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TITLE: Effects of Resistance Training and Aerobic Exercise on Insulin Sensitivity in Overweight Korean Adolescents: A Controlled Randomized Trial ABSTRACT.BACKGROUND: Data on the impact of resistance training on insulin resistance in overweight or obese children are inconclusive. ABSTRACT.METHODS: Thirty overweight South Korean adolescents (mean age of 13.10 years) were divided by sex, and then randomly assigned to one of three treatment groups, which were the diet only (DO), diet with aerobic exercise (AE), or diet with resistance training (RT) group. Physiologic and metabolic parameters were assessed at baseline and after 12 weeks of exercise training and diet modification. ABSTRACT.RESULTS: Both exercise groups (aerobic and resistance) showed significant improvements in their insulin area under the curve and insulin sensitivity index values when compared to their baseline values while the DO group showed no significant changes in these variables. Age-, sex-, and body mass index (BMI)-adjusted intergroup comparison analyses showed a marked reduction in BMI and a significant reduction in muscle mass in the AE group when compared to the RT group and the DO group, respectively. ABSTRACT.CONCLUSION: A 12-week exercise training program of either resistance or aerobic activity improved insulin sensitivity in overweight adolescents, although it failed to show superiority over a DO program. Aerobic exercise decreased both body weight and BMI, and it was noted that this group also had a significant reduction in muscle mass when compared to the DO group. BODY.INTRODUCTION: The prevalence of obesity is increasing at an alarming rate [1-3]. Complications of being overweight and obese are becoming major health care issues due to the increasing prevalence of obese and overweight children. The increase in the prevalence of children and adolescents that are overweight or obese implies a substantial increase in related diseases such as type 2 diabetes mellitus (T2DM) in children and adolescents [4-6], which was previously considered a disease of adults. Insulin resistance precedes the development of T2DM [7] and is a reported risk factor for the development of cardiovascular disease. Abdominal adiposity is associated with insulin resistance and T2DM in adults [8-10] and there is growing evidence that this condition is increasingly occurring in childhood [11-14]. Both aerobic and resistance exercise effectively improve insulin sensitivity and lead to better glycemic control in patients with T2DM [15]. While aerobic exercise has been extensively investigated and shown to be beneficial for improving insulin sensitivity [16-19], resistance training has not been researched extensively. Interestingly, resistance training could be more effective than aerobic exercise in improving the glycemic profile [20] of patients because isometric contractions produce insulin-like effects on the glucose uptake in skeletal muscle [21], and skeletal muscle is the primary site of glucose disposal in euglycemia. In addition to improvements in insulin sensitivity, resistance training can enhance several other physiologic parameters related to metabolic health such as total and regional body composition, blood pressure, and high density lipoprotein cholesterol levels [22]. Recently, the American Diabetes Association and the American College of Sports Medicine published a joint position statement that both aerobic exercise and resistance training improve insulin action, at least acutely, and can assist with the management of blood glucose levels, lipid levels, blood pressure, cardiovascular risk, mortality, and quality of life [23]. However, data on the impact of resistance training on insulin resistance in overweight or obese children have been inconclusive [24,25] and triple comparison studies that focus on dietary restriction, and aerobic and resistance exercise are lacking. In the current study, we directly compared the effects of dietary restriction, resistance training and aerobic exercise on insulin sensitivity and body composition in overweight South Korean adolescents. BODY.METHODS.PARTICIPANTS: Thirty (15 male and 15 female) overweight, but otherwise healthy adolescents (as determined by their annual school physical examination), were recruited from the same grade (mean age of 13.10 years) in a middle school in Seoul, South Korea. Identifying an individual as being overweight (body mass index [BMI] >85th percentile) was assessed by using age- and gender-specific BMI reference charts for Korean children and adolescents between 2 to 19 years of age [26]. Two of the enrolled female students had a BMI between the 85th and 75th percentile but were included in this study because there were only 13 girls that had met the criteria for being overweight in the school at the time of the screening procedure. These two female students were separated and assigned in to the diet only group and the resistance exercise group, respectively. This study was approved by an Institutional Ethics Review Board at the Boramae Medical Center. Informed written consents were obtained from the parents of the enrolled children and assent from the children were also obtained. Participants were excluded if they were taking medication or were smokers. Participants who had been diagnosed with a condition that is known to influence body composition or insulin/glucose metabolism, had an orthopedic condition that would limit their ability to perform exercise, or had participated in a structured exercise, nutrition, or weight loss program in the past six months, were also excluded. BODY.METHODS.STUDY PROTOCOL: The participants were first divided by sex, and were then randomly assigned to one of the three treatment groups with random number table, which were the diet only (DO), diet with aerobic exercise (AE), and diet with resistance training (RT) group. Anthropometric variables (body weight, height and waist circumference), fasting glucose, insulin and 75 g oral glucose tolerance test (OGTT) values were measured prior to and after the 12 weeks of exercise training and diet protocol. Body weight was measured on a balance scale calibrated to 0.1 kg. Barefoot standing height was measured to the nearest 0.1 cm using a wall-mounted stadiometer. The same investigator measured waist circumference before and after the study with a Gullick II measuring tape. Waist circumference was measured, during expiration, at the narrowest point between the lower rib and the iliac crest. BMI was calculated as weight in kilograms divided by the square of the height in meters. Measurements were carried out one week before and after the exercise program. Percent body fat and total body muscle mass were measured by using an Inbody 4.0 bioelectrical impedance meter (Biospace Co., Seoul, Korea). Visceral, thigh and intramuscular fat areas were measured by computed tomography (CT) with a Somatom Sensation 16 (Siemens Medical Solutions, Forchheim, Germany). The CT measurement protocol for fat area was similar to that used in a study by Poehlman et al. [15]. Total thigh fat area (TTFA) and intramuscular fat area (IMFA) were measured using a method that has been reported by Goodpaster et al. [27]. The abdominal bioelectrical impedance analysis (BIA) method [28] was also used to evaluate the visceral fat accumulation in each subject. The insulin sensitivity index was obtained by using the results of the OGTT and Stumvoll's equations [29]. BODY.METHODS.DIETARY PROTOCOL: Each study participant received an individualized dietary education program that was provided by a professional nutritionist twice a week throughout the 12 weeks of the study. The detailed program is described in Table 1. All three of the groups (DO, AE, and RT) participated in the dietary education program. The basic guidelines of the dietary education program were calorie restriction to avoid obesity (at least >1,200 kcal per day to prevent malnutrition), limiting dietary fat intake to improve blood lipid profiles, having low-salt intake to improve hypertension, eating three regular meals a day (breakfast, lunch and dinner) and avoiding snacks as much as possible. Participants were asked to write daily dietary records and these were reviewed weekly by the nutritionist. BODY.METHODS.EXERCISE PROTOCOL: The 12-week exercise training program was fully supervised by a trained exercise physiologist from the Department of Physical Education in the College of Education at the Seoul National University. In addition to the dietary energy restriction, ten participants in the AE group performed aerobic exercise three days a week. The students in the AE group were also told to perform gymnastics every day throughout the second and third month of the program. Modes of aerobic exercise consisted of jumping rope, walking or running on a treadmill and stationary cycling. The daily training duration was 40 minutes which included a 5 minute warm-up and a cool down period with stretching. The training intensity progressed from 60% to 70% of the maximal oxygen consumption (60%, 1st month; 65%, 2nd month; 70%, 3rd month). Maximal oxygen consumption was determined during a ramp treadmill test. The training volume of each session progressed from 300 to 400 kcal/session (300 kcal, 1st month; 350 kcal, 2nd month; 400 kcal, 3rd month) [30]. Another 10 participants in the RT group performed resistance training three days a week in addition to their dietary energy restriction. Training sessions began with a 15 minute warm-up period of jogging, gymnastics, and stretching (5 minutes for each type of exercise). One repetition maximum (1RM) is defined as the maximum amount of resistance that can be moved through the full range of motion of an exercise for no more than one repetition. The 1RM was calculated by using the formula derived by Kuramoto and Payne [31] and resistance training was performed at approximately 60% of 1RM. The resistance training program consisted of one set of each of the following ten exercises: squats, leg extension, lying leg curls, military press, leg press, lateral pulldowns, bench press, crunch, leg raise, and dead lift. These exercises provided a total body resistance training program for all of the major muscle groups of the body. Each subject was given a target load range and they were asked to attempt to keep each set (n=2 to 3) within the target range by adjusting the load to allow the prescribed number (n=10 to 12) of repetitions. The curriculum was personalized to match the skill level of each of the participants and was progressive in nature, by increasing the number of sets, repetitions, and the amount of resistance used as the participant's technique and strength improved. Resting periods were 1 to 1.5 minutes between sets. Each training session lasted approximately 60 minutes, which included a final 5 minute cooling down period of stretching. BODY.METHODS.BIOCHEMICAL ANALYSIS: A standard OGTT was performed in the overweight children by using 1.75 g/kg of a maximum flavored solution containing 75 g of glucose (Glu-Orange; Lotte Pharm, Seoul, Korea). Samples were drawn at 0, 30, 60, 90, and 120 minutes after the OGTT and the levels of glucose and insulin were measured. Serum insulin levels (μU/mL) were measured using an insulin immunoradiometric assay kit (Biosource Europe S.A., Nivelles, Belgium), which had a cross-reactivity with proinsulin of 0.3%. The reported intra-assay variation was 1.6% to 6.2% and the interassay variation was 6.1% to 6.5%. BODY.METHODS.STATISTICAL ANALYSIS: Data were analyzed using the PASW Statistics version 18.0 for Windows (SPSS Inc., Chicago, IL, USA). Values are presented as mean±standard deviation. For all statistical analyses a P value of less than 0.05 (two-sided) was considered to be statistically significant. The study was powered to detect up to a 25% differences in insulin sensitivity by using a conservative estimate of the standard deviation of the insulin sensitivity index, and using a power of 80%. Statistical significance was tested using the one-way ANOVA with Bonferroni correction in order to evaluate group differences at baseline. Changes in variables following the exercise program were compared with baseline values by using the repeated measures ANOVA for each of the three groups. One-way ANOVA with Bonferroni correction was applied to evaluate the main effects and interactions on all of the dependent variables in each of the three groups by time (prior to and after the 12-week program). Differences in the inter-group analysis were tested with the ANCOVA and the post-hoc analysis was performed by using the Tukey method for multiple comparison tests. All values were adjusted for baseline age, sex, and BMI in the inter-group analysis. BODY.RESULTS: Remarkably, none of the participants dropped out of the study and all of the participants had attended all of the exercise and dietary education sessions, yielding a compliance rate of 100%. The descriptive characteristics for all of the groups are given in Table 2. The three groups did not significantly differ at baseline with respect to any of the anthropometric or metabolic variables, and this suggests a successful randomization of the study participants. Anthropometric and body composition data across the groups are shown in Table 3. All of the participants had a significant growth in height during the study. Significant increases in weight, BMI, body fat, waist circumference, and visceral fat area (VFA) as measured by CT were observed in the DO group while their glucose area under the curve (AUC) decreased from the baseline value. The AE group showed a definite decrease in their glucose AUC when compared to their values measured at baseline. The RT group had increases in body weight, BMI, body fat, muscle mass and VFA as measured by CT following the 12-week program. Both exercise groups (aerobic and resistance) had significant improvements in their insulin AUC and insulin sensitivity index versus their baseline values while the DO group showed no significant changes in these variables. Individual changes in insulin AUC for all three groups are illustrated in Fig. 1. We found significant across group differences in BMI and muscle mass after adjusting for age, sex, and baseline BMI. Waist circumference, VFA as measured by various methods, insulin and glucose AUC, insulin sensitivity index, and the insulinogenic index did not show statistically meaningful distinctions across the groups. Age-, sex-, and BMI-adjusted intergroup comparison analyses showed a marked reduction in BMI and a significant reduction in muscle mass in the AE group when compared to the RT group and the DO group, respectively. BODY.DISCUSSION: The primary finding of this 12-week randomized, controlled exercise trial involving overweight South Korean adolescents is that both resistance training and aerobic exercise improved insulin sensitivity; however, the benefit does not appear to be greater than that of diet alone. In addition, aerobic training produced beneficial effects on body weight and BMI when compared to that of diet alone or resistance training. Successful randomization was accomplished as the three groups did not differ significantly at baseline with respect to any anthropometric or metabolic variables. Body weight along with BMI increased in the DO and RT groups, while both of these variables remained constant in the AE group. This relative weight reduction indicates that aerobic exercise is good for lowering body weight in overweight adolescents that may lead to an improvement in their insulin sensitivity. However, this relative weight loss also resulted in reduced muscle mass in the AE group when compared to that of the DO group. This finding implies that even if the RT group failed to lose weight or VFA, resistance training may provide an important way to counteract the decrease in resting metabolic rate and loss of muscle mass often accompanying dietary restrictions [32,33]. Muscle mass increased in the RT group (P=0.039), but this benefit did not reach significance in the post-hoc analysis with the Tukey method due to the rather small number of participants (n=10) in the group. We presume that with a larger sample size, this increase in muscle mass could have reached significance. Increased BMI in both the DO and RT groups may be due to the normal growth of adolescents or due to poor diet control. As a matter of fact, every participant grew in height during the 12-week study. Although little is known about the effects of energy compensation in response to exercise in children, research in adults have shown that when individuals exercise they often compensate with increased energy intake [34] or reduced physical activity outside of the training session [35]. Thus, the participants in the exercise groups may have altered their intake in response to the exercise they were performing throughout the program. However, the dietary records collected during the intervention would have minimized this acute compensatory intake. Insulin resistance is thought to be a critical factor in the pathogenesis of T2DM in both adults [9] and children [36]. Therefore, identifying interventions that can improve insulin sensitivity are critical for defining effective approaches for preventing metabolic diseases associated with obesity, especially in at-risk individuals, such as overweight adolescents. In this study, insulin sensitivity improved in both of the exercise groups (AE and RT) when compared to their baseline values, as reflected by adecreased insulin AUC values and an increased insulin sensitivity index. This improvement in the RT group is consistent with the results published by Bell et al. [37] which reported that increases in insulin sensitivity is independent of changes in body composition. However, inter-group analysis did not show significant differences when compared to the other groups, but we presume that this is due to the small number of participants in each of the three groups. Puberty is normally associated with a mild increase in insulin resistance, and in susceptible children it is a high-risk developmental period for both obesity and T2DM [38]. An assumption could have been made that three months after starting the intervention the insulin resistance would have increased as the children matured, if it was assumed that puberty is likely to influence the results of this study since the groups were comparable in sex distribution and age. We admit that there are limitations to our selected method of evaluating insulin sensitivity. It is known that the euglycemic clamp technique is the recognized gold standard for measurement of insulin sensitivity. Even though fasting or OGTT insulin levels are strong correlates of insulin sensitivity when measured by the euglycemic clamp method, the explained variance is approximately 50% [39]. Thus, our results of how a given exercise influences OGTT insulin values provide only an estimate of how insulin acts. However, if we had measured the OGTT insulin response within 24 hours of the last exercise session (instead of the following week), then it would have been likely that the enhanced insulin action observed in response to diet and exercise would have been even more impressive. Because insulin responsiveness is higher in trained individuals compared to untrained individuals 24 hours after exercise [40]. In addition, we did not observe significant changes in the insulinogenic index. This is not surprising because the youth in this study were all non-diabetic and presumably had, for the most part, healthy β-cells. We attempted to balance the limitations of this study with corresponding advantages. Maturation and/or age should be considered during stratification and during analysis in order to minimize potential confounding variables since they may influence metabolic factors in growing children and adolescents. Therefore, we adjusted for age, sex, and baseline BMI in our statistical analysis. Aerobic components (warm up and cool down periods) in the resistance training protocol and dietary manipulations in both of the exercising group may have led to some changes that make it more difficult to distinguish the independent effects of each group. Furthermore, data on dietary outcomes such as total energy intake and dietary fat were not available for use in the analysis. To further validate our results, we recommend additional studies that include a larger sample size, longer program duration and consideration of the maturation level and age of the study subjects. The strength of this study includes the involvement of a trained exercise physiologist and the enrollment of enthusiastic students that had succeeded in a remarkable 100% attendance rate. Unlike other studies offering rewards or gifts, we did not offer any kind of economic compensation or penalty. In conclusion, a 12-week exercise training (both resistance and aerobic) program improved insulin sensitivity in overweight adolescents, although it failed to show superiority over a DO program. Aerobic exercise decreased both body weight and BMI, and a significant reduction in muscle mass was also noted when compared to the DO group.

TITLE: Comparative pharmacokinetics, safety, and tolerability of two sources of ch14.18 in pediatric patients with high-risk neuroblastoma following myeloablative therapy ABSTRACT.PURPOSE: Dinutuximab (UnituxinTM; ch14.18), a monoclonal antibody against disialoganglioside, improved survival as part of post-consolidation therapy for high-risk neuroblastoma. United Therapeutics Corporation (UTC) assumed ch14.18 production from the National Cancer Institute (NCI); this study evaluates pharmacokinetic comparability, safety, and tolerability of UTC and NCI products. ABSTRACT.METHODS: In this randomized, two-sequence crossover study, 28 patients aged ≤8 years with high-risk neuroblastoma received equivalent ch14.18-UTC or ch14.18-NCI doses. Despite comparable protein content, nominal doses differed: 17.5 mg/m2/day (ch14.18-UTC) and 25 mg/m2/day (ch14.18-NCI). Patients received one product during therapy cycles 1 and 2, the other during cycles 3–5. Ch14.18 pharmacokinetic profile characterization used population modeling (NONMEM® version 7.2). A two-compartment model with first-order distribution and elimination processes described pharmacokinetic data. Estimated product parameters were normalized to UTC nominal dose. For pharmacokinetic comparability, the final model was used to estimate exposure ratios (UTC/NCI) and associated 90 % confidence intervals (CIs) for area under the curve from time zero to infinity (AUCinf) and maximum concentration (Cmax). All comparisons were based on a standardized single-dose regimen (17.5 mg/m2 over 10 h). ABSTRACT.RESULTS: Final-model pharmacokinetic parameters were similar to previously published ch14.18-NCI parameters and comparable for UTC and NCI products. Products' systemic exposures were comparable, with 90 % CIs around ratios for AUCinf (0.96; 90 % CI 0.88–1.04) and Cmax (1.04; 90 % CI 0.98–1.11) within standard bioequivalence bounds (90 % CI 0.80–1.25). Products' adverse events were similar and consistent with those previously reported. ABSTRACT.CONCLUSIONS: Equivalent actual ch14.18-UTC and ch14.18-NCI doses produced comparable exposures, with no notable safety or tolerability differences. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00280-015-2955-9) contains supplementary material, which is available to authorized users. BODY.INTRODUCTION: Neuroblastoma, which is a tumor of the autonomic nervous system, accounts for approximately 7 % of cancers in children <15 years of age [1]; ~90 % of patients are <5 years of age at diagnosis [1]. The disease is heterogeneous, complex, and frequently aggressive [2]. At diagnosis, approximately 40 % of patients have high-risk disease [3], based on factors such as age, disease stage, and biologic markers (e.g., unfavorable histopathology, tumor amplification of MYCN oncogene) [4]. High-risk neuroblastoma is treated with dose-intensive chemotherapy and surgery, followed by myeloablative chemotherapy with autologous stem cell transplantation (ASCT), local radiation therapy, and maintenance with isotretinoin [5, 6]. Despite this intensive treatment, many patients relapse or have treatment-refractory disease, and 5-year event-free survival rates are ≤50 % [4, 7]. Disialoganglioside (GD2) is a surface glycolipid antigen that is strongly expressed on neuroblastoma tumor cells, with limited expression in normal human tissues [8]. GD2 is an important molecular target for immunotherapeutic approaches to treating neuroblastoma, and anti-GD2 monoclonal antibodies are efficacious in patients with high-risk neuroblastoma. Dinutuximab (UnituxinTM), formerly called ch14.18, is a murine–human chimeric anti-GD2 monoclonal antibody [9]. Initial trials demonstrated that ch14.18 at a dose of 25 mg/m2 infused over 10 h daily for 4 consecutive days could be incorporated into treatment regimens containing isotretinoin and the immunomodulators sargramostim and aldesleukin [10–12]. Subsequently, the Children's Oncology Group (COG) conducted a randomized phase 3 clinical trial (ANBL0032) comparing ch14.18 administered with isotretinoin, sargramostim, and aldesleukin versus isotretinoin alone in patients with high-risk neuroblastoma who had responded to induction therapy, surgery, ASCT, and radiotherapy [12]. The trial demonstrated improved event-free survival (p = 0.01) and overall survival (p = 0.02) at 2 years on the immunotherapy arm [12]. Based on the results of this trial, dinutuximab received US Food and Drug Administration (FDA), and European Medicines Agency (EMA) approval for the treatment of high-risk neuroblastoma. As part of a collaborative research agreement and development agreement (CRADA) with the National Cancer Institute (NCI), United Therapeutics Corporation (UTC) has licensed ch14.18 and assumed production. The nominal (i.e., labeled) dose of the UTC product (17.5 mg/m2) differs from the dose of the prior NCI product (25 mg/m2) because of a difference in the extinction coefficient used to determine the protein concentration during the manufacturing process. Despite the change in nominal dosing, the amount of antibody delivered per dose is equivalent for the two products. Corrections for this difference in the products' nominal dose must be made when comparing dose-dependent pharmacokinetic parameters, such as clearance and volumes of distribution. The primary objective of this study was to compare the pharmacokinetics of ch14.18 manufactured by these two independent facilities (i.e., NCI and UTC). The secondary objective was to compare the products' safety and tolerability profiles. BODY.MATERIALS AND METHODS.STUDY DESIGN: Study DIV-NB-201 was a phase 2 randomized, open-label, two-sequence crossover trial evaluating ch14.18 in patients with high-risk neuroblastoma scheduled to receive immunotherapy. The clinical trial was conducted in accordance with the ethical principles of the Declaration of Helsinki and the International Conference on Harmonization E6 Good Clinical Practice Guideline. The protocol was approved by the Institutional Review Board at each participating site, and the parents or guardians provided written informed consent with patient assent, as appropriate. Patients were randomized 1:1 to receive either ch14.18-UTC or ch14.18-NCI during cycles 1 and 2, followed by ch14.18 from the other product during cycles 3–5. Eligible patients were randomized between 56 and 105 days after ASCT. Randomization must have occurred after the completion of tumor assessments post-ASCT and radiotherapy, if applicable. BODY.MATERIALS AND METHODS.PATIENTS AND TREATMENT: Eligible patients were ≤8 years of age, had a diagnosis of high-risk neuroblastoma, and had completed standard induction therapy, surgery, myeloablative therapy and ASCT, and local radiotherapy to the primary tumor if indicated. Patients must have achieved a partial response or better per International Neuroblastoma Response Criteria (INRC) [13] at the primary site, soft tissue metastases, bone metastases, and bone marrow response at the pre-ASCT evaluation. Prior to enrollment, a determination of residual disease was performed, and patients could not have progressive disease per INRC except for protocol-specified bone marrow response to account for sampling errors. Patients were also required to have a Lansky performance status of ≥50 %; a total absolute phagocyte count ≥1000/μL; adequate renal, hepatic, cardiac, pulmonary, and central nervous system function; and a life expectancy of ≥2 months. Patients were excluded if they had received prior anti-GD2 antibody therapy or had prior vaccine therapy for the treatment of neuroblastoma. Patients were also excluded if they had received or planned to receive anticancer therapies, cytokines, or growth factors not included in the prescribed protocol therapy during the study or immunosuppressive drugs other than for acute allergic reactions and anaphylaxis during the study. The study material manufactured by NCI used a theoretical extinction coefficient of 1.00 to calculate the concentration of antibody, whereas UTC material used an actual extinction coefficient of 1.41 to determine the antibody concentration. Thus, each 25 mg/m2 dose of ch14.18-NCI contains the same amount of ch14.18 as a 17.5-mg/m2 dose of ch14.18-UTC, making the respective dosing equivalent despite differences in the nominal doses. The dosing schema in this study is summarized in Table 1. During the first five cycles, patients received ch14.18-UTC or ch14.18-NCI intravenously (IV) over 10–20 h daily for four consecutive days repeated every 28 days, with one product administered during cycles 1 and 2 and the other product during cycles 3, 4, and 5. All patients received sargramostim IV or subcutaneously (SC) (250 mcg/m2/day for 14 days) on cycles 1, 3, and 5 prior to, during, and following ch14.18, and aldesleukin IV (3 MIU/m2/day for 4 days as a continuous infusion for the first week, followed by 4.5 MIU/m2/day for 4 days as a continuous infusion concurrently with ch14.18 for the second week) during cycles 2 and 4. In addition, all patients received six cycles of isotretinoin over 14 days (80 mg/m2/day orally twice daily for >12 kg and 2.67 mg/kg orally twice daily for ≤12 kg) after completion of ch14.18 therapy.Table 1Dosing schema and pharmacokinetic assessment scheduleCycles 1, 3, and 5 (24 days in duration)Cycle day0123456789101112131415–24Sargramostim X X X X X X X X X X X X X X ch14.18 X X X X Isotretinoin X X X X X Pharmacokinetic assessments (cycles 1 and 3 only) X a X b,c X c X c X c X d X d X d X d X e X e Cycles 2, 4, and 6 (32 days in duration)Cycle day(cycles 2 and 4)0123456789101112–1415–2829–32Aldesleukin X X X X X X X X ch14.18 X X X X Isotretinoin X Pharmacokinetic assessments(cycles 2, 4, and 6) X f X g X h X i aDay 0, up to 3 days prior to initial dose of sargramostim. b End of infusion, within 15 min of completion (days 3 and 59), preinfusion (day 59). c End of infusion, within 15 min of completion (days 4–6, 60–62). d 10–14 h post-completion of ch14.18 infusion (days 7 and 63). e Single sample between days 9–11, 14–17, 65–67, and 70–73. f Single sample, prior to aldesleukin (days 24 and 80). g Preinfusion of the first ch14.18 dose (days 31 and 87). h Immediately following the fourth daily ch14.18 infusion (day 90). i End of study within 2 weeks of the final isotretinoin dose (cycle 6, day 163) In addition to study treatment, supportive care measures, including the use of narcotics and other concomitant medications, were required for the treatment of anticipated toxicities. BODY.MATERIALS AND METHODS.ASSESSMENTS: Pharmacokinetic blood samples for the determination of ch14.18 plasma concentrations were obtained at 22 time points over the course of the treatment (Table 1). Samples were drawn prior to the first dose of sargramostim on cycle 1 and the first ch14.18 infusion on cycle 3; after the end of each daily ch14.18 infusion; and 10–14 h, 3–5 days, and 8–11 days after the fourth ch14.18 infusion on cycles 1 and 3. Sampling during cycles 2 and 4 occurred prior to aldesleukin, prior to the first ch14.18 dose, and at the end of the fourth daily ch14.18 infusion on cycle 4 only. A final sample was obtained at study end, within 2 weeks of the final isotretinoin dose on cycle 6. Blood samples for the analysis of human anti-chimeric antibody (HACA) were obtained prior to ch14.18 dosing in each cycle. Plasma from each sample was isolated by centrifugation, frozen, and shipped to Burleson Research Technologies, Morrisville, NC, for storage. Samples were shipped to BioAgilytix, Durham, NC, where ch14.18 and HACA plasma concentrations were measured using validated assays: a sandwich immunoassay employing an electrochemiluminescence platform to measure ch14.18, and a Meso Scale Discovery electrochemiluminescent assay (Meso Scale Diagnostics, Rockville, MD) with a lower limit of quantification of a titer of 10 to measure HACA. Because of the study complexity and data limitations, a model-based approach, rather than a traditional noncompartmental bioequivalence analysis, was used to assess the pharmacokinetic comparability of ch14.18-UTC and ch14.18-NCI. Detailed pharmacokinetic data from nine patients with high-risk neuroblastoma enrolled in a previous pharmacokinetic study of ch14.18-NCI at the Children's Hospital of Philadelphia by Desai [14] were used to develop a structural population pharmacokinetic model. The final model was a two-compartment model with first-order distribution and elimination processes. To account for the effects of body size on pharmacokinetic parameters, actual body weight was included as a predetermined allometric covariate on all clearance and volume of distribution parameters. Estimated pharmacokinetic parameters included clearance from the central compartment (CL), distributional clearance (Q), volume of the central compartment (V1), volume of the peripheral compartment (V2), steady-state volume of distribution (Vss), first-order elimination rate constant (Kel), and first-order distribution rate constants (central-to-peripheral [Kcp]; peripheral-to-central [Kpc]). The final structural model for the data from the prior Desai study was then used to estimate pharmacokinetic parameters for ch14.18-UTC and ch14.18-NCI in the formal comparability study (DIV-NB-201). This staged analysis approach was taken to avoid the potential for inflated alpha error associated with iterative model development using the formal comparability data from DIV-NB-201. Because calculations of some pharmacokinetic parameters are dependent on the nominal dose of drug, differences in the nominal doses of ch14.18-UTC and ch14.18-NCI will affect pharmacokinetic parameter estimates. In the Desai study [14], pharmacokinetic parameter estimates were based on nominal dosing units of ch14.18-NCI. Therefore, when comparing dose-dependent pharmacokinetic parameters generated using UTC and NCI nominal doses, appropriate corrections were made. To formally assess the pharmacokinetic bioequivalence of the UTC and NCI products, maximum concentration (Cmax) and area under the curve from time zero to infinity (AUCinf) were calculated using the population pharmacokinetic parameter estimates of CL and V from DIV-NB-201 and appropriate closed-form equations for Cmax and AUCinf. Variability in exposure estimates was captured by sampling the posterior distribution of pharmacokinetic parameter estimates using the NONMEM® Markov Chain Monte Carlo methodology. To allow for valid comparisons, all calculations were based on a standardized single dose of 17.5 mg/m2 infused over 10 h, using nominal UTC doses. Pharmacokinetic bioequivalence was assessed by calculation of ratios (UTC/NCI) for AUCinf and Cmax with 90 % confidence interval (CI) of the ratios. AUCinf was the primary comparability end point. Bioequivalence was established if the 90 % CIs for the exposure ratios were completely contained within accepted bioequivalence bounds (0.80–1.25). Safety assessments included adverse event reporting, physical examinations, clinical laboratory assessments, and treatment-related changes in electrocardiograms (ECGs). Safety analyses were performed on all patients receiving at least one study drug dose. No inferential statistical analyses of safety data were planned. BODY.RESULTS: Twenty-eight patients were enrolled, and 14 patients were randomized to each treatment sequence. Patient characteristics are summarized in Table 2 with no differences in demographics by sequence. A summary of concomitant medications used by patients during cycles 1–5 is presented in Supplemental Table 1. Patients' completion or discontinuation of study therapy is summarized in Table 3. One patient was excluded from the pharmacokinetic analysis because of a neutralizing antibody response that interfered with measurement of ch14.18 concentration. All 28 patients received at least one dose of ch14.18 and were included in the safety analysis. Seven patients discontinued the study prior to completion of the planned six cycles; reasons were disease progression (n = 2), adverse events (n = 2), or withdrawal of consent (n = 1). Two patients received cycles 1–5 in the USA and returned to their home country to complete cycle 6. Ch14.18 was administered over a median duration of 11 h (range 10–20 h).Table 2Patient demographic and baseline characteristicsCharacteristicSequence 1 (n = 14)(UTC/NCI)Sequence 2 (n = 14)(NCI/UTC)Mean age at randomization (range) (years)4 (2–7)4 (1–9)Male gender, n (%)8 (57)8 (57)Ethnicity, n (%) Hispanic4 (29)2 (14) Not Hispanic10 (71)12 (86)Race, n (%) White12 (86)11 (79) Asian01 (7) Black/African American2 (14)1 (7) Unknown01 (7)Pre-ASCT response, n (%) Complete response5 (36)3 (21) Very good partial response5 (36)4 (29) Partial response4 (29)7 (50)Number of ASCT, n (%) Single13 (93)14 (100) Tandema 1 (7)0Prior chemotherapy, n (%)14 (100)14 (100)Radiotherapy, n (%)12 (86)b 