Third, UGT1A1*28 polymorphism does not appear to affect the pharmacokinetics of axitinib. The ratio of urinary 6, β-hydroxycortisol/cortisol has been used previously as a measure of CYP3A4 induction [23]. In this study, the ratio of urinary 6, β-hydroxycortisol/cortisol concentrations in subjects was consistent with maximal induction of CYP3A4 and returned to normal following washout after the end of rifampin dosing. The maximally observed effect was similar between Japanese and Caucasian subjects after 8 days of rifampin dosing. In addition, there was no difference in the 6, β-hydroxycortisol/cortisol ratios between Caucasian and Japanese subjects. These results are consistent with effective CYP3A induction with rifampin in both Caucasian and Japanese subjects. Results from this study indicate that geometric mean AUCinf and Cmax for axitinib were decreased by 79 and 71%, respectively, when co-administered with rifampin. Induction of CYP3A4 with rifampin 600 mg/day has been demonstrated in previous studies using the ratio of 6, β-hydroxycortisol/cortisol in urine [23]. In addition, rifampin has also been shown to induce UGT1A1 and other drug-metabolizing enzymes in clinical studies [11, 24]. Induction of axitinib metabolism as mediated by CYP3A4 in healthy, non-smoking volunteers resulted in a substantial decrease in axitinib plasma concentration. This study therefore provides the lower bound for (minimum) plasma concentrations expected following an oral dose of 5 mg axitinib, i.e., in the presence of a potent metabolic inducer. First-generation Japanese subjects recruited in this study were required to have been born in Japan, not lived outside of Asia for more than 5 years, and should have had both parents of Japanese descent. Ethnic differences in axitinib pharmacokinetics between Japanese and Caucasian subjects were minimal; the geometric mean ratios for axitinib AUCinf and Cmax were not statistically different between Caucasian and Japanese subjects and pharmacokinetic parameters were similar between the subjects, indicating that these ethnicities do not contribute significantly to the axitinib pharmacokinetic variability observed in this study. In addition, safety data were similar for Caucasian and Japanese subjects. Previous in vitro studies with human liver microsomes have indicated the involvement of UGT1A1 in the glucuronidation of axitinib, and so the effect of the UGT1A1*28 genotype on axitinib pharmacokinetics was also explored in Caucasian and Japanese subjects. Subjects who are variant for UGT1A1*28 have the potential for reducing glucuronidation of axitinib. The frequency of variant UGT1A1*28 is 12% (range 9–16%) for Caucasians and 4% (range 1–6%) for Japanese populations [12–22]. Although this study was not statistically powered to detect genotype-mediated changes, axitinib pharmacokinetic parameters were similar across UGT1A1*28 genotypes and ethnicity, implying that this genotype does not likely contribute to the axitinib pharmacokinetic variability observed in this study. Single-dose axitinib, alone or in combination with rifampin, was well tolerated by healthy volunteers in this study. Combination treatment with rifampin for 8 days did not produce any drug-related AEs. All observed AEs were deemed not related to study drug by the investigator. No serious or clinically significant AEs were reported. BODY.CONCLUSIONS: A reduction in axitinib exposure is observed when co-administered with repeated doses of rifampin. Concomitant treatment with axitinib and potent inducers of CYP3A4 would require axitinib dose adjustment for optimal clinical efficacy. The similarity of axitinib pharmacokinetics in Caucasian and Japanese subjects supports a common starting dose and regimen in these populations.

TITLE: Taekwondo Training Improves Mood and Sociability in Children from Multicultural Families in South Korea: A Randomized Controlled Pilot Study ABSTRACT: Purpose: Children from multicultural families face physical, social, mental, and intellectual hurdles; however, relative interventions are lacking in South Korea (hereafter Korea) in this regard. The purpose of this study was to investigate the effects of regular Taekwondo (TKD) training on physical fitness, mood, sociability, and cognitive functions in these children. Methods: This study included 30 children from multicultural families in Korea who were randomly assigned to a TKD group (n = 15) and control group (n = 15). The children in TKD group underwent 16 weeks of TKD training once a week for 60 min. Each participant underwent a basic fitness test and sociability questionnaire before and after the intervention. Furthermore, we examined the changes in the mood and cognitive function by determining the profile of mood states (POMS), and Stroop color and word test, respectively. Results: Results of the Stork test of balance were significantly higher in the TKD group after intervention (p < 0.05). In terms of sub-variables, POMS, tension, and depression scores were significantly lower (p < 0.05) after the intervention, while the vigor score was significantly higher in the intervention group than those in the control group (p < 0.05). Furthermore, sociability and 'being left out' score, a sub-variable of sociability, was significantly lower (p < 0.05) after the intervention, while sociability score was significantly higher (p < 0.05). Conclusions: Our findings suggest that participation in regular TKD training can be effective for balanced improvements in variables of basic fitness and that it exerts a positive effect on the mood and development of sociability. BODY.1. INTRODUCTION: The number of children from multicultural families due to international marriages and immigrant workers is rapidly increasing in South Korea (hereafter referred to as Korea). In 2016, the number of children from multicultural families was estimated to be approximately 200,000, a significant increase from the estimated 25,000 in 2006 [1,2]. Furthermore, it was estimated that 113,506 were pre-school children (age < 6 years, 56.4%), 56,768 were elementary school students (aged 7–12 years, 28.3%), and 31,059 were middle- and high school students (aged 13–18 years, 15.4%) [2], indicating that the number of children from multicultural families in Korea will increase exponentially over the next 5–10 years. However, it has been reported that multicultural families and the increasing number of children are likely to experience severe difficulties in comparison with mono-cultural Korean children due to different cultural and historical backgrounds, language, culture, and educational methods, communication problems, and low socioeconomic status of the parents [1]. According to recent studies, bi-ethnic adolescents (adolescent children born of marriages of migrants or naturalized Koreans) can be vulnerable to violence at school as Koreans have strong intolerance towards other ethnicities, suggesting that experiencing violence at school and being left out will be closely related to depression [3,4]. Furthermore, Kim et al. suggested that the low acculturation level of immigrant children and adolescents is connected to behavioral problems, such as adolescent delinquency [5]. Moon and An further reported that these children are likely to demonstrate hostility, phobic anxiety, and angering, which are tendencies of children from multicultural families to express anger [6]. The problems faced by children from multicultural families in Korea are not limited to social and emotional aspects. For example, it has been reported that children from multicultural families are vulnerable to verbal development delay because of communication problems with mothers who have difficulty communicating in Korean, resulting in limitations in communication with their peers at school, thus exerting a bad influence on cognitive development and comprehension [7]. Among the many combat sports, the Korean martial art of Taekwondo (TKD) is a popular sport that is characterized by high speed, high tension, and full-contact combat. It has been estimated that more than eighty million people worldwide participate in this sport [8,9,10] with a wide range in the age of the participants in more than 180 countries, especially children and adolescents. It has also been reported that the number of children participating in TKD is increasing globally [11,12,13]. Several recent studies have reported the significant health benefits of regular TKD training [11,13,14,15]. For example, Fong and Ng (2011) critically reviewed 23 TKD-related papers and investigated the effect of regular TKD training on the improvements in physical fitness [11]. Their analyses revealed no conclusive results in relation to the improvements in anaerobic fitness or muscle strength; however, the results did indicate improvements in aerobic capacity, body composition, fat loss, and flexibility [11]. Since then, Kim et al. reported that just 12 weeks of TKD training can induce positive improvements in terms of body composition and flexibility in female adolescents as well as a gain in skeletal muscle fitness [14]. Furthermore, Lee and Kim reported that TKD training can be effective in improving physical fitness and growth and development in children [13]. By using functional magnetic resonance imaging to observe the brains of children who participated in regular TKD training, Kim et al. reported a significant improvement in the activation of the brain, implying that TKD training can have a positive effect on the growth and development, fitness, and certain aspects of the health of the brain including cognitive function [15]. However, most TKD training-related studies have been limited to the physiological changes in children, although such studies have confirmed a positive correlation between exercise/physical activity and cognition, academic achievements, behavior, and psychosocial function in children [16]. Thus, the present study aimed to investigate the effect of TKD training on physical fitness, mood, sociability, and cognitive function in children from multicultural families who have relatively poor health-related factors compared with mono-cultural Korean children. BODY.2. METHODS.2.1. SUBJECTS: This study included 30 children from multicultural families in Korea. Their fathers were Korean and their mothers were Chinese, Vietnamese, or Japanese. The children were randomly assigned to a control group (n = 15) and TKD group (n = 15). They attended elementary school at grades 4–6 and did not participate in regular exercises except for the physical education class at school (2–3 times a week) and did not have any mental or physical illnesses. All the participants were born and raised in Korea. The characteristics of the subjects are shown in Table 1. We excluded children who already had experience with TKD or lacked the ability to communicate in interviews. The study purpose, method, and procedures were explained to the parents of all subjects and they signed a consent form that included information such as voluntary withdrawal of participation. The study protocol was approved by the Institutional Review Board of Dong-A University (ID: 2-104709-AB-N-01-201609-HR-035-06). BODY.2. METHODS.2.2. PRE-INTERVENTIONAL AND POST-INTERVENTIONAL TESTING: Testing was conducted before (pre-) and after (post-) 16 weeks of intervention by selecting indices that could analyze the physique, fitness, mood state, sociability, and cognitive function of each subject. Physique was assessed by the height, weight, body mass index (BMI), and percentage of body fat. Height was measured using a stadiometer (SECA213, SECA, Hamburg, Germany), while weight, BMI, and the percentage of body fat were measured using a body composition analyzer (Inbody720, Biospace, Seoul, Korea). Physical fitness was measured by cardiorespiratory endurance (VO2max), strength (grip strength and back strength), flexibility (sit-and-reach), power (Sargent jump), and balance (Stork test). Specifically, the cardiorespiratory endurance was measured by estimating VO2max with the Nemeth protocol on a treadmill (Q65, Quinton, Milwaukee, WI, USA) while wearing a wireless heart rate measuring equipment (Polar-a5, Polar, Kempele, Finland) [17]. The formula used for cardiac endurance was as follows: [VO2max = −1772.81 + 318.64 × gender (girl = 0, boy = 1) + 18.34 × weight (kg) + 24.45 × height (cm) − 8.74 × 4 min heart rate − 0.15 × weight (kg) × heart rate difference + 4.41 × treadmill speed (mph) × heart rate difference]. Grip and back strength were measured twice up to tenth of a kg using a digital grip (GRIP-D, Takei, Tokyo, Japan) and back strength (BACK-D, Takei, Tokyo, Japan) measuring equipment, respectively, and the highest score was recorded for analysis. Sit-and-reach, Sargent jump, and Stork test were measured twice using basic fitness measuring equipment (Helmas-III, O2run, Seoul, Korea), and the best score was recorded for analysis. The mood state was estimated using the Korean Version of the Profile of Mood State-Brief (K-POMS-B) described by Yeun and Shin-Park, which is an adaptation of the profile of mood states (POMS) originally described by McNair et al. for Korea, and verified its reliability and validity [18,19]. This questionnaire consists of a total of 30 items, which are rated on a 5-point Likert scale (0 = not at all, 1 = a little, 2 = moderately, 3 = quite a bit, and 4 = extremely) and is divided into factors of 6 sub-areas: tension-anxiety, depression-dejection, anger-hostility, vigor-activity, fatigue-inertia, and confusion-bewilderment. Sociability was evaluated using the sociability measuring model for juveniles developed by Lim and Lee that consists of 24 items, such as leadership, group life, being left out, sociability, expressiveness, and patience, each of which consists of four items [20]. In this sociability measurement model, the higher the sub-area score, the better the sociability, and the lower the sub-area score, the worse the sociability, except for the 'being left out' item. Cognitive function was measured using the Korean version of the children's version of the Stroop color and word test, which was originally developed by Golden et al. and adapted for Koreans by Shin and Park [21,22]. The Stroop color and word test consists of three conditions: word reading (Word), color reading (Color), and color-word reading (Color-Word). These conditions are used to evaluate the executive function of the frontal lobe that is involved in learning, attention, and problem-solving skills. The higher the recorded score, the better the cognitive function. Each condition consists of 100 items per page with five columns and 20 rows. In the word reading condition, "Red, Blue, Green" were randomly written in black ink. In the color reading condition, "XXXX" was written in blue, red, and green color ink, which was randomly assigned. The color-word condition consisted of 100 colored words that were written using the same word and color or a different word and color. The subject was asked to read the words aloud within 45 s and to read as many as possible under each condition (word, color, and color-word). In this study, the raw score of the subject that was correctly read aloud for each condition within the allocated time limit was recorded. BODY.2. METHODS.2.3. TAEKWONDO-BASED TRAINING INTERVENTION METHOD: TKD-based training consisted of a training session of 60 min: 5 min of warmup and cool-down and 50 min of the main exercise. This session was performed once a week at 50–80% HRmax for 16 weeks, as shown in Table 2. All interventions were conducted by a TKD expert instructor by demonstration and coaching, and the main exercise consisted of 10 min of basic fitness such as shuttle runs, the Burpee test, and leapfrog, 5 min of six basic TKD motions and body punching in the horse-riding stance, 10 min of Poomsae from the 1st to 8th chapters, 10 min of kicking sessions with basic kicking and steps as well as mitt kicks, and 15 min of Taekwon gymnastics. BODY.2. METHODS.2.4. STATISTICAL ANALYSES: The mean and standard deviation of all variables were calculated by SPSS Version 23.0 for Windows (IBM Corp., Armonk, NY, USA). Two-way repeated measures analysis of variance (ANOVA) was subsequently performed to investigate the differences in the dependent variable group (between the control and TKD groups) and time (before and after intervention). Independent and dependent t-tests were also conducted to identify statistically significant interactions. Statistical significance was set at 0.05. BODY.3. RESULTS.3.1. CHANGES IN PHYSIQUE AND FITNESS: Changes in physique (height, weight, BMI, and percentage of body fat) and fitness (VO2max, grip strength, back strength, sit-and-reach, Sargent jump, and Stork test) before and after the intervention in the control and TKD groups are shown in Table 3. Two-way repeated measures ANOVA for physique and fitness revealed a significant difference in the interaction for the Stork test score (F = 5.027, p = 0.033) between the intervention time (before and after) and groups (control and TKD). According to the post-hoc test, the Stork test score was not significantly different before and after the intervention in the control group. In contrast, the TKD group showed significantly improved sit-and-reach scores before and after the intervention (p < 0.05); however, except for the Stork test, there were no significant differences in terms of height, weight, BMI, percentage of body fat, VO2max, grip strength, back strength, sit-and-reach, or Sargent jump (p > 0.05). BODY.3. RESULTS.3.2. CHANGES IN MOOD STATE: Changes in the mood state before and after the intervention in the control and TKD groups are shown in Table 4. Two-way repeated measures ANOVA for mood state revealed no significant interaction between intervention time (before and after) and group (control and TKD) for tension (F = 7.127, p = 0.012), depression (F = 6.964, p = 0.013), and vigor (F = 7.258, p = 0.012). Post-hoc tests showed no significant difference before and after the intervention in the control group. In contrast, the TKD group showed a significant reduction in tension and depression after the intervention and a significant increase in the vigor score (p < 0.05). Furthermore, after the intervention, the TKD group had a significantly higher vigor score than did the control group (p < 0.05). However, there were no significant differences in the scores for anger, fatigue, or confusion (p > 0.05). BODY.3. RESULTS.3.3. CHANGES IN SOCIABILITY: Changes in sociability before and after the intervention in the control and TKD groups are shown in Table 5. Two-way repeated measures ANOVA for sociability revealed a significant interaction for being left out (F = 10.036, p = 0.004) and sociability (F = 5.090, p = 0.032) between the intervention time (before and after) and group (control and TKD group). Post-hoc tests showed no significant difference before and after the intervention in the control group. In contrast, the TKD group showed a significantly lower 'being left out' score after than before the intervention and a significantly higher sociability score (p < 0.05). However, there was no significant difference in terms of leadership, group life, patience, or expressiveness (p > 0.05). BODY.3. RESULTS.3.4. CHANGES IN COGNITIVE FUNCTION: Changes in cognitive function before and after the intervention in the control and TKD groups are shown in Table 6. Two-way repeated measures ANOVA for cognitive function showed no significant interaction for the word (F = 0.971, p = 0.333), color (F = 2.301, p = 0.140), or color-word (F = 0.260, p = 0.614) tests between the intervention time (before and after) and group (control and TKD). BODY.4. DISCUSSION: Numerous recent studies have proved that regular physical activity such as exercising is the most effective, safe, accessible, and inexpensive strategy for skeletal development [23] and is essential for the improvement of body composition and health-related fitness in children and adolescents [24,25]. TKD training is also expected to have a positive effect on growth and development, as well as improve the general fitness, such as body composition, aerobic capacity, strength, power, and flexibility in both children and adolescents [11,13,14]. However, the present study revealed no significant difference in any of the variables, except for the Stork test score, related to physique and physical fitness, indicating that these results are from the weekly exercise frequency of TKD training. According to the American College of Sports Medicine guidelines, exercise is required 3–5 times a week or at least more than three times a week (at least more than twice a week for flexibility) in order to improve various aspects of fitness, such as cardiorespiratory endurance, strength and muscular endurance, and flexibility [26]. Furthermore, by reviewing 573 exercise-related papers for evidence of improvements in bone mineral accumulation, which is closely associated with growth in children and adolescents, Hind and Burrows reported that a frequency of exercise of three sessions per week was effective [27]. Recent studies have reported improvement in fitness and body composition in response to TKD training and suggested an exercise frequency of at least twice a week [14,28]. Kim et al. reported that TKD training twice a week for 12 weeks in female adolescents led to improvements in isokinetic strength, standing long jump, and sit-and-reach performance, as well as a significant reduction in the percentage of body fat and fat body mass [14]. Furthermore, our study showed that five sessions of TKD training per week were effective in improving the cardiorespiratory endurance (VO2max) in children [28]. TKD training intervention once a week does not appear to induce sufficient stimulation to develop the physique and improve fitness. The Stork test, which determines balance, demonstrated significant improvements after 16 weeks of TKD training. These results support those of previous studies that reported an improvement in balance following TKD training, indicating that the unique motions associated with TKD effectively improve balance [29,30]. Pons et al. reported that TKD includes various movements, such as jumping, gymnastics, and weight shifting and kicking exercises, and indicated that these highly dynamic movements can work effectively to improve the balance [30]. Furthermore, previous studies have reported that frequent jumps and spinning kicks in TKD training can stimulate the development of the vestibular system, thus supporting the present findings. Multiple previous studies have reported that regular exercise is associated with physiological benefits, including an improvement in fitness and growth and development of the body as well as mental and social development in children during their major growth periods [31,32]. For example, Biddle and Asare suggested a relationship between physical activity and mental health in young people because physical activity can alleviate anxiety, although specific evidence for this relationship is limited [31]. Garcia et al. further reported that there is a close relationship between moderate-to-vigorous physical activity and social factors such as sociability in children [32]. Notably, it has been reported that participation in TKD training during childhood can be effective for emotional and social development by alleviating anxiety, promoting independence and leadership, and controlling aggression [15,33]. In the present study, we examined the changes in mood state and sociability in response to TKD training in children from multicultural families in Korea using a sociability questionnaire that examined the POMS, leadership, group life, being left out, patience, sociability, and expressiveness. The present study showed that TKD led to significant reduction in tension and depression scores (sub-variables of POMS) and significant increases in the vigor score. Additionally, the 'being left out' score, a sub-variable of sociability, was significantly reduced, while the sociability score was significantly increased. This suggests that TKD training can be effective in improving the mood state and developing sociability in children from multicultural families and supports earlier studies that suggested that TKD exercise improved mood state, and that traditional TKD training was effective in enhancing sociability of adolescents [34,35,36]. For example, Toskovic reported that dynamic TKD practice induced a positive change in the mood state of 20 university students who showed significant improvements in tension, depression, anger, fatigue, confusion, and vigor, which are all sub-variables of POMS [35]. Trulson assigned juvenile delinquents to a traditional TKD group and a modern martial art (martial art as a competitive sport) group and trained them for six months [36]. The results revealed that the TKD group showed reduced aggression and anxiety in addition to improvements in social ability and self-esteem, indicating that TKD is based on self-control and self-defense while featuring key characteristics such as respect, humility, responsibility, perseverance, and honor [34,36]. This study also suggested that the moral cultural effect of TKD training could work effectively to improve the mood state and sociability in children from multicultural families. In contrast, despite some previous studies that reported improvements in cognitive function secondary to regular TKD training [37,38], we observed no significant changes in the Stroop color and word test score, which were the assessment scales for cognitive function used in this study. It is believed that the lack of weekly TKD training would not lead to an improvement in aerobic fitness and fail to induce increases in neuroplasticity-related growth factors such as insulin-like growth factor (IGF)-1, vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF). The fact that these factors were not directly examined represents a key limitation of the present study. Previous studies have reported that increased expression of neurotrophic and growth factors during exercise training is likely to contribute to improvements in cognitive function, although the specific mechanisms involved are not clearly understood [39,40], thus verifying the fact that the improvement in aerobic fitness is related to the increased expression of neuroplasticity-related growth factors [41,42,43]. In particular, IGF-1, which regulates the expression of VEGF and BDNF, showed a significant positive correlation with the level of aerobic fitness [43] and our own previous study showed that five sessions of TKD training for 16 weeks was effective in improving the cognitive function as it resulted in improved aerobic fitness (VO2max) and increased levels of serum IGF-1, VEGF, and BDNF [28]. This study has additional limitations. First, the number of subjects was small as it was a pilot study in a single institution. Korean discrimination against foreigners is based on the economic power of the corresponding country as lower economic power is linked to higher neglect, discrimination, and exclusivism [44], and the subjects in this study were children from Asian multicultural families despite the fact that there is racial inequality [45] such that children from Arab and South-East Asian multicultural families also have learning disabilities due to the lack of positive educational feedback compared with children from multicultural white (US and Great Britain) families. Second, sociodemographic characteristics of the parents of the subjects such as occupation and economic statuses could not be considered even though it is reported that psychosocial adaptation such as social withdrawal, depression/anxiety, and behavior problems such as delinquency and aggression are significantly affected by the degree of social relation network and economic status (monthly income) of parents in multicultural Korean children [46]. Third, it is hard to suggest the exact mechanism of how TKD training, despite the weekly frequency, was effective in improving the mood state and sociability, and it is hard to assert whether these positive effects are from regular participation in a physical activity or TKD training alone. BODY.5. CONCLUSIONS: Our data indicate that participation in regular TKD training can be effective in improving the mood state and developing sociability in children from multicultural families. Within this cohort, however, TKD training did not result in significant changes in the physique, cardiorespiratory endurance, strength, power, flexibility, and cognitive function. Future studies are needed to investigate the positive effects of TKD training based on socio-economic status and cultural data (skin color, occupation, economic level such as monthly income, and national power and economic power of the country of origin) as variables, while simultaneous using latest technique such as functional magnetic resonance imaging for neurotransmitters and stress hormones that are involved in control of the mood, and by using a normal exercise group such as aerobic exercises as the control group.

TITLE: Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer ABSTRACT.BACKGROUND: Proliferation may predict response to neoadjuvant therapy of breast cancer and is commonly assessed by manual scoring of slides stained by immunohistochemistry (IHC) for Ki-67 similar to ER and PgR. This method carries significant intra- and inter-observer variability. Automatic scoring of Ki-67 with digital image analysis (qIHC) or assessment of MKI67 gene expression with RT-qPCR may improve diagnostic accuracy. ABSTRACT.METHODS: Ki-67 IHC visual assessment was compared to the IHC nuclear tool (AperioTM) on core biopsies from a randomized neoadjuvant clinical trial. Expression of ESR1, PGR and MKI67 by RT-qPCR was performed on RNA extracted from the same formalin-fixed paraffin-embedded tissue. Concordance between the three methods (vIHC, qIHC and RT-qPCR) was assessed for all 3 markers. The potential of Ki-67 IHC and RT-qPCR to predict pathological complete response (pCR) was evaluated using ROC analysis and non-parametric Mann-Whitney Test. ABSTRACT.RESULTS: Correlation between methods (qIHC versus RT-qPCR) was high for ER and PgR (spearman ́s r = 0.82, p < 0.0001 and r = 0.86, p < 0.0001, respectively) resulting in high levels of concordance using predefined cut-offs. When comparing qIHC of ER and PgR with RT-qPCR of ESR1 and PGR the overall agreement was 96.6 and 91.4%, respectively, while overall agreement of visual IHC with RT-qPCR was slightly lower for ER/ESR1 and PR/PGR (91.2 and 92.9%, respectively). In contrast, only a moderate correlation was observed between qIHC and RT-qPCR continuous data for Ki-67/MKI67 (Spearman's r = 0.50, p = 0.0001). Up to now no predictive cut-off for Ki-67 assessment by IHC has been established to predict response to neoadjuvant chemotherapy. Setting the desired sensitivity at 100%, specificity for the prediction of pCR (ypT0ypN0) was significantly higher for mRNA than for protein (68.9% vs. 22.2%). Moreover, the proliferation levels in patients achieving a pCR versus not differed significantly using MKI67 RNA expression (Mann-Whitney p = 0.002), but not with qIHC of Ki-67 (Mann-Whitney p = 0.097) or vIHC of Ki-67 (p = 0.131). ABSTRACT.CONCLUSION: Digital image analysis can successfully be implemented for assessing ER, PR and Ki-67. IHC for ER and PR reveals high concordance with RT-qPCR. However, RT-qPCR displays a broader dynamic range and higher sensitivity than IHC. Moreover, correlation between Ki-67 qIHC and RT-qPCR is only moderate and RT-qPCR with MammaTyper® outperforms qIHC in predicting pCR. Both methods yield improvements to error-prone manual scoring of Ki-67. However, RT-qPCR was significantly more specific. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-017-3111-1) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: The proliferative activity of individual cells is a hallmark of tumor biological aggressiveness and a key determinant of sensitivity to (neo)adjuvant chemotherapy, thus being among the principal factors guiding clinical management in primary breast cancer [1, 2]. The most widely used method to assess proliferation as well as hormone receptor expression is immunohistochemistry (IHC). Nuclear staining of the nuclear antigen Ki-67 is most widely used as a surrogate for proliferative activity. Ki-67 is present in the cell nucleus throughout all stages of the cell-cycle excluding the resting phase G0 [3]. The recently proposed St Gallen recommendations for the identification of the intrinsic subtypes using surrogate pathologic-based definitions have underlined the value of Ki-67 as a clinical tool in routine clinical practice [2]. Ki-67 is recommended as a valuable factor to distinguish between Luminal A- and B-like tumors, a fundamental distinction in clinical decision-making today. [4–6]. Despite its widespread use, driven by the premise of solving delicate therapeutic dilemmas combined with several advantages such as universal accessibility, easy application and low cost, the assessment of Ki-67, ER and PR is affected by technical and observer-based variabilities of the IHC method [7, 8]. This can be illustrated by observations, that tumors with as little as 1% positive nuclei still respond to anti-hormonal treatment, which indicates that tumor cells lacking nuclear ER staining within in these tumors do have some extend of ER expression rendering them sensitive to estrogen deprivation or estrogen receptor blockade [9]. While the clinical role particularly of ER testing by IHC is well established, the clinical utility of Ki-67 is still controversial [10]. The reason lies in a series of analytical and preanalytical factors, but also in staining interpretation and scoring [11]. Importantly, attempts to reduce the high discordance rates either by means of formal counting quantification methods (as opposed to simple eyeballing) or by training of individuals have not been successful [12]. Despite methodological concerns, overall a strong correlation of Ki-67 with breast cancer outcome is sufficiently supported, particularly by data originating from randomized clinical trials with central review of biomarkers [10]. This has also been shown in the neoadjuvant setting, where higher Ki-67 values are consistently associated with higher rates of pathological complete response (pCR) [13], a finding which reflects the fundamental link between tumor replication fraction and activity of cytotoxic agents. Still it remains difficult to identify a reasonable cut-off to predict pCR [14]. Two techniques which could circumvent the inter- or intra-observer variability of Ki-67 manual microscopic assessment are automated image analysis and reverse transcription quantitative real-time PCR (RT-qPCR). A trained human eye may achieve an excellent understanding of images and patterns, but is less accurate when it comes to quantification. Computer-based vision methods could represent a solution to this problem by offering standardized image processing and reliable quantification [15]. However with regard to the assessment of tumor proliferation, the areas of interest and staining intensities have to be defined, and measured reproducibly. Also, it is still under debate how to deal with areas of increased proliferative activity (hot spots), and if low intensity staining should be taken into account [16]. Therefore, automated estimations of Ki-67 are highly correlated with manual assessments, but it is not yet certain whether or not they can improve prediction and prognostication [17–19]. RT-qPCR has a series of widely acknowledged methodological advantages over IHC, which appear particularly beneficial in the context of reducing the bias of routine Ki67 assessment; it is quantitative by nature with much wider dynamic range, it does not require an experienced eye, and results are not affected by subjective interpretations [20]. Moreover, access to standardized protocols and automation ensures accurate performance and fast turn-around. In recent years highly specific and sensitive techniques have been developed, which allow for fast and efficient extraction of high-quality nucleic acids from FFPE overcoming the challenges posed by fixation and embedding [21]. Validation of the various available methods for the assessment of Ki-67 requires comparative testing preferably in a specifically defined clinical context. In the present study we used the neoadjuvant setting of a phase II trial randomizing patients receiving anthracyclin/taxane based standard treatment between pemetrexed and cyclophospamide in order to directly compare the assessment of Ki-67 with automatic, quantitative read-out of IHC (qIHC) and the determination of tumor MKI67 mRNA with RT-qPCR on FFPE tissue extracted RNA. BODY.METHODS.STUDY POPULATION: Core needle biopsies from 101 out of 105 patients (96,2%) with primary invasive breast cancer, that had been enrolled in the H3E-MC-S080 (NCT00149214, Sponsor: Eli Lilly and Company) neoadjuvant phase II study [1], were obtained. All patients had been diagnosed with operable (T2-T4/N0-2/M0) breast cancer at a single institution (National Center for Tumor Diseases, University-Hospital, Heidelberg) had been randomized to receive sequential anthracycline/taxane-based regimens containing either pemetrexed or cyclophosphamide in combination with epirubicin. A written informed consent for the research use of patient biological material was granted at the time of enrolment. The study was approved by the local ethics committee. Complete molecular data (including RT-qPCR data) and clinical follow-up information were available in 83 out of 105 (79%) patients (statistics data set #1). Ki-67, ER, and PR IHC slides were available in 54 (51%) patients for quantitative IHC (statistics data set #2). BODY.METHODS.ISOLATION OF TUMOR RNA: For RNA extraction from FFPE tissue, a single 10 μm curl was processed according to a commercially available bead-based extraction method (RNXtract® kit; BioNTech Diagnostics GmbH, Mainz, Germany). In brief, a lysis buffer was used to liquefy FFPE tissue slices while melting of paraffin was carried out in a thermo-mixer. Tissue lysis was accomplished with a proteinase K solution. Thereafter, lysates were admixed with germanium-coated magnetic particles in the presence of special buffers, which promote the binding of nucleic acids. Purification was carried out by means of consecutive cycles of mixing, magnetization, centrifugation and removal of contaminants. RNA was eluated with 100 μl elution buffer and RNA eluates were then stored at −80 °C until use. BODY.METHODS.GENE EXPRESSION BY RT-QPCR: The MammaTyper® is a molecular in vitro diagnostic tool for the assessment of the gene expression levels of the four cancer biomarkers that are required for the clinical management of breast cancer patients in daily routine clinical practice. Instead of using IHC to assess protein expression of HER2, ERα, PR, and Ki-67, with MammaTyper®, it is possible to measure the mRNA transcripts of the corresponding genes (ERBB2, ESR1, PGR, and MKI67), doing so by using routine FFPE material and by achieving accurate, reproducible and objective results. The gene expression data may be then integrated so as to assign individual samples to a molecular subtype of breast cancer. The mRNA expression levels of ERBB2, ESR1, PGR, and MKI67 as well as of two reference genes (REF), namely B2M and CALM2, were determined by RT-qPCR, which involves reverse transcription of RNA and subsequent amplification of cDNA executed successively as a 1-step reaction. In MammaTyper®, the 6 assays (assay = primer pair and probe specific for the respective target sequence) are duplexed into three assay mixes, each using a pair of hydrolysis probes labelled with different fluorophores for separate detection of the duplexed assays [22]. Each patient sample or control was analyzed with each assay mix in triplicates. The experiments were run on a Versant kPCR Molecular System (Siemens Healthcare, Erlangen, Germany) according to the following protocol: 5 min at 50 ° C, 20 sec at 95 ° C followed by 40 cycles of 15 sec at 95 ° C and 60 s at 60 ° C and according to MammaTyper® instructions for use 140603-90020-EU Rev 2.0. Forty amplification cycles were applied and the cycle quantification threshold (Cq) values of MKI67 and the two REF genes for each sample (S) were estimated as the median of the triplicate measurements. These were then normalized against the mean expression of the REF genes and set off against a calibrator (PC), to correct for inter-run variations (ΔΔCq method) (Livak et al. 2001). The final values were generated by subtracting ΔΔCq from the total number of cycles to ensure that normalized gene expression obtained by the test is proportional to the corresponding mRNA expression levels, a method that facilitates interpretation of data and clinicopathological correlations. The various calculation steps are summarized in the following formula:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ 40\hbox{-} \Delta \Delta \mathrm{Cq}\left(\mathrm{MKI}67\right)\mathrm{S} = 40\hbox{-} \left(\left(\mathrm{Cq}\left[\mathrm{MKI}67\right]\mathrm{S}\ \hbox{--}\ \mathrm{meanCq}\left[\mathrm{REF}\right]\mathrm{S}\right)\ \hbox{--}\ \left(\mathrm{Cq}\left[\mathrm{MKI}67\right]\mathrm{pc}\ \hbox{--}\ \mathrm{meanCq}\left[\mathrm{REF}\right]\mathrm{pc}\right)\right) $$\end{document}40‐ΔΔCqMKI67S=40‐CqMKI67S–meanCqREFS–CqMKI67pc–meanCqREFpc In 18 patients the MammaTyper® assay failed, because the required amount of RNA was not sufficient for analysis according to pre-specified criteria as described in the instructions for use. BODY.METHODS.PATHOLOGY AND IMMUNOHISTOCHEMISTRY: Tumor grading, tumor typing and immunohistochemistry (ER, PR, Ki-67) was performed on the pretreatment core biopsies on all patients. Pathological complete response (pCR) was determined on tumor resection specimens after completion of neoadjuvant chemotherapy, and was defined as no evidence of residual invasive and ductal disease in the breast and lymph nodes (ypT0,ypN0). Immunohistochemistry was performed according to previously standardized protocols on an automated IHC platform (Dako Techmate 500) with citrate buffer for antigen retrieval [23] and observing the ASCO/CAP guidelines for immunohistochemistry [7]. The following primary antibodies and corresponding dilutions were used (DakoCytomation, Glostrup, Denmark): ER (clone 1D5, 1:100), PR (clone PgR636, 1:100) and Ki-67 (MIB-1, 1:200). Slides were assessed by quantitative image analysis (qIHC) using the Aperio Image Analysis toolbox (Leica Biosystems, Nussloch, Germany). Staining intensity and percentage of positive nuclei were recorded after manually segmenting tumor from adjacent stroma. Tumors with ER/PR Remmele scores greater than 3 or positive nuclei greater than 1% were considered hormone receptor positive. BODY.METHODS.STATISTICAL METHODS: The Spearman correlation coefficient r was used as a measure of the strength and direction of the linear relationship between variables. 2×2 contingency tables were used to calculate positive percent agreement (PPA) and negative percent agreement (NPA) as a measure of agreement: PPA = 100% x a/(a + c), NPA = 100% x d/(b + d). Receiver Operating Characteristics (ROC) analysis was performed to determine the optimal cut-off for MammaTyper® gene and qIHC protein measurements with pCR as the endpoint. ROC analysis instead of comparing odds ratios to take into account the ratios of clinically relevant false positive and false negative determinations and to identify cut points for each method at clinically relevant prerequisites (i.e., detect all responding tumours). ROC analysis has been used to objectively address each method providing different result codings in a non-parametric manner [24]. On the other hand ROC analysis bears the risk of misinterpreting clinical validity when analyzing heterogeneous populations [25]. However, we have analyzed the response to neoadjuvant chemotherapy within controlled, randomized phase II trial which has defined inclusion and exclusion criteria to have the most comparable basic risk situation. As optimal cut-off for the identification of complete response by the methodologies the point of highest sensitivity still retaining 100% specificity was chosen. The p value reported for evaluating the ROC curve tests the null hypothesis that the area under the curve really equals 0.50 as provided by the statistical program used (GraphPad Prism). The non-parametric Mann-Whitney test was used to confirm the statistical significance when comparing responding versus non-responding tumors and box plots were used to illustrate each case of responding and non-responding tumor above and below the cut-off value. Statistical analyses were performed with JMP SAS (SAS Institute, Cary, NC, USA) and Graph Pad Prism software (Version 5.04; Graph Pad Software Inc., La Jolla, CA, USA). BODY.RESULTS.PATIENT POPULATION: Biopsy tissue was available from 101 out of 105 patients. Gene expression analysis by MammaTyper® was successful in 83 biopsy specimens with full clinical data out of a total of 105 trial participants (Fig. 1). 12 patients out of this group had achieved complete pathological remission (pCR). Basic clinicopathological characteristics of statistics data set #2 that includes quantitative IHC data is listed in Table 1.Fig. 1Remark diagram of sample selection Table 1Basic tumor characteristicsno-pCRpCRHistological Type p= 0.39 Invasive ductal NOS3782.2%9100.0% Invasive lobular715.6%00.0% Other12.2%00.0%Histological Grade p= 1.00 Grade 21533.3%333.3% Grade 33066.7%666.7%ypT Category p= 0.81 ypT000.0%9100.0% ypT12750.0%00.0% ypT2920.0%00.0% ypT3817.8%00.0% ypT412.0%00.0%ypN Category p= 0.81 ypN02750.0%9100.0% ypN+2546.3%00.0% ypNX23.7%HER2 status p= 0.68 Neg3666.7%713.0% Pos713.0%23.7% BODY.RESULTS.COMPARISON OF IHC WITH RT-QPCR BASED ASSESSMENT OF ER, PR AND KI-67: Comparing qIHC of ER with RT-qPCR of ESR1 demonstrated a good overall agreement of 96,6% (PPA 100%; NPA 92.3%) as well as a good correlation looking at the continuous data (spearman's r = 0.82, p < 0.0001). Correlation between vIHC of ER and RT-qPCR for ESR1 (spearman's r = 0.85, p < 0.0001) and between vIHC and qIHC ER (spearman's r = 0.88, p < 0.0001) was high, too. Overall agreement for PR protein and PGR mRNA expression was 91.4% (PPA 83.3%; NPA 100%) comparing qIHC and RT-qPCR and there was a high correlation for the continuous data (r = 0.86, p < 0.0001). Correlation between vIHC and RT-qPCR and between vIHC and qIHC was very high, too (r = 0.88, p < 0.0001 and r = 0.90, p < 0.0001, respectively). Concordance when comparing visual IHC protein with RT-qPCR RNA expression was good for ESR1 as well as for PGR, although slightly lower compared to the agreement between qIHC and RT-qPCR (OPA 91.2%; PPA 90.9%; NPA 91.7%) and for PGR (OPA 92.9%; PPA 88.0%; NPA 96.9%, respectively). For both, ESR1 and PGR, only 4 cases were discordant, with 3 cases each positive by vIHC and negative by RT-qPCR, while 1 case was negative by vIHC and positive by RT-qPCR (Fig. 2). However, several of these discrepancies could be resolved by using quantitative IHC, as these cases were also discrepant when comparing vIHC with qIHC. Moreover, qIHC could delineate quantitative differences of hormone receptor expression at the highest Remmele Score value of 12, where vIHC could not resolve expression differences. In addition, at the lower range of expression levels RT-qPCR based assessment could still determine substantial differences of mRNA levels while the IHC based assessment could not detect any protein expression. The inter-gene spearman correlation was moderate for ESR1 and PGR (r = 0.59, p < 0.0001), while Ki-67 correlated negatively with PGR (−0.37, p = 0.007). While the correlation for ESR1 and PGR protein and RNA expression was high when comparing IHC with RT-qPCR results (spearman's r = 0.82, p < 0.0001 and r = 0.86, p < 0.0001, respectively), the correlation between MKI67 protein and RNA expression was only moderate (spearman's r = 0.56 for vIHC and r = 0.47 for qIHC). In contrast the correlation between both methods for Ki-67 assessment by IHC was high (r = 0.80, p < 0.0001). The median value of Ki-67 proliferation index by image analysis IHC (qIHC) was 23.4%, by conventional visual IHC (vIHC) 35.0%, and 37.01 for RT-qPCR, clearly reflecting the inclusion criteria of the S080 trial which targeted clinically higher-risk patients. Scatter plot analysis displays the positive correlation between RT-qPCR and visual as well as quantitative IHC assessment in Fig. 4.Fig. 2Correlation of RT-qPCR for ESR1, PGR and MKI67 with quantitative IHC by image analysis (a, c, e) and visual IHC assessment (b, d, f) BODY.RESULTS.PREDICTION OF PCR: To compare the clinical utility we performed a ROC analysis to determine the optimal cutoff for predicting the pCR. The results of the ROC analysis are presented in the graphical plots of Fig. 3. With RT-qPCR, 100% of responders could be detected with a specificity of 68.9% at a 40-ddCT level of 37.31 which almost reflected the median mRNA expression in this cohort (Fig. 3a, b). Conversely, no responder was below RT-qPCR of 37.31 (Fig. 4a). For IHC assessment, it was difficult to determine a reliable cutoff reaching high sensitivity and specificity. With RT-qPCR the area under the curve was 0.78 for the overall cohort and 0.80 for the IHC cohort (Statistics #2) (p = 0.002 and p = 0.004). For both IHC methods, the ROC was not significant.Fig. 3ROC analysis for prediction of pathological complete response by quantifying MKI67/Ki-67 expression by RT-qPCR (a, b) and IHC (c, d) showing overall increased ability of mRNA assessment to correctly identify responders versus non-responders Fig. 4Scatter plots illustrating the distribution of RT-qPCR mRNA (upper panel) and qIHC (lower panel) and vIHC (right panel) protein measurements in relation to the groups of responders (green dots) and non-responders (blue dots). Differences were tested with the Mann-Whitney test (a = data set 1, n = 83, b, c, d = common data set, n = 54) Since maximum sensitivity was a pre-requisite, the two methods were compared with respect to specificity, which was found to be substantially higher for MammaTyper® (68.9%) compared to qIHC (22.2%). Using the cut-offs indicated by ROC analysis (37.31 for RT-qPCR, 13.2% for qIHC and 3.5% for vIHC), tumors were characterized as bearing either high or low MKI67 RNA or Ki-67 protein expression, respectively. However, as illustrated in Fig. 4, statistically significant differences between groups were found for the RT-qPCR but not for IHC methods when patients were stratified according to proliferation and pCR (Mann–Whitney p = 0.003 and p = 0,005 for RT-qPCR and p = 0.099 for qIHC and p = 0.133 for vIHC). Using the cut-offs obtained by ROC analysis, pCR was observed in 9 of 24 patients (37.5%, p < 0.001) with high MKI67 RNA expression but in no patient with low RNA expression. Accordingly for qIHC, pCR was observed in 9 of 44 patients (20.5%, p = 0.27) with high Ki-67 labelling index and similarly it was entirely lacking in patients with low proliferation. For RT-qPCR the ROC analysis was also highly significant when only luminal tumors had been assessed, though the sample size was small in this subset (data not shown). BODY.DISCUSSION: In this study we have validated clinical performance of hormone receptor gene expression by RT-qPCR by comparing predefined cut-offs in a blinded fashion with the current standard of IHC. Furthermore, we have investigated the diagnostic performance of two methods for assessing MKI67 gene expression, namely IHC with computerized quantification of protein and RT-qPCR RNA quantification with the MammaTyper® IVD kit in the setting of pCR prediction. When continuous data were dichotomized to reflect high- and low-MKI67 categories with cut-offs obtained by ROC curve analysis after considering 100% sensitivity, RT-qPCR was significantly more specific than qIHC. To the best of our knowledge this is the first direct comparison of this kind in the context of a clinical trial. For the mRNA estimation we used the MammaTyper®, a novel in vitro diagnostic test for breast cancer molecular subtyping. To prove the clinical utility of mRNA based assessment, we compared RT-qPCR with conventional visual assessment as well as digital image analysis based determination at a reference pathology lab in the context of a clinical trial. Moreover, the methods were examined with respect to their ability to predict pCR according to Ki-67 protein or MKI67 mRNA expression levels measured on pretreatment core biopsies. Our results indicate, that, when using RT-qPCR valid cut-offs for mRNA expression, which reliably distinguish between non-responding and responding tumors as determined by pCR (ypT0 ypN0) can be identified. Pathological complete response has gained wide acceptance as one of the strongest predictors of prolonged survival in the setting of neoadjuvant chemotherapy [26, 27]. Therefore, laboratory assays that can efficiently predict a patient's response to a given preoperative chemotherapeutic combination may serve as tools for individualizing treatment and improving long-term outcomes [28]. As with adjuvant chemotherapy, neoadjuvant regimens also suffer from the fact that substantial therapeutic benefit is restricted only to a fraction of those treated, whereas all patients will experience adverse events because of toxicity [29]. In several neoadjuvant studies Ki-67 protein expression has been investigated in pre-operative biopsies in relation to the response to treatment and in most cases a high Ki-67 proliferation rate was predictive of higher probability of pCR [13]. Fasching et al. analyzed Ki-67 by conventional IHC in core biopsies from 552 patients from a single German institution and showed that a pre-defined 13% cut-off could predict pCR with 94% sensitivity and 36% specificity [30]. Interestingly, our ROC analysis for qIHC requiring 100% sensitivity with the least possible loss on specificity led to an identical cut-off (13.2%) for Ki-67. However, this finding requires careful interpretation, due to differences characterizing the clinical settings between the two neoadjuvant studies and the original work by Cheang [31]. In the latter case, the Ki-67 cut-off was fine-tuned against gene expression profiling in order to distinguish Luminal A from Luminal B tumors in a population containing both high- and low-risk breast cancers, whereas in the neoadjuvant setting the same cut-off was intended to identify the majority of, mainly high-risk, patients that would most likely benefit from preoperative cytotoxic therapy. Alike what has been repeatedly shown in the adjuvant setting, it appears that the molecular architecture of tumors as defined by the expression of hormone receptors and HER2/neu may act as a modifier of the association between Ki-67 and response to neoadjuvant treatment and between pCR and long-term outcomes [14, 32]. While 101 tumors were available for analysis, the inclusion of 83 or 54 tumors in this study was not based on a statistical rational but was dictated by the availability of tumor tissue with complete RT-qPCR and qIHC data. A novel aspect of the present work is the comparison between protein-based and mRNA-based methods for the assessment of tumor proliferation. Our findings highlight the feasibility of using RT-qPCR for the routine assessment of ESR1, PGR & MKI67 in order to assist the selection of breast cancer patients for neoadjuvant treatment. Even though both RT-qPCR and qIHC of MKI67/Ki-67 could be calibrated to maximize negative predictive value, only with the former this was achieved whilst ensuring sufficient specificity, which if validated would signify that MammaTyper® could help a considerable number of patients safely forego unnecessary treatment. These data collectively indicate that MammaTyper®MKI67 RNA was overall more representative of the true proliferation state of the tumor than was computer assisted Ki-67 protein estimation, a finding that is worth validating in larger datasets. Significant correlations between conventional Ki-67 visual assessment and RT-qPCR have been previously reported [33, 34], indicating a strong biological link between mRNA and protein expression despite methodological variations, as is further indicated by comparable prognostic hazard ratios obtained by both methods [35]. To the best of our knowledge however, our study is the first to compare image analysis with RT-qPCR for the assessment of tumor proliferation with the additional advantage of using material from a randomized clinical trial. The correlation between mRNA and protein was significant but moderate, a finding which may reflect post translational modifications or may be related to the increased dynamic range of RT-qPCR as compared to IHC. Another possible explanation might be that mRNA levels are a reflection of the average gene expression in the entire FFPE slice, whereas IHC may be biased in favor of selected "representative" tumor areas. Even in the case of image analysis systems, inspection of digitalized images and manual identification of tumor areas is necessary before automatic scoring. Computerized methods have been recommended as a solution to the problem of subjectivity in the visual assessment and scoring of IHC-stained slides. Not surprisingly, Ki-67 scores from image analysis systems are generally in close agreement with those of manual methods because manual scoring for research purposes is customarily performed by a pathologist with longstanding experience in the field [17, 36]. It is worth mentioning, however, that in a routine decentralized setting, digital processing and scoring of slides would probably outperform manual assessment which is prone to considerable subjectivity often not improved upon standardization [37]. Digital analysis yields more reproducible results with regard to staining intensity, by facilitation the definition of low grade staining intensities. Definitive conclusions would require comparisons between all three methods (central versus local versus automatic) performed preferably in the prospective retrospective setting of a large multi-center trial. Multi-gene molecular signatures have also been tested as a way for predicting pCR in patients with breast cancer [38–40]. However, generalized use of these commercialized assays is limited by their increased cost and the requirement to run in centralized platforms or both. Interestingly, proliferation genes, including MKI67, are often heavily weighted in multi-gene scores which serve as estimators of a patients' risk of developing recurrences. This is perhaps one of the reasons why multi-gene tests do not always prove to be convincingly superior to conventional or less sophisticated methods for tumor risk stratification [35, 41, 42], leading some authors to question their cost-effectiveness [43]. Moreover, for several commercially available tests, neither doctors nor consumers can gain access to the continuous expression data of individual proliferation markers that make up the final risk scores. This restriction overall minimizes the possibility of potentially interesting comparisons between proliferation motifs or scores and single proliferation markers based on RT-qPCR or IHC. Strikingly, our ROC curve analysis of MKI67 40-ΔΔCq values for the prediction of pCR displayed performance characteristics that are comparable with those of a 50-gene predictor of tumor recurrence risk developed by supervised training of Cox models [39]. Along these lines, single-gene MKI67 RT-qPCR may be worth considering as a golden means for assessing tumor proliferation due to its unique ability to combine technical advancements and diagnostic accuracy with more affordable pricing. BODY.CONCLUSIONS: Image analysis-assisted scoring of ER, PR and Ki-67 IHC and quantification of ESR1, PGR and MIKI67 RNA expression with RT-qPCR both represent promising alternatives to conventional visual estimation and may assist in improving reproducibility and accuracy in the field. However, RT-qPCR assessment of tumor proliferation was overall more accurate than quantitative IHC. This is the first study to compare tumor MKI67 gene expression by RNA and protein assessment in a prospective retrospective neoadjuvant setting. Due to the relatively small sample size, these data should be considered preliminary and worth validating in larger datasets.

TITLE: Hernias- Is it a primary defect or a systemic disorder? Role of collagen III in all hernias- A case control study ABSTRACT.INTRODUCTION: The need of this study is to assess the role of collagen III in all hernias which include primary inguinal hernias ventral and recurrent abdominal hernias. Collagen type III represents the mechanically instable, less cross-linked collagen synthesized during the early days of wound healing. Quantitative assessment of collagen III in scar tissue on transversalis fascia as tissue obtained from cases operated for various hernias and compared to that of patients operated for abdominal surgeries for indications other than hernia was compared. ABSTRACT.MATERIALS AND METHODS: In this study we had a total of 90 patients, of which 45 patients underwent mesh repair for the various hernias and 45 patients who underwent laparotomies for various reasons were included as controls. Size of 1 × 1cm transversalis fascia was taken in both subjects and was sent for quantitative assessment using Immunohistochemistry test. All the above cases were randomized as per age,sex,BMI, co morbidities and materials used for repair. ABSTRACT.RESULTS: Results were analysed quantitatively and classified into following groups:Based on intensity of staining into Mild, Intermediate and Well stained and based on Quantity of Collagen III into Grade 0---NIL, Grade 1--1-25%, Grade 2-26-50%, Grade 3--51-75%, Grade 4--76-100% (Table 1). In the case group we had 52.4%,35.7% and 11.9% of the cases in Grade 4, Grade 3 and Grade 2 which proved that there was increased presence of Collagen 3, where as 84.4%,4.4% and 11.1% of patients in the control group were classified as Grade 1, Grade 2, Grade 0. For the quantitative study -Chi square test value −81.279 and the p value < 0.001. For the intensity of staining -Chi square test value −57.64 and p value is < 0.001. ABSTRACT.CONCLUSION: This study signifies that ventral, recurrent and primary inguinal hernias are not just caused because of a primary defect but an acquired disorder with respect to collagen distribution. BODY.DISCUSSION: 1 The etiology of hernias are multi factorial, the components of the extracellular matrix, and the importance of collagen which promote a loss of resistance and elasticity of the transversalis fascia determines the onset and recurrences of hernias should be taken into account in the decision-making process for the surgical repair of inguinal hernias [1]. Collagen type I is characteristic for mature scars or fascial tissues while collagen type III represents the mechanically instable less cross linked collagen synthesized during the early days of wound healing [2], [3]. Its already proved that the presence of collagen in adequate amounts and its properties are essential to render the transversalis fascia functional and give it adequate strength [4], [5], [6]. In the study by –Antonio Britto Casanova et al., results showed 17.3% less total collagen in patients with hernias compared with the control group (P < 0.01). Type I collagen in patients with indirect inguinal hernias was 23.7% less than the control group (P < 0.01), type III collagen was 6.4% less in the controls (P < 0.01) [7]. Wagh et al. suggested that diminished collagen, in the sheath of the rectus abdominis muscles of patients causes indirect or direct inguinal hernias [8], [9]. KlingeU found out that immunohistochemistry analysis revealed a decrease in the ratio of collagen I/III when compared to controls. The decreased tensile strength of collagen type III play a key role in the development of incisional hernias [10]. Peeters E1, DeHertoghG, JungeK, −2014 they compared the collagen type I/III ratio in patients with primary inguinal, recurrent inguinal, primary incisional and recurrent incisional hernia patients with controls and demonstrated that the ratio was significantly lower in skin and anterior rectus sheath fascia of the cases and also showed that incisional and recurrent inguinal hernia had lower ratio than primary inguinal hernia patients. And the important point noticed was that collagen type I/III ratio in skin was representative for that in abdominal wall fascia [11]. A study by Henriksen NA in which fifty-two patients were included showed that collagen alteration was also dependant on the types of hernias presented like there was more pronounced changes in patients with direct inguinal hernias than in indirect and other hernias. Consistent findings showed a significant increase in the immature collagen type III with relation to the stronger collagen type I in those patients with a hernia, which further resulted in thinner collagen fibres with a decreased biomechanical strength [12]. Rosch r demonstrated that there was a significant lesser amount of collagen and higher amounts of elastic fibres in transversalis fascia from those patients with direct inguinal hernia as compared to those patients with indirect inguinal hernia [13]. Even though genetic factors was thought to be responsible for the elevation of collagen III was not clear, this study conducted in 1993 on "Increases in type III collagen gene expression and protein synthesis in patients with inguinal hernias" in the Annals of Surgery was the first attempt which showed the world that individuals with the abnormality in the collagen production have a higher chance for developing hernias. It has been showed that constitutive systemic increase in type III collagen synthesis may result in reduced collagen fibril assembly in the abdominal wall, eventually leading to the development of herniation [14]. In our Case Control study, 45 different types of hernia were operated in our hospital and 45 cases selected as controls were included which satisfied our inclusion and exclusion criteria. A piece of transversalis fascia of size 0.5 × 0.5 cm was obtained from all the patients with prior permission from the patients before the surgery and was taken in a formalin container to the Central Research Laboratory and was processed and then taken for immuno histochemistry test. Of the 45 patients in cases study, the youngest was 25 years old and the oldest was 60. The mean age was 42.7 years. There were 35males (77.7%) and 10 females (22.2%) among the 45 patients studied in case group. We had a variety of bilateral, unilateral inguinal hernias, lower midline, umbilical and a spigelian hernia. Of the 45 patients in control group, the youngest was 22 and oldest was 59. The mean age was 43.7 years. There were 30males (66.6%) and 15 females (33.3%) among the 45 patients studied in control group. Included cases were appendicectomy, intestinal obstruction, abdominoperineal resection, carcinoma stomach, whipples procedure and colostomy closure in the control group and we took a piece of fascia transversalis from all the patients with prior permission and was processed in the same way and taken up for immunohistochemistry (see Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5).Fig. 1Grade 4.Fig. 1Fig. 2Grade 3- collagen.Fig. 2Fig. 3Grade 2- collagen.Fig. 3Fig. 4Grade 1- collagen.Fig. 4Fig. 5Grade 0.Fig. 5 52.4% of the cases in Grade 4 and 35.7% of the cases in Grade 3 in the cases, here we can see the increased amount of collagen and the intensity of staining by the dye used in the immunohistochemistry test. In the control group 84.4% of patients in the control group with Grade 1, 4.4% of the patients with Grade 2 and 11.1% with Grade 0 showed significant amount of decrease in the grading and intensity of staining of collagen. What was different in this study was since we evaluated only collagen III when compared to all other studies cited, a ratio of collagen I: collagen III could not be assessed but the results and analysis showed a significant increase in collagen III in hernia patients when compared to the controls. Limitation of this milestone concept it's a quantitative analysis and not a standardized way of calculating the amount of collagen keeping in mind the human errors. BODY.RESULTS: 2 A total of 90 patients of which 45 represented cases and 45 represented controls were recruited into our study based on the inclusion and exclusion criteria. All slides were assessed and mounted on high power and low power microscope. Results were analysed quantitatively and classified into following groups: BODY.QUANTITY OF COLLAGEN III (SEE : 3 Grade 0NILGrade 11–25%Grade 226–50%Grade 351–75%Grade 476–100% BODY.QUANTITY OF COLLAGEN III (SEE .STATISTICAL ANALYSIS OF THE GRADING OF STAINING : 3.1 Assessing the results in the case group we had 52.4% of the cases in Grade 4 and 35.7% of the cases in Grade 3 and 11.9% cases in Grade 2 which signifies that there is an increased presence of Collagen 3 in the patients which we have included in our study where as we had an 84.4% of patients in the control group with Grade 1, 4.4% of the patients with Grade 2 and 11.1% with Grade 0. Chi square test value is 81.279 and the p value < 0.001 which is significant.Table 1Results.Table 145 cases45 Controls22 Slides- Grade 438 slides-Grade 115 slides- Grade 32 Slides Grade25 slides – Grade 25 Slides- Grade 03 slides – Improper stainingTable 2Statistical analysis.Table 2GradeCasesControlsTotal00550.0%11.1%5.7%1038380.0%84.4%43.7%252711.9%4.4%8.0%31501535.7%0.0%17.2%42202252.4%0.0%25.3%Total424587100.0%100.0%100.0%Chi-square = 81.279, p < 0.001. BODY.INTENSITY OF STAINING: 4 They were classified into Mild, Intermediate and Well stained groups. Out of the 22 slides(in cases) included in Grade 4, 20 of them was included in the group of well stained and 2 of them was included in intermediate group. 15 of them included in Grade 3 had 7 with well stained slides and 8 with intermediate stain. 5 slides were included as mild stain. Out of the 38 slides included in Grade 1 (in Controls) most of them were included in mild stain with 2 of the slides included in intermediate stain. BODY.INTENSITY OF STAINING.STATISTICAL ANALYSIS OF INTENSITY OF STAINING : 4.1 Assessing the intensity of staining 64.3% of the cases were included in well stained group, 23.8% were included in the intermediate group and 11.9% were included in the mild group.Table 3Intensity of staining.Table 3IntensityCasesControlsTotalWell Stained2702764.3%0.0%32.9%Intermediate1021223.8%5.0%14.6%Mild5384311.9%95.0%52.4%Total424082100.0%100.0%100.0%Chi-square = 57.64, p < 0.001. In the control group 95% of the slides were included in the mild group and 5% were not stained. Chi square test value was 57.64 and p value is < 0.001 which was significant. BODY.CONCLUSION: 5 Despite sincere efforts made to optimize the patients and after taking all the precautionary measures prior to elective surgical procedure incisional hernias, recurrent hernias still remains a challenge to all surgeons. Presence of high quantity of collagen III in the tissue obtained from cases operated for hernia and poor staining in those operated for other than hernia indicates that there is an intrinsic and inherent weakness in the tissue which makes the individual more prone for developing hernia that is primary, incisional or recurrent hernias. The staining and quantitative assessment has proved that ventral, recurrent and primary inguinal hernias are not just caused because of a primary defect but an acquired disorder with respect to collagen distribution. BODY.ETHICAL APPROVAL: By MS Ramaiah Medical College, Ethical Committee. BODY.SOURCES OF FUNDING: Personal. BODY.AUTHOR CONTRIBUTION: Both authors equally helped in collecting data and gathering information about the study. BODY.CONFLICTS OF INTEREST: No. BODY.GUARANTOR: DR. Sam Koruth. BODY.RESEARCH REGISTRATION UNIQUE IDENTIFYING NUMBER (UIN): researchregistry2256.

TITLE: Effects of lemon verbena extract (Recoverben®) supplementation on muscle strength and recovery after exhaustive exercise: a randomized, placebo-controlled trial ABSTRACT.BACKGROUND: Exhaustive exercise causes muscle damage accompanied by oxidative stress and inflammation leading to muscle fatigue and muscle soreness. Lemon verbena leaves, commonly used as tea and refreshing beverage, demonstrated antioxidant and anti-inflammatory properties. The aim of this study was to investigate the effects of a proprietary lemon verbena extract (Recoverben®) on muscle strength and recovery after exhaustive exercise in comparison to a placebo product. ABSTRACT.METHODS: The study was performed as a randomized, placebo-controlled, double-blind study with parallel design. Forty-four healthy males and females, which were 22–50 years old and active in sports, were randomized to 400 mg lemon verbena extract once daily or placebo. The 15 days intervention was divided into 10 days supplementation prior to the exhaustive exercise day (intensive jump-protocol), one day during the test and four days after. Muscle strength (MVC), muscle damage (CK), oxidative stress (GPx), inflammation (IL6) and volunteer-reported muscle soreness intensity were assessed pre and post exercise. ABSTRACT.RESULTS: Participants in the lemon verbena group benefited from less muscle damage as well as faster and full recovery. Compared to placebo, lemon verbena extract receiving participants had significantly less exercise-related loss of muscle strength (p = 0.0311) over all timepoints, improved glutathione peroxidase activity by trend (p = 0.0681) and less movement induced pain (p = 0.0788) by trend. Creatine kinase and IL-6 didn't show significant discrimmination between groups. ABSTRACT.CONCLUSION: Lemon verbena extract (Recoverben®) has been shown to be a safe and well-tolerated natural sports ingredient, by reducing muscle damage after exhaustive exercise. ABSTRACT.TRIAL REGISTRATION: The trial was registered in the clinical trials registry (clinical trial.gov NCT02923102). Registered 28 September 2016 BODY.BACKGROUND: All kinds of training, moderate, exhaustive or unaccustomed, cause so-called exercise-induced muscle damage (eiMD) accompanied by oxidative stress and inflammation [1]. Delayed onset muscle soreness (DOMS) is the most common symptom of eiMD, whereas histological evidence of disruption of the myofibrillar structure and myofibre necrosis, as well as inflammation, are the ultimate signs of eiMD [1]. DOMS is associated with muscle fatigue and muscle soreness, symptoms, which last for a number of days and have a negative impact on the exercise performance of athletes and amateur sports people, especially when they carry out long-term training programs [1]. Incomplete regeneration of myofibrillar structures and metabolic processes before the next training increases the risk for secondary injuries. Therefore, it is important for the adherence to training plans and for the training success that regeneration is as effective and as short as possible. A product that could accelerate recovery from DOMS or muscular fatigue would be beneficial not only for high performance athletes, but also for amateur athletes, enabling them to train more frequently or reduce the risk of injury. As inflammation and ROS (reactive oxygen species) are presumably the main cause of DOMS [1], it is hypothesized that natural ingredients with anti-inflammatory and antioxidative properties may help in accelerating or supporting the regeneration after muscle damage inducing exercise [2–4]. The use of supplements with antioxidative or anti-inflammatory effects in the sports nutrition is already widespread. Many of these ingredients and products have been investigated in recent years; for example, curcumin [5], omega-3 fatty acids [6, 7] and polyphenols [2, 8–10]. Lemon verbena (Aloysia citriodora (L.)) is an annual eatable herbaceous plant native to South America commonly used as tea, refreshing beverage, food, or spice. Traditional medicinal applications are related to digestion and nervous discomfort [11]. A literature search focusing on peer-reviewed publications showed that only limited data are available for lemon verbena in connection with muscular recovery after exercise. PubMed listed over 80 publications in June 2017, that investigated lemon verbena as stand-alone product or in combination with other ingredients in any context (keywords for search were Aloysia citriodora, Lippia citriodora, Lippia triphylla, Aloysia triphylla, and lemon verbena). Analytical references demonstrate that water-based extracts out of lemon verbena leaves are high in polyphenols [12, 13]. Several in vitro and in vivo studies have shown antioxidative and anti-inflammatory effects [11, 14–18]. Out of six human studies [19–24], only one human pilot study was published investigating lemon verbena extract on the muscular damage biomarker, creatine kinase and liver biomarker related to oxidative stress [19]. This study showed some effects on cytokines and oxidative stress markers in neutrophils, but no functional parameters like muscle strength or muscle soreness were essessed. Furthermore, this study used a high dosage of 1800 mg/day which is not suitable for application in food supplements as it would require the intake of approximatley nine capsules per day. Therefore, further research to observe effects of lemon verbena on muscle damage, muscle soreness, and recovery needs to be performed. A proprietary lemon verbena extract (Recoverben®), high in polyphenols, was developed and recently identified as an anti-inflammatory agent [25]. One mode of action responsible for the anti-inflammatory properties is the inhibition of cyclooxygenase (COX) [26]. COX inhibition properties have also been shown for curcumin [27], which is also an natural ingredient shown to be beneficial in recovery [5, 28]. Based on these data and on data from literature, we hypothesized that a lemon verbena extract (Recoverben®) could have beneficial effects on exercise induced muscle damage, muscle soreness, and recovery. The aim of this study was to investigate effects on functional, metabolic, and subjective parameters of recovery. Furthermore, parameters indicating antioxidative and anti-inflammatory properties were included to document mode of action. BODY.METHODS.STUDY DESIGN: This study was a double blind, randomized controlled trial with a parallel-group design that investigated the effects of a proprietary lemon verbena extract (Recoverben®) supplementation on muscle strength and recovery after exhausive exercise. It was conducted in orientation to the ICH-GCP guidelines, in compliance with the declaration of Helsinki, and was reviewed by the Institutional Review Board (IRB) "Landesärztekammer Baden Württemberg" without concerns (F-2016-080 September 13th, 2016). All subjects signed the IRB-approved informed consent prior to any procedures. The study was performed from October 2016 to March 2017 at BioTeSys GmbH, Esslingen, Germany, an independent study site which is focused on nutritional research. BODY.METHODS.SUBJECTS: Subjects were recruited from internal database of the study site, advertisements in local newspapers, and notice boards in public buildings. Seventy subjects responded to the advertising campaign and received detailed information about the study. Out of these, 45 subjects were invited for screening visits. Forty-four healthy, non-smoking, moderately active (exercise 1–3 times per week) men and women with an age between 22 and 50 years and a BMI between 19 and 30 kg/m2 were deemed eligible for the current study. Subjects had a usual intake of five or less portions of fruits plus vegetables per day. Detailed inclusion- and exclusion criteria are presented in Table 1. Eligibility was evaluated by medical history, concomitant medication, physical examination, electrocardiogram, blood pressure, and anamnesis. Physical activity was assessed using the International Physical Activity Questionnaire (IPAQ) [29]. Nutrition pattern of subjects was determined during the screening visit using a subjective, retrospective, semi-quantitative nutrition frequency questionnaire. The score ranges from 0 to 100, whereby 0 means a very good nutrition pattern and 100 a very poor nutrition pattern [30]. The questionnaire was developed for the German-speaking area and tested in a neutral and geriatric collective. Adequate determination of supply of micronutrient was validated by comparing with a detailed quantitative food frequency questionnaire [30]. A score around 50 can be interpreted as balanced nutrition. Amount of fruits and vegetables portions taken per day was asked during screening, whereby one portion was specified to be around 150 g. Subjects were requested to refrain from intake of anti-inflammatory or antioxidative drugs or supplements during the study, potentially interfering with this trial. They were asked not to change their dietary habits and physical activity during the study. The evening before the study days, subjects ate a standardized dinner low in polyphenols (noodles with cheese sauce). During the study days, they received a standardized breakfast (cereal bars) and a standardized snack (white wheat roll with butter and cheese). Amount and time were also standardized. Alcohol intake and exhaustive activities were prohibited 48 h before the study days until 96 h after the exhaustive exercise. Subjects were asked for changes in nutrition habits and sportive activity at the end of the study.Table 1In- and exclusion criteriaInclusion criteriaExclusion criteriaSubject is able and willing to sign the Informed Consent Form prior to screening evaluationsRelevant history, presence of any medical disorder or chronic intake of medication/dietary supplements (e.g. polyphenols, anti-inflammatory or antioxidative drugs or supplements, antihypertensive drugs) potentially interfering with this study at screeningHealthy subjects: Subject is in good physical and mental health as established by medical history, physical examination, electrocardiogram, vital signs, results of biochemistry and haematologyFor this study clinically relevant abnormal laboratory, vital signs or physical findings at screeningMen and womenDiabetes or serious cardiovascular diseasesAge ≥ 22 and ≤50 yearsChange of dietary habits within the 2 weeks prior to screening (for instance start of a diet high in vegetables and fruits (≥ 5 portions per day)BMI: 19–30 kg/m2Diet high in vegetables and fruits ≥ 5 portions per dayPhysically active 1–3 times per weekParticipants anticipating a change in their lifestyle or physical activity levels during the studyNonsmokerSubjects not willing to abstain from intake of analgesic medication (e.g. Aspirin) 24 h prior to visit 2 until visit 5Able and willing to follow the study protocol proceduresSubjects with history of drug, alcohol or other substances abuse, or other factors limiting their ability to co-operate during the studyKnown hypersensitivity to the study preparation or to single ingredientsPregnant subject or subject planning to become pregnant during the study; breast-feeding subjectKnown HIV-infectionKnown acute or chronic hepatitis B and C infectionBlood donation within 4 weeks prior to visit 1 or during the studySubject involved in any clinical or food study within the preceding month Subjects were randomly assigned to the study groups and stratified by gender after assessing eligibility during screening. To ensure double-blind performance, the randomization scheme was created by the sponsor using the software Randlist. All subjects, the investigator, and study staff involved in study performance, and data analysis were blinded until database lock. Disposition of subjects is summarized in Fig. 1.Fig. 1Dispostion of subjects following Concort BODY.METHODS.STUDY PRODUCT AND SUPPLEMENTATION: The investigated commercial product Recoverben® (batch number 16P0007) is a proprietary lemon verbena extract obtained by water extraction out of organic dried lemon verbena leaves. Lemon verbena (Aloysia citriodora (L.)) is a member of the family Verbenaceae. The product is a native extract without any additives, standardized to more than 18% polyphenols. The extract is hydrophilic and can easily be dissolved in water, and has a high ORAC level of 170.000 μmol TE/100 g. Lemon verbena extract (Recoverben®) as well as the placebo were formulated in capsules, matching in size and color, and were supplied by Vital Solutions GmbH, Germany. Each capsule contained 200 mg lemon verbena extract or placebo (maltodextrin). All subjects were instructed to take two capsules daily in the morning. Products were consumed 10 days before an exhaustive exercise test, during the test day and four days after the test. The ingredient is safe for human consumption and its quality complies with EU legislation concerning hygiene, contaminants, and maximum residue levels of pesticides of foodstuff [31, 32]. Additionally, the extract has been tested for and shown to be free of banned substances by a specialist anti-doping laboratory (LGC Limited, UK), therefore demonstrating suitability as a sports nutrition product. BODY.METHODS.EXHAUSTIVE EXERCISE PROTOCOL: In the current study, maximal eccentric loading of the lower extremity was induced by an intensive jump-protocol 10 days after the start of supplementation, which was modified based on a jump protocol used previously [33]. The protocol comprised 200 countermovement jumps with an additional load of 10% of the participant's body weight. The 200 countermovement jumps were performed in 10 sets of 20 jumps every four seconds with 90 s rest between sets. Knee joint angle between the jumps had to be 90°, which was controlled by the observer. At the end of the test, rating of perceived exertion (RPE) was obtained using the Borg RPE scale [34]. Borg RPE scale and jump repetitions were used to ensure subjective exhaustion after exercise and to control comparability of jump protocol between groups. Massive deviation from jump protocol was defined as exclusion criteria from VCAS analysis for exclusion of confounding factors. BODY.METHODS.MUSCLE FUNCTION TESTING - MAXIMAL VOLUNTARY CONTRACTION (MVC): Changes in muscle function appear to be the best marker for the degree of exercise induced muscle damage [1]. Therefore, in the current study, MVC was investigated by assessing isometric strength of the M. quadriceps femoris, with 90° knee angle, using a dynamometer KM40 (2kN) from ME-Messsysteme GmbH and a strength chair from fasttwitch, TTI GmbH. Subjects were fixed in a seated position with a hip belt, had their arms crossed in front of the chest, and had the free leg hanging without contact to any surface to reduce support by other body parts during the test. Before each measurement, subjects performed a 3 min warm-up (level 7, 70 rpm) on a cycle ergometer (Crane Power Studio Ergometer). The strength measurement was performed for the dominant leg. This was identified at screening by a shove-test, where the subject was pushed unexpected by the observer and the leg which was used by subject to balance was defined as dominant leg. All examinations were performed in triplicates, immediately before and 3, 24, and 48 h after the exhaustive exercise protocol. The highest value was used for analysis. Subjects were familiarized to the measurement at screening. For each subject, the settings of the strength chair (position of back rest, leg rest and position of measurement arm) were documented. All further measurements were performed with the individual settings. First measurement was performed during screening visit to avoid training effects during study visits. Variability was checked between screening and pre-exercise measurement. BODY.METHODS.PERCEIVED MUSCLE SORENESS: Muscle soreness was measured using two different methods. BODY.METHODS.PERCEIVED MUSCLE SORENESS.MOVEMENT INDUCED PAIN (VAS): Subjects were asked to sit down into and get up from a chair and to rate the pain they experienced in doing so using a 100 mm visual analogue scale (VAS), which consisted of a from zero mm (no pain) to 100 mm (worst imaginable pain). This assessment was conducted immediately before and 3, 24, 48, 72, and 96 h after the exhaustive exercise protocol. Using VAS is frequently described in literature for assessing acute exercise induced pain [2, 33, 35]. BODY.METHODS.PERCEIVED MUSCLE SORENESS.RETROSPECTIVE PAIN (LIKERT SCALE): A seven point retrospective pain questionnaire (7 point Likert-scale for muscle soreness) by Vickers et al. was used to evaluate retrospective perceived pain during daily life activities with zero "a complete absence of pain" and six "a severe pain that limits my ability to move" [36]. The subjects were asked to answer the questionnaire immediately before the exhaustive exercise and 24, 48, 72, and 96 h after the jump test. BODY.METHODS.BIOCHEMICAL ANALYSIS: Different biomarkers were analyzed to evaluate muscle damage and antioxidative capacity to describe exercise-induced oxidative stress. BODY.METHODS.BIOCHEMICAL ANALYSIS.CREATINE KINASE (CK): CK is a biomarker for muscle damage typically increased after intense exervcise. In our study, CK was determined from blood samples obtained before and 3, 24, and 48 h after the exhausting exercise protocol. Analyses were carried out at Synlab Medizinisches Versorgungszentrum Leinfelden using an enzymatic-kinetic test method [37]. BODY.METHODS.BIOCHEMICAL ANALYSIS.GLUTATHIONE PEROXIDASE (GPX): Exercise training is accompanied with oxidative stress via production of reactive oxygen species (ROS), and modulating the endogenous antioxidant defense system, including GPx. In a healthy organism, exercise induces GPx levels, inactivating ROS and maintain them in physiological levels [38, 39]. The determination in plasma (GPxP) was carried out at the study lab via GPx-Assay-Kit (Cayman Chemical Company, Ann Arbor, MI, USA) pre-exercise and 3, 24, and 48 h post exercise. BODY.METHODS.BIOCHEMICAL ANALYSIS.INTERLEUKIN-6 (IL-6): IL-6 is a multifunctional cytokine involved in pro- as well as anti-inflammatory processes. Exercise-induced IL6 response is dependent on intensity and duration of the exercise [40]. The determination in serum was carried out at the study lab via Quantikine® HS Human IL-6 Immunoassay Kit (R&D Systems, Inc., MN, USA) pre-exercise and 3, 24, and 48 h post exercise. BODY.METHODS.SAFETY AND TOLERABILITY: At each visit, changes in physical conditions since the last visit were reviewed with subjects. Based on entries in subject diaries, complete blood count, and adverse events were assessed. Tolerability of the study product was assessed 96 h after exercise, and at the end of study. The subjects rated overall tolerability by selecting out of three categories: "well tolerated", "slightly unpleasant", and "very unpleasant". BODY.METHODS.STATISTICS: The study was planned as an exploratory trial. Sample size was calculated based on different studies observing the effects of lemon verbena and using a similar design [19, 35]. Therefore, a sample size calculation was performed with effect size f = 0.2, significance level 0.05, power 80%, two number of groups and four measurement time points (pre, 2 h, 24 h, 48 h), correlation among repeated measures 0.5 and with nonsphericity correction 1. With these suggestions, a total sample size of 36 subjects was calculated. Considering a drop-out rate of 10%, 40 subjects were planned to be included in the study. Objectives were the difference of muscle strength, muscle soreness, retrospective pain, CK, IL-6, and glutathione peroxidase after exhaustive exercise under lemon verbena in comparison to placebo. Data were analyzed using SAS Version 9.3 and GraphPad Prism Version 5.04. All statistical tests were performed two-sided. Significance level was set to 0.05. For evaluation of treatment effects, a linear mixed model with repeated measures was used. For retrospective pain, a generalized linear mixed model with Poisson-distribution for count data was used. Due to explorative data analysis, no correction for multiple comparison was performed. Gender, its interaction with treatment and the respective pre value was included as covariates for the biomarkers and MVC. For movement induced pain and retrospective pain only gender and its interaction with treatment were defined as covariates. Changes within groups were assessed using one way ANOVA or Friedmann test, as appropriate. All efficacy parameters were checked for baseline differences. Results presented below refer on valid case analysis set (VCAS). VCAS criteria were pre-defined in the protocol: missing data, adverse events or concomitant medication interfering with study results, extreme outliers, and major protocol violation (compliance <85%, > 115%, drop outs / withdrawals, major deviation of study performance). BODY.RESULTS.GROUP CHARACTERISTICS: Out of 44 subjects allocated to intervention, 40 subjects (19 men and 21 women) completed the study according to the protocol (full analysis set; FAS). Four subjects dropped out after start of supplementation and before efficacy testing. Three of these suffered from a common cold, which did not allow them to perform the exhaustive exercise test, and one dropped out due to personal decision. Thirty-seven subjects were analyzed for valid case analysis set (VCAS). One subject had a strong common cold during the exhaustive exercise test, which may have influenced biomarker results. Another subject did not meet compliance criteria for study product intake and the third subject had major deviation of the exhaustive exercise protocol. As these three subjects did meet exclusion criteria for VCAS analysis, they were not considered in final analysis (VCAS). Gender, age, BMI, blood routine markers, and blood pressure did not differ significantly between the groups prior to the study. Subject characteristics are summarized in Table 2. The nutrition frequency questionnaire scores were 45.35 ± 10.51 points in the lemon verbena group and 42.65 ± 11.33 points in the placebo group (p = 0.4597). Most subjects consumed between two and four portions fruits plus vegetables per day (lemon verbena: 82%, placebo: 70%). Intake of fruit and vegetables, as supplier for polyphenols, were comparable between groups. The protocol compliance of study product intake was very good. For VCAS, all subjects met the compliance criterion ≥85% and ≤115% of study preparation consumed (lemon verbena group: 102 ± 5%, placebo: 101 ± 2%). For FAS, one subject had a compliance >115% since much less products remained, bringing up uncertainty about correct intake, which was the reason for exclusion from VCAS. Borg scale data after exhaustive exercise and jump repetitions were not significantly different between groups (p = 0.8997, p = 0.1561). Therefore, burden of subjects by the exhaustive exercise and jump protocol was comparable between groups, so a comparable stress could be expected.Table 2Subject characteristics at screening for FAS (N = 40, 19 men, 21 women)Parameter[unit]Lemon verbena(N = 20 59% men, 41% women)Placebo(N = 20 40% men, 60% women)Inclusion criteria/Reference rangeAge[years]Mean31.730.622–50Sd8.89.3BMI[kg/m2]Mean22.6723.019–30Sd2.32.5Activity[MET min/week]Mean32092470.0–Sd3957.02521CHOL[mg/dL]Mean172.5180.0100–200Sd23.031.85TG[mg/dL]Mean89.6882.14<160Sd43.5324.71HDL[mg/dL]Mean58.9158.95>40Sd6.48714.24LDL[mg/dL]Mean93.86109.5<150Sd16.3228.7Glucose[mg/dL]Mean87.5586.1460–100Sd8.4054.96SBP[mmhg]Mean121.6121.6120–129Sd12.812.8DBP[mmhg]Mean75.870.680–84Sd8.810.5BMI body mass index, MET metabolic equivalent of task, CHOL cholesterol, TG triglycerides, HDL high-density lipoprotein, LDL low-density lipoprotein, SBP systolic blood pressure, DBP diastolic blood pressure BODY.RESULTS.MUSCLE FUNCTION TESTING - MAXIMAL VOLUNTARY CONTRACTION: MVC was significantly less reduced after exercise in the lemon verbena group than in the placebo group (p = 0.0311), with significant time effect (p = 0.0051), (Fig. 2, Table 3). In the lemon verbena group, muscle strength was completely back to baseline after 48 h, whereas strength was still reduced at that time point in the placebo group.Fig. 2Maximal voluntary contraction. Delta MVC [N] (Mean ± 95% CI; product effect: p = 0.0311) * indicating significance against baseline; & indicating significant group differenecesTable 3Statistical results within (one way-ANOVA) and between groups (Linear mixed models with repeated measures or in case of Ret. pain generalized linear mixed model with poisson distribution for count data) for maximal voluntary contraction (MVC), movement induced pain (VAS), retrospective pain (Ret. pain), creatin kinase (CK), gluthation peroxidase (GPxP) and interleukine-6 (IL-6)Within groupBetween groupLemon verbenaPlaceboProduct effectTime effectMVC0.005<0.00010.03110.0051VAS<0.0001<0.00010.0788<0.0001Ret. pain<0.0001<0.00010.782<0.0001CK<0.0001<0.00010.9412<0.0001GPxP0.2040.58950.06810.0624 BODY.RESULTS.PERCEIVED MUSCLE SORENESS: Movement induced pain (VAS) in the lemon verbena group was less pronounced by trend (p = 0.0788) with a significant time effect (p < 0.0001) in comparison to the placebo group (Fig. 3, Table 3). Retrospective pain was comparable between groups without significant difference (Fig. 4). Both groups showed significant increase in soreness 24, 48, and 72 h after exercise in relation to pre-exercise.Fig. 3Movement induced pain. Movement induced pain (VAS) [cm] (Mean ± 95% CI; product effect: p = 0.0788) * indicating significance against baselineFig. 4Retrospective pain. Retrospective pain [score] (Mean ± 95% CI; product effect: p = 0.7820) * indicating significance against baseline BODY.RESULTS.BIOCHEMICAL ANALYSES.CREATINE KINASE: Exercise-induced CK was not significantly different between groups (p = 0.9412 with significant time effect (p < 0.0001) (Fig. 5, Table 3).Fig. 5Creatine kinase. Delta Creatine kinase [U/L] (Mean ± 95% CI; product effect: p = 0.9412) * indicating significance against baseline In the lemon verbena group, CK was significantly increased above baseline at 24 h but not 3- or 48-h post-exercise. In contrast, the placebo group showed significant elevations of CK at all three time points relative to baseline. BODY.RESULTS.BIOCHEMICAL ANALYSES.GLUTATHIONE PEROXIDASE: The GPxP activity within lemon verbena group was increased by trend compared to placebo group (p = 0.0681), with time effect also by trend (p = 0.0624), (Fig. 6, Table 3).Fig. 6Glutathione peroxidase. Delta Glutathione peroxidase [U/L] (Mean ± 95% CI; product effect: p = 0.0681) * indicating significance against baseline BODY.RESULTS.BIOCHEMICAL ANALYSES.INTERLEUKIN 6: Only distinct increase of IL-6 could be observed after exercise, without significant differences but high inter-individual variability. Differences between groups were not significant (p = 0.5824). BODY.RESULTS.SAFETY AND TOLERABILITY: Blood chemistry, vital signs, adverse events, and concomitant medication did not indicate any safety concerns over 15 days. There was no statistical difference between the two groups for adverse events (p = 0.231). The most frequent adverse events were headache (lemon verbena: 5%, placebo: 18%) and common cold (lemon verbena 23%, placebo: 23%). None of the adverse events were serious or related to the study products. The intervention was well tolerated. BODY.DISCUSSION: The aim of this study was to investigate the effects of supplementing with 400 mg of lemon verbena extract (Recoverben®) on muscle strength and recovery in healthy, moderately active adults. We found that consumption of lemon verbena significantly attenuated loss of muscle strength compared to placebo. Muscle strength loss is considered a reliable and valid functional marker for assessing muscle damage [1]. Therefore, our preliminary findings suggest that lemon verbena may reduce exercise-induced muscle damage. Muscle strength was reduced by 21% in placebo group, which is within the expected range of 20–50% and recovery was not completed until two days post exercise. It is generally accepted that two to seven days are necessary for full recovery following exercise induced muscle damage [1]. Compared to placebo, lemon verbena extract significantly (p = 0.0311) buffered strength loss after exercise. MVC in the lemon verbena group was reduced by 11%, which is defined as mild muscle damage [1]. Furthermore, complete recovery was reached after 48 h. Based on these findings, lemon verbena appears to not only speed recovery, but also reduce fatigue directly after exercise. These results were reflected by findings for perceived muscle soreness. Movement induced pain, which estimated actual perceived pain showed discrimination between study groups with slight superiority of lemon verbena extract by trend. The less pronounced muscle damage, seen by significantly less reduction of MVC, seems to be reflected by less perceived pain under lemon verbena extract if compared to placebo. Maximum of muscle soreness was reported 48 h after exercise, fitting to the general knowledge that muscle soreness peaks 24 h or 48 h after damaging exercise [1]. The extent of muscle soreness was medium for both groups, supporting that the exercise protocol caused mild to moderate muscle damage. After 96 h, subjects were, on average, not completely painless, even if muscle strength at that time was already recovered in this group. However, the same phenomenon has already been observed by others [9, 33]. Increasing concentration of CK in the blood is an indication of muscle damage, being frequently used in sports nutrition studies [9, 35]. The time course of CK increase peaked at 24 h after exercise, which is comparable to findings reported in literature [9, 35]. Exercise-induced increases in CK are known to exhibit high interindividual variability, with some people showing large increases (responders) and others showing only moderate increases (non-responders) [1]. In our study, high levels of interindividual variation in CK concentrations were present, which could explain why we failed to observe a significant between-group difference despite other markers of muscle damage, such as MVC, favoring the lemon verbena group. Many research studies have shown that supplementation with dietary polyphenols has the potential to positively influence symptoms of exercise-induced muscle damage [2, 5, 8–10, 41, 19]. However, underlying processes are still unclear and it is not sure if antioxidative effects are the primarily mechanisms [41]. Furthermore, the benefit of reducing oxidative stress has been discussed diversely [41, 42]. Increased of oxidative stress can lead to progressive cell damage and decline in physical function [42, 43]. However, ROS act as biological stimuli in cellular processes of adaption to training [41, 42] and cells can adapt to repetitive increases of ROS by improving antioxidant capacity [44, 45]. During the current study, glutathione peroxidase in plasma was selected as a parameter to supply information about antioxidative capacity. Our results indicate an activation of the antioxidative defense under lemon verbena extract by up-regulating GPxP shortly after exercise. In contrast to this, baseline GPxP was not increased by supplementation with lemon verbena extract. Therefore, it appears, that supplementation with lemon verbena extract strengthens the antioxidative defense system and enables effective counteraction of oxidative stress, but only if needed. Both groups experienced significant exercise-induced increases in IL-6 without significant difference between one another. Some evidence suggests that changes in IL-6 depend in part on exercise intensity and duration [1, 46, 40]. It is possible that the exhaustive exercise protocol used in our study was not intense and/or long enough to elicit meaningful changes in IL-6 that could have been effected by lemon verbena supplementation. Similar results were found in other human studies investigating natural ingredients for effects of muscle strength and muscle damage, such as ashwagandha extract [47], curcumin [48], pomegranate extract [10], and blueberry [2]. These natural ingredients are high in polyphenols, a trait shared by lemon verbena. It has been proposed, that polyphenols could be useful to prevent muscle damage or improve recovery [4]. The major biological functions of polyphenols are as antioxidants and anti-inflammatory agents. Enhanced production of vasodilation factors and the inhibition of synthesis of vasoconstrictors have also been shown [49]. These could be additionally beneficial by improving tissue oxygen supply and removal of metabolic waste products. The proprietary lemon verbena extract (Recoverben®) investigated in the current study has shown anti-inflammatory effects [26] and is characterized by a high polyphenol content and high ORAC level. Therefore, the observed reduction in muscle strength loss and indicated accelerated recovery in the present study might be explained by lemon verbenas' constituents and their ability to prevent or reduce inflammatory processes or reduce oxidative stress. BODY.CONCLUSION: In summary, our study showed that ingestion of a 400 mg/day proprietary lemon verbena extract (Recoverben®) resulted in significantly less muscle strength loss in healthy, moderately active adults. Influences by trend on muscle soreness and antioxidative capacity emphasizes the potential of the product accelerating recovery after exhaustive exercise. Larger studies could provide statistical evidence also for the parameter, which only showed improvements by trend in the study.

TITLE: The Lateralization of Intrinsic Networks in the Aging Brain Implicates the Effects of Cognitive Training ABSTRACT: Lateralization of function is an important organization of the human brain. The distribution of intrinsic networks in the resting brain is strongly related to cognitive function, gender and age. In this study, a longitudinal design with 1 year's duration was used to evaluate the cognitive training effects on the lateralization of intrinsic networks among healthy older adults. The subjects were divided into two groups randomly: one with multi-domain cognitive training over 3 months and the other as a wait-list control group. Resting state fMRI data were acquired before training and 1 year after training. We analyzed the functional lateralization in 10 common resting state fMRI networks. We observed statically significant training effects on the lateralization of two important RSNs related to high-level cognition: right- and left- frontoparietal networks (FPNs). The lateralization of the left-FPN was retained especially well in the training group but decreased in the control group. The increased lateralization with aging was observed in the cerebellum network (CereN), in which the lateralization was significantly increased in the control group, although the same change tendency was observed in the training group. These findings indicate that the lateralization of the high-level cognitive intrinsic networks is sensitive to multi-domain cognitive training. This study provides neuroimaging evidence to support the hypothesis that cognitive training should have an advantage in preventing cognitive decline in healthy older adults. BODY.INTRODUCTION: The human brain is the most complex system in nature. The interhemispheric interaction is crucial to brain functions such as motor and cognitive processes. However, hemispheric specialization is also an obvious organizing principle for efficient information processing, such as leftward lateralization for speech and rightward lateralization for visuospatial attention (Corballis, 2014). Postmortem and neuroimaging studies have displayed the asymmetries of the human brain (Goldberg et al., 2013). Recently, resting state functional magnetic resonance imaging (fMRI) has been widely used in the studies of the human brain in which it was organized into several intrinsic resting state networks (RSNs). More and more studies have provided the evidence to support the importance of these RSNs on the brain function, such as default mode and attention. In general, the spatial pattern of these RSNs is relatively stable across subjects. These RSNs demonstrate either spatial hemispheric symmetry, such as the default mode network (DMN) and sensorimotor network (SMN), or hemispheric asymmetry, such as the frontoparietal network (FPN) and attention network (Fox et al., 2006). Hemispheric symmetry, which is represented by the spatial lateralization of RSNs, may reflect the slight changes of RSNs in different states of the brain. For example, the lateralization is influenced by development (Kelly et al., 2009), gender (Agcaoglu et al., 2015) and diseases (Luo et al., 2014b). The lateralization of RSNs is also observed to be significantly altered in healthy older adults compared with younger adults (Agcaoglu et al., 2015; Seidler et al., 2015). Neurodegeneration is associated with increasing age (Seidler et al., 2010), i.e., declining cognition and decreases in motor performance (Cheng et al., 2012; Brown et al., 2013). Physical activity and exercise have positive influences on neurodegeneration (Brown et al., 2013). It has been observed that directed cognitive training interventions will increase mental activity in older adults, helping them to resist age-related cognitive decline and even potentially reducing the risk of dementia (Gates and Valenzuela, 2010). In a previous behavioral study, we observed that cognitive training can improve multi-cognition in community-living older adults, including memory, attention and neuropsychological status (Cheng et al., 2012). However, the underlying brain mechanism allowing the training to enhance function has been unclear till now. Noninvasive neuroimaging techniques, especially fMRI, are used to investigate the association between the intervention and brain function. For example, musical training would enhance the functional connectivity in perceptual and motor systems (Luo et al., 2012a) and salience network (Luo et al., 2014a). Additionally, the motor-related exercise increased the integration of motor performance and imaging systems (Gong et al., 2015, 2016; Li et al., 2015a). MRI studies of aging adults have shown enlarged local cortex thickness and enhanced functional integration related to the expertise in cognitive and sensorimotor interventions (Boyke et al., 2008; Lustig et al., 2009; Cao et al., 2014; Li et al., 2014). Therefore, we hypothesized that the lateralization of RSNs found in resting state fMRI scanning might provide an approach to reflect the influence of cognitive training on healthy older adults. In this study, to test our hypothesis, a longitudinal design with 1 year's duration was used to evaluate the cognitive training effects on the lateralization of intrinsic networks among healthy older adults. Healthy older adults with community-living were recruited and divided into two groups randomly: one received a multi-domain cognitive training over 3 months; the other was included in a wait-list control group. Resting state fMRI data were acquired before training and 1 year after training. The functional lateralization of 10 common resting state fMRI networks was evaluated between two groups using repeated measures analysis of variance (ANOVA). BODY.MATERIALS AND METHODS.PARTICIPANTS: Forty healthy older adults were recruited from three community centers around Tongji Hospital in Shanghai via a dispatched notice/broadcasting by the local institute of community service from March 2008 to April 2008. All participants were admitted to the study after a personal interview according to the inclusion criteria as follows: normal functional capacity; independent living in the community; age (range: 65–75 years); educational level (more than 1 year); no abnormality in hearing, vision, or communication status; a score of 19 or above (the lower normal cut-off point of the MMSE score is due to the lower educational level in China than in the US; Li et al., 2006) using the Chinese version of the Mini-Mental State Examination (MMSE); and no physical disease or psychotic disorder. The subjects with obvious cognitive decline, a diagnosis of AD, a brain tumor, or serious neurological and/or psychiatric disorders such as major depressive disorder and schizophrenia were excluded in this study. All of participants underwent cognitive measurements and fMRI scanning at baseline and at 1 year after training. They were divided randomly into two groups, including the multi-domain training group (n = 23) and the control group (n = 17). One participant out of 23 in the multi-domain training group and one in the single-domain training group were excluded because of left-handedness, which would interrupt the study of the lateralization of brain (Mackey et al., 2013). This study was reviewed and approved by the Ethical Committee of the East China Normal University. This study was performed according to the recommendation of the Ethical Committee of the East China Normal University with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. BODY.MATERIALS AND METHODS.COGNITIVE INTERVENTIONS AND NEUROPSYCHOLOGICAL TESTS: We conducted a randomized, controlled design to determine the effect of cognitive training on brain function and cognitive function (Cheng et al., 2012). The neuroimaging data and cognitive measurement were collected at baseline and at 1 year after training. The multi-domain training group received 24 sessions (each session was 60 min) of cognitive training at a frequency of twice a week over a 12-week period, and the training procedure took place in a classroom in Tongji Hospital. The multi-domain cognitive training targeted memory, reasoning, problem-solving strategies, visual-spatial map reading skill developments, handcraft making, and health and physical exercise. A lecture about a common disease in aging people was presented during the first 15 min of each session. Then, the trainer taught the participants about a special cognitive strategy or technique via slide lecture during the second 30 min. The newly practiced skills were consolidated by dealing with some real-life problems during the last 15 min. The wait-list control group served as a match for the social contact associated with cognitive training. The multi-domain training group and the control group attended a lecture about healthy living every 2 months (the training details are found in our previous study; Cheng et al., 2012). To evaluate the effects of intervention on cognitive function, all measurements were administered at baseline and at 1 year after intervention, including the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS, Form A), which has good validity and reliability in a Chinese community-living elderly sample (Lim et al., 2010; Cheng et al., 2011), the trail making test (TMT; Ashendorf et al., 2008), the visual reasoning test (Xiao et al., 2002) and the Color Word Stroop test (CWST; van Boxtel et al., 2001). BODY.MATERIALS AND METHODS.DATA ACQUISITION: All participants were scanned using a Siemens 3T MRI Scanner (Erlangen, Germany) at baseline and at 1 year after training ending at East China Normal University, Shanghai, China. To minimize head motion, foam pads were used to fix the subjects' heads. High-resolution T1-weighted images were acquired using a magnetization-prepared rapid gradient-echo sequence, generating 160 slices (repetition time (TR) = 1900 ms, echo time (TE) = 3.43 ms, flip angle (FA) = 90 degrees, matrix size = 256 × 256, field of view (FOV) = 240 × 240 mm2, slice thickness = 1 mm; no gap). Resting state functional images were acquired using a single-shot, gradient-recalled echo planar imaging sequence (TR = 2000 ms, TE = 25 ms and FA = 90 degrees, FOV = 240 × 240 mm2, matrix = 64 × 64, slice thickness = 5 mm (no gap), 32 slices per volume). All subjects underwent a 310 s scanning to yield a total of 155 volumes. The subjects were instructed to rest with their eyes closed, not to think of anything in particular, and not to fall asleep. BODY.MATERIALS AND METHODS.DATA PRE-PROCESS ANALYSIS: Pre-processing of fMRI data were conducted using the SPM8 Software Package [statistical parametric mapping1]. The slice time correction, 3D motion detection and correction, spatial normalization and resample (3 mm × 3 mm × 3 mm), and spatial smoothing using an isotropic Gaussian kernel (6 mm full width at half maximum) were applied. The processing is identical to that used in prior studies (Chen et al., 2015; Jiang et al., 2016). Only the subjects with head motion less than 1.5 mm and 1.5° during fMRI acquisition were included in the following preprocessing. In addition, the translation and rotation of the subjects were assessed by frame wise displacement (FD), FDi=|Δdix|+|Δdiy|+|Δdiz|+|Δαi|+|Δβi|+|Δγi|, where i is the i-th time point, and Δdix=d(i−1)x−dix (similarly for the other head motion/rotation parameters; Power et al., 2012). No significant differences were found between groups in FD. Additional preprocessing in preparation included voxelwise nuisance correction by regressing out six motion signals. BODY.MATERIALS AND METHODS.ICA DECOMPOSITION: Similar to the approaches in our previous studies (Luo et al., 2012c; Li et al., 2015b), we first conducted spatial group ICA in which two times of data of all subjects were included in one group, using the GIFT Software, version 2.0a2. The time courses were temporally concatenated across subjects and reduced by means of principal component analysis in temporal domain, followed by an IC estimation using the FastICA algorithm. This algorithm was repeated 20 times in ICASSO3, and the resulting components were clustered to estimate the reliability of the decomposition (Himberg et al., 2004). Dimension estimation on all subjects was performed using the minimum description length (MDL) criterion to determine the number of independent components (ICs). The ICs were back-constructed for each scans and each subject using the dual-regression approach (Zhang et al., 2010). Finally, we interrogated all components to identify RSNs. Ten RSNs were chosen based on the average power spectra and spatial map of the components such as in the previous studies (Luo et al., 2012c; Li et al., 2015b). Here, the power spectra of time course of ICs was analyzed, and the power spectra of the selected IC should show the frequency content was mainly concentrated below 0.1 Hz (Beckmann et al., 2005; Luo et al., 2012b). BODY.MATERIALS AND METHODS.SPATIAL NORMALIZATION TO SYMMETRIC TEMPLATES: To acquire the hemispheric symmetries' RSNs, the warping step was performed from the resulted RSNs to the symmetries' templates. First, the MNI template was warped to a symmetrized version of the MNI template because all subjects' functional images were normalized into the MNI template. Then, the MNI template was normalized to the symmetrized template. Finally, the warping parameters were applied to all components for the symmetrizing RSNs in all subjects. BODY.MATERIALS AND METHODS.CALCULATION OF VOXELWISE HOMOTOPIC MAPS: The component values were initially normalized across voxels using z-score. The differences (Bv) between component values of both homotopic voxels on both sides of cerebral cortex were calculated using the following formula 1. As with our previous study (Agcaoglu et al., 2015), the difference was shown in a map with a positive difference (R > L) on the right side of the brain and negative difference (L > R) on the left side of the brain. (1)Bv={​​​(Rv−Lv)ifRv>Lv(Lv−Rv)ifLv>Rv0 otherwise where R and L represents the right hemisphere and left hemisphere for each pairs of homotopic voxels v. Hence, the voxelwise homotopic map was produced for each subject each RSN. BODY.MATERIALS AND METHODS.LATERALITY COFACTOR: The laterality cofactor (LCF) was defined based on a voxelwise homotopic map, as in the previous study (Agcaoglu et al., 2015). The amount of laterality for a given RSN was calculated in the global laterality metric. The LCF was acquired by taking the differences between the sum of all intensities of laterality on the right and left hemispheres with respect to the sum of all intensities across the brain (Formula 2). The LCF was computed for the average models for each group. In addition, individual LCF was calculated to evaluate the alteration of LCFs related with training. (2)LCF=Sl−SrSl+Sr Where Sl and Sr represents the sum of all intensities of laterality on the left and right hemispheres. BODY.MATERIALS AND METHODS.STATISTICAL TEST: For each of the components, one sample t-test was performed in voxelwise homotopic maps in three groups. Here, the pretraining fMRI data from all the subjects were included in one group; the second data were divided into training and control groups on the basis of receiving the training or not. For the three t-value maps, we then applied a mask to retain those voxels whose t-values exceed one standard deviation of the t-value across voxels. The thresholded t-maps were used to compute the LCFs of group level (Global LCF) according to the same formula to calculate the LCF (Formula 2). According to the formula 2, the LCF value range was between −1 and 1. The Fisher-z-translation of individual LCF was performed for the distribution normality for each RSN. The individual LCF of each RSN was also evaluated used the ANOVA for repeated measurement data with post hoc, pair-wise Scheffe test in SPSS v20 (IBM Corp., Somer, NY, USA). BODY.RESULTS.DEMOGRAPHIC INFORMATION: At baseline, 23 subjects from the multi-domain training group and 17 subjects from the control group underwent cognitive measurements and fMRI scanning. Eighteen of the multi-domain training group and 14 of the control group finished the cognitive measurements and fMRI scanning at 1 year after the intervention. A total of eight participants withdrew, one due to death, two due to intestinal cancer, one due to her husband's death, one due to operation and three due to rejecting the scanning. No significant differences with regard to age, gender, education and MMSE score were found between the multi-domain training group and the control group (Table 1). Table 1 Demographic information of the subjects . Multi-domain training group Control group p Age (year) Baseline 70.61 ± 3.29 68.59 ± 3.24 0.838 One-year Posttest 72.39 ± 3.43 70.85 ± 4.05 0.782 Gender (male) Baseline 23 (16) 17 (9) 0.283 One-year Posttest 18 (13) 14 (9) 0.631 Education (year) Baseline 10.91 ± 3.65 10.64 ± 3.06 0.452 MMSE Baseline 27.57 ± 2.57 28.17 ± 1.94 0.505 One-year Posttest 27.72 ± 2.16 27.85 ± 2.31 0.900 BODY.RESULTS.IDENTIFICATION OF RSN AND VOXELWISE HOMOTOPIC MAPS: There were 45 components resulting from the group ICA. Ten components were selected as nonartifactual, relevant networks by visual inspection in accordance with published results (Luo et al., 2012c; Li et al., 2015b). The spatial maps of these RSNs were highly stable (reliability index >0.83) as determined by ICASSO. The 10 RSNs were shown in Figure 1 and labeled as follows: Figure 1Ten resting state networks (RSN) were chosen. The one sample t-test in the group included all subjects in the pre-training scans was performed. V1N: primary visual network. The visual cortex is often apparent in two separate components. This network showed spatial patterns consisting of primary visual area (Damoiseaux et al., 2006; Luo et al., 2012c).V2N: the second visual network illustrated spatial patterns consisting of more lateral visual arear in the occipital lobe which were previously known to be involved in visual processing (Damoiseaux et al., 2006; Luo et al., 2012c).AN: auditory network (AN) primarily encompassed bilateral middle, superior temporal gyrus, temporal pole and insular, corresponded to the auditory system (Damoiseaux et al., 2006; Luo et al., 2012c).SMN: sensorimotor network (SMN) was a network corresponding to sensory-motor function. This network includes pre- and postcentral gyrus, the primary sensorimotor cortices, and the supplementary motor area (Damoiseaux et al., 2006; Luo et al., 2012c).CereN: cerebellum network included bilateral cerebellum hemispheres.DMN: default mode network (DMN) mainly encompasses posterior cingulate cortex, medial prefrontal gyrus, bilateral superior frontal gyrus, and bilateral angular gyri (Raichle et al., 2001; Damoiseaux et al., 2006; Luo et al., 2012c).lFPN: left lateral FPN (lFPN) along with right lateral FPN showed the similar spatial patterns with DAN consisting of regions previously known to be involved in goal-directed top-down processing (Damoiseaux et al., 2006; Luo et al., 2012c). This network primarily involved precuneus, inferior parietal lobule, middle frontal gyrus, superior parietal lobule.rFPN: right lateral FPN (rFPN) including clusters lateralized to the right hemisphere putatively associated with DAN. lFPN and rFPN were the only maps to be strongly lateralized and were largely left–right mirrors of each other (Damoiseaux et al., 2006; Luo et al., 2012c).CEN: central executive network showed spatial patterns consisting of the superior and middle prefrontal cortices, anterior cingulate and paracingulate gyri, and ventrolateral prefrontal cortex (Beckmann et al., 2005).BGN: basal ganglia network, encompassed middle temporal gyrus, superior temporal gyrus, insular and temporal pole and corresponded to the auditory system (Luo et al., 2012b). For 10 RSNs, their voxelwise homotopic maps were calculated for each group. Because the homotopic maps were similar to each other among groups, the group level maps resulted from the pre-training scans were illustrated in the Figure 2. Figure 2The homotopic maps of the 10 RSN. The group level maps resulted from one sample t-test in the groups including the pre-training scans (before cognitive training) of all subjects. BODY.RESULTS.LATERALITY COFACTORS: In the three groups, the global LCF of each RSN were illustrated in Figure 3. According to the previous criteria (Agcaoglu et al., 2015), the LCFs are called highly lateralized when it has absolute value above 0.75 or has lateralized with absolute value above 0.2; the LCFs in rFPN and lFPN were highly lateralized in three groups. The remaining eight RSNs did not have marked lateralization, in which the LCFs have absolute value less than 0.2. Figure 3The group level laterality cofactor (LCF) resulted from the three group Bv homotopic maps for each RSN. Three groups included the pre-training group including all subjects, the post-training group and the control group of the second scans. To investigate the influence of the multiple cognitive training on the laterality of RSN, the LCFs of each RSN and each subject were also evaluated (Table 2, Figure 4). After the Fisher r-to-z translation, a repeated measure ANOVA revealed that both groups improved their LCFs in CereN as indicated by a significant main effect of time (F = 6.903, p = 0.015). In addition, a significant training main effect on the LCF in rFPN (F = 5.897, p = 0.021) and lFPN (F = 7.641, p = 0.01), as well as interactions of LCF of lFPN (F = 8.908, p = 0.006), were observed. Post hoc analysis showed significantly increased LCF of lFPN in the training group compared with the control group at the second scan (T = 3.48, p = 0.001). Paired-sample t-tests revealed that the global LCF of CereN (T = 2.30, p = 0.03) was significantly increased after the training scan compared with the before-training scan in the control group but not in the training group. Similarly, compared with the before-training scan, the after-training scan showed the decreased LCF of lFPN (T = 2.21, p = 0.04) in the control group, whereas a marginal significant increase (T = 2.02, p = 0.06) was observed in the training group. Table 2 The results of the repeated measure ANOVA for the laterality cofactors of individual Bv maps in 10 RSNs . V1N V2N AN SMN CereN BGN DMN CEN lFPN rFPN Time main effects F value 3.498 0.279 0.111 3.888 6.903 1.044 0.677 0.901 0.006 1.183 P value 0.071 0.601 0.741 0.058 0.015* 0.315 0.417 0.35 0.939 0.285 Training main effects F value 0.192 0.264 0.048 0.699 0.499 3.808 1.088 0.026 7.641 5.897 P value 0.664 0.611 0.828 0.41 0.485 0.06 0.305 0.872 0.01* 0.021* Interaction effects F value 0.229 0.012 0.821 1.858 0.945 0.27 1.057 0.102 8.908 0.649 P value 0.636 0.915 0.372 0.183 0.339 0.607 0.312 0.752 0.006* 0.427 Note: *Represented the statistical significance p < 0.05 . Figure 4The LCFs of individual Bv maps in the lFPN, rFPN and CereN. The repeated measure ANOVA were performed. The significance of statistical test was demonstrated in the Table 2. BODY.DISCUSSION: The lateralization of human brain function is an obvious sign reflecting functional specialization. Neurodegeneration would alter the functional specialization in aging. In this study, we used the LCF (Agcaoglu et al., 2015) of intrinsic networks resulting from the resting fMRI to investigate the effects of multi-domain cognitive training on healthy older adults. We observed statistically significant training effects on the lateralization of two important RSNs related to high-level cognition: right- and left- FPNs. In particular, the lateralization of lFPN were retained well in the training group but decreased in the control group. The increased lateralization with aging was observed in the CereN, in which the lateralization was significantly increased in the control group, although the same change tendency was observed in the training group. These findings indicate that the lateralization of the high-level cognitive intrinsic networks is sensitive to multi-domain cognitive training. This study provides neuroimaging evidence to support the idea that cognitive training should have advantages in preventing the cognitive decline in healthy older adults. Both human and animal studies indicates neural plasticity across the lifespan (Ball et al., 2002; Papp et al., 2009). A number of studies support the protective effects of late-life cognitive training on dementia (Ball et al., 2002; Wilson et al., 2002; Snowball et al., 2013). Our previous study has illustrated positive effects of multi-domain cognitive training interventions in healthy older adults. Here, the findings from neuroimaging were provided to uncover the potential brain changes to response the effects of interventions. The functional neuroimaging biomarkers can play an important role in detecting, assessing and diagnosing neurodegeneration (Horwitz and Rowe, 2011). In addition, there was altered intrinsic connectivity in the special targeted networks such as DMN and salience network to different neurodegenerative disorders (Seeley et al., 2009). Recently, the lateralization of RSNs was observed as an alteration accompanied by increased aging (Wilson et al., 2002). The hemispheric lateralization was also associated with enhanced cognitive ability (Gotts et al., 2013). Thus, the spatial pattern of intrinsic networks would be a candidate feature for the cognitive training intervention in older adults. Enhancement and maintenance of memory, visuospatial/construction and attention endured in healthy older adults with multi-domain cognitive interventions (Cheng et al., 2012). The changed lateralization of FPN and CereN which was observed in the cohort might be related to the brain mechanism of these behavior improvements. The FPNs are associated with attention, cognitive performance and control processes. In the studies of resting state fMRI, the FPN is often identified using ICA. FPNs have marked asymmetry, which is involved in a multitude of cognitions; the right is more involved in monitoring processes whereas the left is putatively more involved in production processes (Cabeza et al., 2003). Combining lFPN and rFPN showed a similar spatial pattern, with a dorsal attention network consisting of regions previously known to be involved in goal-directed top-down processing (Damoiseaux et al., 2006; Luo et al., 2012c). Significant interactions of global LCF of lFPN were observed in this study. Global LCF of lFPN was significantly decreased in healthy controls whereas a marginal significant increase was observed in the training group. We presumed that the lateralization of lFPN would be an important predictor for maintenance of attention functioning and production processes. These findings implicated that the multi-domain cognitive training would contribute to the top-down attention function in the healthy older adults. Although the training main effects were also observed in the rFPN using repeated measure ANOVA, as indicated by the difference between the training group and control group, the training main effects might be stained by the initial distinction between groups (Figure 4). Thus the influence of training on the rFPN would not be evaluated directly in this study. In general, the FPN would be a target to multi-domain cognitive training. Motor-related function decline is another physiological sign in old adults compared with younger adults. Accumulating evidence demonstrates decreased functional connectivity in the motor-related networks in aging, including the CereN and sensory-motor network (Tomasi and Volkow, 2012). Consistent with the previous studies, we found that the CereN is symmetrical at baseline (first scanning). However, improved lateralization of the CereN was observed in both group as indicated by a significant main effect of time in repeated measure ANOVA. The laterality significantly increased between two times of scans with interval of 1 year in healthy control groups whereas it did not in the training group. This finding reflected that the symmetry of the CereN was maintained in healthy older adults with multi-domain cognitive training. Recently, the functional connectivity studies indicate that the cerebellum participates in functional networks with motor control and cognitive processes (Habas et al., 2009; Krienen and Buckner, 2009). Thus, our findings might implicate that the multi-domain cognitive intervention contributed to the improvement of motor control in older adults. Actually, SMN would be directly related with motor performance (Zhang et al., 2015). Seidler et al. (2015) found greater network interactivity of SMN in older adults and suggested the protection against age declines in motor performance. However, no difference of the lateralization was found in the SMN in this study. It did not mean that the negative effects of cognitive training on the motor performance and motor prediction which are related with SMN are responsible. Future studies should focus on the various aspects of motor function associated with the cognitive intervention. There are several limitations which should be mentioned here. First, the longitudinal design with 1-year duration was adopted in this study. The subjects were randomly grouped, and the behavior scores and genders were matched between groups. However, some connectivity features from the resting state fMRI might not match because their features could be calculated according to different methods. Thus, some features whose difference illustrated by repeated measure ANOVA might be stained by the mismatch in baseline (the first time) measures. Second, the relative small sample size would be another shortage of our study. In the future, more subjects should be recruited into the longitudinal study. Finally, the Group ICA was used to investigate the symmetry of the spatial pattern of RSNs. However, the number of ICs might be an underlying confounding factor. Till now, there has been no validated way to identify the number of ICs in ICA analysis. In this work, the MDL criteria implanted in GIFT software was used to determine the number of ICs. In conclusion, lateralization of function is an important organization of the human brain. The distribution of intrinsic networks in the resting brain and their association with the multi-domain cognitive training is analyzed in this study. The lateralization of bilateral FPN and CereN was related to both aging and the multi-domain cognitive training in healthy older adults. These findings provide a neuroimaging evidence to support the positive effects of the cognitive training on enhancement and maintenance cognitive and motor function in healthy older adults. BODY.AUTHOR CONTRIBUTIONS: Conceived and designed the work: CLi, CLuo and DY; Acquired the data: XC, TL, YC and LJ; Analyzed the data: WC, XZ, CLuo, YG, XC and YC; Wrote the article: CLuo, CLi and DY. All authors revised the work for important intellectual content. All of the authors have read and approved the manuscript. BODY.CONFLICT OF INTEREST STATEMENT: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