13 (93)Cancer-related surgery, n (%)12 (86)c 11 (79) ASCT autologous stem cell transplantation, NCI National Cancer Institute, UTC United Therapeutics Corporation aPatients were required to undergo ASCT (first transplant for tandem transplant patients) within 9 months after starting the first induction chemotherapy for high-risk neuroblastoma. In addition, patients were required to enroll in the study within 105 days post-ASCT (date of second transplant for tandem patients) such that study day 0 (first dose of sargramostim) occurred within 110 days post-transplantation bRadiotherapy may have been waived for patients who either had a small adrenal mass that was completely resected initially or who never had an identifiable primary tumor cPatients may not have had an identifiable primary tumorTable 3Patients' completion or discontinuation of study therapyDispositionSequence 1 (UTC/NCI), n (%)Sequence 2 (NCI/UTC), n (%)Safety population, n 1414Pharmacokinetic population, n 13a 14Completed all study therapy9 (64)12 (86)Discontinued study therapy Cycle 11 (7)0 Cycle 201 (7) Cycle 32 (14)0 Cycle 401 (7) Cycle 52 (14)b 0Reason for study discontinuation Disease progression1 (7)1 (7) Adverse event1 (7)c 1 (7)d Consent withdrawn1 (7)0 Moved out of country2 (14)b 0 NCI National Cancer Institute, UTC United Therapeutics Corporation aOne patient excluded because of interfering human anti-chimeric antibodies, for the pharmacokinetic assay bPatients completed cycles 1–5 and went on to complete scheduled course of isotretinoin in their country cPatient discontinued during cycle 3 due to serum sickness dPatient discontinued during cycle 2 due to neuropathy BODY.RESULTS.IMMUNOGENICITY: Six of 27 patients had detectable HACA during the study. Only one patient (17 %) had a pharmacokinetic-neutralizing response (detected in cycle 3) and was therefore excluded from the pharmacokinetic analysis. BODY.RESULTS.PHARMACOKINETICS: Representative concentration time profiles for ch14.18-UTC and ch14.18-NCI from a single patient are presented in Fig. 1a (semilog), b (linear). A comparison of the pharmacokinetic profiles indicates similar exposures for both products. Population mean concentration time profiles are shown in Supplemental Figure 1. Summary statistics of post hoc pharmacokinetic parameters are presented in Table 4 for each product, separately and combined. Dose-dependent pharmacokinetic parameters were normalized to the nominal ch14.18-UTC dose. Clearance, volumes of distribution, and rate constants were equivalent for the NCI- and UTC-manufactured products.Fig. 1Representative semilog (a) and linear (b) concentration–time profiles from a single patient for ch14.18-UTC and ch14.18-NCITable 4Summary of individual post hoc pharmacokinetic parameter estimates for ch14.18Parameter, mean (SD)ch14.18-UTC(n = 26)ch14.18-NCI(n = 25)ch14.18-UTC and ch14.18-NCI combined(n = 27)CL (L/d)0.683 (0.307)0.75 (0.32)0.709 (0.315)CL (L/d/m2)1.09 (0.457)1.17 (0.455)1.12 (0.457) Q (L/d)0.767 (0.163)0.956 (0.213)0.857 (0.198) Q (L/d/m2)1.20 (0.026)1.46 (0.023)1.32 (0.056)V1 (L)1.43 (0.403)1.36 (0.42)1.40 (0.404)V1 (L/m2)2.23 (0.29)2.05 (0.28)2.15 (0.28)V2 (L)3.94 (1.1)3.76 (1.09)3.85 (1.07)V2 (L/m2)6.10 (0.42)5.65 (0.40)5.87 (0.39) V ss (L)5.38 (1.47)5.12 (1.49)5.25 (1.45) V ss (L/m2)8.32 (0.57)7.70 (0.57)8.03 (0.54)Kel (1/d)0.489 (0.211)0.569 (0.232)0.520 (0.221)Kcp (1/d)0.552 (0.073)0.723 (0.103)0.630 (0.090)Kpc (1/d)0.199 (0.018)0.259 (0.021)0.228 (0.018) CL clearance from the central compartment, Kcp first-order distribution rate constant (central-to-peripheral), Kel first-order elimination rate constant, Kpc first-order distribution rate constant (peripheral-to-central), NCI National Cancer Institute, Q distributional clearance, UTC United Therapeutics Corporation, V1 volume of the central compartment, V2 volume of the peripheral compartment, V ss steady-state volume of distribution Following standardized single-dose regimens, population pharmacokinetic estimates for AUCinf were 431 μg·h/mL for ch14.18-UTC and 413 μg·h/mL for ch14.18-NCI (ratio = 1.04; 90 % CI 0.98–1.11). Population pharmacokinetic estimates for Cmax were 6.57 μg/mL and 6.88 μg/mL, respectively (ratio = 0.96; 90 % CI 0.88–1.04). The 90 % CIs for exposure ratios of AUCinf and Cmax were contained within the standard bioequivalence bounds (0.80–1.25), consistent with comparable exposure between products. BODY.RESULTS.SAFETY: All 28 randomized patients were included in the safety analyses and had at least one treatment-related adverse event (TRAE) overall and at least one TRAE attributed to ch14.18. Overall, a total of 1945 TRAEs were reported, with most being grades 1–3. The most commonly reported TRAEs included pyrexia (100 %), hypoalbuminemia (96 %), hypokalemia (96 %), hyponatremia (82 %), cough (75 %), increased alanine aminotransferase (ALT) (68 %), anemia (68 %), hypocalcemia (68 %), pain (68 %), pruritus (68 %), increased aspartate aminotransferase (AST) (64 %), hypertriglyceridemia (64 %), and abdominal pain (61 %). Although differences between products were seen for individual adverse events, evaluation over the entire study showed no notable differences in TRAE incidence by manufacturer, either overall or attributable to ch14.18. Pain-related TRAEs were generally similar between treatment sequences; the most commonly reported pain-related events in the ch14.18-UTC and ch14.18-NCI groups were pain (59 and 44 %), abdominal pain (48 and 41 %), and pain in extremity (33 and 41 %), respectively. Pain-related TRAEs were most commonly reported during cycles 1, 2, and 4 (93, 74, and 71 %, respectively). Allergic-type adverse events were also generally similar between ch14.18-UTC and ch14.18-NCI groups, with the most commonly reported events (i.e., ≥15 % of patients) including urticaria (30 vs. 26 %), peripheral edema (15 vs. 11 %), pruritus (63 vs. 52 %), and rash (26 vs. 26 %), respectively, and occurred most frequently in cycles 1, 2, and 4. Table 5 summarizes grade 3 or higher TRAEs considered by the investigator to be attributable to ch14.18 in ≥10 % of patients. The most common grade ≥3 events were pyrexia, anemia, hypokalemia, and hyponatremia, with no discernible differences between study drugs. Other safety assessments, including clinical laboratories, physical examinations, and ECGs, were generally consistent between UTC- and NCI-manufactured ch14.18.Table 5Grade 3 or higher treatment-related adverse events (≥10 %)Adverse events, n (%)ch14.18-UTC (n = 27)ch14.18-NCI (n = 27)≥1 Adverse event22 (81.5)23 (85.2)Pyrexia13 (48.1)12 (44.4)Anemia6 (22.2)9 (33.3)Hypokalemia7 (25.9)7 (25.9)Hyponatremia5 (18.5)5 (18.5)Platelet count decreased4 (14.8)5 (18.5)ALT increaseda 4 (14.8)1 (3.7)Lymphocyte count decreased2 (7.4)3 (11.1)Pain-related adverse events Pain5 (18.5)2 (7.4) Pain in extremity2 (7.4)3 (11.1) Abdominal pain2 (7.4)3 (11.1)Hypocalcemia3 (11.1)2 (7.4)Hypotension2 (7.4)3 (11.1)Neutrophil count decreased2 (7.4)3 (11.1)Hypoxia1 (3.7)3 (11.1)Urine output decreased3 (11.1)1 (3.7) ALT alanine aminotransferase, NCI National Cancer Institute, UTC United Therapeutics Corporation aALT increases were transient BODY.DISCUSSION: The NCI-manufactured ch14.18 was used in the pivotal randomized phase 3 trial that demonstrated the efficacy of ch14.18 combined with sargramostim, aldesleukin, and isotretinoin administered as continuation therapy for high-risk neuroblastoma [12]. This randomized crossover study comparing equivalent doses of ch14.18-UTC (17.5 mg/m2) and ch14.18-NCI (25 mg/m2) was conducted to confirm equivalence of the pharmacokinetic and safety profiles of the two products. Pharmacokinetic parameters were estimated by fitting a two-compartment pharmacokinetic model to individual concentration–time data. The products were formally compared using the results of a model-based bioequivalence analysis, which showed equivalent systemic exposures as measured by the AUCinf for ch14.18-UTC and ch14.18-NCI, and 90 % CIs about the geometric least-squares mean ratios for AUCinf and Cmax within standard bioequivalence bounds (90 % CI 0.80–1.25). Clearance, volume of distribution, and rate constants were also equivalent between the UTC and NCI products. For this analysis, dose-dependent pharmacokinetic parameters were derived using the ch14.18-UTC nominal dose of 17.5 mg/m2. Historical parameters, such as CL and volumes of distribution, for the ch14.18-NCI material were derived using a 25-mg/m2 dose and require a correction factor of 0.7 for comparison with ch14.18-UTC material. Data from an independent study (CHP1002) were used to develop a pharmacokinetic model for ch14.18. This model was then used to estimate pharmacokinetic parameters for the comparability study (DIV-NB-201). This approach was taken to avoid the potential for inflated alpha error associated with iterative model development based on the formal comparability data (DIV-NB-201). A model-based approach (rather than a traditional noncompartmental bioequivalence analysis) was chosen to assess the pharmacokinetic comparability of ch14.18-UTC and ch14.18-NCI because of data limitations and the complexity of DIV-NB-201. The pharmacokinetic parameters derived from the population model for the CHP1002 study were comparable to those previously published by Desai et al. [14]. The Desai analysis was also based on a two-compartment structural model; however, the methodology used for parameter estimation differed from the current analysis, which utilized a population pharmacokinetic approach. In addition, Desai et al. used different pharmacokinetic software (MLAB; Civilized Software, Silver Spring, MD). Despite these differences, mean pharmacokinetic parameters for the CHP1002 study were comparable between the Desai analysis and the population model. When pharmacokinetic parameters are expressed in terms of nominal ch14.18-NCI dosing units, estimates for the Desai analysis and the population pharmacokinetic analysis, respectively, are similar (CL, 2.1 vs. 1.9 L/d/m2; Vl, 2.2 vs. 1.9 L; Kel, 0.026 vs. 0.027 1/h; Kcp, 0.021 vs. 0.023 1/h; and Kpc, 0.010 vs. 0.015 1/h). Overall, the TRAEs (including allergic-type events) observed in this study were consistent with those reported in other studies in which ch14.18 was administered with growth factors and cytokines [12, 15]. The most commonly reported adverse events in >60 % of patients, regardless of manufacturer, were pyrexia, hypoalbuminemia, hypokalemia, hyponatremia, cough, increased ALT, anemia, hypocalcemia, pain, pruritus, increased AST, hypertriglyceridemia, and abdominal pain. The ANBL0032 study demonstrated that approximately 17 % of patients treated with ch14.18 develop HACA, with <5 % having a neutralizing antibody response in a biological assay (data on file, United Therapeutics Corporation). In this study, 6/27 (22 %) of patients had confirmed HACA, and only one patient was excluded from the pharmacokinetic analysis due to a neutralizing antibody response. In summary, the current analysis confirms that the ch14.18-UTC dose of 17.5 mg/m2 is comparable to the ch14.18-NCI dose of 25 mg/m2 in terms of systemic exposure and with no notable safety and tolerability differences. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material 1 (DOCX 148 kb)

TITLE: Effect of magnesium infusion on thoracic epidural analgesia ABSTRACT.INTRODUCTION:: Patients of lung volume reduction surgery (LVRS) having an ASA status III or more are likely to be further downgraded by surgery to critical levels of pulmonary function. ABSTRACT.AIM:: To compare the efficacy of thoracic epidural block with (0.125%) bupivacaine, fentanyl combination and (0.125%) bupivacaine, fentanyl combination with adjunctive intravenous magnesium infusion for the relief of postoperative pain in patients undergoing LVRS. ABSTRACT.METHODS:: Patients were operated under general anesthesia. Thirty minutes before the anticipated completion of skin closure in both groups, (Group A and Group B) 7 ml of (0.125%) bupivacaine calculated as 1.5 ml/thoracic segment space for achieving analgesia in dermatomes of T4, T5, T6, T7, and T8 segments, along with fentanyl 50 μg (0.5 ml), was administered through the catheter, activating the epidural block, and the time was noted. Thereafter, in patients of Group A, magnesium sulfate injection 30 mg/kg i.v. bolus was followed by infusion of magnesium sulfate at 10 mg/kg/hr and continued up to 24 hours. Group B was treated as control. ABSTRACT.RESULTS AND ANALYSIS:: A significant increase in the mean and maximum duration of analgesia in Group A in comparison with Group B (P<0.05) was observed. Total epidural dose of fentanyl and bupivacaine required in Group A was significantly lower in comparison with Group B in 24 hours. ABSTRACT.DISCUSSION:: Requirement of total doses of local anesthetics along with opioids could be minimized by magnesium infusion; therefore, the further downgradation of patients of LVRS may be prevented. ABSTRACT.CONCLUSION:: Intravenous magnesium can prolong opioid-induced analgesia while minimizing nausea, pruritus, and somnolence. BODY.INTRODUCTION: Lung volume reduction surgery (LVRS) is a nonanatomic resection of lung tissue which removes approximately 20 to 30% of the poorly functioning emphysematous space occupying lung tissue from each lung. By reducing the lung size, the remaining lung and the surrounding muscles (intercostals and diaphragm) are able to work more efficiently, makes breathing easier, and thus the patients achieve a better quality of life. The improvement in lung function is greater with bilateral LVRS than unilateral, without any increase in morbidity or mortality. The bilateral approach is best conducted through a central incision.[1] Pain management must be prophylactic, integral to surgery, and proactive than retroactive. LVRS is a modern procedure to which these principles apply.[2] An effective regimen of postoperative analgesia not only shortens the time to recovery, but also helps in avoiding the pulmonary, hemodynamic, and metabolic complications of acute postsurgical pain.[3] Patients of LVRS having an ASA status III or more are likely to be further downgraded by surgery to critical levels of pulmonary function,[4] need special postoperative care as postoperative pain may lead to hypoventilation, areas of low ventilation to perfusion ratio, an increased intrapulmonary right-to-left shunt, atelectasis, functional disruption of respiratory muscle, temporary diaphragmatic dysfunction, inadequate cough leading to impairment of pulmonary secretion clearance, requirement of high FiO2, reduction in vital capacity and functional residual capacity (FRC).[5] Thoracic epidural analgesia is considered by most to be the best and is effectively performed in various thoracic surgery centers.[6] Although opioids have been traditionally used as an adjunct,[7] they have their inherent dependent side effects like respiratory depression, diminished cough reflexes, changes in gastrointestinal motility, urinary retention, nausea, and vomiting.[8] A comparative study conducted for post-thoracotomy analgesia in infants with bupivacaine and bupivacaine-fentanyl combination through thoracic epidural catheter showed that pain scores decreased significantly in the first 24 hours in the group with bupivacaine-fentanyl compared with bupivacaine alone.[9] It has been concluded from previous studies that intravenous magnesium sulfate infusion with regional blockade decreases the analgesic requirement without enhancing the side effects of opioids or potentiating the side effects of regional blockade.[10–12] So, an endeavor was made to compare the efficacy of thoracic epidural blockade with bupivacaine (0.125%), fentanyl 50 μg and bupivacaine (0.125%) combination, fentanyl 50 μg in combination with intravenous magnesium infusion for postoperative pain relief in adult patients undergoing LVRS. BODY.INTRODUCTION.AIMS AND OBJECTIVES: To compare the efficacy of thoracic epidural block with (0.125%) bupivacaine, fentanyl combination and (0.125%) bupivacaine, fentanyl combination with adjunctive intravenous magnesium infusion for the relief of postoperative pain in patients undergoing LVRS.To note the complications if any. BODY.METHODS: This single center, prospective, randomized, controlled, double-blind study was carried out in the Department of Anaesthesiology of IPGME and R, SSKM Hospital, Kolkata, after obtaining permission from the Institutional Ethical Committee in the period between June 2007 and May 2010. BODY.METHODS.SELECTION CRITERIA.INCLUSION CRITERIA: A total of 60 adult patients of either gender aged between 35 to 60 years of ASA status II and III, scheduled for LVRS with ≥50% of predicted forced expiratory volume in 1st second (FEV1), FEV1 /FVC (forced vital capacity), and mid-expiratory flow rate (MEFR), were included after obtaining informed consent from each patient. BODY.METHODS.SELECTION CRITERIA.EXCLUSION CRITERIA: Patients of ASA status IV, with signs and symptoms of systemic infection or local sepsis, bleeding diathesis, coagulation abnormalities, diseases like diabetes mellitus, ischemic heart disease, hypertension, malignancy, renal or hepatic compromise or any major systemic illness, ongoing antiplatelet or anticoagulant therapy, hemodynamically unstable, hypersensitivity to the study drugs, spinal deformity, progressive neurological disease, mental retardation, psychiatric illness, pregnancy, or those who on awakening from general anesthesia complained of pain or not responding to vocal command were excluded from the study. BODY.METHODS.TECHNIQUE: Sample size was estimated using the PS (Power and sample size calculation version 2.1.30 February 2003). The sample size required for correctly rejecting the null hypothesis (of equal mean duration of effective analgesia—the primary end point) with a probability of 90% (i.e., power 0.90 or 90%) was calculated based on the following assumptions. Clinically important difference in mean duration of analgesia is 20 minutes with α = 0.05 or 5% (probability of type I error), σ = 35.26 min (within group SD obtained by a previous study by Kumar and Rajendran),[13] m = 1.2 (ratio of number of control to experimental patients); it was determined that 30 patients were required in each group (Group A and B). BODY.METHODS.TECHNIQUE.ANESTHESIA TECHNIQUE: Following a detailed history and physical examination, breathing exercises and incentive spirometry were begun preoperatively. Fourth hourly nebulization with ipratropium bromide and salbutamol were started. All patients were instructed not to consume solid food after midnight on the day of surgery, but clear fluids were permitted till 4 hours before the scheduled time of operation. Patients were explained about the anesthetic technique, the blocks to be placed, the interpretation of visual analog scale (VAS), the method of postoperative analgesia, and to demand rescue analgesic at the onset of breakthrough pain (VAS>30 mm). The patients were given alprazolam tablet 0.25 mg and omeprazole tablet 40 mg 2 hours before surgery. On arrival in the operating room, all the monitors were attached. Intravenous infusion of lactated Ringer's solution was started; premedications of ondansetron injection 4 mg and glycopyrrolate injection 0.2 mg were given. Under strict aseptic precautions, on infiltration with local anesthetic (LA), an epidural 18G multiorifice catheter was placed 2 cm inside the epidural space through a 16G Tuohy epidural needle by paramedian approach at T5–T6 interspace. The epidural space was identified by loss of resistance technique using saline. A test dose of 3 ml of (1.5%) lignocaine with 1 : 200 000 adrenaline was administered through the epidural catheter on negative aspiration of CSF and blood to rule out accidental intrathecal or intravascular placement.[14a] Furthermore, the patients were put back to horizontal supine position. Fentanyl injection 2 μg/kg bodyweight i.v. was given 4 minutes before induction. After preoxygenation, the patients were induced with thiopentone injection 5 mg/kg i.v., or till the loss of eyelash reflex. Tracheal intubation was facilitated using double lumen tube after achieving full relaxation with vecuronium injection 0.1 mg/kg. Anesthesia was maintained using isoflurane 0.5 to 1% in 50% N2 O and O2, along with supplemental doses of vecuronium injection 0.02 mg/kg and fentanyl injection 0.5 μg/kg bodyweight i.v. top up doses, to maintain bispectral index between 40 and 60. Thirty minutes before the completion of skin closure, all the 60 patients were randomized into two groups (Group A and B), generated by the statistical software 'Microsoft Excel XPTM (2003).' Thirty minutes before the anticipated completion of skin closure in both groups (Group A, Group B), 7 ml of (0.125%) bupivacaine calculated as 1.5 ml/thoracic segment space for achieving analgesia in dermatomes of T4, T5, T6, T7, and T8 segments,[14b] along with fentanyl 50 μg (0.5 ml), was administered through the catheter, activating the epidural block, and the time was noted. Furthermore, in patients of Group A, magnesium sulfate injection 30 mg/kg (10 ml) i.v. bolus was followed by infusion of magnesium sulfate (12 gm) in 50 ml normal saline at 2 ml/h (i.e.,10 mg/kg/h) and continued up to 24 hours. In patients of Group B, normal saline injection (10 ml) i.v. bolus was followed by infusion of normal saline at 2 ml/h and continued up to 24 hours. The nurse was engaged in the preparation of the infusions and the observer was blinded. At the end of surgery, anesthetic agents were discontinued and neuromuscular blockade was reversed with neostigmine injection 50 μg/kg along with glycopyrrolate injection 10 μg/kg, after fulfilling the criteria for extubation. Patients complaining of pain immediately after termination of general anesthesia were offered systemic opioid analgesia with tramadol injection 2 mg/kg bodyweight i.v. and excluded from the study. The time interval between the activation of the epidural blocks in both the groups and the requirement of the first top up dose, when VAS >30 mm, was regarded as the effective duration of analgesia. Top up dose preparation contained bupivacaine (0.125% at 1.5 ml/thoracic segment space) along with fentanyl 50 μg (0.5 ml). All patients received top up epidural doses for the maintenance of postoperative analgesia for 72 hours. Hemodynamic parameters, that is, noninvasive blood pressure monitoring for systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), heart rate, urine output, respiratory rate, and patellar reflexes, were recorded from the immediate postextubation period to 24 hours postoperative period. Incidence of untoward events like nausea, vomiting, pruritus, and respiratory depression were noted for the first 24 hours postoperative period, which were assessed using the evaluation scores noted in Table 1, as per scoring system of postoperative checkpoints.[15] Nausea was considered to be present if score was ≥2. Pruritus was considered present when score was ≥2. The patients were considered as participants of the study if alertness score was ≤2. Table 1 Scoring system of postoperative checkpoints[ 15 ] Nausea/vomiting No nausea 1 Complains of nausea but tolerable 2 Severe nausea needs medication 3 Pruritus (itching)  No itching 1  Complain of itching but tolerable 2  Severe itching, needs medication 3 Respiratory depression  None detected 1  Exist (RR <8/min) 2 Alertness  Clear mentality 1  Good response to verbal command, but drowsy 2  Poor response to repeated verbal command 3 RR: Respiratory rate The assessment was carried out by other anesthesiologists who were not involved in the care of the patients and blinded to the group assignment. Rescue medication was administered after any untoward event. Primary outcome of the study was to determine the duration of effective analgesia (the time interval between the activation of the block and the first request for analgesic). Secondary outcomes were VAS, complaints of pruritus, nausea, vomiting, respiratory depression, and hypotension (30% fall of blood pressure from baseline value) noted for the first 24 postoperative hours. BODY.METHODS.STATISTICAL ANALYSIS OF DATA: All the data were entered into Excel Spreadsheet and analyzed using statistical software 'SPSS and Statistica.' Parametric data was presented as Mean (SD) and was compared by student t-test; nonparametric data by Mann Whitney U Test; and categorical data was compared by Fisher's exact test. P value <0.05 was considered statistically significant. BODY.RESULTS: A total of 66 patients were needed for the study; as six patients complained of pain immediately after extubation (VAS >30 mm), they had to be excluded. Table 2 shows the demographic profile (age, gender, BMI), the duration of surgery, VAS after extubation, and serum magnesium, urea, and creatinine levels to be comparable between the two groups (P>0.05). Table 2 Comparison of demographic profile, duration of surgery, VAS after extubation, and serum magnesium, urea, and creatinine levels between the groups Group A Group B P value Age (year) 47(10.6) 45 (10) >0.05 Sex (M : F) 16 : 14 19 : 11 BMI (kg/m 2 ) 28 (5.7) 27 (4.1) >0.05 Duration of surgery (min) 130 (25.55) 125 (20.25) >0.05 VAS after extubation (mm) 24.28 (4.3) 25.45 (3.9) >0.05 Serum magnesium level (mEq/l) 1.66 (0.22) 1.72 (0.18) >0.05 Serum urea level (mg/dl) 26.33 (2.63) 29.12 (2.77) >0.05 Serum creatinine level (mg/dl) 0.86 (0.12) 0.84 (0.14) >0.05 BMI: Body mass index, VAS: Visual analog scale, Values are in Mean (SD) unless otherwise specified The comparison of SBP, DBP, and MAP between the groups at various time points as seen in Table 3 were found to be comparable (P>0.05) in the immediate postoperative period and in the 1st, 2nd, 3rd, and 4th hours. However in the 5th hour, there was a significant difference (P<0.05) between the groups where Group B shows higher values. Also, though the readings were comparable in the 6th and 9th postoperative hours, the difference becomes statistically significant (P<0.05) in the 12th hour, with Group B showing higher values. Table 3 Comparison of systolic blood pressure, diastolic blood pressure, and mean arterial pressure (in mm Hg) between roups SBP DBP MAP Immediate postoperative  Group A 122.2 (12.12) (9.64) 88.2 (10.2)  Group B 127.28 (9.84) 74.4 (9.98) 91.9 (11) 1 h postoperative  Group A 117 (16.84) (10.2) 87.2 (11.2)  Group B 123.64 (14.25) 72.24 (10.34) 92.24 (10.8) 2 h postoperative  Group A 136.33(11.62) (11.2) 95.33 (11.4)  Group B 137.5 (12.64) 78.22 (12.22) 97.83 (12.44) 3 h postoperative  Group A 120.6 (11.32) (11.3) (11.34)  Group B 120 (11.62) 78.26 (11.4) 92.24 (11.66) 4 h postoperative  Group A 122.8 (11.62) (10.1) 88.1 (11.2)  Group B 121 (12.62) 75.24 (11.24) 93.6 (11.8) 5 h postoperative  Group A 122.2 (11.12) 72.84 (9.83) 93.2 (10.8)  Group B 148.28 (9.84) # 96.8 (11.24) # 131.12 (10.6) # 6 h postoperative  Group A 122.2 (11.22) (10.2) 89.8 (10.66)  Group B (9.94) 77.88 (11.84) 93.8 (11.2) 9 h postoperative  Group A (10.22) (11.2) (10.8)  Group B (12.24) (11.64) 93.2 (12.2) 12 h postoperative  Group A 122.84 (10.2) 70.6 (9.8) 93.24 (11)  Group B 152.82 (10.34) # 98.8 (9.2) # 134.86 (11.8) # # P <0.05, Values given as Mean (SD) unless mentioned otherwise, SBP: Systolic blood pressure, DBP: Diastolic blood pressure, and MAP: Mean arterial pressure Table 4 compares the pulse (/min) and VAS (mm) between the groups at various time points. The readings are comparable in the immediate postoperative period, 1st, 2nd, 3rd, and the 4th hours postoperative period in both the groups (P>0.05). The 5th postoperative hour however shows significant difference (P<0.05), with Group B showing higher values. Furthermore, the 6th, 9th, and 12th hour postoperative period readings are comparable. The 12th hour reading shows an increase in pulse rates as well as VAS in both the groups. Table 4 Comparison of postoperative pulse rates (/min) and VAS (mm) between the groups at various time points Pulse/min VAS in mm Immediate postoperative  Group A 93 (8.2) 10.2 (3.1)  Group B 96 (9.88) 10.6 (4.4) 1 h postoperative  Group A 80.44 (10.2) 11.6 (3.2)  Group B 88.22 (10.44) 12.4 (3.28) 2 h postoperative  Group A 78.2 (9.4) 11.8 (2.8)  Group B 86.8 (9.12) 12.6 (3.98) 3 h postoperative  Group A 79.82 (8.34) 11.4 (3.66)  Group B 84.8 (9.8) 12.88 (4.6) 4 h postoperative  Group A 80.2 (8.8) 12.8(3.51)  Group B 85.6 (9.2) 22.8 (4.46) 5 h postoperative  Group A 80.2 (8.22) 12.2 (3.44)  Group B 110.4 (8.2) # 38.88 (4.82) # 6 h postoperative  Group A 82.1 (7.4) 15.2 (3.02)  Group B 89.6 (9.2) 33.56 (4.26) 9 h postoperative  Group A 82.2 (7.4) 25.4 (3.98)  Group B 84.2 (9.2) 22.22 (3.4) 12 h postoperative  Group A 85.2 (8.88) 30.88 (4.2)  Group B 88.2 (9.88) 32.2 (5.66) # P <0.05, Values are in Mean (SD) unless specified, VAS: Visual analogue scale A statistically significant (P<0.05) increase in the mean and maximum duration of analgesia (minutes) was found in Group A (571.66 [34.313]) in comparison with Group B (305.83 [77.313]) as seen in Table 5 and Figure 1. Table 5 Mean effective duration of analgesia (min) in mean (SD) Group N Mean (SD) A 30 571.66 (34.313) B 30 305.83 (77.313) P value <0.05 SD = Standard deviation Figure 1Comparison of mean effective duration of analgesia The total intravenous dosage of fentanyl required in the intraoperative period is comparable in both the groups. However, the total epidural dose of required fentanyl (μg) is significantly higher in Group B (160.75 [20.75]) compared with Group A (70.55 [15.65]), which is statistically significant (P<0.05). Similarly, the total epidural dose of required bupivacaine (mg) is also higher in Group B (32.75 [5.67]) in comparison with Group A (15.75 [5.75]), which is also statistically significant. Pruritus was significantly less in Group A compared with Group B. The incidence of nausea and vomiting was also significantly less in Group A in comparison with the control. Respiratory depression was not observed in any patient. Patellar reflexes were normal in both the groups [Table 6] [Figures 2 and 3]. Table 6 Secondary outcome variables Group A Group B P value Total i.v. dose of fentanyl in intraoperative period (μg) 110 (15.25) 115 (15.25) >0.05 Total epidural dose of fentanyl in 24 h (μg) 70.55 (15.65) 160.75 (20.75) <0.05 Total dose of bupivacaine in 24 h (mg) 15.75 (5.75) 32.75 (5.67) <0.05 Patients with pruritus during first 24 h (%) 2 (7) 10 (33) <0.05 Patients with nausea during first 24 h (%) 3 (10) 14 (47) <0.05 Patients with vomiting during first 24 h (%) 1 (3) 9 (30) <0.05 Patients with respiratory depression during first 24 h (%) 0 0 Urine output in 24 h (ml) 1375.44 (178.88) 1299.26 (222.78) >0.05 Absence of Patellar reflex (%) 0 0 >0.05 Values are in Mean (SD) unless otherwise specified Figure 2Comparison of total epidural dose of fentanyl (μg) in 24 hours Figure 3Comparison of total epidural dose of bupivacaine (mg) in 24 hours BODY.DISCUSSION: Emphysema is a chronic, progressive disease of the lungs, most commonly caused by smoking. There occurs a breakdown in the walls of the air sacs of the lung which become abnormally enlarged, functioning poorly for oxygenation, also the small airways which carry air to and from the air sacs collapse during breathing, especially exhalation. These abnormally enlarged air sacs fill easily with air during inspiration but lose their ability to empty through the small airways during exhalation. This resembles airways obstruction. Thus, the term chronic obstructive pulmonary disease is used to describe emphysema. The problem of easy filling and poor emptying leads to progressive hyperexpansion of lungs. Many people suffering from emphysema have portions of the lung more affected than others. This finding led to the development of a surgical approach for treatment, that is, LVRS, a procedure which removes portions of the poorly functioning, space-occupying tissue from each lung, which thereby improves the respiratory mechanics in severe emphysema by re-expansion of the functional lung tissue which was compressed by overdistended emphysematous alveoli. This restores the diaphragmatic mobility and improves the bellows function of the chest wall structures which work more efficiently making breathing easier and thus lead to a greater quality of life. Ineffective postoperative pain management may lead to deep vein thrombosis, pulmonary embolism, coronary stress, atelectasis, pneumonia, poor wound healing, insomnia, and demoralization. Prevention and effective relief of acute pain may improve clinical outcomes, avoid clinical complications, reduce hospital stay, and thus save health care resources and improve quality of life. Thoracic epidural anesthesia with LA and opioids appears to be the ideal means of achieving optimal intra- and postoperative analgesia with minimum central nervous system and respiratory depression. But it also has potential complications like hypotension, urinary retention, incomplete or failed block, and in rare instances paraplegia.[16] A study by Tauzin-Fin et al. showed that intravenous magnesium infusion in radical prostatectomy under general anesthesia reduced the analgesic requirement.[10] Another study by Tramer and Glynn showed that in ambulatory inguinal hernia or varicose vein operations under general anesthesia with analgesic adjuvants, pretreatment with intravenous magnesium had no impact on postoperative pain or analgesic requirement.[11] Apan et al. showed that the addition of magnesium infusion as adjunct in spinal anesthesia reduced postoperative analgesic consumption.[12] This prospective randomized double-blind study including 60 adult patients between 35 to 60 years of ASA physical status II and III, scheduled for LVRS with ≥50% of predicted FEV1, FEV1/FVC, and MEFR, revealed a significant increase in the mean and maximum duration of analgesia (min) in magnesium recipients, that is, Group A [571.66 (34.313)] in comparison with control (i.e., Group B) (305.83 [77.313]), which was statistically significant (P<0.05). The total epidural dose of fentanyl required (μg) in Group A (70.55 [15.65]) was significantly lower in comparison with Group B (160.75 [20.75]) in 24 hours. Simultaneously, the total epidural dose of bupivacaine required (mg) in Group A (15.75 [15.65]) was also significantly (P<0.05) lower compared with Group B (32.75 [5.67]) in 24 hours. Associated complications related to opioids like pruritus, nausea, and vomiting were significantly lowered in the magnesium recipient group in comparison with the control group. In thoracotomy patients, the motor effects of full concentrations of LA blockade result in increased FRC due to the caudad movement of the diaphragm and decrease in intrathoracic fluid volume which is beneficial, but the weakened inspiratory effort which accompanies is disadvantageous. Low-dose LAs come close to achieving an ideal situation by allowing for caudad movement of the diaphragm and recruitment of lung volume, without reducing the power of the intercostals necessary for coughing. In practice, motor block is reduced, almost to clinical insignificance, by the use of dilute solutions, without the loss of sensory block.[17] Magnesium inhibits calcium entry into the cell by noncompetitive blockade of the N-methyl D-aspartate (NMDA) receptor.[18] Magnesium and NMDA receptor are thought to be involved in the modulation of pain.[19] The NMDA receptor antagonism inhibits induction and maintenance of central sensitization after nociceptive stimuli.[20] Magnesium is also a physiological calcium antagonist at different voltage-gated channels,[21] which may be important in the mechanisms of antinociception.[22] Thus, it potentiates the action of low-dose LAs and therefore may prevent the further downgradation of patients of LVRS with ASA status III or more to critical levels of pulmonary function. Serum magnesium comprises only approximately 0.3% of total body magnesium, where it is present in three states—ionized (62%), protein bound (33%), and those bound mainly to albumin and complexed to anions such as citrate and phosphate. Equilibrium between tissue pools is reached slowly with a half-life varying between 41 and 181 days. Therefore, serum magnesium estimation may not provide representative information on the status of other stores.[23] Post-thoracotomy pain management for LVRS thus poses a significant challenge to the anesthesiologist due to the high-risk patient population and nature of surgery. Management requires good understanding of the pathophysiology of the disease and the surgical procedure. Close co-ordination between the anesthesiologist and the critical care specialist with support staff is of paramount importance. In addition to pain management, nebulized bronchodilator therapy and aggressive chest physiotherapy should begin in the immediate postoperative period to maximize pulmonary function. BODY.CONCLUSION: Effective pain relief is vital to the success of LVRS and dictates the course of perioperative outcome. Considering the fact that thoracic epidural opioids have become the 'Gold Standard' for pain relief despite their potential side effects, coadministration of intravenous magnesium infusion may negate their undesired effects to reduce postoperative morbidity and mortality. However, the benefit of this therapy must be weighed against the potential adverse effects and the requirement of close observation. Thus, it may be concluded that intravenous magnesium can prolong opioid-induced analgesia while minimizing nausea, pruritus, and somnolence. However, we cannot state clearly whether this is the ideal dose or higher doses might produce fewer side effects while prolonging analgesia. Furthermore, it may be suggested that magnesium may be considered as one of the ingredients of multimodal analgesic stratagems in reducing the severity of post-thoracotomy pain. BODY.CONCLUSION.LIMITATIONS OF THE STUDY: Perioperative magnesium assay was not done, and target-controlled infusion for accurate dose delivery of opioids for the maintenance of analgesia was not available.