TITLE: Effectiveness of lifestyle change plus dental care (LCDC) program on improving glycemic and periodontal status in the elderly with type 2 diabetes ABSTRACT.BACKGROUND: Currently, there is an increased prevalence of diabetes mellitus among the elderly. To minimize adverse effects on glycemic control, prevention and management of general and oral complications in diabetic patients is essential. The purpose of the present study is to assess the effectiveness of a Lifestyle Change plus Dental Care (LCDC) program to improve glycemic and periodontal status in the elderly with type 2 diabetes. ABSTRACT.METHODS: A quasi-experimental study was conducted in Health Centers 54 (intervention) and 59 (control) from October 2013 to January 2014. 66 diabetic patients per health center were included. At baseline, the intervention group attended a 20 minute lifestyle and oral health education program, individual lifestyle counseling using motivational interviewing (MI), application of self regulation manual, and individual oral hygiene instruction. The intervention group received booster education every visit by viewing a 15 minute educational video. The control group received a routine program. Participants were assessed at baseline and 3 month follow up for glycosylated hemoglobin (HbA1c), fasting plasma glucose (FPG), body mass index (BMI), periodontal status, knowledge, attitude and practice of oral health and diabetes mellitus. Data were analyzed by using descriptive statistic, Chi-square test, Fisher's exact test, t-test, and multiple linear regression. ABSTRACT.RESULTS: After the 3 month follow up, a multiple linear regression analysis showed that the intervention group was significantly negatively correlated in both glycemic and periodontal status. Participants in the intervention group had significantly lower glycosylated hemoglobin (HbA1c), fasting plasma glucose (FPG), plaque index score, gingival index score, pocket depth, clinical attachment level (CAL), and percentage of bleeding on probing (BOP) when compared to the control group. ABSTRACT.CONCLUSIONS: The combination of lifestyle change and dental care in one program improved both glycemic and periodontal status in the elderly with type 2 diabetes. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.in.th: TCTR20140602001. BODY.BACKGROUND: The elderly are at high risk of chronic conditions including diabetes mellitus (DM), arthritis, congestive heart failure, and dementia [1]. Furthermore, the prevalence of DM and oral manifestations of DM including periodontal disease also increase in the elderly [2]. Currently, many countries face the problem of increased prevalence of DM, which is a chronic, systemic metabolic disorder. DM causes morbidity and mortality due to long-term complications, which affect the important organs [2]. Clinical complications of DM include retinopathy, nephropathy, neuropathy, macro-vascular disease, delayed wound healing, and periodontal disease [3]. Patients with type 2 diabetes have to make multiple choices about the management of their condition, such as, appropriate dietary intake, physical activity, and adherence to drugs [4]. Periodontal disease, an inflammatory disease affecting the periodontium, is a complication of type 2 diabetes associated with health outcomes due to systemic inflammation [2]. Periodontal disease and DM have a bidirectional relationship [2]. The effects of DM on periodontal health and periodontal infection also affect glycemic control. Furthermore, periodontal infection increases the risk for developing DM complications [2]. It is important to minimize the adverse effects of oral complications on glycemic control in diabetic patients, particularly periodontal disease, through prevention and management [2]. There have been many studies of diabetes intervention programs to prevent and control diabetic complications by decreasing HbA1c including using nurse practitioners for lifestyle intervention in Dutch primary care [5], using delivery meals and dietary counseling to reduce body weight in Japan [6], using lifestyle change programs (dietary counseling and exercise) to reduce the proportion of metabolic syndrome [7] and using diabetes empowerment to increase medication adherence and self-care behavior [8]. Furthermore, there have been periodontal intervention programs in others countries which have emphasized periodontal therapy including tooth brushing instruction, oral health education, and supra-gingival scaling. All of these programs reportedly improved glycemic control by decreasing HbA1c and periodontal status with statistically significant differences [9-11]. However, Promsudthi et al. [12], who carried out their study in Thailand, found a decrease of HbA1c 3 months after periodontal treatment of scaling and root planing without a statistically significant difference [12]. Although, diabetes intervention programs can prevent and control diabetic complications by decreasing HbA1c and periodontal intervention programs also decreased HbA1c and periodontal status, decreasing HbA1c from lifestyle change alone may not prevent periodontal disease; it only decreases the risk of periodontal infection [2]. The most important method for treating periodontal disease is intensive oral hygiene instructions including teaching tooth brushing, flossing, and the use of others devices [13]. Periodontal treatment programs improved periodontal status [9-12]. To control DM, patients should maintain healthy lifestyles and routinely control health levels, healthy eating habits, regular physical activity, and take diabetes medicine [14]. Combined lifestyle change and periodontal care intervention are needed to prevent dental complication. The objective of the present study was to assess the effectiveness of the Lifestyle Change plus Dental Care (LCDC) program to improve glycemic and periodontal status in the elderly with type 2 diabetes. BODY.METHODS: The quasi-experimental study was conducted in two health centers located in Bangkok, Thailand from October 2013 to January 2014. Health Centers 54 and 59 were selected from 68 health centers in Bangkok because these health centers serve a population with similar socio-demographic characteristics, have scheduled appointments and have at least 500 patients in their Diabetes Clinics. The two health centers were randomly assigned to intervention (Health Center 54) and control (Health Center 59) groups. Systematic sampling was used to select 66 participants in each health center. The sample size was calculated based on a previous study [12]. The average HbA1c level of the intervention and the control groups were 8.78% and 9.28%, respectively and the pooled variance was 0.88 [12]. The sample size required in each group in the current study was 55 with 80% power at the 5% significant level. 20% was increased for refusal and attrition in each group so the total sample size in each group was 66 and overall sample size was 132 participants. The inclusion criteria for both male and female participants included patient age over 60 years with type 2 diabetes and at least 16 natural teeth (acceptable for scoring plaque and gingival index). The patients who had serious systemic diseases or complications, blood disease, liver damage, kidney disease, severe chronic periodontitis, communicable disorder, could not speak Thai language, or did not agree to participate, were excluded. BODY.METHODS.TRAINING OF INTERVIEWERS: The interviewers were standardized by attending a 2–day training program. Two nurse practitioners were trained in Motivational Interviewing (MI) for lifestyle change and dental care including dietary counseling, physical activity, quiting smoking, and oral health care, by experts in this field. Moreover, the same two nurse practitioners and two dental assistants attended a one day training program for the education and teaching technique, by experts in education, diabetes, and oral health. BODY.METHODS.INTERVENTION: Lifestyle Change plus Dental Care (LCDC) program is based on the health belief model, social cognitive theory, and cognitive-behavioral theory [15]. At baseline, participants received a 20 minute lifestyle and oral health education program, by trained nurse practitioners, which emphasized type 2 diabetes complications, the prevention of general and oral health complications, the relationship between type 2 diabetes and oral complications, and oral health care. Then participants received individual lifestyle counseling by MI, application of self regulation manual and selected their goal of lifestyle and oral health care change with trained nurse practitioners. The content of the lifestyle counseling and self regulation manual were consistent with lifestyle change and oral health education. The goals included loss of body weight, eating healthy food (fruits and vegetables), eating more high-fiber foods, eating less sugar, exercising for more than 30 minutes at least 3–5 times/week, quiting smoking, tooth brushing after meals, and using dental floss at least 1 time/day. Individual oral hygiene instruction by trained dental assistants was also conducted in the dental room. The content included tooth brushing with fluoride toothpaste, using dental floss or other devices such as inter-proximal brush, cleaning dentures, and how to check oral health by themselves. In the 1st and 2nd month, participants received an educational booster by viewing a 15 minute educational video covering all of the abovementioned points. Furthermore, the goal of lifestyle and oral health care change was boosted by nurse practitioners. A focus group discussion was used to develop a slide presentation for lifestyle change and oral health education, self regulation manual, and the 15 minute educational video by brainstorming ideas from doctors, nurse practitioners, dentists, dental assistants, and a representative of diabetic patients. The slide presentation, self regulation manual, and educational video were validated by an expert in education, an expert in diabetes and an expert in dentistry. A pretest of the three items was also conducted. BODY.METHODS.CONTROL GROUP: The routine program in the diabetes clinic, which the participants in the control group attended, included seeing a doctor, measuring fasting plasma glucose (FPG), collecting diabetic medicine from a pharmacist, and making an appointment for their next visit. BODY.METHODS.OUTCOME MEASURES: Participants in both groups received oral examination, blood sample testing, and face-to-face interview at baseline and 3 month follow up. The single-blind technique was used. The participants did not know if they were in the intervention or the control group. Oral examination was done by two calibrated dentists. Inter-examiner reliability was tested by using Cronbach's Coefficient Alpha. Another 5 diabetic patients were examined for periodontal status (plaque index, gingival index, pocket depth, gingival margin, and percentage of bleeding on probing (BOP)) by three dentists included one expert in periodontics (gold standard) and the other two dentists who conducted the present study to measure the agreement between examiners. The Cronbach's Coefficient Alpha was 0.85. Plaque index score, gingival index score, pocket depth, clinical attachment level (CAL), and percentage of bleeding on probing (BOP) [16] were used to find periodontal status. Pocket depth was measured by a level from the gingival margin to the most coronal extension of the epithelial attachment [17]. Gingival recession was measured by a level from cemento-enamel junction to the gingival margin [17]. The pocket depth and gingival recession of the six surfaces (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distobuccal) of every tooth in the diabetic patient's mouth were recorded by using a periodontal probe [18]. Clinical attachment level (CAL) was calculated by measuring pocket depth plus gingival recession [18]. Blood sample testing was done by nurse practitioners. Biomedical outcomes included glycosylated hemoglobin (HbA1c), fasting plasma glucose (FPG), and body mass index (BMI). The questionnaire was validated by three experts in public health. The Item-Objective Congruence Index (IOC) was 0.83. A pilot study was carried out to test the reliability of the questionnaire. The Cronbach's Coefficient Alpha was divided into 4 parts; knowledge toward oral health and DM: 0.84, attitude toward oral health and DM: 0.87, oral health behaviors: 0.77, and practice toward DM: 0.89. The questionnaire included general characteristics, BMI, knowledge and attitude toward oral health and DM, oral health behaviors, and practice toward DM. BODY.METHODS.STATISTICAL ANALYSIS: Descriptive statistic, Chi-square test, Fisher's exact test, and t-test were used to compare the difference between the intervention and the control groups at baseline. T-test was used to compare the difference of biomedical outcomes, periodontal status, knowledge and attitude toward oral health and DM at baseline and 3 months follow up between the intervention and the control groups. Chi-square test and Fisher's exact test were used to explore the effect of the intervention on oral health behaviors and practice toward DM. Multiple linear regression was also used to measure the relationship. The Enter Method was used to include variables in the regression models. Group affiliation, age, gender, smoking, BMI, and the respective baseline measures were adjusted for glycemic status (FPG and HbA1c). In regard to periodontal status (plaque index, gingival index, pocket depth, CAL, and BOP), group affiliation, age, gender, smoking, and the respective baseline measures were adjusted. Data were analyzed by SPSS statistical package version 16.0. All analysis used a 95% confidence interval (CI), and statistically significant p-value of less than 0.05. BODY.METHODS.ETHICAL CONSIDERATION: Ethics approval was granted from the Ethics Review Committee for Research Involving Human Research Subjects, Health Science Group, Chulalongkorn University (No. 123.1/56). Informed consent was signed by all participants. BODY.RESULTS.STUDY POPULATION: Of the 132 participants who enrolled at baseline (66 intervention and 66 control), 130 (98.5%) were eligible for follow up at 3 months. Of those who were not eligible, 1 participant in the intervention group was too ill to follow up (paralysis) and 1 participant in the control group moved to live in another province. BODY.RESULTS.BASELINE CHARACTERISTICS: Among the 132 diabetic patients (66 diabetic patients per group), most of the participants in each group were female. The average age of the intervention and the control groups were 63.83 (4.51) years and 64.06 (5.53) years, respectively. There were no statistically significant differences of gender, age, educational level, BMI, health insurance, duration of having diabetes, and smoking between the intervention and the control groups (p = 0.856, 0.357, 0.790, 0.057, 0.643, 0.118 and 0.474, respectively) (The details are demonstrated in Table 1). Table 1 Baseline characteristics (N = 132) Variables Intervention group Control group p-value* (n = 66) (%) (n = 66) (%) Gender        Male 23 (34.8) 24 (36.4) 0.856  Female 43 (65.2) 42 (63.6)   Age        60–69 years 56 (84.8) 55 (83.3) 0.357  70–79 years 10 (15.2) 9 (13.6)    ≥80 years 0 (0.0) 2 (3.1)   Educational level        Illiteracy 2 (3.0) 3 (4.5) 0.790  Primary school 53 (80.3) 48 (72.8)    Secondary school 8 (12.2) 9 (13.7)    Vocational school 2 (3.0) 3 (4.5)    Bachelor degree 1 (1.5) 3 (4.5)   BMI (kg/m 2 )        Mean (S.D.) 25.30 (3.57) 26.63 (4.37) 0.057  Min-max 17.95–34.60 18.93–36.50   Health insurance        Universal coverage 55 (83.4) 59 (89.4) 0.643  Universal coverage (other) 3 (4.5) 2 (3.0)    Government/state enterprise officer 7 (10.6) 5 (7.6)    No 1 (1.5) 0 (0.0)   Duration of being diabetes (years)        Mean (S.D.) 6.86 (5.16) 8.42 (6.19) 0.118  Min-max 1–20 1–25   Smoking        Never 57 (86.4) 58 (87.8) 0.474  Ever 7 (10.6) 4 (6.1)    Current smoker 2 (3.0) 4 (6.1)   p by chi-square test or t-test. *Statistical significances of difference: p < 0.05. BODY.RESULTS.BIOMEDICAL OUTCOMES: Among the remaining 130 participants (65 intervention and 65 control), HbA1c in the intervention group decreased from 7.39 (1.19) % at baseline to 7.10 (1.04) % at 3 month follow up. Whereas, in the control group, HbA1c increased from 7.68 (1.48) % at baseline to 7.77 (1.46) % at 3 month follow up. FPG in the intervention group decreased from 143.83 (38.78) mmol/l at baseline to 129.57 (21.24) mmol/l at 3 month follow up. Whereas, in the control group, FPG increased from 152.91 (51.35) mmol/l at baseline to 158.32 (47.28) mmol/l at 3 month follow up. There were statistically significant differences of both HbA1c and FPG between the intervention and the control groups at 3 month follow up (p = 0.003 and <0.001, respectively). However, there was no statistically significant difference in BMI between the intervention and the control groups at 3 month follow up (p = 0.057) (Table 2). Table 2 T-test for biomedical outcomes, periodontal status, knowledge and attitude toward oral health and DM (N = 130) Variables Intervention group Control group p-value* n = 65 n = 65 Mean Standard deviation Mean Standard deviation HbA1c            Baseline 7.39 1.19 7.68 1.48 0.217  3 months 7.10 1.04 7.77 1.46 0.003 FPG            Baseline 143.83 38.78 152.91 51.35 0.258  3 months 129.57 21.24 158.32 47.28 <0.001 BMI            Baseline 25.35 3.57 26.66 4.40 0.064  3 months 25.57 3.64 26.88 4.11 0.057 Plaque index score            Baseline 0.59 0.42 0.63 0.41 0.544  3 months 0.26 0.31 0.45 0.45 0.006 Gingival index score            Baseline 0.64 0.41 0.77 0.48 0.139  3 months 0.27 0.29 0.48 0.40 0.001 Pocket depth            Baseline 2.35 0.55 2.39 0.81 0.719  3 months 2.04 0.47 2.54 0.88 <0.001 CAL            Baseline 3.33 0.86 3.68 1.31 0.079  3 months 2.96 0.85 3.64 1.37 0.001 BOP            Baseline 35.36 25.83 37.32 22.17 0.644  3 months 17.66 22.18 38.22 33.36 <0.001 Overall knowledge            Baseline 7.19 2.36 7.04 2.27 0.705  3 months 9.48 0.70 7.29 2.23 <0.001 Oral knowledge            Baseline 3.68 1.73 3.51 1.60 0.564  3 months 4.77 0.52 3.63 1.36 <0.001 DM knowledge            Baseline 3.53 1.09 3.58 0.97 0.799  3 months 4.70 0.28 3.66 1.17 <0.001 Overall attitude            Baseline 43.75 4.42 41.80 6.57 0.057  3 months 47.82 3.41 41.44 5.27 <0.001 Oral attitude            Baseline 21.54 2.95 20.78 3.15 0.161  3 months 23.85 1.80 20.58 3.18 <0.001 DM attitude            Baseline 22.25 2.32 21.74 2.57 0.239  3 months 23.92 1.81 20.86 2.62 <0.001 p by t-test. *Statistical significances of difference: p < 0.05. BODY.RESULTS.PERIODONTAL STATUS: Among the 130 participants (65 intervention and 65 control), the plaque index score and gingival index score decreased from baseline to 3 month follow up in both the intervention and the control groups with statistically significant differences between the intervention and the control groups at 3 month follow up (p = 0.006 and 0.001, respectively). Pocket depth and CAL decreased from baseline to 3 month follow up only in the intervention group. Whereas, in the control group, pocket depth increased and CAL was equal at baseline and 3 month follow up. The percentage of BOP decreased from baseline to 3 month follow up in the intervention group. Whereas, in the control group, the percentage of BOP increased from baseline to 3 month follow up. There were statistically significant differences between the intervention and the control groups at 3 month follow up of pocket depth, CAL, and BOP (p < 0.001, 0.001 and, <0.001, respectively) (Table 2). BODY.RESULTS.KNOWLEDGE AND ATTITUDE TOWARD ORAL HEALTH AND DM: The average score of overall knowledge, oral health knowledge, and diabetes knowledge increased from baseline to 3 month follow up only in the intervention group with statistically significant differences between the intervention and the control groups at 3 month follow up (p < 0.001, <0.001 and <0.001, respectively) (Table 2). The average score of overall attitude, oral health attitude, and diabetes attitude increased from baseline to 3 month follow up only in the intervention group with statistically significant differences between the intervention and the control groups at 3 month follow up (p < 0.001, <0.001 and <0.001, respectively) (Table 2). BODY.RESULTS.ORAL HEALTH BEHAVIORS: Every participant (100%) in both the intervention and the control groups reported regular cleaning of their oral cavity by tooth brushing at baseline and 3 month follow up (Table 3). Table 3 Chi-square test and Fisher-exact test for oral health behaviors and practice toward DM, at 3 month follow up (N = 130) Variables Intervention group (n = 65) Control group (n = 65) p-value* no. (%) no. (%) Oral health behaviors       Tooth brushing (yes) 65 (100.0) 65 (100.0) - Mouth rinse (yes) 32 (49.2) 24 (36.9) 0.157 Salt solution (yes) 32 (49.2) 19 (29.2) 0.020 Dental floss (yes) 46 (70.8) 5 (7.7) <0.001 Tooth pick (yes) 33 (50.8) 40 (61.5) 0.216 Inter-proximal brush (yes) 23 (35.4) 20 (30.8) 0.576 Have had dental treatment, previous 3 months (yes) 24 (36.9) 15 (23.1) 0.085 Practice toward DM Exercise        > 5times/week 19 (29.2) 6 (9.2) < 0.001  2-5 times/week 24 (36.9) 22 (33.8)    1 time/week 12 (18.5) 9 (13.8)    2-3 times/month 7 (10.8) 5 (7.7)    Never 3 (4.6) 23 (35.5)   Tested weight        > 1 time/month 22 (33.8) 13 (20.0) 0.075  1 time/month 43 (66.2) 52 (80.0)   Diet modification (yes) 61 (93.8) 46 (70.8) 0.001 Forgot to take any drugs prescribed (yes) 28 (43.1) 30 (46.2) 0.724 Eye examination, last year (yes) 56 (86.2) 50 (76.9) 0.175 Foot examination, last year (yes) 57 (87.7) 16 (24.6) < 0.001 Always wear covered shoes (yes) 41 (63.1) 19 (29.2) < 0.001 Screening of feet       Everyday 50 (76.9) 39 (60.0) 0.044 Sometimes 12 (18.5) 15 (23.1)   Rarely/never 3 (4.6) 11 (16.9)   p by chi-square test or Fisher-exact test. *Statistical significances of difference: p < 0.05. At baseline, there were no statistically significant differences in the use of mouth rinse, salt solution, dental floss, toothpick, inter-proximal brush, and having had dental treatment between the intervention and the control groups (p = 0.856, 0.291, 0.804, 0.722, 0.176, and 0.148, respectively). After 3 month follow up, the participants in the intervention group were more likely to use mouth rinse, salt solution, dental floss, and inter-proximal brush. However, only salt solution and dental floss had statistically significant differences (p = 0.020 and < 0.001, respectively). The participants in the intervention group were less likely to use toothpicks with no statistically significant difference (p = 0.216). Furthermore, the participants in the intervention group were more likely to have had dental treatment in the previous 3 months with no statistically significant difference (p = 0.085) (Table 3). BODY.RESULTS.PRACTICE TOWARD DM: At baseline, there were no statistically significant differences in exercise, tested weight, diet modification, forgetting to take any prescribed drugs, eye examination, foot examination, always wearing covered shoes, and screening of feet between the intervention and the control groups (p = 0.310, 0.397, 0.518, .0384, 0.394, 0.170, 0.282, and 0.337, respectively). After 3 month follow up, the percentage of participants who exercised more than 5 times/week in the intervention group (29.2%) was more than the control group (9.2%). Moreover, the participants who never exercise in the intervention group (4.6%) was less than the control group (35.5%). Exercise had a statistically significant difference between the intervention and the control groups (p < 0.001). The percentage of participants in the intervention group (93.8%) who modified diet was higher than the control group (70.8%) with a statistically significant difference (p = 0.001). The percentage of participants in the intervention group who received foot examination, always wore covered shoes, and participated in screening of feet were higher than the control group with statistically significant differences (p <0.001, <0.001, 0.044) (Table 3). BODY.RESULTS.MULTIPLE LINEAR REGRESSION ANALYSIS: In the multiple linear regression analysis, the intervention group was significantly negatively correlated in both glycemic and periodontal status at 3 month follow up. FPG at 3 month follow up was significantly correlated with group affiliation and FPG at baseline (R2 = 0.539, p <0.001). HbA1c at 3 month follow up was significantly correlated with group affiliation, smoking, and HbA1c at baseline (R2 = 0.757, p <0.001) (Table 4). The plaque index score at 3 month follow up was significantly correlated with group affiliation, smoking, and the plaque index score at baseline (R2 = 0.238, p <0.001). The gingival index score at 3 month follow up was significantly correlated with group affiliation and the gingival index score at baseline (R2 = 0.200, p <0.001). Pocket depth at 3 month follow up was significantly correlated with group affiliation and pocket depth at baseline (R2 = 0.533, p <0.001). CAL at 3 month follow up was significantly correlated with group affiliation and CAL at baseline (R2 = 0.721, p <0.001). The percentage of BOP at 3 month follow up was significantly correlated with group affiliation and BOP at baseline (R2 = 0.318, p <0.001) (Table 5). Table 4 Multiple linear regression analysis for glycemic status (FPG and HbA1c) (N = 130) Variables FPG (3rd month) HbA1c (3rd month) Parameter estimate * (Standard error), p-value Group affiliation (ref. control) −23.537 (4.831), <0.001 −0.403 (0.117), 0.001 Age −0.568 (0.481), 0.607 −0.004 (0.012), 0.719 Gender (ref. female) −8.188 (4.912), 0.098 0.070 (0.119), 0.557 Smoking (ref. no) 2.062 (4.912), 0.675 0.283 (0.118), 0.018 BMI 0.310 (0.602), 0.607 −0.005 (0.015), 0.738 FPG at baseline 0.541 (0.053), <0.001 0.795 (0.044), <0.001 HbA1c at baseline R 2  = 0.539, p <0.001 R 2  = 0.757, p <0.001 * Adjusted for group affiliation, age, gender, smoking, BMI, and the respective baseline measures. Table 5 Multiple linear regression analysis for periodontal status (plaque index, gingival index, pocket depth, CAL, and BOP) (N = 130) Variables Plaque index Gingival index Pocket depth CAL BOP (3rd month) (3rd month) (3rd month) (3rd month) (3rd month) Parameter estimate* (Standard error), p-value Group affiliation (ref. control) −0.148 (0.062), 0.018 −0.172 (0.058), 0.004 −0.480 (0.091), <0.001 −0.374 (0.111), 0.001 −18.938 (4.406), <0.001 Age 0.008 (0.006), 0.170 −0.002 (0.006), 0.712 0.002 (0.009), 0.826 0.010 (0.011), 0.372 −0.245 (0.434), 0.573 Gender (ref. female) 0.072 (0.064), 0.261 0.008 (0.060), 0.888 0.101 (0.094), 0.284 0.148 (0.117), 0.209 6.366 (4.587), 0.168 Smoking (ref. no) 0.166 (0.064), 0.011 0.011 (0.060), 0.857 −0.091 (0.097), 0.352 0.029 (0.118), 0.808 2.437 (4.549), 0.593 Plaque index at baseline 0.346 (0.075), <0.001         Gingival index at baseline   0.313 (0.066), <0.001       Pocket depth at baseline     0.732 (0.068), <0.001     CAL at baseline       0.836 (0.053), <0.001   BOP at baseline         0.585 (0.092), <0.001   R 2  = 0.238, p <0.001 R 2  = 0.200, p <0.001 R 2  = 0.533, p <0.001 R 2  = 0.721, p <0.001 R 2  = 0.318, p <0.001 *Adjusted for group affiliation, age, gender, smoking, and the respective baseline measures. BODY.DISCUSSION: The present study shows that the combination of lifestyle change and dental care in one program, improved both glycemic and periodontal status in the elderly with type 2 diabetes. As mentioned in the introduction, combined lifestyle change and periodontal care intervention are needed to prevent dental complications. The multiple linear regression analysis showed that the LCDC program was significantly negatively correlated in both glycemic (FPG and HbA1c) and periodontal status (plaque index, gingival index, pocket depth, CAL, and BOP). Furthermore, the multiple linear regression analysis also found HbA1c and the plaque index score significantly correlated with smoking which is consistent with a previous study that found that smoking is a risk factor for both periodontal disease and DM [19,20]. After the LCDC program, glycemic status including FPG and HbA1c decreased with statistically significant differences between the intervention and the control groups at 3 month follow up. Whereas, in the control group, both FPG and HbA1c increased from baseline to 3 month follow up. The results showed an improvement of glycemic status in the intervention group and deterioration of glycemic status in the control group. The difference of HbA1c in the intervention and the control groups were −0.29% and +0.09%, respectively with a statistically significant difference consistent with a previous study regarding the effect of changes in diet on HbA1c for 3 months which found the difference of HbA1c in the intervention was −0.83% with a statistically significant difference [21]. In other previous studies, meal preparation training also decreased HbA1c (0.3%) after 6 month follow up [22] and lifestyle counseling in the primary care setting also decreased HbA1c [23]. However, the effect of changes in supportive telephone counseling on HbA1c for 18 months did not present a statistically significant difference between the intervention and the control groups [24]. After the LCDC program, periodontal status including plaque index, gingival index, pocket depth, CAL, and BOP decreased from baseline to 3 month follow up. The slight decrease in mean differences of periodontal status highlighted the first step of the periodontal disease improvement with statistically significant differences between the intervention and the control groups at 3 month follow up. The present study found the significant correlation between periodontal status (BOP) and glycemic status (FPG) after the LCDC program was consistent with the previous studies that found periodontal disease is associated with the progression of HbA1c [25,26]. Many studies in other countries have found periodontal therapy including tooth brushing instruction, oral health education, and supra-gingival scaling improved glycemic control by decreasing HbA1c and periodontal status with statistically significant differences [9,10]. However, a previous study in Thailand found a decrease of HbA1c after periodontal treatment by scaling and root planing without a statistically significant difference [12]. Previous research papers which studied knowledge and attitude toward oral health and DM in type 2 diabetes have found the scores of knowledge and attitude in diabetic patients were low to moderate [27-30]. The present study found knowledge and attitude toward oral health and DM increased after the intervention. These results showed the effectiveness of the LCDC program to increase and maintain knowledge and attitude of the elderly with type 2 diabetes for 3 months. This is consistent with a previous study which found diabetic patients who received oral health information related to diabetes by health professionals, knowledge scored 2.9 times higher, compared to participants who did not receive that information [31]. The present study found the participants in the intervention group more likely to exercise, modify diet, have foot examinations, always wear covered shoes, and participate in screening of feet than the participants in the control group. This is inconsistent with a previous research paper, which studied a structured group diabetes education program of 6 hours, which was delivered in the community, with follow up for 3 years. The research found no statistically significant difference of physical activity [8]. The difference between the results of the current study and the abovementioned study is due to the fact that the abovementioned study used group education, did not use educational boosters, and used long term follow up. However, the present study used a mix of individual and group education, which was boosted every month and used short term follow up. The results of the present study are consistent with a previous study, which found improvements in eating control and step counts after receiving meal preparation training [22]. Yet another previous study found the association between the knowledge of preventive behaviors regarding foot ulcers and actual preventive behaviors [32] to be consistent with the present study which found the participants in the intervention group increased their knowledge score and improved their foot behaviors after receiving the LCDC program. The results of the present study show knowledge and attitude toward oral health and DM in the elderly with type 2 diabetes translated to practice in both oral health and DM. The strengths of the present study are the high response rate (98.5%) and that it used biomarkers including HbA1c, FPG, plaque index score, gingival index score, pocket depth, and CAL to examine the outcomes. The limitations of the present study are a lack of random assignment due to a quasi-experimental design, selection bias from the willingness to participate and it was not representative of the entire elderly population with type 2 diabetes because of the small number of centers in which the present study was conducted. However, the LCDC program had the effectiveness and acceptability that could be adapted into routine work by staff in the health centers which could be implemented in the other health centers. The single-blind technique might cause measurement bias. Furthermore, the use of participant reports to estimate practice toward DM and oral health behaviors are subject to some degree of measurement error. Future studies need to incorporate a longer follow-up period to generate understanding of intervention effects, adherence and sustainability, over time, by randomized controlled trial. BODY.CONCLUSION: The LCDC program was significantly negatively correlated in both glycemic and periodontal status that improved the glycemic status (HbA1c and FPG) and periodontal status (plaque index score, gingival index score, pocket depth, CAL, and BOP), which were maintained for 3 months. Furthermore, the results of the present study show the effectiveness of the LCDC program by increasing knowledge, attitude and practice toward oral health and DM of the elderly with type 2 diabetes. BODY.ABBREVIATIONS: BOP: Bleeding on probing; DM: Diabetes mellitus; HbA1c: Glycosylated hemoglobin; FPG: Fasting plasma glucose; MI: Motivational interviewing; BMI: Body mass index; CAL: Clinical attachment level; CI: Confidence interval; LCDC program: Lifestyle change plus dental care program. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: SS participated in the sequence alignment, drafted the manuscript, designed the study and performed the statistical analysis. ST conceived the study, participated in its design and coordination, and helped to draft the manuscript. Both authors read and approved the final manuscript. BODY.AUTHORS’ INFORMATION: Saruta Saengtipbovorn is a dentist at Health Center 54 and a doctoral student in Public Health at the College of Public Health Sciences, Chulalongkorn University. Surasak Taneepanichskul is a medical doctor, Professor of Obstetrics and Gynaecology and Dean of the College of Public Health Sciences, Chulalongkorn University. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1472-6831/14/72/prepub

TITLE: The effect of stretching exercise and walking on changes of blood pressure in nulliparous women ABSTRACT.BACKGROUND:: Hypertension in pregnancy is one of the prevalent disorder resulting in maternal death. The aim of this study was to investigate the effect of stretching exercise and walking on changes of blood pressure in nulliparous women during pregnancy. ABSTRACT.MATERIALS AND METHODS:: This was a quasi-experimental trial that consisted three groups of women who took part in pre- and post-tests. We used a simple randomized sample, including 118 pregnant females (walking: 29 subjects, stretching exercise: 30 subjects control: 59 subjects). The data were collected using the demographic checklist and blood pressure was measured every week. SPSS 16 was used to analyze the data by one-way analysis of variance (ANOVA) and repeated measure ANOVA. ABSTRACT.RESULTS:: No significant difference was found in the demographic characteristics of the three groups of women. Mean systolic and diastolic blood pressure in the three groups (stretching exercises, walking, and routine care) at three intervals (pre-test, first post-test, and second post-test) were significantly different (P < 0.05). In this case, Tukey's test showed significant improvement of systolic and diastolic blood pressure in stretching exercise group. Walking and control groups showed no change or significant reduction (P < 0.05). No significant difference was found between the walking and control groups (P > 0.05). ABSTRACT.CONCLUSIONS:: The results of the study showed that stretching exercise versus walking reduces systolic and diastolic blood pressure in the second trimester of pregnancy and controls it in the third trimester of pregnancy. In contrast, walking has no effect on blood pressure during pregnancy. BODY.I: Pregnancy is often a stressful period with great biochemical, physiological, and anatomical changes in the body. Sometimes, these physiological changes cause pathological conditions, and problems and diseases for the pregnant mother. Hypertensive disorder is one of the most common complications during pregnancy and it occurs in 5–10% of pregnancies.[1] Based on the latest classification system, these disorders are divided into five classes of gestational hypertension, preeclampsia, eclampsia, chronic hypertension, and superimposed preeclampsia.[2] Gestational hypertension and preeclampsia can occur with pregnancy. Only definitive treatment is termination of the pregnancy.[3] Small numbers of risk factors for hypertensive disorders during pregnancy are known. Recent studies have suggested that height body mass index and weight gain during pregnancy are important risk factors for hypertensive disorders in pregnancy. Studies have shown that the risk of these disorders increases with obesity, being overweight before pregnancy, and excessive weight gain during pregnancy.[45] These conditions are associated with increased risk of preterm delivery, neonatal intensive care unit (NICU) admission, and fetal death. Moreover, these conditions form the second leading cause of death for pregnant women.[6] It was also shown that 19% of maternal deaths after childbirth and 20% of deaths after stillbirth are due to these disorders.[7] High blood pressure during pregnancy can cause serious problems during childbirth, such as stroke, premature birth, or low birth weight.[8] In addition, gestational hypertension and preeclampsia are the risk factors for future metabolic syndrome, insulin resistance, and cardiovascular diseases.[9] Antihypertensive agents that seem to have no effect on pregnancy outcome are used for gestational hypertension treatment. However, it must be borne in mind that these studies were limited to the use of these drugs during pregnancy. On the other hand, these treatments are used after mid-pregnancy, when the potential risks of congenital anomalies have passed. However, complications such as fetal intrauterine growth restriction, fetal and neonatal bradycardia, and rare cases of thrombocytopenia have been reported due to the use of antihypertensive medications.[10] Today, due to the limitations of therapies, much attention has been paid to the prevention of hypertensive disorders in pregnancy.[11] Recently, a large number of women, according to the recommendations of the American Board of Obstetrics and Gynecologists and other organizations, engage in exercise during pregnancy.[12] Evidence shows that exercise during pregnancy, even for women who have been sedentary before pregnancy, has beneficial effects.[13] Exercise is one of the most effective ways to reduce the adverse effects of pregnancy, such as excessive weight gain[1415] and hypertension.[16] Most pregnant women choose walking as their exercise because it is easy to implement,[17] but stretching exercise as physical activity could be done easier than walking; therefore, women adherence to this activity.[18] Stretching exercises increase muscle flexibility that was measured in a previous study and could reduce the most common complaints such as back pain after childbirth.[19] Some studies on the relationship between physical activity and hypertensive disorders in pregnancy have shown that physical activity and exercise before or during pregnancy can reduce the risk of hypertensive disorders and preeclampsia.[202122] However, Heggard et al.[23] and Vollebregt et al.[24] have not reported any relationship between these factors in their studies. In addition, the study results of Østerdal et al. showed that high-intensity physical activity in the first trimester of pregnancy may increase the risk of preeclampsia.[25] In total, there are discrepancies between the present studies, and most of the studies are cohort and case–control studies. To investigate the effect of physical activity on the prevention of high blood pressure during pregnancy, further studies are needed with better quality and semi-experimental trial designs. The purpose of the present study was to investigate the effects of walking and stretching exercises on blood pressure during pregnancy. BODY.M: The present study was a quasi-experimental study with pre- and post-test design. The participants were randomly selected from two health centers in Isfahan, Iran, from April 2011 until November 2012. 118 healthy pregnant women in their second trimester were selected. Furthermore, they were randomly placed in three groups of walking (n = 29), stretching exercises (n = 30), and routine care (n = 59). A written informed consent was obtained from every participant. This study was approved by the Ethics Committee of Isfahan University of Medical Sciences. The inclusion criteria of subjects in the present study included: First pregnancy, gestational age 18-22 weeks, willingness to participate in the study, a live fetus, women aged between 19 and 35 years, body mass index less than 30 kg/m2, absence of a family history of preeclampsia (mother and sister), no history of stretching exercises before pregnancy, and noprohibition to participating in stretching and walking exercises including movement disabilities, obstetric contraindications (history of threatened abortion, preterm birth risk in the current pregnancy, vaginal bleeding, history of preterm delivery, placenta previa, any problem in the waist or hips, hypertension, cardiovascular disease, incompetent cervix, leaking amniotic fluid, dizziness, headache,), and any kind of problem due to which inactivity is prescribed by the physician. Subjects from two public health centers in Isfahan were interviewed via phone calls, and were updated regarding the exercises and walking after the completion of the questionnaire and consent form. They were placed in three groups of stretching exercises, walking and routine care, based on their own preference. For the group with stretching exercises, the program lasted 20 weeks; each week consisted of a maximum of five and a minimum of three sessions and each session lasted about 45 minutes. Each session consisted of three parts: Warm-up, stretching (1. back stretch to correct the curvature, 2. unilateral neck stretch, 3. neck rotating, 4. up and down movement of the head, 5. moving the chin upward with stretch, 6. stretching the chest with the hands crossing over the chest, 7. hand and arm stretch, 8. unilateral stretch of the body while seated, 9. child pose, 10. cat pose, 11. hamstring stretch, 12. pelvis tilt, 13. groin stretching, and 14. V stretching of the thighs), and cooling down (including relaxation and breathing exercises). Stretching exercises were copied on a compact disc and were handed to the group with stretching exercises. Pressure and the repetition of each exercise were determined based on individual abilities. Incremental exercise was performed in three stages. Due to lack of space, two training sessions per week were performed at home and one session per week at the health center by exercise rehabilitator. Emergency exercise stopping criteria were: Shortness of breath, palpitations, dizziness, headache, nausea, severe or sudden abdominal pain, chest pain, back pain, pain in pubic area, vaginal bleeding, and reduced fetal movements after training.[26] Due to lack of space in the health centers, the 3-days-a-week exercises were limited to one session training in the health center and a minimum of two and maximum of four sessions at home. The exercises along with the warnings and necessary awareness were provided for the subjects on a compact disc. The exercises were performed at home for 30 to 45 minutes. The participants visited the health centers 1 day a week and their blood pressure was measured before the exercise and 15 min after the exercise. The group with walking exercise walked slowly for a maximum of 20 weeks, with a minimum of three sessions and a maximum of five sessions in each week, and each session lasted for 30–45 minutes. The pressure of walking was adjusted by a breathing test. The participants were taught to repeat a standard sentence while walking without shortness of breath. The sentence was "my Lord is the greatest, the most merciful, the most knowledgeable, and the most capable." At the end of each week, the subjects referred to the related relevant health center for blood pressure measurement and monitoring for signs of preeclampsia and possible problems associated with it. The volunteers' blood pressure was recorded after each weekly session. Moreover, the mean blood pressure was used for comparison of the second trimester and third trimester of pregnancy. The routine care group was asked to refer to the health center as routine clinical visits for measurement of hypertension and severity of preeclampsia. Prenatal care performed by midwives or physician at health centers consisted of: Pressure control, body temperature, fetal heart rate, health and nutritional counseling and trainings, and routine tests during pregnancy according to the integrated national guidelines for maternal care. The frequency of meetings for low-risk mothers was eight times, during 6–10, 16–20, 26–30, 31–34, 35–37, 38, 39, and 40 weeks of gestation.[27] Since blood pressure had to be measured in the second half of the pregnancy to the end of pregnancy in this study, the researcher measured blood pressure of the control group seven times, and the subjects' blood pressure was recorded and controlled based on their medical records in health centers. Data were analyzed using SPSS for Windows (version 16; SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) test was used to compare demographic characteristics and fertility. ANOVA with repeated data was used to compare blood pressure in the three groups. BODY.R: In the present study, 118 patients were selected, of whom 2 in the stretching exercise group, 3 in the walking group, and 5 in the routine care group were excluded during the study. Thus, statistical analysis was performed on 108 subjects. Age, gestational age, body mass index, and systolic and diastolic blood pressure in all three groups showed no significant difference (P > 0.05) [Table 1]. Mean systolic and diastolic blood pressure in all three groups of stretching exercises, walking, and routine care during the three periods (pre-test, post-test I, and post-test II) are shown in Table 2. The results showed that systolic and diastolic blood pressure in the three study groups had a significant difference (P < 0.05). Tukey's test showed that the difference between systolic and diastolic blood pressure of stretching exercises group and both groups of walking (P < 0.05) and routine care (P < 0.05) was significant. Nevertheless, the difference between walking group and routine care (P > 0.05) was not significant. Figure 1 shows the comparison of changes in systolic and diastolic blood pressure of the three groups of stretching exercises, walking, and routine care in pre-test and post-test I and II. Table 1 Characteristics of the pregnant women included in the sample Table 2 Repeated measure ANOVA results of systolic and diastolic blood pressure Figure 1A comparison of the blood pressure changes during pregnancy among the research groups BODY.D: Comparison of demographic characteristics revealed that the groups were similar in terms of age, gestational age, body mass index, and systolic and diastolic blood pressure. Therefore, the sampling method was good, and the results of this study seem reliable. To answer the aim of the present study, which was the comparative effectiveness of stretching exercises and walking on systolic and diastolic blood pressure of pregnant women during pregnancy, the results showed that stretching exercises compared to walking and routine care (no exercise) resulted in better control of systolic and diastolic blood pressure during pregnancy. In other words, it was shown that stretching exercises compared to walking could better decrease systolic and diastolic blood pressure in the second trimester and control their increase in the third trimester. In contrast, it was shown that the curve of systolic and diastolic blood pressure changes in the walking group was similar to the routine care group. Therefore, in this study it was concluded that walking does not affect systolic and diastolic blood pressure. Pregnancy causes an increase in blood volume, an increase in cardiac output, and a slight increase in stroke volume; but with the increase in blood volume, blood pressure does not change significantly during pregnancy. However, studies have shown that a slight decrease in blood pressure occurs especially in the second trimester of pregnancy.[1] It was also observed in our study that the systolic and diastolic blood pressure had fallen slightly in the second trimester. This might be due to the fact that in addition to the physiological nature of the pregnancy in the second trimester, exercise decreases blood pressure. In contrast, during the third trimester, systolic and diastolic blood pressure in all three groups showed a slight increase. One possible reason for this was that the patients were approaching the time of delivery and, thus, their psychological stress and fear of childbirth increased. However, stretching exercises could control systolic and diastolic blood pressure better than walking in the third trimester of pregnancy. According to previous studies, oxidative stress will possibly increase in this period, which is expected to increase blood pressure. However, studies have shown that stretching reduces oxidative stress, or in other words, produces anti-oxidative activity.[2829] Based on the present results, it seems that the proposed mechanism for the control of hypertension in the third trimester of pregnancy in the stretching exercises group is that regular stretching exercises is associated with increased endogenous antioxidants.[30] Sorenson et al. concluded in their study that the increase in energy expenditure during mild to moderate exercise during pregnancy reduces the risk of preeclampsia.[20] The results of other studies show the fact that physical activity before and during pregnancy reduces the risk of preeclampsia and hypertensive disorders of pregnancy.[2122313233] Yeo, in his clinical trials, experimented on the effects of walking and stretching exercises on the risk of preeclampsia. Systolic blood pressure had significantly increased in the walking group (P < 0.01), while for the stretching group, this change was not significant. Diastolic blood pressure decreased significantly in the stretching group (P < 0.05), but there was no significant change in the walking group.[30] In another report of Yeo et al., the incidence of preeclampsia in the walking group was 14.6% and in the stretching exercise group was 2.6%. In addition, the mean transferrin levels, as an indicator of antioxidants, were significantly higher in the stretching group compared to the walking group (P < 0.05).[33] This indicates that the stretching exercises had better effect on reducing blood pressure during pregnancy. This can be due to the endogenous antioxidant produced by regular stretching exercises, and was consistent with the current research. Tyidum et al.,[34] Rudra et al.,[35] Heggard et al.,[23] and Vollebregt et al.[24] showed that physical activity before pregnancy does not lower the risk of preeclampsia or gestational hypertension. This was consistent with the results of the present study in the walking group. The reasons for the inconsistency of the results of these researches with that of the stretching exercises group could be due to not having a single definition for physical activity, different instruments used to measure physical activity, and the study method which was the prospective cohort method. BODY.C: The results of this study have shown that stretching exercise intervention can prevent the risk of hypertensive disorders during pregnancy. Accordingly, it is recommended that pregnant women engage in stretching exercises during pregnancy, and use these exercises more often during this period. Since it is easier to perform stretching exercises than to walk, they can be more committed to these exercises. As mentioned, despite the ease of performing stretching exercises, they have benefits such as controlling hypertension during pregnancy. Therefore, health and fitness professionals should pay more attention to preparation and delivery of a variety of sports programs with different stretching techniques and encourage pregnant women to engage in this kind of exercise.

TITLE: South Asian Heart Risk Assessment (SAHARA): Randomized Controlled Trial Design and Pilot Study ABSTRACT.BACKGROUND: People of South Asian origin suffer a high burden of premature myocardial infarction (MI). South Asians form a growing proportion of the Canadian population and preventive strategies to mitigate the risk of MI in this group are needed. Prior studies have shown that multimedia interventions are effective and feasible in inducing health behavior changes among the obese, smokers, and among those who are sedentary. ABSTRACT.OBJECTIVE: Among at-risk South Asians living in Canada, our objectives are to determine: (1) the feasibility of a culturally tailored multimedia intervention to induce positive behavioral changes associated with reduced MI risk factors, and (2) the effectiveness and acceptability of information communicated by individualized MI and genetic risk score (GRS) reports. ABSTRACT.METHODS: The South Asian HeArt Risk Assessment (SAHARA) pilot study enrolled 367 individuals of South Asian origin recruited from places of worship and community centers in Ontario, Canada. MI risk factors including the 9p21 genetic variant status were provided to all participants after the baseline visit. Participants were randomly allocated to receive a multimedia intervention or control. The intervention group selected health goals and received personalized health messages to promote adherence to their selected goals. After 6 months, all participants had their MI risk factors repeated. The methods and results of this study are reported based on the CONSORT-EHEALTH guidelines. ABSTRACT.RESULTS: The mean age of participants was 53.8 years (SD 11.4), 52.0% (191/367) were women, and 97.5% (358/367) were immigrants to Canada. The mean INTERHEART risk score was 13.0 (SD 5.8) and 73.3% (269/367) had one or two copies of the risk allele for the 9p21 genetic variant. Both the intervention and control groups made some progress in health behavior changes related to diet and physical activity over 6 months. Participants reported that their risk score reports motivated behavioral changes, although half of the participants could not recall their risk scores at the end of study evaluation. Some components of the multimedia intervention were not widely used such as logging onto the website to set new health goals, and participants requested having more personal interactions with the study team. ABSTRACT.CONCLUSIONS: Some, but not all, components of the multimedia intervention are feasible and have the potential to induce positive health behavior changes. MI and GRS reports are desired by participants although their impact on inducing sustained health behavior change requires further evaluation. Information generated from this pilot study has directly informed the design of another randomized trial designed to reduce MI risk among South Asians. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT01577719; http://clinicaltrials.gov/ct2/show/NCT01577719 (Archived by WebCite at http://www.webcitation.org/6J11uYXgJ). BODY.INTRODUCTION.BACKGROUND: Myocardial infarction (MI) due to coronary artery disease (CAD) remains a major cause of death globally [1]. The rising prevalence of overweight, obesity, and type 2 diabetes is predicted to potentiate the CAD epidemic in developing countries [2]. South Asians, people who originate from the Indian subcontinent, suffer a high burden of premature MI [3,4], and are projected to account for 40% of the global CAD burden by 2020 [5]. More than 1.2 million South Asians live in Canada and are the fastest growing group of non-white Canadians [6]. Our previous study has shown that, compared to white Caucasians in Canada, South Asians suffer a 2.5 times excess prevalence of elevated glucose (dysglycemia), and CAD [7], and develop cardiometabolic risk factors (ie, abnormal glucose and lipids) at significantly lower body mass index (BMI) values [8]. Despite several previous studies showing excess cardiometabolic risk [4,9,10] and increased premature MI among South Asians [11], there is no routine screening process of South Asians for CAD despite the Canadian Cardiovascular Society recommendations to screen "high-risk" groups including South Asians [12]. Therefore, there is a need for routine screening of CAD risk factors in South Asian adults and to develop and test interventions to improve risk factors among South Asians. This is critical because collectively common risk factors (abnormal lipids, elevated glucose, elevated blood pressure, and abdominal obesity) account for over two-thirds of the population attributable risk of MI [4]. Several studies have shown that multimedia interventions to manage risk factors of common disorders and to modify health behaviors are effective [13-19]. Multimedia interventions include use of email messaging, text messaging, video- or computer-based education, and electronic personalized health records, which are attractive because they involve components of goal setting and feedback—key components of health behavior modification, are relatively cost efficient, and have the potential to be scalable to large numbers of individuals [20-25]. The use of MI risk tools to guide risk factor modification in cardiovascular prevention is increasing [26]. More recently the addition of genetic information into these risk tools has been evaluated. This has been made possible by the recent large-scale genetic studies that have identified common genetic variants associated with MI risk. The most robust genetic variant associated with increased risk for MI is a common polymorphism located on the short arm of chromosome 9 (9p21) [27,28]. This genetic variant is common in the general population, with 50% of people carrying one copy of the risk allele, which increases MI by 15-20%, and 25% of the population carrying two copies of the risk allele, which increases MI risk by 30-40% [29]. Further there is evidence to suggest that the MI risk associated with 9p21 may be modified by healthy dietary patterns [30]. While some recent studies have evaluated whether knowledge of genetic risk of a condition influences individuals' behavior change [31,32], the results remain inconclusive. To our knowledge there have been no multimedia health behavior modification interventions, which have incorporated genetic risk information among South Asians at risk for MI. BODY.INTRODUCTION.OBJECTIVE: To address this gap we conducted a pilot study, the South Asian HeArt Risk Assessment (SAHARA) among at-risk South Asians living in Canada, to determine: (1) the feasibility of a culturally tailored multimedia intervention to induce positive behavioral changes associated with reduced MI risk factors, and (2) the effectiveness and acceptability of information communicated by individualized MI and genetic risk score (GRS) reports. Information generated from the SAHARA pilot study will directly inform the design of another randomized trial designed to test the effectiveness of this intervention to reduce MI risk among South Asians. BODY.METHODS.STUDY DESIGN AND RECRUITMENT: The study is a randomized controlled pilot trial that was approved by the McMaster/Hamilton Health Sciences Research Ethics Board on June 3, 2009 (09-225). Individuals were recruited from places of worship and community centers in Southwestern Ontario, Canada, during the period from January 16, 2011 to January 29, 2012. Recruitment clinics were setup in these "high-yield" locations at high yield times (following weekly ceremonies and scheduled activities) to maximize enrollment. The study team contacted community leaders in the recruitment locations to obtain permission to inform the congregation about the study, and this was done 1-2 weeks prior to the screening event. BODY.METHODS.ELIGIBILITY: Men and women ≥30 years of age of South Asian ancestry, defined as people whose ancestors originate from the Indian subcontinent (India, Pakistan, Bangladesh, and Sri Lanka), were eligible for inclusion in the SAHARA pilot study. All participants were required to have access to email, cell phone with text messaging capability, or a smart phone (ie, a handheld device capable of sending and receiving text messages and searching the Internet such as an iPhone or Blackberry). BODY.METHODS.EXCLUSION CRITERIA: Individuals who had suffered a previous MI, had coronary artery bypass graft (CABG) surgery, coronary angioplasty, or stroke, who were not permanent residents of Ontario, and who did not have an Ontario health card were excluded. BODY.METHODS.CONSENT AND BASELINE DATA COLLECTION: Written informed consent, including consent to use of the health card number to facilitate future record linkage with health services databases, and to analyze DNA for genetic variants, was obtained from each participant. Information on risk factors including cholesterol status, diabetes, hypertension, current, former, and second-hand exposure to tobacco smoke, diet, physical activity, sedentary behaviors, and psychosocial stress questions was collected. Stages of change information based on Prochaska's model of change [33] were also obtained for diet, physical activity, sedentary behavior, and smoking. Blood pressure (two measures 3 minutes apart using an automated OMRON device), body weight and height (to calculate BMI), waist and hip circumference, and body fat percentage using a digital bioelectrical impedance scale were measured. A 30-mL nonfasting blood sample was collected from all participants, and was processed onsite within 2 hours of collection. The blood samples were analyzed for apolipoprotein A1 and B, HbA1C, and the 9p21 single nucleotide polymorphism (SNP) (rs1333049) genotype using Taqman. All genotypes were in Hardy-Weinberg equilibrium (HWE) for the total sample (P>.05). Previous studies have reported a minimal difference in apolipoproteins' levels when comparing fasting to nonfasting levels [34]. The remaining serum and plasma aliquots were placed in long-term storage for future study-related analysis. BODY.METHODS.RISK PROFILE: Using the information collected at the baseline visit, a MI risk report was generated for each participant using the INTERHEART risk score (IHRS; Multimedia Appendix 1, which is a simple and valid risk factor scoring system developed and validated from the INTERHEART case-control study to assess MI risk in adult men and women [35]. This risk model included the following factors: apolipoprotein B-to-A1 ratio, smoking, second-hand smoke exposure, hypertension, HbA1c, abdominal obesity, physical inactivity, diet, and psychosocial factors. As part of the SAHARA pilot study the risk score report was pretested and modified in an easy to understand format that classifies individuals as low (0-9), moderate (10-15), or high (16-48) risk using a color visual display (Figure 1) (also see website [36]). In addition to IHRS, a GRS based on 9p21 genotype information was generated. The GRS was developed, pretested, and modified in an easy to understand format and classifies individuals who have 0, 1, or 2 risk alleles using a color visual display (Figure 2). The contents of the report were pretested in 2 focus groups conducted at a South Asian temple, and modified to the grade 5 reading level. BODY.METHODS.RANDOMIZATION.MYOSCAR-SAHARA: Approximately 4-6 weeks after the screening visit was completed, participants were sent an email asking them to log onto the secured MyOSCAR-SAHARA website [37], to access their risk score results. If they were eligible for randomization (based on study inclusion criteria), they were prompted to click on a button that took them to a Web portal to be randomized to intervention or control (usual advice) groups using a computer-generated algorithm in OSCAR (Open Source Clinical Applications and Resources)—an open source software project launched by the Department of Family Medicine at McMaster University in Hamilton, ON, Canada, in 2002, designed for the delivery of evidence-based resources and decision support at the point of care for both patients and providers [37]. We used a specially constructed MyOSCAR-SAHARA personalized website to enable study participants' to retrieve their results, and to set goals which triggered a series of goal-tailored health messages they received by email or text message (for screenshots, see Figures 3-7). Intervention and control groups received the same baseline assessment and usual care while only the intervention group received the study intervention. Figure 1IHRS risk report example. Figure 2Genetic risk score. Through your blood work, we looked for a specific SNP in your DNA which has been shown to be a marker for heart attack risk. This SNP is located on chromosome 9, and is known as 9p21. The SNP is not within a gene itself, but is likely closely related to a gene which causes coronary artery disease. The 9p21 SNP has been shown to increase heart attack risk in several different ethnic groups, including South Asians. Based on your blood work, we determined if you did not have this SNP, only had it on one chromosome (inherited from one parent), or had it on two chromosomes (inherited from both parents). Having either one or two copies of this marker increases your genetic risk of having a heart attack. Figure 3SAHARA home page. Figure 4Welcome email. Figure 5Second welcome email. Figure 6MyOSCAR Web login. Figure 7SAHARA Web consent and personal health record page. BODY.METHODS.RANDOMIZATION.INTERVENTION: Participants randomized to the intervention were prompted to choose a health goal on the website in the areas of (1) healthy diet, (2) physical activity, (3) reducing sedentary behaviors, and (4) smoking cessation, and were prompted to update their goals weekly on the website. Participants then received health messages via email or text, tailored to their chosen health goal on a daily basis. The messages were based on self-efficacy and social support concepts [38-41] to motivate subjects to make health behavior changes including providing advice and support regarding reduction of energy-dense, nutrient-poor foods (ie, fried, fast foods, sugary beverages, and desserts), advocating increased consumption of fruits and vegetables, encouraging sedentary individuals to minimize sedentary behaviors and increase regular physical activity, and encouraging smokers to quit smoking. Participants were given a choice of methods to receive the health messages by: (1) email sent to an account using a personal computer or a handheld device (eg, BlackBerry, iPhone, or other smartphones), or (2) text message (short message service, SMS) to a handheld device (any cell phone). In addition to the health messages, a weekly health tip was sent to all intervention participants by email outlining a particular health topic related to healthy lifestyle or an analysis of a recent medical study reported in the press. All of these health messages were then posted on our public website (Figure 3). The components of the intervention are listed in Textbox 1 BODY.METHODS.RANDOMIZATION.CONTROL: Participants randomized to the control group were provided with advice on how to interpret their risk report, and if any significantly abnormal results were identified, they were encouraged to discuss them with their family doctor. All participants had access to the SAHARA website that contained health information regarding cardiovascular risk factors from a South Asian perspective [42]. This website includes information on culturally relevant healthy dietary habits, and the health benefits of regular physical activity. The site also includes a frequently asked questions section, and a mechanism for participants to ask our study team study-related questions and receive feedback. Multimedia intervention components of the SAHARA pilot study.Components for intervention participants:MyOSCAR-SAHARA Goal selection program: a tool which permits participants to select biweekly goals related to improving diet, increasing activity, decreasing smoking, and reducing sedentary behaviorsDaily health messages—sent via email or text: messages provided tips on how to counter unhealthy habits and maintain healthy onesBiweekly reminders to pick a health goal and monitor progress on the goalAccess to latest health information through personal MyOSCAR-SAHARA accountAccess to healthy living videos, such as yoga and other exercise regimens BODY.METHODS.WEBSITE USAGE AND ADHERENCE TO INTERVENTION “FIDELITY”: Participants usage of the goal setting website was monitored centrally, and for those participants who did not log on to access their risk score reports or for intervention participants who had not set goals 2 weeks from the time they were prompted by email, a study team member attempted to reach them by telephone to encourage them to access their results and set goals. After 4 weeks if results had not been accessed from the website, a printed MI and genetic risk score report was mailed to participants' home. BODY.METHODS.PILOT STUDY OUTCOME MEASURES: The two outcome measures of the pilot study included: First, feasibility of the intervention, defined by: (1) success at transmitting risk score information and health messages via electronic media (website, email, and cell phone), (2) success at participants returning to use the website and set health goals, as this reflects the uptake of the intervention and helps to assess the effect of intervention on health behaviors, and (3) trend in the risk score change to indicate if the intervention leads to progressive health behavior change. Second, effectiveness and acceptability of risk score information was measured by: (1) participants' knowledge of their risk score over time, (2) if this information induced positive behavior change, and (3) participants' satisfaction with the information received. BODY.METHODS.FOLLOW-UP: All participants were followed up for a minimum of 6 months after randomization and repeat risk factor assessment was collected at the end of the study. End of study data were collected via face-to-face reassessment at the recruitment sites (238/324, 73.5%) and by telephone or mail (86/324, 26.5%). Repeat HbA1C and apolipoproteins A1 and B were also collected from participants who attended the face-to-face reassessment visit. The reporting of this study follows the CONSORT-EHEALTH [43] guidelines Multimedia Appendix 2. BODY.RESULTS.SUMMARY: Participants (n=412) were screened from 11 centers between January 2011 and January 2012. Among them, 41 were ineligible (5 had cardiovascular disease—CVD, 23 had no email accounts, 13 were missing information required for the risk score, and 4 were eligible but not randomized due to a clerical error), leaving 367 participants randomized into the pilot study. Follow-up data collection occurred between October 28, 2011 and November 11, 2012. The median time of follow-up is 280 days with the interquartile range (IQR) of 252-319 days follow-up. As shown in Figure 8, there were 43 participants (21/167, 12.6% and 22/204, 10.8% of the intervention and control group, respectively) who did not complete the follow-up (21 were not contactable and 22 participants withdrew from the study). BODY.RESULTS.DEMOGRAPHIC AND SOCIAL CHARACTERISTICS: Participants' characteristics are shown in Table 1. Briefly the mean age is 53.8 years (minimum age=30.0 years, maximum age=82.0 years, and median age=53.0 years), approximately half are women, and the majority of participants are immigrants to Canada. More than half reported speaking English at home, 88.7% (323/364) received more than secondary school education and 69.2% (254/367) are actively employed. More than 52.0% (191/367) are vegetarian; few (4/367, 1.1%) use or are exposed to tobacco, and approximately one-quarter (27.5%, 101/367) engage in regular physical activity. Further, more than 32.0% (117/367) are exposed to more than 2 hours of screen time per day. The mean BMI at baseline is 26.4 (SD 3.5) for men and 26.5 (SD 4.1) for women. Three quarters of participants have one or two risk alleles for the 9p21 genetic variant. BODY.RESULTS.RISK FACTOR INFORMATION AT BASELINE AND END OF STUDY: Table 2 shows participants' risk factors at baseline and follow-up. Over a quarter of all participants had hypertension and elevated cholesterol at baseline, and 13.8% (44/319) reported having type 2 diabetes. Over two thirds of participants are inactive at leisure time, the mean servings of fruits and vegetables consumed daily are 2 and 3, respectively, and more than 27.1% (86/317) of participants reported having stress and depressive symptoms. Objective study measures including HbA1c (mean 5.9, SD 0.8; apolipoprotein B-to-A1 ratio: mean 0.68, SD 0.18) and waist-to-hip ratio (WHR, men: mean 0.95, SD 0.05; women: mean 0.88, SD 0.07) indicate that the cohort has a moderate risk for MI, with the mean IHRS being 13.0 (SD 5.8). BODY.RESULTS.FEASIBILITY: SUCCESS AT TRANSMITTING MESSAGES VIA ELECTRONIC MEDIA: All participants were required to have an email access (including shared family email if they choose to use this email account) to be eligible for this study. The majority of participants (352/367, 96%) had personal email access (Table 3). Most participants had no difficulty logging into website or viewing their results, although 23% (74/324) reported having some technical problems with the website, which inhibited the risk score report and health messages delivery. BODY.RESULTS.SUCCESS AT PARTICIPANTS RETURNING TO USE THE WEBSITE AND SET HEALTH GOALS: Participants use of the MyOSCAR-SAHARA website was monitored to determine how many participants logged on to the website to view their risk score reports and in the case of intervention participants, to set health goals. The login to the study MyOSCAR-SAHARA website was low for both groups (82/182, 45.1% of intervention and 115/185, 62.7% of control groups did not logon or used the website only once). The mean number of login attempts of the intervention group mean was 2.64 (SD 3.17, median 2.0) and the control group was 1.63 (SD 2.14, median 1.0). The difference between intervention and control groups login was statistically significant as expected since the intervention group was asked to set goals (Wilcoxon two-sample test, P=.0003). Figure 9 shows the frequency of login by intervention and control groups. On average the intervention group selected 1.12 goals (SD 1.67, median 1.0). Figure 8Participants' flow diagram. Table 1 Demographic characteristics. Characteristics Overall Intervention Control Number of participants 367 182 185 Age in years, mean (SD) 53.8 (11.4) 54.6 (11.5) 53.0 (11.3) Median age (min, max) 53.0 (30.0, 82.0) 55.0 (31.0, 81.0) 53.0 (30.0, 82.0) Male/female (%) 176 (48.0)/191 (52) 84 (46.2)/98 (53.8) 92 (49.7)/93 (50.3) Ancestral country of origin (%) India 327 (89.1) 163 (89.6) 164 (88.6) Pakistan 4 (1.1) 2 (1.1) 2 (1.1) Sri Lanka 4 (1.1) 2 (1.1) 2 (1.1) Other 32 (8.7) 15 (8.2) 17 (9.2) Place of birth—Canada, (%) 9 (2.5) 1 (0.6) 8 (4.4) Language spoken at home—English, (%) 188 (52.2) 93 (52.0) 95 (52.5) Married (%) 337 (92.1) 167 (91.8) 170 (92.4) Post-secondary education (%) 323 (88.7) 156 (86.7) 167 (90.8) Employed (%) 254 (69.2) 119 (65.4) 135 (73.0) Household income >CDN$ 60,000/year (%) 218 (61.6) 106 (59.6) 112 (63.6) Alcohol consumption ≥1 drink per day (%) 23 (6.4) 11 (6.0) 12 (6.8) Vegetarian (%) 191 (52.5) 90 (49.5) 101 (55.5) Daily activity mild/none (%) 266 (72.5) 125 (69.8) 141 (77.0) Screen time mean minutes/day (SD) 140.1 (130.7) 133.8 (121.5) 146.3 (139.2) BMI—male, mean (SD) 26.4 (3.5) 26.5 (3.6) 26.4 (3.3) BMI—female, mean (SD) 26.5 (4.1) 26.2 (3.7) 26.8 (4.5) One or two risk alleles of 9p21 (%) 261 (73.3) 130 (74.3) 131 (72.4) Table 2 Baseline and follow-up risk factors in intervention and control groups. Risk factor Intervention Control Baseline Follow-up P value a Baseline Follow-up P value a Apolipoprotein B/apolipoprotein A1 ratio (SD) 0.66 (0.19) 0.67 (0.18) .42 0.70 (0.18) 0.71 (0.20) .59 HbA1c (SD) 5.9 (0.8) 5.9 (0.8) .81 5.8 (0.8) 5.9 (0.9) .01 Self-reported diabetes, b n c (%) 31 (20.0) 36 (23.2) .03 13 (7.9) 16 (9.7) .08 Self-reported hypertension, n c (%) 43 (28.1) 46 (30.1) .08 36 (22.4) 42 (26.1) .01 Elevated BP, d n (%) 35 (30.4) 25 (21.7) .06 31 (27.0) 22 (19.1) .08 Mean SBP (SD) mm Hg 128 (18) 124 (16) .008 127 (19) 123 (17) .003 Mean DBP (SD) mm Hg 81 (10) 79 (10) .006 82 (11) 79 (11) <.0001 Waist-to-hip ratio—male, mean (SD) 0.95 (0.06) 0.96 (0.06) .68 0.95 (0.05) 0.94 (0.06) .07 Waist-to-hip ratio—female, mean (SD) 0.89 (0.07) 0.88 (0.07) .12 0.87 (0.06) 0.87 (0.06) .62 Stress in last year at baseline and in last 6 months at follow-up, n (%) 48 (30.8) 29 (18.6) .001 52 (32.1) 33 (20.4) .002 Depression for ≥2 weeks in last year at baseline and last 6 months at follow-up, n (%) 50 (32.3) 24 (15.5) .0001 36 (22.2) 16 (9.9) .0009 Mean servings of fruits/day (SD) 2.0 (1.2) 2.0 (1.2) .90 2.0 (1.2) 1.9 (1.4) .67 Mean servings of vegetables/day (SD) 3.0 (1.7) 2.8 (1.8) .24 3.0 (1.9) 2.9 (1.9) .83 Mean servings of deep fried foods/snacks per day (SD) 0.3 (0.5) 0.2 (0.2) .0005 0.3 (0.4) 0.2 (0.4) .003 Moderate/very active in leisure time, n (%) 58 (37.9) 88 (57.5) <.0001 47 (29.0) 77 (47.5) <.0001 Self-reported high cholesterol, c n (%) 42 (28.2) 49 (32.9) .008 37 (22.6) 44 (26.8) .008 IHRS e,f (SD) 13.4 (5.8) 12.0 (5.8) .002 12.6 (5.9) 11.7 (5.9) .05 a Pairwise comparison of data using paired t -test for continuous measures and McNemar’s test for categorical measures. b Prevalence of events at follow-up includes baseline plus additional new events since baseline. Therefore the prevalence of diabetes, hypertension, and high cholesterol are higher at follow-up. c Number of participants with data available for the specific variable. d Bloodpressure (BP) was measured at baseline and follow-up. Elevated BP is >140/90. e IHRS: INTERHEART risk score. f No significant difference in change between the intervention and control group ( P =.70). Table 3 Electronic access and reported technical difficulties. Baseline Overall, n=367 n (%) Intervention, n=182 n (%) Control, n=185 n (%) Personal email access 353 (96.2) 176 (96.7) 177 (95.7) Smart phone access 73 (19.9) 41 (22.5) 32 (17.3) Cell phone access 177 (48.2) 98 (53.8) 79 (42.7) Receive and send text messages 127 (34.6) 141 (38.5) 57 (30.8) Check email multiple times per day 148 (40.3) 79 (43.4) 69 (37.3) Participants who completed follow-up 324 (88.3) 159 (87.4) 165 (89.2) Problems accessing results on website 25 (7.7) 9 (5.7) 16 (9.7) Logon difficulties to website 37 (11.4) 17 (10.7) 20 (12.1) Did not receive email with instruction on logon 11 (3.4) 4 (2.5) 7 (4.2) Instructions were unclear 10 (3.1) 4 (2.5) 6 (3.6) MyOSCAR-SAHARA website was difficult to use 13 (4.0) 4 (2.5) 9 (5.5) Total problems with MyOSCAR-SAHARA website 74 (22.8) 31 (19.5) 43 (26.1) Figure 9Summary of login attempts to website over the course of the pilot study. BODY.RESULTS.SIGNAL THAT INTERVENTION LEADS TO BEHAVIORAL CHANGES: Both the intervention and control group showed a reduction at follow-up in blood pressure, and reported less stress and depression compared to baseline. There was also an improvement in physical activity and reduction in fried food and snacks consumption in both groups at follow-up. Comparing follow-up to baseline score change, the intervention group had a significant reduction in their IHRS score at follow-up (intervention group baseline IHRS: mean 13.4, SD 5.8; follow-up IHRS: mean 12.1, SD 5.9, P=.002), and a trend was seen in the control group (baseline IHRS: mean 12.6, SD 5.8; follow-up IHRS: mean 11.7, SD 5.9, P=.05) (see Table 2), though these results were not statistically significant. BODY.RESULTS.RISK SCORE.RISK REPORT FEEDBACK: Participants were asked to acquire their risk score reports (IHRS and GRS) following the baseline assessment by logging onto the MyOSCAR-SAHARA website. If they did not retrieve it, it was mailed to their homes. At the end of the study, participants were asked about their knowledge and recall of their risk scores (Tables 4 and 5). Overall while participants reported appreciating receiving their risk information, the recall between baseline and end of study of risk status was low. For example, of 68 participants who were told they were high-risk at baseline, only 11 recalled this correctly (11/68, 16.2%), 17 recalled being moderate risk (17/68, 25.0%), 3 recalled it being low risk (4/68, 4.4%), and 37 could not recall their risk score (37/38, 54.4%) at all. Similarly only 7.3% (5/68) of participants recalled their increased genetic risk score accurately at follow-up (Table 5). BODY.RESULTS.RISK SCORE.RISK SCORES AND MOTIVATION TO CHANGE: Figure 10 shows individuals reporting that knowledge of their risk for MI could be a motivator to change health behaviors including diet and physical activity. There was a trend (P=.06) showing the intervention group as compared to the control group was more likely to agree that the risk score was a motivator for increasing health behaviors especially for physical activity, though these results were not statistically significant (Figure 10). BODY.RESULTS.STAGES OF CHANGE: We assessed the stages of change for three main domains: diet, physical activity, and weight loss. Although we also included smoking, only three individuals are current smokers in this sample. Overall, more than 13.9% (51/367) of participants progressed from inactive (precontemplation, contemplation, and preparation) to the active (action, maintenance) stage in diet and physical activity, and 12.5% (46/367) progressed to the active stage in weight loss plans; however, no significant differences were observed between intervention and control groups (Table 6). BODY.RESULTS.EXIT SURVEY: At the end of the pilot study, feedback from the study participants was obtained by asking all participants about their experiences of participating in the pilot study. The main feedback included: (1) daily messages were too frequent which could potentially lead them to ignore the messages; (2) phone calls to remind participants to login to the study website were too frequent, while others reported that there was not enough in-person contact and would have liked to have a mid-program visit that with more face-to-face contact with the study team; and (3) IHRS and GRS reports should be sent via email and remove the website login component. Most of the participants reported that participation in the SAHARA study was worthwhile for them. Table 7 shows summary of the exit survey. Table 4 Agreement between MI risk score results and participants recall of risk score at follow-up. Actual IHRS score category at baseline Recall of risk score category at follow-up Low Moderate High Do not know Total Low 47 15 2 63 127 Moderate 11 46 6 59 122 High 3 17 11 37 68 Total 61 78 19 160 318 Table 5 Agreement between genetic risk score results and participants recall of risk score at follow-up. Actual GRS score category at baseline Genetic risk category recall at follow-up Not increased Increased Do not know Total Not increased (0 risk alleles) 22 6 56 84 Increase (1 or 2 risk alleles) 38 16 172 226 Total 60 22 228 310 Figure 10Motivation to change behavior based on risk score reports. Table 6 Stages of change a : moving from inactive to active stage by intervention at follow-up. Domain Overall, % Intervention,% Control, % P value b Diet 14.9 15.7 14.1 0.69 Weight loss 12.5 13.7 11.4 0.53 Physical activity 14.2 15.5 12.9 0.51 a Stages of change levels: 1=precontemplation, 2=contemplation, 3=preparation, 4=action, 5=maintenance. Inactive=levels 1-3, active=levels 4-5. b These results were obtained from chi-square tests. Table 7 Exit survey (n=317). Rank Intervention, n (%) Control, n (%) (A) Did you find participation in SAHARA to be worthwhile? a Number of participants 155 (48.8) 162 (51.1) Very worthwhile 75 (48.4) 78 (48.2) 4 47 (30.3) 45 (27.8) 3 24 (15.5) 19 (11.7) 2 8 (5.2) 10 (6.2) Not at all worthwhile 1 (0.6) 10 (6.2) (B) Did you succeed in setting and achieving your health goals? b Number of participants 153 (48.3) 157 (49.5) Very successful 27 (17.7) 27 (17.2) 4 60 (39.2) 50 (31.9) 3 51 (33.3) 38 (24.2) 2 9 (5.9) 25 (15.9) Not at all successful 6 (3.9) 17 (10.8) a There was no significant statistical difference between intervention and control groups in their view of study participation ( P =.09; obtained from chi-square tests). b The intervention group reported that they were more likely to be successful in achieving their goals than the control group ( P =.004; obtained from chi-square tests). BODY.DISCUSSION.PRINCIPAL FINDINGS: We observed that a multimedia health behavior intervention is feasible in a South Asian population at risk for MI. While participants reported being motivated by receiving the risk score information, a number of features of the SAHARA intervention require optimization prior to assessing its effectiveness in MI risk factor reduction. Most participants had access to email, Internet, and text messages and had no difficulty receiving email or text messages. However, our requirement of participants to proactively logon to the website to receive their risk reports, and to set goals was problematic with 23.9% (88/367) of the study participants reporting technical difficulties. It is likely that this contributed to the low number of goals chosen over the course of the follow-up, and reduced the interventions potential impact on changing health behaviors. In addition, participants received the study messages either by email/text checked on a mobile device or emails checked on a fixed device. These different methods of receiving messages may have also impacted the uptake of the study intervention. The anticipated difference would be based on the fact that the mobile device message would likely be received in real time or close to it, whereas the fixed device message might not be received immediately, although it may reach people when they're more ready to act on the information (ie, they have specifically chosen to sit down at the computer, as compared to a mobile device when the email/text may arrive when the person is doing something else). In this study it is not known the impact of receiving messages via mobile or a fixed device on the intervention uptake and outcome. Despite these technical challenges, the intervention group showed a significant reduction in the IHRS score at follow-up, and were more likely than control subjects to report that their personalized risk scores motivated them to increase their healthy dietary choices, physical activity, and reduce sedentary behaviors. The greater engagement of the intervention group in the study, their receipt of regular messages and reminders to change their health behaviors, may explain this difference. Based on the participants' feedback from the exit survey, the use of a website health behavior intervention, which requires participants to logon to a website, reduces the chances that participants will be engaged in the study. In our study, 54.9% (100/182) of the intervention group logged onto the website at least twice, which is in keeping with previous studies using Internet-based intervention to aid smoking cessation [44]. A systematic review and meta-analysis of Web-based intervention studies to induce behavioral changes reported that the average logon to website/person/study duration in weeks varies from 2.6 logons/person/32 weeks in a study of depression to 1008 logons/person/36 weeks in a study of HIV. In addition the average time spent on website in minutes per person varied from 4.5 to 45 minutes/person [45]. Furthermore, even when information is sent directly to participants by email, the rate of opening the email is variable. For example in a study of 345 men and women where daily email messages were sent to improve employees' diet and physical activity behaviors in the workplace, only 68.9% (238/345) of the emails were opened by study participants [23], even though all study participants worked in the same office and had a computer at their desk. This is consistent with other studies using Web-based interventions where an uptake of only 62% was reported [46]. To optimize the uptake of the intervention for the main SAHARA trial we will ask participants to set their goals at the baseline interview, we will remove the logon to website requirement to access risk score reports, and we will deliver the reports directly to participants by emails. These components will be followed by telephone calls and one face-to-face visit mid-way through the study, to ensure receipt and knowledge of risk scores, and to maintain participant interest in the study. Individuals who participated in this pilot study were at moderate risk of MI based on their baseline IHRS compared to risk score values reported in the validation study [35]. Both the intervention and control groups made some progress in changing their health behaviors and in general participants reported the information they received was useful. Despite participants claiming that their risk reports motivated behavioral changes, half of the participants could not recall their risk report at 6 months. The poor risk score recall may reflect low health literacy (ie, the degree to which individuals can obtain, process, and understand the basic health information) and numeracy (how individuals interpret medical risk information) [47]. However, our sample was of high socioeconomic status, well educated, and we pretested our risk score information in focus groups and presented the information (Figure 2) at the grade 5 reading level. Thus, we attempted to minimize low health literacy and numeracy as possible barriers to understanding risk score information. It is also possible that the active phenomenon of resistance to retain negative information about one's health to maintain an optimistic view of future health was at play. Such views have been described as psychological defense mechanisms [48]; however, it is difficult to confirm if such views hold in the current study. In addition, the low risk score recall may also represent the phenomenon of "unrealistic optimism" whereby individuals display an optimism bias when evaluating own susceptibility to risk [49]. However, this view does not explain the poor recall of low-risk reports. The low recall rate of health information received, including in face-to-face counseling, is not uncommon. In a large study investigating the recall of health advice given face-to-face to patients (n=3261) who participated in the EuroHeart Failure Survey 12 weeks following discharge, only 57.8% (1885/3261) of patients recalled advice on exercise, 54.9% (1793/3261) recalled advice on diet, 41.9% (1369/3261) recalled advice on smoking, and only 38.9% (1271/3261) recalled advice on alcohol [50]. Nonetheless in our study, participants reported that knowledge of their risk factor and genetic risk score were motivators to improve their health behaviors even if they could not recall their exact risk category. It is possible that recall may vary by the type of information provided to participants, and recall may decrease over time. For example, patients with type 2 diabetes are more likely to recall health recommendations regarding medications than regarding health behaviors [51], and provision of genetic risk information to smokers regarding their risk of lung cancer showed early high recall of risk status yet lower recall with increasing duration of follow-up [52]. We hypothesized that genetic information may motivate behavior change differently than nongenetic health information because of the highly personalized nature of the information [47]. In a recent study among patients with type 2 diabetes who participated in a lifestyle modification trial in which genetic information was provided in a gene score, almost all participants (98%) reported that high-risk genetic results would increase their motivation for lifestyle modification. On the other hand their response to receiving low-risk genetic results varied, with some reporting that low-risk genetic status would decrease their motivation to take on lifestyle changes. However, their reported response has not yet been correlated to their actual change in risk factors as this study is on-going [53]. Future studies, including the main SAHARA trial must assess if provision of genetic risk information is strongly correlated with changes in risk factors and clinical outcomes. We assessed the stage of change transition over the course of the follow-up. It is known now that the stages of change are not linear and individuals do not progress from one stage to the next as originally proposed [33]. Rather, these stages follow a spiral model with relapses that resets the process back to the precontemplation stage [54]. Despite these challenges, the stages of change model is widely used and accepted as a useful measure to assess motivation to change and the impact of this motivation on achieving the desired behavioral modification [55]. We observed that 25.8% (94/367) of the participants progressed forward in stages of change relating to physical activity, while 18.2% (67/367) regressed in their stages of change. Overall however more than 12.5% (46/367) progressed from the inactive to an active stage at the end of study in all three domains (physical activity, diet, and weight loss). No difference between intervention and control subjects in stages of change transitions was observed. BODY.DISCUSSION.LIMITATIONS: Our pilot study had a number of strengths, which include recruitment of an apparently healthy population sample of reasonable size, and prospective measurement of health behaviors that included objective measurements (ie, lipid, blood pressure, and anthropometric measurements). Some limitations of our intervention occurred including the technical challenges of logging onto the website, the low rate of logons to set health goals, and relatively poor recall of personal risk at follow-up. Despite these, a significant reduction in the MI risk score was observed in follow-up. In addition the SAHARA study population may not be representative of all South Asians in Canada; however, the socioeconomic characteristics of SAHARA participants are similar to findings from previous health surveys in Ontario [56]. BODY.DISCUSSION.MODIFICATIONS TO THE SAHARA TRIAL INTERVENTION: We have taken a number of steps to optimize the SAHARA intervention prior to testing its effectiveness in MI risk reduction in a future trial. These changes include: (1) risk reports and randomization status will be emailed directly to participants, (2) the number of health goals participants can focus on has been reduced from 4 to 2, with only one being chosen at one time for a 6-month duration, (3) the duration of follow-up will be extended to 12 months with baseline, 6 months and 12 months face-to-face visits occurring, (4) increasing the frequency of in-person contacts to improve adherence to the intervention and interest in the program, (5) health tips will be tailored to each participant based on the goal selected and their readiness to change, and (6) the frequency of messages will be reduced from daily to weekly and sent at a time of day chosen by participants. BODY.DISCUSSION.CONCLUSION: A multimedia intervention is feasible and has the potential to induce positive health behavior changes aimed at reducing MI risk. Information generated from the SAHARA pilot has directly informed the design of the main randomized trial designed to test the effectiveness of a multimedia behavioral intervention to reduce MI risk among South Asians.

TITLE: MEK and the inhibitors: from bench to bedside ABSTRACT: Four distinct MAP kinase signaling pathways involving 7 MEK enzymes have been identified. MEK1 and MEK2 are the prototype members of MEK family proteins. Several MEK inhibitors are in clinical trials. Trametinib is being evaluated by FDA for the treatment of metastatic melanoma with BRAF V600 mutation. Selumetinib has been studied in combination with docetaxel in phase II randomized trial in previously treated patients with advanced lung cancer. Selumetinib group had better response rate and progression-free survival. This review also summarized new MEK inhibitors in clinical development, including pimasertib, refametinib, PD-0325901, TAK733, MEK162 (ARRY 438162), RO5126766, WX-554, RO4987655 (CH4987655), GDC-0973 (XL518), and AZD8330. BODY.INTRODUCTION: The mitogen-activated protein kinase (MAPK) signaling pathways involve a family of protein kinases that play critical roles in regulation of diverse cellular activities, including cell proliferation, survival, differentiation, motility, and angiogenesis. The MAPK pathways transduce signals from various extracellular stimuli (growth factors, hormones, cytokines and environmental stresses), leading to distinct intracellular responses via a series of phosphorylation events and protein-protein interactions [1]. Four distinct MAPK cascades have been identified and named according to their MAPK module. These are extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and ERK5. Each of these cascades comprised of three sequentially acting kinases, activating one after the other (MAPKKK/MAP3K, MAPKK/MAP2K, and MAPK). These signaling cascades are often dysregulated in human cancer cells. Many small molecule inhibitors targeting various component of these cascades are moving quickly from bench to bedside [2-4]. For instance, vemurafenib is the first B-RAF inhibitor that received FDA approval in 2011 for the treatment of BRAF V600E/K mutation positive metastatic melanoma [5,6]. This review focuses on MAP2K or MAPKK component of each of the four MAPK cascades with their characteristics and the small molecule inhibitors targeting these proteins/enzymes. Mitogen-activated protein kinase or MAP2K or MAPKK are commonly known as MEK proteins. BODY.MEK PROTEINS: MEK proteins belong to a family of enzymes that lie upstream to their specific MAPK targets in each of the four MAP kinase signaling pathways and so far 7 MEK enzymes have been identified (Figure 1). These MEK enzymes selectively phosphorylate serine/threonine and tyrosine residues within the activation loop of their specific MAP kinase substrates [1]. Figure 1MEK proteins and their signaling pathways. In human, four distinct MAP kinase signaling pathways involving 7 MEK enzymes have been identified. The corresponding MEK enyzmes and their associated signaling pathways are shown in the diagram. The molecular weight of MEK proteins ranges between 43 and 50 kDa. Like all protein kinases, they display a similar structural organization consisting of an amino-terminal domain, a catalytic domain which is also called the kinase domain, and the carboxyl-terminal domain (Figure 2). MEKs share extensive homology in their kinase domain while the amino- and carboxy-termini are more diverse. Figure 2The structures of 7 MEK proteins. All 7 MEK proteins display a similar structural organization consisting of an amino-terminal domain, a kinase domain, and the carboxyl-terminal domain. MEKs share extensive homology in their kinase domain while the amino- and carboxy-termini are more diverse. MEK1 and MEK2 are closely related (Figure 2). They participate in the Ras/Raf/MEK/ERK signal transduction cascade. MEK 1, also designated as MAPKK-1, is the prototype member of MEK family proteins. It is encoded by the gene MAP2K1 located on chromosome 15q22.31. The gene, MAP2K2, encoding MEK 2 protein, resides on chromosome 19p13.3. MEK 1/2 proteins consist of a N-terminal sequence, a protein kinase domain, and a C-terminal sequence [7]. The N-terminal sequence contains an inhibitory/allosteric segment, a nuclear export sequence (a unique feature not shared with other MAPKK family members), and a docking site (D-domain) that aids in binding ERK substrates. The kinase domain contains the ATP binding site and catalytic apparatus. The C-terminus houses the domain for versatile docking (DVD) which serves as a major determinant binding site for upstream components of the Ras/Raf/MEK/ERK cascade [8]. MEK 1/2 signaling cascade is activated by ligand binding to receptor tyrosine kinases (RTK), leading to dimerization of the receptors and autophosphorylation of specific tyrosine residues in its C-terminal region. These activated receptors recruit and phosphorylate adaptor proteins Grb2 and SOS, which then interact with membrane-bound GTPase Ras and cause its activation [9,10]. H-Ras, K-Ras, and N-Ras function as molecular switches when an inactive Ras-GDP is converted into an active Ras-GTP [11]. In its GTP-bound form, Ras recruits and activates Raf kinases (A-Raf, B-Raf, and C-Raf/RaF-1) [12]. The activated Raf kinases interact and activate MEK 1/2, which in turn catalyze the phosphorylation of threonine and tyrosine residues in the activation sequence Thr-Glu-Tyr of ERK1/2 [10]. Unlike Raf and MEK 1/2 kinases which have narrow substrate specificity, ERK1 and ERK2 have a wide variety of cytosolic and nuclear substrates. Activated ERKs can translocate into the nucleus to initiate diverse cellular responses, such as cell proliferation, survival, differentiation, motility, and angiogenesis. For instance, ERK1/2 signaling promotes the progression of cells from the G0/G1 to S phase by activation of positive cell cycle regulators cyclin D1 and c-Myc [13,14], and down-regulation of anti-proliferative proteins such as Tob1, FOXO3a and p21 [15,16]. Similarly the Raf/MEK/ERK MAP kinase pathway promotes cell survival by blocking NF-kB, leading to increased transcription of anti-apoptotic and pro-survival genes like Bcl-2 and Mcl-1 [17]. The Ras/Raf/MEK/ERK signaling is activated in human cancers via several different mechanisms. Increased ERK 1/2 signaling is often due to direct mutational activation or amplification of genes encoding key components of the Ras/Raf/MEK/ERK pathway such as Ras and B-Raf. A large-scale cancer genome sequencing study revealed that B-Raf is mutated in about 20% of all cancers and in more than 60% of melanomas [18]. Less commonly ERK 1/2 cascade can also be activated by MEKs in solid tumors including melanoma, colon, and lung carcinomas [19,20]. MEK3 and MEK6 are functionally similar and encoded by MAP2K3 and MAP2K6 genes, respectively. The genes are both located on chromosome 17q. MEK3 and MEK6 consist of 347 and 334 amino acids residues respectively [21]. Structurally MEK6 differs from MEK3 in terms of C- and N- terminal regions. However, the ATP binding sites, and serine/threonine and tyrosine catalytic sites are conserved [22,23]. MEK3/6 signaling pathway is activated by growth factor stimulation through RTKs. Additionally, the cascade can also be activated by G-protein coupled receptors, intracellular receptors, and toll-like receptors [24], in response to numerous stimuli including physical and chemical stresses, hormones, UV irradiation, and cytokines, such as interleukin-1 and tumor necrosis factor. These stimuli activate different MAPK kinase kinases (MAPKKKs), which include TAK1, ASK1/2, DLK, MEKK4, TAO1/2/3 and MLK2/3 [25]. Active MAPKKKs phosphorylate and activate MEK3/6, which in turn catalyzes the concomitant phosphorylation of a threonine/serine and a tyrosine residue in the p38 MAPK. MEK6 activates all the four isoforms of p38 MAP kinase (α, β, γ and δ) whereas MEK3 can only activate p38α and p38β isoforms [25]. p38 MAP kinase inhibits G1/S and G2/M cell cycle progression through down-regulation of cyclin D1 and Cdc25 expression respectively, both at the level of gene transcription and post-translation [26-28]. In addition, MEK3/6-p38 MAPK cascade promotes p53-dependent growth arrest by phosphorylating p53 at serine 33 and 46 [25]. Together, these targets of MEK3/6-p38 MAPK pathway (cyclin D1, Cdc25, and p53) cooperate to arrest the cell cycle. Thus decreased p38 activity may play an important role in carcinogenesis. For example, p38 activity has been shown to be reduced in hepatocellular carcinoma in comparison to adjacent normal tissue, with tumor size inversely related to p38 activity [29]. MEK4 and MEK7 are members of the stress-activated protein kinase (SAPK) signaling cascade. MEK4, a product of MAP2K4 gene (chromosome 17p11.2) is composed of 399 amino acids residues, whereas MEK7 is encoded by MAP2K7 gene that maps to chromosome 19p13.3 [21]. MEK4 and MEK7 are homologous in their kinase domains which contain 11 subdomains, but their N- and C- terminal subunits are different [30]. Upon activation by upstream kinases, MAP3Ks including MEKKs (MEKK1–4), MLK2/3, Tpl-2, DLK, TAO1/2, TAK1 and ASK1/2 catalyze the phosphorylation of threonine residues in the activation segment of either MEK4 and MEK7 or MEK4 only [8,31]. Activated MEK4/7 work synergistically and activate JNK protein kinases, including JNK1, JNK2, and JNK3. To execute their functions, JNKs activate several transcription factors, including c-Jun, ATF-2, NF-ATc1, HSF-1 and STAT3 [32,33]. MEK4/7-JNK signaling pathway acts as a key tumor suppressive pathway [34,35]. It has also been reported that MEK4/7 along with its substrate JNK may promote apoptosis by phosphorylating and inactivating anti-apoptotic proteins Bcl2, Bcl-XL and Mcl-1 [36]. The MEK-JNK signaling also play an important role during embryogenesis [37]. Transgenic mice studies have shown that MEK4 activity is required for normal hepatogenesis, B and T-cell lymphopoiesis, and erythropoiesis [38,39]. There is considerable evidence that MEK4-JNK signaling cascade is also a critical mediator of cardiac hypertrophy in response to preload and afterload changes [40]. MEK4, in addition to its principal target, JNK, also crosstalks with MEK3/6-p38MAPK pathway by activating p38α and p38β [41-43]. MEK4 has been consistently observed to be inactivated by non-sense, missense or deletion mutations in many solid tumors [44]. The expression of MEK4 was shown to be down-regulated in 75% of cases of serous ovarian cancer [45]. It has been hypothesized that loss of MEK4-p38MAPK signaling cascade may be a relevant pathway associated with tumorigenesis [46]. MEK5 has 448 amino acid residues, and shares 40% identity with other protein kinases [47,48]. The upstream kinases are MEKK2 and MEKK3. Growth factors, oxidative stress and hyperosmotic conditions lead to activation of MEK5 via dual phosphorylation of its serine 311 and threonine 315 residues [49-52]. The best characterized downstream target of MEK5 is ERK5, also known as big MAP kinase 1 (BMK1) because it is twice the size of other MAPKs. The interaction of MEK5 with MEKK2, MEKK3 or ERK5 is mediated by the PB1 domain of MEK5 [53]. Upon activation, ERK5 translocates to the nucleus to stimulate the activity of a number of transcription factors [54-57]. MEK5-ERK5 signaling enhances progression through the cell cycle [58,59]. ERK5 also plays a role in cardiovascular development and neural differentiation [60,61]. Overexpression of MEK5 has been reported in cancers of the colon [62], prostate [63], breast [64], lymphoma [65], and in malignant mesothelioma [66]. BODY.MEK INHIBITORS IN CLINICAL TRIALS: A number of MEK inhibitors have progressed into clinical trials since the first MEK inhibitor (PD098059) was described in the literature in 1995 (Table 1). Currently thirteen MEK inhibitors have been tested clinically but only trametinib (GSK1120212), a selective inhibitor of MEK 1 and 2, has emerged as the first MEK inhibitor to show favorable clinical efficacy in a phase III trial [67]. Table 1 MEK inhibitors in clinical trials MEK Inhibitors Target Clinical trial Common toxicities Tumors References Trametinib (GSK1120212) MEK1/2 III Rash, diarrhea, retinopathy Melanoma, colorectal cancer [ 67 ] Pimasertib (AST03026) MEK1/2 I Nausea, rash, visual disturbance, asthenia Colorectal, multiple myeloma [ 74 ] Selumetinib (AZD6244) MEK1 II Nausea, rash, xerostomia Melanoma, NSCLC [ 76 - 85 ] PD-0325901 MEK1/2 I Rash, fatigue, blurry vision, diarrhea Melanoma, NSCLC [ 89 - 91 ] Refametinib (RDEA119) MEK1/2 II Rash Hepatocellular cancer, melanoma, colorectal cancer [ 94 , 95 ] TAK733 MEK1/2 I Not available Melanoma, NSCLC, colorectal, breast cancer [ 96 , 97 ] MEK162 MEK1/2 I/II Rash, dermatitis, CPK elevation N-Ras melanoma, NSCLC, pancreatic cancer [ 100 ] RO5126766 Raf/MEK1/2 I Rash, diarrhea, CPK elevation Melanoma [ 101 ] WX-554 MEK1/2 II Not available Advanced solid tumors [ 102 ] RO4987655 MEK1 I Rash, GI disorders Melanoma [ 104 ] GDC-0973 MEK1 I Rash, nausea, dysguesia, elevated CK Melanoma, Pancreatic cancer, endometrial cancer [ 108 ] AZD8330 MEK1/2 I Mental status change, rash, nausea Advanced solid tumors [ 110 ] MEK inhibitors are sub-divided into two major classes, ATP non-competitive and ATP competitive inhibitors (Figure 3). Most of the known MEK inhibitors are noncompetitive i.e. they do not directly compete for the ATP–binding site. Rather they bind to a unique allosteric site adjacent to the ATP site. This explains the high specificity of the non-competitive MEK inhibitors [68]. Figure 3The chemical structures of 9 MEK inhibitors in clinical development. Nine MEK inhibitors are shown in the diagram. Among them, trametinib is being evaluated by FDA for treatment of advanced metastatic melanoma. BODY.TRAMETINIB (GSK1120212, JTP 74057): Trametinib (GSK1120212, JTP-74057) is a potent small molecule inhibitor of MEK kinase. It is an allosteric, second generation, ATP non-competitive inhibitor with nanomolar activity against purified MEK 1 and MEK 2 kinases [69]. Preclinical studies showed efficient inhibition of p-ERK 1/2 which correlates with potent cell growth inhibition in tumor lines with mutant B-RAF or Ras. By this mechanism, trametinib induces cell-cycle arrest. In xenograft models of HT-29 and COLO205 colorectal tumor cell lines, trametinib demonstrated robust anticancer activity when administered daily for 14 days [69,70]. An early phase I dose-escalation trial of trametinib (≤3 mg/day continuous or intermittent dosing schedule) enrolled 206 patients with advanced solid tumors. Dose limiting toxicities included rash, serous central retinopathy and diarrhea. Dose of 2 mg/day was chosen for further studies. Overall objective response rate was 10%. However, B-Raf mutant melanoma had a response rate of 33% [71]. These encouraging results led to several phase II/III clinical trials of trametinib alone or in combination with other agents [NCT01553851, NCT01682083, NCT01362296, NCT01619774, NCT01245062, details are available on clinicaltrials.gov]. In the first published phase III trial of trametinib, 322 previously treated (interferon or chemotherapy) patients with advanced melanoma with V600E or V600K B-Raf mutations were randomly assigned in a 2:1 ratio to receive oral trametinib (2 mg once daily) or intravenous chemotherapy consisting of either dacarbazine (1000 mg/m2) or paclitaxel (175 mg/m2), every 3 weeks [67]. The median progression-free survival (PFS) of patients who received trametinib (4.8 months) was significantly longer than that of patients who received chemotherapy (1.5 months) (hazard ratio [HR] 0.45; P < 0.001). At 6 months, the rate of overall survival was 81% in the trametinib group versus 67% in the chemotherapy group (HR 0.54; P = 0.01). BODY.PIMASERTIB (AS703026, MSC1936369B): Pimasertib, also known as AS703026, MSC1936369B, is a highly potent ATP noncompetitive second generation inhibitor of MEK1 and MEK2 [72,73]. Pimasertib selectively binds to the distinctive allosteric site on MEK1/2 [73]. In xenograft models, pimasertib demonstrated significant tumor growth inhibition in a human plasmacytoma H929 MM cell line at 15 and 30 mg/kg for 21 days [72]. Tumor regression was also observed at 10 mg/kg in a mouse model of D-MUT colorectal tumor [73]. A multicenter phase I/II clinical trial of pimasertib plus FOLFIRI as a second line treatment in K-Ras mutated metastatic colorectal cancer (mCRC) enrolled 16 patients [74]. Initially no DLT was observed at 45 mg/day which allowed dose escalation to 60 mg/day. At this dose, 2 of 5 patients experienced grade 3 mucositis/stomatitis leading the expansion of 45 mg/day cohort. Most common treatment-emergent adverse events (TEAEs) after 3 cycles of treatment were asthenia, diarrhea, mucositis, ocular events, nausea, rash and vomiting. These TEAEs were observed in more than one third of the treated subjects. Currently, a few phase I/II studies are underway to test pimasertib (MSC1936369B) in the setting of advanced or metastatic solid tumors including melanoma [NCT01016483, NCT01016483, NCT016968017, NCT01453387]. BODY.SELUMETINIB (AZD6244, ARRY-142886): Selumetinib is a non-ATP competitive highly selective MEK 1/2 inhibitor with IC50 of 14 nm [75]. In xenograft models, its antitumor activity correlates with decrease in phosphorylated ERK1/2 levels. In a phase I dose escalation study of 57 patients with advanced cancers, a total daily dose of 200 mg was suggested for subsequent trials [76]. Rash, diarrhea and hypoxia were reported as major DLTs. At the recommended dose of 100 mg bid most of these TEAEs were grade 1 or 2. Other common TEAEs were nausea, fatigue, peripheral edema, transaminitis and blurry vision. Best response was stable disease and achieved in 33% of patient at the end of 2nd cycle. Patients with mutated Ras or Raf remained longer in the study with higher response rate but analysis of statistical significance could not be performed due to small number of patients. Multiple phase II studies were conducted in patients with papillary thyroid, lung, liver, pancreatic, colorectal cancers and melanoma [77-82]. Patients in these trials received selumetinib irrespective of Ras/Raf mutation status and none of these trials met their primary end points. However, patients harboring Ras/Raf mutations had higher objective response rate, indicating the need of proper patient selection in subsequent studies evaluating selumetinib. A randomized placebo controlled phase II trial was done in previously treated patients with K-Ras mutant stage III-IV non-small cell lung cancer (NSCLC) [83]. Patients were randomized to receive docetaxel plus either placebo or selumetinib (75 mg twice daily q21 days), with overall survival (OS) being the primary end point. Median OS was 9.4 months (m) in selumetinib arm vs 5.2 m in control arm, yet the difference was statistically non-significant (HR 0.8, 80% CI 0.56-1.14, p = 0.21). However, median progression free survival was significantly prolonged in selumetinib arm (5.3 m) compared to control arm (2.1 m). Overall response rate was also better in selumetinib group. The combination of docetaxel and selumetinib had higher toxicity than docetaxel alone. Selumitinib was also studied in recurrent low grade serous carcinoma of the ovary/peritoneum in a single arm phase II study and in mitigating radioactive iodine refractoriness in metastatic thyroid cancer [84,85]. BODY.PD-0325901: PD-0325901 is a highly specific and potent synthetic analog of MEK inhibitor CI-1040. It has subnanomolar and non-competitive inhibitory activity (IC50 = 0.33 nM) against purified MEK 1 and MEK 2 [86]. PD-0325901 inhibited phosphorylation of ERK1/2 in melanoma and papillary thyroid cancer (PTC) cell lines harboring B-Raf mutation [87]. In xenograft models, PD-0325901 demonstrated significant antitumor activity at a dose of 20–25 mg/kg/day with tumor shrinkage by 58% in PTC cells with the RET/PTC1 rearrangement [88]. In a phase I, dose-escalation study of 30 patients with multiple solid tumors, the DLTs were acneiform rash involving face, trunk and arms at 30 mg twice daily. Transient and reversible visual effects characterized by blurred vision and halos were observed at ≥15 mg BID [89,90]. The most frequent treatment-emergent adverse events (TEAE) included rash, fatigue, diarrhea, nausea, and vomiting. There were 1 PR (melanoma) and 5 SD (4 melanomas, 1 NSCLC). In an open-label, phase II study, patients with progressive, recurrent, or advanced NSCLC were treated with15 mg PD-0325901 twice daily [91]. There were no objective responses during the trial period. Due to a lack of responses coupled with the safety issues, the trial was closed after the first stage. However, Pfizer initiated a new multi-arm phase 1 study in 2012 to test PF-04691502 and PF-05212384, dual PI3K/mTOR Inhibitors in combination with PD0325901 or irinotecan in patients with advanced cancer [NCT01347866]. BODY.REFAMETINIB (RDEA119, BAY 869766): Refametinib is the only cyclopropane-1-sulfonamide derivative, and exhibits a highly selective allosteric inhibition of MEK 1/2 [92]. When dosed once daily for 14 days, refametinib showed potent activity in preclinical xenografts of human melanoma A375, colon carcinoma Colo205 and HT-29, pancreatic cancer OCIP19, 21, and 23, and skin carcinoma A431 tumor models [92,93]. In a phase I/II study of patients with advanced solid tumors, refametinib was well tolerated at doses 100 mg daily. Rash was the most common TEAE [94]. Subsequently, a phase II study enrolled seventy patients to evaluate refametinib in combination with sorafenib as first-line treatment for unresectable hepatocellular carcinoma (HCC) [95]. Of sixty-five patients analyzed for efficacy per protocol, three (5%) had PR, and the median time-to-progression was 4.1 months. BODY.TAK733: TAK733 is a novel second-generation, allosteric kinase inhibitor with potent anti-MEK 1/2 activity [96]. In xenograft models, TAK-733 exhibited broad antitumor properties [96,97]. Phase I/II trials using TAK733 alone and in combination with alisertib in advanced non-hematologic malignancies are still accruing [NCT00948467, NCT01613261]. BODY.MEK162 (ARRY 438162): MEK162 (ARRY 438162) is another novel, second generation inhibitor that targets MEK 1/2 [98,99]. A phase II study examined MEK162 in 71 patients with N-Ras and B-Raf mutated advanced melanoma patients. It was given as 45 mg twice daily. Disease control rates of 63% and 51% were noticed in N-Ras and B-Raf mutant melanoma patients, respectively. No complete response was observed. Grade 3–4 adverse events include rash, diarrhea, fluid retention and creatinine phosphokinase (CPK) elevation [100]. A MEK162 analog, ARRY 300, recently completed phase I testing in healthy volunteers in the United States (NCT00828165). BODY.RO5126766: As a novel, highly potent, first-in-class dual MEK/Raf inhibitor, RO5126766 selectively binds to MEK 1/2 to form a stable Raf-MEK-RO5126766 complex. Cell cycle arrest was shown to be the primary mechanism for the growth-inhibitory properties of RO5126766 in a panel of human tumor cell lines [101]. A phase I open-label, dose-escalation study of RO5126766 was undertaken in 52 patients with advanced cancers [101]. Tolerability of RO5126766 was similar to that of other MEK inhibitors and the most common toxicities included rash-related disorders, elevated CPK, and diarrhea. The overall objective response rate was 40% in forty-five patients. Of 21 patients with metastatic melanoma included in the study, three PR were seen in two B-Raf mutant melanomas and one in an N-Ras mutant melanoma. The dose recommended for phase II investigation was 2.7 mg daily 4 days on/3 days off. BODY.WX-554: WX-554 is another MEK 1/2 inhibitor. To determine pharmacokinetic and pharmacodynamic parameters, WX-554 is planned to be administered intravenously as single doses in the range of 0.05 mg/kg to 5.0 mg/kg to healthy volunteers in dose escalated manner [102]. Results of this study are not available yet. An oral formulation of this inhibitor is being tested in a phase I/II trial in patients with advanced solid tumors (NCT01581060). BODY.RO4987655 (CH4987655): RO4987655 is a highly selective, small molecule MEK inhibitor. The unique 3-oxo-oxazinane ring structure of RO4987655 confers metabolic stability, This compound showed slow dissociation from MEK with remarkable antitumor efficacy, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human [103]. In a recently published phase I study of RO4987655, MEK 1 inhibition in cancers was demonstrated by decreased ERK1/2 phosphorylation. Partial responses and stable disease were achieved below MTD (8.5 mg twice daily) mainly in patients with skin melanomas [104]. DLTs were reversible grade 3 blurry vision and grade 3–4 elevation of CPK. The compound alone is currently undergoing further clinical development in an expansion of this study [NCT00817518]. BODY.GDC-0973 (XL518): A derivative of methanone, GDC-0973 is a potent, orally bioavailable, small-molecule inhibitor of MEK 1 [105]. GDC-0973 showed strong antineoplastic activity in a B-Raf and K-Ras mutant cancer cell lines [105-107]. In a phase I clinical trial of 46 evaluable patients, GDC-0973 in combination with GDC-0941 induced PR in 3 patients (B-Raf melanoma, B-Raf pancreatic cancer, K-Ras endometrioid cancer) and stable disease in 5 [108]. Safety data showed that the DLTs were increase in serum lipase and CK enzymes. Additional phase I-III clinical trials are ongoing [NCT01689519, NCT01271803, NCT00996892, NCT01271803, NCT01562275]. BODY.AZD8330 (ARRY-424704, ARRY-704): AZD8330 (ARRY 704) represents a new member of MEK1/2 inhibitors [109]. A large phase I trial of 82 patients with advanced solid tumors defined the MTD to be 40 mg/day. Change in mental status was the dose limiting toxicity. Other common TEAEs include rash, fatigue, diarrhea and vomiting. Disease control rate of 40% was demonstrated in this study. Mutation analysis of Ras/Raf genes were not mandated by the study [110]. BODY.CONCLUSIONS AND FUTURE DIRECTIONS: Four distinct MAP kinase signaling pathways involving 7 MEK enzymes have been identified. MEK1 and MEK2 are the prototype members of MEK family proteins. Several MEK inhibitors are in clinical trials. Trametinib is being evaluated by FDA for the treatment of metastatic melanoma. Targeted therapies with small molecular inhibitors for solid tumors and hematological malignancies are moving quickly from bench to bedside [111-113]. Combination of targeting agents against different signaling pathways may provide additional benefits and warrant further clinical studies [114,115]. BODY.COMPETING INTERESTS: The authors have no relevant competing interest. BODY.AUTHORS’ CONTRIBUTIONS: All authors have contributed to data preparation, drafting and revising the manuscripts. All authors have read and approved the final manuscript.

TITLE: Comparison of an injectable toltrazuril-gleptoferron (Forceris®) and an oral toltrazuril (Baycox®) + injectable iron dextran for the control of experimentally induced piglet cystoisosporosis ABSTRACT.BACKGROUND: Cystoisospora suis causes diarrhoeal disease and reduced weight gain in suckling piglets, and a toltrazuril-based oral suspension is available for treatment. Recently a combinatorial product with toltrazuril plus iron has been developed for parenteral application. In this study we compared the efficacy of the injectable product with the oral suspension against experimentally induced piglet cystoisosporosis. ABSTRACT.METHODS: In a randomised controlled study, three groups of piglets (n = 10–13) were treated either with a fixed dose of 45 mg toltrazuril + 200 mg gleptoferron i.m. per piglet (Forceris®) on the second day of life (study day 2; SD 2) or with 20 mg toltrazuril/kg body weight as an oral suspension (Baycox® 5%) on SD 4 or left untreated (Control group). The Baycox® and the Control group received 200 mg of iron dextran/piglet on SD 2. All piglets were infected with 1000 sporulated C. suis oocysts on SD 3. Faecal samples were taken daily from SD 7 to SD 20 to determine faecal consistency, oocyst shedding and other diarrhoeal pathogens. Body weight was recorded on SD 1 and then weekly until SD 29. Animals were observed daily for general health and after treatment for possible adverse events. ABSTRACT.RESULTS: In the Control group all animals shed oocysts for 3.1 days on average and all animals showed diarrhoea for an average of five days. Excretion peaked on SD 9 (max. 48,618 oocysts per gram of faeces). Treatment with Forceris® completely suppressed oocyst excretion. In the Baycox® group, low levels of excretion could be detected. Diarrhoea was reduced to single piglets in the treated groups. Body weight development was reduced in the Control group compared to the treated groups. Enteropathogenic bacteria (Escherichia coli, Clostridium perfringens) could be detected. All parameters related to oocyst excretion, faecal consistency and weight gain were significantly improved in the treated groups compared to the Control group without significant differences between the treated groups. Both products were safe to use. ABSTRACT.CONCLUSIONS: Treatment with both the injectable (Forceris®) and the oral (Baycox®) formulation of toltrazuril in the prepatent period were safe and highly effective against experimental infection with C. suis in newborn piglets. BODY.BACKGROUND: Porcine neonatal coccidiosis caused by Cystoisospora suis (syn. Isospora suis) is a major cause of diarrhoea and unthriftiness in piglets worldwide [1–6] and induces substantial economic losses in the pig breeding industry [7–10]. Currently, the only effective chemotherapeutic drug available for treatment is the triazinetrione toltrazuril [11]. It is available as a 5% suspension to be applied as a single oral treatment during the third to fifth day of life. Oral treatment with toltrazuril is highly effective in controlling oocyst excretion and diarrhoea both in experimental and natural infections of suckling piglets [12–17]. Recently an injectable combination product, toltrazuril + iron (30 mg toltrazuril/ml; 133.4 mg iron/ml as gleptoferron) has been developed for the prevention of piglet coccidiosis together with a prevention of iron deficiency (Forceris®, Ceva, Libourne, France). Treatment is scheduled from the first to the third day of life (i.e. between 24 and 96 h after birth) as a single intramuscular injection of 1.5 ml/piglet corresponding to 45 mg of toltrazuril and 200 mg of iron. Forceris® is the first toltrazuril product to be marketed in an injectable formulation for the control of C. suis in piglets from the first day of life, and this study aimed to evaluate its efficacy against porcine coccidiosis in an experimental infection model [18] in comparison to an established reference product, an oral toltrazuril suspension (Baycox® 5%, Bayer Animal Health, Monheim, Germany). BODY.METHODS.ANIMALS AND HUSBANDRY: A total of 35 piglets from four litters were enrolled in the trial on study day (SD) 1 and finished it on SD 29. Sows (Landrace × Large White) were moved to the large animal facilities of the Institute of Parasitology of the Vetmeduni Vienna two weeks before farrowing to acclimatise to the housing conditions. Animals were kept on straw with a heat lamp for the piglets and fed with conventional feed free of coccidiostats. Water was provided ad libitum. On the first day of the study (SD 1; within 24 h after birth of the piglets) animals were marked individually and enrolled in the study if they were clinically healthy and weighed at least 900 g. They were randomly allocated to one of three groups according to body weight on SD 1 in the order of the birth of the litters. The general health of sows and their piglets was observed and recorded daily from SD 1 to SD 29. BODY.METHODS.TREATMENT: Animals were treated either with toltrazuril + gleptoferron (investigational product: Forceris®, Ceva, France) at a fixed dose of 1.5 ml/piglet corresponding to 45 mg of toltrazuril and 200 mg of iron, i.m. on SD 2 (Forceris® group, n =13 piglets) or with a toltrazuril suspension (reference product: Baycox® 5%, Bayer Animal Health, Monheim, Germany) at a dose of 20 mg/kg orally on SD 4 (Baycox® group, n = 12 piglets). A third group (Control group, n = 10 piglets) remained untreated. The Baycox® and the Control groups received iron dextran (Uniferon® 200, Virbac, Kolding, Denmark) at a fixed dose of 200 mg/piglet, i.m. on SD 2. Allocation to treatment groups and treatment were carried out under blinded conditions, i.e. only the dispenser was aware of the allocation of piglets to a treatment group during the course of the study. Post-treatment observations for any adverse events (swelling/bleeding of the injection site, inability to stand, walk, suckle or other abnormal behaviour, including dyspnoea, vomiting, limping, lateral recumbency, signs of pain, distress or neurological alterations, [19]) were conducted under blinded conditions by a veterinarian at 2, 6 and 24 h after treatment, and after that daily until SD 8. BODY.METHODS.EXPERIMENTAL INFECTION: Each piglet was orally infected with a single dose of approximately 1000 sporulated oocysts of C. suis, strain Wien-I [15] on SD 3. BODY.METHODS.DETERMINATION OF CONCOMITANT BACTERIAL AND VIRAL INFECTIONS: On the first day of faecal sampling (SD 7) pooled faecal samples from each litter (n = 4) were submitted for bacteriological and virological examination to the Institute of Microbiology and Institute of Virology, respectively, of the Vetmeduni Vienna. A general qualitative/semiquantitative bacteriological examination, followed by specific differentiation and virulence factor/toxin typing in positive cases, was conducted. Porcine rotavirus A antigen and nucleic acids of porcine coronaviruses TGEV and PRCoV were determined from the faecal material. In case of diarrhoea after treatment with toltrazuril, individual samples were taken on the first day of diarrhoea and examined for bacterial pathogens as well. BODY.METHODS.EFFICACY PARAMETERS: Individual faecal samples were taken daily from SD 7 to SD 20. Efficacy of toltrazuril was evaluated by qualitative (autofluorescence; [20]) and quantitative (McMaster counting of oocysts per gram of faeces, OpG) determination of oocysts and faecal consistency as previously described [18]. Faecal consistency of each sample was scored immediately with faecal score (FS) 1 describing firm, FS 2 pasty, FS 3 semi-liquid and FS 4 liquid faeces, with FS 3 and FS 4 considered as diarrhoea. Individual body weight was recorded for each enrolled piglet on SD 1, 8, 15, 22 and 29. To evaluate the efficacy of treatment, oocyst excretion, faecal consistency and body weight development were analysed statistically. This included the area under the curve (AUC) for quantitative oocyst excretion and FS, the number of days with oocyst excretion/diarrhoea, and the number of animals that showed excretion/diarrhoea in each group, as well as the body weight on each weighing day and body weight gain. Data were evaluated for homogeneity of the groups and analysed by means of the Kruskal-Wallis H-test (for continuous parameters) or by means of the Chi-square test (for discrete parameters). Pairwise comparisons were performed either using Fisher's exact test (excretion/diarrhoea present or not) or Wilcoxon-Mann-Whitney U-test (other parameters). For all tests, P-value correction with Bonferroni was applied. BODY.RESULTS.SAFETY: No animal showed treatment-related adverse events that required veterinary intervention. Two animals from the Forceris® group showed a slight temporary swelling at the injection site within the first day of observation after treatment. Two animals from the Control group had to be treated with Ringer's lactate due to dehydration. BODY.RESULTS.QUALITATIVE OOCYST EXCRETION DETERMINED BY AUTOFLUORESCENCE: All piglets of the Control group and 25% of the piglets in the Baycox® group excreted oocysts detectable by autofluorescence (AF), while none of the animals in the Forceris® group shed oocysts. The mean duration of oocyst excretion was 1.3 days in the Baycox® group and 3.1 days in the Control group (Table 1).The number of days with AF detectable excretion and the number of piglets that excreted AF detectable oocysts were significantly reduced in the Forceris® and Baycox® groups compared to the Control group without statistical difference between the treatment groups (Table 2).Table 1McMaster countable oocyst excretion, autofluorescence detectable oocyst excretion and diarrhoea values per groupForceris®Baycox®Control No. of piglets131210 No. of samples over all sampling days181168140McMaster countable oocyst excretion No. (%) positive piglets0 (0)2 (16.7)9 (90.0) No. (%) excretion days0 (0)3 (1.8)21 (15.0)Autofluorescence detectable oocyst excretion No. (%) positive piglets0 (0)3 (25.0)10 (100) No. (%) excretion days0 (0)4 (2.4)31 (22.1)Diarrhoea No. (%) positive piglets2 (15.4)4 (33.3)10 (100) No. (%) diarrhoea days3 (1.7)6 (3.6)50 (35.7)Liquid diarrhoea - faecal score (FS) 4 No. (%) FS 4 piglets1 (7.7)1 (8.3)7 (70.0) No. (%) FS 4 days1 (0.6)3 (1.8)22 (15.7)Table 2P-values (given as -log10) are given for the parameters of oocyst excretion and faecal score. Differences at P < 0.05 are indicated in boldParameterForceris® vs ControlBaycox® vs ControlForceris® vs Baycoxχ2, dfArea under the Curve for OpG 3.89 3.19 0.3524.87, 2No. of days with McMaster countable excretion 3.89 2.70 0.3523.17, 2McMaster countable excretion present or not 4.43 2.24 0.1823.09, 2No. of days with AF detectable excretion 4.62 3.80 0.7028.29, 2AF detectable excretion present or not 5.52 2.85 0.5425.36, 2Area under the Curve for FS 3.72 4.72 0.0020.64, 2No. of days with diarrhoea 4.13 3.60 0.0024.46, 2Diarrhoea present or not 3.70 2.29 0.0017.44, 2Daily body weight gain SD 1–29 1.56 2.59 0.8814.59, 2Abbreviations: OpG oocysts per gram of faeces, AF autofluorescence, FS faecal score, χ2 and df statistics and degrees of freedom according to Kruskal-Wallis rank sum test or Chi-square test BODY.RESULTS.QUANTITATIVE OOCYST EXCRETION DETERMINED BY MCMASTER COUNTING: McMaster countable oocyst excretion was observed in nine out of ten piglets of the Control group, in two animals of the Baycox® group and in none of the piglets of the Forceris® group. When excretion days were evaluated, positive animals in the Control group excreted McMaster countable oocysts between one and four days (mean 2.3 days), whereas in the Baycox® group two positive animals excreted for one and two days, respectively (Table 1). The maximum OpG value in the Control group was 48,618 OpG on SD 9 (Fig. 1); on that day, the prevalence of McMaster countable excretion also reached its peak at 50% (Fig. 2), while in the Baycox® group the OpGs did not exceed 333.Fig. 1Mean oocyst excretion given as ln (OpG+1) per group during the whole study periodFig. 2Prevalence of McMaster countable excretion The area under the curve for OpG, the number of days with McMaster countable excretion and the number of piglets that excreted countable oocyst numbers were significantly reduced in the Forceris® and Baycox® groups compared to the Control group without statistical difference between the treatment groups (Table 2). BODY.RESULTS.FAECAL CONSISTENCY AND DIARRHOEA: The average faecal score increased above 2 in the Control group from SD 8 to SD 14, while in the treated groups the mean FS never exceeded 2. The maximum prevalence of diarrhoea was 100% in the Control group (SD 11 and SD 12) with an average duration of 5.0 days, while in the Baycox® group 33.3% of the animals showed diarrhoea for an average of 1.5 days, and in the Forceris® group two animals (15.2%) showed diarrhoea for one and two days, respectively. FS 4 (watery diarrhoea) was observed in 70% of the Control animals (average duration: 3.1 days) while in the Baycox® group one animal had watery diarrhoea for three days and in the Forceris® group one animal for one day (Table 1). The peak of diarrhoea was observed on SD 11 to SD 12 when all animals in the Control group showed diarrhoea with a mean FS of 3.6 (Figs. 3 and 4). The area under the curve for the FS, the number of days with diarrhoea and the number of piglets that had diarrhoea were significantly reduced in the Forceris® and Baycox® groups compared to the Control group without statistical difference between the treatment groups (Table 2).Fig. 3Course of diarrhoea (FS 3 and 4) in the different groupsFig. 4Course of the average faecal score (FS) in the different groups BODY.RESULTS.BODY WEIGHT DEVELOPMENT: Body weights were not significantly different between the groups on SD 1, the day of randomisation (Kruskal-Wallis H-test, χ2 = 95145, df = 2, P = 0.62). Daily body weight gain and total weight gain from SD 1 to SD 29 were lowest in the Control group due to a severe depression of weight gain in the acute phase of infection (from SD 8 to SD 15) during which the Control group only gained 975.8 g on average compared to 2008.3 g in the Baycox® group and 1976.9 g in the Forceris® group (Fig. 5).Fig. 5Body weight development during the study (study days 1–29). Vertical lines depict the upper standard deviations The daily body weight gain from SD 1 to SD 29 was significantly higher in the Forceris® and Baycox® groups compared to the Control group without statistical difference between the treatment groups (Fig. 5). BODY.RESULTS.CONCOMITANT INFECTIONS: All litters showed infections with a similar pattern of bacteria. Haemolytic Escherichia coli which were positive for virulence factors fimH, papC, iucD, and cnf1 and β-2 toxin positive Clostridium perfringens Type A were detected in high amounts at the beginning of sampling and were also diagnosed in those animals that showed diarrhoea after treatment. No viral infections were detected. BODY.DISCUSSION: Piglets experimentally infected with C. suis were treated with toltrazuril either parenterally or orally before the onset of oocyst excretion and diarrhoea. Both, Forceris® and Baycox® had a comparable effect on oocyst shedding, faecal consistency and body weight development. Oocyst shedding was significantly suppressed in both treated groups compared to the Control group. Despite the intra-litter randomisation which entailed a high environmental contamination in all litters due to the presence of untreated control animals, no piglets of the Forceris® group excreted oocysts during the trial. This indicates that the treatment was highly effective and the level of false-positive samples due to coprophagy [21] in such an experimental setting is likely to be low. In the Baycox® group three samples were positive with one oocyst in McMaster counting each, resulting in OpG values of 333. It is difficult to unequivocally conclude that these findings are derived from true infections for the reason stated above; however, one of the two animals that excreted countable oocysts did so on two consecutive days indicating that at least this individual shed oocysts due to infection and not due to coprophagy, but data on the extent of false positive samples due to this in experimentally infected mixed litters are not available. Low levels of oocyst shedding can occur despite treatment with Baycox® [14] while in other experimentally induced infections suppression of oocyst development was complete [15]. Despite low levels of oocyst shedding the reduction of environmental recontamination by disruption of the parasite's life-cycle was highly effective. Diarrhoea is a hallmark of porcine cystoisosporosis [2, 3, 18, 22]; it can be induced by parasite infection in the absence of other enteropathogens [23, 24] but exacerbated by rotavirus [24] or Cl. perfringens [25, 26], and probably by other enteropathogens as well. Early studies on the use of toltrazuril in pigs indicated that C. suis has a forerunner role for bacterial infections as anticoccidial treatment reduced diarrhoea and simultaneously the amount of antibiotics required to control bacterial infections on affected farms, but a low level of diarrhoea was still seen after toltrazuril application [12]. In the light of these findings, it is conceivable that the E. coli and clostridia that were circulating in the litters (and also detected in the animals with diarrhoea after treatment) contributed to the clinical outcome in some individuals despite successful treatment of cystoisosporosis. Body weight development and diarrhoea are inversely related [18] and consequently body weight gain was increased in treated animals (which showed less diarrhoea), with the result of higher weights around weaning on SD 29 compared to the Control group. Improved weight gain has also been shown upon treatment in C. suis-positive herds with no apparent clinical signs [27], indicating that the presence of the parasite may influence performance of piglets even at low infection levels. Parasite shedding, diarrhoea and body weight development showed no significant differences between the two toltrazuril formulations; both were highly efficacious. Forceris® can be used to simultaneously treat metaphylactically against C. suis infection and supply the iron required to support the rapid growth and increase in blood volume during the first days of life of the piglets, thus reducing the required handling of the piglets. Time needed for oral treatment of one piglet was estimated at 10 seconds with a labour cost estimation of € 18/hour [27], significantly contributing to the costs of coccidiosis control. Forceris® can be applied from the first day of life (Baycox®: third to fifth day of life) and may thus also be more useful in cases where C. suis infections occur very early after birth and are often seriously exacerbated by bacterial infections, such as with Cl. perfringens [17]. Accurate dosing of piglets by injection may also be of advantage when vomiting occurs for a variety of reasons, including infection with C. suis [28]. BODY.CONCLUSIONS: Oocyst shedding was reduced to a minimum in the Baycox® group and suppressed completely in the Forceris® group. Diarrhoea was also reduced significantly in both treatment groups, resulting in significantly better weight gain. The latter was depressed during the acute stage of infection (in the second week of life) in animals that were clinically affected by pasty to watery diarrhoea. Both applications were safe to use and effective in a single application. Early (metaphylactic) treatment of piglet coccidiosis during the prepatent phase of infection can control infection and significantly improve piglet health when C. suis is present on a farm. Treatment with toltrazuril together with iron by injection was safe and effective. Handling of animals for medication was reduced by the combination product without interfering with the efficacy of toltrazuril treatment.

TITLE: Subfoveal Choroidal Thickness after Panretinal Photocoagulation with Red and Green Laser in Bilateral Proliferative Diabetic Retinopathy Patients: Short Term Results ABSTRACT: Purpose. To compare subfoveal choroidal, central retinal, and peripapillary nerve fiber layer (RNFL) thickness after panretinal photocoagulation (PRP) with red and green laser in diabetic patients. Study Design. Randomized clinical trial. Methods. A total of 50 patients with bilateral proliferative diabetic retinopathy and no diabetic macular edema underwent PRP. One eye was randomly assigned to red or green laser. Subfoveal choroidal, central retinal, and RNFL thicknesses were evaluated at baseline and 6 weeks after treatment. Results. The mean subfoveal choroidal, central retinal, and peripapillary nerve fiber layer (RNFL) thickness increased significantly in each eye 6 weeks after PRP (P values in red laser group: <0.01, 0.03, and <0.01, resp., and in green laser group <0.01, <0.01, and <0.01). There was no difference between red and green laser considering subfoveal choroidal, central retinal, and peripapillary nerve fiber layer (RNFL) thickness increase after PRP (P values: 0.184, 0.404, and 0.726, resp.). Conclusion. Both red and green lasers increased mean subfoveal choroidal, central retinal, and peripapillary nerve fiber layer (RNFL) thickness significantly 6 weeks after PRP, but there is no difference between these two modalities in this regard. BODY.1. INTRODUCTION: Diabetic retinopathy is one of the most common causes of visual loss worldwide. According to Early Treatment of Diabetic Retinopathy Study (ETDRS), protocol panretinal photocoagulation (PRP) can prevent severe visual loss in proliferative diabetic retinopathy (PDR) [1]. Several studies have shown histopathologic changes in choroid of diabetic patients [2–4]. Meanwhile, using panretinal photocoagulation (PRP), as was suggested beneficially by diabetic retinopathy study for proliferative diabetic retinopathy, has gotten general acceptance [1]. With the advent of enhanced depth imaging optical coherence tomography (EDI-OCT), it is now possible to visualize and measure choroidal changes easily. Two different laser types for PRP are common, red and green laser. To the best of our knowledge, no other study has compared these two laser modalities in changing choroidal or central retinal thickness. In this study, we have evaluated the subfoveal choroidal, central retinal, and peripapillary retinal nerve fiber layer (RNFL) thickness changes before and after PRP using EDI-OCT and we compare the green and red laser in this regard. BODY.2. METHODS: This clinical trial was approved by the Institutional Review Board/Ethics Committee. Written informed consents were obtained from all participants. Eligible cases were 50 patients (100 eyes) with bilateral proliferative diabetic retinopathy. Exclusion criteria were advanced proliferative diabetic retinopathy, a history of any laser treatment (panretinal or focal laser photocoagulation), any history of intravitreal drug injection, ocular surgery, significant media opacity, myopia, or hyperopia with refractive error of more than 3 diopters. Primary objective was to determine subfoveal choroidal and retinal thickness and peripapillary nerve fiber layer thickness changes 6 weeks after red versus PRP laser. A complete ophthalmic examination including best corrected visual acuity (BCVA) using Snellen chart, Goldmann applanation tonometry, and dilated indirect ophthalmoscopy was conducted. Diabetic retinopathy grading (DRG) was performed according to the ETDRS definition [5]. Enhanced depth imaging optical coherence tomography (EDI-OCT) mapping was performed using SD-OCT (Spectralis®, Heidelberg Engineering, Heidelberg, Germany). All patients were treated on an outpatient basis. PRP was performed in 3 sessions with one week interval. A total number of 1600 to 2000 spots per eye were applied to ablate retina using Ellex Integre Pro Scan laser photocoagulator (82 Gilbert Street, Adelaide, SA 5000, Australia). The order of treated areas was as follows: nasal, inferior, superior, and then temporal retina. Time exposure was 100 milliseconds. To have threshold laser photocoagulation, power setting was chosen to have a mild gray-white burn (Grade 2) according to ETDRS guidelines [1]. Laser setting parameters did not differ significantly between two groups (mean power 303 mWatts in red and 290 mWatts in green laser group, P value: 0.14; mean laser spots 1789 spots in red and 1869 spots in green laser group, P value: 0.13). One eye of all patients was randomly assigned to red laser and the other eye to green laser. Spectral domain optical coherence tomography images were obtained using Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany) to measure central retinal thickness and enhanced depth imaging (EDI) mode to measure subfoveal choroidal thickness. BCVA measurement and an OCT scan were performed at baseline and 6 weeks after completion of PRP. Subfoveal choroidal thickness was measured using apparatus measurement tool at baseline and 6 weeks after PRP. Choroidal segmentation was done manually, moving the reference lines from the retinal borders to the choroidal borders. The internal limiting membrane line was moved to the base of the retinal pigment epithelium. The basement membrane line was moved to chorioscleral junction. Also retinal nerve fiber layer (RNFL) thickness analysis was done at baseline and 6 weeks after treatment. BODY.2. METHODS.2.1. STATISTICAL ANALYSIS: Data were analyzed using SPSS software (version 16, SPSS, Inc.). Data normality was assessed using Shapiro-Wilk test. A paired sample t-test was used to compare macula and choroid thickness before and after treatment. Independent sample t-test was performed to compare macula and choroid thickness changes from baseline between two groups. General Linear Model was applied to assess differences within and between group. P values of <0.05 were considered significant. BODY.3. RESULTS: A total number of 50 patients with PDR entered the study. The mean age of the patients ± standard deviation was 55.6 ± 8.9 years. Nineteen patients were male (38%). The mean visual acuity of patients was 0.32 ± 0.28 logMAR and 0.27 ± 0.23 logMAR in red laser group and green laser group, respectively. The mean HbA1C was 8.47 ± 1.57%. The baseline characteristics of the patients are presented in Table 1. No complication occurred in both groups during laser and in follow-up period. The mean subfoveal CT increased significantly in each group at 6 weeks follow-up (in red laser group, 202.14 ± 24.5 micron at baseline and 211.7 ± 26.6 micron at 6 weeks, P value < 0.00, showing 4.86% CT increase compared to baseline) (in green group, 201.9 ± 21.13 micron at baseline and 208.9 ± 25.0 at 6 weeks, P value < 0.00, showing 3.54% increase compared to baseline) (Table 2). There was not a significant difference between red and green laser in terms of choroidal thickness changes from baseline (P value 0.184). In red laser group, the mean central retinal thickness was 271.6 ± 30.33 micron at baseline and 298.5 ± 62.14 micron at week 6 of follow-up (P value 0.03). In green laser group, the mean central retinal thickness was 267.0 ± 36.9 micron at baseline and 306.4 ± 73.3 micron at week 6 of follow-up, (P value 0.000 for both groups). There was no significant difference between macular thickness increase between two groups (P value: 0.404) (Table 2). There was no significant association between choroidal thickness change and retinal thickness change in each group (P values: 0.051 and 0.52, resp.). The mean peripapillary RNFL thickness was 100.5 ± 20.1 micron in red laser group at baseline which increased to 108.2 ± 23.1 micron at 6 weeks (P value 0.000) (7.8% as compared to baseline). In green laser group, it was 103.5 ± 19.8 micron at baseline which increased to 112.7 ± 22.9 in week 6 of follow-up (P value 0.000) (9.2% as compared to baseline). RNFL change between two groups was not significant (P value: 0.762) (Table 2). BODY.4. DISCUSSION: In this study, in eyes with proliferative diabetic retinopathy and without significant macular edema, the mean subfoveal choroidal, central retinal, and peripapillary RNFL thickness increased significantly after both red and green argon laser treatments. This is in accordance with some of previously published studies that confirm this finding [6–9]. Takahashi et al. measured changes in choroidal blood flow in the foveal area one month after PRP in patients with severe diabetic retinopathy and no macular edema using a laser-Doppler flowmetry. They reported that PRP increases both the choroidal blood flow and the choroidal blood volume. Also, Cho et al. in their case series reported that PRP induced increases in both SFCT and macular thickness. Changes in subfoveal choroidal thickness did not correlate with changes in macular thickness in their study. Two possible mechanisms are proposed by which this increase occurs. First, it has been attributed to redistribution of choroidal blood flow [10–14]. After PRP, following degradation of photoreceptors, blood flow of treated areas decreases. This leads to redistribution of blood in other untreated areas, especially the macula. Following blood flow increase, choroidal thickness increases in the macular area. This redistribution mechanism is documented in animals in some prior studies [10–17]. The second hypothesis is the inflammation triggered by PRP. This transient inflammation probably causes release of cytokines and leads to increase of blood flow and choroidal thickness [18, 19]. Mean CT increase in both groups in this study is similar to what have been reported before in other trials [7–10]. It is noteworthy that, in some other trials, the measured subfoveal CT showed decreased thickness after PRP in short term follow-up. They have hypothesized that the reason may be due to the destruction of choroidal vasculature after thermal damage of PRP [20, 21]. Also, we found that central macular thickness and RNFL thickness increased significantly in both groups; this is also similar to what has been found in other studies [20–24]. This finding is similar to what has been reported before about the increase of retinal thickness after PRP and it was attributed to the release of proinflammatory cytokines or hypoxia-induced macular edema [20–24]. Also, we did not find any correlation between changes of choroidal thickness and central retinal thickness. This is similar to what has been reported by Cho et al. [7]. One of the novel analyses done in this study is the comparison between red and green laser in changing retinal, choroidal, and RNFL thickness. Although all three measured variables showed significant increase after treatment in each eye, intereye comparisons were not statistically significant considering central retinal, subfoveal choroidal, and RNFL thickness changes (P values: 0.404, 0.184, and 0.762, resp.) that may indicate no considerable difference between red and green laser in this regard. To the best of our knowledge, there is only one report by Ghassemi et al. [24] that compared the difference between red and green laser in PDR patients. They reported significant increase of RNFL thickness after PRP without any difference between red and green laser. This is in accordance with our findings in this study. One of the advantages of this study is selection of both eyes of one patient which can exclude the effect of systemic factors on retinal, choroidal, and RNFL thickness. This study has some limitations including low sample size and short follow-up time. Nonetheless, this study showed significant increase of subfoveal choroidal, foveal retinal, and peripapillary RNFL thickness after red and green laser PRP and no significant difference between these two lasers in aforementioned measurements.