TITLE: Post-market outcome of an extract of traditional Cretan herbs on upper respiratory tract infections: a pragmatic, prospective observational study ABSTRACT.BACKGROUND: The beneficial effects of traditional herbs of Crete, Greece for the treatment of upper respiratory tract infections have been reported in observational and laboratory studies. Following a published, double blind, randomized, placebo controlled trial, this study aimed to assess the effectiveness of an extract of three Cretan herbs on the treatment of upper respiratory tract infections, upon its market release. ABSTRACT.METHODS: An observational study was conducted in Heraklion, Crete, Greece. Participants were patients presenting at selected pharmacies with symptoms of upper respiratory tract infection, choosing to receive the extract for their treatment. Patients' symptoms (local, general, total) where recorded at three time points within 1 week, using a questionnaire developed based on the Wisconsin Upper Respiratory System Survey. For each patient, symptoms were scored on a 0–7 Likert scale and three indexes were calculated: the score of local symptoms, the score of general symptoms and the total score of symptoms. Effectiveness was assessed by examining the reduction in these indexes over the 1-week observation period. ABSTRACT.RESULTS: Mean score of general symptoms was 19.1 (SE: 0.9) in day 1, dropping to 8.6 (SE: 0.6) and 3.1 (SE: 0.4) in days 4 and 7 respectively. Mean score of local symptoms declined from 7.9 (SE: 0.5) in day 1 to 2.3 (SE: 0.3) in day 4 and to 0.5 (SE: 0.1) in day 7. Total score of symptoms reached 27.0 (SE: 1.2) in day 1, decreasing to 10.9 (SE: 0.8) in day 4 and to 3.5 (SE: 0.5) in day 7. The percentage of participants reporting fever was 82.1% at baseline, 8.0% in day 4 and 2.0% in day 7 (p < 0.0001 for paired differences). Multiple regression models indicated that supplementary medication intake did not seem to affect symptoms' severity or the day patients reported that their symptoms ceased completely. ABSTRACT.CONCLUSIONS: This pragmatic study added evidence about the potential therapeutic effects of an extract of Cretan herbs on the amelioration of upper respiratory tract infection symptoms. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12906-017-1978-7) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Aromatic plants have been historically used as remedies in the island of Crete, Greece [1]. Anthropological and laboratory research has explored their antioxidant activity and their effects on the alleviation of common cold symptoms [2, 3]. Such evidence led to the conduction of a double blind, randomized, placebo controlled trial aiming to test the effectiveness of an extract of three Cretan herbs, namely thyme (Coridothymus capitatus (L.) Rchb. f. synonym of Thymbra capitata (L.) Cav.), dictamnus (Origanum dictamnus L.) and sage (Salvia fruticosa Mill., Salvia pomifera L.), on the treatment of upper respiratory tract infections. The results indicated amelioration in the severity of symptoms, with 90% of treated patients being symptom-free at the last day of observation [4]. The extract was released in the market in 2015, in the form of nutritional supplement (extracts at a dilution of 15 ml/L in extra virgin olive oil, formulated as 0.5 ml soft-gel capsules, for a daily dose of two capsules). The aim of this observational study was to explore the extract's effectiveness on the treatment of upper respiratory tract infections, after its market release, through a questionnaire-based inquiry of participants. The primary objective was to assess the severity and duration of symptoms upon treatment. Secondary objectives included the investigation of the extract's effectiveness on the presence of fever and in cases of supplementary medication intake. BODY.METHODS.STUDY DESIGN: An observational study was conducted in the prefecture of Heraklion, Crete, Greece (February – June 2016). Sixteen pharmacies, purposively selected, participated as recruiting sites. The preparation of the extract is extensively described elsewhere [4]. BODY.METHODS.SAMPLE: Patients presenting at the pharmacies with symptoms of upper respiratory tract infection, who voluntarily requested and choose to purchase the extract as part of their treatment were recruited. Eligibility for inclusion in the study was based on the Jackson criteria for the identification of patients suffering from common cold [5]. In order also to increase the possibility that the infection was viral, and not of any other type, the presence of fever was considered as a perquisite for inclusion in the study. As such, inclusion criteria were: age over 18 years, sudden emergence of fever within 48 h from the visit in the pharmacy and presence of at least one of the symptoms: cough, sore throat, nasal discharge or ingestion, headache, muscle pain, sweating, rigors and fatigue. Exclusion criteria were: age under 18 years, absence of fever within 48 h from the visit to the pharmacy, daily consumption of aspirin (≥100 mg), presence of malignancies, immune system disorders, liver damage and pregnancy. Setting as primary outcome the day patients reported that their symptoms ceased completely and assuming 5% probability of type-I error, a sample size of 185 patients had 90% power to detect an average time of symptoms' cessation of 6 days, with standard deviation of ±1.2 days and accuracy of 0.17 days. BODY.METHODS.DATA COLLECTION: Upon presenting at the pharmacies, eligible patients were invited to complete an anonymous questionnaire (Additional file 1: Questionnaire.doc), developed based on the Wisconsin Upper Respiratory System Survey (WURSS-21), which assessed symptoms' severity [6]. This tool has been previously used with reliable results [4]. Symptoms were recorded at the day of visit at the pharmacy (day 1), as well as at the fourth (day 4) and seventh (day 7) day of extract intake, via phone calls performed by the pharmacists. Symptoms were classified as local (cough, sore throat, itchy throat, hoarseness, nasal congestion, excreta, sneezing and headache) and general (fatigue, muscle pain and rigors). They were scored in a Likert scale, with 0 denoting absence of symptom and 7 denoting maximal severity of the respective symptom. For each patient, three indexes were calculated: The score of local symptoms, the score of general symptoms and the total score of symptoms, defined as the sum of general and local symptoms' scores. Presence of fever was recorded as a dichotomous variable (yes/no). Collected socio-demographic and clinical characteristics included age (years), gender (male/female), BMI (Kg/m2), smoking (currently/former/never) and presence of chronic conditions (yes/no). Any supplementary medication received for the treatment of patients' upper respiratory tract infection was also recorded (type and days of intake). The day patients reported feeling better and the day patients reported that their symptoms ceased completely were recorded as well. BODY.METHODS.STATISTICAL ANALYSIS: Sample characteristics were summarized using descriptive statistics. For comparisons between categorical variables Pearson's χ2 tests were performed. Paired Samples T-test and McNemar tests were used for comparisons of paired differences. To assess the severity of all symptoms over the observation period, the Area under the Curve (AuC) was computed. Multiple linear regression models were performed with dependent variables: the AuC, the day patients reported feeling better and the day patients reported their symptoms ceased completely. Independent variables were the socio-demographic and clinical characteristics and supplementary medication intake (yes/no). Linear mixed models with dependent variables the total score of symptoms and presence of fever and independent variables the day of observation (1, 4, 7), supplementary medication intake (by day of observation), socio-demographic and clinical characteristics were used to assess symptoms' alleviation over time. Confidence level was a = 0.05. Statistical software used were IBM SPSS version 21 and Stata SE version 12. BODY.RESULTS.SAMPLE CHARACTERISTICS: A total of 493 patients with upper respiratory tract infection symptoms visited the selected pharmacies during recruitment. Among those, 315 (63.9%) met the inclusion criteria, 157 (50.2%) agreed to participate and 6 (3.8%) dropped-out. Data from 151 patients were analyzed (81.6% of the estimated target of 185 patients). Participants' mean age was 43.6 (±17.6) years, while 95 (62.9%) were females. Mean BMI was 26.3 (±5.6) kg/m2, 68 (45%) participants were current smokers and 44 (29.1%) reported at least one chronic condition. A total of 85 (56.3%) participants received supplementary medication for the treatment of their upper respiratory tract infection at day 1. The respective percentage for the whole observation period was 64.9% (n = 98). BODY.RESULTS.SYMPTOMS’ SEVERITY: Table 1 presents the mean scores of general and local symptoms and the mean total score of symptoms during the observation period, along with the means of individual changes in symptoms' scores between baseline and end of follow-up. Mean score of general symptoms was 19.1 (SE: 0.9) in day one, dropping to 8.6 (SE: 0.6) and 3.1 (SE: 0.4) in days 4 and 7 respectively, illustrating a decrease by 55.0% in day 4 and by 83.8% in day 7 (p < 0.001 for all paired differences). Mean score of local symptoms declined from 7.9 (SE: 0.5) in day 1 to 2.3 (SE: 0.3) in day 4 and to 0.5 (SE: 0.1) in day 7, showing a reduction by 70.9% in day 4 and by 93.7% in day 7 (p < 0.001 for all paired differences). Mean total score of symptoms reached 27.0 (SE: 1.2) in day 1, falling to 10.9 (SE: 0.8) in day 4 and to 3.5 (SE: 0.5) in day 7, indicating a decrease by 59.6% in day 4 and by 87.0% in day 7 (p < 0.001 for all paired differences; Fig. 1). On the first day, 124 (82.1%) patients reported having fever. This rate dropped to 8.0% at day 4 and to 2.0% at day 7 (p < 0.0001 for paired differences).Table 1Mean symptoms' scores over the observation periodSymptomsMeanStandard errorMinimumMaximumGeneral symptoms Day 119.10.9145 Day 48.60.6035 Day 73.10.4035 Mean of individual change (day 1- day 7)16.10.8045Local symptoms Day 17.90.5021 Day 42.30.3019 Day 70.50.1014 Mean of individual change (day 1- day 7)7.40.5021Total Day 127.01.2266 Day 410.90.8042 Day 73.50.5035 Mean of individual change (day 1- day 7)23.51.1265 Fig. 1Mean symptoms' scores and standard errors per day of observation Patients reported feeling better on the third day of extract intake (median: 3.0, 95%CI: 3.0–3.5). They also reported that their symptoms had ceased completely by the sixth day of intake (median: 6.0, 95% CI: 5.0–6.0). By the end of the observation period 81.7% of our participants reported that their symptoms had ceased completely. The mean AuC for all symptoms was 26.2 (±17.4). Multiple linear regressions did not show any significant predictors (including supplementary medication intake) for the AuC, the day patients reported feeling better or the day patients reported that their symptoms ceased completely. BODY.RESULTS.SYMPTOMS’ SEVERITY OVER TIME: Linear mixed models showed a significant decrease in the total score of symptoms over time (b = −14.6 in day 4 and b = −21.6 in day 7; p < 0.0001 for both) and the presence of fever (b = −0.8, p < 0.001 both for days 4 and 7). A borderline positive association between the total score of symptoms and supplementary medication intake was observed (b = 2.9; p = 0.065), indicating that patients with more severe symptoms tended to consume extra medication. Results indicated no significant interactions between supplementary medication intake and day of observation in the prediction of the total score of symptoms (b = −2.03; p = 0.362 for day 4 and b = −3.0; p = 0.306 for day 7). Post-hoc analyses indicated no significant differences in the total score of symptoms between patients consuming supplementary medication and those who did not (mean difference = 0.87; 95% CI: −2.5 - 4.26; p = 0.611 for day 4 and mean difference = −0.1; 95% CI: −5.1-5.0; p = 0.980 for day 7). BODY.DISCUSSION: The present study follows a previously published work [4] on the effectiveness of a combination of Cretan herbs on the amelioration of upper respiratory tract infections. Indeed, our data indicated significant decrease in symptoms' scores in days 4 and 7 of observation, compared to baseline. We documented a reduction of 90.3% in the percentage of people reporting fever between days 1 and 4 and 97.6% between days 1 and 7, a result that has not been reported previously. Supplementary medication intake did not seem to affect either the reduction on the severity of symptoms or the day patients reported that their symptoms ceased completely. A literature review study reported 16 trials of herbal remedies for respiratory tract infections [7]. Trials have also examined the efficacy of Salvia officinalis products regarding certain respiratory tract infections, including acute sore throats [8] and acute pharyngitis [9], presenting positive effects in the treated groups. Our narrative search did not identify studies regarding the effectiveness of similar products upon their market release. However, we viewed our results in parallel to the results from the placebo group of a trial with similar population, as well as the placebo group of this extracts' clinical trial. The first trial explored the efficacy of a Pelargonium sidoides preparation in common cold and reported that 11.8% of participants in the placebo group had complete clinical cure (according to cold intensity score) within 10 days of observation [10]. In the extract's clinical trial, 70% of the participants in the placebo group had symptoms' cessation by day 7 [4]. In the present study, 81.7% of the participants reported that their symptoms had ceased completely by day 7. Although results cannot be compared directly due to the differences in the design of the studies, this may provide an indication about the performance of the extract in relation to no actual treatment. This study is prone to the limitations of its observational design, including the fact that there was no control group for comparable results. Additionally, information about the daily course of patients' treatment could not be reported, since data was gathered at distinct time points. The presence of fever was, also, not recorded as exact temperature but rather as a dichotomous variable (yes/no). Almost two out of three patients received supplementary medication for their treatment. In all regression models, supplementary medication intake was modelled as dichotomous on each observation, since types and duration of intake differed for each patient. Thus, the effect of competitive active ingredients and types of medication could not be documented. Moreover, it was not possible to determine the extracts' effectiveness on specific viral infections, since there was no identification of patients' viral strains and viral loads. Finally, our sample was confined only to patients who voluntary requested the extract and were able to afford its purchase. Despite these limitations, this study reflected the potential therapeutic effects of the extract, out of the controlled clinical trial setting, in the daily practice scenario where patients consume several medications at different time points for their treatment. In a period were debate regarding the cost-effectiveness of antiviral therapy is ongoing [11], further research on the use of aromatic plants may yield promising results. BODY.CONCLUSIONS: The findings of this study affirmed those of a previously published, double blind, randomized, placebo controlled trial, providing additional evidence about the potential therapeutic role of Cretan herbs in cases of upper respiratory tract infections.

TITLE: Ultra fast-track extubation in heart transplant surgery patients ABSTRACT.BACKGROUND:: Heart transplant surgeries using cardiopulmonary bypass (CPB) typically requires mechanical ventilation in intensive care units (ICU) in post-operation period. Ultra fast-track extubation (UFE) have been described in patients undergoing various cardiac surgeries. ABSTRACT.AIM:: To determine the possibility of ultra-fast-track extubation instead of late extubation in post heart transplant patients. ABSTRACT.MATERIALS AND METHODS:: Patients randomly assigned into two groups; Ultra fast-track extubation (UFE) group was defined by extubation inside operating room right after surgery. Late extubation group was defined by patients who were not extubated in operating room and transferred to post operation cardiac care unit (CCU) to extubate. ABSTRACT.RESULTS:: The mean cardiopulmonary bypass time was 136.8 ± 25.7 minutes in ultra-fast extubation and 145.3 ± 29.8 minutes in late extubation patients (P > 0.05). Mechanical ventilation duration (days) was 0 days in ultra-fast and 2.31 ± 1.8 days in late extubation. Length of ICU stay was significantly higher in late extubation group (4.2 ± 1.2 days) than the UFE group (1.72 ± 1.5 days) (P = 0.02). In survival analysis there was no significant difference between ultra-fast and late extubation groups (Log-rank test, P = 0.9). ABSTRACT.CONCLUSIONS:: Patients undergoing cardiac transplant could be managed with "ultra-fast-track extubation", without increased morbidity and mortality. BODY.INTRODUCTION: Heart transplant surgeries using cardiopulmonary bypass (CPB) typically requires mechanical ventilation in intensive care units (ICUs) during post-operation period. Some centers attempt early endotracheal extubation as part of a fast-tracking strategy, whereas others prefer mechanical ventilation in ICU as their routine patient management in the postoperative period.[1] Generally, the term "early extubation" is applied when the endotracheal tube is removed within 6 to 8 hours after the surgery.[2] Fast-tracking and early endotracheal extubation have been described in patients undergoing various cardiac surgeries; however, criteria for patient selection have not been validated in a prospective manner.[3] Ultra Fast-track extubation (UFE) has been applied when patient is extubated in operating room (OR). The desire to reduce hospital costs by shortening ICU and hospital stay interest in early extubation has become oblige to current cardiothoracic surgery. Besides, late extubation expose patients to multiple risk such as infectious complications, ventilator associated pneumonia, and atelectasis. The purpose of the present study was to determine the possibility of fast extubation instead of late extubation in post heart transplant patients. BODY.MATERIALS AND METHODS: The study was reviewed and approved by the University Review Board and hospital ethics committee and has been performed in accordance with the ethical standards laid down in an appropriate version of the 2000 Declaration of Helsinki (http://www.wma.net/e/policy/b3.htm). Information about trial was given comprehensively both orally and in written form. All patients gave their informed written consents prior to their inclusion in the study according to University Hospital Ethical Board Committee. BODY.MATERIALS AND METHODS.PATIENT SELECTION: Patients were selected in a prospective, randomized, controlled trial at a 1-year time period from those who underwent heart transplantation. Patients were eligible if they had undergone cardiac transplant, higher than 18 years of age. Randomization was performed based on odd number to ultra-fast-track extubation and evens to standard extubation. Ultra-fast-track extubation group were defined by extubation inside OR right after surgery. Late extubation group was defined by patients who were not extubated in OR and transferred to post-operation cardiac care unit (CCU). Patient in late extubation group had successfully passed mechanical ventilation weaning screening test or spontaneous breathing trial on the day of extubation. Exclusion criteria were any patient with lung problems (impaired preoperative pulmonary function test), younger than 18 years or older than 70 years, pregnant, had a Simplified Acute Physiology Score II greater than 80, mechanical ventilation preoperatively, surgical procedure complexity assessed with the Risk Adjustment for Congenital Heart Surgery (RACHS) score <4.[4] Contraindications to ultra-fast-track extubation were (except for failure to meet standard extubation criteria) hemodynamic instability and persistent bleeding at the end of operation. Demographic, preoperative and perioperative physiologic and radiographic features, coexisting conditions, and mechanical ventilation characteristics at randomization were recorded. Preoperative predicted operative mortality was calculated using the European System for Cardiac Operative Risk Evaluation. BODY.MATERIALS AND METHODS.ULTRA FAST-TRACK EXTUBATION: Patients underwent ultra-fast-track extubation in OR in case of stable hemodynamics (blood pressure, heart rate, and respiratory rate), warm peripheries, adequate blood gas exchange, adequate muscle strength, satisfactory urine output, acceptable hematocrit chest tube drainage, and adequate pain control. Postoperative transthoracic echocardiogram was done in all patients before extubation. The reasons given by the anesthesiologists for deferring extubation are listed in Table 1. Significant pulmonary hypertension (PHT) after the discontinuation of cardio-CPB had been completed (independent of preoperative PHT). it was the most common cause for deferring OR extubation, followed by hypoxemia, coagulopathy, and hemodynamic instability. Table 1 Characteristics and pump time and hospital discharge days of patients with BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: All multiple comparison tests were two-tailed. Direct comparisons between two treatment groups were performed with the unpaired Student t-test or the nonparametric Mann–Whitney test when the data sets were not normally distributed. A survival analysis was performed with Kaplan-Meier and Cox proportional hazards (COX PH) analysis. A P value of 0.05 or less was considered significant. All statistical analyzes were performed with Statistical Package for the Social Sciences (SPSS) 18. BODY.RESULTS: Eleven patients were ultra-fast-track extubated (UFE) within operating room and 12 patients were late-extubated (LE) after 24 hours in CCU. Mean duration of ventilation among UFE patients was 36.2 ± 12.3 minutes and among LE patients was 28.7 ± 9.6 hours. The mean age of UFE patient was 35.7 ± 14.6 years and 39.5 ± 16.8 years in LE (P > 0.05). The mean weight of UFE patient was 76.8 ± 15.3 kg and 72.4 ± 18.4 in LE patients (P > 0.05). The mean CPB time was 136.8 ± 25.7 minutes in UFE and 145.3 ± 29.8 in LE patients (P > 0.05). Various aspects of UFE patients are provided in Table 1. However, patients in the prolonged intubation group had more frequent unscheduled extubation and reintubations or recannulations. After adjustment for repeated operations, heart transplantations, and renal replacement therapy at base line remained similar between group differences in primary and secondary outcomes [Table 2]. Table 2 Comparison of patients outcomes characteristics between Ultra-fast-track extubation and late extubation Finally using Kaplan-Meier and COX PH analysis, we considered death in follow-ups as status variable (event). Patients were censored from study if they failed to follow-up. The length of survival was the time between operation and death or end of follow-up time at 400 days. In survival analysis, there was no significant difference between UFE and LE groups (Log-rank test P = 0.9) [Figure 1]. Figure 1Kaplan Meier survival curve for both fast-track and late extubation groups survival were not significantly different (Log-rank test P value = 0.9) BODY.DISCUSSION: Late extubation and mechanical ventilation is a part of postoperative care in cardiac surgery. Recent advances in surgical and anesthetic techniques have facilitated early extubation following cardiac surgery and heart transplant.[5] Potential benefits of an early extubation are decreasing cardiac and respiratory morbidity,[6] increasing cardiac performance, and lower rate of nosocomial pneumonia.[7] Early extubation has been shown to expedite ICU discharge[8] as well as overall length of stay, thus leads to reduce cancellation of surgery and decrease cost of patient care.[2] Studies have shown that early extubation after elective cardiac surgery of patients has not increased perioperative morbidity.[910] In this study, ultra-fast-track extubation has provided much benefit for patients, who require prolonged mechanical ventilation after cardiac surgery, in terms of mechanical ventilation duration, length of ICU stay, mortality rate, or frequency of infectious complications like ventilator associated pneumonia (VAP). None of the patients had any severe underlying disease to compromise our study. The major factor that caused delay in UFE strategy was deep sedation and confusion. The reason for deep sedation was variable doses of intraoperative fentanyl and midazolam. Other groups employ UFE strategy for all patients undergoing elective coronary artery bypass grafting (CABG) surgery, fulfilling the fast track criteria with the aim to wean and extubate patients within the first 6 hours following surgery.[11] In another study on 109 patients, no patient required reintubation within the first 24 hours after operation and only one patient required reintubation 3 days after operation for sputum retention.[12] Ultra-fast-track anesthesia using an ultra-short acting opiate remifentanil was used in 160 unselected patients undergoing off-pump coronary artery bypass grafting. Extubation within 10 minutes of the end of operation was feasible in 150 patients (94%). Five patients (3%) were extubated within 2 hours, and the remaining 5 patients (3%) were converted to standard anesthesia.[1314] Another study on 304 patients receiving either a typical fentanyl/isoflurane/propofol regimen or a remifentanil/isoflurane/propofol regimen for ultra-fast-track cardiac anesthesia showed the efficacy of utilization of remifentanil and fentanyl in fast-track.[15] In another study, it has been showed that CPB time and aortic cross clamp time are two factors which delay the early extubation.[16] Age, weight, sex, severity of pulmonary artery hypertension, and type of ventricular septal defect (VSD) did not affect early extubation.[17] Longer CPB time is consistently associated with prolonged mechanical ventilation after coronary artery disease (CHD) surgery.[3] This is not surprising, because longer CPB time is required for more complex cases or if unexpected difficulties occur.[18] Despite this, the complexity of the cases alone did not predict successful extubation in the OR in our analysis. Furthermore, longer CPB time is associated with an increased risk of inflammatory response syndrome with generalized edema, decreased respiratory compliance, acute lung injury, and coagulopathy, all of which affect the ability to extubate a patient immediately after surgery.[19] In conclusion, patients undergoing elective repair may be managed with "ultra-fast-track extubation," without increased morbidity and mortality. Prolonged unnecessary hospitalization contributes little, if any, to a more favorable therapeutic outcome. Family reliability and ready access to the cardiac surgical team will result in satisfactory outpatient resolution of most late-onset complications. Implementation of ultra-fast-track extubation provided adequate hemodynamic control and facilitated OR extubation in all patients.