TITLE: Combined treatment with headgear and the Frog appliance for maxillary molar distalization: a randomized controlled trial ABSTRACT.OBJECTIVE: To evaluate the efficiency of the Frog appliance (FA) alone or in combination with headgear for distalizing the maxillary molars. ABSTRACT.METHODS: Fifty patients (25 males and 25 females) aged 12.6 - 16.7 years who received treatment for Class II malocclusion at the Orthodontic Clinic of Al-Baath University were selected for this study and randomly divided into 2 equal groups. Maxillary molar distalization was achieved using the FA alone (group 1) or a combination of the FA with high-pull headgear worn at night (group 2). Lateral cephalograms were obtained before and after treatment. ABSTRACT.RESULTS: The maxillary molars moved distally by 5.51 and 5.93 mm in groups 1 and 2, respectively. Distal movements were associated with axial tipping by 4.96° and 1.25°, and with loss of anchorage by mesial movement of the second maxillary premolars by 2.70 and 0.90 mm in groups 1 and 2, respectively. The combined use of the FA and nighttime high-pull headgear decreased the distalization time and improved the ratio of maxillary molar distalization movement relative to the overall opening space between the first maxillary molars and second premolars. ABSTRACT.CONCLUSIONS: The FA can effectively distalize the maxillary molars, this distalization associates with some unfavorable changes. Nighttime use of high-pull headgear combined with the FA can reduce these unfavorable changes and improve treatment outcomes. BODY.INTRODUCTION: Maxillary molar distalization is an increasingly popular option for orthodontic resolution of Class II malocclusions because it enables correction of increased overjet while facilitates space regaining.1,2 Following molar distalization, the premolars, canines, and incisors may be retracted with different techniques.2 Molar distalization appliances can be intraoral or extraoral. Extraoral appliances such as headgear are efficient in improving the jaw relationship,3,4 but they require the patient's cooperation and accuracy during application. In fact, studies on patient cooperation, appliance wearing time, and esthetics5,6 have shown that patient compliance decreases with time7 and that non-compliance is unpredictable.8,9 In contrast, intraoral appliances reduce or even eliminate the need for patient compliance. This has led to the development of many devices such as repulsing magnets,10 NiTi springs,11 the Jones jig,3 pendulum appliances,12 distal jets,13 and superelastic wires,14,15 all of which are efficient in achieving maxillary molar distalization. In 2006, Walde developed an intraoral molar distalizing appliance named the Frog appliance.16 The Frog appliance consists of a special distalizing expansion screw with an anterior activation head that can be easily reached intraorally; additionally, a wired spring is attached to the expansion screw and is inserted into the palatal tubes of the molars. However, intraoral appliances have disadvantages too, including anchorage loss, molar tipping, extrusion with subsequent bite opening, and increased overjet. To overcome these drawbacks, many researchers have suggested the use of intraoral appliances in combination with uprighting bends, utility arches, and headgear to produce more favorable results, including vertical control of the molars, uprighting of the roots, and prevention of anterior movements of the maxilla.17,18 To date, very few studies have evaluated the efficiency of the recently developed intraoral Frog appliance, and no studies have evaluated the combined use of this appliance with extraoral devices. In this study, we describe the efficiency of the Frog appliance - alone or in combination with headgear wear - in achieving maxillary molar distalization. BODY.MATERIALS AND METHODS.PATIENT SELECTION: Before undertaking this randomized clinical trial, we estimated the sample size required to achieve statistical significance. Power analysis using the Minitab software (version 15; Minitab Inc., State College, PA, USA) showed that 19 subjects per group would be required to achieve a statistical power of 90% at a significance level of 0.05. We increased the number of subjects per experimental group up to 25 to cover possible withdrawals. The research sample consisted of 50 patients (25 males and 25 females) with a mean age of 14.3 ± 1.8 years (range, 12.6 - 16.7 years) who sought treatment at the Orthodontic Clinic of Al-Baath University (Hama, Syria). All the patients had Angle's Class II malocclusion resulting from dentoalveolar deformities or simple skeletal discrepancies in the maxilla. All patients had an ANB angle ≤5°, and they were chosen based on the following criteria: permanent occlusion with fully erupted second maxillary molars; sufficient posterior space for the molars, according to Ricketts et al.;19 the presence of third molar buds, which have erupted only to the level of the middle of the roots of the maxillary second molars; a well-aligned mandibular dental arch, with minimal crowding; normal or horizontal growth pattern; acceptable facial profile (not requiring modification); no history of previous orthodontic treatment; absence of extraction in the treatment plan; and adequate oral hygiene. After ensuring that the inclusion criteria were fulfilled, informed consent was obtained from the patients' parents after acquainting them with the aims of the research and the relevant procedures. A computer-generated randomization list was used to randomly divide the patients into 2 equal groups of 25 patients each. Group 1 was treated with the Frog appliance (Forestadent, Pforzheim, Germany) alone, while group 2 was treated with the Frog appliance in combination with high-pull headgear worn at night (Table 1, Figure 1). Both the patients and the researcher were aware of the allocation during treatment, but the researcher was kept blinded during data analysis, as described below. Distalizing appliances were not combined with any other appliances during this stage of the treatment. The Institutional Review Board approval was achieved for this research. BODY.MATERIALS AND METHODS.FROG AND HEADGEAR APPLIANCES: The Frog appliance kit consists of a special expansion screw that is activated anteriorly by using a special key. It also contains a prefabricated palatal arch that has been adapted to fit the posterior end of the expansion screw (Figure 2). To fix the appliance, wiry spurs were used on the maxillary first premolars, and bands were used on the maxillary second premolars. The parts of the appliance were gathered with an acrylic button based on the palate. The opening of the expansion screw pushes the palatal arch distally, leading to distalization of the maxillary molars (Figure 3). In group 2, besides the Frog appliance, face bows were applied to the buccal tubes of the maxillary first molar bands with high-pull headgear, which was only used at night (Figure 4). Treatment continued until Class I occlusion with an overcorrection of 2 mm was achieved (Figure 5). All appliances were then removed, and records were obtained immediately. BODY.MATERIALS AND METHODS.CEPHALOGRAM IMAGE ANALYSIS: Pre- and post-treatment lateral cephalograms were obtained in centric occlusion. Important skeletal and dental changes were studied. SPP (the line drawn between the anterior nasal spine and the posterior nasal spine; constructed X-axis) and PTV (the perpendicular line to Frankfurt plane through Ricketts' pterygoid point; constructed Y-axis) lines were drawn. The constructed X-axis was used to describe vertical and angular measurements, whereas the constructed Y-axis was used to describe sagittal measurements. The incisal edges of the central incisors and the central points of the crowns of the molars and premolars represented the middle points of the lines that connected the mesial and distal maximum convexities of the crowns (Figure 6). Angular changes were measured using teeth axes that were perpendicular to the lines that connected the mesial and distal maximum convexity points of the crowns (Figure 6). During cephalogram image analysis, the researcher was blinded to the identity of the patients, i.e., pre- and post-treatment cephalograms were analyzed after replacing the patients' names with codes. To determine the error associated with the method used for cephalogram image analysis, 10 randomly selected cephalograms were traced at least 1 month after the first tracing, and separate sheets were used to collect the new data. The total method error (ME) was calculated using the Dahlberg equation:20 ME = √Σd2/2n where d is the difference between the 2 measurements and n is the number of duplicate measurements. The ME never exceeded 0.4 mm or 0.5°. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: For determining the changes induced by the appliances in each group, we tested the null hypothesis, which suggested that there would be no significant changes associated with the use of the Frog appliance, nor with its combination with high-pull headgear. For determining the differences between the 2 groups, we tested the null hypothesis, which suggested that there would be no significant differences between the 2 treatments. All variables were obtained on pre-treatment cephalograms (T1) and post-treatment cephalograms (T2). The data were then entered into SPSS 17.0 (SPSS Inc., Chicago, IL, USA) to process pre-treatment variables, the treatment effects on the studied variables in each group, the relationship between the used appliance and the amount of changes in the studied variables, treatment time, distalization speed, and the ratio of maxillary molar distalization movement relative to the overall opening space between the first maxillary molars and second premolars. Paired-sample t-tests were used to determine the significant changes induced by each treatment, and 2-sample t-tests were used to compare the differences between the 2 treatments. All the results were judged at a 5% confidence level. BODY.RESULTS: First, we determined whether the 2 groups were similar at baseline. The 2-sample t-test showed that p-values were greater than 0.05 for all the studied variables, that is, at the 95% confidence level, there were no statistically significant differences in any variable before treatment (Table 2). These results indicated that the 2 groups were equivalent. Then, we used a paired-sample t-test to determine the effects of each type of treatment; we also employed a 2-sample t-test to compare treatment efficacy between the 2 groups (Table 3). The maxillary molars moved distally by 5.51 ± 2.56 mm and 5.93 ± 1.46 mm in groups 1 and 2, respectively. The change was significant in each of the 2 groups (pre-versus post-treatment), but there was no significant difference between the 2 groups. Molar distalization was associated with tipping of molar axes by 4.96 ± 1.41° and 1.25 ± 2.02° in groups 1 and 2, respectively. The tipping was only significant in group 1, and it was significantly larger in this group than in group 2 (p < 0.001). Mesial movement of the second premolars was 2.70 ± 1.37 mm and 0.90 ± 1.38 mm in groups 1 and 2, respectively. Although the change was significant in each of the 2 groups, mesial movement was significantly higher in group 1 than in group 2 (p = 0.008). Two-sample t-tests were used to study the differences in overall treatment time, speed of maxillary molar distalization, and the ratio of the maxillary molar distalization movement relative to the overall opening space between the molars and second premolars between the 2 groups (Table 4). Treatment time was significantly greater in group 1 than in group 2 (7.44 ± 1.30 months versus 6.27 ± 1.11 months; p < 0.001). The speed of maxillary molar distalization was also significantly lower in group 1 than in group 2 (0.68 ± 0.40 mm/month versus 0.86 ± 0.32 mm/month; p < 0.001). Finally, the ratio of the maxillary molar distalization movement relative to the overall opening space between the molars and second premolars was significantly lower in group 1 than in group 2 (70.74% ± 7.25% versus 90.97% ± 5.51%; p < 0.001). BODY.DISCUSSION: In this study, we showed that the intraoral Frog appliance is an effective tool for promoting maxillary molar distalization, even more so when used in combination with extraoral high-pull headgear. A review of the orthodontic literature reveals the lack of studies that have tested the efficiency of the Frog appliance, with only one case report published.21 Another study presented 2 case reports for the application of a modified Frog appliance, the so-called skeletal Frog appliance.16 No study has hitherto tested the effectiveness of the combined Frog appliance and headgear; this hinders the comparison of the present results with previously reported data. In this study, the Frog appliance - alone or in combination with headgear - significantly promoted the distalization of the maxillary molars. Our displacement values were similar to those obtained by Chiu et al.22 and Bussick and McNamara23 using the pendulum appliance (6.10 mm and 5.70 mm, respectively). However, our values were greater than those obtained by Bayram et al.21 with the Frog appliance (4.00 mm), Bondemark and Karlsson2 with headgear (1.00 mm), and Patel et al.24 with the Jones jig appliance (3.20 mm). These differences may be due to the longer duration of the present treatment as well as its endpoint (the attainment of overcorrection). Tipping of the molar axes using the Frog appliance alone was similar to that obtained by Chiu et al.22 with the distal jet (5.00°) and by Kinzinger et al.25 with the pendulum K (4.65°). Bussick and McNamara23 and Patel et al.24 achieved better tipping with the pendulum (10.60°) and the Jones jig (9.54°). These differences may be due to the differences in appliance design and in the point of force application relative to the center of resistance of the molars. However, the molar tipping found in the current study was greater than that in the studies by Bayram et al.21 and Bolla et al.1 (3.00° with the Frog appliance and 3.10° with the distal jet). In addition to the difference in appliance design, these differences may have resulted from differences in the extent of molar distalization. The amount of molar tipping associated with molar distalization was lesser in the combination group than in all previous studies. This may be due to the uprighting of the molars, accomplished by the high-pull headgear, which may have contributed to adjust distalization-associated tipping. Premolar mesial movement values found in the present study in the Frog appliance group were similar to those found by Chiu et al.22 with the distal jet (2.60 mm), and by Ghosh and Nanda12 with the pendulum (2.25 mm). However, the values were higher than those found by Chiu et al.22 with the pendulum (1.40 mm). These differences may be due to differences in the extent of molar movement, which is positively proportional to the amount of anchorage loss. The design of the appliances may also play a role. Premolar mesial movement was lesser in the group that underwent treatment with combined Frog appliance and headgear. This may be due to the influence of the headgear itself, which may adjust the mesial forces applied to the premolar teeth by the Frog appliance. However, in other studies that used only headgear to achieve molar distalization, the premolars moved distally, such as in the study by Brickman et al.26 with cervical pull headgear. Despite the lack of significant skeletal changes in the sagittal plane in both studied groups, some significant differences were noted between the 2 groups. A slight progress in point A and a non-significant increase in the ANB angle were observed in the Frog appliance group, while a slight decline in point A and in the ANB angle were observed in the combination group, leading to significant differences between the 2 groups. The associated skeletal changes in the vertical plane were evident in the posterior rotation of the mandible in the Frog appliance group; this resulted in significant differences in mandible position between the Frog appliance group and the Frog appliance plus headgear group. In the current study, the combination of the Frog appliance and the application of high-pull headgear at night was able to reduce the time required for maxillary molar distalization, with an average treatment time of 7.44 months and 6.27 months in the Frog appliance group and the combination group, respectively. These results are similar to those obtained by Ngantung et al.27 with the distal jet (6.70 months) and to those by Bussick and McNamara23 with the pendulum (7.00 months). However, the average treatment time was greater than that in the studies by Bolla et al.1 with the distal jet (5.00 months) or by Haydar and Uner3 with the Jones jig (2.50 months). These differences may be due to the greater molar movement observed in the current study and the differences in appliance design. BODY.CONCLUSION: The Frog appliance effectively distalized the maxillary molars in patients with Class II malocclusion and fully erupted second molars. This distalization was associated with an acceptable degree of loss of anchorage and with some unfavorable changes, such as axial tipping of maxillary molars, mesial movement of the anchor teeth, and backward rotation of the mandible. Nighttime application of high-pull headgear together with the Frog appliance can greatly reduce or even eliminate most of these unfavorable changes. The combined use of these 2 appliances can also reduce treatment time, increase the speed of distalization, and improve treatment outcomes by increasing the ratio of maxillary molar distalization movement relative to the overall opening space between the first maxillary molars and second maxillary premolars.

TITLE: High strength extrafine pMDI beclometasone/formoterol (200/6 μg) is effective in asthma patients not adequately controlled on medium-high dose of inhaled corticosteroids ABSTRACT.BACKGROUND: A high strength of beclomethasone/formoterol fumarate (BDP/FF) in a pressurised metered dose inhaler (pMDI), which contains extrafine BDP (200 μg/actuation) and FF (6 μg/actuation) has been developed to treat those asthmatics who are not adequately controlled on previous treatments. ABSTRACT.METHODS: A 12-week, randomized, double-blind, parallel group study was performed to compare the efficacy and safety of pMDI BDP/FF 200/6 (two actuations bid) with BDP 100 μg (four actuation bid) in a population of 376 randomized adult asthmatics not adequately controlled with high dose of inhaled corticosteroids (ICS) or medium dose of ICS plus long acting β2agonists (LABA). ABSTRACT.RESULTS: The primary endpoint [change from baseline over the entire treatment period in average pre-dose morning peak expiratory flow (PEF)] demonstrated the superiority of BDP/FF over BDP monotherapy, with an adjusted mean difference of 19 L/min, which is above the minimal important clinical difference reported for this parameter. Overall, BDP/FF and BDP showed a similar improvement of symptom-based parameters and of the use of rescue medication after 3-month treatment. The safety profile of the two drugs was comparable, although BDP monotherapy, but not BDP/FF, slightly reduced the levels of serum cortisol. ABSTRACT.CONCLUSIONS: The study proved that pMDI BDP/FF 200/6 μg was superior to BDP alone in improving lung function with comparable safety profiles. Therefore it may be considered as an effective treatment for adults with asthma not adequately controlled with high dose of ICS monotherapy or medium dose of ICS/LABA combinations. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov: NCT01577082, date 06/04/2012. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12890-016-0335-9) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: In spite of the availability of effective treatments, many people with asthma continue to suffer from significant symptoms and impaired lung function and not well controlled asthma remains a significant social and economic burden [1, 2]. International guidelines recommend a step-wise approach to treat asthma, which is linked to increasing severity of the condition and the need to achieve disease control. According to this approach, if asthma is not adequately controlled on current therapies, treatment should be stepped up until control is achieved. The preferred treatment at step 4 is to combine a medium/high dose of ICS + LABA when control cannot be achieved with a medium dose of ICS combined with LABA, or with a high dose of ICS (http://www.ginasthma.com/). A pressurised metered dose inhaler (pMDI) extrafine formulation containing a fixed combination of 100 μg BDP and 6 μg FF is currently available for the treatment of asthma where use of an ICS/LABA combination product is appropriate. With this combination, the usual recommended regimens are one or two inhalations twice daily [thus providing a medium total daily dose of ICS, according to The Global Initiative for Asthma (GINA) guidelines], and this schedule has been demonstrated as effective as other ICS/LABA combinations in improving symptoms and pulmonary function in asthmatics not adequately controlled with ICS monotherapy [3, 4] or in maintaining asthma control in patients already treated with ICS/LABA combination [5]. In the effort to provide caregivers more flexibility to adapt treatments to specific patients' condition, a high strength of extrafine pMDI BDP/FF containing a higher dose of BDP (200 μg/actuation) and the same dose of FF (6 μg/actuation) has been developed. This new fixed dose combination, like the medium strength (100/6 μg) is characterized by an extrafine [i.e. mean mass aerodynamic diameter (MMAD) 1.5 μm] formulation of both active ingredients. The dosing schedule of pMDI BDP/FF 200/6 μg is 2 actuations twice daily, for a total daily dose of 800 and 24 μg for BDP and FF, respectively. The aim of the present study was to compare the efficacy and safety of BDP/FF 200/6 μg at a daily dose of 800/24 μg with that of BDP monotherapy at a daily dose of 800 μg in asthmatic patients not adequately controlled on high dose of ICS or medium dose of ICS in ICS/LABA combinations. BODY.METHODS.PATIENTS: Asthmatics aged ≥18 years, with a forced expiratory volume in the first second (FEV1) ≥40 and <80% of predicted normal value and with a documented positive reversibility test, defined as change in FEV1 ≥ 12% and ≥200 mL over baseline within 30 min after administration of 400 μg of salbutamol, were enrolled. All patients had to be partly or not controlled (based on GINA asthma control parameters) and with an asthma control questionnaire (ACQ) > 0.75, despite previous treatment with high dose of ICS in monotherapy (BDP non-extrafine >1000 μg/day, or equivalent) or medium dose of ICS (BDP non-extrafine 500–1000 μg/day or equivalent) in combination with LABA. Patients were eligible for randomization if their asthma was still not fully controlled at the end of a 2-week run-in period. Patients were excluded if their asthma deteriorated, resulting in a change of asthma therapy in the 4 weeks before screening. The study was performed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. All institutions participating to the study were granted approval by their respective Ethics Committees and Competent Authorities (the complete list in the Additional file 1). All patients gave written informed consent for their participation in the study. BODY.METHODS.STUDY DESIGN: This was a phase III, multinational, multicentre, randomised, double-blind, double-dummy, active-control, 2-arm parallel group study designed to demonstrate the superiority of BDP/FF 200/6 μg pMDI extrafine (two puffs twice a day) vs. BDP 100 μg (four puffs twice a day) in change in pre-dose morning PEF. A total of 9 visits, 2 weeks apart from each other, were performed during the study: pre-screening visit (Visit 0), screening visit (Visit 1), randomisation visit (Visit 2) and visits at Weeks 2, 4, 6, 8, 10 and 12 (Visit 3 to 8). Screening visit was followed by a 2-week run-in period (open-label), during which the patients received extrafine BDP (800 μg/day). At the randomisation visit, patients were randomised to receive either BDP/FF (800/24 μg/day) or BDP (800 μg/day) for 12 weeks. Patients recorded daily, pre-dose morning and evening PEF, rescue and study medication use and asthma symptom (cough, wheeze, chest tightness and breathlessness) scores (score 0–3) on their electronic peak flow meter (In2itive, Vitalograph Ltd, UK); safety variables [treatment-emergent adverse events (TEAEs), adverse drug reactions (ADRs), heart rate (HR) and blood pressure (BP)] were assessed at each study visit. Compliance was evaluated on the basis of the information recorded daily by the patient in the electronic peak flow meter, dividing the total number of doses by the number of scheduled doses. A range 75–125% was considered as a satisfactory level of compliance. The sponsor (Chiesi) developed the protocol, with guidance from the other academic authors. The first and second authors wrote the first draft of the manuscript. All the authors reviewed and edited the manuscript and made the decision to submit the manuscript for publication. All the authors contributed to the interpretation of the data and had access to the full data. BODY.METHODS.EFFICACY ASSESSMENTS: PEF measurements were performed at approximately the same time every day and before the intake of run-in or study medication; an alarm reminded the patients to perform measurements. During each measurement session, a series of messages were displayed on the device to guide the patient and ensure a high quality test as follows: i) "Blow-out faster": if the time to PEF was >120 msec; ii) "Do not cough": if a cough was detected in the first second of the test; iii) "Blow again": if the test was not within 40 L/min of the best PEF (to ensure repeatability). The quality criteria were based on the American Thoracic Society/European Respiratory Society (ERS) guidelines for standardisation of spirometry [6]. At each visit, data were downloaded and reviewed by the Investigator, who verified the correct use of the device, PEF values and checked patient's compliance. Spirometry was performed at all clinic visits at approximately the same time of day, in accordance with the ERS recommendations [6]. All sites were provided with the same spirometer and a specific procedure for centralised spirometric reading was applied. A moderate/severe asthma exacerbation was defined as a significant deterioration of asthma and signalled by one or more of the following: i) need for an oral/parenteral corticosteroid course; ii) unscheduled medical visit for an asthma exacerbation; iii) hospitalisation or emergency room attendance for asthma. BODY.METHODS.SAFETY ASSESSMENTS: The safety of all treatments was assessed over the entire treatment period. Electrocardiogram (ECG), haematology and blood chemistry measurements were assessed at study entry and at weeks 12. A subset of 15% of randomised patients underwent cortisol assessment. Serum cortisol was collected at randomization and at week 12 and the corresponding area under the curve (AUC)0-24h, was assessed by a central laboratory. BODY.METHODS.STATISTICAL ANALYSIS: The primary efficacy variable of this study was the change from baseline to the entire treatment period in average pre-dose morning PEF. Baseline was calculated as the mean of the pre-dose morning PEF measurements recorded during the run-in period. Comparison between groups was performed using an analysis of covariance (ANCOVA) model with change from baseline to the entire treatment period in average pre-dose morning PEF as dependent variable and treatment, country and gender as factors and baseline as a covariate. The sample size was calculated to demonstrate the superiority of BDP/FF over BDP in the primary efficacy variable. Assuming a mean difference of 15 L/min between treatments and a standard deviation of 40 L/min, it was calculated that 151 evaluable patients per treatment group would assure 90% power to demonstrate the superiority, with a two-side significance level of 0.050. Estimating a non-evaluable rate of 20%, a total of about 378 patients were required to be randomised. For all the endpoints measured repeatedly during the randomised treatment by period, the change from baseline has been analysed by a Multiple Repeated Measurement Model (MRMM) including treatment, country, gender, visit/period, treatment by visit/period interaction as fixed effects and baseline and baseline by visit/period interaction as covariates. When the endpoint was a measure of the entire treatment period, the change from baseline was analysed using the same ANCOVA analysis applied for the primary endpoint. The primary analysis has been performed in the intention to treat (ITT) population (all randomised patients who received at least one administration of the study drug and with at least one available evaluation of efficacy after baseline) and the per-protocol (PP) population (all patients from the ITT population without any major protocol deviations), while the secondary endpoints were analysed in the ITT population only. A post-hoc analysis of the change from baseline to each visit and over the treatment period in pre-dose morning FEV1 was also performed considering the reversibility before randomisation [change in FEV1 (mL) from pre-salbutamol] as factor. Secondary endpoints were: evening pre-dose PEF, PEF daily variability, pre-dose clinic FEV1, rescue medication use, day-time and night-time symptom score, asthma control days percentage, ACQ and moderate/severe exacerbation rate. Daily PEF variability was calculated for each day using the following formula: 100x [(best PEF evening-best PEF morning)/(best PEF evening + best PEF morning)/2]. Safety variables have been descriptively analysed. Cortisol assessments have been log-transformed and the change from baseline analysed as ratio and 95% CI. BODY.RESULTS.PATIENT’S CHARACTERISTICS: A total of 542 patients was screened, of whom 376 were randomised to receive BDP/FF (n = 192) or BDP (n = 184). The main reason for screening failure was violation of inclusion or exclusion criteria (n = 142). The majority of patients completed the study (178 [92.7%] and 164 [89.1%] in the BDP/FF group and BDP group, respectively). Fourteen (7.3%) patients in the BDP/FF group and 20 (10.9%) patients in the BDP group discontinued the study mainly due to protocol violations (Fig. 1).Fig. 1Patients' disposition The demographics and clinical characteristics were similar in the two treatment groups (Table 1). In both treatment groups, there was a prevalence of females. The mean compliance to the study drug was similar in the two treatment groups was above 92%.Table 1Demographic and clinical characteristics - ITT populationBDP/FF N = 184BDP N = 175Age (years)Mean (SD)49.5 (13.7)49.1 (14.1)Gender n (%)Male84 (45.7)63 (36)Female100 (54.3)112 (64)BMI (kg/m2)Mean (SD)26.8 (4.6)27.1 (5.2)Smoking n (%)Non-smoker151 (82.1)147 (84)Ex-smoker33 (17.9)28 (16)Therapy at study entry n (%)ICS alone16 (8.7)15 (8.6)ICS/LABA168 (91.3)160 (91.4)FEV1 (L)a Mean (SD)2.1 (0.6)1.9 (0.5)FEV1 % of pred. normal valuea Mean (SD)64.7 (8.1)64.3 (9.5)Reversibility: FEV1 change (L)b Mean (SD)0.53 (0.3)0.56 (0.3)Reversibility: FEV1 % changeb Mean (SD)27.7 (15.7)30.2 (19.3)PEFa Mean (SD)310.4 (107.6)312.6 (102.6)ACQa Mean (SD)2.12 (0.6)2.12 (0.6) aMeasured at randomization, bMeasured at screening BODY.RESULTS.EFFICACY: In the ITT population pre-morning PEF (assessed during run in period) was similar in the two treatment groups [310.4 (108)] L/min in the BDP/FF group and [313 (103)] L/min in the BDP group) (Table 1). In terms of adjusted mean change of PEF from baseline over the entire study, treatment period was positive in the BDP/FF group and unchanged in the BDP group (18 L/min [95% CI: 12, 24; p < 0.001] and -1 L/min [95% CI: -7, 5; p = 0.781], respectively) (Fig. 2). The difference in the adjusted mean change from baseline between the two treatment groups was statistically significant in favour of the BDP/FF group (19 L/min, 95% CI: 10, 27; p < 0.001), indicating superiority of BDP/FF treatment vs. BDP (Table 2). Similar differences were also observed for evening PEF (data not shown).Fig. 2Change from baseline in average pre-dose morning PEF (L/min) [95% CI] – ITT population Table 2Pre-dose morning PEF (L/min) - Change from baseline to the entire treatment periodITT populationBDP/FF N = 184BDP N = 175Change from baseline to the entire treatment periodn182170Adjusted mean (95% CI)17.63 (11.58, 23.68)-0.90 (-7.26, 5.46)Adjusted mean difference BDP/FF vs. BDP (95% CI), p-value18.53 (10.33, 26.73), < 0.001PP populationBDP/FF N = 176BDP N = 164Change from baseline to the entire treatment periodn176163Adjusted mean (95% CI)17.85 (11.60, 24.10),-0.21 (-6.76, 6.35)Adjusted mean difference BDP/FF vs. BDP (95% CI), p-value18.06 (9.63, 26.49), < 0.001 The mean daily PEF variability, at the end of run in period, was similar in BDP/FF and BDP groups (11.6 and 12.1%, respectively) and both treatments induced a reduction in PEF variability during the study, although statistically significant in BDP/FF only. In addition, the adjusted mean difference between treatment groups in daily PEF variability was statistically significant in favour of the BDP/FF group over the entire treatment period (p = 0.010). Both treatments showed a statistically significant increase in pre-dose morning FEV1 from baseline to each clinic visit and over the entire treatment period (p < 0.001) with a difference of 0.071 L in favour of BDP/FF group (p = 0.058). When reversibility test results were added as covariate (post-hoc analysis), the magnitude of the FEV1 difference between groups were even larger 0.087 L (p = 0.016) in favour of BDP/FF (Fig. 3).Fig. 3Change from baseline in pre-dose morning FEV1 (L) [95% CI] – Post-hoc analysis - ITT population The mean average use of rescue medication was similar in the BDP/FF and BDP groups at baseline (2.7 and 2.9 puffs/day respectively) and decreased significantly in both groups at the end of the treatment period (1.4 and 1.7 puffs/day respectively; p < 0.001 vs baseline). There were no statistically significant differences between the adjusted mean changes in use of rescue medication across treatment groups at each inter-visit period (p-value ranging from 0.051 to 0.586) and over the entire treatment period (p = 0.143). The mean number of rescue-use free days increased significantly both with BDP/FF (from 33.0 at baseline to 54.5 at end of treatment; p < 0.001) and BDP (from 29.4 at baseline to 50.4 at end of treatment; p < 0.001) with no statistically significant differences between the two groups. The mean total day-time and night-time asthma symptom scores at baseline were similar in the BDP/FF and BDP groups (day-time asthma symptom scores: 4.03 and 4.08, respectively; night-time asthma symptom scores: 3.75 and 3.67, respectively). There were no statistically significant differences between the adjusted mean changes across treatment groups at each inter-visit period and over the entire treatment period. The percentage of asthma symptom free days increased from 5.2 to 15.3 with BDP/FF and from 4.7 to 16.1 with BDP (p < 0.001 vs baseline for both groups). Similarly, the percentage of asthma control days increased from 4.6 to 15.0 with BDP/FF and from 4.4 to 15.4 with BDP (p < 0.001 vs baseline for both groups). No difference was observed between groups. The percentage of asthma control days was similar in the BDP/FF and BDP groups at baseline (4.64% and 4.39%, respectively). The ACQ scores were similar in the BDP/FF and BDP at baseline (2.12 and 2.16, respectively). Both treatments resulted in a statistically significant reduction in ACQ score from baseline to Week12 (p < 0.001), above the minimal clinical difference of 0.5 points. The extent of the reduction was similar across treatments. The number of patients who experienced asthma exacerbations and the corresponding number of exacerbations were slightly lower in the BDP/FF group than in the BDP group: 4 (2.2%) patients with 4 exacerbations vs. 6 (3.4%) patients with 7 exacerbations. BODY.RESULTS.SAFETY ASSESSMENTS: Overall, TEAEs were reported with similar frequency in the two treatment groups: 35 TEAEs in 29 (15.3%) patients in the CHF 1535 group and 40 TEAEs in 30 (16.7%) patients in the BDP group. Treatment-emergent ADRs were reported slightly less frequently in the CHF 1535 group than in the BDP group: 3 events in 2 (1.1%) patients vs. 5 events in 5 (2.8%) patients. Table 3 presents the ADRs reported during the study, in decreasing order of frequency.Table 3Summary of Adverse Drug Reactions (ADR)BDP/FF N = 189BDP N = 180Number of patients (%)Number of eventsNumber of patients (%)Number of eventsAny ADR2 (1.1)35 (2.8)5Respiratory, thoracic and mediastinal disorders0 (0.0)03 (1.7)3Asthma0 (0.0)01 (0.6)1Dysphonia0 (0.0)01 (0.6)1Throat irritation0 (0.0)01 (0.6)1Skin and subcutaneous tissue disorders1 (0.5)11 (0.6)1Acne1 (0.5)10 (0.0)0Dermatitis contact0 (0.0)01 (0.6)1Gastrointestinal disorders0 (0.0)01 (0.6)1Nausea0 (0.0)01 (0.6)1General disorders and administration site conditions1 (0.5)10 (0.0)0Chest discomfort1 (0.5)10 (0.0)0Infections and infestations1 (0.5)10 (0.0)0Oral candidiasis1 (0.5)10 (0.0)0 Overall, in the BDP/FF group, serum cortisol levels remained stable from baseline to the end of the treatment period (geometric mean ratios [95% CI] for change from baseline: 1.0 [0.9, 1.2] for both AUC0-24h and minimum plasma concentration (Cmin) [0.7, 1.5]), while they decreased in the BDP group (geometric mean ratios [95% CI] for change from baseline: 0.8 [0.7, 0.9] for AUC0-24h and 0.6 [0.4, 0.7] for Cmin (Fig. 4).Fig. 4Log-transformed 24–hour serum cortisol ratio (a) and Cmin serum cortisol ratio (b) to baseline for end of treatment period No clinically significant abnormalities in haematology or blood chemistry parameters were reported during the study. With the exception of 1 patient in the BDP group, for whom the ECG reading was abnormal at the end of the treatment period, ECG readings were considered normal or not clinically significant abnormal. Overall, QTc abnormalities were infrequent and observed in a similar percentage of patients in the two treatment groups (change from screening in QTcF between 30 and 60 msec observed for 7 (3.7%) and 7 (3.9%) patients in the BDP/FF and BDP groups, respectively). BODY.DISCUSSION: The primary efficacy analysis showed that BDP/FF was superior to BDP in terms of change from baseline to the entire treatment period in average pre-dose morning PEF; the target population was a group of patients with party or uncontrolled asthma despite their medium dose of ICS/LABA or high dose of ICS, which could benefit from a step up pharmacological approach. The mean difference vs. BDP was 19 L/min, which is in line with the mean value reported in the reviews of Li et al. [7] (17.9 L/min) and Cochrane [8] (19.6 L/min) and with the minimal patient perceivable improvement differences (MCID) of 18.8 L.min-1 which was reported by Santanello et al [9] in a similar asthma population. The observed difference in morning PEF between BDP/FF and BDP is consistent with that observed in clinical studies when fluticasone/salmeterol high dose (500/50 μg bid) was compared to high dose of fluticasone (500 μg bid) as observed by Aubier et al [10], Boyd et al [11] and Van Noord et al [12] (22, 21 and 23 L/min, respectively). This is due to the fact that a dose of 50 μg salmeterol twice daily (as in fluticasone/salmeterol combination) is comparable to 12 μg formoterol twice daily [13, 14]. For the budesonide/formoterol (combination, the difference in morning PEF between high dose budesonide/formoterol and high dose of budesonide observed by Jenkins et al [15] and Peters et al [16] were higher than those observed in this study (33 and 34 L/min, respectively). However, the total daily dose of formoterol which is used in high strength budesonide/formoterol was 48 μg, that is twice than that used in BDP/FF and dose-response studies have shown that the degree of bronchodilation is related to the dose of inhaled FF [17]. Therefore, data from high strength budesonide/formoterol are difficult to be compared with BDP/FF and may explain the larger differences in morning PEF which have been reported by budesonide/formoterol studies. Importantly, it should be noted that in the present trial the vast majority of patients were already on a fixed dose ICS/LABA combination at screening (i.e. > 90%, see Table 1) which is significantly more than in similar studies with other combinations, and this might have reduced the room for improvement. Overall, compared to BDP, BDP/FF resulted in a greater and statistically significant improvement of other lung function parameters: average pre-dose morning PEF (from baseline to each inter-visit period), average pre-dose evening PEF and daily PEF variability. In addition, treatment with BDP/FF showed an improvement in FEV1 from baseline to each visit and throughout the treatment period compared to BDP with an increase in FEV1 at the end of the study period which was more than 200 ml, a cut off used to define airway reversibility [18]. There was a difference of 0.071 L in favour of BDP/FF very close to statistical significance (p = 0.058) and this result is in line with previous studies comparing the superiority of fixed combination vs. ICS in patients with asthma ranging in severity from mild to moderate to severe [19]. It is interesting to note that after using FEV1 reversibility to salbutamol as covariate in the statistical model, the change from baseline to the overall treatment period was statistically significant and the observed difference of ~0.09 L is the same as the mean value reported in the review of Lin et al [7] and also by some budesonide/formoterol data [20]. Overall, BDP/FF and BDP resulted in a similar improvement of symptom-based parameters and of the use of rescue medication. Again, these results were in part expected when looking at high dose fluticasone/salmeterol data. Aubier et al [10] showed that combination therapy and fluticasone alone all increased the mean percentage symptom-free days, symptom-free nights, rescue-free days and rescue-free nights over weeks 1–12. However, there was no significant difference between the combination and concurrent therapies for any of these measurements over 12 weeks. Our results are in line also with the data from mometasone/FF high strength. Weinstein et al. performed a study in patients who were uncontrolled with high dose of ICS [21]. At 12 weeks, the difference between mometasone 400 μg /FF 10 μg bid and mometasone 400 μg bid in ACQ was less than 0.5, thus not clinically relevant. At week 12, there were no significant between-group differences in the mean change from baseline in AQLQ(S) total score. A possible further explanation of the similar magnitudes of asthma control between treatments may be the highly uncontrolled disease in this cohort, with the high ACQ scores. BDP/FF and BDP showed a comparable safety profile, with no adverse events of clinical concern observed with either treatment. Overall, TEAEs and treatment-emergent ADRs were reported with low frequency in both treatment groups. In addition, all events were mild or moderate and the large majority resolved by the end of the study. Only one TEAE leading to discontinuation was reported during the study (asthma exacerbation in the BDP group which was not related to the study drug); that event was considered as not related to the study drug and resolved by the end of the study. Of note, no serious TEAEs, serious treatment-emergent ADRs or TEAEs leading to death were reported during the study. BODY.CONCLUSIONS: In conclusion, the study successfully demonstrated its primary objective i.e. that BDP/FF is superior to BDP alone in improving lung function in uncontrolled asthmatic patients with an acceptable safety profile. Because pulmonary function is still considered the primary outcome of all short-term studies assessing the efficacy of bronchodilator treatment in asthma, this study confirm that this new extrafine BDP/FF combination may represent a useful option in the management of moderate/severe asthma.

TITLE: Crystalloid and colloid preload for maintaining cardiac output in elderly patients undergoing total hip replacement under spinal anesthesia ABSTRACT: The aim of the present study was to compare the effects of colloid and crystalloid preload on cardiac output (CO) and incidence of hypotension in elderly patients under spinal anesthesia (SA). A randomized, double-blinded study was conducted including 47 elderly patients undergoing scheduled total hip replacement (THR), who were randomized to three groups: the control group (C group, n = 15), crystalloid (RS group, n =16) and colloid group (HES group, n = 16). An intravenous preload of 8 mL/kg of either lactated Ringer's solution in the RS group or 6% hydroxyethyl starch in the HES group was infused within 20 min before SA induction, while no intravenous preload was given in the C group. There was a trend of decrease in CO and systolic blood pressure after SA with time in the C group. In the RS and HES groups, CO increased significantly after fluid preloading as compared with baseline (P < 0.01). Thereafter, CO remained higher than baseline until 30 min after SA in the HES group. The change of systolic blood pressure was similar to CO, but no significant difference from baseline was observed in each group. Hypotension occurred in 3 patients in the C group and one each in the RS and HES group, respectively (P = 0.362). Intravascular volume preload with colloid is more effective than crystalloid solution in maintaining CO, which may be improved the hemodynamic stability in elderly patients during SA. BODY.INTRODUCTION: Hip fracture is a common disease in the elderly and often requires surgical treatment such as total hip replacement[1],[2]. However, there are predictable physiological and metabolic changes in elderly patients that limit their ability to respond to surgical and anesthetic stress. Furthermore, comorbidities such as cardiovascular disease increase with age and may contribute to perioperative adverse events and mortality[3]–[5]. The selection of anesthesia for total hip replacement can either be general or regional anesthesia. Studies have shown that regional anesthesia for total hip replacement results in better postoperative outcomes, including improved respiratory function, less nausea and vomiting, less pain, and lower incidence of deep vein thrombosis[6]–[8]. Among regional anesthetic techniques, spinal anesthesia has become the preferred technique because it offers a fast, profound, and high quality sensory and motor block with reduced risk of local anesthetic toxicity. However, hypotension remains the most common complication after spinal anesthesia and may increase the risk of myocardial ischaemia, especially in the elderly patients[3],[9],[10]. To prevent spinal anesthesia-induced hypotension, intravenous fluid preload is commonly used. However, based on previous studies in which only blood pressure and heart rate were used to assess the efficacy of fluid preload, the type and amount of fluid preload for preventing spinal hypotension are still controversial[11]-[13]. On the other hand, the true goal of hemodynamic management during anesthesia is to maintain tissue perfusion. Cardiac output has been documented to be a better indicator of cardiac function and tissue perfusion than arterial blood pressure[14]. Thus, a better understanding of cardiac output changes during spinal anesthesia would help improve hemodynamic stability in elderly patients. The aim of this randomized controlled study was to compare the effects of crystalloid and colloid preload on cardiac output (CO) during spinal anesthesia (SA) in elderly patients undergoing scheduled total hip replacement using the FloTrac/Vigileo system. BODY.MATERIALS AND METHODS.PATIENTS: The study protocol was approved by the medical ethics committee of Nanjing First Hospital and a written informed consent was obtained from all study participants. We recruited a total of 48 American Society of Anesthesiologists (ASA) physical status I-III patients (age > 60 years) scheduled for total hip replacement under SA between September 2009 and June 2010 at Nanjing First Hospital. Exclusion criteria included congestive heart failure (NYHA III-IV), unstable angina, significant aortic stenosis, recent myocardial infarction or severe cardiac arrhythmia, and contraindications to spinal anesthesia. Patients were randomly allocated to one of three groups, the control (C group), the crystalloid (RS group) and the colloid group (HES group) by computer-generated codes that were kept in sealed, numbered envelopes. BODY.MATERIALS AND METHODS.ANESTHESIA: Patients were premedicated with intramuscular administration of luminal sodium 0.1 g and atropine 0.5 mg. A 20-gauge intravenous catheter was inserted into the forearm vein, and an intravenous preload of 8 mL/kg of either lactated Ringer's solution in the RS group or hydroxyethyl starch solution (6% HES 130/0.4; Voluven, Fresenius Kabi, Homburg, Germany) in the HES group was infused over 20 min before SA while no fluid preload was given in the C group. Thereafter, all patients received intravenous lactated Ringer's solution at approximately 2 mL/(kg·h) except for the additional volume for blood loss during the operation. Once preloading was completed, spinal anesthesia was performed at the L3–L4 or L4–L5 intervertebral space using a 25-gauge pencil-point needle with the patient placed in the lateral position. After clear, free flow of the cerebrospinal fluid was obtained, 2 mL hyperbaric 0.5% bupivacaine (10 mg) was injected intrathecally over 10 s. The patient was then immediately turned to the supine position. The upper sensory block level was measured by assessing loss of pinprick discrimination until the level of sensory block was satisfactory for the operation. BODY.MATERIALS AND METHODS.MEASUREMENTS: A 20-gauge intraarterial line was inserted into the radial artery for continuous monitoring of the cardiac output, systolic blood pressure and heart rate. The FloTrac/Vigileo system was connected to the arterial line, and the transducer was adjusted to the level of the left atrium according to the instructions of the manufacturer[15]. The relevant variables were recorded before fluid preloading (baseline, T1), immediately after preloading completion (T2), after spinal anesthesia reached a peak sensory block of Th9 (T3), 15 min (T4), 30 min (T5), and 45 min (T6) post spinal block, and the end of operation (T7), respectively. Hypotension was defined as a decrease in systolic blood pressure to <90 mm Hg or <75% of the baseline value, and was treated with an intravenous bolus of ephedrine (6 mg). Bolus was repeated if needed. Bradycardia was defined as heart rate <50 beats/min and was treated with intravenous atropine (0.5 mg). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Data were expressed as mean±SD or number (percentage). The primary outcome variable was CO change. Sample size calculations indicated that inclusion of 15 patients per group would have 80% power at the 5% significance level to detect a difference in CO of 1.0 L/min between groups, assuming an SD of CO of 1.1 L/min (obtained from a pilot study). Secondary outcome variable was the incidence of hypotension. Continuous variables were compared with one-way ANOVA. Hemodynamic values over time were compared using analysis of variance for repeated measures. Post-hoc testing was performed with the Bonferroni method. Categorical variables were compared with the χ2 or Fisher exact test as appropriate. All analysis was performed using SPSS (Version 13.0) for Windows statistical software (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant. BODY.RESULTS: One patient in the control group was excluded from the study because of inadequate spinal anesthesia and required general anesthesia. Patient demographic characteristics were similar among the groups (P > 0.05). The main comorbid disease was hypertension in 9 patients in the control group, 6 in the RS group and 8 in the HES group (P > 0.05, Table 1). Table 1 Patient characteristics and baseline hemodynamic data Parameters Control ( n =15) HES ( n =16) RS ( n =16) Age(years) 72 ± 7 69 ± 8 71 ± 7 Weight(kg) 61 ± 7 62 ± 7 64 ± 8 Height(cm) 159 ± 9 161 ± 8 161 ± 7 Sex(M/F) 4/12 4/12 5/11 Hypertension, [ n (%)] 9(60) 8(50) 6(38) HR(beats/min) 79 ± 13 74 ± 9 76 ± 11 SBP(mmHg) 141.9 ± 15.4 144.9 ± 17.9 139.3 ± 17.0 CO(L/min) 5.3 ± 0.8 5.2 ± 0.6 4.9 ± 0.8 Values are mean±SD or number (percentage). CO: cardiac output; HES: the colloid group; HR: heart rate; RS: the crystalloid group; SBP: systolic blood pressure. Changes in the cardiac output, systolic blood pressure and heart rate over time are shown in Table 2 and Fig. 1-3. Baseline hemodynamic variables were comparable among the groups (P > 0.05). Overall, there was a trend toward gradual decrease in cardiac output in the control group, while a trend toward an initial increase and a subsequent decrease in the cardiac output in both the RS and HES group. Repeated-measures ANOVA showed that the cardiac output at T2 was significantly higher than baseline value (T1) in both the RS and HES group (P < 0.01). The cardiac output remained significantly higher than baseline until 30 min after induction of spinal anesthesia in the HES group. In contrast, the CO was not different from baseline at any time during spinal anesthesia in the RS group. Intergroup comparison showed that the CO in the HES group was significantly higher than that in the RS and C groups at the corresponding time point during spinal anesthesia, but no significant difference existed between the RS and C groups (Fig. 1). Table 2 Mean cardiac output in the three groups at different time-points Group Tl T2 T3 T4 T5 T6 T7 Control 5.3 ± 0.9 5.2 ± 0.7 4.7 ± 0.8 4.6±0.8 4.3 ± 0.6 4.2 ± 0.7 4.2 ± 0.8 RS 4.9 ± 0.8 5.5 ± 0.6* 5.0±0.8 4.8±0.7 4.6 ± 0.6 4.6 ± 0.9 4.4±0.7 HES 5.2 ± 0.6 5.9 ± 0.7* 5.6±0.8* 5.5±0.8* 5.5 ± 0.7* 5.3 ± 0.7 5.2 ± 0.7 HES: the colloid group; RS: the crystalloid group; T1: before fluid administration; T2: after fluid administration; T3: after spinal anesthesia reached a peak sensory block of Th9; T4: 15 min after spinal block; T5: 30 min after spinal block; T6: 45 min after spinal block; T7: the end of surgery. * P < 0.01 compared with T1. (mean±SD) Fig.1Cardiac output (CO) changes over time in control patients (C) and in patients who received an intravenous preload of 8 mL/kg of either lactated Ringer's solution (RS) or hydroxyethyl starch solution (HES). A change trend similar to that of the cardiac output was found with systolic blood pressure. However, there were no significant differences from baseline during spinal anesthesia within each group, or at the corresponding time points among three groups (Fig. 2). Hypotension occurred in 3 patients in the C group and one each in the RS group and HES group, respectively (P = 0.362). In addition, there was a nonsignificant trend toward decrease in the heart rate after spinal anesthesia in all patients, with no significant difference among the three groups (Fig. 3). Fig. 2Systolic blood pressure (SBP) changes over time in control patients (C) and in patients who received an intravenous preload of 8 mL/kg of either lactated Ringer's solution (RS) or hydroxyethyl starch solution (HES). Fig. 3Heart rate changes over time in control patients (C) and in patients who received an intravenous preload of 8 mL/kg of either lactated Ringer's solution (RS) or hydroxyethyl starch solution (HES). BODY.DISCUSSION: The present study showed that fluid preload alleviated the trend toward decrease in the cardiac output and systolic blood pressure after induction of spinal anesthesia in elderly patients. Furthermore, this effect was more evident in patients who received colloid as preload than those who received crystalloid solution. Total hip replacement is usually performed in elderly patients who often have functional limitations and concomitant diseases, which compromise cardiovascular stability, leading to decreased organ perfusion and subsequently to organ dysfunction or cardiovascular complications[16]. Although routine blood pressure and heart rate measurements are of value in assessing the safety of anesthetic technique, cardiac output changes may be more important in terms of the goal of maintaining organ perfusion because the change in cardiac output has been shown to correlate better with tissue blood flow than blood pressure[17]–[20]. In addition, an increased systemic vascular resistance (SVR) in elderly patients could also render blood pressure a poor indicator of cardiac output and organ flow[21]. We therefore decided to investigate the effects of fluid preload on cardiac output changes associated with spinal anesthesia in elderly patients in this study. Measurements of CO have previously been assessed by thermodilution, dye dilution, or Doppler ultrasound. These methods, however, may bring certain disadvantages with regard to their use during anesthesia. For example, thermodilution or dye dilution techniques require catheterization of the pulmonary artery, which may incur serious complications and limit their routine use[22]. Transthoracic Doppler echocardiography is operator-dependent and time-consuming and cannot be used for continuous measurements[23]. On the other hand, the FloTrac/Vigileo system, which is based on the standard deviation of the pulse pressure waveform, is minimally invasive, can be easily applied and may provide continuous real-time cardiac output data. Studies have confirmed a good agreement between continuous cardiac output measurements using FloTrac/Vigileo system with other methods including the "gold standard" pulmonary artery thermodilution in numerous clinical situations[24]–[27]. Therefore, the use of FloTrac/Vigileo system offers the occasion to conveniently detect immediate cardiac output changes and then improves hemodynamic management in elderly patients during anesthesia. Hypotension results in inadequate organ perfusion and can ultimately end with the loss of consciousness and cardiovascular collapse, particularly in elderly patients. Induction of spinal anesthesia induces sympathetic block, which causes blood pressure to fall as a result of decreased systemic vascular resistance and cardiac output, the latter being secondary to reduced venous return and sometimes decreased heart rate[21]. Our results also reflected this tendency (Fig. 2). Therefore, optimizing cardiac preload may be fundamental to preventing spinal hypotension and organ dysfunction in elderly patients. However, to date, there are many literatures debating the type and amount of fluid preload[11]–[13]. The present study showed that a fluid preload of 8 mL/kg increased cardiac output. The increased cardiac output should be attributed to increase in stroke volume (i.e., cardiac preload) because heart rate did not increase corresponding at the same time. Furthermore, in accordance with a previous study[28], colloid preload was more effective than crystalloids for maintaining cardiac output. This is not surprising because colloid increases more intravascular volume and stays in the vascular space longer due to the larger molecular weight compared with crystalloid. As a result, blood pressure tended to decrease less in patients receiving colloid preload, although it did not reach statistical significance. Of note, the incidence of hypotension was similar among the three groups in this study. Other studies have shown that fluid preload reduces the incidence of spinal-related hypotension[29]. However, the lack of significance in the incidence of hypotension may be the result of a type II statistical error due to the small sample in this study. Additionally, an alternative explanation may be the relatively lower volume of fluid preload in our elderly patients as compared with younger patients in other studies (8 mL/kg vs 15-20 mL/kg)[12],[30]. The volume of 8 mL/kg for fluid preload chosen in this study was mainly based on the clinical concern that more fluid preloading may put the elderly at increased risk of cardiovascular complications and pulmonary oedema[9]. A further larger prospective study then is needed to better clarify the efficacy of fluid preloading and to evaluate the optimal dose for the prevention of spinal hypotension. However, despite colloid preload at the volume studied did not significantly prevent spinal hypotension, the increase in CO should be beneficial for elderly patients during anesthesia as discussed above. In conclusion, our data suggest that use of moderate volume of colloid preload is more effective than crystalloids in maintaining the CO and hemodynamic stability in elderly patients.

TITLE: Analgesic efficacy of transversus abdominis plane block in providing effective perioperative analgesia in patients undergoing total abdominal hysterectomy: A randomized controlled trial ABSTRACT.BACKGROUND AND AIMS:: Transversus abdominis plane (TAP) block has been shown to provide postoperative pain relief following various abdominal and inguinal surgeries, but few studies have evaluated its analgesic efficacy for intraoperative analgesia. We evaluated the efficacy of TAP block in providing effective perioperative analgesia in total abdominal hysterectomy in a randomized double-blind controlled clinical trial. ABSTRACT.MATERIALS AND METHODS:: A total of 90 adult female patients American Society of Anesthesiologists physical status I or II were randomized to Group B (n = 45) receiving TAP block with 0.25% bupivacaine and Group N (n = 45) with normal saline followed by general anesthesia. Hemodynamic responses to surgical incision and intraoperative fentanyl consumption were noted. Visual analog scale (VAS) scores were assessed on the emergence, at 1, 2, 3, 4, 5, 6 and 24 h. Time to first rescue analgesic (when VAS ≥4 cm or on demand), duration of postoperative analgesia, incidence of postoperative nausea-vomiting were also noted. ABSTRACT.RESULTS:: Pulse rate (95.9 ± 11.2 bpm vs. 102.9 ± 8.8 bpm, P = 0.001) systolic and diastolic BP were significantly higher in Group N. Median intraoperative fentanyl requirement was significantly higher in Group N (81 mcg vs. 114 mcg, P = 0.000). VAS scores on emergence at rest (median VAS 3 mm vs 27 mm), with activity (median 8 mm vs. 35 mm) were significantly lower in Group B. Median duration of analgesia was significantly higher in Group B (290 min vs. 16 min, P = 0.000). No complication or opioid related side effect attributed to TAP block were noted in any patient. ABSTRACT.CONCLUSION:: Preincisional TAP block decreases intraoperative fentanyl requirements, prevents hemodynamic responses to surgical stimuli and provides effective postoperative analgesia. BODY.INTRODUCTION: Transversus abdominis plane (TAP) block is a widely practiced peripheral nerve block, utilized to anesthetize the somatic nerves supplying the anterior abdominal wall by depositing local anesthetic in the neurovascular plane between internal oblique and transversus abdominis muscle layers. It was introduced in anesthesia practice in 2001 by Rafi utilizing the traditional anatomical land marks.[1] TAP block has subsequently been used as a component of multimodal analgesia for postoperative pain relief following various surgical procedures such as large bowel resection,[2] open appendectomy,[3] retropubic prostatectomy,[4] nephrectomy,[5] hernia repair,[6] laparoscopic cholecystectomy[78] and cesarean section.[9] Although Carney et al.[10] and Atim et al.[11] have observed analgesic benefit of TAP block in total abdominal hysterectomy by landmark based approach and ultrasound guided (USG) approach respectively, Griffith et al. found that TAP block does not confer any definite analgesic benefit in major gynecological procedures[12] over a multimodal analgesic regimen. Furthermore, the effect of preincisional TAP block on intraoperative as well as postoperative analgesia in patients undergoing total abdominal hysterectomy remains yet to be elucidated. Based on these observations, this study was conceptualized to elucidate the efficacy of bilateral preincisional TAP block as a component of multimodal analgesia for providing perioperative pain relief in patients undergoing total abdominal hysterectomy. BODY.MATERIALS AND METHODS: After obtaining approval by the institute ethics committee and written informed consent, 90 adult female patients of American Society of Anesthesiologists (ASA) physical status (PS) I or II, scheduled for total abdominal hysterectomy by a lower abdominal transverse incision were recruited in this randomized double-blind controlled clinical trial. Unwilling patients and patients with body mass index >30 kg/m2, compromised renal and liver function, uncontrolled diabetes, severe cardiovascular, respiratory disease, having a history of allergy to any of the study drug, and history of abdominal surgery were excluded from the study. Primary outcome of our study was immediate postoperative visual analogue scale (VAS). Secondary outcomes were intraoperative fentanyl requirement and hemodynamic changes, postoperative hemodynamic changes and time to request first postoperative analgesic. A thorough review of related literature was performed from standard textbooks and an internet search of related articles was performed PubMed. Sample size was calculated on the basis that a 20 mm difference in the mean VAS between the two groups would be clinically useful. We used PS Power and Sample Size Calculations Software, version 3.0 [Department of Biostatistics, Vanderbilt School of Medicine, Nashville, TN]. We assumed a standard deviation of 30 mm as a standard deviation of VAS score in the population. Forty one patients in each group would be needed, assuming the probability of alpha (α) error is 5% and a power of the study is 85%. Assuming a probable drop out of 10%, 90 patients were recruited. Patients were randomly allocated into two equal groups of 45 patients in each group using a random number generators in Microsoft ExcelTM 2003 [Microsoft Corporation, Redmond, WA] and allocation concealment were maintained by using an opaque sealed envelope technique. Standard ASA monitoring was used. Baseline parameters such as heart rate, continuous electrocardiogram, noninvasive blood pressure, SpO2 were noted down. Patients were randomly allocated into two groups, Group B or Group N to receive one of the following solutions for bilateral TAP block. Group B: Injection bupivacaine, 0.25% (0.5 ml/kg body weight on each side).Group N: Injection normal saline (0.5 ml/kg body weight on each side). The anesthesiologist, who prepared the solution in identical syringes, remained unaware of the nature of the study and was not involved in further data collection. The lumbar triangle of Petit, located just anterior to the latissimus dorsi muscle was identified by palpating the iliac crest in an anterior to posterior direction, until the edge of the latissimus dorsi was felt. The skin was pierced just cephalic to the iliac crest over the triangle of Petit with a blunt 18 gauge Tuhoy needle [Smiths Medical International Limited, Hythe, Kent, UK] after infiltration with 2% lignocaine. The block was administered following the technique advocated by McDonnell et al.[2] The needle was advanced perpendicular to the skin in the coronal plane until the first resistance of external oblique muscle was encountered. Gentle advancement of the needle resulted in a pop sensation as the needle entered the plane between the external and internal oblique fascial layers. A second resistance was felt as the needle passed through the internal oblique muscle. A second loss of resistance was encountered when the needle reaches the transversus abdominis fascial plane between the internal oblique and transversus abdominis muscle. A test dose of 1 ml was injected to determine resistance to flow and confirm the needle tip placement within the neurovascular plane. After this one of the study solutions was injected on each side following careful aspiration to exclude vascular puncture. Ten min after TAP block, all patients received a standardized general anesthesia with fentanyl 1 mcg/kg, propofol and atracurium. Bispectral index (BIS) was maintained within a range of 40-60. They were monitored for any signs of inadequate analgesia in the intraoperative period such as sweating, lacrimation, tachycardia (>100/min) and hypertension (>20% elevation of baseline mean arterial pressure) and supplemental doses of injection fentanyl 0.5 mcg/kg were given as needed. An anesthesiologist who was unaware about the patient allocation did recording of intraoperative hemodynamic data and other anesthesia management. Intravenous infusion of paracetamol (1 g to patients with body weight >40 kg and 750 mg to patients with body weight <40 kg) was given 30 min prior to completion of surgery. Prophylactic antiemetics were not given in any patients. Fluid deficit arising from preoperative fasting was corrected by maintenance fluid. Blood loss and other plasma losses were approximately calculated from mops and suction drain bottles. Blood losses up to the transfusion threshold were replaced with 3 ml of Ringer's lactate for each ml of blood loss. After the patient had adequately recovered from anesthesia, and was able to assess pain, postoperative analgesia was assessed with VAS 0-100 mm in the immediate postoperative period (when the patient was able to communicate in the postanesthesia care unit), at 1, 2, 3, 4, 5, 6 and 24 h and whenever the patient complained of pain. The time of administration of rescue analgesic in the form of injection tramadol 2 mg/kg intravenous (IV) was noted when VAS >40 mm. VAS score was assessed in both rest and movement (knee flexion) by an independent observer who was unaware about the allocation. After administration of rescue analgesic, patients were shifted to a postoperative analgesic regimen of injection tramadol IV 2 mg/kg 8 hourly and injection paracetamol 6 hourly up to 24 h. The incidence of postoperative nausea and vomiting was noted during the first 24 h. Rescue antiemetics were given to any patient who complained of nausea or vomiting. Any signs of adverse effects of the technique like local site infection, hematoma formation, local anesthetic toxicity due to intravascular injection of anesthetic (like dizziness, tinnitus, perioral numbness and tingling, lethargy, seizures, signs of cardiac toxicity like atrio-ventricular conduction block, arrhythmias, myocardial depression and cardiac arrest), peritoneal perforation, bowel perforation, difficulty ambulating or fall and injury secondary to spread of local anesthetic to nerves of the buttock, lateral thigh or leg in the distribution of the femoral nerve were sought for.[13] All raw data were entered into a Microsoft Excel spreadsheet and analyzed using standard statistical software. Continuous numerical data were expressed as mean and standard deviation (for normally distributed data), or median and inter-quartile range (for data that are not normally distributed). Categorical data were expressed as frequencies and percentages. Normally distributed numerical data between groups were analyzed using the Student's t-test. Skewed data between groups were analyzed using the Mann–Whitney U-test. Categorical variables were analyzed using the Fisher's exact test or the Pearson's Chi-square test as applicable. All tests were two-tailed. P < 0.05 were considered statistically significant. BODY.RESULTS: Ninety patients were recruited for the trial and data from all of them has been analyzed. A CONSORT flow diagram depicting the passage of participants through the trial has been provided in Figure 1. The two groups were comparable in terms of baseline demographic parameters (age, sex and body weight), duration of surgery and anesthesia and preoperative hemodynamic parameters (pulse rate, systolic and diastolic blood pressure, respiratory rate) and the volume of the study drug required in TAP block. A summary of base line characteristics of the patients has been furnished in Table 1. Figure 1CONSORT flow diagram for patient selection Table 1 Baseline characteristics of the patients in each group From the analysis of the intraoperative hemodynamic parameters it was found that pulse rate was significantly higher in patients receiving placebo (95.9 ± 11.2 bpm vs. 102.9 ± 8.8 bpm, mean the difference 7.0 s, P = 0.001) after surgical skin incision. Both systolic and diastolic blood pressure after surgical skin incision was also significantly higher in patients receiving placebo, but similar at all other time points. Intraoperative hemodynamic changes have been graphically plotted in Figures 2 and 3. Figure 2Comparison of mean preoperative and intraoperative pulse rate, B = Group B, N = Group N, PR1 = 10 min after TAP block, PR2 = Before induction, PR3 = After induction, PR4 = After incision, PR5 = 15 min intraoperative, PR6 = 30 min intraoperative, PR7 = 60 min intraoperative, PR8 = 90 min intraoperative, PR9 = 120 min intraoperative Figure 3Comparison of mean preoperative and intraoperative systolic blood pressure and diastolic blood pressure. BSBP = Systolic blood pressure of Group B, NSBP = Systolic blood pressure of Group N, BDBP = Diastolic blood pressure of Group B, NDBP = Diastolic blood pressure of Group N, BP1 = 10 min after TAP block, BP2 = Before induction, BP3=after induction, BP4 = After incision, BP5 = 15 min intraoperative, BP6 = 30 min intraoperative, BP7 = 60 min intraoperative, BP8 = 90 min intraoperative, BP9 = 120 min intraoperative Median requirement of intraoperative fentanyl was significantly higher in patients receiving placebo in comparison to bupivacaine in TAP block (81 mcg vs. 114 mcg, P = 0.000, Mann–Whitney U-test). The difference between the medians is 32.0 mcg with a 95% confidence interval (CI) of 22.0, 42.5 (Hodges–Lehman median difference). There was no statistically significant difference between the two groups in terms of median values of immediate postoperative pulse rate, systolic blood pressure and diastolic blood pressure (Mann–Whitney U-test). Postoperative oxygen saturation varied from 97% to 100% in all patients of both Groups B and N. VAS scores in the immediate postoperative period both at rest (median VAS 3 mm vs. 27 mm) and with activity (median 8 mm vs. 35 mm) were significantly lower in patients who received TAP block. Median duration of analgesia was significantly higher in patients belonging to Group B (290 min vs. 16 min, P = 0.000, Mann–Whitney U-test). The difference in median duration of analgesia is 275 min with 95% CI of 250–307 min (Hodges–Lehman median difference). These findings have summarized in Table 2. A Kaplan–Meier survival analysis for the cumulative duration of analgesia in first 24 h shows a significantly longer duration of analgesia in patients receiving TAP block [Figure 4]. Table 2 Comparison of quality of analgesia Figure 4Duration of analgesia in either group has been depicted in by Kaplan Meyer survival analysis. The survival graph shows significant cumulative analgesia in patients receiving transversus abdominis plane block Incidence of postoperative nausea-vomiting was also similar in both groups. No opioid related side effects such as respiratory depression, pruritus or urinary retention was noted in any of the patients. None of the patients in either group had any complication that can be attributed to TAP block. Median value of pulse rate, systolic blood pressure, diastolic blood pressure, VAS scores at 1, 2, 3, 4, 5, 6 and 24 h were not compared as >30% patients in Group N received rescue analgesic during the immediate postoperative period. BODY.DISCUSSION: The principal finding of our study is that bupivacaine in TAP block provides effective intraoperative and immediate postoperative analgesia in patients undergoing total abdominal hysterectomy. Our finding of preincisional TAP block reducing intraoperative fentanyl requirement was consistent with those of Mukhtar and Khattak[14] who reported a significant reduction in intraoperative morphine consumption in patients receiving TAP block with 0.5% bupivacaine in renal transplant recipients (0.4 ± 1.2 mg vs. 9.3 ± 1.4 mg; P < 0.0001). El-Dawlatly et al.[7] reported a similar significant reduction in intraoperative sufentanil consumption in patients undergoing laparoscopic cholecystectomy (8.6 ± 3.5 mcg vs. 23.0 ± 4.8 mcg, P < 0.01). Similar findings were reported in a study by Ra et al.[15] in patients undergoing laparoscopic cholecystectomy where intraoperative remifentanil use was significantly lower in patients receiving either 0.5% or 0.25% bupivacaine in comparison to placebo. However, no other RCT, to the best of our knowledge has addressed the efficacy of preincisional TAP block in preventing hemodynamic response to surgical stimuli. We have found the superiority of TAP block in providing immediate postoperative analgesia reflected by a lower VAS score both at rest and with activity. The current literature on TAP block is not unanimous in the matter that whether it improves postoperative pain score or not. Our finding is consistent with those of McDonnell et al.[2] in abdominal surgery and Carney et al.[3] in open appendicectomy. In 2008, Carney et al.[10] found that anatomical TAP block in total abdominal hysterectomy patients significantly reduces postoperative pain scores up to 48 h period. Postoperative morphine consumption also decreased at 12 h, 36 h and 48 h time period. However, the authors did not address intraoperative opioid requirement. Recently, Sharma et al.[16] also found that TAP block by landmark technique improves VAS score in first 24 h in patients undergoing major abdominal surgery. Petersen et al.[8] in 2012 also found that US guided bilateral TAP block in patients undergoing laparoscopic cholecystectomy provides superior postoperative pain scores. Petersen et al.[17] in 2013 found that TAP block does not provide superior analgesia in comparison to placebo after inguinal hernia repair. A previous Cochrane review[18] and a meta-analysis[19] in 2012 failed to demonstrate the beneficial effect of TAP block on postoperative pain scores. In this context, it is worth mentioning that the meta-analysis found that TAP block decreases postoperative opioid consumption, which may be a more important parameter to decide an analgesic regimen. The median duration of effective postoperative analgesia from our study was 290 min in patients receiving TAP block, and we did not use any additive in TAP block. Clonidine in peripheral nerve block has been shown to significantly increase the duration[20] and may be considered here also. Time to the requirement of first postoperative analgesic is also significantly increased in patients received TAP block (290 min vs. 16 min). This was consistent with McDonnell et al.,[21] who demonstrated in their anatomic study that TAP block with 0.5% lignocaine may provide analgesia for 4-6 h. Only three patients in bupivacaine TAP group required rescue analgesic in first two postoperative hours whereas 43 patients in the saline group required the same. Four patients in bupivacaine group did not require rescue analgesia in first 24 h postoperative period. The cause of prolonged duration of analgesic effect following single shot TAP block is not entirely clear. This may be explained by the fact that the TAP is relatively poorly vascularized, and therefore drug clearance may be slowed.[10] Inadequate analgesia even after TAP block may be either due to technical failure or due to visceral pain component, which is not addressed by TAP block. We found a 6.67% of inadequate analgesia in first 2 h postoperative period. As such, until now, all local anesthetic techniques carry an inherent failure rate of 5-20%, depending on the skill of the operator.[22] The most important clinical implication of our findings is the significant opioid sparing effects of TAP block both in the intraoperative as well as the postoperative period. Opioids, though very effective in perioperative pain management, may be associated with nausea-vomiting, pruritus and respiratory depression. Moreover, some patients who are morbidly obese or having obstructive sleep apnea will be maximally benefitted from TAP block as it provides opioid sparing effects. Besides this, TAP block also prevents the hemodynamic responses of surgical incision. Patients having ischemic heart disease or stenotic valvular lesion like mitral or aortic stenosis, where tachycardia is undesirable, will also be benefitted from preincisional TAP block. It may be a relatively safer alternative to neuraxial block for intra and postoperative analgesia in patients having coagulopathy. Our study has a few limitations. First, it is difficult to define inadequate analgesia in the intraoperative period. Though we controlled the depth of anesthesia by BIS monitoring, ensured adequate muscle relaxation, prevented hypovolemia, indirect assessment of intraoperative pain by hemodynamic parameters may be unreliable. Second use of real time USG for TAP block is increasing; we used a landmark based anatomical approach. However, as real time US guidance may increase the efficacy of TAP block, it won't change the primary finding of our study. Third, use of patient controlled analgesia in the postoperative period could have accurately delineated postoperative opioid consumption. BODY.CONCLUSIONS: Preincisional TAP block decreases intraoperative fentanyl requirements, prevents hemodynamic responses to surgical stimuli and also provides effective postoperative analgesia. The anatomical approach of TAP block is also very safe and reasonably effective.

TITLE: The effect of acute pomegranate extract supplementation on oxygen uptake in highly-trained cyclists during high-intensity exercise in a high altitude environment ABSTRACT.BACKGROUND: Recent research has indicated that pomegranate extract (POMx) may improve performance during aerobic exercise by enhancing the matching of vascular oxygen (O2) provision to muscular requirements. POMx is rich in ellagitannin polyphenols and nitrates (NO3 −), which are both associated with improvements in blood flow and O2 delivery. Primarily, this study aimed to determine whether POMx improves performance in a cycling time trial to exhaustion at 100%VO2max (TTE100%) in highly-trained cyclists. In addition, we investigated if the O2 cost (VO2) of submaximal exercise was lower with POMx, and whether any changes were greater at high altitude where O2 delivery is impaired. ABSTRACT.METHODS: Eight cyclists exercised at three submaximal intensities before completing a TTE100% at sea-level (SEA) and at 1657 m of altitude (ALT), with pre-exercise consumption of 1000 mg of POMx or a placebo (PLAC) in a randomized, double-blind, crossover design. Data were analysed using a three way (treatment x altitude x intensity) or two-way (treatment x altitude) repeated measures ANOVA with a Fisher's LSD post-hoc analysis. Significance was set at p ≤ 0.05. The effect size of significant interactions was calculated using Cohen's d. ABSTRACT.RESULTS: TTE100% performance was reduced in ALT but was not influenced by POMx (p > 0.05). Plasma NO3 − were 10.3 μmol greater with POMx vs. PLAC (95% CI, 0.8, 19.7,F 1,7 = 7.83, p < 0.04). VO2 measured at five minutes into the TTE100% was significantly increased in ALTPOMx vs. ALTPLAC (+3.8 ml.min−1kg−1, 95% CI, −5.7, 9.5, F1,7 = 29.2, p = 0.001, ES = 0.6) but unchanged in SEAPOMx vs. SEAPLAC (p > 0.05). Submaximal VO2 values were not affected by POMx (p ≥ 0.05). ABSTRACT.CONCLUSIONS: The restoration of SEA VO2 values at ALT is likely driven by the high polyphenol content of POMx, which is proposed to improve nitric oxide bioavailability. Despite an increase in VO2, no change in exercise performance occurred and therefore this study does not support the use of POMx as an ergogenic supplement. BODY.BACKGROUND: Pomegranate (Punica granatum) (POM), is a seeded, red, fleshy fruit of Middle East origin which was used in traditional medicine to treat a variety of inflammatory conditions [1]. In modern-day research, the health benefits of POM have been attributed to its high concentration of nitrates (NO3 −) and polyphenol compounds, and consumption of POM juice (POMJ) or extract (POMx) has been linked to a decline in cancer proliferation [2], the amelioration of cardiovascular disease markers [3] and decreases in gut and joint inflammation [4, 5]. Recent research has indicated that POM-based supplements can also improve performance during aerobic exercise by enhancing the matching of vascular O2 provision to muscular requirements [6, 7]. Polyphenols are a group of phytochemicals with antioxidant properties that contain one or more aromatic rings and at least two hydroxyl groups [8]. POMJ contains a greater concentration of polyphenols (~3.8 mg.ml−1) than other polyphenol-rich beverages such as red wine (~3.5 mg.ml−1), Concord grape juice (~2.6 mg.ml−1) and cranberry juice (~1.7 mg.ml−1) [9]. These are predominantly from the ellagitannin (ET) subclass (80–90%) with smaller amounts of anthocyanins (8–15%) [10]. Consumption of ET or anthocyanin-rich foods is associated with a decrease in systolic blood pressure (SBP) and an increase in vessel diameter and blood flow [6, 11, 12] suggesting a link between POM consumption and O2 delivery. Supplementation with dietary NO3 − influences O2 delivery during exercise through its conversion to the potent vasodilator, nitric oxide (NO). During resting conditions, NO is primarily produced endogenously by NO synthases (NOS) [13]. However, in hypoxic conditions, such as those present locally in the muscles during exercise, activity of this pathway is limited, placing greater importance on the secondary NO3 −-nitrite (NO2 −)-NO pathway [14]. Polyphenols further enhance the effects of dietary NO3 − by promoting their conversion into NO [15, 16], and protecting NO from damage caused by reactive oxygen species (ROS) [17]. Previous research involving NO3 − supplementation has predominantly used beetroot juice (BRJ), which contains ~11 mmol.L−1 NO3 − [18] in comparison to 12.93 ppm.L−1 (~0.2 mmol.L−1) in POMJ and 109 ppm.L−1 (~1.76 mmol.L−1) in POMx, as reported by Roelofs et al. [6]. Acute or short-term BRJ supplementation in low to moderately-trained individuals is associated with a large increase (>92%) in plasma NO2 −, a 3–5% reduction in O2 uptake (VO2) during submaximal exercise and a 15–25% improvement in performance during cycling, running and knee extension time to exhaustion protocols [18–22]. In contrast, BRJ has little or no benefit on these parameters in highly-trained athletes (VO2max >60 ml.min−1kg−1), who may have greater NOS activity and consequently a lower reliance on NO production via NO3 − [23–29]. However, BRJ appears to benefit these athletes during exercise at high altitude, where the lower atmospheric pressure of O2 (PO2) impairs O2 transport and places greater reliance on the non-aerobic pathway to produce NO [30, 31]. This is significant because exercise at high altitude features in a number of major sporting competitions, such as the Tour de France. While BRJ clearly has an effect on VO2, the high concentration of ET in POM may provide a source of dietary NO3 − with greater bioavailability, despite the lower total NO3 − content. Currently, only two studies have investigated the effect of POM supplementation on endurance exercise performance at sea-level, with no previous research investigating its effects in low PO2 conditions. In moderately-trained individuals, acute POMx intake (1000 mg) increased pre-exercise blood flow and time to exhaustion during treadmill running at 90-100% of peak velocity [7]. Conversely, following seven days of POMJ supplementation (1000 ml.day−1) in a trained cohort, no change in performance was observed during a ten minute cycling time trial or time to exhaustion protocol in hot conditions [32]. Thus, further research is warranted to determine whether POM improves O2 transport and endurance exercise performance. The outcomes of the current study are primarily to further explore the effect of acute POMx supplementation on endurance exercise performance, and secondarily, to determine the effect of POMx on O2 transport parameters. In addition, the study will investigate whether any observed effects are greater during exercise in a high altitude environment. We hypothesize that acute POMx supplementation will reduce the submaximal O2 cost of exercise, and in doing so, improve performance in an environment (altitude) where O2 availability may be limiting. BODY.METHODS.PARTICIPANTS: All participants provided written consent after being informed about the study requirements and benefits and risks of participating. A health questionnaire was also completed. Eight highly-trained cyclists, including seven males and one female, were recruited from the regional cycle community. This sample size was chosen based on previous studies which have successfully shown a decrease in VO2 following NO3 − supplementation with a sample size of 8 participants [18, 21]. While the sample size is small, this was unavoidable given the highly-trained status of participants that we wished to study. All participants were current or past members of the national cycling or triathlon junior development programmes. Their age, height, body mass and peak aerobic capacity (VO2max) were 17–18 years, 67.6 ± 7 kg, 180 ± 9 cm and 74.4 ± 6.2 ml.min−1kg−1 respectively. The study was approved by the Massey University Human Ethics Committee (Southern A 15/54) in accordance with the Declaration of Helsinki. BODY.METHODS.EXPERIMENTAL DESIGN.VO: In order to determine workloads for the supplemented trials, participants completed a VO2max test in thermoneutral conditions (18–20 °C) at sea-level, on an electronically-braked cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands), which was set up as closely as possible to the participant's own bike. This session doubled as an initial familiarization session to ensure participants were familiar with the equipment and protocols involved in the experimental sessions. Following a five minute warm-up at 100 W, participants completed four × 7-min stages of increasing workload (e.g., 150, 200, 250, 300 W) with expired air being collected into Douglas Bags during the last minute of each stage and analysed for O2 and CO2 concentrations and volume. Following a five minute active rest period, an incremental "ramp" protocol was used to determine VO2max. Power began at 100 W and increased linearly with time (25 W.min−1). Participants cycled for as long as possible and verbal encouragement was given to elicit maximal effort. As the participant's VO2max was approached (as indicated by a change in breathing pattern), expired air was captured in Douglas bags until exhaustion. Analysis of Douglas bags was done using a calibrated gas analysing system (AD Instruments, Dunedin, New Zealand). The gas analyser was calibrated using gases of known concentration (15.01% O2, 5.01% CO2). Minute ventilation (VE) and concentrations of O2 and CO2 values were used to calculate the volume of inspired air (VI) using the Haldane transformation, where VE was corrected for barometric pressure, ambient temperature and atmospheric water saturation. Subsequently, VO2 and expired CO2 (VCO2) could be determined and are reported as standard temperature and pressure dry (STPD). The respiratory exchange ratio (RER) was calculated using VCO2/VO2 and attainment of VO2max was confirmed with RER ≥1.1. A relationship between steady-state workload and VO2 values was drawn through creation of a power curve and generation of a linear line equation y = mx + c, where m = gradient, x = power and c = start point. The equation was used to estimate power output at 50, 65 and 80% of VO2max. BODY.METHODS.EXPERIMENTAL DESIGN.EXPERIMENTAL PROTOCOL: The experimental protocol (Fig. 1) was a randomized, double-blind, crossover design, which was completed on four occasions: twice in sea-level conditions (SEA) and twice in high-altitude conditions (ALT, 1657 m, ~17% O2). The testing location was the Turoa ski-field carpark, Ohakune, which was chosen due to being the highest road accessible to our mobile laboratory in the North Island of New Zealand. Prior to each trial, participants ingested 1000 mg of either a POMx supplement (POM Wonderful LLC, USA) in capsule form or a placebo capsule of the same colour, size and shape as POMx (PLAC, brown sugar). The quantity of POMx was based on Trexler et al. [7] who showed an association between acute POMx supplementation and improved running performance. Previous analysis of the POM Wonderful LLC supplement have shown it to contain 1800 ppm polyphenols, comprised of 95.5% ellagitannins, 3.5% ellagic acid and 1% anthocyanins [32]. Participants completed a POMx trial and a PLAC trial in each environmental condition in a randomized order; the supplement blinded to the participant and the researcher. The participant was instructed to swallow the capsule whole, without tasting it, to avoid an expectation bias. In accordance with previous POMx research, the supplement was ingested 2.5 h prior to each experimental trial to allow maximal absorption prior to exercise [9]. During the 48 h prior to each session, participants were asked to limit consumption of NO3 − and polyphenol-rich foods and avoid strenuous exercise and antibacterial substances, such as mouthwash, which destroy NO3 − -NO2 − converting bacteria on the tongue [33]. Participants consumed a standardized meal (524 cal, 8 g protein, 11 g fat, 51 g carbohydrate) three hours prior to exercise. Participants were asked to arrive at the testing session in a hydrated state, and consumed water ad libitum throughout the trial.Fig. 1Schematic representation of the study design On arrival to the laboratory, body mass was obtained and a heart rate (HR) monitor (Garmin, Kansas, USA) was applied and recorded HR at a sampling rate of two seconds throughout the trial. After sitting in a supine position for five minutes, blood pressure of the brachial artery was measured using an automated sphygmomanometer (Japan Precision Instruments, Gumma, Japan). Blood was collected via fingerprick sample (~5 μL) and analysed for blood lactate concentration ([La−]b) using a blood lactate test meter (Lactate Pro, Arkray KDK, Japan). To determine haematocrit (Hct), additional blood (~35 μL) was collected from the fingertips in heparinized capillary microtubes and immediately spun in a microhaematocrit centifuge (Thermo IEC MB, Bellport, USA) at 14,000 rpm for two minutes. Hct was calculated as the length of red blood cells as a percentage of the total length of blood in the tube. Samples were taken in duplicate to allow calculation of an average value. In the SEA trials, a venous blood sample was also collected via venepuncture from the antecubital vein in heparinized vacutainers and immediately spun in a centrifuge (Eppendorf AG, Hamburg, Germany) at 2500 rpm for 12 min at 4 °C. Plasma was transferred into epindorphs and stored at −80 °C until analysis. The venous sample was not collected in ALT as samples could not be stored or analysed in the mobile laboratory. Following the pre-exercise measures, the participant mounted the ergometer to begin cycle exercise. The experimental protocol began with three x six minute stages of stationary cycling exercise at power outputs corresponding to 50, 65 and 80% of their previously determined VO2max. In the last minute of each stage, VO2 was measured as previously described. Perceived exertion (RPE) was also recorded using Borg G [34] Scale of Perceived Exertion. Then, following a five minute rest, the load on the ergometer was increased to a workload calculated to elicit VO2max and participants were instructed to ride at this intensity, at a cadence of ≥ 80 rpm for as long as possible, with the trial being terminated once the participant could not maintain the required cadence (for the previous 10 s) or at volitional exhaustion. Time to fatigue at 100%VO2max (TTE100%) was chosen in this study as a performance measure rather than a self-paced time trial to enable physiological data to be collected and compared at the five minute point. Accordingly, five minutes into the TTE100% VO2 was measured. BODY.METHODS.BLOOD ANALYSIS: Plasma samples were analysed using a Nitric Oxide Colorimetric Assay Kit (BioVision Incorporated, Milpitas, California, USA) to measure NO3 − and NO2 −. This method is a two-step process, in which nitrate reductase is used to convert NO3 − to NO2 −, and then Greiss Reagents convert NO2 − to a deep purple azo compound. Absorbance is read at 540 nm and plotted as a function of NO3 − and NO2 − concentration. BODY.METHODS.STATISTICAL ANALYSES: Statistical analyses to compare the values of all variables measured were done using statistical computer software (SPSS Statistics, Version 23, IBM Corporation, New York). Normal distribution of data was confirmed with the Shapiro-Wilks test. Variables measured during the submaximal exercise stages were analysed using three-way repeated measures ANOVA to test the significance level for main effects of, and interactions between, altitude (SEA or ALT), workload (50, 65 and 80%VO2max) and treatment (PLAC or POMx). Similarly, two-way (altitude x treatment) repeated measures ANOVA was performed on all variables measured during the TTE100%. Where significant interactions (p ≤ 0.05) were observed, two-tailed paired t-tests with a Fisher's LSD post-hoc analysis were used to identify the location of the significance. The effect sizes (ES) of significant interactions were calculated using Cohen's d (0.1 small; 0.5 medium; 0.8 large) [35]. The relationship between VO2 and performance during the TTE100% was analysed using the Pearson correlation coefficient. Data is presented as mean ± SD or mean change and 95% CI, as appropriate. BODY.RESULTS: Participants' self-reported adherence to supplement intake was 100% and no side effects of supplementation were reported. There was no order effect present for any of the significant results (p > 0.05). BODY.RESULTS.TTE100% PERFORMANCE: Average performances during each condition were: SEAPLAC: 10.7 ± 2 min, SEAPOMx: 12.6 ± 8.7 min, ALTPLAC: 7.0 ± 2.3 min and ALTPOMx: 8.2 ± 4.5 min. There was no main effect of treatment on performance (F 1,7 = 0.776, p > 0.05). Performance was significantly decreased at ALT compared to SEA (−4.1 min, 95% CI, −6.9, −1.3, F 1,7 = 12.3, p < 0.02, ES = 0.8). The individual performance responses in the TTE100% to POMx in SEA and ALT are displayed in Fig. 2. Despite no overall significant effect of POMx, there appear to be two participants (HW and LS) who increased their performance with POMx in both altitudes.Fig. 2Individual TTE100% performance responses to POMx in (a) SEA and (b) ALT BODY.RESULTS.RESTING MEASURES: Plasma NO3 − was greater following POMx compared to PLAC (+10.3 μmol, 95% CI, 0.8, 19.7, F 1,7 = 7.83, p < 0.04, ES = 0.9). However, the assay used was not sensitive enough to detect changes in NO2 −. SBP was not significantly affected by POMx or ALT (p > 0.05). However, there was a trend towards an increase in SBP with POMx vs. PLAC (+3.9 bpm, 95% CI, −0.6, 8.5, F 1,7 = 4.28, p = 0.08, ES = 0.3). There was a strong trend towards an increase in DBP with ALT, which showed a moderate effect size (+5 mmHg, F 1,7 = 1.18, p = 0.054, ES = 0.6) and a significant treatment x altitude x time interaction (F 1,7 = 7.64, p < 0.03). However, post-hoc analyses revealed no significant differences between pre or post-exercise DBP with either treatment at either altitude (p > 0.05). Hct was significantly increased by ALT (+1.4%95% CI, 0.2, 2.6, F 1,7 = 7.56, p < 0.03, ES = 0.4) and significantly decreased by POMx, although the effect size was small (−0.76%, 95% CI, −1.3, −0.2, F 1,7 = 10.4, p < 0.02, ES = 0.2). BODY.RESULTS.PULMONARY GAS EXCHANGE, VENTILATORY AND BLOOD LACTATE RESPONSES TO SUBMAXIMAL EXERCISE: There was no significant main effect of POMx nor any significant altitude x treatment interaction on VO2, VCO2, HR or [La−]b (p > 0.5). There was a trend towards an increase in HR with POMx, but this had a small effect size (+2.3 bpm, 95% CI, −5.0, 0.5, F1,7 = 3.80, p = 0.09, ES = 0.1). There was a significant main effect of altitude on [La−]b (−0.8 μmol,95% CI, 0.3, 1.3, F 1,7 = 2.95, p < 0.01, ES = 0.6). BODY.RESULTS.PULMONARY GAS EXCHANGE, VENTILATORY AND BLOOD LACTATE RESPONSES TO THE TIME TRIAL TO EXHAUSTION AT 100%VO: The variables measured during the TTE100% are summarized in Table 1. All data was recorded five minutes into the TTE100%. The two-way ANOVA showed a significant altitude x treatment interaction for VO2 (F 1,7 = 12.5, p = 0.01). Post-hoc analysis identified that POMx significantly increased VO2 at ALT (+3.8 ml.min−1kg−1, 95% CI, −5.7, 9.5, F1,7 = 29.2, p = 0.001) but not at SEA (F1,7 = 0.95, p > 0.05, ES = 0.2). Cohen's d showed a moderate effect size of 0.6. There was no correlation between VO2 during the TTE and performance (r 2 = 0.0001, p ≥ 0.05). There was a significant altitude x treatment interaction for VCO2 (F1,7 = 6.32, p = < 0.04). However, post-hoc analysis identified no significant differences between treatments at SEA (F1,7 = 1.76, p > 0.5, ES = 0.2), and a trend towards an increase in VCO2 with POMx at ALT with a moderate effect size (+3.6, 95% CI, −0.665, 7.82, F1,7 = 3.98, p = 0.09, ES = 0.5). HR and [La−]b were not affected by POMx or altitude (p > 0.5).Table 1Variables measured during the TTE100%SEA-PLACSEA-POMxALT-PLACALT-POMxVO2 (ml.min.kg−1)70.0 ± 6.368.5 ± 7.263.2 ± 5.6a 66.9 ± 5.3bc VCO2 (ml.min.kg−1)72.4 ± 7.870.6 ± 8.969.6 ± 7.173.2 ± 8.0HR (bpm)186 ± 8175 ± 8184 ± 7184 ± 5[La−]b (μmol.L−1)11.0 ± 3.710.2 ± 2.912.2 ± 3.911.7 ± 3.9Data are presented as means ± SD VO 2 oxygen uptake, VCO 2 expired carbon dioxide, HR heart rate, [La − ] b blood lactate concentration, SEA-PLAC sea-level placebo, SEA-POMx sea-level pomegranate extract, ALT-PLAC high-altitude placebo, ALT-POMx high-altitude pomegranate extract aindicates P < 0.05 compared to SEA-PLAC bindicates p < 0.05 compared to SEA-POMx cindicates p < 0.05 compared to ALT-PLAC BODY.DISCUSSION: Although acute POMx supplementation was associated with a 10.3 μmol increase in plasma NO3 − (95% CI, 0.8, 19.7, p < 0.04, ES = 0.9), its use as an acutely-ingested ergogenic supplement by highly-trained athletes is not supported by the current study, as neither performance nor submaximal VO2 were significantly altered by POMx ingestion. However, our data indicated that POMx does allow maintenance of VO2 at a workload prescribed to elicit 100% VO2max (at sea level) during high intensity exercise under low PO2 conditions, despite no significant performance effect. The absence of changes in performance during the TTE100% is in agreement with Trinity et al. [32] who demonstrated no effect of acute POMJ supplementation on a cycling time trial or time to exhaustion protocol in hot conditions in moderately-trained individuals. However, Trexler et al. [7] found an increase in time to exhaustion during treadmill running at 90–100% of peak velocity, in moderately trained participants, following acute supplementation of POMx (1000 mg, 30 min before exercise) at sea-level. In addition, POMx has been shown to improve performance and recovery from resistance and sprint cycling exercise [36, 37]. The current study is the first study to test POMx supplementation in highly-trained endurance athletes and is in accordance with previous research involving acute BRJ supplementation in this cohort, which found no change in performance in a running or cycling time trial in hypoxic conditions [28, 29]. However, two studies involving multi-day periods of NO3 − supplementation in low PO2 conditions (11–13% O2) showed changes in both O2 parameters and exercise performance in moderately-trained individuals (VO2max 58–61 ml.min−1kg−1). Kelly et al. [31] found that three days of BRJ supplementation (~8.4 mmol.day−1) decreased steady-state VO2 during a bout of moderate-intensity cycle exercise by 7.6% and this resulted in an 8% improvement in a subsequent high intensity time to exhaustion protocol, compared to exercise done following the intake of a placebo. Masschelein et al. [30] found that six days of BRJ supplementation (~5 mmol.day−1) increased arterial O2 saturation by 2.7% and the muscle tissue oxygenation index in the vastus lateralis by 4% during submaximal cycling. This resulted in a 5% increase in performance during a subsequent cycling graded exercise test. Thus, the small increase in VO2 with acute supplementation of POMx may not be sufficient to produce an ergogenic effect during a 100%TTE in highly-trained athletes and a longer period of supplementation may be necessary. Alternatively, the absence of change in performance despite an increase in VO2 may reflect the lack of correlation between VO2 and performance times (r2 = 0.0001), which indicates that VO2 was not the determining factor in the TTE100%. Rather, it is likely that other factors, such as anaerobic capacity, contribute more greatly to performance in a high-intensity TTE. To the authors' knowledge, the current research is the first study to have measured VO2 during cycling exercise following supplementation with POMx. The lack of effect of POMx on submaximal VO2 or VCO2 values in either environment despite an increase in plasma NO3 − is in accordance with previous research involving acute supplementation with NO3 −-rich BRJ in highly-trained athletes (VO2max >66 ml −1min−1kg−1) conducted at sea-level [23, 24, 26, 38] or in low PO2 conditions (13–15% O2) [28, 29]. In addition, the absence of change in VO2 during the TTE100% at SEA correlated with Boorsma et al. [39] who conducted a running protocol involving similar intensities to the current study, and found no significant difference in VO2 values during a 1500 m TT. The restoration of an ALT-induced lowering in VO2 during intense exercise following POMx compared to SEA (+3.8 Lmin−1, 95% CI, −5.7, 9.5, p = 0.001, ES = 0.6) differed from previous NO3 −-based research involving highly-trained participants, although no other studies using hypoxia and VO2 measurement have utilised a performance test protocol similar to ours. Masschelein et al. [30] found no change in VO2max recorded during a graded exercise test to exhaustion in hypoxic conditions (11% O2, ~5000 m altitude) despite a 4% increase in the muscle tissue oxygenation index. However, in that study, exercise intensity was not stable, as it was in the current study and VO2 was measured at exhaustion, rather than part-way into the exercise. In addition, MacLeod et al. [29] recorded no change in VO2 during a 10 km cycling time trial completed in a normobaric hypoxic chamber (~2440 m altitude). However, the duration of this test was significantly longer than ours, which may have influenced potential effects on VO2. The changes in VO2 in the current study were likely driven by the high polyphenol content of POMx, as POMx has a lower NO3 − concentration than BRJ. While BRJ-based research in highly-trained athletes has recorded increases in plasma NO3 − of 31–1907% [27, 31, 39], the current study measured a 44% increase in NO3 − with POMx. Although polyphenolic compounds are also present in BRJ, these are predominantly from the quercetin subclass, in comparison to the high ET component in POMx [40]. Previous research has generally found no link between quercetin intake, and changes in VO2 or performance parameters [41, 42]. However an increase in pre or post-exercise blood flow and vessel diameter has been observed with consumption of ET or anthocyanin-containing fruit juices and extracts [6, 7, 11]. Further, Ignarro et al. [17] demonstrated that POMJ is more effective at protecting NO from breakdown than other polyphenol-containing juices and red wine, with significant antioxidant actions occurring at dilutions greater than 1000-fold. Thus, the increased VO2 during the TTE100% following POMx supplementation may be due to a polyphenol-induced greater NO bioavailability. Despite the large amount of research indicating a relationship between a supplement-induced increase in NO and changes in VO2 during exercise, presumably through an increase in the efficiency of O2 transport, the mechanisms behind these changes are currently irresolute. Although NO is known to mediate vasoactivity, an overall increase in vasodilation does not explain the reduction in VO2 during submaximal exercise in normoxia which has been observed in other studies [18–22]. Rather, the predominant mechanistic theories to explain the effects of NO on VO2 involve an increase in either the efficiency of mitochondrial O2 usage or in the muscular use of ATP, affording a lower VO2 requirement to sustain a given work rate [21, 43]. However, during exercise under hypoxic conditions, a NO-induced augmentation in vasodilation has been shown to be important in maintaining blood flow to the active muscles [44]. Thus, it is likely that under hypoxic conditions, the vasoactive role of NO contributes more greatly to the level of VO2 which can be achieved during intense exercise. In accordance with previous research [26, 28], POMx did not affect [La−]b, indicating that any changes observed were probably not due to a change in fuel usage. Several reasons have been proposed for the lower efficacy of NO3 − supplementation on submaximal VO2 and performance in trained compared to untrained individuals. Firstly, as a response to training, athletes tend to have elevated NOS activity [45], and higher resting NO2 − and NO3 − values [46]. Consequently, they may have a lower requirement for NO3 −-NO2 −-NO pathway, and have sufficient NO3 − present in the blood to use it when needed. Further, training adaptations which aid in O2 transport and energy production, such as increased capillarization and mitochondrial density, may reduce the incidence of acidic and hypoxic muscular environments, which decrease NOS activity, and increase reliance on the NO3 − pathway [25, 47]. Finally, research in rats has suggested that the NO3 − pathway is predominantly used in type II muscle fibres, which work more frequently under acidic or hypoxic conditions [48]. If this is the case, NO3 − supplementation may be more effective in untrained individuals who tend to have a greater percentage of type II fibres [49]. However, currently, there is no direct evidence that type II fibres are preferentially affected in humans. Alternatively, the lack of overall performance response may be due to the presence of 'responders' and 'non-responders' to POMx. Despite no overall significant effect of POMx on performance in the current study, there were two participants who increased performance in both altitudes. Thus, a larger sample size of participants may be needed to determine the ratio of responders vs. non-responders in a highly-trained population. In addition to the results already presented in the discussion, the current study produced two results which are difficult to explain. Firstly, in contrast to previous research which has reported a lowering in SBP following acute BRJ or ET polyphenols from grapes in sedentary or moderately-trained individuals [11, 18, 50], and no effect on SBP following BRJ supplementation in highly-trained individuals [25, 27, 29, 38], this study found a trend towards an increase in SBP with POMx (+3.9 bpm, 95% CI, −0.6, 8.5, p = 0.08). The only explanation we can give for this result is that the increase in SBP was a reactive response by the vasculatare to ensure that the mean arterial pressure and thus, vessel perfusion and blood flow, were maintained, despite the NO-induced vasodilation. Further, being an acute intervention, we did not anticipate changes in Hct with POMx, but surprisingly, there was a significant decrease in Hct following POMx compared to PLAC (−0.76%, 95% CI, −1.3, −0.2), which indicated a small reduction in the O2-carrying capacity of the blood. However, despite being significant, this change had a small effect size (ES = 0.2) and considering the increase in VO2 with POMx at ALT, did not appear to affect VO2 capacity. Hct varies from day to day by ~3% [51] and it is possible that this result was due to differences in hydration between tests, as we did not measure pre-exercise hydration status, or standardize water intake during exercise. However both these explanations are purely speculative and further research is required to determine whether POMx supplementation consistently results in similar effects on SBP and Hct. A limitation to the current study is the relatively low altitude used in the hypoxic condition compared to the altitude generally associated with physiological changes (2500–3000 m) [52] and that simulated in previous studies involving NO3 − supplementation in hypoxia (~2500–5000 m) [28, 30, 31]. Due to our study being conducted in a mobile laboratory, we were restricted to areas with vehicle access in the North Island of New Zealand, and conducted the study at the highest altitude possible under these conditions. However, future studies could investigate the potential ergogenic benefit of POMx at a higher altitude of >2500 m. In addition, the current study was limited by a relatively small sample size (n = 8), which was unavoidable due to the research taking place during a high-performance junior training camp. While previous research has demonstrated a reduction in VO2 during submaximal cycling exercise [18, 21], it is acknowledged that the lack of effect of POMx on submaximal VO2 in the current study may have been due to an insufficient number of participants. BODY.CONCLUSION: In conclusion, acute POMx supplementation allowed a partial restoration of VO2 during intense exercise in a hypoxic environment. However, no significant changes in VO2 occurred during submaximal exercise and there was no effect of POMx on performance in either environment. Thus, the results from the current study do not support POMx as an ergogenic supplement when ingested acutely prior to exercise.

TITLE: Better Glasgow outcome score, cerebral perfusion pressure and focal brain oxygenation in severely traumatized brain following direct regional brain hypothermia therapy: A prospective randomized study ABSTRACT.BACKGROUND:: Induced hypothermia for treatment of traumatic brain injury is controversial. Since many pathways involved in the pathophysiology of secondary brain injury are temperature dependent, regional brain hypothermia is thought capable to mitigate those processes. The objectives of this study are to assess the therapeutic effects and complications of regional brain cooling in severe head injury with Glasgow coma scale (GCS) 6-7. ABSTRACT.MATERIALS AND METHODS:: A prospective randomized controlled pilot study involving patients with severe traumatic brain injury with GCS 6 and 7 who required decompressive craniectomy. Patients were randomized into two groups: Cooling and no cooling. For the cooling group, analysis was made by dividing the group into mild and deep cooling. Brain was cooled by irrigating the brain continuously with cold Hartmann solution for 24-48 h. Main outcome assessments were a dichotomized Glasgow outcome score (GOS) at 6 months posttrauma. ABSTRACT.RESULTS:: A total of 32 patients were recruited. The cooling-treated patients did better than no cooling. There were 63.2% of patients in cooling group attained good GOS at 6 months compared to only 15.4% in noncooling group (P = 0.007). Interestingly, the analysis at 6 months post-trauma disclosed mild-cooling-treated patients did better than no cooling (70% vs. 15.4% attained good GOS, P = 0.013) and apparently, the deep-cooling-treated patients failed to be better than either no cooling (P = 0.074) or mild cooling group (P = 0.650). ABSTRACT.CONCLUSION:: Data from this pilot study imply direct regional brain hypothermia appears safe, feasible and maybe beneficial in treating severely head-injured patients. BODY.INTRODUCTION: Despite current standards of treatment and care for the severely head injured patients, the desirable outcome for this patient group is hampered by the high morbidity and mortality rates. Conventional treatment routinely involves surgical evacuation of significant hematomas, efforts to restore and maintain adequate brain perfusion and prompt management of cerebral edema and raised intracranial pressure (ICP). Other treatment modality, in particular systemic hypothermia, had shown promising beneficial effect, though results appeared inconsistent in numerous trials.[1234567] Notwithstanding, induced hypothermia had also been reported in the management of stroke, hypoxic encephalopathy and seizures.[891011] The therapeutic basis of inducing hypothermia is supported by a neuroprotective effect on the brain following trauma or ischemic insults. Several mechanisms thought to underlie this effect which include the reduction in the metabolic rate and energy expenditure, attenuation in excitatory amino acids release and free radicals synthesis, suppression of excessive ischemia-induced and post-traumatic inflammatory reactions, and prevention of blood-brain barrier disruption and brain edema.[1213] Despite our greater understanding in the scientific basis of brain hypothermia, an optimal method of brain cooling remains an issue that needs to be vigorously studied. It is apparent that method of inducing hypothermia does influence the effectiveness of delivering the cooling effect onto the injured brain. Methodically, brain cooling can be divided into peripheral and core cooling.[14] In our opinion, the direct cooling effect on the injured brain via both methods could still remain suboptimal, and frequently limited by complications. Such complications include pneumonia, systemic infections and cardiac arrhythmias in a systemic body cooling, and focal soft tissue injuries over the head in a helmet cooling.[141516] Given so much controversy in inducing hypothermia for the injured brain, we sought to design a prospective, randomized pilot study to assess efficacy of new method in brain cooling called "direct regional brain hypothermia." In this article, we present our preliminary experience with direct focal or regional brain cooling, obtained via direct irrigation of cold fluid onto the surface of severely injured brain for trauma patients who require decompressive craniectomy with a Glasgow coma scale (GCS) of 6-7. BODY.MATERIALS AND METHODS.STUDY DESIGN: This was a randomized controlled trial, designed to assess the effect of direct regional brain cooling treatment in severely head injured patients. The study has been approved by our local research and ethics committee (USMKK/PPP/JEPem [225.3 (13)]). Patients referred to the Department of Neurosciences from January 1, 2010 to January 1, 2012 and fulfilled the criteria were recruited into the trial. The sample size was calculated using two proportions formula with alpha of 0.05 and power of 80% with expected total sample size of 36 patients. BODY.MATERIALS AND METHODS.SUBJECT CRITERIA: All patients with traumatic brain injury were screened prior to recruitment. The inclusion criteria were: (a) Age 12 and above; (b) severe head injury with GCS 6-7; (c) require decompressive craniectomy; (d) able to be followed-up after 6 months being discharged from the hospital and (e) consented by next of kins or guardians. Patients with the following criteria were excluded: (a) Penetrating brain injury; (b) significant drop in blood pressure (systolic blood pressure of <90 or diastolic blood pressure of <60 mmHg) and/or significant hypoxia prior to admission; (c) bilateral fixed and dilated pupils; (d) severe injury to other organ systems which may lead to marked morbidity or even mortality; (e) concomitant traumatic spinal cord injury; (f) known pre-morbid immune or neurological diseases; (g) severe head injury with only extradural hematoma, and (h) known pre-morbid condition prior to the accident, including history of seizures. All recruited patients were then randomized to either group A (cooling group) or B (no cooling or standard treatment group). BODY.MATERIALS AND METHODS.RANDOMIZATION AND THERAPY: Potential patients were identified and screened by the principal investigators who are also the treating surgeons (Z.I and M.S.Z). They explained the randomization process, surgical procedures and cooling method, required neuromonitorings, imaging and follow-up to all potential candidates' legal representative in details. Once they agreed to participate, informed consent was obtained, and the patient was randomized to one of the two-treatment arms: Cooling versus no cooling. Sealed envelopes, initially blinded to both consenting individuals (on patients' behalf) and clinicians containing either paper A (for cooling group) and B (for no cooling or standard treatment group) were randomly chosen. Group A consisted of patients who had therapy with direct regional brain cooling and group B consisted of patients who did not have direct regional brain cooling therapy. There was no blinding done after assignment to interventions. All recruited patients received monitoring for ICP, brain oxygen and brain temperature using ICP and Licox probes (GMS, Kiel-Mielkendorf, Germany). ICP probe was inserted into the ventricle or brain parenchyma whilst Licox probe for brain oxygen and temperature was placed into the damaged brain areas. In addition, the cooling group (group A) had bloods taken for immunological parameters pre- and post-cooling therapy. The studied immunological parameters were CD3, CD4, CD8, CD19, CD16, and 56, interleukins (IL-1), IL-6, tumor necrosis factor (TNF) and total white blood cells count. The duration for monitoring and period of cooling therapy was for at least 24 h, although in case-by-case basis, longer therapy and monitoring was considered for persistently raised ICP without obvious surgical lesion on repeated computed tomography (CT) images. The body temperature was also monitored during the treatment period. The recruited patients had CT brain prior to surgery and were categorized into different grade of severity of brain or whole body injury based on: (a) GCS; (b) Marshall score and (c) injury severity score (ISS). The unilateral decompressive craniectomy was the standard operation indicated for pathology causing midline shift, while bifrontal decompression was done for diffused pathology that causing cerebral swelling. The monitoring [Figure 1a] and therapies given after the surgery were the standard therapy for severely injured brain patients which include the following: (a) On ventilator support; (b) sedated with or without muscle paralysis agents; (c) draining of cerebrospinal fluid and/or hypertonic saline or mannitol therapy for persistently raised ICP of >20 mmHg; and (d) thiopentone coma therapy as a final step to treat postoperative refractory intracranial hypertension. For the cooling group (group A), direct regional brain cooling therapy was given after decompressive craniectomy [Figure 1b] by persistent irrigation of the swollen brain with a cold Hartmann's solution. The temperature range of initial infused fluid was used as the basis to analyze and to further divide Group A into two groups: (1) Deep cooling at a temperature of 20-29°C and (2) mild cooling at a temperature of 30-36°C. The cold infusion was achieved via neurojaf external ventricular drainage (EVD) with multiple side-holes catheter, which was inserted superior to the dura flap and at the inner surface of the dura, sprinkled onto the surface of the swollen brain. The catheter was in contact with the surface of the brain. The 500 ml of Hartmann's solution infusion rate was scheduled within 7 h (70 ml/h). Owing to patients' head position setting in the Intensive Care Unit, a second larger draining tube was inserted at the lower part of the craniectomy flap outside the dura, which was loosely closed to drain the excess fluid with a low suction pressure. The temperature of the infused Hartmann's solution was regularly monitored through the three way connector draining the fluid out to the collection port for temperature assessment. If the drained solution's temperature was under or above the intended value (s), the preceding infusion was replaced by a new solution with the correct intended temperature. An immediate CT scan of the brain was indicated if patients' ICP showed persistently raised values despite standard therapies being given. This was to exclude any new surgical lesions and/or the retention of infused solution as a cause of raised ICP. However, if the ICP hold within normal values, the CT scan was repeated after 48 h of therapy. Figure 1(a) Neurointensive care monitoring and therapy for patients in this study. (b) External ventricular drainage with multiple side-holes for surface irrigation of the brain with cold solution BODY.MATERIALS AND METHODS.OUTCOME MEASURES: The assessment of outcomes was performed through a dichotomized Glasgow outcome score (GOS) at discharge and mainly at 6 months after trauma as: (a) Good neurological outcome group (GOS 4 and 5), and (b) poor neurological outcome group (GOS 1, 2 and 3). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Data entry and analysis was done using Statistical Package for Social Sciences (SPSS; IBM, Chicago, Illinois USA) version 18.0. The level of statistical significant was set at 0.05 in a two-tailed fashion. In the descriptive analysis, the frequency, percentages, mean, median and confidence interval (CI) were reported for numerical variables. For inferential statistical analysis, the following tests were used for each objective: (a) Pearson Chi-squared test was used to compare the dichotomized GOS (good and poor) at discharge and at 6 months post-therapy for the studied groups, (b) general linear model for repeated measures ANOVA was used to calculate and depict the trends for ICP, cerebral perfusion pressure (CPP), brain oxygenation and brain-body temperature and their gradients, (c) trends and Wilcoxon signed rank test for comparison of immunological parameters, before and after cooling therapy, (d) Spearman rank correlation coefficient was used in correlation analysis between studied parameters with outcome scores and (e) Kruskal Wallis test comparing the studied groups for complication. BODY.RESULTS.DEMOGRAPHIC AND CLINICAL CHARACTERISTICS: A total of 32 patients were recruited into the 2 years study period from 1st January 2010 to 1st January 2012, aged between 14 and 74 years old. Of these, five patients aged more than 60 years old (19.2%). There were 27 (84.4%) males and 5 (15.6%) females. Patients were randomized into 2 groups; cooling group consisted of 19 patients (59.4%) and no cooling group consisted of 13 patients (40.6%). Finally, further stratification of cooling group based on the temperature of infused solution created 3 analyzable groups; mild cooling consisted of 10 patients (31.3%), deep cooling of 9 patients (28.1%) and no cooling of 13 patients (40.6%). The mean ages of the recruited patients were 28.9, 26.7 and 45.5 years old for the mild cooling, deep cooling and no cooling groups, respectively. Fifteen patients were admitted with GCS of 6 (46.9%) and 17 patients with GCS of 7 (53.1%). Among these, 28 patients (87.5%) underwent unilateral decompresive craniectomy, while four patients had bifrontal decompressive craniectomy (12.5%). Table 1 illustrates those parameters where all groups were comparable with non-statistical difference shown for gender, GCS, ISS, Marshall score and clotting profiles. Age was the only basic parameters that differed among the three studied groups (P = 0.02). Even though the highest mean age was found in the control group, the 95% CIs (95% CI) for all three groups were still within the age of <60 years old. Table 1 Basic parameters comparison among the 3 studied groups BODY.RESULTS.EFFECT OF REGIONAL BRAIN COOLING ON GLASGOW OUTCOME SCORE: There was a strong significant difference at 6 months post-trauma outcomes with P = 0.007 between the two studied groups: Cooling versus no cooling [Table 2]. There were 63.2% of patients (12 patients) in cooling group attained good GOS at 6 months compared with only 15.4% in non-cooling group (2 patients). There was no significant difference between the two groups when outcomes analysis was made at time of discharge. Further analysis at 6 months post-trauma was made after stratifying the cooling group into 2: Mild and deep cooling. Table 3 disclosed presence of significant difference among the three analyzed groups at 6 months post-trauma (P = 0.023). When comparison was only made between 2 groups (no cooling vs mild cooling; no cooling vs deep cooling and mild cooling vs deep cooling), it seems that the mild-cooling-treated patients fared better than no cooling (70% of mild cooling attained good GOS compared with only 15.4% attained good GOS in no cooling group, P = 0.013); and apparently, the deep-cooling-treated patients failed to be better than either no cooling (P = 0.074) or mild cooling group (P = 0.650). Table 2 Effect of regional brain cooling on GOS at discharge and at 6 months Table 3 Effect of regional brain cooling on GOS at 6 months after stratifying the cooling group into mild and deep cooling BODY.RESULTS.EFFECT OF REGIONAL BRAIN COOLING ON TRENDS OF INTRACRANIAL PRESSURE, CEREBRAL PERFUSION PRESSURE, BRAIN OXYGENATION, BRAIN AND BODY TEMPERATURE AND BRAIN-BODY TEMPERATURE GRADIENT: All recruited patients did have ICP monitored but there were one patient in deep cooling, two patients in mild cooling and three patients in no cooling groups did not have neuromonitoring for focal brain oxygenation (PtiO2) and temperature due to unavailability at certain time of the study period (26 patients did have those two specific neuromonitorings). Cooling the severely injured brain which had underwent decompressive craniectomy did not reduce the ICP further, but instead shown marked improvements in CPP and Licox PtiO2 after 12 h of cooling. Figure 2 discloses mean ICP values and patterns for the three studied groups. All ICP readings stayed below 25 mmHg, and marked fluctuations were noted in cooling groups. Figures 3 and 4 reveal mean values and trends for CPP and Licox PtiO2, respectively. Mild cooling group had ascending trends of CPP with mean values above 60 mmHg but <75 mmHg together with PtiO2 mean values of above 40 mmHg after 12 h of cooling therapy. Interestingly, the deep cooling group did have similar ascending trends for CPP and PtiO2 but their mean values after 12 h of cooling therapy were above 75 mmHg and <30 mmHg, respectively. Figure 5 gives additional information on brain-body (axillary) temperature gradient and types of cooling therapy. Patients in mild cooling group appeared to have a larger difference in brain-body (axillary) temperature gradient. Figure 6 shows, even during brain cooling therapy for severely injured brain, the brain temperature is still higher than body (axillary) temperature. Figure 2Trends of mean intracranial pressures for three different groups within 48 h of monitoring and therapy Figure 3Trends of mean cerebral perfusion pressures for three different groups within 48 h of monitoring and therapy Figure 4Trends of mean focal brain oxygenation for three different groups within 48 h of monitoring and therapy Figure 5Trends of mean difference between brain and body temperature (brain – body temperature) for three different groups within 48 h of monitoring and therapy Figure 6Brain temperature is higher (0.1-2°C higher) than body (axillary) temperature. The figures depicted in such way that the scales were aligned and therefore, the figures were comparable BODY.RESULTS.EFFECT OF REGIONAL BRAIN COOLING ON IMMUNOLOGICAL BIOMARKERS: Due to inadequate volume of blood taken for immunological analysis for either before or after cooling therapy, immunological data of three patients in mild cooling and one patient in deep cooling were excluded from the analysis. Trend analysis of seven patients in mild cooling and eight patients in deep cooling showed obvious decrements in values for both, T cell markers and pro-inflammatory cytokines after cooling therapy. Intriguingly, markers for pro-inflammatory cytokines (IL-1, IL-6 and TNF) did show more marked decrement than markers for T-cell [Table 4]. Despite of marked decrement in most studied immunological biomarkers after cooling therapy, the Wilcoxon Signed Ranked test for two related samples disclosed insignificant difference between pre- and post-cooling for all immunological parameters. This presumably related to our small sample size, which incapable to detect significant differences in those studied immunological parameters. Table 4 Effects of regional brain cooling (both mild and deep cooling groups combined together) on immunological parameters BODY.RESULTS.CORRELATION BETWEEN STUDIED PARAMETERS WITH GLASGOW OUTCOME SCORE AT 6 MONTHS POST-TRAUMA: Correlation analysis using Spearman rank correlation coefficient discloses age (r = 0.46) and Licox brain oxygenation (r = 0.40) as the only two studied parameters that reasonably correlated with outcome score at 6 months post-trauma. Table 5 illustrates the results. Table 5 Correlation analysis between studied parameters with outcome score at 6 months BODY.RESULTS.INCIDENCE OF COMPLICATIONS: Of the 32 patients, 50% (n = 16) had developed complications as shown in Table 6. The complications included wound infection in 21.9% (n = 7), CSF infection in 9.4% (n = 3), pneumonia in 6.25% (n = 2), hydrocephalus in 3.12% (n = 1) and brain infarct in 9.4% (n = 3). The number of patients who developed complications was higher in no cooling, and deep cooling groups compared with mild cooling group. Kruskal Wallis non-parametric statistical test comparing the three groups for complication disclosed insignificant difference with P = 0.405. In conclusion, no significant difference is found in the incidence of complication among the three groups. Table 6 Association between treatment groups and incidence of complication BODY.DISCUSSION.PATHOPHYSIOLOGY OF BRAIN HYPOTHERMIA, METHOD OF COOLING AND SAFETY ISSUE: Induced hypothermia for the treatment of traumatic brain injury remains a moot point. Therapeutic hypothermia was first studied in 1943 and since then, many studies reported inconsistent clinical results that dissuade its adoption as a routine evidence-based practice.[1234561718] Our current, in-depth understanding on the pathophysiology of secondary brain injury is limited, but many animal studies have shown benefits of induced hypothermia for the injured brain.[19202122] Poldermann published a review article on this issue and highlighted the benefits of induced hypothermia on the injured brain.[23] He noted pathways that promote cerebral ischemic-hypoxic events which can lead to cell death or apoptosis are mostly temperature dependent and, therefore, can be mitigated with cerebral hypothermia. The proposed neuroprotective mechanisms include: (a) Hypothermia can inhibit the activation of caspase enzymes, (b) prevents or mitigates mitochondrial dysfunction, (c) decreases the metabolism as well as decrease the overload of excitatory neurotransmitters such as glutamate and free oxygen radicals, (d) modifies the cellular disorders of intracellular ion concentrations, (e) suppresses the inflammatory and immunological responses and epileptic activity, (f) reduces the disruption in blood brain barrier, vascular permeability and edema, (g) improves the microcirculatory circuits and intra- and extra-cellular acidosis, (h) corrects the hyperthermia after brain injury and influence the local secretion of various vasoactive mediators secreted by the endothelium, and (i) enhances expression of immediate early genes and cold shock proteins. Building on the vast benefits of cerebral hypothermia, we did a prospective study to test the null hypothesis, "cerebral hypothermia has no benefit on severely injured traumatic brain with initial GCS of 6 and 7". In the design of our study, several important issues related to induced hypothermia were put into context. First, the area of injury should be cooled. Therefore, brain cooling is the target, not body or scalp cooling. This was thought possible in patients who had decompressive craniectomy and irrigating the brain surface with cold Hartmann's solution and with second drainage tube situated outside the dura which was loosely closed. Furthermore, our experience from endoscopic intraventricular neurosurgery, by routinely irrigating the brain with a large amount of fluid seems safe, without inducing high rate of infectious complications. Second, brain temperature can be different from measured temperature of cooling method or therapy given to the patients. Statement saying "the brain was cooled to certain temperature degree needs to be stated cautiously." Measured brain temperature depends on many factors, including the location or site of the measuring tip as the inflamed area of the brain would manifest higher brain temperature than non- or less-inflamed brain area.[24] Third, by applying the concept of increase brain pulsation during compensatory phase of raise ICP, the irrigated regional fluid hopefully will be distributed to other brain regions via principles of vascular and brain pulsations.[25] Finally, induced hypothermia for the brain can be made below 32°C. An animal study by Oku et al. in 2009, disclosed focal brain cooling above 0°C did not induce irreversible histological change or cortical damage.[22] This study was carried out based on these four aspects of brain hypothermia and found the complication rate was indeed no difference between the cooling and control groups [Table 6]. BODY.DISCUSSION.GROUP COMPARISON: It is essential to ensure the studied groups were comparable. Table 1 reveals only age has a significant difference between the studied groups. Mean age for no cooling or standard treatment group was higher than cooling groups. Nonetheless, the 95% CIs for those groups were still within non-elderly category. In terms of other parameters: Gender, GCS, ISS, Marshall score and clotting parameters, these were comparable. We purposely limited patient's inclusion to those with severely head injured patients with GCS 6 and 7 only. Since one of our main objectives is to study the true effect of cerebral hypothermia on outcomes, we thought by restricting patients to GCS 6 and 7, we can eliminate other prognostic indicators that may greatly influence the outcomes such as poor motor responses (decerebrate or decorticate posturing). By doing so, we can ascertain the "true benefit of brain cooling". BODY.DISCUSSION.PROVEN BENEFITS AND REASONS: Patients in cooling group had a higher percentage of good GOS and statistically had a significant difference at 6 months post-trauma when compared to non-cooling group. This finding may conclude that cooling the injured brain has indeed the potential benefits. The benefits of brain hypothermia for the severely injured brain can be explained based upon the pathophysiology of secondary brain injury as discussed above and also because of our method of cooling-our study applied direct regional brain cooling which means the cooling effect was directed toward the region of interests and perhaps via CSF pulsation, the residual cooling effect might have been distributed to the whole brain surfaces. This direct method of brain cooling is currently thought feasible for patients who had decompressive craniectomy. Further analysis on cooling-treated patients after stratifying them into two groups disclosed interesting findings: (a) Mild cooling group, treated at temperature of 30-36°C had fared better 6 months outcomes when compared to no cooling group and b) there was no significant difference for the 6 months post-trauma outcomes when the deep cooling group (temperature ranges from 20°C to 29°C) was compared to the non-cooling group. This signifies mild cooling-treated patients has the most significant benefits; and despite no apparent difference in the incidence of treatment complications, the deep cooling group surprisingly failed to be equally better as mild cooling group. The beneficial effects of mild brain cooling at temperature 30-36°C can partly be explained by our monitored parameters as discussed below. BODY.DISCUSSION.MONITORED AND STUDIED PARAMETERS; AND BRAIN COOLING: Various study on hypothermia did notice reduction in ICPs, improvement in CPPs or PtiO2 after cerebral hypothermia, which translated to better outcomes.[262728] Since our study recruited only trauma patients with severe head injury and required decompressive craniectomy, reduction in ICP was not expected to be a major determinant correlated with outcomes, obviously because decompressive craniectomy itself has been shown to cause significant reduction in ICPs.[293031] This could also be a reason why, majority of our treated patients with direct regional brain cooling had induced cerebral hypothermia for period up to 48 h only (short period). Besides ICP, cerebral blood flow (CBF) is another important parameter that must be optimized during monitoring the severely injured brain patients in neurointensive care. Interesting to note, the cooling-treated patients did have ascending or improved trends in CPP and brain oxygenation [Figures 3 and 4]. These two parameters could indirectly reflect the status of CBF. The mild cooling group did have mean CPP within accepted range of 60-75 mmHg [Figure 3] and mean PtiO2 ranged from 35 to 48 mmHg [Figure 4] which is within normal range for a normal individual and proven to correlate well with outcome score [r = 0.4; Table 5]. As noted in other studies, reduction in ICP and optimal CBF parameters could have been contributed to the best long term outcomes for our mild-cooling-treated patients compared to deep- and no-cooling-treated patients. Since the neuroinflammatory and metabolic responses happened inside and on the surface of the brain parenchyma, the irrigated cold fluid acts as an exogenous factor to exert mitigating effects on those responses. Therefore, the temperature of the brain should be higher and not be the same as the temperature of the irrigated fluid. This study disclosed that the intended temperature of induced cerebral hypothermia with direct regional brain cooling obviously did not correlate with brain temperature. One new observation noted from this study is brain-body temperature gradient; in mild cooling group, great gradient exists between brain-body temperature at 2-4 h after the therapy, whereas in deep cooling group, the gradient seems to become greater only after 24 h of cooling when both were compared with no cooling group [Figure 5]. The importance of this observation is not yet known and, therefore, need to be reconfirmed by other studies. Finally, findings in Figure 6 proved the previous reported study on brain temperature, which was always higher than the body temperature [0.5-1.5°C; Figure 6].[2431] Severe brain injury will induce significant and protracted inflammatory responses, beginning approximately 1 h after the injury and continuing for several days. Pro-inflammatory mediators such as IL-1, IL-6 and TNF are released in large quantities by astrocytes, microglia and endothelial cells. These inflammatory responses can cause significant additional brain injury via synthesis of various toxic products, activation of complements and further stimulating immune reactions in what may become a vicious circle.[121432] In our study, direct regional brain hypothermia seems to suppress all these excessive inflammatory responses. The obvious benefit noted in this study was marked suppression of pro-inflammatory cytokines such as IL-1, IL-6 and TNF [Table 4]. Therefore, our findings are in agreement with previous animal and clinical studies, which showed hypothermia decreases the levels of pro-inflammatory cytokines following brain injury.[333435] Nonetheless, to know the true effect of regional brain cooling on immunological biomarkers, one needs also to consider levels in non-cooling group, which was not done in this study. BODY.DISCUSSION.LIMITATION AND RECOMMENDATION: The limitation of this pilot study lies with its small sample size, and irrespective of patient randomization, recruitment of younger patients in the cooling group. Despite of the above mentioned drawbacks, this study could provide a nice foundation for future work on direct hypothermia, which can explore benefits in terms of GOS or other scoring outcomes. Future study to prove the clinical benefits of the present technique clearly need multicenter trials before inferring recommendation for the therapy as a standard of care. In addition, it is also important to consider a longer cooling period of >48 h even in the presence of normalized intracranial pressure and to do future study for patients even with GCS of <6. BODY.CONCLUSIONS: This preliminary or pilot study found that direct regional brain hypothermia may have potential benefits in treating the severely head injured patients with initial GCS of 6 or 7. Other than a safe and practicable approach, this direct regional brain cooling therapy may serve as an added therapy for patients who require urgent decompressive craniectomy, irrespective of the underlying etiologies in the future.