TITLE: The effects of different loading doses of dexmedetomidine on sedation ABSTRACT.BACKGROUND: Dexmedetomidine is a useful sedative drug with various uses. We designed this study to investigate the clinical effects and complications of different loading doses, 0.5 and 1.0 μg/kg. ABSTRACT.METHODS: Forty six patients, of American Society of Anesthesiologists physical status I and II, who required elective and emergency operation under spinal anesthesia were randomly assigned to group L or group H. Group L received a loading dose of 0.5 μg/kg for 10 minutes while group H received 1.0 μg/kg. Bispectral index (BIS), systolic blood pressure, heart rate, and Ramsay score were recorded at T0 (before loading), TL (just after loading) and T10, 20, 30 (10, 20, 30 minutes after TL). Complications, drug use, lowest BIS and time to reach BIS 80 after termination of dexmedetomidine were recorded during this study. ABSTRACT.RESULTS: In group H, BIS value decreased significantly after TL compared to the baseline (T0), while in group L after T10. Between two groups, BIS values showed a significant differences only at T10, BIS of group H was lower than that of group L. Ramsay score showed no significant differences except in TL; the score of group L was significantly lower than that of group H. Other vital signs and complications showed a minimal differences between two groups. ABSTRACT.CONCLUSIONS: Higher loading dose (1.0 μg/kg) of dexmedetomidine can lead to faster sedation without any severe complications. BODY.INTRODUCTION: Dexmedetomidine is a highly selective alpha 2 agonist [1]. This drug activates alpha 2 receptors in the locus ceruleus of the brain, resulting in very effective sedation of the patient [2]. Unlike many other sedative drugs that cause respiratory depression during sedation, dexmedetomidine is free from such side effects [3,4,5,6]. This advantage contributes to the variable use of dexmedetomidine, not only in the critical care unit as originally intended, but also in sedation and anesthesia in both preoperative and postoperative periods [7]. However, this alpha 2 agonist has its own side effects. Activation of the alpha 2 receptors in peripheral vessels causes unexpected hypertension and bradycardia during the first few minutes of infusion, and the converse may occur after this period: rebound of the autonomic nervous system can result in hypotension and tachycardia [8,9]. The dosing of dexmedetomidine varies based on its purpose, although clinical use usually suggests a loading dose of 0.5-1.0 μg/kg for 10 minutes and a maintenance dose of 0.2-0.7 μg/kg/min [7]. We designed this study to investigate the clinical effects and complications at two different loading doses, 0.5 and 1.0 μg/kg. BODY.MATERIALS AND METHODS: Forty six patients, of American Society of Anesthesiologists physical status I and II, who required elective and emergency operation under spinal anesthesia were enrolled in this prospective, randomized, double-blind study. The exclusion criteria were underlying neurologic and cardiovascular disease, renal failure, liver failure and contraindication cases of spinal anesthesia such as bleeding tendency, patient refusal. The study was approved by the Institutional Review Board and written informed consent was obtained from each patient. All patients fasted for 8 hours before the operation, and no premedications were administered. In the operating room, 5 L/min of oxygen was provided to patients via an oxygen mask and an intravenous line was placed in each patient's forearm. Before spinal anesthesia, Ringer's lactate solution 10 ml/kg was administered intravenously as pre-hydration. The patients were placed in the lateral decubitus position and spinal punctures were performed using a 25-gauge Quincke spinal needle at L3-4 or L4-5. Hyperbaric 0.5% bupivacaine 12 mg was administered intrathecally and lied down immediately. In all patients, spinal anesthesia was successfully conducted, and sensory block level was evaluated in pin prick test by using 25 G needle. After wiping the patient's forehead with alcohol gauze, a BIS monitor (Model A 3000, Aspect Medical systems, Natick, MA, USA) was placed and the initial BIS value was recorded. Heart rate (HR), oxygen saturation (SpO2), non-invasive blood pressure, Ramsay score (Table 1) and BIS were closely monitored in all patients, before administration of the loading dose of dexmedetomidine (T0), just after loading (TL), and at 10 min intervals thereafter (T10, T20, T30) [10]. Loading was started immediately after 20 minutes in a manner corresponding to the loading dose of each group: group H with 1.0 μg/kg and group L with 0.5 μg/kg. After 10 minutes of loading, dosing was changed to a maintenance dose, 0.5 μg/kg/min. All data were recorded every 10 minutes until the end of the operation. After the operation, infusion of dexmedetomidine was stopped. The lowest BIS score during the operation and the time to reach BIS 80 after termination of infusion were recorded. Aside from the primary data, all complications were recorded when they occurred. Complications were defined as hypertension (systolic blood pressure or mean arterial pressure > 20% of baseline), hypotension (systolic blood pressure < 80 mmHg), bradycardia (heart rate < 50 beats/min), tachycardia (heart rate > 100 beats/min for 5 minutes), hypoxemia (SpO2< 95%) and oral dryness. Ephedrine and atropine used for the rescue of such complications were also recorded after each use. All measures were displayed as mean ± standard deviation (SD). When BIS had a difference of 10 or more, the α value was set at 0.05, the β value was set at 0.2 and the sample size was set at 23. Statistical analysis was performed using R 3.0.0. for Windows. Enumeration data such as sex, incidence of side effects and use of drugs were compared with Chi-square analysis. Nonenumeration data such as BIS, SBP, and HR were compared by Student's t-test. P values less than 0.05 were considered statistically significant. BODY.RESULTS: Forty-six patients were enrolled in this study. No patients were excluded and patient characteristics such as sex, age, height, weight and sensory block level showed no significant differences between two groups (Table 2). In group H, BIS value decreased significantly after TL compared to the baseline (T0), while in group L after T10 (Fig. 1). Between two groups, BIS values showed a significant differences only at T10, BIS of group H was lower than that of group L. However, the other BIS parameters (the lowest BIS value and time to reach BIS 80) did not show any significant differences between two groups (Table 3). Throughout this study, heart rate decreased significantly rather than T0 value in the both groups. But, there were no significant differences between two groups (Fig. 2). Systolic blood pressure did not show any significant differences except at T30 in group L, where SBP was significantly lower rather than T0 (Fig. 3). Between two groups, SBP value in both groups showed a significant difference only at TL, SBP value of group L was lower than that of group H (Fig. 3). There were no significant differences between two groups in the incidence of bradycardia, hypoxemia, hypotension and hypertension (Table 3). Incidence of oral dryness, tachycardia, excessive bradycardia and use of atropine and ephedrine did not show any significant differences. Ramsay score did not show any significant differences except at TL when the score of group L was significantly lower than the score of group H (Fig. 4). BODY.DISCUSSION: Dexmedetomidine is a highly selective alpha 2-agonist that is used in the operating room and ICU [7]. Its unique characteristics of powerful sedation without respiratory depression make dexmedetomidine one of the most useful drugs in sedative fields [7,11]. In our study, both groups showed significantly lower BIS values compared to baseline after loading, although the breakpoint was different. In group L, the significant difference revealed itself 10 minutes after loading, while in group H it occurred right after loading (Fig. 1). BIS values between the two groups did not show any significant differences except at T10, where the BIS value of group H was lower than that of group L. Previous investigations commented that an increase in loading dose and plasma concentration of dexmedetomidine resulted in improved sedation [12]. In addition to affirmative results in the past, it has been suggested that dexmedetomidine has a dose-dependent sedation effect, which may be altered by the loading dose [13]. These results indicate that a higher loading dose of dexmedetomidine may lead to more rapid sedation. As faster sedation is one of the major goals in the use of dexmedetomidine, a loading dose of 1.0 μg/kg may be far superior to 0.5 μg/kg in terms of sedation. A recent trial indicated that a BIS value of 65 to 85 may be an optimal standard for sedation [14]. According to our data, group H showed faster entrance to BIS below 85 than group L. Ramsay score differed significantly only at TL, where the value of group L was lower than that of group H. Group H showed successful entrance to the Ramsay score above 3 after TL, while group L after T10. We have found reports that Ramsay score of 3 to 4 is optimal for sedation [15]. Also, this score roughly corresponds to BIS 69 to 81, which is acceptable to our result as well [16]. The superiority of the higher loading dose in faster sedation is well reflected in our data. HR values were significantly lower than the baseline value at all points, while no significant differences were shown between two groups. These data are consistent with previous investigations that dexmedetomidine has a great relation to the complication of bradycardia [7]. The SBP of the two groups showed significant differences at TL, when the SBP of group H was higher than that of group L. This corresponds to results of past research that indicated that a greater loading dose of dexmedetomidine may result in faster incidence of transient hypertension [17,18]. In total, use of a higher loading dose of dexmedetomidine results in faster effects, whether sedation or cardiovascular features. These data suggest that the cardiovascular effects of dexmedetomidine are related to its concentration and dosing [19]. The subjectivity of the Ramsay score calls for attention however. Values such as SBP, HR, and BIS are perfectly numerated and can be defined objectively by anyone. However, the Ramsay score requires a small 'interview' and may differ by patient or questioner. In conclusion, the 1 μg/kg loading dose of dexmedetomidine can lead to faster sedation without any severe complication. So, for rapid sedation after spinal anesthesia, a higher loading dose such as 1 μg/kg may be an optimal choice for this drug.

TITLE: An interactive workshop plus locally adapted guidelines can improve General Practitioners asthma management and knowledge: A cluster randomised trial in the Australian setting ABSTRACT.BACKGROUND: A cluster randomised trial was conducted to determine the effectiveness of locally adapted practice guidelines and education about paediatric asthma management, delivered to general practitioners (GPs) in small group interactive workshops. ABSTRACT.METHODS: Twenty-nine practices were randomly allocated to one of three study arms. Australian asthma management guidelines were adapted to accommodate characteristics of the local area. GPs in the intervention arm (Group 1, n = 18 GPs) participated in a small group based education program and were provided with the adapted guidelines. One control arm (Group 2, n = 18 GPs) received only the adapted guidelines, while the other control arm (Group 3, n = 15 GPs) received an unrelated education intervention. GPs' knowledge, attitudes and management of paediatric asthma was assessed. ABSTRACT.RESULTS: Post intervention, intervention arm GPs were no more likely to provide a written asthma action plan, but were better able to assess the severity of asthma attack (Group 1vs Group 2 p = 0.05 and Group 1 vs Group 3 p = 0.01), better able to identify patients at high risk of severe attack (Group 1vs Group 3 p = 0.06), and tended to score higher on the asthma knowledge questionnaire (Group 1 vs Group 2 p = 0.06 and Group 1 vs Group 3 p = 0.2). Most intervention arm GPs felt more confident than control GPs to manage acute asthma attack and ongoing management of infrequent episodic asthma. ABSTRACT.CONCLUSION: Using interactive small group workshops to disseminate locally adapted guidelines was associated with improvement in GP's knowledge and confidence to manage asthma, but did not change GP's self-reported provision of written action plans. BODY.BACKGROUND: The prevalence of asthma in Australia is one of the highest in the world [1,2]. Asthma accounts for about one fifth of the total disease burden in Australian children aged 0 – 14 years [3]. Self-management education together with effective drug therapy can reduce morbidity and mortality [4]. Australian General Practitioners (GPs) see 90% of Australians at least once annually [5] and are ideally placed to coordinate the care of patients with asthma; however, they are still under-diagnosing asthma and under-treating with inhaled steroids. In an attempt to standardise asthma education and management, and reduce variability in patient care, clinical guidelines have been developed in Australia [6,7] and internationally. Despite this, recent studies in Australia indicate that recommendations are often not implemented in clinical practice [8], suggesting a need for more effective and innovative dissemination and implementation strategies [9]. The local adaptation of national guidelines has been proposed as a way of reversing this trend as it provides the benefit of local ownership, while maintaining scientific validity [10]. The International Drug Education randomised controlled trial [11] suggested that improvements in asthma treatment are possible with an educational program for providers based on self-learning in small peer groups. A recent systematic review concluded that interactive workshops could result in moderate changes in professional practice [12]. However, the few randomised controlled trials (RCTs) involving continuing medical education (CME) for asthma management alone, have had limited success in improving health outcomes for patients [13]. Generally, multifaceted strategies have been shown to be most effective [14]. This study brings together two key strategies for improving management of chronic disease: small group workshops plus locally-adapted clinical guidelines. The aim of this paper is to examine the effect of this intervention on General Practitioners (GPs) knowledge and management of paediatric asthma. BODY.METHODS.STUDY DESIGN: A cluster randomised controlled trial (RCT) design was used, with practices randomised to one of three study arms (Figure 1). Group 1 participated in small group asthma education workshops and received locally adapted asthma management guidelines; Group 2 received the locally adapted asthma guidelines only and Group 3 received an alternative education program consisting of information about management of paediatric ear, nose and throat (ENT) problems. Group 3 did not receive any adapted asthma resource materials until the end of the trial. Approval for this study was given by the University of Melbourne Human Research Ethics Committee. Figure 1Randomisation of General Practices and General Practitioners. BODY.METHODS.STUDY POPULATION AND RECRUITMENT: The trial took place between February and November 2001 in Melbourne, Australia. The Northern and North West Melbourne Divisions of General Practice and the Royal Australian College of General Practitioners (RACGP) promoted the trial via their newsletters, fax and e-mail circulars. GPs on the Divisions' membership lists were sent a personal invitation with a fax-back reply sheet. A telephone reminder from the study investigators followed this one-week later if the fax-back form had not been returned. Patients with asthma aged between 2 and 14 years were recruited from each participating practice to assess the effect of the intervention on patient outcomes. Patient recruitment and outcomes were also assessed but are not reported in this paper. They will be published in an upcoming publication. BODY.METHODS.RANDOMISATION AND BLINDING: The unit of randomisation was the general practice clinic. A table of random numbers was used to assign GP practices to study groups. It was not possible to blind GPs to which study group they had been assigned, however, patients were not informed by the investigators as to their GPs group allocation. Investigators were unable to be blinded to the group allocation of GPs, but were blind to the group allocation of patients. BODY.METHODS.DESCRIPTION OF THE EDUCATION PROGRAM AND GUIDELINES: The paediatric asthma education program was developed using the resources available from the Royal Children's Hospital (RCH) Asthma Interest Group and the National Asthma Council [7], including the 6-step asthma management plan and adherence booklet. The education program included two workshops of approximately 3-hours duration that were held on a Saturday afternoon and Sunday morning. Presenters at the workshops included Paediatricians from the Melbourne Royal Children's Hospital, the Northern Hospital, and GPs. Twelve of eighteen eligible GPs attended the first program and a condensed program was provided to the remainder. The paediatric asthma guidelines were adapted to the local context, a low socioeconomic area with a high proportion of culturally and linguistically diverse (CALD) groups, by an inter-Divisional group of GPs and the investigators. The guidelines were presented as flow-charts and dot points in three laminated A4 pages, printed on both sides. Approximately 12-hours of group discussion and several hours of individual review were required to achieve consensus on the guidelines. BODY.METHODS.GP OUTCOMES AND INSTRUMENTS: GPs completed the survey instruments before the intervention and 6 months later. The primary outcome was GPs self reported use of Written Asthma Action Plans (WAAP) as part of the management of children with asthma. Secondary outcomes included knowledge of paediatric asthma that comprised 21 true/false statements about triggers of asthma in children and treatment of paediatric asthma and was adapted from the paediatric asthma knowledge questionnaire of Fitzclarence and Henry [15]. Additional questions were asked about knowledge of the severity of an acute asthma attack, which was made up of a checklist of 11 clinical features that GPs ranked as either a reliable indicator of severity, useful but less reliable, or unreliable or unrealistic feature. GPs were also asked to identify 7 indicators out of a list of 10 items that would make them concerned that a child was at 'high risk' of a severe attack. For each of these 3 outcomes, the number of correct responses were summed and transformed to percentage score. GPs were also asked to rate their confidence (very confident, not very confident, not confident at all) in managing a mild, moderate, or severe/critical asthma exacerbation in children, and their confidence in managing infrequent episodic, frequent episodic, and persistent asthma. Other questions were asked about patterns of asthma, and diagnosis and management based on three case scenarios. The case scenarios presented paediatric patients presenting with three levels of asthma severity (infrequent episodic, frequent episodic and persistent). The education package, guidelines and questionnaire are available online [16]. BODY.METHODS.SAMPLE SIZE CALCULATIONS: Sample size calculations for the trial were based on patient outcomes. The primary outcome was the proportion of patients with a written asthma action plan at 6 months post-intervention. We assumed an intra-cluster correlation (ICC) of 0.02 for the provision of asthma action plans in the sample size calculations, as at the time of the sample size calculations there was no published literature for the ICC estimate. To show a difference of 20% in the proportion of patients with written asthma action plans between intervention and control group, assuming that 50% of patients would have a written asthma plan in the control group, 80% power and significance level of 5% for a 2-sided test, we required 13 patients from each GP practice given that there were 10 GP practices in each arm. After allowing for non-response and loss to follow-up throughout the trial we required 17 patients per practice. BODY.METHODS.DATA ANALYSIS: The data were analysed using Stata Statistical Software: Release 8.0 (Stata Corporation, College Station, Tx, USA). Demographic variables were summarised using frequencies and percentages for categorical data and median and inter-quartile range (IQR) for years in general practice. Fisher's Exact test was used to examine GPs level of confidence across the three study groups. For the primary outcome of whether the GP had provided patients with a written asthma action plan (WAAP yes/no), logistic regression was used to calculate the odds ratio and respective 95% confidence interval (CI) and p-value comparing the intervention group to each control group. No adjustments were required for the clustering effect (arising from more than one GP participating from some Practices), as the estimated intra-cluster correlation for provision of WAAP was very close to zero. Linear regression was used to examine the difference in mean knowledge scores between the intervention group and each control group, adjusting for baseline outcome measure. In the regression analysis, the mean cluster score for each practice was used to allow for the clustering effect. Results were reported as difference in means with respective 95% confidence intervals (CI) and p-values. BODY.RESULTS: GPs from 32 practices (n = 63 GPs) were initially enrolled, though three practices and 12 GPs dropped out of the study after patient recruitment. The flow of practices and GPs through the study is shown in Figure 1. Demographic and other background information about the participating GPs is presented in Table 1 (see additional file 1). Practice factors and GP characteristics were generally well balanced across the three study groups, except for years in general practice where GPs in Group 3 tended to have more years in general practice than GPs allocated to Groups 1 and 2. Table 1 Demographic and other background information about the participating practices and GPs at baseline Group 1 Group 2 Group 3 n (%) n (%) n (%) Practice characteristics (N) 10 9 10 Number of GPs participating from each practice (GP cluster size) 1 3 (30) 4 (44) 6 (60) 2 6 (60) 2 (22) 3 (30) 3–4 1 (10) 3 (33) 1 (10) Number of Solo GPs 2 (20) 4 (44) 4 (40) GP Characteristics (N) 18 18 15 Gender (proportion of male GPs) 12 (67) 12 (67) 10 (67) Graduated in Australia 10 (56) 10 (56) 10 (67) Years in General Practice in Australia – Median (IQR) 11 (7, 16) 15.5 (10, 20) 19 (14, 25) Years in General Practice in overseas- Median (IQR) 0.5 (0, 5) 1 (0, 5) 0 (0, 5) Qualifications and Membership* Vocational registration 11 (61) 13 (72) 14 (93) Fellowship/GP Training Program 15 (61) 7 (28) 4 (20) Diploma/Master/PhD 2 (11) 6 (33) 5 (33) Other 2 (11) 2 (11) 3 (20) Sessions usually worked per week – Median (IQR) 9 (6, 10) 8 (6, 10) 8 (6, 10) * Groups not mutually exclusive There was no evidence supporting the hypothesis that GPs in the intervention group (education + guidelines) were more likely to report providing a written asthma action plan to children compared to GPs in the two control groups (Group 1 vs Group 2: OR = 1.15, 95% CI: 0.14 to 9.4, P = 0.89; and Group 1 vs Group 3: OR = 3.8, 95% CI 0.50 to 28.4, P = 0.20) (see additional file 2). At baseline (pre-intervention) there were no differences between the groups in their asthma knowledge, assessment of asthma severity, or assessment of high-risk asthma (see additional file 3). Post-intervention, there was some evidence suggesting that GPs in the intervention group had increased their knowledge about asthma and were better able to assess the severity of asthma attack. Likewise, there was evidence suggesting that these GPs were better able to identify patients at high risk of severe attack compared to the control groups that received the alternate intervention (see additional file 3). At baseline there were no differences between intervention and control groups in GPs self-reported confidence in managing acute asthma or routine management of asthma. The majority of GPs (> 87%) in all 3 groups felt 'confident' or 'very confident' treating and managing mild or moderate acute attacks and infrequent or frequent episodic asthma. However, 38% of GPs felt 'not very confident' or 'not confident at all' managing a severe/critical attack of asthma and 20% were 'not confident' about the ongoing management of persistent asthma. Post-intervention, a higher proportion of GPs in the intervention arm (Group 1) felt 'very confident' compared to control arm GPs for ongoing management of infrequent episodic asthma (p = 0.03), but there was no evidence to suggest that they were more confident managing acute attacks or ongoing management of frequent episodic or persistent asthma (see additional file 4). No adverse events were encountered during the trial. BODY.DISCUSSION.SUMMARY OF MAIN FINDINGS: This study found that simple, locally adapted best practice guidelines for paediatric asthma, disseminated during small group interactive workshops, can improve General Practitioners knowledge of asthma and confidence in triaging and managing patients with asthma, especially infrequent episodic asthma. However, certain elements of asthma management, in particular providing patients with a written asthma action plan, were not changed. This may be due to the relatively high self-reported use of written action plans (66%) in the intervention group at baseline and the characteristics of the GPs in the intervention group who tended to be relatively younger GPs who were more engaged with the Royal Australian College of GPs training program. The result may also be partly due to the nature of the intervention which focussed on education more so than behaviour change. Behaviour change may require more complex and multifaceted interventions that include practice organisation strategies to initiate, support and maintain change. BODY.DISCUSSION.STRENGTHS AND THE LIMITATIONS OF THIS STUDY: This paper focuses on the GP outcomes; however, the number of GPs in each arm is small. The small number of practices means our estimates for the provision of written asthma action plans, asthma knowledge and confidence scores are less precise than we would ideally like (as indicated by the wide confidence intervals), however, the effect sizes suggest that the intervention was associated with real improvement in knowledge and confidence in the intervention group when compared to the control group, and the proportion of GPs in the intervention group who reported using written asthma action plans increased (as hypothesised) although this change was not statistically greater than the change observed in the control groups. A larger sample size would be needed to determine if changes are a result of the intervention or due to chance. The intervention was delivered at the practice level and main outcomes are self-reported, so we are unable to determine whether changes in knowledge and confidence translate into better clinical practice. The results may also be influenced by GP recall and positive responding (particularly in regard to provision of a written asthma action plan by the intervention group); however, no statistically significant differences were found in the self reported provision of a written asthma action plan. An alternative strategy would have been to audit practice medical records, but these can not be considered a gold standard since the practice records may be incomplete or inaccurate [17,18], and in any case, are not appropriate to assess GPs knowledge and confidence and so the additional expense of conducting an audit of records was not appropriate. A common methodological issue in general practice and primary care trials is the Hawthorne effect [19]. This was addressed through the use of control practices that received an alternative intervention (ENT education in a similar guidelines and workshop format). In addition, the control group practices were motivated to collect data and discouraged from seeking other CME activities on asthma during the trial period. Despite this, 6 GPs in this control arm did not complete the trial. Another potential problem for primary care based RCTs is that patients may not return to see the GP who participated in the educational intervention. To minimise the risk of this, one of our selection criteria was that at least 50% of GPs from any clinic must be enrolled in and complete the study. Hence recruitment occurred at clinic level with clinics selected and randomised, instead of individual GPs. This, however, also proved to be a barrier to participation, as some GPs who expressed interest in the project could not participate because partner GPs were either not interested or could not find the time. This was particularly true for the practices with three or more GPs. Finally, GPs were self-selected and represented a small proportion of the 400-strong membership of the Northern and North West Melbourne Divisions of General Practice. This may limit the generalisability of this study. BODY.DISCUSSION.IMPLICATIONS FOR FUTURE RESEARCH OR CLINICAL PRACTICE: Despite the development of a large number of clinical practice guidelines in the last 25 years, only a few have been rigorously evaluated in primary care settings with randomised controlled trials [20]. Similarly, reviews of the education literature show that while education can lead to improvements in confidence and competence of practitioners [21], there is a dearth of quality RCTs that have investigated the effectiveness of education interventions for asthma in primary care settings [13]. This is consistent with the education literature generally, where there is little high quality evidence on the impact of health care professional education on the quality of services or on patient health outcomes. Further analyses of data collected as part of our study are planned to determine if there are benefits to patients that flow on from the changes in knowledge and confidence reported by their GPs here. BODY.CONCLUSION: Evidence based clinical practice guidelines for the management of asthma have been available in Australia for more than a decade. However, there is evidence that care is still not in line with these guidelines. Our approach, which utilised small group education and dissemination of locally adapted guidelines, was associated with some benefits. GPs in this study were drawn from practices located in areas of high cultural diversity, and so this approach will be particularly useful to others seeking to improve GPs asthma knowledge and confidence in similar settings. Understandably, this RCT which involved an educational intervention demonstrated improvements primarily in knowledge and confidence, but did not produce a statistically significant change in behaviour (i.e. the use of written asthma action plans), although a greater proportion of intervention group GPs reported utilising written asthma action plans 6 months after the intervention. It seems likely that further behaviour change may require an understanding of the readiness to change as well as ongoing support at the point of care. BODY.COMPETING INTERESTS: The author(s) declares that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: CH, SL, NS and SD contributed to conception and design of the study, interpretation of results, and critical review of the manuscript. CH was primarily responsible for the development and delivery of the intervention. CB was involved in the acquisition of data, some data analysis, interpretation of findings, and drafting the manuscript. SS was involved in the development of the intervention, interpretation of results, and critical review of the manuscript. PC provided advice on study design, conducted the statistical analysis and contributed to drafting the manuscript. All authors have read and provided comment on the final draft of the manuscript. Table 2 GPs prescription of written asthma action plans and how they were used. Group 1 Group 2 Group 3 n (%) n (%) n (%) P Pre-intervention number of GPs 18 17 15 Do you use Written Asthma Action Plans for children with asthma? Yes 11 (61.1) 10 (58.8) 12 (80.0) 0.37 If yes, how do you usually write an Asthma Action plan? Standardised pre-printed form 4 (36.4) 3 (30.0) 5 (41.7) Computerised proforma 2 (18.2) 0 (0) 3 (25.0) Blank page 5 (45.5) 6 (60.0) 2 (16.7) Other 0 (0) 1 (10.0) 2 (16.7) Post-intervention number of GPs 17 15 9 Do you use Written Asthma Action Plans for children with asthma? Yes 15 (88.2) 13 (86.7) 6 (66.7) 0.43 If yes, how do you usually write an Asthma Action plan? Standardised pre-printed form 7 (46.7) 6 (46.2) 2 (33.3) Computerised proforma 6 (40.0) 2 (15.4) 3 (50.0) Blank page 1 (6.7) 5 (38.5) 1 (16.7) Other 1 (6.7) 0 (0) 0 (0) Note one GP in Group 2 did not have baseline information (excluded from the denominator) Group 1 = Asthma Education + guidelines Group 2 = Guidelines only Group 3 = Alternative (ear, nose and throat) education only Table 3 Pre and Post intervention GP asthma knowledge in the intervention and control groups. Pre intervention Post Intervention Group N * Mean(SD) N Mean(SD) Diff (95% CI) P Knowledge about asthma – Percentage correct responses out of 21 statements Group 1 18 63.5(12.3) 16 80.8(13.1) 0 Group 2 17 62.8(12.4) 15 71.1(11.6) -10.7 (-21.9, 0.56) 0.06 Group 3 15 61.8(10.1) 9 70.2(14.5) -7.6 (-19.6, 4.4) 0.20 Assessment of severity of acute attack in children – Percentage of correct responses out of 11 statements Group 1 18 30.9(11.9) 16 56.3(17.9) 0 Group 2 17 35.6(13.1) 15 40.1(17.3) -14.1 (-28.1, -0.01) 0.05 Group 3 14 33.1(13.3) 9 33.8(13.3) -20.9 (-35.7, -6.2) 0.01 Identification of a child with asthma that may be at high risk – Percentage of correct response out of 10 statements Group 1 18 82.8(8.3) 17 89.4(9.0) 0 Group 2 17 84.7(10.7) 15 89.3(9.6) -0.02 (-6.9, 6.8) 1.00 Group 3 15 81.3(8.3) 9 82.2(8.3) -7.2 (-14.5, 0.17) 0.06 Diff = Difference between means, adjusted for outcome at baseline; Reference group is Group 1 n = Number of GPs SD = Standard deviation 95%CI = 95% Confidence Interval. Linear regression was fitted on the mean score for each practice. Nb. One GP in Group 2 did not have baseline information (excluded from the total) Group 1 = Asthma Education + guidelines Group 2 = Guidelines only Group 3 = Alternative (ear, nose and throat) education only Table 4 GPs self reported confidence treating children with asthma Post – intervention Group 1 (N = 17) Group 2 (N = 15) Group 3 (N = 9) P * Management of n (%) n (%) n (%) Acute attack Very confident 16 (94.1) 12 (80.0) 5 (55.6) 0.09 Confident 1 (5.9) 3 (20.0) 3 (33.3) Not very confident 0 0 1 (11.1) Not confident at all 0 0 0 Moderate acute attack Very confident 13 (81.3) 11 (78.6) 4 (50.0) 0.30 Confident 3 (18.8) 3 (21.4) 3 (37.5) Not very confident 0 0 1 (12.5) Not confident at all 0 0 0 Severe/critical attack Very confident 2 (11.8) 3 (20.0) 3 (33.3) 0.58 Confident 11 (64.7) 7 (46.7) 3 (33.3) Not very confident 4 (23.5) 5 (33.3) 3 (33.3) Not confident at all 0 0 0 Ongoing management of Infrequent episodic asthma Very confident 14 (87.5) 9 (60.0) 3 (33.3) 0.03 Confident 2 (12.5) 6 (40.0) 5 (55.6) Not very confident 0 0 11 (11.1) Not confident at all 0 0 0 Frequent episodic asthma Very confident 9 (33.3) 6 (40.0) 3 (33.3) 0.49 Confident 8 (55.6) 9 (60.0) 5 (55.6) Not very confident 0 0 1 (11.1) Not confident at all 0 0 0 Persistent asthma Very confident 7 (43.8) 5 (33.3) 3 (33.3) 0.73 Confident 9 (56.3) 8 (53.3) 5 (55.6) Not very confident 0 2 (13.3) 1 (11.1) Not confident at all 0 0 0 BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here:

TITLE: The Effects of Single Long and Accumulated Short Bouts of Exercise on Cardiovascular Risks in Male Japanese Workers: A Randomized Controlled Study ABSTRACT: The aim of this study was to determine whether accumulated short bouts of exercise can achieve the same cardiovascular benefits as a single long bout of exercise in sedentary male Japanese workers and to compare the programs' relative effects on oxidative stress. Twenty-three sedentary male workers were randomly assigned into 2 different exercise programs: a Long-bout group, which performed a single period of continuous exercise (Long-bout group: 30 min × 1) 3 d per week, and a Short-bouts group, which performed 3 short bouts of exercise (Short-bouts group: 10 min × 3) 3 d per week. Cardiovascular risk factors, including the plasma thiobarbituric acid-reactive substances (TBARS) level, were examined at baseline and after both 10 and 20 wk. In the Long-bout group, waist circumference and maximum oxygen uptake (VO2max) significantly improved after 20 wk. The Short-bouts group demonstrated significant increases in VO2max after 10 weeks and in HDL-C after 20 wk. Plasma TBARS significantly decreased after 20 weeks in the Long-bout group and tended to decrease (but not significantly) in the Short-bouts group. These results indicate that accumulated short bouts of exercise are an effective option, especially for busy workers, for incorporating exercise into one's lifestyle. BODY.INTRODUCTION: With the aging of the workforce, the incidence of cardiovascular risk factors such as dyslipidemia and hypertension has increased in Japan1). One recommended approach to address these issues is an increase in physical activity2). However, according to the Survey on State of Employees' Health 2007 by the Ministry of Health, Labour, and Welfare, Japan, 30.6% of Japanese employees report regular mild or moderate physical activity, including brisk walking, calisthenics, and jogging3). This represented a slight increase compared with the previous survey (28.9% in 2002). However, adherence to an exercise program in Japanese employees is less than desirable4). One reason for employees' low adherence to exercise programs is lack of available time5). Many studies have shown that for improvements in physical fitness and body weight, exercise programs comprising an accumulation of several short sessions per day are as effective as those comprising longer continuous sessions6,7,8,9,10,11). The Active Guide 2013, issued by the Ministry of Health, Labour, and Welfare, Japan, also emphasizes that the accumulation of physical activity throughout the week is important for health promotion2). An exercise program that includes short bouts can be performed more frequently, especially by employees with limited time available for daily exercise. In their review, Murphy et al. found that most studies reported no difference in the effect on cardiovascular fitness between accumulated and continuous patterns of exercise12). However, whether accumulated exercise is as effective as continuous exercise in terms of adiposity, the lipid profile, and glucose metabolism remains unclear. Oxidative stress is associated with the pathogenesis and progression of such cardiovascular risk factors as adiposity, high blood pressure, dyslipidemia, and glucose intolerance and is detectable before the onset of clinically significant disease13,14,15,16). Continuous moderate-intensity exercise potently reduces CV risk factors and oxidative stress17, 18). However, there is no study reporting whether accumulated exercise has the same effect as continuous exercise on oxidative stress. The aim of this study was to determine if several short bouts of exercise can achieve the same effects on cardiovascular risk factors in sedentary male Japanese workers as single long bouts of exercise during a period of 20 weeks and to compare the relative effects of these exercise programs on oxidative stress. BODY.METHODS.SUBJECTS: This was a non-blinded randomized controlled study. Twenty-three sedentary male Japanese workers with a mean ( ± SD) age of 43.9 ± 11.6 years were recruited through the university newsletter. The inclusion criteria specified men who did not participate in any vigorous physical activity (reported exercising <20 min·d−1 on <3 d·wk−1 for the previous 6 months). We also included workers who had some cardiovascular risk factors (e.g., obesity, mild hypertension, controlled diabetes, and dyslipidemia) but not a history of cardiovascular disease or of musculoskeletal problems preventing the use of a cycle ergometer. All subjects had sedentary jobs. Medication levels were not modified during the study period. Workers were randomly assigned using random numbers generated by a personal computer to one of the following exercise programs: the Long-bout group, which performed a single period of continuous exercise for 30 min on 3 days per week, or the Short-bouts group, which performed 3 short bouts of exercise, 10 min each, on 3 days per week. A flowchart depicting the participant selection is shown in Fig. 1 Fig. 1.Flowchart for participants.. All subjects provided written informed consent to participate in the present study. This study was approved by the ethics committee of the University of Occupational and Environmental Health, Japan. BODY.METHODS.CLINICAL ASSESSMENT: All subjects underwent complete clinical examination at baseline, after 10 weeks, and again after 20 weeks. Body weight and height were determined using a portable scale and body mass index (BMI) was calculated by dividing the weight (kg) by the square of the height (m2). Waist circumference was measured using a standard protocol19). Resting blood pressure with the subject seated was measured 3 times between 8:00 AM and 10:00 AM using an automatic sphygmomanometer (BP-203RVIII; Nihon Colin, Tokyo, Japan). The mean of the 3 blood pressure readings was used for further analysis. Blood samples were collected after nocturnal fasting for at least 12 h. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), fasting plasma insulin (FPI), and fasting plasma glucose (FPG) were measured. Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula (LDL-C=TC − HDL-C − 1/5 × TG). Insulin resistance was calculated using the homeostasis model assessment of insulin resistance (HOMA-IR) with the formula: HOMA-IR=FPI (μU·ml−1) × FPG (mg·dL−1) / 405. The plasma level of thiobarbituric acid-reactive substances (TBARS) was assayed using the fluorescence method, as previously described20, 21). The level of TBARS, which are byproducts of lipid peroxidation, is a biomarker of the systemic oxidative stress level and is correlated with cardiovascular disease and its risk factors22). Maximum oxygen uptake (VO2max) was estimated using a 3-stage graded submaximal test with a cycle ergometer (ML-1800, Fukuda Denshi, Tokyo, Japan)23). A 6-lead electrocardiogram was recorded and blood pressure was monitored every 2 min during the test. The workload was increased by 35, 40, or 50 W every 3 min depending on the subject's age and physical constitution. VO2max can be used as an index for cardiorespiratory endurance. BODY.METHODS.PROTOCOL: Both the Long-bout and Short-bouts exercise programs were 20 wk long and consisted of 30 min of cycle ergometer exercise per day on 3 days per week. An exercise intensity of 50% VO2max was established for each subject based on the result of the VO2max test. Exercise intensity could be calculated in watts, and the participants were instructed to practice exercising by themselves with the cycle ergometer at the proper intensity. Cycle ergometers were placed near their workplaces so that each participant could attain access to an ergometer within 5 min. VO2max was reassessed during the tenth week, and the exercise intensity was adjusted according to the 10-wk VO2max test for the remaining 10 wk. Subjects in the Short-bouts group were instructed to accumulate 30 min of cycle ergometer activity per day in 3 10-min sessions separated by intervals of ≥2 h on 3 d per week. Subjects in the Long-bout group were instructed to perform a single 30-min continuous bout of cycle ergometer activity 3 d per week. Both exercise programs were self-monitored, and the participants were encouraged to schedule their exercise times into their daily routines while at work. The subjects recorded the time of day for each exercise bout and agreed not to make changes to their diet. BODY.METHODS.STATISTICAL ANALYSIS: Statistical calculations were performed with personal computer statistical software (JMP 9.0; SAS Institute, Inc., Cary, USA). Differences in mean values were assessed using unpaired and paired t-tests for comparisons of 2 variables after confirmation of the normality of each data distribution using the Shapiro-Wilk W-test. Some data that were not normally distributed were subjected to logarithmic conversion prior to analysis. Changes in the cardiovascular risk factors and oxidative stress data were assessed using multivariate analysis of variance (MANOVA) with 2 factors, time (baseline, 10 weeks, and 20 weeks) and pattern of exercise (Short-bouts vs. Long-bout). The level of statistical significance was set at p<0.05. In addition, the effect size (ES) was calculated using G*Power 3, which is a power analysis program designed as a standalone application to handle several types of statistical tests24). The indices of ES were Cohen's d for the paired t-tests and f or f 2 for MANOVA25). According to standard practice, the thresholds for small, medium, and large effects were defined as d values below 0.20, 0.50, and 0.80; f values below 0.10, 0.25, and 0.40; and f 2 values below 0.02, 0.15, and 0.35, respectively25). BODY.RESULTS.CHARACTERISTICS OF THE SUBJECTS: No medical complications occurred during the study. As shown in Table 1Table 1.Baseline characteristicsShort-bouts, n=12, mean ± SD(range)Long-bout , n=11, mean ± SD(range)pbAge (yr)43.3 ± 12.4(27–63)44.5 ± 11.1(27–59)0.822 SBP (mm Hg)122.5 ± 11.2(105.3–140.6)118.6 ± 8.9(104.3–131.7)0.374 DBP (mm Hg)77.5 ± 6.8(67.0–89.7)75.1 ± 6.1(67.0–85.0)0.370 PR (bpm)76.8 ± 12.4(60.3–115.7)73.7 ± 12.4(60.0–90)0.567 BW (kg)74.1 ± 8.5(59.7–87.0)73.3 ± 11.4(57.5–89.7)0.856 BMI (kg·m–2)26 ± 2.4(22.7–32.0)25.6 ± 3.9(20.8–32.1)0.760 Waist circumference (cm)89.5 ± 5.1(83.5–101.2)89.9 ± 7.6(79.8–99.4)0.895 V·O2max (mL·kg–1·min–1)a35.1 ± 4.5(26.6–46.4)38 ± 7.5(29.8–53.3)0.280 TC (mg·dL–1)212.8 ± 32.4(169–281)201.5 ± 20(167–234)0.331 HDL-C (mg·dL–1) a51.8 ± 13.9(33–81)47.5 ± 9.2(38–62)0.451 TG (mg·dL–1) a150.4 ± 82.5(66–356)143.1 ± 79.4(74–348)0.787 LDL-C (mg·dL–1)130.8 ± 29.5(92.8–176.2)125.3 ± 27.1(71.4–159.2)0.645 FPI (μU·mL–1) a8.9 ± 3.6(3.4–16.4)7.7 ± 3(2.4–13.8)0.449 FPG (mg·dL–1) a124.7 ± 46.1(96–266)100.5 ± 7.1(91–112)0.049 HOMA-IR a2.64 ± 1.00(0.81–4.41)1.95 ± 0.9(0.54–3.82)0.127 TBARS (μmol·L–1) a0.28 ± 0.05(0.20–0.36)0.28 ± 0.05(0.22–0.37)0.999 Smoking0.465 Current smoker10Ex-smoker54Non-smoker67Alcohol consumption0.901 Almost every day98Occasional drinker33Medication–Antihypertensive agent32Oral anti-diabetes agent10Lipid-lowering agent01Type of a Job0.509 Researcher67Clerical worker64Short-bouts = three 10-min exercises d–1 on 3 d week–1; Long-bout = 30-min exercises d–1 on 3 d weeks–1.SBP: systolic blood pressure, DBP: diastolic blood pressure, PR: pulse rate, BW: body weight, BMI: body mass index, V·O2max: maximum oxygen uptake, TC: total cholesterol, HDL-C: high-density lipoprotein cholesterol, TG: triglyceride, LDL-C: low-density lipoprotein cholesterol, FPI: fasting plasma insulin, FPG: fasting plasma glucose, HOMA-IR: Homeostasis model assessment of insulin resistance, TBARS: thiobarbituric acid reactive substances. a: for analyses these continuous values were log-transformed. b: p for unpaired t-test or χ2 test., with the exception of FPG, there were no significant differences in age, blood pressure, heart rate, body weight, BMI, waist circumference, VO2max, lipid profile, FPI, HOMA-IR, or plasma TBARS between the groups at baseline. There were also no differences between the groups with respect to the subjects' smoking and drinking habits. Three subjects in the Short-bouts group and 2 subjects in the Long-bout group were taking antihypertensive medication, 1 in the Short-bouts group was using an oral anti-diabetes agent, and 1 in the Long-bout group was taking medication for hyperlipidemia (Table 1). An additional 2 subjects were taking medication for other diseases. Subjects did not alter their medications during the intervention. Thirteen of the subjects were researchers, and the others were clerical workers in the university; there was no significant difference in job type between the 2 groups (p=0.51). The percentages of the actual total exercise duration (of the 1,800 min prescribed) reported by subjects in the Long and Short-bouts groups over the entire 20 weeks were 68.9% (1,239 ± 670 min) and 55.5% (999 ± 528 min), respectively (p=0.35). BODY.RESULTS.GROUP DIFFERENCES: In the Long-bout group, waist circumference and VO2max significantly improved after 20 wk, whereas the DBP after 10 weeks showed a significant increase compared with the baseline value (Table 2Table 2.Changes in anthropometric measures, blood pressure, predicted maximum oxygen uptake, and glucose and lipid metabolism of subjects from pre- (baseline) to mid- (10 wk) and post- (20 wk) training for the long-bout and short-bouts groupsShort-bouts (n=12)Long-bout (n=11)TimeTime × Group10 wk-baselinepbES (d)20 wk-baselinepbES (d)10 wk-baselinepbES (d)20 wk-baselinepbES (d)pcES (f)pcES (f 2)Δ SBP (mm Hg)2.6 ± 7.10.2290.37 9.6 ± 10.50.0090.91 1 ± 7.90.6830.13 0.7 ± 11.30.8430.06 0.1090.50 0.1830.39 Δ DBP (mm Hg)3.2 ± 4.30.0240.74 8.1 ± 7.40.0231.09 2.1 ± 2.60.0230.81 1.8 ± 6.20.3680.29 0.0040.87 0.1110.44 Δ PR (bpm)–3.5 ± 11.80.3290.30 –5.7 ± 210.3720.27 –0.8 ± 7.80.7420.10 –1.4 ± 12.90.7170.11 0.6110.22 0.6740.19 Δ BW (kg)–0.15 ± 1.30.6980.12 –0.48 ± 2.860.570.17 –0.62 ± 1.280.140.48 –0.49 ± 1.710.3630.29 0.3960.31 0.5510.20 Δ BMI (kg·m–2)–0.05 ± 0.470.7190.11 –0.22 ± 1.030.4710.21 –0.28 ± 0.490.0880.57 –0.23 ± 0.710.3010.32 0.2970.36 0.3360.43 Δ Waist (cm) –0.66 ± 1.630.190.40 –1.31 ± 3.30.1960.40 –1.41 ± 2.250.0650.63 –2.19 ± 2.470.0150.89 0.0330.64 0.6720.20 Δ Ln V·O2max(mL·kg–1·min–1)a0.024 ± 0.0360.0420.67 0.065 ± 0.1180.0830.55 0.055 ± 0.0720.030.76 0.073 ± 0.0810.0140.90 0.0070.88 0.5580.00 ΔTC (mg·dL–1)–1.5 ± 19.60.7960.08 2.9 ± 18.30.5930.16 –0.3 ± 10.10.9310.03 1.2 ± 16.70.8190.07 0.7860.16 0.9420.08 Δ Ln HDL-C (mg·dL–1)a0.013 ± 0.1050.6790.12 0.083 ± 0.1180.0340.70 –0.044 ± 0.1030.1920.43 0.036 ± 0.0930.2280.39 0.0030.96 0.2910.00 Δ Ln TG (mg·dL–1)a–0.005 ± 0.2670.9520.02 –0.163 ± 0.4210.2080.39 –0.074 ± 0.4370.5830.17 –0.122 ± 0.2380.1190.51 0.2090.44 0.4240.16 ΔLDL-C (mg·dL–1)–2.4 ± 210.6980.11 4.1 ± 230.5460.18 6 ± 15.60.2290.38 3.3 ± 14.60.4770.23 0.6780.20 0.4120.29 Δ Ln FPI (μU·mL–1)a–0.079 ± 0.2530.3060.31 0.001 ± 0.3770.9950.00 0.082 ± 0.3910.5050.16 0.055 ± 0.3810.6430.01 0.9260.09 0.5020.18 Δ Ln FPG (mg·dL–1)a–0.044 ± 0.0860.1060.51 –0.007 ± 0.0650.7290.11 0.003 ± 0.0490.860.06 0.005 ± 0.0690.8310.07 0.4160.33 0.3650.31 Δ Ln HOMA-IRa–0.122 ± 0.2550.1260.48 –0.006 ± 0.3870.9590.02 0.084 ± 0.4110.5130.20 0.059 ± 0.4250.6530.14 0.8160.14 0.3220.34 Δ Ln TBARS (μmol·L–1) a–0.006 ± 0.090.820.07 –0.071 ± 0.1230.0710.58 –0.059 ± 0.1110.1120.53 –0.085 ± 0.1240.0470.69 0.0110.71 0.2080.30 Short-bouts = three 10-min exercises d–1 on 3 d week–1; Long-bout = 30-min exercises d–1 on 3 d weeks–1. SBP: systolic blood pressure, DBP: diastolic blood pressure, PR; pulse rate, BW: body weight, BMI: body mass index, V·O2max: maximum oxygen uptake, TC: total cholesterol, HDL-C: high-density lipoprotein cholesterol, TG: triglyceride, LDL-C: low-density lipoprotein cholesterol, FPI: fasting plasma insulin, FPG: fasting plasma glucose, HOMA-IR: Homeostasis model assessment of insulin resistance, TBARS: thiobarbituric acid reactive substances, ES: effect size (these values represent as absolute values). The thresholds for small, medium, and large effects were defined as d values below 0.20, 0.50, and 0.80; f values below 0.10, 0.25, and 0.40; and f 2 values below 0.02, 0.15, and 0.35, respectively. a: for analyses there values were log-transformed. b: p for paired t-test, versus baseline. c: p for MANOVA.). The Short-bouts group demonstrated significant increases in VO2max after 10 weeks and in HDL-C after 20 weeks, whereas the DBP after both 10 and 20 wk and the SBP after 20 wk had significantly increased (Table 2). The VO2max after 20 wk showed a trend toward an increase in the Short-bouts group, with a medium ES (d=0.55), but this change did not reach statistical significance (p=0.08). Physical fitness levels increased with both multiple short bouts (2.4 ml·kg−1·min−1, 7.0%) and a single long bout (2.7 ml·kg−1·min−1, 7.1%) of exercise. Plasma levels of TBARS significantly decreased after 20 wk in the Long-bout group (Table 2). A trend towards decreased plasma levels of TBARS with a medium ES (d=0.58) was observed after 20 wk in the Short-bouts group but did not reach statistical significance (p=0.07). As shown in Table 2, comparison [p (time × group)] between the long and short bouts of exercise showed no significant difference in the effects of the interventions on the overall variables. Moreover, the ES (f 2) values for VO2max, TC, and HDL-C were also below the medium level (0.15). Waist circumference, HDL-C, VO2max, and TBARS significantly improved in all subjects over the 20 wk, whereas diastolic blood pressure significantly increased [p (time) in Table 2]. BODY.DISCUSSION: This study highlights 2 major findings in sedentary male Japanese workers. First, the results of this study demonstrated no significant differences in cardiorespiratory fitness, anthropometry, or the lipid profile between accumulated short bouts of exercise and single long bouts of exercise even over a long-term intervention. Second, the subjects overall showed a significant reduction in oxidative stress during the exercise programs, and no significant difference in these effects was observed between the exercise programs. These findings suggest that accumulation of total exercise duration per week is more important than the length of each exercise session and thus provide an alternative for busy workers. BODY.DISCUSSION.EFFECT ON PHYSICAL FITNESS: High levels of physical fitness have been shown to reduce the risks of cardiovascular disease, diabetes mellitus, and some types of cancer in male Japanese workers26,27,28). Therefore, it is important for occupational health to promote physical fitness among workers to prevent in-office deaths and decreased productivity due to these diseases29,30,31). The present study demonstrated that physical fitness levels, as indicated by VO2max, increased in response to both short bouts (2.4 ml·kg−1·min−1, 7.0%, p=0.08, d=0.55 [a medium ES]) and long bouts (2.7 ml·kg−1·min−1, 7.1%, p=0.01, d=0.90 [a large ES]) of exercise. In a long-term intervention study by Jakicic, et al., physical fitness levels in overweight women increased by 6.3% and 9.9% after 72 wk of short-bouts or long-bout exercise, respectively, with no difference between the exercise groups7). Recently, Osei-Tutu et al., reported that physical fitness levels increased by 7.2% in the Short-bouts group and by 6.7% in the Long-bout group in studies conducted over 8 weeks in sedentary men10). The results of the present study investigating the improvements in physical fitness over 20 wk in sedentary male workers are consistent with those of these previous studies. On the other hand, a previous study reported that physical fitness in sedentary healthy men who jogged for 30 min/d on 5 days per week for 8 wk increased by 7.6% in a Short-bouts group and 13.9% in a Long-bout group, a significantly greater increase in the Long-bout group6). Murphy et al., have shown improvements in physical fitness in response to either 3 short bouts (14.2%) or 1 long bout (3.8%) of brisk walking for 10 weeks in sedentary women, but this increase was greater in the group walking for short bouts8). These differences may be due to the exercise intensities used in each study. For example, the exercise intensities (>70% maximum heart rate) in the studies cited above were higher than the intensity used in the current study (50% of maximal oxygen consumption, ≤70% maximum heart rate). Further study is needed to clarify the effects of the mode and intensity of exercise on physical fitness. BODY.DISCUSSION.EFFECTS ON FAT DISTRIBUTION, LIPID PROFILE, AND OTHER CARDIOVASCULAR RISK FACTORS: The Long-bout group demonstrated a significant decrease in waist circumference after 20 wk compared with the baseline value, but the decrease in waist circumference in the Short-bouts group showed a small ES (d=0.40) and did not reach statistical significance. BMI was unaltered in both groups. The decreases in waist circumference in the Long-bout group could indicate a potential alteration in the distribution of body fat away from the waist, which may lower the risk of cardiovascular disease. An interventional study by Osei-Tutu et al., also reported that only long-bout walking produced significant reductions in the percentage of body fat10). However, there are some reports that exercise patterns do not influence the magnitudes of anthropometric changes7,8,9). These inconsistencies may be attributed to population differences in parameters such as the pre-intervention waist circumference. The Short-bouts group in the current study demonstrated a significant increase in the HDL-C level after 20 wk, but the HDL-C level in the Long-bout group showed a small ES (d=0.39) and was not significantly different. Increases in HDL-C may reduce cardiovascular risk. However, findings from other studies vary regarding the effects of brisk walking (whether accumulated through short bouts or through longer sessions) on serum lipid levels. While some studies report changes in response to both patterns of exercise9), others do not32). These inconsistencies may also be attributed to population differences in parameters such as the pre-intervention lipid profile12). No changes in other cardiovascular risk factors (TC, TG, LDL-C, or glucose metabolism) were observed in the 2 exercise groups in this study. Eriksen and colleagues have reported that short bouts of exercise are preferable to a single continuous exercise period with regard to their effects on glucose metabolism32), but the intensity of exercise in that study (60% to 65% of maximal oxygen consumption) was higher than that used in the current study, suggesting that this difference in exercise intensity might explain the inconsistencies. BODY.DISCUSSION.EFFECT ON OXIDATIVE STRESS: The current study showed almost identical reductions in plasma levels of TBARS between the exercise groups over the 20-wk study period, based on the findings of no significant difference in the effects of the interventions when the long and short bouts of exercise were compared and on significant improvements in plasma levels of TBARS in all subjects over the 20 wk. The plasma levels of TBARS after 20 wk tended to be lower in the short-bouts group; although this trend did not reach statistical significance (p=0.07), the ES was medium (d=0.58), suggesting possible practical implications. Despite other studies that have reported the efficacy of 1 continuous exercise period33, 34), to our knowledge, no other studies have examined whether the total accumulated exercise duration could suppress oxidative stress. Oxidative stress is closely associated with the pathogenesis and the progression of cardiovascular risk factors13,14,15,16). Thus, from the perspective of occupational health for busy workers, it is highly significant that the total accumulated exercise duration may limit cardiovascular risk through the reduction of oxidative stress. BODY.DISCUSSION.STRENGTH AND LIMITATIONS: This study has several strengths. First, this was a randomized controlled study. Second, the subjects of this study were middle-aged male Japanese workers. There have been few studies that focused on the comparison between short and long bouts of exercise using data from Japanese workers. Third, the exercise programs used in this study were tailor-made exercise programs of moderate intensity, which was measured objectively by the cycle ergometer. However, this study also has several limitations. First, this study did not have a control group despite the random assignment of the interventional groups. The aim of this study was to compare the effects of short-bout and long-bout exercise on cardiovascular risk factors, and a single continuous exercise session lasting 30 min is generally recognized as a non-pharmacologic therapy for lifestyle-related diseases. Therefore, in this study, the Long-bout group was considered the control group. Second, in the Short-bouts group, a significant increase in blood pressure in comparison with baseline was observed after 20 wk. This interventional study began in June, and the laboratory follow-ups were held in September (after 10 wk) and December (after 20 wk). Blood pressure is known to vary with the time of year, and the odds ratio for a systolic blood pressure ≥ 120 mm Hg is 1.24 for winter versus summer months35). Such seasonal changes in blood pressure may explain the unexpected increase associated with short bouts of exercise. However, the blood pressure levels in the Long-bout group were unchanged, suggesting a suppressive effect of continuous exercise on the seasonal increase in blood pressure. Third, this study is limited by the relatively small sample size. A larger sample would be necessary to establish full equivalence between the effects of 3 short bouts of exercise and 1 longer bout of exercise36, 37). Moreover, all of the ES (f 2) values in Table 2 except those for VO2max, TC, and HDL-C were either large or medium. Therefore, based on the results of the current study, exercise in short bouts cannot be deemed as effective as long-bout exercise. Finally, the rates of adherence to the prescribed exercise programs were 60% to 70%, but there was no dropout. Low exercise compliance may explain the small effects of exercise on cardiovascular risk factors, including the smaller-than-expected improvements in lipid profiles and glucose metabolism. Moreover, the adherence rate in the Short-bouts group was unexpectedly lower than that in the Long-bout group, although the difference was not significant. Because the participants in this study included researchers from the university who make frequent business trips, it may have been difficult to practically implement the short-bouts exercise program using a fixed cycle ergometer. These results also imply that occupational practitioners should match the exercise program, either short or long bouts of exercise, to the participant's work-style. BODY.CONCLUSIONS: We have demonstrated in this study that it is the performance of an exercise program itself rather than the exact nature of the program that is effective for improving physical fitness, the lipid profile, and oxidative stress and that short-bout exercise protocols are an option, especially for busy workers, for incorporating exercise into one's lifestyle. A larger-scale randomized controlled test should be planned to determine the equivalence between the effects of 3 short bouts of exercise versus 1 longer bout of exercise.