TITLE: The usability and preliminary effectiveness of a web-based physical activity intervention in patients with knee and/or hip osteoarthritis ABSTRACT.BACKGROUND: A large proportion of patients with knee and/or hip osteoarthritis (OA) do not meet the recommended levels of physical activity (PA). Therefore, we developed a web-based intervention that provides a tailored PA program for patients with knee and/or hip OA, entitled Join2move. The intervention incorporates core principles of the behaviour graded activity theory (BGA). The aim of this study was to investigate the preliminary effectiveness, feasibility and acceptability of Join2move in patients with knee and/or hip OA. ABSTRACT.METHODS: A non-randomized pilot study was performed among patients with knee and/or hip OA. Primary outcomes were PA (SQUASH Questionnaire), physical function (HOOS and KOOS questionnaires) and self-perceived effect (7-point Likert scale). Baseline, 6 and 12 week follow-up data were collected via online questionnaires. To assess feasibility and acceptability, program usage (modules completed) and user satisfaction (SUS questionnaire) were measured as secondary outcomes. Participants from the pilot study were invited to be interviewed. The interviews focused on users' experiences with Join2move. Besides the pilot study we performed two usability tests to determine the feasibility and acceptability of Join2move. In the first usability test, software experts evaluated the website from a list of usability concepts. In the second test, users were asked to verbalize thoughts during the execution of multiple tasks. ABSTRACT.RESULTS: Twenty OA patients with knee and/or hip OA between 50 and 80 years of age participated in the pilot study. After six weeks, pain scores increased from 5.3 to 6.6 (p=0.04). After 12 weeks this difference disappeared (p=0.5). Overall, users were enthusiastic about Join2move. In particular, performing exercise at one's own pace without time or travel restrictions was cited as convenient. However, some minor flaws were observed. Users perceived some difficulties in completing the entire introduction module and rated the inability to edit and undo actions as annoying. ABSTRACT.CONCLUSIONS: This paper outlines the preliminary effectiveness, feasibility and acceptability of a web-based PA intervention. Preliminary results from the pilot study revealed that PA scores increased, although differences were not statistically significant. Interviews and usability tests suggest that the intervention is feasible and acceptable in promoting PA in patients with knee and/or hip OA. The intervention was easy to use and the satisfaction with the program was high. ABSTRACT.TRIAL REGISTRATION: The Netherlands National Trial Register. Trial number: NTR2483 BODY.BACKGROUND: Osteoarthritis (OA) in the knee and hip is a degenerative joint disorder with a high prevalence that increases with age. The disease is associated with pain, functional disability and impaired quality of life [1,2]. OA is considered one of the major disabling diseases in the western world, affecting 10% of men and 18% of women over the age of 60 [3]. It has been recognized that regular physical activity (PA) is an effective lifestyle strategy in the management of OA [4-6]. However, to date the vast majority of OA patients remain sedentary [7-9]. In the long term, physical inactivity may lead to functional decline [10,11]. To maintain and improve physical function, the promotion of PA is a cornerstone in the treatment of OA [12]. Since general practitioners (GP) are considered the first and main point of contact for people with OA, the general practice is ideally situated to promote PA. In practice, however, a GP's ability to encourage physical exercise is limited by time constraints and lack of standard protocols [13,14]. In particular, core elements concerning the risks of sedentary behaviour are insufficiently emphasized. At the same time, it is unlikely that OA patients will receive help elsewhere, since 90% are not referred to other health care professionals such as a physical therapist, orthopaedic doctor, rheumatologist or rheumatology trained nurse [15]. In this study, we call this group 'outside-care patients' and define them as those patients who did not have 'face-to-face' contact with a health care provider, other than a GP, for OA in the last six months. The World Wide Web provides an alternative medium for reaching outside care patients. In Europe 61% and in North America 79% of the population have internet access [16]. Although the rate is lower in younger age groups [17], recent trends show that older people are among the fastest-growing internet users. To illustrate, in the Netherlands 95% of adults (55–65 years) and 75% of older adults (65–75 years) have access to internet in their home [18]. The internet is convenient, anonymous and appealing for those who want to work in their own environment and in their own time [19]. In particular, web-based interventions without the involvement of professionals have the potential to reach large populations, with a minimal burden on scarce health resources [20]. In recent years, several reviews reported that web-based interventions can be effective in promoting PA. Internet programs for patients with diabetes [21], multiple sclerosis [22] and heart failure [23] have led to the improvement of PA outcomes, even though effect sizes are small. Considering the potential of high reach and low costs [19], even these small effect sizes have large public health consequences. Given the advantages of the internet and its unique ability to reach outside care OA patients, we developed Join2move. Over the course of one year, we used an iterative design methodology to test, analyse and refine the Join2move program. As part of the iterative development process, this paper focuses on the preliminary effectiveness and the usability of Join2move. BODY.BACKGROUND.JOIN2MOVE: Development was based on a systematic review [24] and a previously developed Behavioral Graded Activity (BGA) intervention [25]. The framework of the BGA program incorporates a baseline test, goal setting, time-contingent PA objectives (i.e. on fixed time points) and text messages to promote PA. An essential feature of the BGA program is the positive reinforcement of gradual PA, despite the presence of pain. The gradual increase in activities changes the perception that PA is related to pain and reinforces confidence to improve PA performance. This may lead to positive physical (e.g. physical capacity, muscle strength and joint mobility) and psychological changes (e.g. self-esteem, pain perception and anxiety). Due to the highly structured format of the BGA intervention, the internet constitutes a promising platform for translating BGA into a self-help format. The Join2move intervention is a fully-automated web-based intervention which contains automatic functions (automatic text messaging and automatic e-mails) without human support. Participants are initially presented with the homepage http://www.artroseinbeweging.nl. The password-secured PA program is available 24/7 from the homepage and is provided without charge. In keeping with the BGA treatment, the Join2move intervention is a self-paced nine week PA program in which patients' favourite recreational activity is gradually increased in a time-contingent way. In the first week of the program, users select a central activity (e.g. cycling, walking or gardening), perform a 3-day self-test and determine a short term goal for the next eight weeks. Based on test performances and a short term goal, eight tailored weekly modules are automatically generated. Every week, new weekly assignments and evaluation forms (pain and performance) are posted on the password-secured website. If a scheduled weekly module is missed, users can choose to repeat the module, adapt the difficulty or continue with the next module. Since personal messages are updated on a weekly basis, users are encouraged to log in once a week. Automatic e-mails are generated if participants do not visit the website regularly. A description of the intervention is provided in Table 1. Table 1 Description of the Join2move intervention     1. Filling out a PA Readiness Questionnaire (PARQ) If participants answered “YES” to any of the seven PARQ, they were advised to see their GP before participation. If patients answered ‘NO’ to all of the questions, it was considered safe for them to engage in Join2move. 2. Provision of educational messages Core elements of the program are presented on the personal website, including 1) focus on improving physical function rather than pain reduction; 2) first weeks can be accompanied by more pain;3) participant shares responsibility and has an active role. 3. Selection of a central PA A favourite and a problematic activity are selected from an activity list, including walking, cycling, swimming etc. 4. Determination of baseline value via a 3-day self-test To determine the baseline value, participants were requested to perform the selected activity three times a week until the pain threshold was reached. PA performances (minutes) and pain scores (1 to 10) were recorded in an online diary and stored on the website. 5. Setting a short and long term goal In accordance with the baseline values, a range of goals is generated and presented on the website. Between the lower and upper limit of goals, patients could select a short term goal (9 weeks). Furthermore, a long term goal was set for 1 year. 6. Signing an agreement form Participants sign an online agreement form. This form presents the short term goal and, again, core elements of the program. 7. Gradually increase selected activity (8 weekly modules) Based on the short term goal, a tailored schedule of eight weekly modules is made on a time-contingent basis (i.e. fixed time points). The start of the schedule is slightly below the baseline value and increases incrementally towards the short term goal. Patients should not under-perform or over-perform this gradually increasing schedule. Every week, new modules and evaluation forms (pain and performance) are posted. GP general practitioner, PA physical activity. BODY.BACKGROUND.OBJECTIVES: Extensive exploration is needed in order to examine the potential of the Join2move program. Consequently, our research question was:" What is the preliminary effectiveness (PA, physical function and self-perceived effect), feasibility and acceptability of Join2move in patients with knee and/or hip OA?" "Feasibility" concerns whether we are capable of carrying out Join2move in a larger study. "Acceptability" is whether participants support or reject Join2move. BODY.METHODS.PILOT STUDY.STUDY DESIGN AND OBJECTIVE: This pilot study used a non-randomized design. Our primary focus was to determine the preliminary effectiveness of the Join2move intervention. A second purpose was to determine program use and user satisfaction with the Join2move intervention. This pilot study, which aimed to provide a basis for a large Randomized Controlled Trial (RCT), was part of a research protocol which has been approved by the ethics committee of the VU University Medical Center Amsterdam (Dutch Trial Register NTR2483). BODY.METHODS.PILOT STUDY.PARTICIPANTS: Patients with self-reported knee and/or hip OA were recruited through advertisements in Dutch newspapers and online health-related websites. Eligibility criteria were 1) age 50–80; 2) self-reported OA in knee and/or hip; and 3) no physical therapy and/or treatment from a medical specialist for OA in the last six months. Potential participants were excluded if they 1) had no internet access at home, 2) were unable to understand the Dutch language and 3) had contra-indications (loss of consciousness and cardiovascular disease) for PA without medical supervision. To verify self-reported diagnosis, we performed clinical tests to assess the presence of knee and/or hip OA. Assessments were performed by a physiotherapist after the study period, according to the American College of Rheumatology (ACR) [26,27]. BODY.METHODS.PILOT STUDY.PROCEDURE AND MEASURES: Interested patients who met the inclusion criteria were sent an invitation letter requesting informed consent. Once written informed consent was obtained, participants were invited to fill out a baseline questionnaire. After the baseline assessment, participants were assigned to the intervention. We conducted two online posttests at 6 and 12 weeks after baseline. BODY.METHODS.PILOT STUDY.PRELIMINARY EFFECTIVENESS: To assess the potential effectiveness of the Join2move intervention, primary outcome measures in this study were PA, physical function and self-perceived effect. Secondary outcomes were OA symptoms, sport and recreation and quality of life. The first primary outcome, self-reported PA, was measured by the Short Questionnaire to Assess Health-enhancing PA (SQUASH) [28]. Pain scores and physical function were determined through a 10- point likert scale as well as the subscale pain of The Knee Osteoarthritis Outcome Score (KOOS) [29,30] and the Hip Injury Osteoarthritis Outcome Score (HOOS) [31,32]. The three secondary outcomes, symptoms, sport and recreation activity and quality of life, were also collected by using the HOOS and KOOS questionnaire. Descriptive statistics were used to analyse the data. Paired sample t-tests and regression analysis were used to determine the significance of the differences. BODY.METHODS.PILOT STUDY.FEASIBILITY AND ACCEPTABILITY: To assess the feasibility and acceptability of the intervention, program usage and user satisfaction were measured as secondary outcomes. Program usage was measured by the number of weekly modules completed. Once a participant read the weekly assignments and filled out the evaluation form, the module was defined as completed. Adequate exposure to the program was achieved if users interacted at least 75% with the program content. This cut-off point was determined by the research team on the basis of previous research [33]. User satisfaction was measured via the System Usability Scale (SUS) [34]. Besides the usage and satisfaction, patients from the pilot study were invited for interviews to test user experiences. Semi-structured interviews were audio-recorded and transcribed with the interviewee's permission. An interview guide with open questions was employed to provide structure to the interviews. Transcribed texts were read and discussed to gain an overall understanding of the usability and user satisfaction. BODY.METHODS.USABILITY TESTS.PARTICIPANTS: Two qualitative tests were performed to determine the usability of the Join2move intervention, viz.,1) heuristic evaluation, and 2) the Thinking Aloud approach. For the heuristic evaluation, four software experts from Netherlands Institute for Health Services Research (NIVEL) were invited to participate. With respect to the Thinking Aloud approach, five patients between the ages of 50–80 years with self-reported knee and/or hip OA were recruited via the Dutch Arthritis Foundation. The sample size for the Thinking Aloud approach was based on previous research by Nielsen [35]. The author claims that five users are enough to catch 85% of the usability problems. BODY.METHODS.USABILITY TESTS.PROCEDURES AND MEASURES: The first usability test, the heuristic evaluation, was performed by means of a set of usability criteria created by Jakob Nielsen [36] and Dana Chisnell [37]. Nielsen [38] described heuristic evaluation as an informal method of usability testing that consists of a number of evaluators who are presented with an interface design and are then asked to comment on the errors and effectiveness of the product. Heuristics includes concepts such as "Does the system behave consistently?", "Does the site use words that older adults know?", "Is the program perceived as helpful?"(see Appendix 1 for the full list of heuristics). Software experts individually evaluated the website, based on the list of heuristics. Subsequent discussion yielded a list of usability issues. The second instrument, the Thinking Aloud approach [39], was used to consider how end-users interact with the intervention. In a home-based setting, test subjects were encouraged to verbalize their thoughts during the execution of multiple tasks. These tasks represented the major functionality of the intervention. Evaluations were carried out by two moderators. The procedure was video-recorded and transcribed afterwards. BODY.RESULTS.PILOT STUDY.PARTICIPANTS: Of the 47 registered patients, fifteen (32%) did not meet the inclusion criteria. Reasons for exclusion were: no OA symptoms (n=3); receiving treatment from a physical therapist for OA (n=2); OA in other joints than knee or hip (n=7); and not meeting the age criteria of 50–80 years (n=3). Furthermore, seven (15%) participants did not return the informed consent document and five (11%) participants withdrew after returning informed consent. A total of twenty (42%) participants were finally included. Sixteen (80%) participants agreed to be interviewed. According to the ACR criteria, thirteen of the sixteen participants (81%) had clinical knee and/or hip OA, and three participants (19%) had no OA. Participants' demographic characteristics are shown in Table 2. Table 2 Demographic and clinical characteristics Participants (N, %)     Gender     Male 5 25 Female 15 75 Age (years, SD) 64 6.6 Location OA (N, %)     Knee 7 35 Hip 5 25 Knee and hip 8 40 Duration OA symptoms (years, SD) 9.3 11.4 OA, osteoarthritis; SD, standard deviation. BODY.RESULTS.PILOT STUDY.PRELIMINARY EFFECTIVENESS: PA results at baseline, six weeks and twelve weeks are given in Table 3. Over the twelve week period, the total time spent on PA increased from 1,697 to 2,044 min/week, and the time spent on moderate intensity increased from 323 to 553 minutes a week. These results, did not however, attain statistical significance (p= 0.3 and p=0.43, respectively). At 6 weeks, patients did report significantly higher levels of pain compared to the baseline - from 5.3 to 6.6 (p=0.04). After twelve weeks the differences were no longer statistically significant (p=0.5). With regard to physical function, a small, non-significant increase was observed (Table 4). Table 3 Comparison of change in PA levels (mean and SD) PA (mean, SD) Baseline Baseline Baseline   (n=20) (n=20) (n=20) Total PA (min) 1697 (1174) 2108 (1206) 2044 (1369) Moderate PA (min) 323 (330) 539 (549) 553 (673) Pain (0–10) 5.3 (1.7) 6.6 (2.0)* 5.2 (1.8) *p<0.05 compared with baseline. PA Physical Activity. For (moderate) PA a higher score indicates an improvement. For pain, a lower score indicates an improvement. Table 4 HOOS and KOOS scores (mean and SD)   HOOS HOOS HOOS KOOS KOOS KOOS   baseline 6 weeks 12 weeks Baseline 6 weeks 12 weeks Pain (0–100) 54.2 (19.2) 55 (16.0) 59.3 (17.1) 45.6 (18.5) 47.8 (17.4) 49.1 (15.1) Symptoms (0–100) 49.6 (16.5) 48.9 (13.7) 58.8 (16.2) 61 (16.8) 55.2* (16.0) 62.6 (14.9) ADL (0–100) 53.2 (20.3) 49.2 (14.9) 54.9 (17.4) 46.8 (20.1) 46 (14.9) 47.5 (20.6) Sport (0–100) 33.3 (23.4) 18.8* (18.0) 45.1 (33.9) 18.2 (16.1) 16.3 (18.6) 15 (19.1) QOL (0–100) 37 (18.8) 38.5 (13.7) 41 (12.9) 27.9 (17.7) 32.9 (14.1) 34.1 (12.0) p<0.05; HOOS/KOOS, The Hip/Knee Osteoarthritis Outcome Score; ADL, activities of daily life; QOL, Quality of life. For all outcomes a higher score indicates an improvement. BODY.RESULTS.PILOT STUDY.FEASIBILITY AND ACCEPTABILITY: The majority of participants (n=12, 60%) selected walking as the central activity. Other selected activities were floor exercises (n=3, 15%), cycling (n=1, 5%), domestic tasks (n=1, 5%), gardening (n=1, 5%), and rowing (n=1, 5%). A total of twenty participants commenced the intervention with the program introduction. Login-file analyses revealed that 100% (n=20) of the users completed the introduction module. Overall, 55% (n=11) of the participants completed at least 75% of the program (≥7 week assignments). 70% (n=14) achieved 60% program exposure and 30% (n=6) were exposed to at least 30% of the intervention. The exposure percentage declined over time. The most listed reasons for skipping a weekly PA were other commitments or of lack of time. Adverse events, such as extreme pain or injuries, were not reported during the program. The 16 interviews revealed that performing the activities in one's own time and at one's own pace was regarded as convenient. In general, participants perceived the website as an additional motivation to perform PA. However, the interviews also revealed an important usability issue. It became clear that patients were dissatisfied with the rigid character of Join2move. As one user commented "When I skipped my weekly PA exercise due to other commitments, I had no opportunity to repeat that exercise. That was frustrating". The results from the SUS among 15 participants revealed an average score of 73 points (SD 15) on a 100-point scale questionnaire. According to the study of Bangor et al. [40], this score can be considered "good". Only two patients disagreed with the statement "The website was easy to use" and nearly all patients disagreed with the statement "I think I would need technical support to be able to use the program". BODY.RESULTS.USABILITY TESTS: Experts in heuristic evaluation rated the rigid character of the intervention as a disadvantage. This was in accordance with results from the interviews. Results of the Thinking Aloud test are given in Table 5. The majority of tasks were completed as expected. Of the 15 tasks presented, on average, 12 (80%) were completed successfully. However, several usability problems were identified. Respondents had difficulties in logging (task 4), completing the introduction module (task 5) and establishing their personal starting level (task 14). On all occasions, navigation to Aim of the Program (task 10) was not executable due to an error in the system. Table 5 Thinking Aloud test among 5 participants Tasks Average time (sec) Task correct 1) What is the moderator's telephone number? 15.60 100% (n=5) 2) Register yourself for the program 308.40 100% (n=5) 3) Search for information about healthy weight and osteoarthritis 68.60 80% (n=4) 4) Login (with your username and password) 85.60 60% (n=3) 5) Complete module 1 (introduction) 352.40 40% (n=2) 6) Navigate to the webpage ‘Symptoms’ 48.20 60% (n=3) 7) Navigate to the webpage ‘My profile’ 12.20 100% (n=5) 8) Watch home exercise video No. 4 23.00 80% (n=4) 9) Write something in your workbook 84.60 100% (n=5) 10) Navigate to the webpage ‘Programme Aim ” 82.80 0% (n=0) 11) Log out 2.00 100% (n=5) 12) Log in, once again. 59.40 100% (n=5) 13) Fill in the evaluation form (performance and perceived pain) 62.00 100% (n=5) 14) Check the starting point of your programme in minutes 73.80 60% (n=3) 15) Check your most recent update in your workbook 16.20 100% (n=5) BODY.RESULTS.USABILITY TESTS.ADJUSTMENTS: Based on the results of the interviews and the heuristic evaluation, we changed the program's time contingent structure (i.e. fixed time periods) into a more flexible format. In the most recent version, options have been included which give users the choice of repeating modules and adapting the difficulty of the modules. The usability errors from the Thinking Aloud approach had more to do with the design of the website and the location of several buttons. These relatively minor problems were also addressed. BODY.DISCUSSION: Results from this study indicate that Join2move is a plausible, feasible and acceptable program for patients with knee and/or hip OA. Although effectiveness was not proved due to the lack of power, results do indicate that Join2move has the potential to increase PA levels in patients with knee and/or hip OA. Participants reported higher levels of PA, particularly (and as expected) involving moderate activities like walking and cycling (200 minutes). In line with other research [41], walking was by far the most frequently selected activity. Our positive results correspond with a comparable face-to-face intervention, showing a moderate PA increase of 170 minutes [25]. In the first three weeks, the increase was accompanied by more pain. Fortunately, after twelve weeks the pain scores declined towards baseline levels. Although the intervention focused on improving PA rather than on pain reduction, the increased pain was certainly a reason for concern. The precise cause of observed elevated pain scores is unclear. A possible explanation is the increased PA which may generate more muscle and joint pain. However, it is important to note that higher levels of pain are not associated with deterioration of OA [42,43]. Providing an intervention does not automatically mean that patients will use it, particularly when it is self-directed, with minimum personal contact. Since the success of web-based interventions requires active participation, non-usage attrition has been pointed out as a common concern in the field of web-based education. In line with other studies, [21,22,44], the number of users gradually decreased during the nine-week program. Overall, 55% (n=11) of the participants completed at least 75% of the program. This exposure percentage corresponds with the study of Steele et al. [33] and can be rated as reasonably high for web-based interventions without human interference. The delivery of personal information on a weekly basis is a possible explanation for this relatively low non-usage attrition. In this respect, it was not possible for users to run the entire program at one time. Although we did not examine the specific strategies of engagement, the authors assume that the week-by-week basis provided an incentive to return to the website. With respect to usability, the involvement of end-users was extremely valuable for identifying usability issues and system flaws. Along the way, we incorporated greater flexibility into the program. The implemented changes resulted in a less rigid version with more options tailored to the performance of the individual user. The findings from this study need to be interpreted in light of the study's limitations. The small sample size, single group design and lack of long-term assessments limit conclusions of causality, long-term effects and generalizability. Furthermore, the potential presence of the so-called Hawthorne effect may have contributed to an overestimation of PA scores. This implies that observed PA changes may be partly the result of study participation. Besides the Hawthorne effect, self-reported PA measures may also contribute to an over-estimation of PA levels in this study. This may be a consequence of recall error, perceived social desirability and other biases. To obtain the best results, a combination of validated questionnaires with objective measures would be preferable in future studies. Another limitation concerns outside-care patients who lack computer skills or internet access. These groups are mostly excluded from web-based interventions. Unfortunately, this disadvantage applies also to the Join2move intervention. Typically, these patients are disproportionately less educated and have a lower income. Particularly with regard to these under-served populations, GPs should refer sedentary OA patients more frequently to a physical therapist or other health care provider. Further, it will be important to translate Join2move for other self-help platforms, such as videos, brochures and self-help books. A final limitation is that we only performed one Thinking Aloud test to detect and resolve usability issues. Unfortunately, we did not retest the redesigned intervention. In order to optimize usability for the implementation phase, a repetition of this procedure is advised. BODY.CONCLUSIONS: Strong evidence indicates that regular PA is important in the management of OA. To date, however, many patients with knee and/or hip OA remain sedentary. Unfortunately, the vast majority of these patients do not receive any help in the promotion of PA. Low-cost, effective and accessible PA interventions are needed. Although our results are not conclusive, this study suggests that Join2move has the potential to contribute to meeting this need. The intervention is unique, since this is the first web-based PA intervention focusing on outside-care patients with OA. Moreover, while most web-based PA interventions have additional human contact, the Join2move intervention is fully computerized. Given the fully automatic character, the program has the potential to reach large populations while placing a minimal burden on our scarce health resources. This paper illustrates how involving end-users and experts can contribute successfully to the development of a web-based self-help intervention. The results suggest that the intervention is feasible and acceptable in promoting PA among patients with knee and/or hip OA. The intervention was easy to use and satisfaction with the program was high. This suggests that the intervention is acceptable for patients with knee and hip OA. Preliminary results from the pilot study revealed that PA scores increased, although differences were not statistically significant. A randomized controlled trial is needed to determine the effectiveness of the Join2move program. BODY.APPENDIX 1: Usability items used for the heuristic evaluation Interaction 1) Are the links to websites consistent throughout the website? 2) Do buttons and links show that they have been clicked? 3) Does the 'back' button appear on the browser toolbar on every page? 4) Are error pages descriptive, and did they provide a solution to the user? 5) Does the system inform users what is going on through appropriate feedback within a reasonable time frame? 6) Does the system behave consistently? 7) Does the system eliminate error-prone conditions and present users with confirmation options before they commit to the action? Information and architecture 8) Is the path for any given task a reasonable length (2–5 clicks)? Visual design 9) Is the default font size 12-point or larger? If not, is there an obvious way on the page to increase the font size? If not, does changing the font size in the browser enlarge all of the text? 10) Are text and interaction elements a different colour from the background? Are clickable items highlighted differently from other non-clickable highlighted items? Information design 11) Has the amount of text been minimized; is only necessary information presented? 12) Is the content written in the active voice, directed to "you"? 13) Does the site use words that most older adults know? Are instructions written in plain language? 14) Is a relevant help button provided? Does the system provide documentation about the website? Persuasive principles 15) Can users relate to and feel familiar with the context, images and figures that appear in the program? 16) Does the system contain the knowledge to be learned? 17) Is the program easy to use and are the tasks easy to perform with a small number of steps and keystrokes? 18) Can users learn about how they solved the tasks on previous occasions when the system was used? 19) Are users aware that the moderator can observe and see the results? 20) Do users get rewards or praise when a task is performed correctly 21) Is the program perceived as helpful? 22) Does the program act as a coach? BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: DB was responsible for day to day management of the project, developed the web-based intervention, collected all data, analysed the data and wrote the paper. CV had the idea for the study and managed the project. JD and DdB contributed to the intellectual content of the manuscript. All authors read and approved the final document. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1472-6947/13/61/prepub

TITLE: Pharmacokinetics and efficacy of intravenous famotidine in adult cattle ABSTRACT.BACKGROUND: Abomasal ulceration is recognized in neonatal and adult cattle, but research regarding treatment is limited. Histamine‐2 receptor antagonists (H2RA), such as famotidine, are used clinically with little evidence‐based research about efficacy in adult cattle. ABSTRACT.HYPOTHESIS AND OBJECTIVES: Intravenous famotidine administered at 0.4 mg/kg will increase the pH of abomasal outflow digesta compared to saline control in adult cattle. The objectives were to assess the effect of famotidine, administered as a single dose and as multiple doses, on abomasal outflow fluid pH in adult cattle. A third objective was to describe the pharmacokinetic parameters of IV famotidine in cattle. ABSTRACT.ANIMALS: Four clinically healthy adult Angus‐cross steers previously fitted with duodenal cannulae placed orad to the biliary and pancreatic ducts. ABSTRACT.METHODS: Randomized, 2‐way cross‐over clinical trial. Steers received IV famotidine (0.4 mg/kg) as a single and 3‐dose regimen (every 8 hours) versus saline control. Blood for analysis of serum famotidine concentration was collected intermittently for 12 hours, and abomasal outflow fluid pH was measured at intervals for a 24‐hour period. After a 34‐hour washout period, the opposite treatments were administered and the sampling repeated. ABSTRACT.RESULTS: Abomasal outflow fluid pH was higher in steers treated with famotidine for up to 4 hours after a single dose but the effect decreased with subsequent doses. The median (range) elimination half‐life was 3.33 (3.21‐3.54) hours. ABSTRACT.CONCLUSIONS AND CLINICAL IMPORTANCE: Famotidine may be useful for treatment or prevention of abomasal ulceration in adult cattle, but the duration of effect may decrease with time. BODY: AbbreviationsAUCarea under the curveH2RAhistamine type‐2 receptor antagonistLSleast squarePPIproton pump inhibitorSDstandard deviation BODY.INTRODUCTION: 1 Abomasal ulceration is a disease syndrome recognized in calves and adult cattle. Approximately 2%‐20% of both dairy and beef cattle have abomasal ulcerations of variable severity identified at slaughter.1, 2, 3 Clinical signs of abomasal ulceration vary with severity of disease and include anorexia, bruxism, cranial abdominal pain, melena, signs of localized or generalized peritonitis, anemia, and death. In adult cattle, suggested risk factors include abomasal lymphoma, stress from periparturient disease, diets high in carbohydrates, and use of nonsteroidal anti‐inflammatory drugs.4 Cattle admitted to veterinary hospitals often have a combination of these risk factors, and thus are predisposed to either having ulceration at admission or developing ulceration during hospitalization. One of the challenges of diagnosing and managing abomasal ulceration is the lack of sensitive and specific definitive diagnostic tests. Presence of melena, positive fecal occult blood tests, and typical clinical signs of bruxism and cranial abdominal pain are suggestive of abomasal ulceration. Thus, empirical treatment often is recommended as preventative or presumptive therapy. Treatment of gastric ulceration in other species includes increasing gastric pH by use of PO alkalinizing agents or suppression of gastric acid production. The use of PO alkalinizing agents such as magnesium hydroxide and PO gastric acid suppressants including histamine type‐2 receptor antagonists (H2RA) and proton pump inhibitors (PPI) has been studied in pre‐ruminant calves.5, 6, 7 These agents have variable efficacy in increasing abomasal pH in pre‐ruminants. Administration of PO therapies may be ineffective in adult cattle because of the buffering and diluting effect of the rumen, unpredictable bioavailability, and delivery to the abomasum.8 Limitations of parenteral administration of H2RA or PPI include the need for IV access, frequent administration, relatively large doses required, and associated cost. Famotidine is an H2RA that competitively inhibits histamine binding to the H2‐receptor on the basolateral membrane of the parietal cells, and thus decreases stimulation of the H+/K+ ATPase pump at the luminal surface of the cell, which decreases acid secretion. It is used in humans with gastric or duodenal ulceration, as well as in those with gastroesophageal reflux disease.9 Dosage rates in dogs have been suggested at 0.5 mg/kg IV q12h10 and in horses at 0.2‐0.4 mg/kg IV q6h‐q8h.11 Evidence for parental administration of gastric acid suppressants in adult cattle is lacking, although anecdotal descriptions of a variety of treatments are available.4, 12 The dosage rate and frequency of administration chosen for our study utilized the upper end of the dosage for horses (q8h) to be able to detect an effect, if any, with a feasible time for sampling. We hypothesized that the abomasal outflow of adult cattle receiving IV famotidine will have a higher pH compared to the abomasal outflow of cattle treated with saline control. The objectives of our study were to: (1) assess the effect of a single dose of famotidine administered at a dosage of 0.4 mg/kg IV on the pH of abomasal outflow of adult cattle; (2) assess the effect of famotidine administered at a dosage of 0.4 mg/kg IV on the pH of abomasal outflow of adult cattle when administered as 3 doses q8h to mimic clinical treatment; and (3) to describe serum concentrations and pharmacokinetic parameters of famotidine in adult cattle. BODY.MATERIALS AND METHODS: 2 BODY.MATERIALS AND METHODS.ANIMALS: 2.1 This study was a randomized 2‐way cross‐over clinical trial utilizing 4 adult Angus steers fitted previously with surgically implanted duodenal cannulae placed orad to the biliary and pancreatic ducts. The steers were approximately 34 months of age and weighed 800 ± 24.22 kg. They were determined to be healthy based on clinical examination before the study, with no previous history of ulceration. They were housed in tie‐stall stanchions for the 2 consecutive 48‐hour sampling periods. The steers were fed 2.5 kg per head of an equal mixture of whole corn and pelleted soybean hulls twice daily at approximately 0700 and 1900 hours, starting 5 days before and during the study. Steers also were offered ad libitum alfalfa hay and water. All animal procedures were approved by the University of Missouri Institutional Animal Care and Use Committee (ACUC) Protocol #8331. BODY.MATERIALS AND METHODS.FAMOTIDINE ADMINISTRATION: 2.2 Intravenous jugular catheters (BD Angiocathm, Becton and Dickson Therapy Systems, Inc., 9450 South State Street, Sandy, Utah) were placed aseptically before each 48‐hour study period for IV drug administration and blood sample collection. Famotidine (Famotidine Injection USP [10 mg/mL], West‐ward, Eatontown, New Jersey) was administered at a dosage of 0.4 mg/kg IV, or an equivalent volume of saline control, as a single dose or multiple doses as described below, and the catheter irrigated with 10 mL of heparinized saline after administration. The interval between the single dose and multiple dose studies was 22 hours. After a 34‐hour washout period from the last treatment, the opposite treatment was administered and the study was repeated. Time 0 represents the time period immediately preceding administration of either famotidine or saline control, and ranged from 2 to 4 hours after feeding concentrate. BODY.MATERIALS AND METHODS.FAMOTIDINE ADMINISTRATION.SINGLE DOSE STUDY: 2.2.1 Famotidine (0.4 mg/kg; n = 4) or an equivalent volume of saline (n = 4) was administered via IV jugular catheter once. Blood samples from the jugular catheter were obtained at 0, 1, 2, 3, 4, 8, 12 hours and abomasal outflow samples were obtained hourly from 0 to 12 hours (Figure 1). Figure 1Schematic of the single dose study: a single dose of intravenous famotidine (0.4 mg/kg) (n = 2) or an equivalent volume of saline (n = 2) administered at 0 hours. Blood samples were obtained at 0, 1, 2, 3, 4, 8, 12 hours. Abomasal outflow fluid samples were obtained hourly at 0–12 hours BODY.MATERIALS AND METHODS.FAMOTIDINE ADMINISTRATION.MULTIPLE DOSE STUDY: 2.2.2 Famotidine (0.4 mg/kg; n = 4) or an equivalent volume of saline (n = 4) was administered every 8 hours at 0, 8, and 16 hours. Jugular blood samples were obtained at 0, 8, 16, and 24 hours. Abomasal outflow samples were obtained at 0, 1, 2, 4, 6, 8, 10, 12, 16, 17, 18, 20, 22, 24 hours (Figure 2). Figure 2Schematic of the multi‐dose study: Three doses of intravenous famotidine (0.4 mg/kg) (n = 2) or an equivalent volume of saline (n = 2) administered at 0, 8, and 16 hours. Blood samples were obtained at 0, 8, 16, and 24 hours. Abomasal outflow fluid samples were obtained at 0, 2, 4, 6, 8, 10, 12, 14, 16, 17, 18, 20, and 24 hours BODY.MATERIALS AND METHODS.BLOOD SAMPLING: 2.3 Whole blood samples were obtained after 20 mL of blood and heparinized saline was aspirated from the catheter to clear the catheter of any residual drug or heparinized saline. Approximately 10 mL of whole blood was collected into a serum separator tube containing no anticoagulant (BD Vaccutainer SST Plus blood collection tubes; Becton Dickson and Company, Franklin Lakes, New Jersey). The 20 mL of blood withdrawn to clear the catheter then was given back to the animal and the catheters were irrigated with 10 mL heparinized saline. Blood samples were allowed to clot at room temperature and stored on ice for < 8 hours before centrifugation at 1500g for 30 min at 4°C. Serum was separated and frozen at −20°C and shipped to the laboratory for famotidine analysis. BODY.MATERIALS AND METHODS.DRUG CONCENTRATION DETERMINATION AND PHARMACOKINETIC ANALYSIS: 2.4 Famotidine was quantified in bovine serum by liquid chromatography‐tandem mass spectrometry using a previously published method13 and d‐4 famotidine as the internal standard. A partial validation was performed using bovine serum as the matrix. Calibration curves and negative control samples were prepared fresh for each quantitative assay and quality control samples (bovine serum fortified with analyte at 4 concentrations within the standard curve) were included as an additional check of accuracy. The response for famotidine was linear and gave a coefficient of determination (R 2) of .99. The precision and accuracy of the assay were determined by assaying famotidine quality control samples in replicates (n = 6). The accuracy (% nominal concentration) was 115, 104, 111, and 106% at 0.3, 25, 100, and 700 ng/mL, respectively. Precision (% relative standard deviation [SD]) was 6, 3, 5, and 2% for 0.3, 25, 100, and 700 ng/mL, respectively. The assay was optimized to provide a limit of quantitation of 0.2 ng/mL and a limit of detection of approximately 0.1 ng/mL. To assess the potential impact of the silicone plug in the serum separator tubes on famotidine concentrations (ie, drug binding), control bovine serum was collected into both serum separator tubes and serum tubes without the silicone plug. Famotidine, at 1 of 3 concentrations (1 ng/mL, 5 ng/mL, and 10 ng/mL), was added to serum in the different tube types. Each concentration was spiked in triplicate. Tubes containing drug were allowed to sit at room temperature for 1 hour. Drug concentrations in each tube were measured as described above for the in vivo samples. Pharmacokinetic analysis was performed on serum famotidine concentrations using non‐compartmental analysis and a commercially available software program (Phoenix WinNonlin Version 6.2; Pharsight, Cary, North Carolina). The sampling time points in this study were more fitting for non‐compartmental versus compartmental methods to determine pharmacokinetic parameters. The elimination rate constant (lambdaz) was calculated by determination of the slope of the terminal portion of the plasma concentration versus time curve and the plasma elimination half‐life (HL) using the formula (ln 2)/the elimination rate constant. The area under the curve (AUC) from 0 to infinity (0‐∞) was calculated using the log‐linear trapezoidal method. The AUC0‐∞ % extrapolated was calculated using the formula [(AUC0‐∞ − AUC0–12)/AUC0‐∞] × 100. BODY.MATERIALS AND METHODS.ABOMASAL OUTFLOW FLUID SAMPLING AND PH MEASUREMENT: 2.5 Abomasal outflow fluid sampled from the duodenal cannulae was used as an indicator of abomasal pH changes due to orad placement from biliary and pancreatic ducts. Approximately 50 mL of duodenal fluid was allowed to drain from the cannula before collecting 20 mL in to a plastic collection bag (Whirl‐Pak bags; Nasco, Modesto, California) by gravity flow. The operator obtaining the samples and measuring pH was not blinded to the treatment or sample time. Abomasal outflow pH was analyzed directly after sampling (within 5 minutes) using a bench‐top pH analyzer (Fisher Science Accumet; Thermo Fisher Scientific, Inc, Blk 55 Ayer Rajah Crescent, Singapore). The pH meter was calibrated according to manufacturer's instructions with 4.01 and 7.0 pH solutions at the start of the study and once every 12 hours. Outflow samples that appeared grossly contaminated with bile, flowed very slowly, or had a pH > 5.0 were resampled within a 10‐minute period. If the second sample had a pH measurement within 0.25 units of the initial reading, the original sample value was recorded. If the second sample had a pH that was > 0.25 units more acidic than the initial sample, the new sample pH value was recorded. Any samples that had a pH > 5.0, or had gross contamination with bile or mucus were recorded, but not included in data analysis because they were considered an inaccurate reflection of abomasal outflow pH. BODY.MATERIALS AND METHODS.ABOMASAL OUTFLOW PH STATISTICAL ANALYSIS: 2.6 For abomasal outflow pH, a mixed model analysis with commercial software (SAS Version 9.4; SAS Institute Inc, Cary, North Carolina) was used. The MIXED procedure was used with treatment, sampling hour, and treatment × hour as the fixed effects and steer and period as random effects in the model. Least square means were separated using least significant difference and analyzed using analysis of variance. P < .05 was considered statistically significant. BODY.RESULTS: 3 The treatment × sampling hour interaction affected abomasal outflow pH for both a single dose and multiple doses of famotidine (P < .001). A single dose of famotidine at 0.4 mg/kg significantly increased the pH of abomasal outflow fluid for 4 hours (P < .05) compared with saline control (Figure 3). The greatest difference was observed at 2 hours post‐treatment with least square (LS) means ± SD pH values 3.90 ± 0.12 (control) versus 6.01 ± 0.18 (famotidine; P < .001). When administered every 8 hours, famotidine significantly increased the pH of abomasal outflow fluid for 3 hours after the first dose, 2 hours after the second dose, and only 1 hour after the third dose (Figure 4). The greatest difference was observed 2 hours after the first treatment with LS means ± SD pH values of 4.49 ± 0.30 (control) versus 5.67 ± 0.27 (famotidine; P < .001). The pH of outflow in the control group was less at 9 hours than at hours 1, 4, and 12 (P ≤ .04). Otherwise, the outflow pH of the control group did not change over time (P > .10). Figure 3Least square means and standard deviation pH of abomasal outflow fluid from adult cattle (n = 4) administered a single intravenous dose of 0.4 mg/kg famotidine (circles) compared with adult cattle (n = 4) administered an equivalent volume of saline (squares). *Denotes statistical significance (P < .05) Figure 4Least square means and standard deviation pH of abomasal outflow fluid from adult cattle (n = 4) administered multiple intravenous doses of 0.4 mg/kg famotidine (circles) every 8 hours (time 0, 8, 16 hours) compared with adult cattle (n = 4) administered an equivalent volume of saline (squares). *Denotes statistical significance (P < .05) Serum famotidine concentrations with respect to time after a single administration are summarized in Table 1 and shown in Figure 5. Measured serum famotidine concentrations from spiked serum separator tubes were 93%‐95% of the concentrations measured in serum tubes without the silicone plug at all concentrations studied. Figure 5Semi‐log plot of serum concentrations of famotidine after administration of a single intravenous dose of famotidine (0.4 mg/kg) to adult cattle (n = 4). Data presented as mean ± standard deviation Table 1 Serum concentration of famotidine when administered as a single dose of 0.4 mg/kg IV at 0 hour to adult cattle (n = 4) Serum concentration (ng/mL) Time (hour) 0 1 2 3 4 8 12 Median ND a 821.8 43.5 30.4 17.9 9.2 4.1 Range (ng/mL) ND a (154.9–1232) (35.1–50.9) (26.4–35.6) (13.1–22.0) (5.2–12.1) (2.8‐6.2) a Limit of detection of approximately 0.1 ng/mL. Pharmacokinetic parameters after administration of a single dose of famotidine are summarized in Table 2. The median (range) HL (Lambdaz), volume of distribution (Vdss), and clearance (CL) were 3.33 (3.21‐3.54) hours, 0.042 (0.014‐1.89) L/kg, and 1.26 (0.625‐11.5) mL/min/kg, respectively. Serum concentrations with respect to time after multiple doses are depicted in Table 3. Pharmacokinetic analysis was not performed on the multiple dose data because of a limited number of time points and a small number of animals (n = 2 for time 8 and 16 hours and n = 4 for time 0 and 24 hours). Table 2 Median (range) pharmacokinetic parameters after administration of a single IV dose of famotidine (0.4 mg/kg) to adult cattle (n = 4) Parameters Median (range) Lambda z (1/h) 0.208 (0.196‐0.216) HL Lambda z (h) 3.33 (3.21‐3.54) Vdss (L/kg) 0.042 (0.014‐1.89) CL (mL/min/kg) 1.26 (0.625‐11.5) AUC (h × ng/mL) 7,019 (579‐10 673) AUC extrap (%) 0.326 (0.129‐5.46) AUC, area under the curve; CL, clearance; HL, half‐life; V dss , volume of distribution. Table 3 Serum concentration of famotidine when administered as 3 doses of 0.4 mg/kg IV at 0, 8, and 16 hours to adult cattle (n = 4) Serum concentration (ng/mL) Time (hour) 0 8 16 24 Median ND a 935.5 b 4217.4 b 761.8 Range (ng/mL) ND a (411.3–1459.7) b (453.4–7981.3) b (100.2–5858.5) a Limit of detection of approximately 0.1 ng/mL. b n = 2. No adverse clinical effects were observed in the steers for the duration of study period, based on lack of clinical abnormalities such as changes in fecal consistency, appetite, attitude, and physical examination findings at the conclusion of the study. BODY.DISCUSSION: 4 To our knowledge, this study is the first investigation of parenteral administration of famotidine in adult cattle. Our results indicate that famotidine administered at a dosage of 0.4 mg/kg IV is effective at increasing the abomasal outflow fluid pH of adult cattle for up to 4 hours after a single dose compared with saline control. When administered every 8 hours, as is typically done for clinical treatment,11 famotidine increased the pH of the abomasal outflow fluid, but the effect decreased with additional doses. Famotidine increased the abomasal outflow fluid pH for 3 hours after the first dose, 2 hours after the second dose, and for only 1 hour after the third dose. This phenomenon of tachyphylaxis has been described in humans and a decrease in response to treatment with H2RA has been reported after the second dose and with repeated PO dosing of famotidine in dogs.14, 15 However, although the effect on acid secretion was reported to be negligible in these studies of humans, there was a perceived effect of treatment, which may be a result of other mechanisms. In humans, plasma concentration is dose‐related with greater acid suppression correlated with larger doses.16 Similar studies have not been performed in veterinary species to our knowledge. The mechanisms of a decreasing effect on acid secretion in cattle are not known, and may occur through a similar process, such as degradation of parietal cell H2‐receptors with time.17 The presence of tachyphylaxis is clinically important when recommending dosing interval or frequency.15 Previous reports describing use of other H2RA in ruminants have been published. In 1 report, abomasal pH increased for 1 hour after administration of ranitidine (6.6 mg/kg IM) in cattle.12 Other studies have reported a dose‐dependent effect of PO cimetidine and ranitidine in pre‐ruminant calves.5 The effect of IV ranitidine has been investigated in sheep with abomasal cannulae, indicating that a dosing interval of every 8–12 hours is most effective.18 That study, however, also found that sheep receiving ranitidine had increased total serum protein and increased serum creatinine concentrations, increased aspartate aminotransferase activity, and decreased serum pepsinogen concentration, indicating safety concerns for this agent in sheep.18 Drugs that increase gastric pH above 3.0 for up to 75% of a 24‐hour period may be associated with healing of gastric or duodenal ulcers in humans.19 When administered as a single dose, famotidine significantly increased the abomasal outflow pH compared to the controls for up to 4 hours, from a baseline of 4.26 ± 0.81 pretreatment to a high of 6.01 ± 0.18 at 2 hours post‐administration of famotidine. When administered every 8 hours, as has been clinically recommended, the greatest difference between control‐ and famotidine‐treated cattle occurred 2 hours after administration of the first dose. However, the 2 groups were only different for 7 hours of a 24‐hour period, which equates to approximately 30% of the day. This is less than the observed 48.9% of a 24‐hour period in dogs treated with famotidine (0.5 mg/kg IV q12h)10 and less than the 75% of the day recommended in humans.19 This finding suggests that administering H2RA every 8 hours may not be cost‐effective if there is no clinical effect, and perhaps may have the same duration of effect in a 24‐hour period, if administered less frequently. These percentages are less than the suggested interval for treatment of humans, but the optimal degree of gastric acid suppression has not been clearly defined in veterinary species, including cattle. Our study reports higher pH values obtained for fluid from duodenal cannulae as compared with studies with direct luminal abomasal fluid measurement in calves and adult cattle, either from direct cannulation or abomasocentesis,5, 7, 8, 20, 21 which may be a consequence of backflow of biliary and pancreatic secretions and mucus production by small intestinal enterocytes. Correct placement of the duodenal cannulae was determined at the time of surgical implantation by measurement of pH, and thus was unlikely to have caused the difference in our study, especially based on consistency among individuals. Additionally, minimal fluctuation was observed in the abomasal outflow pH of control steers during the study period, suggesting that feeding concentrate did not have an effect on the acid secretion and pH of the abomasal outflow fluid in our study. In our study, the HL of famotidine was just over 3 hours, suggesting that > 99% of the drug would be eliminated within 24 hours (7 elimination HL) post‐administration. However, only serum was obtained in this study and a multi‐dose regimen with tissue samples would be required to determine accurate tissue residues to determine appropriate withdrawal times. Veterinarians are recommended to consult the Food Animal Residue Avoidance Database (FARAD), or other regulatory body, for recommendations on withdrawal times after administration of this medication, because famotidine currently is not approved for use in food‐producing animals in the United States and its use is considered extra‐label. Limitations of our study include small sample size, short study period, and use of a single IV catheter for administration and sampling. A small sample size may increase the effect of individual variation, especially when interpreting the pharmacokinetic data. The short 34‐hour washout period between studies may have allowed an unknown residual effect of the drug on the parietal cells within the abomasum. To more accurately analyze the pharmacokinetic indices of famotidine, more frequent blood sampling would have been necessary directly after administration of the drug, but due to certain constraints, this was not possible for our study. The C max, in particular, would be affected, as the lack of initial data points may have resulted in the failure to identify the true peak concentration, which may have occurred before the first data point. More data points would have changed the shape of the time‐concentration curve, and thus the AUC and CL values are likely to have been underestimated, because of the first data point being an hour after administration. Because of personnel limitations and temperament of the cattle, direct IV administration of the treatment was not practical for this pilot study. Utilizing the same IV catheter for administration of drug and blood sample collection may have altered the measured serum famotidine concentrations because of crystallization of famotidine within the catheter or extension set. Additionally, returning the 20 mL of blood taken before sample collection to the animal post‐sampling may have inadvertently readministered drug remaining within the catheter, also affecting the pharmacokinetic analysis. In particular, 1 steer in the multi‐dose section of the study had repeatedly higher serum famotidine concentrations than the others steers, which may have been caused by individual variation, but the possibility of sampling error as a result of the above‐mentioned issues cannot be ruled out. In addition, some data points for the multi‐dose study were not available, thus making interpretation of the pharmacokinetic analysis of the multi‐dose study problematic. More frequent abomasal outflow fluid samples or continuous sampling by pH electrode through the cannulae would have allowed detection of the initial effects of famotidine on parietal cell secretion and a more accurate detection of the timing of abomasal outflow pH changes. Studies with larger numbers of individuals using direct abomasal cannulation 5, 7, 20 or using repeated percutaneous ultrasound‐guided abomasocentesis21 may have resulted in more accurate measurements of abomasal pH. This pilot study facilitates further studies assessing different dosage rates and dosing intervals of famotidine, as well as investigating the effect of different commercially available gastric acid suppressant therapeutic drugs in cattle and other ruminant species. Further studies investigating the efficacy of famotidine after extravascular administration may be clinically useful. In conclusion, famotidine when administered parenterally at a dosage of 0.4 mg/kg is effective at increasing abomasal outflow pH for at least 4 hours after a single dose, and could be a useful adjunctive treatment for abomasal ulceration in adult cattle, but should not be the sole form of treatment. Further studies are needed to evaluate the use of this drug, especially dose rates and frequency. BODY.CONFLICT OF INTEREST DECLARATION: Authors declare no conflicts of interest. BODY.OFF‐LABEL ANTIMICROBIAL DECLARATION: Famotidine is not labeled for use in food‐producing animals in the United States. BODY.INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION: All animal procedures were approved by the University of Missouri Institutional Animal Care and Use Committee Protocol #8331.

TITLE: Impact of Hydroxychloroquine on Atherosclerosis and Vascular Stiffness in the Presence of Chronic Kidney Disease ABSTRACT: Cardiovascular disease is the largest cause of morbidity and mortality among patients with chronic kidney disease (CKD) and end-stage kidney disease, with nearly half of all deaths attributed to cardiovascular disease. Hydroxychloroquine (HCQ), an anti-inflammatory drug, has been shown to have multiple pleiotropic actions relevant to atherosclerosis. We conducted a proof-of-efficacy study to evaluate the effects of hydroxychloroquine in an animal model of atherosclerosis in ApoE knockout mice with and without chronic kidney disease. Forty male, 6-week-old mice were divided into four groups in a 2 x 2 design: sham placebo group; sham treatment group; CKD placebo group; and CKD treatment group. CKD was induced by a two-step surgical procedure. All mice received a high-fat diet through the study duration and were sacrificed after 16 weeks of therapy. Mice were monitored with ante-mortem ultrasonic echography (AUE) for atherosclerosis and vascular stiffness and with post-mortem histology studies for atherosclerosis. Therapy with HCQ significantly reduced the severity of atherosclerosis in CKD mice and sham treated mice. HCQ reduced the area of aortic atherosclerosis on en face examination by approximately 60% in HCQ treated groups compared to the non-treated groups. Additionally, therapy with HCQ resulted in significant reduction in vascular endothelial dysfunction with improvement in vascular elasticity and flow patterns and better-preserved vascular wall thickness across multiple vascular beds. More importantly, we found that presence of CKD had no mitigating effect on HCQ's anti-atherosclerotic and vasculoprotective effects. These beneficial effects were not due to any significant effect of HCQ on inflammation, renal function, or lipid profile at the end of 16 weeks of therapy. This study, which demonstrates structural and functional protection against atherosclerosis by HCQ, provides a rationale to evaluate its use in CKD patients. Further studies are needed to define the exact mechanisms through which HCQ confers these benefits. BODY.INTRODUCTION: Cardiovascular disease (CVD) is the largest cause of morbidity and mortality[1, 2] among patients with chronic kidney disease (CKD) and end-stage kidney disease (ESKD), with nearly half of all deaths attributed to CVD[1]. Though no single factor thus far has been identified as the driver for this cardiovascular (CV) burden; inflammation, vascular stiffness likely resulting from endothelial dysfunction, and accelerated atherosclerosis are considered prominent factors contributing to the high rates of CVD in CKD. Therapeutic interventions conventionally proven to be effective in CVD among patients with normal renal functions, have limited if any efficacy in presence of significant CKD. For example, HMG coenzyme A inhibitors, statins, when studied in CKD and ESKD populations have failed to show a significant reduction in CV mortality[3–7], even though some data suggest that they might be effective in reducing the rates for CV events[4]. When studied in animal models of atherosclerosis, statins failed to reduce the severity of atherosclerosis in presence of uremia; a sharp departure from their proven efficacy in presence of normal renal function[8]. Similarly, many therapies targeted toward management of renal failure (such as quotidian dialysis)[9], or its consequences (bone-mineral disease, anemia, etc.) have also failed to reduce the high CV mortality associated with CKD[3, 4, 7, 9, 10]. These factors have led to a high CV mortality in CKD[2] that has remained largely unchanged over the last two decades,[11] and at present time we do not have an effective management strategy that reduces the high CV mortality in CKD and ESKD. Thus there is an urgent need to investigate unique therapies that are efficacious against atherosclerosis and vascular disease, not just in presence of normal renal function, but also in presence of uremic milieu. Most patients suffering from CKD have multiple traditional and nontraditional CV risk factors such as chronic inflammation, oxidative stress, endothelial dysfunction, reduced vascular compliance, insulin resistance, and metabolic syndrome[12]and these factors have been repeatedly shown to contribute not only toward the pathogenesis of CVD in CKD but also a state of refractoriness toward conventional therapies[13]. Hydroxychloroquine (HCQ) is an anti-malarial drug that is commonly used in clinical practice for its anti-inflammatory actions. Multiple in vivo, in vitro, and cohort-based reports over the last two decades show that, in addition to being anti-inflammatory, HCQ has multiple other properties that include beneficial effects on vascular compliance and endothelial function,[14] insulin resistance, metabolic syndrome,[15–17] and immune dysfunction.[18, 19] Recently when studying the p-53 based stress signaling in the metabolic syndrome, investigators have found signals for a possible anti-atherosclerosis effect for HCQ in presence of normal renal function[17]. However, as the statin experience suggests, the efficacy in presence of normal renal function does not automatically prove the efficacy in presence of uremic milieu and the impact of HCQ as an anti-atherosclerosis as well as vasculoprotective agent in a uremic milieu has not been examined previously. We present the findings of our proof-of-efficacy animal study in ApoE-/- mice aimed to examine whether HCQ has beneficial effects on atherosclerosis and vascular disease in presence of a uremic state. BODY.MATERIALS AND METHODS: Mice were maintained in the Veterans Administration Medical Unit (VAMU) at Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals and the protocol was approved by the Institutional Animal Care and Use Committee (IACUC #12-11-1) of Central Arkansas Veteran Healthcare System on animal care. Forty-two male, 6- to 8-week-old ApoE-/- mice with a C57BL6 background (Jackson Laboratories, Bar Harbor, ME) were divided into two different groups after an initial week of acclimatization: a CKD group (n = 26) and a sham group (n = 16). Mice in the CKD group underwent a two-step surgical process (cortical electrocoagulation of 80% of the right kidney followed two weeks later by a left kidney total nephrectomy), originally described by Gagnon et al.[20] and validated in our laboratories[21], to establish a reduced renal mass and induce CKD. The sham-operated mice underwent a right-flank incision and closure in the first stage of surgery and a left-flank incision and closure in the second stage of surgery and served as groups with normal renal function. All surgeries were performed under ketamine/xylazine (91.0/9.1 mg/kg, i.p.) anesthesia and all efforts were made to minimize the pain and potential distress by two post-operative analgesic injections of buprenorphine (0.05mg/kg, 0.1 ml) administered subcutaneously for one day. All mice underwent serial blood work obtained by retro-orbital puncture for blood urea nitrogen and creatinine assessment every four weeks during the study period. After surgery, all mice were fed a high-fat diet (Harlan Teklad, Madison, WI) with 21% total fat and 0.15% cholesterol to exacerbate hyperlipidemia and atherosclerosis development. Both CKD and sham groups of mice were divided further into those receiving HCQ (treatment groups) and those treated with placebo (controls), creating four final groups: (1) a sham control group (n = 8); (2) a sham treatment group (n = 8); (3) a CKD control group (n = 13); and (4) a CKD treatment group (n = 11). HCQ was administered in drinking water to achieve a therapeutic dose of 10 mg/kg/day. The dose of HCQ was selected based on the conventional low dose (6–10mg/kg/day) of HCQ used in human populations where the pleiotropic cardioprotective actions of HCQ have been demonstrated[14, 15, 22], and also in a prior animal study in a similar model in the presence of normal renal function[17]. The feasibility of HCQ mixed in the water supply and its effects on daily water intake were tested through a prior pilot observation study in the same strain of mice. Animals were group-housed in an animal care facility with 3–4 animals per cage. Ante-mortem ultrasound echography (AUE) is an excellent tool to visualize the development and progression of atherosclerosis in mice and to evaluate the impact of modulating factors or interventions[23]. We performed an AUE of the aorta and major vessels at baseline, 12 weeks, and 16 weeks (just prior to sacrifice) into the study using the VisualSonics (Toronto, CAN) Vevo 770 in B mode (for the evaluation of the extent of atherosclerotic lesions), in M mode (for wall density and thickness at the pre-specified locations), and in Doppler mode (for rigidity and flow evaluations)[21, 24]. The detailed procedures and technique for this AUE examination have been published previously by our laboratory[21]. All mice were studied for a total duration of 16 weeks, with the last assessment for biochemical parameters and AUE at the time of sacrifice. Vascular wall thickness was obtained by the measurement of arterial wall thickness in 3–5 images taken of the vessel. Measurements and analysis were made separately for the upper and lower walls visible on AUE by an operator blinded to the identity of the animal. Mice were euthanized at the end of sixteen weeks of therapy (at 23–24 weeks of age) with 300 mg/kg ketamine and 30 mg/kg 6 mg/kg xylazine (IP) followed by exsanguinations, and the sera and tissue were collected for the biochemical and histological assessments. Postmortem assessments included en face staining of the entire aortas with Sudan IV stain to detect the extent of atherosclerotic lesions (% of total aortic area with atherosclerotic lesions). Images of en face preparations of the whole aortas with atherosclerotic lesions were captured with a Nikon J1camera (Nikon, Melville, NY). The quantification of atherosclerotic lesions in full-length aorta, aortic arch, innominate artery (from the branching point to the Y-shaped bifurcation), thoracic aorta, and abdominal aorta were compared by using computerized analysis using the Image J. v 1.46 software (National Institutes of Health, Bethesda, MD), with lesions expressed as percentage of total vascular surface for each animal and the result was expressed as the percent of surface area of the entire aorta. For Oil Red O (Sigma, St. Louis, MO) staining for the cross-sectional analysis of lipid and fatty deposits in the aortic roots, the aorta along with the 3-mm lengths of ascending aorta (2 to 3 mm proximal to the brachiocephalic artery) was removed and frozen in OCT compound (Tissue-Tek, Sakura Finetek, Tokyo, Japan). 5 μm thick cryosections were stained with Oil Red O and counterstained with hematoxylin. For quantitative analysis of atherosclerosis, the total lesion area of the cross-sections was captured with a Nikon DS camera and analyzed by Image J. v 1.46 software. Mean plaque area of the aortic sinus was assessed and compared among the groups. Aortic roots (3 mm pieces of ascending aorta) were separated prior to en face staining and used for cryosectioning to assess atherosclerotic lesions in the aortic bulb. Additionally, mice sera were tested for lipids, IL-6 (Rey Biotech, Inc. Norcross, GA, cat. # ELM-IL-6-001), high sensitivity C-reactive protein (hsCRP; Antibodies-online, Inc. Atlanta, GA, cat. # ABIN415420), and soluble VCAM-1 (Quantikine ELISA kit, R&D Systems Inc., Minneapolis, MN cat. #MVC00) according to the manufacturers' instructions. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: All analyses were performed with the use of software GraphPad InStat 3, version 6.0 (GraphPad Software Inc., San Diego, CA) and SAS 9.4 (SAS Institute, Inc., Cary, NC). Continuous variables are expressed as mean ± SD and compared by Student's t-test if it is independent and normally distributed, or Kruskal-Wallis test if it is independent but not normally distributed. The Friedman test was used to compare the significance of differences at multiple time points for elasticity and velocity. A two-way analysis of variance (ANOVA) was used to examine the main effects and the interactions of CKD and drug intervention on atherosclerosis and vascular thickness. Post-hoc Tukey's and Dunn tests were applied when comparing three or more groups. A P value of < 0.05 was considered statistically significant. BODY.RESULTS: Table 1 shows the morphologic and biochemical parameters among all four groups at the end of the 16 weeks of therapy. Two mice died due to anesthesia complications, leaving 8 mice each in both sham groups and eleven and thirteen mice in CKD treatment and control groups, respectively. There was no significant difference among the various groups for weight, daily water intake, and daily food intake. The water intake between all groups was similar and the final HCQ dosing was achieved as desired. 10.1371/journal.pone.0139226.t001 Table 1 Quantification of serum urea nitrogen (BUN), total bilirubin (TBIL), glucose (GLU) among all groups of mice at the time of sacrifice after 16 weeks of therapy with either HCQ or placebo. Sham control (n = 8) Sham treatment (n = 8) CKD control (n = 13) CKD treatment (n = 13) BUN (mg/dl) 20±1.4 26±2.0 90±12.3 * 114±18.0 † TBIL (mg/dl) 0.33±0.0 0.33±0.0 0.35±0.0 0.4±0.0 GLU (mg/dl) 152±6.5 159±13.2 163±10.4 175±6.5 Body weight (g) 28.7 ±0.5 29±0.7 25.6±0.7 25.3±0.5 Food intake (g/24h) 3.3±0.5 3.0±0.7 2.9±0.4 3.0±0.6 Water/HCQ intake (ml/24h) 7.1±1.8 8.4±1 7.7±1.7 8.4±1.5 Body weight and daily food and water intake did not differ among different groups based on either presence or absence of CKD or therapy with HCQ or placebo. Data presented as means ± SD * P <0.001 versus sham control † P <0.01 versus sham treatment by unpaired t-test. BODY.RESULTS.IMPACT OF HCQ ON ATHEROSCLEROSIS: ANTEMORTEM AND POSTMORTEM EVALUATIONS: Fig 1 shows the ante-mortem AUE evaluation for aorta and major vessels in B and M mode echography and Doppler of the major vessels in the CKD groups of mice at the end of 16 weeks of therapy. The AUE studies were performed at baseline and 12 and 16 weeks into therapy just prior to sacrifice. The area of atherosclerosis was measured by identification of echo-dense atherosclerotic plaques on B-mode echography of the great vessels. We found that the extent of atherosclerosis on AUE examination was significantly reduced in mice receiving HCQ therapy (Fig 1B) compared to the controls (Fig 1A) at 12 weeks into the therapy. This continued to be evident on the examination performed at the end of the study at 16 weeks of therapy. The beneficial effects were evident in mice with CKD and with normal renal function. 10.1371/journal.pone.0139226.g001Fig 1Antemortem echography (AUE; B, M, and Doppler mode) in CKD groups of mice at end of study.Representative images of aortic arch/ascending aorta and brachiocephalic arteries obtained by ante-mortem ultrasound echography (AUE) of CKD control (A) and CKD treated (B) mice, using VisualSonics Vevo 770. All mice were fed a high-fat diet and treated with 10 g/kg/day of HCQ for 16 weeks after CKD surgery. Data is shown for four control and four treated mice (n = 13, CKD control; n = 11, CKD treatment) groups. Blood velocity, arterial and aortic wall elasticity measurements in aortic arches/ascending aortas brachiocephalic arteries, left carotid arteries, left subclavian arteries and abdominal aortas were visualized by B-mode, Doppler mode, and M-mode. P, atherosclerotic plaque; AA, aortic arch; BCA, brachiocephalic artery; LCAA, left common carotid artery; LSA, left subclavian artery. We also measured the vascular wall thickness at pre-specified locations at their origin through the M-mode echo examination on AUE at the aortic arch and branching of major vessels (e.g. common carotid artery, left subclavian artery, and innominate artery). The progression of atherosclerosis was associated with progressive thickening of the vascular wall in all groups of mice. The vessel wall thickness was significantly lower among mice treated with HCQ compared to those that were not treated with HCQ (Fig 2), both in mice with normal renal function as well as those with CKD. The results from two-way ANOVA analyses further confirmed that HCQ therapy significantly associated with better preservation of thickness on these vascular walls (P < .0001 for aortic arch, left subclavian artery, and innominate artery). 10.1371/journal.pone.0139226.g002Fig 2Histograms shows the quantitative analysis of mean arterial wall thickness in treated and untreated groups of sham and CKD mice. a-f. Data presented here are mean ± SD. (n = 8 for sham placebo and sham HCQ, n = 13 CKD placebo, and n = 11 CKD HCQ groups), *p<0.01 **p<0.001 as compared to the control group. For normally distributed data, unpaired 1-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons test was used to test wall thicknesses in the 4 different groups of mice when significant (P<0.05). Post hoc analysis with unpaired Student t tests were used when comparing control and treated group. Figs 3–5 show the postmortem data for the extent and severity of atherosclerosis in both CKD and non-CKD groups of mice. HCQ significantly reduced the area of atherosclerosis as evidenced on en face Sudan IV staining of the whole aorta (Figs 3 and 4) in the CKD treatment group (20.75 ± 3.3%) compared to CKD control mice (47.48 ± 4.1%) (P<0.001). This was accompanied by a significant reduction in the severity of atherosclerosis in the aortic bulb on Oil Red O examination in CKD treatment mice compared to control CKD (Fig 5). Similar beneficial effects were confirmed in non-CKD/sham groups, with a reduction in atherosclerotic plaques from 43.18 ± 3.7% (untreated) to 17.53± 2.4% (in treated group (P<0.001), validating the results of a prior study in a non-CKD state (Figs 4 and 5)[16]. This was also confirmed in the two-way ANOVA model (β estimate ± SE: -23.5 ± 5.2, P < .0001). 10.1371/journal.pone.0139226.g003Fig 3 En face analysis of dissected entire aortic specimens in all groups of mice at the end of 16 weeks of therapy.Representative images of the Sudan IV staining for en face analysis of the total aorta. Atherosclerotic plaques are stained in orange/red. (n = 8 sham placebo and sham HCQ, and n = 13 CKD placebo, n = 11 CKD treated groups). 10.1371/journal.pone.0139226.g004Fig 4Total atherosclerosis area for the entire aortas in all groups of mice at the end of 16 weeks of therapy.Graph shows the quantification of total atherosclerotic lesion area on Sudan IV staining of entire separated aortas in all groups of mice. The black horizontal bar represents means. 10.1371/journal.pone.0139226.g005Fig 5Isolated aortic bulb analysis with Oil Red O staining in CKD groups.Representative images of Oil Red O staining for lipid deposits in ascending aortas of all groups of mice. Red indicates the lipid-laden areas representing atherosclerotic lesion coverage. Black arrows represents the aortic root / valve leaflet in the section. BODY.RESULTS.IMPACT OF HCQ ON VASCULAR FUNCTIONS: We measured the vascular endothelial function through the antemortem measurements of vascular elasticity and blood-flow dynamics for all major vessels on AUE using the M mode and Doppler mode. We observed a significant and progressive reduction in vascular elasticity (Figs 6 and 7) and blood flow parameters (Fig 7) in all major vessels over the study period in all groups of mice, in tandem with development of atherosclerosis. Mice receiving HCQ therapy had better preservation of the functional indices of aortic and vascular compliance as determined through the M-mode and Doppler assessments (Fig 7) compared to the mice not receiving HCQ. In parallel with histologic protection, the beneficial effects for HCQ were seen equally potent in the presence of uremic milieu when compared to those with normal renal function (sham groups). 10.1371/journal.pone.0139226.g006Fig 6Quantitative analysis of vascular elasticity in CKD groups.Quantification of vascular wall elasticity as visualized by M-mode intravital ultrasound echography using VisualSonics Vevo 770 in abdominal aortas. The elasticity of the major vessels, i.e., the movement of vessel wall between the systole and diastole, as well as blood velocity within these vessels were significantly better in CKD mice treated with HCQ. *P < 0.05 compared with CKD placebo mice. AA, aortic arch; BCA, brachio-cephalic artery; LCCA, left common carotid artery; LSA, left subclavian artery. 10.1371/journal.pone.0139226.g007Fig 7Progression of vascular elasticity and velocities in aortic arch and brachiocephalic arteries in all groups of mice.Quantitative analysis of serial changes in the elasticity and velocity of the aortas and brachiocephalic arteries in all groups of mice. †p<0.05, ††p<0.01, as compared to the zero time of individual group, **p<0.05 as compared to the untreated control of each group. BODY.RESULTS.IMPACT OF HCQ ON INFLAMMATION AND SELECT BIOCHEMICAL PARAMETERS: HCQ is an anti-inflammatory drug and inflammation has been proposed to have a significant role in many complications of CKD including CVD and renal failure progression. We examined the impact of HCQ on inflammation, renal functions, and lipids parameters. We evaluated the impact of HCQ on systemic inflammation by measuring the serum levels of hsCRP. Therapy with HCQ did not affect the levels of hsCRP in mice with CKD (Table 2). To confirm these results, we conducted CRP measurements in another laboratory at our institution and obtained similar results (data not shown). Treatment with HCQ did not affect IL-6 levels or VCAM levels in the CKD group (Table 2). Similarly, there was no impact of HCQ on the lipid profile in these hyperlipemic mice or on the severity of renal failure between both CKD groups (Table 2). 10.1371/journal.pone.0139226.t002 Table 2 Serum biochemical parameters at the time of sacrifice. Total cholesterol (mg/dL) LDL (mg/dL) IL-6 pg/mL sVCAM-1 ng/mL CRP ng/mL Sham control (n = 8) 883.2±60.9 532±34 494.8±138.8 714.3±50.1 14.6±9 Sham treated (n = 8) 842.9±41.9 526±25.9 470.2±164.6 679.8±42.8 13.6±17 CKD control (n = 13) 957.4±39.5 616.3±44.3 394.8±143.9 838.8±3 33.9±2 * CKD treated (n = 11) 1010.9±60.5 609.7±29.8 369±130.9 826.7±29.5 † 29.9±1 † Serum biochemical parameters for total cholesterol, low-density lipoprotein cholesterol (LDL), interleukin-6 (IL-6), soluble vascular cell-adhesion molecule-1 (sVCAM-1) and high sensitivity C-reactive protein (CRP) at the study end, at the time of sacrifice in all groups of mice. Data presented as means±SD Kruskal-Wallis test followed by Dunn post-hoc test * P <0.01 vs. sham control † P <0.05 vs. sham treated. BODY.DISCUSSION: Our study demonstrates that HCQ confers a significant functional and histological protection against vascular stiffness and atherosclerosis in presence of uremic milieu. HCQ substantially reduced the severity of atherosclerosis (by ~60%) in these atherogenic mice, improved vascular compliance, and prevented vessel wall thickness. More importantly, the anti-atherosclerotic and vasculoprotective effects of HCQ were not mitigated by and appeared to be equally potent in the presence of uremic milieu. The principle outcome of our study is the validation of strong atheroprotective effects for HCQ even in the presence of CKD. HMG Co-A inhibitors (statins), the mainstay for management of atherosclerotic CVD, have strong evidence for their efficacy in animal atherosclerosis models and in human clinical trials in the presence of normal renal function[25–27]. However, when Ivanovski et al. evaluated their efficacy in a similar animal model in the presence of uremia, they found that statins do not reduce the severity of atherosclerosis in the uremic milieu[8]. Clinically as well, statins have repeatedly failed to reduce cardiovascular or all-cause mortality when studied for their efficacy in high CVD and CKD/ESKD populations[5, 6]. Together, these statin data suggest that the therapeutic efficacy of an anti-atherosclerotic agent proven in the presence of normal renal function does not automatically translate into an effective therapy in the presence of uremic milieu and that statins as a sole strategy for the management of CVD in CKD is inadequate. Our study provides evidence for an in vivo anti-atherosclerotic efficacy for HCQ in CKD. Previous studies demonstrating that HCQ is safe to use in the presence of advanced CKD thus make clinical trials feasible. Clinically, endothelial dysfunction with resultant vascular stiffness, inflammation, and accelerated atherosclerosis are prominent factors for CVD associated with CKD[28]: Endothelial injury and dysfunction with reduction in NO availability, and resultant increase in vascular stiffness are some of the earliest events evident in CV injury. Endothelial integrity and NO availability have important roles in the maintenance of vascular tone, control of inflammation, smooth muscle cell proliferation and migration, and thrombogenesis, fibrinolysis,[29] and eventual downstream worsening of atherosclerosis. Ghigo et al. through their in vitro studies have shown that HCQ stimulates vascular endothelial cell NO release[30]. Multiple in vivo studies in animal models of lupus, diabetes, and metabolic syndrome have since shown a beneficial impact of HCQ vascular endothelial cell function, as judged by improvement in endothelial dependent vasodilation[31–34]. Even studies involving human rheumatological cohorts have shown that long-term use of low-dose HCQ is associated with an improvement in indices of vascular compliance, e.g. aortic pulse wave velocity (APWV) and reduction in new-onset hypertension[14, 35, 36]. Endothelial dysfunction as measured by AUE echography has been strongly associated with the development and severity of atherosclerotic plaque in apoE-/- mice models[21, 24], and blinded AUE assessment of major vasculature has excellent validity with the severity of vascular disease and atherosclerosis. Our findings indicate that HCQ in low doses similar to those employed for chronic use in the human population has strong protective effects on the structural and functional aspects of vascular health as reflected by better preserved vascular wall thickness, and vascular flow indices and elasticity across multiple vascular beds. In our study, HCQ failed to reduce the degree of inflammation as reflected by the serum levels of hsCRP among mice with or without CKD. Retrospective database analyses have suggested a possible beneficial role for HCQ on the lipid profile, with some suggesting an improvement in LDL and HDL values[37–39]. In our study, we did not demonstrate a benefit of HCQ on the conventional lipid profile. We also found no significant impact of HCQ on the degree of renal dysfunction among treated vs. control mice with CKD, indicating that the beneficial effect of HCQ was unrelated to improvement in renal function (Table 1). Thus, our results suggest that the beneficial actions of HCQ toward atherosclerosis occurs even in the absence of significant anti-inflammatory, renoprotective, or anti-lipidemic actions, and might be related to alternate mechanisms. Analyses of various in vitro, in vivo, and human cohort data indicate that HCQ has many pleiotropic effects, e.g. reduction in oxidative stress, improvement in insulin sensitivity and metabolic syndrome[14–19, 22], improvement in vascular endothelial function with improved availability of eNOS activity, and inhibition of autophagy[40] with concerns for heightened maladaptive autophagy in endothelial and vascular smooth muscle cells[41–43] in tissues with accelerated atherosclerosis, etc. Razani et al. in their metabolic studies also found that chloroquine, a precursor molecule of HCQ, inhibits atherosclerosis through a p53-dependent stress pathway[17]. More importantly, the reasons for worsening CVD and its refractoriness towards conventional therapies in the presence of uremic milieu are multifactorial and at present unknown. Thus, additional studies are needed to further define the factors associated with the worsening CVD with uremia and the potential mechanisms by which HCQ affords its beneficial effects on atherosclerosis in uremic milieu. Although our study does not provide mechanistic insight, we believe it is clinically relevant because it proposes a therapy for CVD with a drug that has been in use for several decades, even in patients with advanced CKD and ESKD[44, 45]. This suggests a potential for evaluation of HCQ for cardiovascular events in CKD patients. Further studies are needed to clarify the mechanisms through which HCQ confers its benefits.