TITLE: Randomized sham controlled trial of cranial microcurrent stimulation for symptoms of depression, anxiety, pain, fatigue and sleep disturbances in women receiving chemotherapy for early-stage breast cancer ABSTRACT.PURPOSE: Women with breast cancer may experience symptoms of depression, anxiety, pain, fatigue and sleep disturbances during chemotherapy. However, there are few modalities that address multiple, commonly occurring symptoms that may occur in individuals receiving cancer treatment. Cranial electrical stimulation (CES) is a treatment that is FDA cleared for depression, anxiety and insomnia. CES is applied via electrodes placed on the ear that deliver pulsed, low amplitude electrical current to the head. ABSTRACT.METHODS: This phase III randomized, sham-controlled study aimed to examine the effects of cranial microcurrent stimulation on symptoms of depression, anxiety, pain, fatigue, and sleep disturbances in women receiving chemotherapy for early-stage breast cancer. Patients were randomly assigned to either an actual or sham device and used the device daily for 1 h. The study was registered at clinicaltrials.gov, NCT00902330. ABSTRACT.RESULTS: The sample included N = 167 women with early-stage breast cancer. Symptom severity of depression, anxiety, and fatigue and sleep disturbances were generally mild to moderate. Levels of pain were low. Anxiety was highest prior to the initial chemotherapy and decreased over time. The primary outcome assessment (symptoms of depression, anxiety, fatigue, pain, sleep disturbances) revealed no statistically significant differences between the two groups, actual CES vs. sham. ABSTRACT.CONCLUSION: In this study, women receiving chemotherapy for breast cancer experienced multiple symptoms in the mild to moderate range. Although there is no evidence for the routine use of CES during the chemotherapy period for symptom management in women with breast cancer, further symptom management modalities should be evaluated to mitigate symptoms of depression, anxiety, fatigue, pain and sleep disturbances over the course of chemotherapy. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40064-015-1151-z) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: The American Cancer Society in 2015 projected that in the United States, there will be 231,840 women diagnosed with breast cancer (Siegel et al. 2015). Most women will be diagnosed in the early stages of the disease (Stage I and II) and 90% of these individuals can expect to survive at least 5 years due to improvements in adjuvant chemotherapy and targeted hormonal therapies (Siegel et al. 2012). However, cancer treatments, and, perhaps the cancer itself, contribute to a number of distressing symptoms. In particular, the administration of systemic chemotherapy is associated with multiple, co-occurring distressing symptoms (Dodd et al. 2010; Goedendorp et al. 2008), which include depressive symptoms (Badger et al. 2007), anxiety (Badger et al. 2007), fatigue (Berger et al. 1997), and pain (Utne et al. 2010; Valeberg et al. 2008). In women with breast cancer, anxiety and sleep disturbances (Lee et al. 2004) are also common during the adjuvant chemotherapy treatment phase. These symptoms, both individually and collectively, are strongly associated with decreased quality of life. In addition to the distress related to these common symptoms, fatigue, depression, and physical complaints are associated with poor employment outcomes for breast cancer (Hansen et al. 2008). Because symptoms tend to occur together and may have synergistic negative effects on quality of life, there is a movement towards examining "clusters" of co-occurring symptoms in persons with cancer and for testing interventions that might be effective for ameliorating more than one symptom (Dodd et al. 2001). Pain, depression, and fatigue have been identified as components of a notable cluster that may also include anxiety and sleep disturbances. Collectively, these symptoms can be described as "psychoneurological symptoms" (PNS) (Lyon et al. 2013). PNS may result in a significant decline in quality of life by contributing to adverse health outcomes over the active treatment period and into survivorship. To date, conventional therapies have not been effective for treating multiple concurrent symptoms. Given the limitations of conventional modalities for symptom management, many breast cancer patients report using a complementary or alternative medicine (CAM) modality in conjunction with conventional cancer treatment (Saghatchian et al. 2014). CAM is defined as "a group of diverse medical and health care systems, practices, and products that are not presently considered to be part of conventional medicine" (National Cancer Institute website). The most common reason given for using CAM modalities in individuals with cancer is the belief that CAM will assist with relieving pain and controlling side effects related to disease or treatment (Mansky and Wallerstedt 2006). Cranial electrical microcurrent stimulation (CES) is a non-invasive modality that falls under the category described by the National Center for Complementary and Alternative Medicine (NCCAM) as "Veritable Energy Medicine." The putative mechanism of CES is not completely understood; however, there are several interrelated theories of the mechanism of action of applied "energy" modalities. Both high and low intensity energy modalities are thought to initiate neuromodulation. High intensity energy modalities such as repetitive transcranial magnetic stimulation (rTMS) have now entered main stream psychiatric and neurologic practice. The mechanism for rTMS is thought to include the activation or inhibition of cortical activity depending on stimulation parameters. In contrast, the mechanism of action for low-intensity AC stimulation is less clear. CES is a form of alternating current (AC) stimulation that involves the application of current to infra- or supra-auricular structures (e.g., the ear lobes). Evidence from EEG suggests that CES leads to changes in alpha and beta frequency ranges, indicating potential neuroplastic and cognitive effects. Schroeder and Barr (2001) measured EEG activity during sham and AC stimulation and showed increases in low alpha (8–12 Hz) and high theta (3–8 Hz) activity (Schroeder and Barr 2001). A second hypothesis for the effects of CES effects of cranial AC stimulation is that CES has a primary effect on the peripheral nervous system that is secondarily transmitted to the central nervous system (Zaghi et al. 2009). CES may render its effects not by polarizing brain tissue, but rather via rhythmic stimulation that synchronizes and enhances the efficacy of endogenous neurophysiologic activity (Zaghi et al. 2009). Additionally, increases in blood and cerebrospinal fluid levels of specific neurotransmitters, including serotonin, norepinephrine, dopamine, and β-endorphin have been reported when CES was used for both 1 and 2 weeks (Shealy et al. 1998). Although CES has been used as a modality for treating symptoms of depression, anxiety and insomnia in multiple studies over many years, results have not yet reached mainstream acceptance, possibly due to the lack of rigorously designed randomized clinical trials. There are many studies that have found positive effects of CES (Klawansky et al. 1995); however, most studies have not been methodologically rigorous. Most have been open trials, have had relatively small samples, and few have had designs that have included sham devices (Mindes et al. 2015). Yet, CES has several advantages over most current symptom management strategies and other CAM modalities that make its consideration worthwhile. As a self-administered, relatively low-cost, and portable modality, CES can be used in the home without disrupting daily routines already taxed by the demands of cancer treatments. Given the importance of examining innovative symptom management modalities in oncology practice for multiple, concurrent symptoms, the primary aim of this study was to examine the effects of CES on symptoms of depression, anxiety, pain, fatigue and sleep disturbances in women receiving chemotherapy for early-stage breast cancer. BODY.METHODS.STUDY DESIGN: The study was conducted in accordance with the Declaration of Helsinki and was approved by the Virginia Commonwealth University Institutional Review Board. Women with newly diagnosed stage I–IIIA breast cancer, scheduled to receive at least four cycles of adjuvant or neoadjuvant chemotherapy were referred by oncologists in the Massey Cancer Center and its affiliate sites from June 2009 through December 2012. Women were included in the sample if they had a diagnosis of stages I–IIIA breast cancer; a performance score <2 using the Eastern Cooperative Oncology Group criteria; and were scheduled to receive at least four cycles of adjuvant or neoadjuvant chemotherapy. Women were excluded from the study if they had: (1) previous chemotherapy; (2) dementia; (3) active psychosis; (4) history of seizure disorder; (5) any implanted electrical device, or (6) began or changed a medication regimen for depression or other psychiatric condition within 30 days prior to study enrollment. After informed consent was obtained, participants' medical records were abstracted for medical history and current medication use. The timing of the baseline data collection varied depending on patients' start day of chemotherapy, but was always within 48 h of the initial chemotherapy. During the study, each participant was required to use the CES device daily for 1 h until 2 weeks after chemotherapy cessation. Symptom data were collected weekly. All participants completed a log to record CES use and the number of times that the device was used was assessed weekly. The time to complete all questionnaires at each time point was approximately 30 min. All data were collected either at the Massey Cancer Center and its affiliate sites (the initial, midpoint and post-chemotherapy data were collected in person) or via a weekly telephone call with study personnel. BODY.METHODS.INTERVENTION: The researchers conducted th is study using an Alpha-Stim cranial electrotherapy stimulator. Both the active and sham devices were provided on loan by Electromedical Products International, (2201 Garrett Morris Parkway, Mineral Wells, TX 76067-9034). The CES unit (Alpha-Stim® 100 Microcurrent Stimulator) passes microcurrent levels of biphasic electrical stimulation via ear-lobe electrodes. The CES unit was preset to provide 1 h of 100 μA (sub-sensory level), modified square-wave biphasic stimulation on a 50% duty cycle at .5 Hz, and to automatically turn off at the end of 1 h. CES devices were pre-set at the factory to provide a maximum of 60 min of modified square-wave biphasic stimulation at 0.5 Hz and 100 μA, the lowest setting below the level of perception. Sham devices were identical; however, the electrodes did not transmit a current. Because the devices were pre-set at the factory, participants were unable to change the settings. The devices were dispensed by the investigation pharmacy. Participants were instructed to use the device for 1 h each daily. BODY.METHODS.POWER ANALYSIS: Data from our pilot, feasibility study (Lyon et al. 2010) was used to estimate mean values, correlations, and model-fitting mean square errors for power computations. Power calculations were computed using SASv9.1. The sample size for the proposed study was 166 participants, from which we assumed a 10% attrition rate, similar to the attrition rate in our preliminary study, yielding a final sample size of 150. Power analysis was performed for repeated measures assuming an overall a = 0.05 and a power of 90%. A Bonferroni correction was made to adjust for multiplicity resulting from five dependent variables, thus reducing each symptom alpha level to 0.05/5 = 0.01. BODY.METHODS.RANDOMIZATION: Participants were randomized using a computer-generated random number sequence with a block size of four. Randomization was stratified based on two treatment regimen groups: those who received chemotherapy every 2 weeks and those who received chemotherapy every 3 weeks. Treatment assignments were blinded to the study investigators and patients. BODY.METHODS.MEASURES: In addition to a demographic questionnaire that included details about the stage and characteristics of the breast cancer diagnoses, comorbidities and life-style habits, several validated symptom measures were used to collect symptom data. The Hospital Anxiety and Depression Scale (HADS) is a 14-item self-report questionnaire developed to detect the presence and severity of both anxiety and depressive symptoms at the time of reporting. Participants rate (0–3) the severity of each symptom over a 7-day period. The possible score on the subscales is 0–21 for depression or anxiety and a possible total scale score of 0–42. The HADS has well-established reliability and validity for both depression and anxiety in women with breast cancer (Zigmond and Snaith 1983). The brief pain inventory (BPI) short-form is a pain assessment tool that has well-established reliability and validity for adult patients with no cognitive impairment in trajectory studies of cancer and its symptoms. The BPI assesses the severity of pain, location of pain, pain medications, amount of pain relief in the past 24 h or the past week, and the impact of pain on daily functions. The "Usual pain" item was used to measure pain severity over time in this study (Cleeland and Ryan 1994). The brief fatigue inventory (BFI) is a 9-item scale that taps into a single dimension of fatigue severity and the interference fatigue creates in daily life. The BFI is a clinically validated tool used to assess cancer-related fatigue and its impact on daily functioning. The "Usual fatigue" item was used to measure severity of fatigue over time (Mendoza et al. 1999). The General Sleep Disturbance Scale (GSDS) is a 21-item scale that consists of 21 items that evaluate various aspects of sleep disturbance (quality and quantity of sleep, sleep onset latency, number of awakenings, excessive daytime sleepiness, and medication use) over the past week (Lee 1992). Items are rated on a scale ranging from 0 (never) to 7 (every day). The 21 items are summed to produce a total score with a possible range from 0 (no sleep disturbance) to 147 (extreme sleep disturbance). Adverse events (AEs) were assessed over the phone at week weekly and in person at the mid-point chemotherapy data collection and at the final data collection two weeks after completion of chemotherapy by asking the participants the open-ended question: How are you feeling? AEs were reported by the investigator regardless of whether they were deemed to be related to the treatment. AEs were graded using the most current version of the National Cancer Institute Current Toxicity Criteria. BODY.METHODS.STATISTICAL ANALYSIS: Descriptive statistics were used to describe participants' clinical and demographic characteristics, and the level of symptoms at each of the study time points. A longitudinal repeated measures model was used to compare the effects of the CES intervention group to the sham group over the chemotherapy treatment period until final data collection two weeks after the final chemotherapy. Data of all participants was entered into a secure study database. All participants with any post-baseline data were included in the final analyses on a per protocol basis. The primary endpoints of levels of depressive symptoms, anxiety, pain, fatigue and sleep disturbances were analyzed using a longitudinal repeated measures model which included one between subjects variable (Group: CES or Sham), one within subject factor (visit period: baseline period, treatment period, post-treatment period) and the interaction between group and visit. Participants with In addition to these factors, the model included covariates for age (years), menopausal status (pre- or post-menopausal) and body mass index (kg/m2). We tested a variety of variance–covariance structures including compound-symmetry, AR(1) and unstructured; the structure that fit the data and provided the most parsimonious model was used. The outcome variables for the baseline period were constructed from either 1 or 2 observations (number of observations M = 1.02, SD = 0.14) averaged for each subject, while the outcome variables for the treatment period were constructed from the mean of 2–11 (number of observations M = 5.91, SD = 1.62) observations per subject and the outcome variables for the post-treatment period were constructed from the mean of 1–22 (number of observations M = 9.99, SD = 4.53) observations per subject. BODY.RESULTS: A total of 167 women were consented and enrolled to participate in this study. A flow diagram (Additional file 1: Fig. S1) details the progress of the prospective and actual participants through the trial. BODY.RESULTS.SAMPLE CHARACTERISTICS: The mean age of participants was 51 ± 0.78 years. Table 1 describes demographic characteristics of participants and the differences between groups. Other clinical characteristics such as hormone status and treatments (type, timing, and duration of chemotherapy) are reported in Table 2. The majority of women (88.3%) had infiltrating ductal carcinoma and were diagnosed as having stage II (61.4%) breast cancer in accordance with criteria set by the American Joint Committee on Cancer and most participants (53.4%) had grade 3 tumors. The majority of participants were white (61.7%), married (57.7%), had greater than a high school education (82.0%), and were currently non-smokers (80.1%). Group equivalence differences in the active group and sham group for demographic characteristics and clinical factors were examined by conducting t-tests and Chi square analyses and there were no significant differences between groups.Table 1Demographic characteristicsSham (N = 81)Active (N = 82)Total (N = 163)P-valueAge (mean ± standard error)51.91 ± 0.9751.04 ± 1.2151.47 ± 0.780.57*BMI (mean ± standard error)30.61 ± 0.8730.74 ± 0.9930.68 ± 0.660.92*Leisure Activity Score (mean ± standard error)25.30 ± 2.9922.44 ± 2.5623.86 ± 1.960.47*Life Style Scores (mean ± standard error)2.82 ± 0.052.85 ± 0.042.84 ± 0.030.66*Race (count (%)) White53 (65.43%)48 (58.54%)101 (61.96%)0.37** Other28 (34.57%)34 (41.46%)62 (38.04%)Education (count (%)) ≤HS17 (21.52%)12 (14.63%)29 (18.01%)0.26** >HS62 (78.48%)70 (85.37%)132 (81.99%)Marital status (count (%)) Currently married46 (56.79%)48 (58.54%)94 (57.67%)0.82** Not currently married35 (43.21%)34 (41.46%)69 (42.33%)Employment FT44 (55.00%)39 (47.56%)83 (51.23%)0.34** Not FT36 (45.00%)43 (52.44%)79 (48.77%)Menopausal status (count (%)) Pre30 (37.04%)40 (48.78%)70 (42.94%)0.13** Post51 (62.96%)42 (51.22%)93 (57.06%)Smoking status (count (%)) Yes20 (24.69%)11 (13.41%)31 (19.02%)0.07** No61 (75.31%)71 (86.59%)132 (80.98%)Alcohol use (count (%)) Yes41 (50.62%)43 (52.44%)84 (51.53%)0.82** No40 (49.38%)39 (47.56%)79 (48.47%)* t-test.** Chi-square test.Table 2Clinical characteristicsSham (N = 81)Active (N = 82)Total (N = 163)P-valueSurgery (count (%)) Mastectomy25 (31.25%)29 (35.80%)54 (33.54%)0.28* Minimal surgery29 (36.25%)20 (24.69%)49 (30.43%) Other26 (32.50%)32 (39.51%)58 (36.02%)Histology (count (%)) IDC76 (93.83%)68 (82.93%)144 (88.34%)0.03** Other5 (6.17%)14 (17.07%)19 (11.66%)Grade (count (%)) 15 (6.17%)6 (7.32%)11 (6.75%)0.95* 232 (39.51%)33 (40.24%)65 (39.88%) 344 (54.32%)43 (52.44%)87 (53.37%)Stage (count (%)) I21 (25.93%)16 (19.51%)37 (22.70%)0.25* IIA31 (38.27%)27 (32.93%)58 (35.58%) IIB15 (18.52%)27 (32.93%)42 (25.77%) IIIA13 (16.05%)12 (14.63%)25 (15.34%) IIIB1 (1.23%)0 (0.00%)1 (0.61%)Chemotherapy (count (%)) AC4 (4.94%)7 (8.75%)11 (6.83%)0.49* AC followed by Taxane35 (43.21%)29 (36.25%)64 (39.75%) CMF1 (1.23%)0 (0.00%)1 (0.62%) TC31 (38.27%)37 (46.25%)68 (42.24%) Other10 (12.35%)7 (8.75%)17 (10.56%)Neoadjuvant Yes26 (32.00%)23 (28.00%)49 (30.10%)0.50** No55 (68.00%)59 (72.00%)114 (69.90%)Duration of chemotherapy (weeks)15.93 ± 5.3716.47 ± 5.4216.20 ± 5.390.54*Lymphnodes (count (%)) Yes35 (44.30%)44 (53.66%)79 (49.07%)0.24** No44 (55.70%)38 (46.34%)82 (50.93%)Hormonal therapy (count (%)) Yes40 (51.95%)48 (59.26%)88 (55.70%)0.36** No37 (48.05%)33 (40.74%)70 (44.30%)Estrogen receptor (count (%)) Yes42 (51.85%)48 (58.54%)90 (55.21%)0.39** No39 (48.15%)34 (41.46%)73 (44.79%)HER2 NeuExpression (count (%)) Yes15 (18.52%)24 (29.27%)39 (23.93%)0.11** No66 (81.48%)58 (70.73%)124 (76.07%)Progesterone receptor (count (%)) Yes34 (41.98%)33 (40.24%)67 (41.10%)0.82** No47 (58.02%)49 (59.76%)96 (58.90%)LongActingOpiods (count (%)) Yes16 (19.75%)19 (23.17%)35 (21.47%)0.60** No65 (80.25%)63 (76.83%)128 (78.53%)NonSteroidal analgesics (count (%)) Yes20 (24.69%)10 (12.20%)30 (18.40%)0.04** No61 (75.31%)72 (87.80%)133 (81.60%)Anxiolytic (count (%)) Yes24 (29.63%)17 (20.73%)41 (25.15%)0.19** No57 (70.37%)65 (79.27%)122 (74.85%)* t-test.** Chi-square test. BODY.RESULTS.LEVELS OF PSYCHONEUROLOGIC SYMPTOMS OVER TIME: Women in both groups reported having multiple concurrent symptoms across all time points, however, the level of symptoms were relatively low. Anxiety levels were highest at baseline and decreased over time while depressive and fatigue symptoms increased over time. Levels of pain and sleep disturbances fluctuated slightly across time; however they remained relatively stable throughout treatment and after chemotherapy cessation. Fatigue scores were lowest at baseline and trended upward overtime with highest scores at time point three after chemotherapy treatment had ended. Table 3 demonstrates the values of PNS over time for each group and differences between the CES and sham groups.Table 3Outcome measures by time point (Mean (SD))MeasureGroupTime point 1Time point 2Time point 3AnxietyTotal7.34 (4.11)4.69 (3.46)4.29 (3.78)CES7.09 (4.09)4.40 (3.19)4.07 (3.51)Sham7.59 (4.13)4.98 (3.72)4.51 (4.04)DepressionTotal3.04 (2.62)4.13 (3.17)4.55 (3.51)CES3.03 (2.48)4.24 (3.22)4.47 (3.36)Sham3.06 (2.78)4.02 (3.14)4.63 (3.67)FatigueTotal2.29 (2.81)3.14 (2.30)3.33 (2.50)CES1.95 (2.71)3.15 (2.21)3.34 (2.47)Sham2.63 (2.89)3.14 (2.39)3.32 (2.55)PainTotal1.35 (2.01)1.17 (1.50)1.23 (1.72)CES1.24 (2.05)1.25 (1.44)1.14 (1.65)Sham1.45 (1.98)1.09 (1.56)1.32 (1.80)SleepTotal40.50 (24.28)40.55 (20.08)39.70 (20.66)CES40.70 (24.83)38.70 (18.28)38.50 (20.19)Sham40.30 (23.87)42.44 (21.73)40.91 (21.21) BODY.RESULTS.GROUP DIFFERENCES IN PRIMARY OUTCOME MEASURES: There were no statistically significant group differences in levels of depression, anxiety, pain, fatigue and sleep disturbance (Table 4) at any of the study measurement points (pre-chemotherapy, mid-chemotherapy, and/or two weeks after completion of chemotherapy). There was also no time by group interaction.Table 4Analysis of primary outcome variablesEffectPrimary outcome variablesAnxietyDepressionFatiguePainSleep disturbancedfFp-valuedfFp-valuedfFp-valuedfFp-valuedfFp-valueGroup1,1470.770.381,1470.040.841,1470.020.881,1460.000.101,1470.130.72Visit2,14455.97<0.012,14517.11<0.012,14312.77<0.012,1431.140.342,1430.060.94G × V2,1440.010.992,1450.610.552,1431.500.232,1432.550.082,1430.980.38Age1,14712.78<0.011,1474.260.041,14715.340.011,1473.460.071,14718.36<0.01Menopausal status1,1472.680.101,1472.580.111,14716.06<0.011,1473.780.051,14712.48<0.01BMI1,1470.050.821,1470.090.761,1470.580.461,1473.870.051,1470.500.48 BODY.RESULTS.ADVERSE EVENTS: One participant in the actual CES group had a seizure on a day when she did not use the device. The AE was considered to be unrelated to her usage of the CES device. She was removed from the study after the AE occurred. BODY.DISCUSSION: This double-blind, sham-controlled clinical trial did not detect any difference between the CES and sham CES for reducing psychoneurologic symptoms in women with early-stage breast cancer receiving chemotherapy. The low level of symptoms contrasts with prior studies such as a cohort study of 222 women with early breast cancer which found a point prevalence of depression, anxiety, or both (including borderline cases) was 33% at diagnosis (Burgess et al. 2005). In a study of 94 women with early-stage breast cancer that used Paroxetine for decreasing levels of depression and fatigue, 26 (28%) patients were significantly depressed at baseline, using a CES-D score of 19 or greater to indicate depression (Roscoe et al. 2005). Given that levels of symptoms were generally lower than anticipated it is possible that a floor effect mitigated potential benefits of CES in this sample. This contrasts with a double-blinded sham controlled trial of 115 participants with a primary diagnosis of an anxiety disorder, found significant positive effects of CES vs. sham for reducing symptoms of anxiety (p = 0.001, d = 0.94) and depression (p = 0.001, d = 0.78) from baseline to endpoint of the study (Barclay and Barclay 2014). In this study of CES in women with breast cancer, we found that while most symptoms were in the mild range, the symptoms had different trajectories over time. Anxiety levels were highest at baseline, while depressive symptoms increased over time. These levels of similar to a recent longitudinal study of women with early-stage breast cancer that also found that pre-chemotherapy anxiety scores were significantly (p < 0.05) worse at baseline than cycle 4 day 1, whereas depression scores were significantly worse during treatment than at pre-chemotherapy (p < 0.05) (Sanford et al. 2010; Ancoli-Israel et al. 2014) newly diagnosed breast cancer found that depressive symptoms increased after 4 weeks of chemotherapy (Rissling et al. 2011). Levels of fatigue and pain were low at baseline and fatigue increased over time. This finding is consistent with other studies which have reported that fatigue increased in prevalence, severity, and disruptiveness after the start of treatment (Jacobsen et al. 1999). Levels of sleep disturbances were consistent over time, comparable to women in third semester of pregnancy (Lee and Gay 2004). This finding is similar to a recent studies of women with early stage breast cancer that found higher levels of sleep disturbances prior to chemotherapy, at mid-cycle and 6 months after initiating chemotherapy after but little variation in levels over time (Sanford et al. 2013). The utility of CES was not supported in this study. A recent meta-analysis concluded that the effects of CES over sham did not demonstrate statistical significance in reported studies of patients with pain (SMD −0.24, 95% CI −0.48 to 0.01, P = 0.06). The authors concluded that due to this uncertainty, any clinical application of this modality would be most appropriate within a clinical research setting rather than in routine clinical care (O'Connell 2014). Although CES was not effective in this study for reducing symptoms, it is noteworthy that few trials have rigorously examined the use of any modality for reducing symptoms during the chemotherapy treatment period in cancer patients. Under-treatment of symptoms including depression persists in individuals with cancer. A recent study found that one-fourth of the patients had multiple clinically significant symptoms, whereas only 22.5% were free of any clinically significant symptoms (Reece et al. 2013). Despite the widespread use of CAM in persons with cancer, there have been few double-blinded randomized trials to examine the safety, feasibility and efficacy of CAM modalities. BODY.DISCUSSION.LIMITATIONS: Several limitations should be considered. In this trial, we used a "pragmatic" approach, enrolling women with early-stage breast cancer prior to chemotherapy, regardless of their current or anticipated risk for psychoneurologic symptoms (Thorpe et al. 2009). Although this approach led to the recruitment of a racially diverse sample of participants and a high acceptance rate for the study, it did not, by design, target participants with a higher risk for symptoms. Due to the low severity of symptoms, a possible floor effect may have evident, whereby symptoms did not reach the threshold for warranting intervention. In the future, setting a symptom threshold for inclusion criteria should be considered. Secondly, we had participants with differences in the composition of chemotherapy, the number of cycles or components of the adjuvant medication such as anti-nausea or anxiolytic medications. In addition, the use of medications begun after the initiation of chemotherapy was not controlled. Thirdly, in a trial of this length, because CES was used from the initiation to the completion of chemotherapy, there were many different parameters of usage. Some participants used CES for as few as 6 weeks and some for as many as 32 weeks (depending on their type and schedule of chemotherapy). Although there was no statistically significant difference in number of weeks by group assignment, this wide variability is potentially problematic. In addition, the dose for CES was set at the lowest intensity (sub-sensory) so that the sham could be tested. It could be that the dose intensity was not adequate for this population. Alternatively, it could be that a "nocebo" response was induced by the lack of appreciable stimulation delivered by the devices, both actual and sham. Several other potential methodological weaknesses are noted. The question used to monitor adverse events was general, and not specifically focused on side-effects related to CES. However, in the context of cancer treatment, general questions, potentially identifying serious adverse events is expected from researchers conducting clinical trials in patients undergoing active cancer treatment. Adherence to CES was also self-reported: future trials could consider a more robust technological method, such as a counter on the device, to measure adherence. In addition, we cannot exclude the possibility of a placebo effect in participants in both groups resulting from the contact with study personnel. Study personnel communicated with the study participants weekly: this communication could have been an "intervention" by itself. BODY.CONCLUSION: Further testing of CES in individuals with cancer who meet a higher cut-off for levels of symptoms may warrant consideration in the future. Although the level of symptoms in women in this sample of women with early stage breast cancer was relatively mild to moderate, the need for tailored symptom management strategies persists for cancer patients who are in different stages or treatment or who may have elevated psychoneurologic symptoms in the survivorship period. However, there is not yet evidence for the utility of CES for routine use in symptom management regimens for women with early-stage breast cancer during chemotherapy.

TITLE: Glycaemic control and hypoglycaemia with new insulin glargine 300 U/ml versus insulin glargine 100 U/ml in people with type 2 diabetes using basal insulin and oral antihyperglycaemic drugs: the EDITION 2 randomized 12‐month trial including 6‐month extension ABSTRACT.AIMS: To compare the efficacy and safety of new insulin glargine 300 U/ml (Gla‐300) with insulin glargine 100 U/ml (Gla‐100) over 12 months of treatment in people with type 2 diabetes using basal insulin and oral antihyperglycaemic drugs (OADs). ABSTRACT.METHODS: EDITION 2 (NCT01499095) was a randomized, 6‐month, multicentre, open‐label, two‐arm, phase IIIa study investigating once‐daily Gla‐300 versus Gla‐100, plus OADs (excluding sulphonylureas), with a 6‐month safety extension. ABSTRACT.RESULTS: Similar numbers of participants in each group completed 12 months of treatment [Gla‐300, 315 participants (78%); Gla‐100, 314 participants (77%)]. The reduction in glycated haemoglobin was maintained for 12 months with both treatments: least squares (LS) mean (standard error) change from baseline −0.55 (0.06)% for Gla‐300 and −0.50 (0.06)% for Gla‐100; LS mean difference −0.06 [95% confidence interval (CI) −0.22 to 0.10)%]. A significant relative reduction of 37% in the annualized rate of nocturnal confirmed [≤3.9 mmol/l (≤70 mg/dl)] or severe hypoglycaemia was observed with Gla‐300 compared with Gla‐100: rate ratio 0.63 [(95% CI 0.42–0.96); p = 0.031], and fewer participants experienced ≥1 event [relative risk 0.84 (95% CI 0.71–0.99)]. Severe hypoglycaemia was infrequent. Weight gain was significantly lower with Gla‐300 than Gla‐100 [LS mean difference −0.7 (95% CI −1.3 to −0.2) kg; p = 0.009]. Both treatments were well tolerated with a similar pattern of adverse events (incidence of 69 and 60% in the Gla‐300 and Gla‐100 groups). ABSTRACT.CONCLUSIONS: In people with type 2 diabetes treated with Gla‐300 or Gla‐100, and non‐sulphonylurea OADs, glycaemic control was sustained over 12 months, with less nocturnal hypoglycaemia in the Gla‐300 group. BODY.INTRODUCTION: New insulin glargine 300 U/ml (Gla‐300) has a more constant and prolonged pharmacokinetic (PK) and pharmacodynamic (PD) profile compared with insulin glargine 100 U/ml (Gla‐100) 1. The longer duration of action of Gla‐300 could provide effective 24‐h glycaemic control with once‐daily dosing, while allowing flexibility in injection time. In addition, the more even PK/PD profile may reduce the risk of hypoglycaemia, a key barrier to initiation and intensification of insulin therapy 2. To investigate treatment outcomes with Gla‐300, a programme of clinical studies (the EDITION programme) was conducted in people with type 1 or type 2 diabetes. Similar glycaemic control, recorded as change in glycated haemoglobin (HbA1c), with a lower risk of hypoglycaemia, was observed with Gla‐300 compared with Gla‐100 during the main 6‐month treatment periods of the EDITION 1, 2 and 3 studies, conducted in people with type 2 diabetes 3, 4, 5. During the main 6‐month treatment period of EDITION 2 (NCT01499095), which enrolled people with type 2 diabetes who were using basal insulin and oral antihyperglycaemic drugs (OADs), the relative risk of nocturnal confirmed [≤3.9 mmol/l (≤70 mg/dl)] or severe hypoglycaemia was 29% lower with Gla‐300 than with Gla‐100 3. Similarly, fewer participants experienced one or more confirmed or severe hypoglycaemic event at any time (24 h) with Gla‐300 than with Gla‐100. The annualized rates of confirmed or severe hypoglycaemic events were also lower with Gla‐300 than with Gla‐100. Weight gain was low, with statistically (p = 0.015) lower weight gain observed with Gla‐300 compared with Gla‐100 at 6 months. After the main 6‐month treatment period, participants in EDITION 2 continued in a 6‐month safety extension to examine the longer‐term outcomes of treatment with Gla‐300 in people with type 2 diabetes using basal insulin and OADs. The 12‐month results of the EDITION 2 study are reported in the present paper. BODY.PARTICIPANTS AND METHODS.STUDY DESIGN AND PARTICIPANTS: EDITION 2 (NCT01499095) was a multicentre, multinational, randomized, open‐label, two‐arm, parallel‐group, phase IIIa study conducted between 14 December 2011 and 26 April 2013 in 13 countries (two in North America, eight European countries and Chile, Mexico and South Africa). The protocol and study design have been described previously 3. Adults (aged ≥18 years old) with type 2 diabetes treated with ≥42 U/day basal insulin (Gla‐100 or NPH insulin) and OADs (except sulphonylureas) were randomized 1 : 1 to receive Gla‐300 or Gla‐100 for 6 months, with a 6‐month safety extension period 3. Exclusion criteria included: HbA1c <7.0 or >10%; recent (within the past 3 months) use of premixed insulin, insulin detemir or initiation of new glucose‐lowering agents; recent (within the past 2 months) use of sulphonylurea; recent (>10 days in the past 3 months) use of human regular insulin or mealtime insulin; and rapidly progressing diabetic retinopathy, end‐stage renal disease (defined as requiring dialysis or transplantation 6), or clinically significant cardiac, hepatic or other systemic disease. Gla‐300 (using a modified SoloSTAR® pen, sanofi‐aventis U.S. L.L.C., Bridgewater, NJ, USA) or Gla‐100 (using a SoloSTAR® pen) was self‐administered once daily at the same time each day in the evening, defined as the time immediately before the evening meal until bedtime. The insulin dose was to be titrated based on fasting self‐monitored plasma glucose (SMPG) values, according to a prespecified titration algorithm, seeking an SMPG target of 4.4–5.6 mmol/l (80–100 mg/dl). Irrespective of treatment allocation, rescue treatment with a new antihyperglycaemic drug was allowed if the fasting plasma glucose (FPG) or HbA1c levels were above target values and there was no reasonable explanation for insufficient glucose control, or if appropriate action failed to decrease the levels to below threshold values. The choice of rescue therapy (rapid insulin or other antihyperglycaemic medications) was based on the investigator's decision and local approved guidelines. The protocol was approved by local or national ethics committees, and the study was conducted according to Good Clinical Practice and the Declaration of Helsinki. All participants provided written informed consent. BODY.PARTICIPANTS AND METHODS.EFFICACY AND SAFETY ANALYSES: Efficacy analyses included change in HbA1c, FPG, SMPG and daily basal insulin dose from baseline to the end of 12 months' treatment. Hypoglycaemic events were recorded using American Diabetes Association definitions 7. Severe hypoglycaemia and hypoglycaemia confirmed by an SMPG reading [symptomatic or asymptomatic, confirmed using a plasma glucose threshold of ≤3.9 mmol/l (≤70 mg/dl)] were reported in a composite category of confirmed or severe hypoglycaemia. A more stringent plasma glucose threshold of <3.0 mmol/l (<54 mg/dl) was also used. Severe hypoglycaemia was defined as an event requiring the assistance of another person to administer carbohydrate or glucagon or take resuscitative actions. Each category of hypoglycaemia was analysed as the percentage of participants reporting at least one event and as events per participant‐year (annualized rate). Hypoglycaemic events were analysed by occurrence at any time (24 h) and during the night (nocturnal; 00:00–05:59 hours). Change in body weight was assessed as a safety outcome. Adverse events (AEs), including injection site and hypersensitivity reactions, were recorded during the whole study period. Standard safety laboratory assessments for anti‐insulin antibodies (status, titre and cross‐reactivity with human insulin) were performed. The Diabetes Treatment Satisfaction Questionnaire status (DTSQs) was used to assess participant satisfaction with current treatment, including Perceived Frequency of Hyperglycaemia (item 2 of the DTSQs) and Perceived Frequency of Hypoglycaemia (item 3 of the DTSQs) 8, 9. BODY.PARTICIPANTS AND METHODS.DATA ANALYSIS AND STATISTICS: The modified intention‐to‐treat (mITT) population was defined as all randomized participants who received at least one dose of study insulin and had both a baseline and at least one post‐baseline primary or secondary efficacy endpoint assessment. Analyses of glycaemic control used