TITLE: Differential Effects of Adiposity on Pharmacodynamics of Basal Insulins NPH, Glargine, and Detemir in Type 2 Diabetes Mellitus ABSTRACT.OBJECTIVE: To assess the role of adiposity on the pharmacodynamics of basal insulins NPH, detemir, and glargine in type 2 diabetes mellitus (T2DM), as estimated by glucose infusion rate (GIR) and endogenous glucose production (EGP) rate in the euglycemic clamp. ABSTRACT.RESEARCH DESIGN AND METHODS: We examined the variables that best predicted GIR and EGP in 32-h clamp studies after treatment with subcutaneous injection of 0.4 units/kg NPH, detemir, and glargine in 18 T2DM subjects (crossover). ABSTRACT.RESULTS: A multiple regression analysis revealed that BMI best predicted GIR variation during the clamp. BMI was inversely correlated with GIR in all three insulin treatments, but was statistically significant in detemir treatment only. BMI correlated positively with residual suppression of EGP in detemir, but not with glargine and NPH treatments. ABSTRACT.CONCLUSIONS: Adiposity blunts the pharmacodynamics of all basal insulins in T2DM. However, as adiposity increases, the effect of detemir is lower versus NPH and glargine. BODY: We have shown that the pharmacokinetics and pharmacodynamics (PD) of basal insulins NPH, detemir, and glargine, when examined (euglycemic clamp), differ in subjects with type 2 diabetes mellitus (T2DM) (1). The primary PD end point parameter, the glucose infusion rate (GIR) over the 32 h of clamp study, was greater with glargine versus NPH and detemir. We carried out the present analysis to determine the independent variable(s) that best predicted the PD insulin effect, as measured by GIR in clamp studies, after treatments with basal insulins NPH, detemir, and glargine. BODY.RESEARCH DESIGN AND METHODS: After approval by the local ethical committee and informed written consent, 18 T2DM subjects on insulin and/or oral hypoglycemic agents were recruited (Supplementary Table 1) and studied (1). Stepwise regression analyses were done to identify independent variables that best predicted GIR response. As a set of independent variables, we obtained the age of patients, duration of diabetes, plasma C-peptide, and BMI, which we included in the multiple regression analysis. GIR was the dependent variable. Pearson product moment correlation was used to assess association between changes in GIR and other parameters. Data were analyzed by a mixed-model analysis including sequence, treatment, and BMI (<29 and >29 kg/m2, i.e., below and above the median BMI of the overall group) as fixed factors and patients (nested within sequence) as a random effect followed by Bonferroni adjustments for multiple comparisons. Data in the text were expressed as means ± SD and as means ± SE in Fig. 1. All tests were two-sided tests with a nominal significance level of 0.05. Statistical analysis was performed using NCSS 2007 (NCSS, LLC, Kaysville, UT, www.ncss.com). Figure 1Plasma glucose, rates of glucose infusion to maintain euglycemia, and endogenous glucose production in 18 subjects with T2DM given subcutaneous (s.c.) injection of basal insulin NPH or detemir or glargine (1). Left panels: nine subjects with BMI <29 kg/m2; right panels, nine subjects with BMI >29 kg/m2. BODY.RESULTS: A multiple regression analysis revealed a significant model (F4,13 = 4.44, P = 0.017; adjusted R2 = 0.45). The significant predictor variable was BMI (standardized coefficient = −0.575), which accounted for 39% of variation in GIR variable (P = 0.013) (Supplementary Tables 2 and 3). Bivariate correlations of BMI and GIR split by treatment showed an inverse correlation that was significant only with detemir (r = −0.68, P = 0.003), but not with glargine (r = −0.41, P = 0.11) and NPH (r = −0.37, P = 0.15) (Supplementary Fig. 1). In addition, a positive and significant correlation was found between BMI and residual endogenous glucose production (EGP) with NPH and detemir (r = 0.66, P = 0.005, and r = 0.62, P = 0.006, respectively) but not with glargine (r = 0.35, P = 0.17) (Supplementary Fig. 1). An inverse and significant association with duration of action was found for NPH and detemir but not for glargine (Supplementary Table 4). On the basis of BMI status, with all three basal insulins, GIR was lower in people with BMI >29 kg/m2 compared with people with BMI <29 kg/m2 (Fig. 1), but statistical significance was achieved with detemir treatment only (598 ± 604 and 1,564 ± 649 mg/kg, respectively; P = 0.03) and not with NPH (1,058 ± 859 and 1,282 ± 532 mg/kg) and glargine (1,408 ± 563 and 1,668 ± 807 mg/kg) (both P > 0.2). In addition, residual EGP was greater in people with BMI >29 vs. <29 kg/m2, although significance was achieved with detemir (7.8 ± 1.8 vs. 4.1 ± 2.1 μmol/kg/min, P = 0.001) but not with glargine (5.3 ± 1.3 vs. 3.8 ± 2.7 μmol/kg/min, P = 0.231) and tended to be different with NPH (6.6 ± 2.2 vs. 4.0 ± 2.1 μmol/kg/min, P = 0.068). BODY.CONCLUSIONS: In the euglycemic clamp during intravenous infusion of regular insulin, there is an inverse correlation between BMI and GIR in T2DM (2), indicating that the greater the adiposity, the lower the insulin sensitivity. It is reasonable to translate this information to the clinical situation of the subcutaneous injected long-acting insulin, but no study so far has examined the question directly. We recently reported that GIR area under the curve (AUC)0–32 h (a measure of insulin effect on glucose metabolism after subcutaneous injection of long-acting insulins) is greater with glargine than with NPH and detemir (1). However, in that study (1), the factor(s) responsible for GIR variations between T2DM subjects and between the three basal insulins were not assessed. The results of the present analysis indicate that BMI is a factor that may well predict the PD of basal insulins NPH, detemir, and glargine, as indicated by the inverse correlation between BMI and PD (GIR AUC0–32 h). In addition, the results indicate that the PD of the three basal insulins is differentially affected by BMI (i.e., at increasing BMI, the PD was lower for detemir than for NPH and glargine). Likewise, EGP, the well-known primary determinant of fasting blood glucose (3), was more suppressed by glargine and NPH compared with detemir in people with greater BMI. Although all three basal insulins showed a lower suppression of EGP for increasing BMI, the correlations were significant only for NPH and detemir, suggesting that glargine has a greater effect in restraining EGP at increasing adiposity. The reasons for the lower effect of detemir at higher BMI are unclear. We acknowledge that these conclusions have limitations because of the small sample size of the study; nevertheless, the results might explain findings of recent clinical studies. A previous crossover study, longer than 2 months, reported higher dose requirements of the long-acting insulin analogs compared with NPH insulin, in a group (n = 20) of T2DM subjects with BMI >30 kg/m2 (4). Results of studies examining larger groups of T2DM subjects for ≥6 months indicated that higher detemir insulin doses are required versus NPH (5) and glargine for similar efficacy on glycemic control (6–9). However, none of those studies ascertained the impact of BMI on insulin dose requirements. A recent post hoc analysis (10) of a study comparing insulin glargine (once a day) and detemir (twice a day) in T2DM (9) titrated to reach similar glycemic control has shown that the higher dose of insulin detemir needed was directly correlated with BMI (10). In conclusion, BMI largely explains the PD effect of insulins NPH, detemir, and glargine in T2DM subjects previously reported (1). In fact, as BMI increases, the effect of detemir is lower versus NPH and glargine (lower GIR and greater EGP). The current study suggests that it is obesity that primarily drives the larger detemir versus NPH and glargine doses needed in T2DM. BODY.SUPPLEMENTARY MATERIAL: Supplementary Data

TITLE: Results of a Community Randomized Study of a Faith-Based Education Program to Improve Clinical Trial Participation among African Americans ABSTRACT: This is a report of a cluster randomized clinical trial evaluating the effectiveness of a church-based educational intervention aimed at improving African Americans' (AA) participation in clinical trials. Two hundred and twenty-one AA subjects ages ≥50 years from six predominantly AA churches were randomized to intervention or control condition. The intervention included three educational sessions about clinical trials and health disparities; control participants completed questionnaires. Primary endpoints of the study were differences in individual subjects' intentions to obtain clinical trial information and intention to join a clinical trial, as determined by 10 point scale items at baseline, three and six months. A statistically significant increase in the intention to obtain clinical trial information at the three and six month time points was observed in the intervention group, but not the control group. Older participants (65–95 years) were less likely than younger participants (50–64 years) to increase their motivation to seek clinical trial information by the three and six month time points. No significant increases were observed in intention to join clinical trials. This randomized trial shows that AA church-based educational interventions are likely to increase the motivation of AA subjects to obtain clinical trial information and are therefore potentially effective at ameliorating the underrepresentation of AA subjects in clinical trials. BODY.1. INTRODUCTION: Despite decades of significant medical advances that have resulted in approval of novel prevention strategies, therapeutics, and medical devices, clinical trials continue to be challenged by underrepresentation of racial and ethnic minority participants [1,2]. Recent data demonstrate that overall participation rates among racial/ethnic minorities continue to lag behind those of other groups for various types of clinical trials [1,3,4]. Moreover, enrollment rates are lower among the elderly (typically defined as those ≥70 years) when compared to younger participants [5,6,7], and lower among women than men in cardiology trials [1,4]. Older minority populations (age ≥65 years) carry a heavy burden of chronic and infectious disease morbidity and mortality, and are likely to be prime beneficiaries of medical advances if clinical trial results yield generalizable findings for this group [6,8]. Thus, underrepresentation of this group in trials presents a significant issue given the dramatic increases in the aging population that is also a growing consumer segment for pharmaceuticals and medical products [9,10]. With an increasing aging minority population, it is critically important that randomized clinical trials include adequate representation of this segment of the population [6,11]. Thus, underrepresentation has become an issue of social justice due to the potential for realization of significant health inequities in the years ahead [12,13]. Although older persons (ages ≥65 years) comprise 14% of the developed world's population, this group also consumes one-third of pharmaceutical products, most of which were tested with non-representative populations [14]. Enrollment of persons ≥65 years, including racial and ethnic minorities, in clinical trials is therefore of global interest to address health disparities and the achievement of Healthy People 2020 objectives [15,16]. For this reason, the Food and Drug Administration (FDA), the U.S. Centers for Disease Control and Prevention (CDC), and other groups are now strongly advocating for reduction in age-based exclusions when feasible in clinical trials [16,17]. Significant challenges have been previously described in the recruitment of minorities including older adults (50–69 years) and elderly populations (≥70 years) [10,18,19]. These include logistical challenges such as getting to clinical locations, a lack of social support and social norms promoting participation, experiences of perceived stigma in medical environments, researcher distrust, health challenges, and adequate compensation [20,21,22,23,24]. Among those who have examined factors associated with clinical trial participation, perceived health status and personal/social benefit associated with enrollment factored as strong behavioral predictors [25]. In addition, simply knowing about studies recruiting the population has facilitated involvement of older adults, especially if communication was with a family member or health navigator [26]. Thus, awareness of trials open to enrollment and the personal relevance of the health topics addressed by available clinical studies, combined with perceived social support, have been demonstrated as important facilitators for engagement of this population [26]. Other studies have also highlighted the role of African American churches in recruitment of Southern African Americans, as this influence extends beyond religion [27,28]. For many older African Americans, the church is the centerpiece of religious, social, and political life. Additionally, the church provides an effective means to involve Southern African Americans in clinical research, especially women who belong to communities of faith, as they have been shown to be receptive to health messages delivered in this setting [29,30]. With the support and involvement of pastors, community members, and subject matter experts, we developed and tested an intervention entitled "Delivering a Dose of Hope" to address the problem of clinical trial underrepresentation among a specific segment of African Americans [29]. BODY.2. EXPERIMENTAL SECTION.2.1. DESIGN: This study employed a cluster randomized study design to evaluate an educational intervention developed to increase clinical trial participation among African Americans aged 50 years and older [29]. Twenty churches in the Atlanta metropolitan area with ≥30% membership of African American congregants aged 50 and older were identified through ethnographic observation and informant interviews. The identified churches were enumerated based on denomination and estimated congregational membership to allow for matched pair selection. Three pairs of churches were then randomly selected to participate. One church in each pair was assigned to the control group while its match received the intervention. Following church selection, study participants for each arm were recruited from their respective churches. Within churches, recruitment occurred through flyers, outreach, and health minister or pastor referral to the program staff for eligibility screening. To be considered for inclusion in the trial, an individual had to be ≥50 years, identify as Black/African American, and may not have previously participated in any clinical trials. Based on these criteria, we obtained written consent from 221 persons who were eligible to participate. BODY.2. EXPERIMENTAL SECTION.2.2. PARTICIPANTS: A total of 221 subjects between the ages of 50 and 95 were recruited from the six churches. Of these, 109 participants were in the control group and 112 participants were in the intervention group. BODY.2. EXPERIMENTAL SECTION.2.3. INTERVENTION: Members of the intervention group participated in three information sessions about clinical trials and related health issues guided by church leaders, subject matter experts, and clinical researchers. The church leaders included health ministers with medical degrees and/or doctorates. We also invited Center Disease Control (CDC) public health practitioners with doctorates to speak. They developed PowerPoint presentations and handouts, and created interactive group exercises. Our clinicians and public health experts included faculty and staff from the Emory School of Medicine and Rollins School of Public Health and the Grady Healthcare System who held medical (MD) and public health (PhD and MPH) degrees. Special care was taken to recruit racially and ethnically diverse "program faculty" from within and outside the churches to develop and present the material in the designated three-hour time frame per session. Each presentation lasted up to 40 min with approximately 20 min of dedicated group discussion time for that topic with the speaker/subject matter expert program faculty. The sessions included discussions on lack of community participation in clinical trials (and related historical abuses), concerns about participant safety, clinical trial ethics, the influence of social networks, and health concerns relevant to the participants for which clinical trials were available for enrollment [29]. Control group participants completed questionnaires. They did not engage in any information sessions regarding enrollment in clinical trials, but were invited to attend community events such as health fairs and screenings, and educational presentations on health topics unrelated to clinical trials (e.g., mammography screening). Participants in both groups were notified of studies currently recruiting in the community through monthly telephone and email outreach. BODY.2. EXPERIMENTAL SECTION.2.4. SURVEY MEASURES: All study participants also completed questionnaires on attitudes, beliefs, and perceptions towards clinical research upon enrollment, and at three and six months after enrollment. The questionnaires consisted of items related to demographics, relationships with healthcare providers, attitudes about influenza and immunization, social ties and channels of communication, and attitudes, beliefs, and perceptions towards clinical research and participation in clinical trials. Study participants' clinical trial enrollment was followed for an additional 18-month period after the six-month questionnaire. As the primary endpoints, this interim analysis focused on the effect of the intervention on participants' self-reported intentions to contact Emory University's clinical study sites for information about clinical trials and intentions to join clinical trials. Final trial enrollment outcomes, and the effects of attitudes, beliefs, and social networks will be assessed in future studies. The primary hypothesis for this interim analysis was that the intervention would lead to increases in participants' intentions to seek information about and join clinical trials. In future analyses, we will examine how these intentions translate into action. BODY.2. EXPERIMENTAL SECTION.2.5. MEASURES OF INTENTION TO PARTICIPATE IN CLINICAL TRIALS: We examined the intervention's effects on two trial participation intention measures: (1) participants' self-reported intention to seek information about clinical trials and (2) their intention to participate in clinical trials. Intention to participate in clinical trials was measured through two survey items: (1) intention to contact Emory University about participation in clinical trials and (2) intention to join a clinical trial, each measured at baseline, three months, and six months. Intention to seek information was measured by the question "On a scale from 1 (definitely not) to 10 (definitely so), rank your likelihood of contacting Emory University about being in a medical research study in the next 6 months," where participants circled the appropriate number. Intention to join was measured with the question, "On a scale from 1 (definitely not) to 10 (definitely so), rank your likelihood of joining a medical research study within the next 6 months." On the three- and six-month surveys, participants were asked whether they had sought information about clinical trials or joined a clinical trial since the previous survey. Those who reported seeking information about clinical trials were not asked their intentions to seek information, and were instead assigned a likelihood of 10 for intention to seek information for the appropriate time point. Similarly, those who reported that they had enrolled in a clinical trial at three or six months were assigned a likelihood of 10 for intention to join a clinical trial. BODY.2. EXPERIMENTAL SECTION.2.6. STATISTICAL ANALYSIS: Statistical analyses were performed using SPSS version 22.0 for Windows (IBM SPSS Inc., Chicago, IL, USA). Descriptive statistics were calculated for all variables, including means and standard deviations for continuous variables and frequencies and percentages for categorical variables. Chi-square and Mann-Whitney U-tests of independence were used to assess significant differences in sociodemographic variables between the control and intervention groups. Multivariable linear mixed effects models were used to examine the influence of the intervention on self-reported intention to seek information about and intention to join clinical trials while adjusting for several covariates, including age, gender, income, and baseline intentions. A single model was fitted to each outcome at each of the three-month and six-month time points, and included a random effect to account for the correlation between individuals within the same churches due to the cluster sampling methodology. Potential interactions between assigned groups and each of the covariates were examined, and significant interactions were included in the final model. Participants with missing outcomes or covariates were accounted for using casewise deletion. Within the control and intervention groups, additional mixed effects models were used to examine changes in mean intention to seek information about or join clinical trials. These models adjusted for baseline intentions, age, and interaction between baseline intentions and intervention. Estimated marginal mean change in intention was calculated for each group and evaluated for significance using t-tests. All statistical tests were two-tailed and evaluated at a significance level of 0.05. Confidence intervals were calculated at a 95% significance level. BODY.3. RESULTS .3.1. SAMPLE CHARACTERISTICS: A total of 221 subjects participated in the study, 112 in the intervention group and 109 in the control group. The mean age was 64.0 (SD = 7.7) and 78.7% (n = 174) of the participants were female (Table 1). Most participants had at least completed high school (94.1%, n = 208), with 64.2% (n = 142) having completed some form of post-secondary education. The majority of participants (66.1%, n = 146) had a household income of less than $60,000. Slightly less than half of all participants were married (46.2%, n = 102) and another 26.7% (n = 59) were divorced/separated. There were no significant differences in gender, education, or income between the control and intervention groups (Table 2). However, there was a statistically significant difference in the age distribution (Mann-Whitney U-Test; p = 0.03), with the control group having a significantly higher proportion of 70–79 year olds than the intervention group (z-test, p < 0.05). ijerph-13-00041-t001_Table 1 Table 1 Sample characteristics. Characteristics n % Total 221 100 Gender Male 47 21.3 Female 174 78.7 Age (Mean = 64, Median = 64, Standard Deviation = 7.7) 50–59 62 28.1 60–69 108 48.9 70–79 41 18.6 80–89 5 2.3 90–99 2 0.9 Missing 3 1.4 Education Grade K–8 3 1.4 Grade 9–11 10 4.5 High School/GED 66 29.9 Technical/Vocations/Associates 66 29.9 Bachelor’s Degree 37 16.7 Master’s Degree 33 14.9 Doctorate 6 2.7 Income <$20,000 61 27.6 $20,001–$40,000 49 22.2 $40,0001–$60,000 36 16.3 $60,001–$80,000 20 9.0 $80,001–$100,000 19 8.6 >$100,000 13 5.9 Missing 23 10.4 Relationship Status Single/Never Married 24 10.9 Married/Domestic Partner 102 46.2 Divorced/Separated 59 26.7 Widowed 35 15.8 Other 1 0.5 Assigned Group Intervention 112 50.7 Control 109 49.3 ijerph-13-00041-t002_Table 2 Table 2 Participant characteristics by assigned group. Characteristics Control Intervention N % a N % a p –Value b Total 109 49.3 112 50.7 Gender 0.41 Male 22 20.2 25 22.3 Female 87 79.8 87 77.7 Age 0.03 50–59 25 23.1 37 33.6 60–69 53 49.1 55 50.0 70–79 29 26.9 12 10.9 80–89 1 0.9 4 3.6 90–99 0 0 2 1.8 Missing 1 – 2 – Education 0.45 Grade K–8 1 0.9 2 1.8 Grade 9–11 3 2.8 7 6.3 High School/GED 33 30.3 33 29.5 Technical/Vocations/Associates 31 28.4 35 31.3 Bachelor’s Degree 23 21.1 14 12.5 Master’s Degree 14 12.8 19 17.0 Doctorate 4 3.7 2 1.8 Income 0.50 <$20,000 32 33.3 29 28.4 $20,001–$40,000 27 28.1 22 21.6 $40,0001–$60,000 16 16.7 20 19.6 $60,001–$80,000 8 8.3 12 11.8 $80,001–$100,000 6 6.3 13 12.7 >$100,000 7 7.3 6 5.9 Missing 13 – 10 – Relationship Status 0.25 Single/Never Married 10 9.2 14 12.5 Married/Domestic Partner 47 43.1 55 49.1 Divorced/Separated 36 33.0 23 20.5 Widowed 16 14.7 19 17.0 Other 0 0 1 0.9 a Percentage of non-missing responses within study group. b Mann-Whitney U-test for age; chi-square test for all other variables. BODY.3. RESULTS .3.2. INTENTION TO PARTICIPATE IN CLINICAL TRIALS: Changes over time in both mean intention to seek information about, and intention to join clinical trials for each group are shown in Figure 1. Baseline intentions to seek information about clinical trials were similar for the two groups (Figure 1a), with a mean baseline score of 5.5 out of 10 (SD = 2.9, missing = 2) for the control group, and 5.7 out of 10 (SD = 2.9, missing = 3) for the intervention group. At three months, the mean intention to seek information score for the control group was 6.6 out of 10 (SD = 3.3, missing = 4), while the mean score for the intervention group was 7.5 out of 10 (SD = 3.1, missing = 10). At six months, the control group had a mean score of 6.5 out of 10 (SD = 3.6, missing = 5) and the intervention group a mean score of 7.1 out of 10 (SD = 3.1, missing = 9). Figure 1(a) Change in mean intention to seek information over time; (b) change in mean intention to join over time. ijerph-13-00041-t003_Table 3 Table 3 Intervention effect estimates and results from multivariable linear mixed models. Predictor Variable Intention to Seek Information Intention to Join 3 Months (miss = 42) 6 Months (miss = 43) 3 Months (miss = 41) 6 Months (miss = 44) β (95% CI) p −value β (95% CI) p −value β (95% CI) p −value β (95% CI) p −value Intervention (ref = control) 0.4 (−2.27, 3.08) a 0.70 0.12 (−1.91, 2.16) d 0.88 0.11 (−2.58, 2.35) b 0.90 0.12 (−1.44, 1.69) d 0.85 Baseline Intention 0.12 (−0.1, 0.34) c 0.28 0.21 (−0.01, 0.43) c 0.06 0.21 (0.02, 0.40) c 0.03 0.12 (−0.11, 0.32) c 0.33 Age −0.09 (−0.15, −0.02) 0.01 −0.11 (−0.18, −0.05) <0.01 −0.14 (−0.21,−0.07) d <0.01 −0.08 (−0.14, −0.02) 0.01 Gender (ref = male) −0.83 (−1.96, 0.3) 0.15 −0.67 (−1.79, 0.45) 0.24 −0.76 (–1.68, 0.17) 0.12 −0.42 (–1.43, 0.59) 0.41 Income 0.09 (−0.21, 0.39) 0.57 −0.11 (−0.41, 0.2) 0.49 −0.04 (−0.29, 0.21) 0.76 −0.27 (−0.55, 0.001) 0.05 Intervention × Baseline e −0.14 (−0.44, 0.17) 0.38 −0.11 (−0.43, 0.2) 0.48 −0.12 (−0.40, 0.16) 0.39 −0.18 (−0.5, 0.13) 0.26 Intervention × Age f --- --- --- --- −0.11 (−0.22, −0.01) 0.04 --- --- a The effect of the intervention when baseline is 7. b The effect of the intervention when age is 65 and baseline is 7. c The effect of baseline intention in the intervention group. d The effect of age in the intervention group. e Linear interaction of Intervention and Baseline Intention effects (that is, the amount by which the Intervention effect changes for each unit increase in Baseline Intention). f Linear interaction of Intervention and Age effects (that is, the amount by which the Intervention effect changes for each year increase in Age). ijerph-13-00041-t004_Table 4 Table 4 Estimated marginal mean changes in clinical trial intentions. Mean Differences in Intention (95% CI) a Assigned Group Intention to Seek Information Intention to Join Baseline and 3 Months (miss = 42) Baseline and 6 Months (miss = 43) 3 Months and 6 Months b (miss = 48) Baseline and 3 Months (miss = 41) Baseline and 6 Months (miss = 44) 3 Months and 6 Months ( miss = 46 ) Control 1.08 (−0.44,2.6) 1.02 (−0.43, 2.47) 0.07 (−0.76, 0.9) 0.09 (−1.15, 1.32) −0.03 (−1.16, 1.1) −0.18 (−1.54, 1.18) Intervention 1.98 (0.47, 3.5) * 1.49 (0.05, 2.93) * −0.29 (−1.11, 0.54) 0.36 (−0.86, 1.59) 0.52 (−0.61, 1.65) 0.22 (−1.14, 1.57) a All models adjust for baseline intention, age, and intervention * baseline intention. b Used model without church random effect because estimated between-church variance was zero. Intention to join clinical trials at baseline was similar for the two groups as well, with the control group having a mean baseline score of 5.8 out of 10 (SD = 2.7, missing = 4) and the intervention group a score of 5.8 out of 10 (SD = 2.7, missing = 3) (Figure 1b). At 3 months, the mean score for the control group was 5.9 out of 10 (SD = 2.7, missing = 4) and for the intervention group 6.2 out of 10 (SD = 2.8, missing = 9). The mean intention score at 6 months was 5.7 out of 10 (SD = 3.0, missing = 5) for the control group and 6.3 out of 10 (SD = 2.9, missing = 9) for the intervention group. Results from the multivariable linear mixed models for three- and six-month intentions to seek information about and join clinical trials are shown in Table 3. Participation in the intervention group was not associated with greater improvement in intention to seek information about or join clinical trials at follow-up. Age was significantly related to three-month intention to seek information (p < 0.05), six-month intention to seek information (p < 0.01), three-month intention to join (p < 0.01), and six-month intention to join (p < 0.05), with older participants indicating less increase, or greater decrease, in intentions over baseline. There was a significant interaction between age and intervention in the three-month model for intention to join clinical trials: in the intervention group, younger age was significantly associated with more positive change in intention to join clinical trials at three months relative to baseline (p < 0.01), while in the control group, age was not significantly associated with a difference in the change in intention to join from baseline to three months (p = 0.51). A summary of the estimated marginal mean change in the two trial participation intention measures within each group resulting from the multivariable linear mixed models for mean changes are presented in Table 4. In the intervention group, intention to seek information about clinical trials on average increased significantly from baseline to three months (adjusted mean difference = 1.98, p < 0.05) and from baseline to six months (adjusted mean difference = 1.49, p < 0.05), after adjustment for baseline intentions, age, and interaction between baseline intentions and intervention. Control group participants did not see a significant increase or decrease in intention to seek information about clinical trials, on average, between any pair of time points. There were no significant changes in the average intention to join between any pair of time points for either group. BODY.4. DISCUSSION: This study found that the comprehensive intervention "package" characteristics (i.e., church-based, pastor-supported, based in social networks) likely resulted in the initiation of behavioral change consistent with established theoretical models and persuasion frameworks [31,32,33]. Health communication models indicate that persuasion occurs over a continuum and is bolstered by repeated exposure to messages or content [34]. According to previous behavioral communication studies, persuasion likely occurs at later time points after which the credibility of the source is established through information verification processes also known as "assimilation" [35,36]. The results from this study demonstrate that the first step in motivating people to consider participation—intention to seek information about clinical trials—increased significantly by three months in the intervention group, much faster than we anticipated. Thus, with our participants indicating that they were more likely to get information over this initial introductory program period, the findings offered evidence that information seeking would precede behavioral initiation accounting for the presence of "attitudinal ambivalence" [37]. This term refers to a condition we expected would be present due to knowledge of medical abuses via oral history or, for the intervention arm participants, stemming from our discussion of past clinical trials and ethics abuses in medical and public health research. Our participants were likely to feel conflicted about the information we would present, as their beliefs and knowledge would contribute to attitude formation and subsequent participatory decisions to join or forego joining clinical trials. In other words, we expected that we needed three sessions with our participants to establish our "source credibility" and, consequently, to generate any motivation to attend to messages we presented about clinical trials [36]. In reality, we needed only two sessions for participants to formulate their decision to seek additional information and reduce attitudinal ambivalence. The study findings also indicate that intention to join clinical trials also seemed to increase by the three-month time point, yet the increase was not statistically significant. Similarly, the effects resulting from participation in the program diminished after three months. Both intention to seek information about and intention to join clinical trials did not increase significantly between three- and six-month time points. Indeed, previous studies have demonstrated that persuasion is most effective when study participants are exposed to strong messages, have time to think about them, and have the ability to process the message in the face of counter-persuasion (e.g., negative enduring beliefs and/or social norms about clinical trials) [34]. Our findings suggest that most processing likely occurred in the first three months of program involvement and tapered off after that time point. Although our participants indicated their intention to seek more information about clinical trials at the three-month time point, there is insufficient evidence to attribute this observed, significant increase to the intervention itself. It is possible that a portion of this increase is attributable to positive interaction with the researchers, the perceived social influence of the church network involved with the study, and the availability of information about current clinical trials that was provided to both the control and intervention groups. As this program was developed in collaboration with our Community Advisory Board, pastors from participating churches and their health ministers, any effect may also be attributable in part to the Community-Based Participatory Research (CBPR) processes that occurred [38]. Indeed, there is a growing body of literature on specific persuasive effects that motivate older minorities to participate in research studies [39]. Specifically, hearing the recommendations of family members, physicians, and others, combined with perceived social support for participation, serve as powerful motivational queues [39]. Because control intentions also appeared to increase slightly (though not statistically significantly), we believe all of the above likely contributed to increased intentions; however, more experimental arms and additional research are necessary to assess these effects. The findings also correspond with those of previous studies that point to significant challenges associated with the recruitment of elderly populations [10,19,26]. Advanced age (≥65 years) tempered the increase in intentions over the course of the study; elderly participants in both groups displayed smaller increases in intention (or greater decreases in intention) than their younger (50–64 years) counterparts on both intention outcomes. This suggests that our participants were well-aware of their personal limitations associated with participation, including reliance on others for transportation to/from study visits, their physical immobility, and their health restrictions likely resulting in their exclusion from studies. Thus, their intentions reflect an array of sociostructural and personal factors that must be considered in future interventions seeking to enroll this group [40]. We argue that a multi-pronged intervention which accounts for distance to/from clinical trial sites, transportation options, and whose study promotion messages originate from the church would characterize a future intervention model. In addition, it will be worth evaluating this time-intensive approach to other strategies, such as direct patient outreach and mass advertising to determine its cost-benefit ratio. We acknowledge the limitations in this study. The self-reported intentions to seek information about or join clinical trials that are examined as the analytic outcomes are intermediate to the behavioral outcomes of information seeking and joining clinical trials. However, these intermediate outcomes provide important information about both the pathway to action for clinical trial participation and the internal processes that may lead to action. We also recognize that this pilot intervention is limited in its broader application based on these pilot results. Our interventions in urban Atlanta churches may yield different results than those performed in other cities or in rural areas, especially as the easy availability clinical trials relevant to the participants is necessary for intervention success. On-site enrollment and trial implementation could help improve participation where trial access is difficult [41]. We also know that the involvement of pastors and church leaders is critical to recruitment and to congregant engagement in the intervention, yet their influence may also introduce ethical concerns related to coercion. Our participating churches had a variety of educational backgrounds, and on average our participants had a relatively high degree of educational attainment, making results difficult to generalize to churches serving congregants with less formal education. BODY.5. CONCLUSIONS: After three months, participants from the intervention arm showed increased intentions to seek information about clinical trials. These results suggest that the combination effect derived from positive engagement with researchers, health ministers, pastors, and other subject matter experts in a familiar faith-based setting, plus obtaining information about relevant clinical trials quickly, engages older African Americans on research decision-making. Because these increased intentions were not mirrored by participants of advanced age (≥65 years), it is likely that additional efforts must be made to address the special barriers to research participation faced by the elderly. Nonetheless, this study highlights the promise of church and faith networks as avenues through which to influence older African Americans' knowledge and attitudes towards clinical research participation.

TITLE: Psychological intervention reduces self-reported performance anxiety in high school music students ABSTRACT: Music performance anxiety (MPA) can be distressing for many young people studying music, and may negatively impact upon their ability to cope with the demands and stressors of music education. It can also lead young people to give up music or to develop unhealthy coping habits in their adult music careers. Minimal research has examined the effectiveness of psychological programs to address MPA in young musicians. Sixty-two adolescents were pseudo-randomized to a cognitive behavioral (CB) group-delivered intervention or a waitlist condition. The intervention consisted of psychoeducation, cognitive restructuring and relaxation techniques, identification of strengths, goal-setting, imagery and visualization techniques to support three solo performances in front of judges. Significant reductions in self-rated MPA were found in both groups following the intervention and compared to their baseline MPA. This reduction was maintained at 2-months follow-up. There appeared to be inconsistent effects of the intervention upon judge-rated MPA, however the presence of floor effects precluded meaningful reductions in MPA. There appeared to be no effect of the intervention upon judge-rated performance quality. This study highlights the potential for group-based CB programs to be delivered within school music curricula to help young musicians develop skills to overcome the often debilitating effects of MPA. BODY.INTRODUCTION: Music performance anxiety (MPA) is "the experience of intense and persistent anxious apprehension related to musical performance" (Kenny, 2010, p. 433). It can have a serious negative impact on musicians at any age, with research showing it is present in adult (Van Kemenade et al., 1995; James, 1998), adolescent and child musicians alike (Ryan, 2005; Fehm and Schmidt, 2006; Osborne and Kenny, 2008; Boucher and Ryan, 2011). MPA often leads to poor performance and educational outcomes in music (Kubzansky and Stewart, 1999) and to a lack of enjoyment of the performing experience. This, in turn, can lead to unhealthy coping strategies such as an over-reliance on illicit and licit drugs (West, 2004; Park, 2010) or alcohol (Dobson, 2011), which can compromise or prematurely end promising musical careers (McGinnis and Milling, 2005; Ryan and Andrews, 2009). Data from child and adolescent musicians has revealed that their experience of MPA is similar in quality and intensity to that experienced by adult musicians (Smith and Rickard, 2004; Osborne et al., 2005; Ryan, 2005; Kenny and Osborne, 2006; Osborne and Kenny, 2008). Given that most professional musicians begin their training before the age of 12 years (Nagel, 1987), there have been calls to include early interventions for MPA within school music curricula, alongside music theory and instrumental technique (Nagel, 2009; Khalsa et al., 2013; Thomas and Nettelbeck, 2014). This notion of early intervention is supported by research revealing that performance anxiety appears to be a topic of concern for young musicians, but one that they feel is not adequately addressed by teachers and institutions (Fehm and Schmidt, 2006; Ryan and Andrews, 2009). This inadequacy may be partly attributable to the limited number of studies examining the effectiveness of treatment for MPA in musicians, particularly efficacy studies for interventions targeted at younger musicians. There have been a number of recent reviews conducted on existing treatments for MPA, which indicate that a large range of treatment modalities have been developed, namely behavioral, cognitive, cognitive-behavioral, pharmacological, and alternative therapies (Kenny, 2005, 2011; McGinnis and Milling, 2005; Brugues, 2011). Unfortunately, the overall finding from these reviews is that too few studies meet rigorous methodological criteria, limiting the confidence with which these treatments can be recommended to musicians with MPA. For example, many studies have small sample sizes, lack a randomly assigned control group, do not specify the details of treatment, and/or fail to collect follow-up data. The most promising intervention paradigms that emerge from these meta-reviews involve a combination of cognitive and behavioral techniques, including cognitive restructuring, relaxation, and mental skills training (Harris, 1987; Clark and Agras, 1991; Roland, 1993). This is consistent with Kenny and Osborne's (2006) finding that negative beliefs (e.g., "If I make the slightest mistake, they'll think I'm incompetent and I'll get thrown out of school") greatly increased the prediction of MPA over trait anxiety and gender alone in high school musicians, highlighting the need to address these beliefs in the treatment of MPA in young musicians. Research with adult musicians has also demonstrated that cognitive restructuring techniques that identify and challenge self-defeating, task-irrelevant thought patterns, and replace them with more adaptive and realistic views, significantly reduce levels of MPA (Sweeney and Horan, 1982; Harris, 1987; Clark and Agras, 1991; Connolly and Williamon, 2004; Buswell, 2006). Osborne et al. (2007) piloted a cognitive behavioral (CB) program incorporating performance psychology techniques for MPA in elite secondary music students and found that all students reported reductions in MPA, however only significant reductions were reported for students who adhered to the program requirements. Su et al. (2010) found that training in a relaxation breathing technique was associated with a decrease in MPA in young performers of different backgrounds. While such studies have demonstrated the efficacy of CB interventions in reducing self-perceived MPA, there is less evidence that these interventions increase the quality of a musician's performance. It was originally believed that a simple relationship existed between anxiety and performance, namely that anxiety impairs the quality of music performance, summarized by the "inverted U" law of arousal and performance (Samuels and Samuels, 1987; Hancock and Ganey, 2003). It is now understood however, that a moderate amount of anxiety enhances performance when an individual's skill level matches the performance demands of the situation (Jackson and Csikszentmihalyi, 1999) and the individual interprets that anxiety positively (Jones et al., 1993). For example, Hancock and Warm's (1989) dynamic model of stress and sustained attention provides an "extended-U" conceptualization through the inclusion of both physiological and psychological adaptability to account for the complexity of stress on human performance capability (Hancock and Ganey, 2003). Indeed, intervention studies attest to this more complex, extended relationship. Some early studies found that musicians' MPA decreased and performance quality improved following an intervention with psychotherapy (Appel, 1976; Sweeney and Horan, 1982). Similarly, Roland (1993) demonstrated significant reductions in self-reported state anxiety as well as moderate to strong effect sizes for improved performance quality following a CB intervention for MPA in tertiary students. In contrast, other studies have found no improvements in performance quality following behavioral interventions (Wardle, 1975; Mansberger, 1988; Deen, 2000; Reitman, 2001). For example, whilst Osborne et al. (2007) found significant improvement in self-reported MPA following a CB intervention, there appeared to be no effect on performance quality as rated by external judges. Consistent with the dynamic model of stress and performance (Hancock and Warm, 1989; Hancock and Ganey, 2003), recent work has shown that a performance psychology intervention which encourages conservatoire musicians' self-awareness of idiographic, optimal levels of performance energy (physiological adaptability), coupled with strategies to redirect attention away from task-irrelevant to task-relevant musical cues (psychological adaptability), successfully improves performers' capacity to manage distressing performance anxiety and boosts performance resilience (see Osborne et al., 2014). It is even less clear whether such CB interventions have any effect upon an external observer's ratings of a performer's levels of manifest MPA. Only one study, to our knowledge, has included an external observer's perceptions of MPA, however this was not an intervention study. Kubzansky and Stewart (1999) found that, while self-reported ratings of anxiety were not associated with performance outcomes, judges' ratings of anxiety were strongly related to performance evaluations. Those who were perceived as more anxious were rated as having a poorer performance, regardless of how anxious the performers actually felt. A positive but non-significant correlation was also observed between self-perceived MPA and the judges' ratings of MPA, indicating that the performers' own ratings of anxiety did not reliably correlate with the observers' ratings. While CB techniques have been shown to have the strongest evidence base in the management of MPA, there have also been suggestions to call upon already-established techniques in the sports psychology domain, including performance psychology and positive psychology elements, which have become popular in recent years for treating athletes with performance anxiety (Orlick and Partington, 1988; Seligman, 2002). This is largely due to the many known overlapping features of sports and musical performances, with both requiring high levels of motor control and learning, mastery over mind and body, the necessity for implicit recall and smooth performance, and the presence of an audience, which invokes the potential for enjoyment of excellence, but also psychological pressure (Yoshie et al., 2009). Performance psychology techniques place an emphasis on mental rehearsal (visualization), goal setting, focusing on strengths, with the ultimate goal being to achieve a state of "flow" or an experience of complete immersion in an activity (Williams, 2010). Positive psychology draws upon similar techniques, with a particular focus on the utilization of existing strengths, achieving flow, positive visualization, and the close study of what occurs during an optimal performance (Csikszentmihalyi, 1990). Informed by the success of such performance and positive psychology techniques in athletes, as well as by the growing evidence base for the effectiveness of CB techniques in adult musicians, Unleash Your Potential: Thinking Skills for Peak Performance (Brandon and Ivens, 2009) was developed as a preventative skills-based program for adolescents to facilitate optimal performance across a wide range of performance areas. The CB elements of this program include psychoeducation, cognitive restructuring, and relaxation techniques, whilst techniques such as identification of strengths, goal-setting, positive self-talk and thinking, imagery and visualization are drawn from the performance and positive psychology literature. Given the mounting evidence that MPA is a topic of concern for young musicians and the lack of MPA interventions for this age group, there is a need to scientifically evaluate early intervention and preventative approaches that can be delivered in the school system. The aims of this study were three-fold: (1) to assess whether Unleash Your Potential can be effective in reducing MPA in young musicians; (2) to explore whether participation in this program changes external ratings of manifest MPA; and (3) to assess whether the program improves the quality of a music student's performance. It was hypothesized that: Unleash Your Potential would be superior to a wait-list (WL) control condition in reducing self-reported MPA, and that effects would be maintained at 2 month follow-up.Unleash Your Potential would be superior to a WL control condition in reducing judge-rated MPA, and that these effects would be maintained at 2 month follow-up.Unleash Your Potential would be superior to a WL control condition in improving performance quality, and that these effects would be maintained at 2 month follow-up. BODY.MATERIALS AND METHODS.PARTICIPANTS: Sixty-two female instrumental students in Grade 7–9 (M = 13.78 years, SD = 0.85 years), from a Catholic girl's school in Melbourne, Australia volunteered to take part in this study. Instruments included piano (25%), woodwind (22%), string (16%), brass (10%), voice (10%), guitar/bass guitar (6%), and percussion (5%), with two girls combining voice with guitar (3%). Students had learned their instrument for an average of 4 years (SD = 2.6, range 1–10 years). BODY.MATERIALS AND METHODS.MEASURES.DEMOGRAPHICS: Data included age, main instrument, and years of learning the instrument. Eight open-ended questions asked participants about their reasons for joining the program, expected gains from participating and previous techniques used to manage MPA. Responses helped guide the program facilitators to meet the particular needs of students within each group. BODY.MATERIALS AND METHODS.MEASURES.SELF-REPORTED MPA: Self-reported MPA was measured using a modified version of the Music Performance Anxiety Inventory for Adolescents (MPAI-A; Osborne and Kenny, 2005; see Supplementary Material Table 1). Twelve items from the Somatic, Cognitive and Performance Evaluation factors were used to create a state-performance version. Items were modified to describe immediate feelings related to the pre-performance. For example, "Before I perform I get butterflies in my stomach" was changed to "I have butterflies in my stomach," and "When I perform in front of an audience I get sweaty hands" was changed to "I have sweaty hands." Items were answered using a 7-point Likert scale ranging from 0 = "Not at all; Hardly ever" to 6 = "All or most of the time" and summed to yield a total score (range = 0–72), where higher scores indicate higher levels of MPA. Similar to the original scale, this modified measure displayed high internal consistency (Cronbach's alpha = 0.91). BODY.MATERIALS AND METHODS.MEASURES.JUDGE-RATED MPA AND PERFORMANCE QUALITY: Two judge-rated scales measured the behavioral manifestation of MPA and performance quality (adapted from Halls, 2011; see Supplementary Material Table 2). Behavioral manifestations of MPA were scored using a Likert scale of "0—No effect" to "6—Significant effect" where higher scores indicate higher levels of performance anxiety. For items such as sweating, trembling, hyperventilating, tense musculature, and technical mishaps/stumbling, an overall score of how much these behavioral manifestations were affecting performance was also collected. Performance quality (PQ) was graded according to a Likert scale of "0—Very poor (<45%)" to "6—Outstanding (95%+)," where higher scores indicate better performance quality. Similarly, an overall score for PQ was collected based items such as technique, dynamic contrasts, tempo/rhythm, phrasing, sound/tone, and emotional impact. These adapted measures showed moderate to high internal consistency: Cronbach's alpha MPA = 0.66, PQ = 0.86. Correlations between the two judges were treated as separate dependent variables due to low and inconsistent correlations between ratings. The correlations between judges were as follows for MPA: Time 1 r = 0.19 (p > 0.05), Time 2 r = 0.31 (p < 0.05), Time 3 r = 0.24 (p > 0.05); for PQ: Time 1 r = 0.69 (p < 0.05), Time 2 r = 0.66 (p < 0.01), Time 3 r = 0.41 (p < 0.05). BODY.MATERIALS AND METHODS.PROCEDURE: The Human Research Ethics Committee of the University of Melbourne approved the study. After participants completed consent forms, they were allocated to the intervention (n = 30) or the wait-list control group (n = 32) using a pseudo-randomization method. Students were listed in alphabetical order and alternately assigned to either the intervention or the wait-list control group. Two students were non-randomized due to extra-curricular commitment clashes. All participants were sent an email detailing the upcoming performance and the requirements of the piece to be performed. As outlined in the email, the piece had to be approximately 2 min long, reflect their current level of playing, and not have been previously performed in a formal setting. Both groups also completed a self-report baseline measure (approximately 1 week prior to the first performance), which assessed MPA, motivation and resilience in music learning and performance (see Osborne, 2013 for details). After approximately 3 weeks of learning the piece, participants took part in Performance 1 whereby two external judges, blinded to the group conditions, rated the participants' behavioral manifestations of MPA and their PQ. These external judges met a minimum requirement of Master's level training on their main performance instrument, and had at least 20 years experience as music educators and adjudicators. Immediately before the performance students completed the MPAI-A (Performance) questionnaire, reflecting the finding that this is the period when musicians' apprehensions are reported to be greatest (Salmon, 1990). All performances were audio-video recorded. Following this, the 8-week Unleash Your Music Potential program (Table 1) was conducted for the intervention group, administered in a group format by two school psychologists who are also the authors of the program. The delivery of the program was adapted from the 10 sessions outlined in the published workbook (Brandon and Ivens, 2009) to fit within the school term and study objectives. Sessions relating to team harmony and booster shots were not included for this reason. Team harmony was not included because the main outcome measure was a solo performance. Table 1 Unleash Your Music Potential session schedule . Session Title Themes 1 Discover the champion within Peak performance, being the boss of your own thinking, personal strengths 2 Create your future Goals, goal setting, motivation 3 Success is a mindset Success, self-talk, feelings, affirmations 4 It's all in the preparation Routines, relaxation, self-talk, stress reduction 5 The ultimate dress rehearsal Mental imagery, mental rehearsal, visualization 6 Peak condition Stress management, wellbeing 7 The achievement zone Focus, flow, performing “in the zone” 8 Back on track and energized Setbacks, loss, disappointment, resilience, coping, positive thinking After this 8-week period students in both groups participated in Performance 2, using the same procedure as Performance 1. The wait-list control group then took part in a repeated delivery of the 8-week Unleash Your Music Potential program during the subsequent school term. Performance 3 took place following this intervention, serving as the post-intervention measure for the wait-list control as well as a 2-month follow-up measure for the intervention group. The overall time span of the study from Time 1 to Time 3 was 5.5 months (see Figure 1). Figure 1Study design. BODY.MATERIALS AND METHODS.DATA ANALYSIS: Dependent variables were checked for assumptions of normality using the Kolmogorov-Smirnov (K-S) statistic. Assumptions for normality were met for self-rated MPA for both groups at time 1 and 2, and the waitlist control group at time 3. Self-reported MPA of the intervention group violated assumptions at time 3 (K-S = 0.03). This data was included in parametric analyses, as Levene's and Mauchly's tests were not violated. Mixed-model 2 (group) × 3 (time) ANOVAs were conducted to assess the effect of the intervention on decreased MPA (self-rated and judge-rated) and improved PQ. Difference between groups at time 2 were analyzed using independent group t-tests. Maintenance of effects at 2 month follow-up (time 3) were assessed using repeated measure t-tests. Between and within-group effect sizes were measured by eta-squared. Judges' ratings were not highly correlated and are reported separately in the results. Assessment of judge-rated MPA and PQ at each of the three performance times revealed violations of normality for both judges' ratings of MPA and PQ at each performance. When scores were converted to Z scores, this resulted in only three variables being violated (Judge 2-rated MPA at time 1 and 3, Judge 1-rated MPA at time 2). Further investigation of these three variables using Levene's test of Homogeneity of variance revealed that the variances were equal between groups in these variables, respectively [F(1, 60) = 1.04, ns; F(1, 60) = 0.001, ns; F(1, 60) = 2.29, ns]. Therefore, ANOVAs were conducted for each judge separately on judge-rated MPA and PQ. Students who failed to attend two out of three performances were excluded from analyses (n = 4). Data for students who missed one performance (n = 13) were substituted using the mean of their group (intervention or wait-list control) as analyses revealed no significant difference between cases present and cases absent on age, years of playing, and the baseline self-report measure of MPA. BODY.RESULTS.DESCRIPTIVE STATISTICS: Baseline characteristics of each group, including age, years of learning instrument, self-rated MPA and judge ratings of MPA and performance quality indicate that the two groups were comparable, as shown in Table 2. Baseline levels of MPA as measured by the full 15-item MPAI-A measure were within the mean range reported by three studies investigating MPA in females in this age group: range M = 38.24–56.45; SD = 15.21–20 (Osborne and Kenny, 2005; Osborne et al., 2005; Thomas and Nettelbeck, 2014). Table 2 Baseline characteristics by group . Intervention [ n = 30 , M ( SD )] Wait-list control [ n = 32 , M ( SD )] p -value Age 13.70 (0.87) 13.87 (0.84) 0.45 Years playing 3.96 (2.77) 4.10 (2.40) 0.85 MPAI-State self-rated 28.69 (10.12) 26.69 (10.60) 0.45 MPAI-A full scale 44.86 (13.61) 42.37 (15.14) 0.52 MPA JUDGE-RATED Judge 1 1.80 (0.10) 1.79 (0.82) 0.98 Judge 2 0.38 (0.55) 0.31 (.051) 0.61 PERFORMANCE QUALITY Judge 1 2.43 (1.01) 2.60 (0.90) 0.49 Judge 2 3.20 (1.32) 3.60 (1.03) 0.19 MPA, Music performance anxiety. The MPAI-A full-scale score is provided to assist comparisons of the characteristics of this sample to other studies using the full-scale. The effect of the intervention on the full-scale MPAI-A are discussed in Osborne ( 2013 ). BODY.RESULTS.HYPOTHESIS 1: UNLEASH YOUR POTENTIAL REDUCES SELF-REPORTED MPA: Preliminary analyses revealed that there were no significant differences between the intervention and wait-list control groups' self-reported MPA levels at time 1, t(60) = 0.76, p > 0.05, or time 3, t(60) = −1.22, p > 0.05. There were also no significant differences within the intervention group across time 2 and 3, t(29) = 1.6, p > 0.05, and within the wait-list control group across time 1 and 2, t(31) = 0.63, p > 0.05. Therefore, initial analyses investigated the overall effect of the intervention by collapsing pre- or post-treatment values for the wait-list control and intervention group respectively. This showed that the intervention was successful in significantly reducing self-reported MPA [pre-treatment M = 27.39, SD = 9.80, post-treatment M = 18.90, SD = 8.44, F(1) = 45.51, p < 0.001, η2 = 42.7%]. The mixed model ANOVA revealed a significant interaction between group and time [F(2, 120) = 4.99, p < 0.01, η2p = 7.7%]. Assessment of mean values, shown in Figure 2, revealed that the intervention group gained significant benefit from the intervention between time 1 and time 2 (pre vs. post), and the wait-list control group showed a significant benefit from the intervention between time 2 and time 3 (pre vs. post). This effect was supported by the main effect of time [F(2, 120) = 26.35, p < 0.001, η2p = 30.5%] (see Figure 2). There was no main effect for group [F(2, 120) = 1.37, p > 0.05]. Post-hoc analyses showed a significant reduction in self-reported MPA for the intervention group between time 1 and time 2 [t(29) = 4.34, p < 0.001], and for the wait-list control group, between time 2 and time 3 [t(31) = 3.67, p < 0.01]. The intervention group (post-intervention) showed significantly less self-reported MPA than the wait-list control group (pre-intervention) at time 2 [t(60) = −2.4, p < 0.05]. Figure 2Self-rated MPA scores by group and time. Note these are mean scores with standard error bars, where higher scores indicate more anxiety. Sustained benefits of the program were demonstrated by the significant reduction in MPA for the intervention group between time 1 and 3 [t(29) = 5.35, p < 0.001] and a maintenance of this reduced level of MPA from time 2 to time 3 [t(29) = 1.60, p > 0.05]. Overall, this indicates that the intervention was highly effective in reducing self-reported MPA in both groups and that the reduced levels of MPA were maintained 2 months after students completed the program. These results demonstrate that the intervention led to a significant reduction in self-reported MPA, which was sustained for 2 months post-completion of the intervention. BODY.RESULTS.HYPOTHESIS 2: UNLEASH YOUR POTENTIAL REDUCES JUDGE-RATED MPA: The intervention had no therapeutic benefit in terms of Judge 1's ratings of behavioral manifestations of MPA, as the interaction between group and time [F(2, 120) = 2.64, p > 0.05], and main effects of time [F(2, 120) = 1.54, p > 0.05] and group [F(1, 60) = 1.15, p > 0.05] were all non-significant (see Figure 3). Figure 3Judge 1-rated MPA scores by group and time. Note these are mean scores with standard error bars, where higher scores indicate more anxiety. For Judge 2's MPA ratings, the interaction between group and time [F(2, 120) = 0.40, p > 0.05] and main effect for group [F(1, 60) = 0.001, p > 0.05] were both non-significant. There was, however, a main effect of time [F(2, 120) = 12.73, p < 0.001, η2p = 17.51%]. Paired-samples t-tests revealed a significant increase in Judge 2's rating of MPA for the intervention group between time 1 and time 2 [t(29) = −3.28, p < 0.05], and no significant difference in Judge 2's ratings of MPA for the wait-list control group, between time 2 and time 3 [t(31) = 1.25, p > 0.05] (see Figure 4). There was no main effect for group [F(1, 60) = 0.001, p > 0.05]. While paired-samples t-tests indicated that there was a statistically significant reduction in Judge 2's ratings of MPA for the intervention group between time 2 and 3, these ratings were higher than the baseline rating, indicating that the intervention had no therapeutic benefit in terms of Judge 2's ratings of behavioral manifestations of MPA. Figure 4Judge 2-rated MPA scores by group and time. Note these are mean scores with standard error bars, where higher scores indicate more anxiety. Contrary to expectation, the intervention group demonstrated significantly more MPA than the wait-list control at time 2 [t(60) = 2.47, p < 0.05]. In addition, Judge 2 did not find any significant difference in MPA between groups at time 2 [t(60) = 0.20, p > 0.05]. Therefore, levels of behaviorally manifested MPA were equivalent across both groups despite one group having undertaken the intervention. The combined within and between-groups results demonstrate that the intervention did not reliably lead to reductions in judge-rated behavioral manifestations of MPA in music students, both immediately following the intervention and at 2 month follow-up. Notably, however, given these judge ratings of MPA were consistently low, a floor effect might exist, precluding meaningful reductions in judge-rated MPA. BODY.RESULTS.HYPOTHESIS 3: UNLEASH YOUR POTENTIAL INCREASES JUDGE-RATED PERFORMANCE QUALITY: For Judge 1's PQ ratings, there was no significant interaction between group and time [F(2, 120) = 0.48, p > 0.05] nor main effect for group [F(1, 60) = 2.63, p > 0.05]. There was, however, a main effect of time [F(1, 120) = 7.08, p < 0.05, η2p = 10.6% (see Figure 5)]. Paired-samples t-tests revealed no significant difference in Judge 1's rating of PQ for the intervention group between time 1 and time 2 [t(29) = −0.403, p > 0.05], and no significant difference in Judge 1's ratings of PQ for the wait-list control group, between time 2 and time 3 [t(31) = −1.67, p > 0.05]. There was a statistically significant increase in Judge 1's ratings of PQ for the intervention group between time 1 and 3 [t(29) = −2.53, p < 0.05] (see Figure 5), indicating that, despite the intervention having had no immediate significant therapeutic benefit in terms of Judge 1's ratings of PQ, there was a significant increase in PQ over the five and half month period of the study. Figure 5Judge 1-rated performance quality scores by group and time. Note these are mean scores with standard error bars, where higher scores indicate better performance quality. For Judge 2's performance quality ratings, both the interaction between group and time [F(2, 120) = 0.36, p > 0.05] and main effect for group were not significant [F(1, 60) = 1.36, p > 0.05]. There was however a main effect of time [F(2, 120) = 6.10, p < 0.05, η2p = 9.2%]. There was no support for improved ratings of PQ for the intervention group between time 1 and time 2 [t(29) = 1.62, p > 0.05], yet there was a significant increase in Judge 2's ratings of PQ for the wait-list control group between time 2 and time 3 [t(31) = −2.04, p < 0.05]. While a paired-samples t-test indicated a statistically significant increase in Judge 2's ratings of PQ for the intervention group between time 2 and 3 [t(29) = −2.09, p < 0.05], ratings of PQ at time 1 and 3 were equivalent [t(29) = −0.21, p > 0.05] (see Figure 6). These findings indicate that Judge 2 observed an immediate beneficial effect of the intervention on PQ for the wait-list control group and a delayed improvement in PQ from time 2 for the intervention group. Figure 6Judge 2-rated performance quality scores by group and time. Note these are mean scores with standard error bars, where higher scores indicate better performance quality. At time 2, both judges rated the level of PQ as equivalent, despite one group having undertaken the intervention [Judge 1: t(60) = −1.68, p > 0.05; Judge 2 t(60) = −0.86, p > 0.05]. These combined within and between-groups results for hypothesis 3 provide partial support for an improvement in PQ immediately following the intervention and across the full five and a half month research period. BODY.DISCUSSION: This study provides preliminary evidence that an 8-week psychological skills program which teaches the cognitive skills essential for optimal performance in a group format is effective in reducing self-rated MPA in adolescent musicians and is superior to a waitlist control condition. Compared to baseline, participants in both groups demonstrated significantly less self-reported MPA when they had completed the intervention. Moreover, this reduction in MPA was sustained 2 months after the intervention had been completed, indicating its potential for longer-term therapeutic benefits. Furthermore, the intervention group reported significantly less MPA than the wait-list control group at time 2 (intervention group: post-intervention and wait-list control group: pre intervention), adding further weight to evidence that this intervention has a beneficial effect upon students' self-perceived levels of MPA. These findings were accompanied by large effect sizes. This result is consistent with the research of Osborne et al. (2007), who found a CB program incorporating performance psychology techniques to be effective in reducing adolescent student's self-reports of MPA. Apart from Osborne (2013), to our knowledge no other studies have examined the efficacy of a CB intervention for adolescent musicians, although there have been numerous studies that have found such interventions to be effective in reducing MPA in adult musicians (Harris, 1987; Clark and Agras, 1991; Roland, 1993; Connolly and Williamon, 2004; Buswell, 2006). It is not surprising then that CB interventions also show promise in helping reduce MPA in adolescents given the qualitatively similar experience of MPA between adults and adolescents. The intervention, however, did not reliably lead to a decrease in judge ratings of behavioral manifestations of MPA in students. This was true both immediately following the intervention and at 2-month follow-up. This finding should be interpreted with caution given the consistently low ratings of MPA by both judges. Overall, the judges did not appear to detect behavioral signs of MPA in the students, regardless of whether the students had undertaken the intervention or not. This finding conveys an important psychoeducational message: while students may feel anxious and/or perceive themselves to look anxious, this anxiety is not necessarily discernable to audience members, even when these members of the audience have been explicitly requested to be vigilant to signs of MPA. This finding is consistent with a study by Kubzansky and Stewart (1999) in which performer-rated state anxiety and judge-rated MPA during an orchestral audition process were not significantly related. A second psychoeducational message drawn from the poor inter-rater reliability in the judges' ratings of MPA is that one observer's perception of a performer's anxiety can be very different from another's, and therefore is subjective and not necessarily under the performer's control. Thus, while the intervention was not found to reliably reduce judge-rated MPA, these findings highlight the potential discrepancy between how much a performer vs. an observer perceives MPA to be present and, ultimately, point to the subjective nature of observer-rated MPA. In addition, this study found inconsistent support for improved performance quality as a result of participating in the intervention. These findings highlight the subjective and variable nature of an observer's judgment of another's performance. A wide discrepancy between judges' ratings has also been found in other studies employing judge-rated performance quality. For example, Reitman (2001) found such wide inter-rater discrepancies that it was impractical to attempt further analysis on the performance quality variable, measured using error count. Similarly, Clark and Williamon (2011) found such low correlations between judge-rated performance quality on eight items pertaining to overall quality, technical proficiency, musical understanding, communicative ability, level of preparedness, self-assuredness, interpretative imagination and originality, and ability to cope with performance stress, that they deemed this measure invalid and excluded it from their main analyses. Our finding is also consistent with previous studies that failed to find significant improvements in performance quality following CB interventions when targeting MPA (Wardle, 1975; Mansberger, 1988; Deen, 2000; Osborne et al., 2007). One possible explanation for this finding is that CB interventions, like Unleash Your Potential, have been designed to target subjective symptoms of MPA (cognitive, behavioral and physiological), rather than to optimize performance quality. Certainly, the aim of the present study's intervention was to make performance less psychologically distressing and ultimately more enjoyable for the individual, rather than to achieve excellence in performance quality per se. A second explanation, as suggested by Reitman (2001) who reported a similar finding, could be that the relatively short period of time that students had to translate newly learnt coping skills into the performance setting meant that behavioral changes were yet to be demonstrated. This is consistent with other studies that found behavioral changes to lag behind cognitive changes post-treatment (Appel, 1976). Thus, while the intervention did not reliably lead to improvement in students' performance quality, this finding highlights the specificity of the intervention in targeting self-perceived MPA, and in rendering students' experiences of performing more enjoyable, over and above performance quality. While this study demonstrated that a CB intervention program with performance and positive psychology elements shows great promise in helping adolescent music students reduce their experience of feeling anxious during performance, it was beyond the scope of this study to examine specific elements of the program that could account for this therapeutic benefit. It may be that a combination of cognitive, behavioral, performance and positive psychological strategies effected this change in MPA levels. Future studies should attempt to parse out the particular mechanisms that drive improvements in self-rated MPA and compare the impact of each on the cognitive, physiological and behavioral manifestations of MPA. This study also illuminated some of the inherent difficulties in measuring externally-rated behaviors like MPA and performance quality. The lack of inter-rater reliability on both of these variables points to the challenge of reliably measuring (1) physiological signs of MPA that are subtle and thus tend to be apparent to the performer only, and (2) an outcome variable like performance quality which can be notoriously subjective. Given that the judges in the present study were sitting at least 5 m away from the performers, future studies should endeavor to take "close up" video footage of each of the performers, which could potentially make it easier for observers to detect MPA manifestations like trembling, sweating, and blushing. While it is a difficult task to overcome the problem of variability between observers' ratings of performance quality, future researchers should endeavor to train judges to an adequate level of inter-rater reliability. It is also possible that the performance task used in our study had limited ecological validity due to the fact that this particular performance did not contribute to school assessments. Future studies should endeavor to incorporate performances that are delivered as part of the school assessment program, in order to potentially evoke higher levels of MPA. Finally, given the mixed findings in the literature regarding the efficacy of CB interventions for MPA to facilitate improvements in performance quality, future investigators should continue to look at the relationship between reduced MPA and performance quality. They should examine whether longer-term interventions might allow for behavioral changes in performance quality to be demonstrated, once participants have had a greater period of time to assimilate coping skills. We have demonstrated that a CB intervention program can lead to significant reductions in self-perceived performance anxiety in adolescent music students. We also highlight an important and reassuring psychoeducational message—that while you may feel anxious during a performance, it is very unlikely that the audience can detect this. Given the growing evidence for the ubiquity of MPA in children and adolescents, the young age at which the majority of musicians begin their training, and the potential career-devastating effects of untreated MPA in older musicians, it is critical that evidence-based intervention programs, like Unleash Your Potential, are incorporated into school music curricula. This could result in a more well-rounded approach to music education, whereby the goal of musical excellence is balanced by an equal emphasis upon self-perceived confidence, and ultimately, enjoyment in music performance. BODY.DISCUSSION.CONFLICT OF INTEREST STATEMENT: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.