the mITT population. A mixed‐effects model for repeated measures, conducted on rescue‐free measurements, was used to analyse HbA1c and FPG. Descriptive analyses were performed on dose and eight‐point SMPG. Safety analyses included all participants randomized and exposed to at least one dose of study insulin. Hypoglycaemic event rates were analysed using an overdispersed Poisson regression model. Body weight and insulin dose were analysed using an analysis of covariance model. AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA) system. BODY.RESULTS.STUDY POPULATION: Of the 811 randomized participants, 404 were assigned to Gla‐300 and 407 to Gla‐100. In each group, 1 participant was randomized but not treated, and a further participant in the Gla‐100 group was randomized but did not have a baseline or post‐baseline primary or secondary efficacy endpoint measurement. These participants were excluded from the mITT population (Figure 1). A total of 315/404 (78%) and 314/407 (77%) participants in the Gla‐300 and Gla‐100 groups, respectively, completed 12 months of treatment (Figure 1). Figure 1Study flow. *One participant in each group received rescue therapy and withdrew from the study. The upper portion with open boxes shows participant flow during the main 6‐month study period; the lower portion with shaded boxes shows flow during the extension phase up to month 12. Gla‐100, insulin glargine 100 U/ml; Gla‐300, insulin glargine 300 U/ml; mITT, modified intention‐to‐treat.DOM-12532-FIG-0001-b Baseline characteristics have been reported previously and were similar in the Gla‐300 and Gla‐100 groups 3. Participants had a mean [standard deviation (s.d.)] age of 58 (9.2) years, a mean (s.d.) body mass index (BMI) of 35 (6.4) kg/m2 and a mean (s.d.) duration of diabetes of 12.6 (7.0) years. Their mean (s.d.) HbA1c concentration was 8.24 (0.82)% [66.5 (9.0) mmol/mol], FPG concentration 8.0 (2.8) mmol/l [144.7 (51.0) mg/dl] and basal insulin dosage 0.67 (0.24) U/kg/day. Almost all participants used biguanides in the Gla‐300 and Gla‐100 groups, both before the start of treatment [389/404 participants (96.3%) and 383/407 participants (94.1%), respectively] and during the on‐treatment period [385/404 participants (95.3%) and 381/407 participants (93.6%), respectively]. Dipeptidyl peptidase‐4 inhibitors were used before treatment and during the treatment period by 33 participants (8.2%) and 34 participants (8.4%), respectively, in the Gla‐300 group, and by 51 participants (12.5%) and 54 participants (13.3%), respectively, in the Gla‐100 group. Sulphonylureas had been used in the 3 months before randomization by 32 (3.9%) of all randomized participants. Overall, 16 (2.0%) participants used sulphonylureas during the study, including use as rescue therapy by 2 participants in the Gla‐300 group and 1 participant in the Gla‐100 group. During the 12‐month on‐treatment period, 33 participants (8.2%) in the Gla‐300 group and 41 (10.1%) in the Gla‐100 group received rescue therapy; relative risk 0.81 [95% confidence interval (CI) 0.52–1.25; Figure 1], most commonly as rapid‐acting insulin. BODY.RESULTS.INSULIN DOSE AND GLYCAEMIC CONTROL (MITT POPULATION): Over the whole 12‐month study period, the mean basal insulin dosage increased in both groups, primarily during the first 12 weeks and to a greater extent in the Gla‐300 group than the Gla‐100 group (Figure 2A). The mean basal insulin dosage continued to increase gradually up to month 12 in both treatment groups, reaching 0.97 U/kg/day in the Gla‐300 group and 0.87 U/kg/day in the Gla‐100 group [mean (standard error) difference 0.11 (0.02) U/kg/day; p < 0.0001]. Figure 2(A) Mean daily basal insulin dose (B) glycated haemoglobin (HbA1c) ± standard error by visit (modified intention‐to‐treat population). Gla‐100, insulin glargine 100 U/ml; Gla‐300, insulin glargine 300 U/ml; s.e., standard error.DOM-12532-FIG-0002-c The mean HbA1c decreased from baseline to month 12 to a similar extent in each of the two treatment groups, with the greatest decrease occurring between baseline and week 12 in both groups (Figure S1A). The mean (s.d.) HbA1c at month 12 was 7.62 (1.03)% [59.8 (11.3) mmol/mol] in the Gla‐300 group and 7.64 (1.21)% [60.0 (13.2) mmol/mol] in the Gla‐100 group (Figure 2B). The least squares (LS) mean difference in change from baseline to month 12 between groups was −0.06 (95% CI −0.22 to 0.10)% [−0.66 (95% CI −2.4 to 1.1) mmol/mol]. In addition, FPG decreased to a similar extent in the two treatment groups, primarily during the initial 12 weeks of treatment, remaining relatively stable during the remainder of the 12‐month study period (Figure S1B). The LS mean difference in FPG change from baseline to month 12 between the groups was 0.18 (95% CI −0.21 to 0.57) mmol/l [3.3 (95% CI −3.7 to 10.3) mg/dl]. The mean eight‐point SMPG profiles decreased from baseline to month 12 in both treatment groups at all time points. At month 12, mean plasma glucose levels were similar in the two treatment groups from 03:00 hours to post‐lunch, but numerically lower in the Gla‐300 group than in the Gla‐100 group from pre‐dinner to bedtime (Figure S1C). BODY.RESULTS.HYPOGLYCAEMIA (SAFETY POPULATION). : The cumulative mean number of nocturnal (00:00–05:59 hours) confirmed [≤3.9 mmol/l (≤70 mg/dl)] or severe hypoglycaemic events per participant is reported in Figure 3A. There was a significant 37% relative reduction in annualized rate with Gla‐300 compared with Gla‐100 [1.74 vs 2.77, rate ratio 0.63 (95% CI 0.42–0.96); p = 0.0308; Figure 3B], and a 16% relative risk reduction in the percentage of participants experiencing ≥1 nocturnal confirmed or severe hypoglycaemic event during the 12‐month study period [38% vs 45%, relative risk 0.84 (95% CI 0.71–0.99); Table S1]. Figure 3Confirmed [≤3.9 mmol/l (≤70 mg/dl)] or severe hypoglycaemia. (A) Cumulative mean number of nocturnal (00:00–05:59 hours) events per participant. (B) Nocturnal events per participant‐year. (C) Cumulative mean number of events per participant at any time (24 h). (D) Events per participant‐year at any time (24 h; safety population). CI, confidence interval; Gla‐100, insulin glargine 100 U/ml; Gla‐300, insulin glargine 300 U/ml; RR, rate ratio.DOM-12532-FIG-0003-c Considering all hypoglycaemia, there were fewer nocturnal hypoglycaemic events reported with Gla‐300 (668 events or 1.8 events per participant‐year) versus Gla‐100 [1110 events or 2.9 events per participant‐year; rate ratio 0.61 (95% CI 0.41–0.92); Table S1]. A total of 160 participants (40%) in the Gla‐300 group and 187 participants (46%) in the Gla‐100 group experienced at least one nocturnal hypoglycaemic event [relative risk 0.86 (95% CI 0.73–1.01)]. Annualized event rates and percentages of participants experiencing at least one event for these and other categories of hypoglycaemia during the night are shown in Table S1. The cumulative mean number of confirmed or severe hypoglycaemic events per participant at any time (24 h) is reported in Figure 3C. The annualized event rate (Figure 3D) and the percentage of participants with at least one event during 12 months of treatment were numerically lower but not significantly different with Gla‐300 compared with Gla‐100. Annualized rates of confirmed or severe hypoglycemia were 11.6 for Gla‐300 and 13.2 for Gla‐100, and the corresponding percentages of participants with at least one event were 78% for Gla‐300 and 82% for Gla‐100 (Table S1). Over the 24‐h period, the percentage of participants reporting confirmed or severe hypoglycaemia tended to be lower in the Gla‐300 group than in the Gla‐100 group between 00:00 and 14:00 hours, and was similar in the two groups from 14:00 hours until midnight (Figure 4). Figure 4Percentage of participants with ≥1 confirmed [≤3.9 mmol/l (≤70 mg/dl)] or severe hypoglycaemic event during the 12‐month study on‐treatment period by time of day. Gla‐100, insulin glargine 100 U/ml; Gla‐300, insulin glargine 300 U/ml.DOM-12532-FIG-0004-c Other categories of hypoglycaemia, expressed as annualized event rates and percentages of participants experiencing at least one event at any time (24 h) are shown in Table S1. During the 12‐month study period, severe hypoglycaemia at any time (24 h) was reported by 7 (1.7%) participants (10 events) in the Gla‐300 group and 6 (1.5%) participants (13 events) in the Gla‐100 group, corresponding to a rate of 0.03 events per participant‐year in both treatment groups (Table S1). Three participants reported severe nocturnal hypoglycaemic events [1 participant (0.2%) using Gla‐300 and 2 participants (0.5%) using Gla‐100]. BODY.RESULTS.BODY WEIGHT: The significant between‐group weight difference observed at 6 months was maintained at 12 months of treatment (Figure S2) 3. The mean (s.d.) change in body weight, based on the last on‐treatment value, was significantly lower with Gla‐300 than with Gla‐100 [0.4 (4.1) vs 1.2 (3.6) kg]; the LS mean difference between groups at month 12 was −0.7 (95% CI −1.3 to −0.2) kg; p = 0.009. BODY.RESULTS.TREATMENT SATISFACTION: Participants in both treatment groups reported high satisfaction throughout the study; DTSQs treatment satisfaction scores were similar in each group. The improvement in DTSQs scores observed at month 6 was maintained at month 12, resulting in an overall mean (s.d.) increase from baseline to month 12 of 4.3 (6.6) with Gla‐300 and 4.4 (7.5) with Gla‐100. Perceived frequency of hypoglycaemia remained stable with either treatment [mean (s.d.) change from baseline to month 12, 0.05 (1.86) in both treatment groups]. Perceived frequency of hyperglycaemia slightly decreased in both treatment groups [mean (s.d.) change from baseline to month 12, −1.38 (2.18) and −1.37 (2.29) for Gla‐300 and Gla‐100, respectively]. BODY.RESULTS.ADVERSE EVENTS: There was a similar pattern of treatment‐emergent AEs (TEAEs) in each treatment group, with incidences of 69 and 60% in the Gla‐300 and Gla‐100 groups (Table 1). Fourteen (3.5%) TEAEs in the Gla‐300 group and 15 (3.7%) in the Gla‐100 group were considered by the study investigator to be related to study medication. The most commonly reported TEAE was nasopharyngitis (Gla‐300, 12.2%; Gla‐100, 7.6%). Injection site reactions were experienced by 5 (1.2%) participants in the Gla‐300 group and 12 (3.0%) participants in the Gla‐100 group. Hypersensitivity reactions were reported by 19 participants (4.7%) in the Gla‐300 group and 20 participants (4.9%) in the Gla‐100 group. Table 1 Adverse events (safety population) n (%) Gla‐300 (n = 403) Gla‐100 (n = 406) Participants with any TEAE 278 (69.0) 244 (60.1) Participants with any treatment‐emergent SAE 30 (7.4) 30 (7.4) Participants with any TEAE leading to death 4 (1.0) 2 (0.5) Participants with TEAE leading to permanent treatment discontinuation 11 (2.7) 7 (1.7) Any injection site reaction 5 (1.2) 12 (3.0) Any hypersensitivity reaction 19 (4.7) 20 (4.9) TEAE, treatment‐emergent adverse event; SAE, serious adverse event. The percentage of participants with treatment‐emergent serious AEs (SAEs) or TEAEs leading to treatment discontinuation during the 12‐month on‐treatment period were similar in the Gla‐300 and Gla‐100 groups (Table 1). Two (0.5%) SAEs in the Gla‐300 group (one case of hypoglycaemia and one acute myocardial infarction) and one (0.2%) in the Gla‐100 group (hypoglycaemia) were considered possibly related to study medication. Four participants (1.0%) in the Gla‐300 group and 2 participants (0.5%) in the Gla‐100 group died owing to a TEAE. All those who died had pre‐existing, significant‐event‐related pathology and/or had multiple risk factors contributing to the fatal outcome. In the Gla‐300 group, three deaths (one from myocardial infarction, one from metastatic adenocarcinoma of the oesophagus and one from coronary artery disease) were not considered treatment‐related, and one death (attributable to acute myocardial infarction) was considered by the study investigator as possibly related to study medication or non‐study medication (specifically metformin). The two deaths in the Gla‐100 group (one myocardial infarction and one infection) were not considered related to study medication. No differences were found between treatment groups in safety laboratory measurements, physical examination, vital signs or ECGs, or in numbers of participants reporting anti‐insulin antibodies, anti‐insulin antibodies titre or cross‐reactivity to human insulin. BODY.DISCUSSION: These 12‐month treatment results confirm the findings of the 6‐month initial study period 3. Equivalent glycaemic control was attained with both insulins, but less nocturnal confirmed [≤3.9 mmol/l (≤70 mg/dl)] or severe hypoglycaemia and less weight gain was observed with Gla‐300. The safety profiles of Gla‐300 and Gla‐100 were similar. The reduction in HbA1c values observed in both treatment groups from baseline to month 6 3 was maintained at 12 months. When examining the mean eight‐point SMPG profiles, blood glucose was lower with Gla‐300 than with Gla‐100 between the pre‐dinner and bedtime time points. Better glycaemic control with Gla‐300 than with Gla‐100 at the end of a once‐daily evening dosing schedule might be explained by the longer duration of action of Gla‐300, which has been shown in PK/PD studies 1. In addition to providing sustained glycaemic control, there was a 37% relative reduction in the annualized rate of nocturnal confirmed [≤3.9 mmol/l (70 mg/dl)] or severe hypoglycaemic events with Gla‐300 compared with Gla‐100, and a consistently lower proportion of participants experiencing at least one event. While it is necessary to use defined nocturnal and diurnal intervals of time for the purpose of analysis, these arbitrary definitions may not correspond to the daily habits of participants with regard to timing of sleep and the first meal of the day. As well as providing a significant reduction in hypoglycaemia during the predefined nocturnal (00:00–05:59 hours) period, a lower rate of hypoglycaemia compared with Gla‐100 was observed with Gla‐300 until the middle of the next day, in line with its PK/PD profile. In future studies and analyses, the actual timing of the overnight fasting interval should be considered when assessing the risk of hypoglycaemia. Severe hypoglycaemic events were uncommon in both treatment groups. Lower nocturnal hypoglycaemia, with a small but significant improvement in HbA1c, was seen with Gla‐300 compared with Gla‐100 after 12 months of treatment in the EDITION 1 study, in which people with type 2 diabetes were treated with basal plus mealtime insulin 5. Similar glycaemic control with reduced nocturnal hypoglycaemia has been observed with insulin degludec 100 U/ml compared with Gla‐100 in people with type 2 diabetes treated with basal plus mealtime insulin 10, and in insulin‐naïve people 11. By contrast, another study in insulin‐naïve people with type 2 diabetes, using a more concentrated 200 U/ml formulation of insulin degludec, did not show a difference in nocturnal hypoglycaemia compared with Gla‐100 12; however, differences in study populations and definitions of hypoglycaemia limit direct comparisons of hypoglycaemia rates across all of these studies. The mean daily doses of Gla‐300 and Gla‐100 remained relatively consistent throughout the 6‐month extension period of this study. A higher mean daily insulin dose of Gla‐300 was required, with the majority of dose titration occurring during the first 12 weeks of the study, as reported previously 3. The cause of the difference in insulin dose is uncertain, but it may be attributable to greater inactivation of the glargine molecule by tissue proteases as a consequence of the longer residence time of Gla‐300 in the subcutaneous depot. A longer residence time is consistent with the extended absorption of Gla‐300, which results in the more prolonged PK/PD profile of action observed with Gla‐300 compared with Gla‐100 1, 13, 14. Weight gain after 12 months of treatment remained low in both groups and was not felt to be of clinical concern. Furthermore, there was slightly, but statistically significantly, less weight gain with Gla‐300 than with Gla‐100. This was already apparent after 6 months 3. A trend for lower weight gain with Gla‐300 compared with Gla‐100 has also been observed in EDITION studies in other populations 4, 15, 16, 17. Further analyses are warranted to determine whether this difference can be explained by the difference in hypoglycaemia or other factors. Occurrence of TEAEs, treatment discontinuation, injection site reactions and hypersensitivity reactions occurred with a similar pattern with Gla‐300 and Gla‐100. Both treatments were well tolerated and no safety concerns were identified at 12 months; longer‐term observation will be required to continue to monitor the safety profile of Gla‐300. Limitations of the present study include its open‐label nature and a necessary reduction in ascertainment of hypoglycaemia and other events during the extension phase. The EDITION 2 study was restricted to the investigation of clinical outcomes of Gla‐300 treatment versus Gla‐100 in participants using a basal insulin and OAD (excluding sulphonylurea) regimen; however, the EDITION programme of clinical studies is investigating outcomes of Gla‐300 vs Gla‐100 treatment in other populations using different treatment regimens to cover the broader cross‐section of people with diabetes. Analyses of the 6‐month extension phases of EDITION 3 (insulin‐naïve participants), EDITION 4 (people with type 1 diabetes), EDITION JP 1 (Japanese people with type 1 diabetes) and EDITION JP 2 (Japanese people with type 2 diabetes using basal insulin plus OADs) are ongoing. In summary, in a population using basal insulin plus OADs, excluding sulphonylurea, with a long duration of type 2 diabetes, high BMI, long‐term previous insulin use and high baseline HbA1c, findings from 12 months' treatment with Gla‐300 support and extend the EDITION 2 6‐month results. The sustained glycaemic control achieved in this challenging‐to‐treat population along with less nocturnal hypoglycaemia and weight gain compared with Gla‐100 is reassuring and suggests that these favourable results may be applicable to long‐term treatment in clinical practice. BODY.CONFLICT OF INTEREST: H. Y.‐J. has received honoraria for consulting and speaking from Boehringer Ingelheim, Eli Lilly, Merck (MSD) and Sanofi. R. M. B. has received research support and served as a consultant or on the scientific advisory board for Abbott Diabetes Care, Amylin, AstraZeneca/Bristol‐Myers Squibb Alliance, Bayer, Becton Dickinson, Boehringer Ingelheim, Calibra, DexCom, Eli Lilly, Halozyme, Hygieia, Johnson & Johnson, Medtronic, Merck (MSD), Novo Nordisk, Roche, Sanofi and Takeda, is employed by non‐profit Park Nicollet Institute, who contract for his services and pay him no personal income, has inherited Merck (MSD) stock, and has been a volunteer for the American Diabetes Association and Juvenile Diabetes Research Foundation (JDRF). G. B. B. has received honoraria for advising and lecturing from Eli Lilly, Novartis and Sanofi. M. Z., M. W. and I. M.‐B. are employees of Sanofi. M. M. is an employee of Umanis. M. C. R. has received research grant support from Amylin, Eli Lilly and Sanofi, and honoraria for consulting and/or speaking from Amylin, AstraZeneca/Bristol‐Myers Squibb Alliance, Elcelyx, Eli Lilly, Sanofi and Valeritas. These dualities of interest have been reviewed and managed by Oregon Health and Science University. H. Y.‐J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Sanofi was the sponsor of the study, and was responsible for the design and coordination of the trial. Sanofi monitored the clinical sites, collected and managed the data, and performed all statistical analyses. H. Y.‐J., R. M. B., G. B. B. and M. C. R. participated in the design of the study programme and protocol, analysis and interpretation of the data and in the writing, reviewing and editing of the manuscript. M. Z. and I. M.‐B. contributed to the design and treatment considerations for the trial, analysed and interpreted the data, and reviewed and edited the manuscript. M. W. and M. M. analysed and interpreted the data, and reviewed and edited the manuscript. BODY.SUPPORTING INFORMATION: Table S1. Hypoglycaemia over 12 months in the EDITION 2 study (safety population).Click here for additional data file. Figure S1. (A) Glycated haemoglobin mean change from baseline by visit. (B) Fasting plasma glucose mean change from baseline by visit. (C) Eight‐point self‐monitored plasma glucose profile mean change from baseline at 12 months (modified intention‐to‐treat population).Click here for additional data file. Figure S2. Mean change in body weight over the 12‐month study period (safety population).Click here for additional data file.

TITLE: Effect of Oral Health Care Program on Oral Health Status of Elderly People Living in Nursing Homes: a Quasi-experimental Study ABSTRACT.BACKGROUND:: Oral health of elderly people plays a major role in their overall health and quality of life, and is an integral part of personal care. ABSTRACT.AIM:: The aim of this study was to evaluate the effect of implementing the oral health care program (OHCP) on oral health status of elderly people resident in nursing homes. ABSTRACT.MATERIALS AND METHODS:: This quasi-experimental study was carried out using a pretest-posttest design on 101 elderly people (46 in the intervention group and 55 in the control group) resident in two randomly selected nursing homes in Mashhad, Iran. In the intervention group, the OHCP was carried out by caregivers for 8 weeks. The control group received routine care. Using the oral health assessment tool, the oral health status of elderly people was assessed in both groups at three times; onset of the study, 4th, and 8th week after the start of the study. ABSTRACT.RESULTS:: The oral health status of the elderly people in both groups was not statistically significantly different at baseline, but it changed significantly at the 4th, and 8th weeks (p<.001). ABSTRACT.CONCLUSION:: The implementation of the OHCP for elderly people resident in nursing homes may improve their oral health status after 4 weeks. It is recommended that OHCP be included in care plans of all nursing homes to improve the elderly people's oral health status. BODY.1. INTRODUCTION: The increase in the number of elderly people living in developing countries is one of the most important challenges of the twenty-first century. According to the World Health Organization (WHO), the number of people over the age of 60 was 585 million in 2001 and accounted for 9% of the world population; this rate will double by 2025 (1). In Iran, statistical indicators suggest that the elderly population is increasing annually by 1.25%. The annual growth of the population over 60 years old is estimated to be 2.5% (2). Although aging is an inevitable physiological process, it increases the possibility of health problems in elderly people, including oro-dental diseases (3). Oro-dental diseases, as a health priority, and the most prevalent unmet health need, have a great impact on a person's quality of life, and are associated with health care costs (4). Oro-dental health affects the physical health of elderly people, as well as their appearance, body image, self-esteem, psychological and social functioning, and ultimately, their quality of life (5, 6). A toothless oral cavity and dentures are major sources of pathogenic microbes, which can cause serious inflammatory or infectious diseases in elderly people. These pathogenic microbes also have negative effects on acute and chronic diseases, and are associated with over 100 systemic diseases with oral manifestations. Also, chronic diseases can affect oro-dental health, as well as the ability of a person to maintain their oro-dental hygiene (7-9). It has been shown that diabetic patients with oral problems have problem with blood sugar control and are more prone to the loss of teeth (10). Also, the risk of developing pneumonia and other respiratory diseases is related to the level of oral hygiene (11). It has been shown that the oral health status of elderly people is poor, and living in nursing homes may increase the likelihood of oro-dental diseases, as well as the need for oro-dental related evaluation and education (12-13). Since the population of elderly people living in nursing homes is increasing, and their ability to care for themselves may be impaired due to physical and psychological problems, they need support to maintain their oro-dental health. Therefore, providing oro-dental health care by caregivers and nurses is important for elderly people resident in nursing homes (14). However, several studies have suggested that offering oro-dental care to elderly people by healthcare providers have a low priority and also they do not have appropriate level of knowledge about the importance of oro-dental health in elderly people (15, 16). Thus there is a need to implement educational programs for healthcare providers to promote oro-dental health care in elderly people (17). Jablonsky et al. showed that all nursing home employees should take additional training to understand the importance of oro-dental health (18). Gammack et al. showed that, while an oral health education program increased the knowledge of nurses about the oro-dental health of elderly people, it had no effect on the oro-dental health status of elderly people (19). To the best of our knowledge, no study has been done on using an oral health education program to train caregivers and assessing its impact on the oro-dental health status of elderly people in Iran. BODY.2. AIM: The aim of present study was to evaluate the effect of an oral health care program (OHCP) on the oro-dental health status of elderly people resident in nursing homes. BODY.3. MATERIALS AND METHODS: The present quasi-experimental study was carried out using a pretest-posttest design in 2016 on elderly people resident in nursing homes in Mashhad, Northeast Iran. From a total of 20 nursing homes, 2 nursing homes were randomly selected. Of the 31 caregivers who participated in the study, 25 were female, and 15 were in the intervention group. Based on the inclusion criteria, 109 elderly people were selected by convenience sampling to participate in the study. In total, 101 elderly people, 46 in the intervention group and 55 in the control group, completed the study. The inclusion criteria for the elderly people were permanent residence in nursing homes, cooperation during oral examinations, short stay (less than three months) in the nursing homes, and having no oro-dental and medical interventions within the last three months and during the study. People who had serious changes in their physical health status, received any medical and dental interventions during the study, or started new medications during the study were to be excluded the study. Five people from the intervention group and three people from the control group were excluded because they expired, they were sent to hospital for an acute physical problem, or they returned to their homes. The demographic questionnaire was administered to the elderly people and caregivers. Also, the caregivers were asked to complete the knowledge questionnaire. Ten experts in oro-dental health provided comments that were used to determine the validity of the questionnaires. The internal consistency reliability of the knowledge questionnaire was in acceptable range (Cronbach's alpha= 0.852). The validity of the oral health assessment tool (OHAT) for the elderly people was confirmed through consultations with dentist supervisors and advisors. Its reliability was evaluated according to data collection by equivalent forms; the Spearman correlation was calculated to be 0.92 between the data from the interobserver reliability (researcher and the colleague), which was statistically significant (p < 0.001), indicating very reasonable reliability. Demographic information and knowledge questionnaires were completed by the caregivers in both centers to provide baseline measurements (pre-test). Caregivers at the intervention center were invited to participate in a training program, which consisted of four 90 minutes sessions, twice a week, for 2 weeks. At the end of 2 weeks, the knowledge questionnaires were completed again by the caregivers in both centers as a post-test. The researcher completed the demographic questionnaire and the OHAT for the elderly people before intervention in two groups (pre-test). The OHAT contains sections on the tongue, lips, gums, salivary glands, natural teeth, dentures, oral hygiene, and dental pain. Each section can be scored as 0, 1, or 2; thus, the total score ranges from 0 to 16, where a score of 16 indicates the poorest, and a score of 0 indicates the best oral health status. According to the activities of daily living (ADL) index, the elderly people were divided into three categories: independent, need help, and dependent. The ADL index was completed by the researcher. The researcher reassessed their oral health status using the OHAT in both centers after 4 weeks and 8 weeks of the OHCP being implemented by the caregivers (post-test). Proper space and facilities were provided for the elderly people who were able to independently carry out their oral care, and only needed reminders or monitoring. Permission to conduct this study was given by the institution Ethics Committee and informed consent was obtained from the elderly people who participated in the study. Data were analyzed using SPSS software (IBM, Armonk, NY) using repeated measure analysis of variance (ANOVA), independent t-test, and paired t-test. BODY.4. RESULTS: The highest percentage of caregivers who participated in the study were women and their mean age was 37.3 ± 8.75 years. The caregivers of the intervention and control groups were homogenous in terms of gender and educational level (p > 0.05). There was no significant difference between the two groups of elderly people in terms of gender, educational level, marital status, level of independence, underlying disease, and length of stay in the nursing homes (p > 0.05). The mean age of the elderly people in the intervention group was 77.16 ± 8.32 years and 71.78 ± 8.163 years in the control group (p=0.001). Although this difference was clinically negligible, age was considered to be a covariate based on repeated measures ANOVA. The results showed that the difference in the ages of elderly people had no significant effect (p = 0.781). Table 1 demonstrates that the first knowledge scores of the caregivers in the two groups were not significantly different (p = 0.243); however, Table 2 shows that there was a significant difference in the knowledge scores between the two groups after the training sessions (p < 0.001). Table 1 Comparison of the pre-test knowledge scores of caregivers in the intervention and control groups. Mann–Whitney test (z = –1.168, p = 0.243, statistic = 91) Table 2 Comparison of the post-test knowledge scores of caregivers in the intervention and control groups. Mann–Whitney test (z = –0.824, p <0.001, statistic = 2) Table 3 shows there was a statistically significant difference in the mean pretest oral health score between the two groups (p = 0.021). Although the pretest difference was not clinically reliable, based on repeated measures ANOVA, the pretest oral health score was assumed to be control the confounding effect. Table 3 Comparison of the mean pre-test oral health scores of the elderly people in the intervention and control groups. Mann–Whitney test (z = –2.30, p = 0.021, statistic = 1116) A 2x3 factorial ANOVA with repeated measures was conducted to explore the impact of training of caregivers working in nursing homes on oral health status of elderly residents in the nursing homes. The between-subjects variable was whether or not caregivers were trained; within-subject variable was repeated measures of appraisal time (study onset, end of 4th, and 8th week); and the dependent variable was the oral health status scores of the nursing home residents as measured by the OHAT. An alpha level of .05 was used for statistical significance. Means and standard deviations are shown in Table 4, and a line graph of the results is shown in Figure 1. Table 4 The mean oral health scores of the elderly people in the intervention and control groups Figure 1Trend of elderly oral health status score at study onset, end of 4th week and end of 8th weeks in two groups The assumption of normality was met. However, the Mauchly's test indicated that the assumption of sphericity had been violated, [χ2(2) = .844, p <.001]; therefore the degrees of freedom were corrected using Greenhouse-Geisser estimates of sphericity (e= .865). There was a main effect for appraisal time of measurement [F(1.73, 171.28)=464.005, p<001, partial h2=.824], and a main effect for training of caregivers [F(1, 99)=95.903, p<001, partial h2=.492]. There was also a significant interaction between appraisal time and training of caregivers [F(1.730,171.28)=635.699, p<.001]. Therefore, post hoc tests using the Bonferroni correction were conducted to assess the simple effects. Examination of Figure 1 indicates that oral health status of nursing home elderly residents were similar at the study onset, but improved considerably at the end of 4th, and 8th week, in intervention group, suggesting a positive effect of caregivers' training on the oral health status of nursing home elderly residents. Post hoc Bonferroni pairwise comparison tests (α=.005) revealed that the oral health scores were significantly different at the study onset and the end of 4th week (t=36.042, df=45, p<.001), at the study onset and the end of 8th week (t=26.861, df=45, p<.001), and at the end of 4th and 8th week (t=2.817, df=45, p=.007), within the intervention group, indicating large effect sizes (h2=.95). In control group, the oral health mean score even increased significantly from the study onset to the end of 4th week (t=-3.975, df=54, p<.001); and to the end of 8th week (t=-4.550, df=54, p<.001); though, such differences were not clinically significant. Mean score difference at the end of 4th and 8th week were not statistically significant (t=-1.352, df=55, p=.182, h2=.113). Moreover, there was a significant difference of oral health mean scores between the elderly residents of the two nursing homes at the study onset (t=-2.323, df=107, p=.022). Although this difference is indicative of a slightly better oral health status of intervention group, it was not clinically important. Also, oral health status mean scores at the end of 4th (t=-12.781, df=99, p<.001, h2=.623), and 8th (t=-14.370, df=99, p<.001, h2=.676) week were significantly different. This shows a substantial incremental improvement in oral health status of elderly residents only 4 weeks after training their caregivers, in the intervention group. This general pattern of differences among oral health scores of elderly people suggests that training of their caregivers may indirectly improve their oral health status, but not before 4 weeks (Table 5). Table 5 Repeated measures analysis of variance BODY.5. DISCUSSION: The results of this study indicated that the OHCP significantly improved the oral health status of the elderly people living in nursing home. Based on the knowledge questionnaire, the mean knowledge score of the caregivers in the intervention group after two weeks of training sessions increased as compared with control group. Also, the oral health status of the elderly people in the intervention group has been significantly improved. The findings of the present study are in line with the results of the studies reported by McKeown et al. (20), Le al. (21), Kim et al. (22), and De Visschere et al. (23). Le et al. carried out a study on residents in nursing homes. The caregivers were educated about oral care using a 40-minute video, which was a shorter training session than provided in our study. The results showed that the plaque index (PI) and the knowledge of the caregivers improved from the beginning of the oral health education program to 6 months after the intervention (21), which agreed with the results of our study. Visschere et al. evaluated the implementation of an oral hygiene protocol over 5 years. In their study, a one-hour session training was given to caregivers to explain the principles of intervention, and to provide theoretical and practical education about oral hygiene. This time length of single training session was obviously shorter than the training sessions in our study. They found that the dependence level was correlated with the dental plaque score, which is consistent with our results. Also, it has been shown that 2 years after implementation of the OHCP in the nursing home, the dental plaque score reached its lowest value and the oral health status of the elderly people improved (23). These results were consistent with our findings. The reason for the agreement of the results of the two studies is related to the oral hygiene protocol and the education given to the caregivers. Kim et al. examined the effect of an OHCP for stroke patients in an intensive care unit. The OHCP was conducted for 2 weeks. The OHAT was employed to assess the impact of the OHCP on the oral health status of the elderly people. The results showed that the PI and the gingival index (GI) were much lower in the intervention group than in the control group (22), which was similar to our results. One possible reason for this similarity is the implementation of the OHCP, which is a coherent and systematic program. Simon et al. assessed an oral health training program for the caregivers of elderly people in residential homes. The results showed no changes in the status of oral hygiene and the PI, 6 months after the intervention, which was inconsistent with the results of our study (24). This difference between studies may be due to the absence of trained caregivers during the 6 months in the same nursing home, the insufficient time given to provide oral care by the caregivers, and the lack of adequate facilities to provide optimal care. Overall, despite the differences in the duration and types of training in the various studies, the majority of studies showed that training the caregivers improved the oral health of elderly people with varying degrees of dependency living in nursing homes. Some of the limitations of the present study were caregivers who were unwilling to participate in the training courses, failure of the managers of the nursing homes to provide a suitable place for training the caregivers, and failure by the caregivers to follow the instructions correctly. BODY.6. CONCLUSION: Training the caregivers and subsequently implementation of the OHCP by caregivers for 8 weeks significantly improved the oral health status of the elderly people. It is recommended that OHCP be implemented in all nursing homes to improve the oral health status of elderly people.

TITLE: Standardizing the Protocols for Enhanced Recovery From Colorectal Cancer Surgery: Are We a Step Closer to Ideal Recovery? ABSTRACT.PURPOSE: Enhanced recovery protocols are being implemented into the standard of care in surgical practice. This study aimed to insert a steadfast set of elements into the perioperative care pathway to establish an improved recovery program for colorectal cancer patients. ABSTRACT.METHODS: Seventy patients planned for elective laparoscopic colorectal resection were randomized into 2 groups: conventional recovery group (n = 35) and enhanced recovery group (n = 35). The primary outcome was the length of hospital stay. Secondary outcomes included the times of removal of nasogastric tubes (NGTs), successful enteral feeding, and removal of drains, postoperative complications, intra-hospital mortality, and rate of readmission. ABSTRACT.RESULTS: The mean postoperative hospital stay was 4.49 ± 0.85 days vs. 13.31 ± 6.9 days (P < 0.001), the mean time of removal of NGTs was 0.77 ± 1.031 days vs. 3.26 ± 2.737 days (P < 0.001), the mean time of successful enteral feeding was 1.89 ± 1.13 days vs. 5.46 ± 1.67 days (P < 0.001), and the mean time for removal of intra-abdominal drains was 2.94 ± 1.056 days vs. 9.06 ± 3.757 days (P < 0.001) for the enhanced and the conventional groups, respectively. Complications were significantly lower among patients in the enhanced group (25.7% vs. 65.7%) (P = 0.001). The rates of readmission were similar in the 2 groups. ABSTRACT.CONCLUSION: Applying definite evidence-based elements to the colorectal rehabilitation program significantly boosts the recovery pathway with favorable outcomes, including faster recovery of gastrointestinal tract functions, lower morbidities, and eventually earlier discharge from the hospital. BODY.INTRODUCTION: In order to handle the metabolic response of surgery and speed up the recovery of patients, several enhanced recovery protocols have been formulated with encouraging results. Reduced inci-dence of postoperative morbidity and mortality, shorter hospital stay, as well as reduction in healthcare costs were among these results [1]. The steps to achieve an accelerated recovery begin with a thorough preoperative assessment in the first clinic encounter embedded with appropriate patient education and counseling with a physiotherapist and a stoma nurse when feasible. The prolonged period of preoperative overnight fasting is replaced with carbohydrate loading with drinks up to 4 hours prior to induction of anesthesia. For pain management, epidural analgesia is treasured. Careful fluid therapy is essential. Early 'enforced' ambulation, enteral nutrition and rehabilitation are encouraged postoperatively [23]. However, different versions of the enhanced recovery protocols have been established by combining various sets of elements, and no set is considered an 'ideal' protocol of enhanced recovery; thus, authors all around the world have been utilizing a series of programs that focus on specific key components to improve the recovery pathway [4]. This study was designed to implement the concept of fast track care and to set a policy of enhanced recovery after surgery protocols into the surgical management of patients with a colorectal carcinoma at our oncology center. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. BODY.METHODS.PATIENTS: Seventy colorectal cancer patients planned for elective resection were admitted to the Surgical Oncology Unit, Oncology Center – Mansoura University (OCMU) between June 2012 and October 2016. Patients were randomized into 2 groups: 35 patients underwent a conventional recovery pathway and 35 patients underwent an enhanced recovery pathway. Inclusion criteria for participants were the presence of a pathologically confirmed colorectal carcinoma amenable for elective surgery and no severe physical disability (American Society of Anesthesiologists physical status classification I–III). Exclusion criteria included a previous history of abdominal surgery, chronic pain syndrome, and the need for emergency surgery. All patients complied with the procedures after having given written informed consent. This controlled clinical trial was approved by the 'Institutional Review Board' of the Faculty of Medicine, Mansoura University. BODY.METHODS.PREOPERATIVE PREPARATIONS: Standard preoperative health education was delivered to patients within the conventional group while 'individualized' counseling about the proposed fast track care plan (Table 1) and about the daily chores, milestones and expectations regarding the procedure was delivered to patients within the enhanced recovery group. Routine bowel cleansing was done for patients in the conventional group while selective mechanical bowel preparation was performed for patients in the enhanced group (for left colonic and rectal lesions). Prophylaxis against thromboembolism was mandatory in all patients in both groups of this study (special deterrent stockings and a single dose of low molecular weight Heparin [LMWH] in the evening before surgery - 6:00 PM). Patients in the conventional recovery group were instructed to take clear fluids on the day before surgery and to fast overnight until the time of surgery. On the contrary, patients within the enhanced program were supplemented with carbohydrate-rich drinks (3 sachets of Biogainers [Bio Pharma, Giza, Egypt] daily or a similar carbohydrate-rich fluid; Nutrition Advanced Formula [Prime Pharmaceuticals, 10th of Ramadan, Egypt]) starting 2 days before surgery and continuing up to four hours prior to induction of anesthesia to maintain them in a well-fed state. BODY.METHODS.OPERATIVE TECHNIQUE: A standardized anesthetic protocol was applied to all patients. No preanesthetic medications were allowed in the fast track group. Just before induction of anesthesia, all patients, unless they had refused, had low thoracic epidural catheters (T10–12) inserted for postoperative pain control. Similar laparoscopic techniques were advocated in both groups with preference to smaller transverse incisions in assisted resections within the enhanced recovery group. In the enhanced group, active measures, including warming of the infused intravenous fluids and, in some instances, warming of the air in the operating room, were taken to avoid hypothermia. Warmer coats or blankets were also used for 2 hours postoperatively. Nasogastric tubes (NGTs), urinary catheters and intra-abdominal drains were routinely inserted for patients in both groups. BODY.METHODS.POSTOPERATIVE CARE: All patients after surgery were transferred according to their vital signs and pre-existing comorbidities either to the intensive care unit, the high dependency unit, or the normal ward for close monitoring of their vital signs and for proper pain management. The patients in the conventional group were administered a combination of a local anesthetic (bupivacaine 0.125%) and an opiate (morphine, pethidine, or fentanyl) in epidurals with or without dermal patches (Duragesic [Janssen Pharmaceutica, Beerse, Belgium] patch) unless contraindicated, in which case, the patients were only given analgesic doses of nonsteroidal anti-inflammatory drugs (NSAIDs) at timed intervals. Patients in the enhanced recovery group were not allowed to receive opiates in their epidurals during the postoperative period, but were given intravenous acetaminophen (4 g/day) throughout the recovery period. For breakthrough pain episodes, combinations of NSAIDs and acetaminophen were administered. This combination continued to offer pain relief after removal of the epidurals (48 hours from the surgery). Laxatives were used on the same day of surgery in the enhanced group while in the conventional group, they were used only when peristalsis occurred. In the conventional group, patients were allowed to get out of bed on the night of surgery or the next morning, if tolerable. In the enhanced recovery group, enforced mobilization started on the day of surgery (postoperative day [POD] 0) for 2 hours and gradually increased to 6 hours by the time of discharge. In the enhanced recovery group, urinary catheters were usually removed on POD 0 unless ureteric/bladder injury had occurred (continuous drainage for 14 days). Similarly, NGTs were removed on POD 0 unless severe postoperative nausea and vomiting (PONV) occurred. In the conventional recovery group, patients were allowed to start oral intake once they had passed motion or flatus (later than POD 3). On the contrary, in the enhanced recovery group, patients were instructed to start oral sips of a nutritional supplement as soon as POD 1 in gradually increasing amounts until permission was given for a normal meal, usually on POD 3, with intravenous fluid being restricted to a minimum or discontinued. After full oral intake had been maintained in both groups, drains were removed, and patients were ready to be discharged. Predetermined discharge criteria were similar for the 2 studied groups: no complications at time of discharge, tolerance to food intake (solid diet) with normalization of gastrointestinal tract functions, successful oral analgesia, independent mobilization, and acceptance (consent) of hospital discharge. Fulfilling these criteria allowed 'safe' hospital departure. A follow-up date 1 week after the pa-tient's discharge from the hospital was given to all patients. Patients were readmitted when they presented on their follow up date or sooner with a surgical complication that could not be managed in the outpatient clinic, i.e., persistent PONV, severe urinary tract infection (UTI), postoperative ileus, and intra-abdominal collection on follow-up ultrasound. BODY.METHODS.STATISTICAL ANALYSIS: Data were analyzed using IBM SPSS Statistics ver. 23.0 (IBM Co., Armonk, NY, USA). Numerical data were expressed in the form of means ± standard deviations, and comparisons between groups were made using the chi-square test or Fisher exact test while for categorical data independent samples, the t-test was used. A P-value less than 0.05 was considered significant. BODY.RESULTS: Demographic data regarding patients' age, sex, and location of the tumor (Table 2) were similar in the 2 study groups. Operative techniques were standardized with subsequent similar durations of surgery, types of laparoscopic surgery, and types of incisions utilized in assisted laparoscopies (Table 3). Patients in the enhanced recovery group were compliant to enforced mobilization within 24 hours after surgery (POD 0) while only five patients in the control group were ambulated successfully within that period (P < 0.001). Similarly, patients in the en-hanced recovery group tolerated earlier oral feeding (fluids) without severe PONV (P < 0.001). The times of removal of urinary catheters were similar in the 2 groups (P = 0.202). As the removals of NGTs and intra-abdominal drains were linked to successful oral feeding, noticeable significance was noted as regards their times of removal (P < 0.001). Subsequently, enhanced recovery patients met the pre-emptive discharge criteria earlier and thus left the hospital earlier (P < 0.001) (Table 4). The readmission rates were similar in the 2 groups (11.4% each) (Table 4). Four patients in the conventional group were readmitted: one for urine leak (managed by re-exploration and repair), another 2 for anastomotic 'intestinal' leakage (managed by re-exploration and repair), and the last for a complete bursting of the abdominal wound, which required debridement and closure. The enhanced recovery group also had 4 readmissions: 2 for abdominal collections (managed by tubal drainage), another for a UTI and urine retention (managed by catheterization and urine cul-tures), and the last for severe late-onset PONV (managed conservatively without any further surgical intervention). Surgical complications are presented in Table 5. Regarding postoperative complications, patients in the enhanced recovery group had lower incidences of complications some of which were statistically significant, such as wound infection (P < 0.001), respiratory tract infections (P = 0.025) and anastomotic leak (P = 0.05). Two patients in the conventional recovery group died of pulmonary artery embolism within the first 30 days following surgery (intrahospital mortality) while the enhanced group showed no intrahospital mortality (P = 0.493). BODY.DISCUSSION: Metabolic response to injury has been an intriguing topic to surgeons over time. With an aim to lessen the postoperative morbidity and mortality, a pathway was developed for the patients to allow them to benefit from an understanding of the body's response to surgical trauma in order to accelerate that response [5]. Ongoing clinical trials have explored many of the components of these fast track programs with the aim of formulating a standard protocol for enhancing the recovery and thus minimizing the surgical trauma. Several recent randomized trials have reported the use of 4 to 12 enhanced recovery elements, with a mean of 9, following colorectal resections [6]. Kehlet and Wilmore [7] for example proposed a protocol with 15 elements while Wind et al. [1] used 17 elements in their systemic review. Fifteen items were formulated into an enhanced recovery protocol in our study. Our study showed that employing enhanced recovery protocols helped in boosting the recovery pathway with favorable outcomes, including faster return of GIT functions, reduced postoperative morbidities, and an earlier hospital discharge, after a resection of colorectal cancer. Enhanced recovery trials in the literature show a noticeable reduction in the duration of postoperative hospital stay. The enhanced recovery group in our study had a mean hospital stay of 4.5 days (P < 0.001) compared to the mean hospital stays reported by Wang et al. [8] in China (5.1 ± 3.1 days, P = 0.001), Šerclová et al. [9] in the Czech Republic (7.4 ± 1.3 days, P < 0.001), Vlug et al. [10] in the Netherlands (mean, 5 days; P < 0.001), García-Botello et al. [11] in Spain (4.15 ± 2.2 days, P < 0.001) and lastly Greco et al. [12] in Italy who analyzed the outcome of 16 clinical trials (mean, 5.8 days) [89101112]. Preoperative counseling has been considered an indispensible factor by some authors [8]. Education and counseling stimulates patients to actively participate in the different tasks offered to them, such as early food intake and early ambulation postoperatively, and helps reduce the psychological stress and fear of the suggested procedures. Highlighted counseling of patients preoperatively are also a requirement of the enhanced program in the protocol guidelines carried out in the United Kingdom, France, Italy and Sweden [13141516]. Late removal of NGTs was considered common practice following GIT surgery because of the belief that those tubes facilitated gastric emptying, helped avoid pulmonary aspiration, and decreased the risk of leakage from GIT anastomoses. However, their use in the literature is now decreasing after a meta-analysis done by Verma and Nelson [17] that showed an increased complication rate with their routine use. NGTs were removed in 57.1% of the patients in the enhanced recovery group on the day of surgery while they were removed in 62.9% of the patients in the conventional recovery group on POD 3. In our study, an association was found between the time of removal of NGTs in the enhanced recovery group and the duration of hospital stay (P = 0.032), indicating that the earlier the NGTs were removed, the faster the patients in this group were discharged. One of the important discharge criteria to be fulfilled is sufficient enteral nutrition. In our study, the enhanced group patients were allowed to take oral sips, when tolerated, within the first 24 hours, irrespective of the return of bowel functions. Carbohydrate-rich drinks were given to the patients to increase their caloric intake and hasten recovery. Early oral feeding was found to promote the return of GIT functions and to reduce the incidence of ileus and the risk of infections by improving the immunity [1819]. Although uncommon in surgical practice, fast track protocols in the literature did not recommend the routine use of abdominal drains following colorectal surgeries except in selective circumstances, such as severe bleeding and difficult dissection, and when they were used, they were removed as early as POD 1 [20] or POD 2 [11]. The mean time of removal of drains within the enhanced recovery group in our study was 2.94 ± 1.06 days compared to 9.06 ± 3.76 days in the conventional group. Although meta-analyses have reported no value of the routine use of drains for colorectal surgeries regarding the postoperative morbidities [212223], we found that they were useful in detecting anastomotic leaks. Six out of 8 patients, in both groups, with intestinal leakage showed intestinal contents in the drains. The overall postoperative complications in our study were relatively high compared with those of the studies reported in the literature because minor complications, such as mild wound infection and vomiting, were taken into account. Nonetheless, owing to the utilization of an evidence-based set of elements in the protocols of the enhanced recovery group, we observed a remarkable reduction in the incidence of these complications (25.7% in the enhanced recovery group vs. 65.7% in the conventional recovery group, P = 0.001). Certain morbidities, such as wound infection (P < 0.001), respiratory tract infections (P = 0.025) and anastomotic leakage (P = 0.05), were significantly reduced in the enhanced group. Similarly, Greco's meta-analysis of 16 randomized controlled trials reported a significant reduction in the overall morbidity (P = 0.001). However, it only showed a remarkable decrease in the nonsurgical complications (P < 0.001) while the reduction in surgical complications was insignificant (P = 0.13) [12]. Readmission of a patient was defined as hospitalizing that patient for at least one day after successful discharge. In this study, the reason for readmission was either anastomotic leakage, abdominal wall dehiscence, intra-abdominal collection, or persistent PONV. The difference between the groups in our study was not significant (P = 1.000), which was also the result when compared to different clinical trials applying these fast track protocols [101124]. In conclusion, implementation of enhanced recovery protocols within the health care pathway in our oncology center was successful, leading to more rapid recovery and fewer incidences of complications amongst those patients undergoing major abdominal/pelvic surgeries. The implementation of these protocols allowed earlier restoration of gut functions and faster hospital discharge while having no effect on the re-admission or the intrahospital mortality rate.

TITLE: A comparative study to assess the effect of amikacin sulfate bladder wash on catheter-associated urinary tract infection in neurosurgical patients ABSTRACT.BACKGROUND:: The indwelling urinary catheter is an essential part of modern medical care. Unfortunately, when poorly managed, the indwelling catheter may present a hazard to the very patients it is designed to protect. Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in hospitals and nursing homes. ABSTRACT.AIMS AND OBJECTIVES:: The primary objective was to study the effect of amikacin sulfate bladder wash on CAUTI in neurosurgical patients. The other objectives were to study the various organisms causing CAUTI and their antibiotic sensitivity and resistance pattern. ABSTRACT.MATERIALS AND METHODS:: This was a prospective randomized controlled study performed on 60 patients who met the inclusion criteria at the neurosurgical intensive care of the All India Institute of Medical Sciences between June and December 2006. The patients were randomized into two groups – one was the trial group which received amikacin bladder wash, while the other was the control group that did not receive any bladder wash. ABSTRACT.RESULTS:: Forty percent of the subjects in the control group developed CAUTI, while none of the subjects in study group developed CAUTI. (Fisher's exact test, P value < 0.001) Pseudomonas aeruginosa (51%) was the commonest pathogen. ABSTRACT.CONCLUSIONS:: Amikacin sulfate bladder wash was effective in preventing CAUTI. It can thus decrease the antibiotic usage thereby preventing the emergence of antibiotic resistance. BODY.INTRODUCTION: The indwelling urinary catheter is an essential part of modern medical care and a variety of different indwelling urinary catheters can be used for various purposes. Each year, urinary catheters are inserted in more than five million patients in acute-care hospitals and extended-care facilities.[1] Unfortunately, when poorly managed, the indwelling catheter may present a hazard to the very patients it is designed to protect. Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in hospitals and nursing homes, comprising >40% of all institutionally acquired infections.[2–6] Interventions such as topical meatal antimicrobials, disinfectants added to the urinary drainage bag, and antimicrobials coatings for catheters have not been shown to decrease the incidence of UTI.[7] An effective measure to prevent the CAUTI has not yet been developed. The most common organisms responsible for CAUTI are Escherichia coli, Candida spp., Klebsiella pneumoniae, Streptococcus agalactiae, Enterococcus faecalis, and Pseudomonas aeruginosa. Amikacin has proven to be the most effective antibiotic in preventing the growth of all these species.[8] Previous studies have shown that using individualized regimens for bladder washouts minimizes the infection rate and catheter blockage, thus reducing the need for frequent recatheterization.[9] Since there is no significant systemic absorption of antibiotics used for bladder irrigation,[10] prophylactic bladder irrigations are considered to be safe. We performed an extensive review of literature which did not reveal any study done on the effectiveness of amikacin sulfate bladder wash. BODY.MATERIALS AND METHODS: This study was performed on a total of 60 catheterized patients who were admitted to the Neurosurgery Department of AIIMS, New Delhi, during June–December 2006 and met the inclusion criteria [Appendix 1]. The subject data sheet and procedure of amikacin sulfate bladder wash was developed under the guidance of a guide (Author 3) and co-guides (Authors 2 and 4), and was further validated by six experts from the Departments of Neurosurgery, Microbiology, and Nursing, AIIMS. Informed written consent was taken from the patients or patients' relatives after providing appropriate information to the concerned. Confidentiality of the data was ensured. They were randomized after catheterization to either of the two groups – study and the control groups. Then urine samples were sent within 24 hours for culture and sensitivity (C/S), in case of positive C/S, patients were excluded from the study [Appendix 2]. Study group received amikacin sulfate bladder wash twice daily under strict aseptic precautions and the control group did not receive bladder wash [Appendix 3]. Urine C/S was performed on days 3, 7, and then weekly till the removal of catheter or discharge. Both groups received standard catheter care including perineal care; the only difference was the bladder wash. The researcher (Author 1) performed the bladder wash on all patients to eliminate bias. CAUTI was diagnosed when it met the respective criteria [Appendix 4]. BODY.MATERIALS AND METHODS.STATISTICAL METHODS: Descriptive and inferential statistical methods were used. Data were analyzed using SPSS-10th version. A probability of <0.05 was accepted as significant. For continuous variables having normal distribution, data were summarized using mean±SD, and the groups were compared using independent t test. Range and median were used for all continuous variables having non-normal distribution (age and duration of catheterization) and the two groups were compared using Mann-Whitney U test. Frequency and percentage were used for all categorical variables and the groups were compared using Pearson's chi-square test and Fisher's exact test. The study was conducted following the approval of ethics committee of All India Institute of Medical Sciences (AIIMS). BODY.RESULTS.DEMOGRAPHIC PROFILE OF PATIENTS: Age of the study subjects ranged from 18–68 years [Table 1]. The groups were homogenous in terms of age, sex, level of consciousness, size of catheter and duration of catheterization, and systemic usage of steroids and antibiotics. Bladder wash was well tolerated by all the subjects in the study group. Table 1 Age, sex, and duration of catheterization (n=60) Variables Study group n = 30 Control group n = 30 P value Age (years) 19–65 (38) 18–68 (42.5) 0.399 Males (%) 16 (53) 16 (53) 1.0 Females (%) 14 (47) 14 (47) - Duration of catheterization (days) 3–29 (7) 4–20 (8) 0.823 BODY.RESULTS.INCIDENCE OF CATHETER-ASSOCIATED URINARY TRACT INFECTION AND PREDISPOSING FACTORS: Incidence of CAUTI was 40% in the control group [Figure 1]. None of the subjects from the bladder wash group developed CAUTI. Amikacin bladder wash was effective in preventing CAUTI (P < 0.001). Lower levels of consciousness (i.e. the Glasgow coma scale (GCS)) increased the risk of developing CAUTI (P = 0.026) [Table 2]. Figure 1Effect of amikacin sulfate bladder wash on catheter-associated urinary tract infection Table 2 Risk factors for catheter-associated urinary tract infection (n=60) Factors UTI-positive group n = 12 UTI-negative group n = 18 P value Age (years) 18–68 (46.5) 20–55 (41.5) 0.280 Sex 0.457  Females 7 (50) 7 (50)  Males 5 (31.3) 11 (68.7) Motor score of GCS 0.026 *  M 1–3 5 (83.4) 1 (16.6)  M 4–6 7 (29.2) 17 (70.8) Catheter size 0.131  12 0 2 (100)  14 7 (36.8) 12 (63.2)  16 5 (71.4) 2 (28.6)  18 0 2 (100) Duration of catheterization 6–20 (9) 4–14 (7.5) 0.135 Systemic antibiotics 0.255  Yes 12 (44.4) 15 (55.6)  No 0 3 (100) Steroids 0.643  Yes 7 (36.8) 12 (63.2)  No 5 (45.5) 6 (54.5) BODY.RESULTS.ORGANISM AND SENSITIVITY PROFILE: P. aeruginosa was the most common organism responsible for 51% of CAUTI [Figure 2]. P. aeruginosa was completely sensitive to amikacin sulfate, cefaperazone plus sulbactam, and piperacillin plus tazobactam, while it was completely resistant towards ceftazidime. Figure 2Pathogens causing catheter-associated urinary tract infection BODY.DISCUSSION: The present study is the first of its kind in which the effect of amikacin bladder wash has been analyzed. Efficiency of bladder irrigation using various different solutions have been studied in the past in an attempt to reduce the incidence of CAUTI, majority of the investigators found it to be a time consuming and costly procedure that did not have an impact on CAUTI.[11–15] In contrast, the present study revealed that amikacin bladder wash is effective in preventing CAUTI. In previous studies, the incidence of CAUTI ranged from 11.0–73.3%.[1617] In the present study, incidence of CAUTI was 40% since those who were catheterized for less than three days were excluded in the study. This inclusion criterion might be the chief reason for a higher incidence of CAUTI in the present study. CAUTI increases the burden of the patient in terms of increased morbidity and mortality, prolonged hospital stay, and cost of the tests and medicines.[17–19] Tambyah et al.,[20] found that CAUTI had been responsible for an additional of USD589 per CAUTI in diagnostic tests and in medications. The present study reveals that amikacin sulfate bladder wash is effective in preventing CAUTI. As a vial of amikacin sulfate (500 mg) costs INR58 (approximately USD1.4), this is very cost effective especially in a developing country like ours. Puri et al.,[21] in their study said that the risk was significantly higher for females, elderly patients, critically ill patients, and those on prolonged catheterization. The present study showed only severity of the disease (low motor score of GCS) as a statistically significant risk factor. This might be since a lower GCS corresponds to the severity of tissue injury, where there is hypermetabolism and increased protein catabolism, which eventually leads to decreased immunity that makes the person more susceptible to infections. However, the present study did not show any influence of sex, age, catheter size, duration of catheterization, and systemic use of antibiotics and steroids. This might be because of the small sample size and inclusion of long-term catheterized patients only. Pathogenic organisms responsible for CAUTI and their antibiotic sensitivity pattern vary with time. In a study, Jha et al.,[22] found that most common organisms responsible for CAUTI were E. coli (49%), S. aureus (23%), Proteus spp. (3.6%), Klebsiella (9.71%), Pseudomonas (0.8%), and Citrobacter (2.8%). Whereas in the present study, Pseudomonas (51%), E. coli (17%), Proteus spp., Citrobacter, Klebsiella, and Acinetobacter (8% each) were the most common. Similarly, antibiotic resistance pattern also varied. In their study, Taneja et al.,[23] found the highest frequency of antibiotic resistance was for ciprofloxacin (68.6%) followed by netilmicin (60.7%), ceftazidime (58.8%), imipenem (43.7%), amikacin (43.1%), and piperacillin (39.2%). In the present study, the pattern of antibiotic resistance was ceftazidime (100%), netilmicin (83%), imipenem (75%), ciprofloxacin (75%), and meropenem (60%). In this study, we have shown that amikacin sulfate bladder wash is very effective in preventing CAUTI. Thus it can be included in the routine catheter care, especially if catheterization is needed for more than five days. It is easy to implement and cost effective. The main limitation of this study was the small sample size and the fact that the main researcher was not blinded to the study. BODY.CONCLUSIONS: There is a varying pattern of antibiotic sensitivity and resistance in different institutions. The most important thing to note is the fact that the bacteria have started developing resistance to higher antibiotics. This is very important since the indiscriminate use of antibiotics can lead to resistance, thus potentially endangering the life of a patient. This increasing resistance calls for immediate measures to use methods other than oral or parenteral antibiotics in CAUTI. We believe that this study is important because if used can decrease the development of CAUTI.

TITLE: Genetic ancestry in relation to the metabolic response to a U.S. versus traditional Mexican diet: a randomized crossover feeding trial among women of Mexican descent ABSTRACT.BACKGROUND: Certain populations with a large proportion of Indigenous American (IA) genetic ancestry may be evolutionarily adapted to traditional diets high in legumes and complex carbohydrates, and may have a detrimental metabolic response to U.S. diets high in refined carbohydrates and added sugars. We tested whether IA ancestry modified the metabolic response to a U.S. versus traditional Mexican diet in a controlled dietary intervention. ABSTRACT.METHODS: First and second generation Mexican immigrant women (n=53) completed a randomized crossover feeding trial testing the effects of a U.S. versus traditional Mexican diet. The metabolic response to the diets was measured by fasting serum concentrations of glucose, insulin, IGF-1, IGFBP-3, adiponectin, CRP, IL-6, and computed HOMAIR. Blood collected at baseline was used for genotyping and estimation of African, European, and IA ancestries with the use of 214 Ancestry Informative Markers. ABSTRACT.RESULTS: The genetic ancestral background was 56% IA, 38% European, and 6% African. Women in the highest IA ancestry tertile (>62%) were shorter in height, less educated and less acculturated to the U.S. lifestyle, and tended to have higher waist-to-hip ratio compared to women in the middle and lowest IA ancestry tertiles, respectively. Compared to the U.S. diet, the traditional Mexican diet tended to reduce glucose, insulin, IGF-1, IGFBP-3, and HOMAIR among women in the middle IA ancestry group (IA ancestry ≤45–62%); while having no effect on biomarkers related to inflammation. ABSTRACT.CONCLUSIONS: We observed modest interactions between IA ancestry and the metabolic response to a U.S. versus traditional Mexican diet among Mexican immigrant women. BODY.INTRODUCTION: As Mexican immigrants acculturate to the U.S. lifestyle, they tend to transition from consuming traditional Mexican foods to adopting U.S. dietary patterns (1, 2). U.S. diets commonly consumed by the majority of the population are usually high in processed foods, refined carbohydrates, added sugars, and low in plant foods; while traditional Mexican diets are usually high in fruits and vegetables and complex carbohydrates and legumes rich in dietary fiber (2–5). According to the thrifty gene hypothesis (6), populations with a large proportion of Indigenous American (IA) genetic ancestry, may be evolutionarily adapted to diets high in legumes and complex carbohydrates. Consuming an inexpensive and readily available U.S. diet high in processed foods, refined carbohydrates, and added sugars may lead to a detrimental metabolic and/or inflammatory response placing these groups at a disproportionately higher risk of metabolic disease (7–9). The distribution of genetic ancestry among Mexican immigrants is widely variable, ranging from individuals who are indistinguishable from European ancestry populations to individuals who are indistinguishable from IA populations (e.g., Mayan or Pima) (9–13). In this regard, one of the key questions is the degree to which genetics and environment (e.g., socioeconomic status (SES), diet, and physical activity) contribute to the risk of metabolic disease. Among Mexican immigrants, greater adherence to a U.S. diet has been associated with increased risk of metabolic disease, including, obesity, insulin resistance (IR), systemic inflammation, and breast cancer (3, 5). Whether a greater proportion of IA ancestry in this population modifies the metabolic response to specific dietary patterns kept or adopted by Mexican immigrants has not been previously investigated. In a randomized controlled feeding trial we found that compared to a U.S. diet, a traditional Mexican diet reduced IR and circulating concentrations of insulin-like growth factors (IGFs) among Mexican immigrant women (14). Building on this research, we aimed to evaluate the interplay between genetic ancestral background and diet-related risk of metabolic disease. We hypothesized that IA genetic ancestral background would modify the metabolic response to a U.S. versus traditional Mexican diet among first and second generation, healthy Mexican immigrant women. BODY.MATERIALS AND METHODS.SUBJECTS AND STUDY DESIGN: The study participants and study design have been previously described (14). Briefly, 58 healthy, Mexican or Mexican American women (first and second generation), ages 18–45 years, enrolled in the trial. Out of 58 study participants, 53 completed the trial that consisted two 24-day intervention periods, separated by a washout period of 28 days. In one period they consumed a traditional Mexican diet and in the other a typical U.S. diet. The order of the diets was randomized. Exclusion criteria included elevated fasting glucose (≥100 mg/dl), pregnancy, lactation or cessation of menses, BMI <18.5 or >40 kg/m2, smoking, physician-diagnosed disease requiring dietary restrictions or certain medications, or intake of ≥2 alcoholic drinks per day. The Institutional Review Board and Clinical Trials Office of the Fred Hutchinson Cancer Research Center (FHCRC) approved the study and all participants signed written informed consent. The trial was registered at clinicaltrials.gov (identifier: NCT01369173). Participants provided demographic, acculturation, habitual diet and physical activity through self-administered questionnaires and research staff measured height, weight, waist circumference and hip circumference at baseline using standardized protocols (15). All food and beverages were prepared by the FHCRC Human Nutrition Laboratory. Participants came to the study center three times per week for food pick-ups and body weight measures. Participants were instructed to consume only the foods provided and to return any unconsumed food to study staff. Diets were eucaloric (e.g., diets that provided energy content for weight maintenance) and did not differ in macronutrient composition as a percent of total energy (50% from carbohydrates, 35% from fat, and 15% from protein). Experimental diets differed in foods and beverages such that the traditional Mexican diet included corn tortillas, beans, traditional soups, Mexican-mixed dishes, citrus fruits, vegetables, full-fat milk and Mexican cheeses. The U.S. diet, on the other hand, included processed foods, mixed dishes such as mac and cheese and pizza, refined carbohydrates and added sugars and it was based on the proportion of foods and beverages that contributes the most to Americans daily intake as reported in the National Health and Examination Nutrition Survey (NHANES, 2007–2010) (15). Adherence to controlled diets was carefully monitored and participants' energy intakes were controlled and adjusted as needed to maintain their weight within 3% of baseline measures. BODY.MATERIALS AND METHODS.SAMPLE COLLECTION AND ANALYSES: Blood was collected on the first day and last day of each intervention period after a 12–hour fast by trained research staff. Specimens were locally processed and stored at −80°C until analyses. Glucose was measured on a Roche Module P chemistry autoanalyzer (Roche Diagnostic Inc., Indianapolis, IN) at the Northwest Lipid Research Laboratories (University of Washington, WA). Insulin was measured using a Tosoh 2000 autoanalyzer (Tosoh Biosciences Inc., South San Francisco, CA) at the Diabetes Endocrinology Research Center Immunoassay Laboratory (University of Washington, WA). The rest of the biomarkers assessments and the genotyping were conducted at the FHCRC Biomarker Core Laboratory and the Molecular Epidemiology Laboratories. Immunoassays were used to measured total adiponectin (Total Adiponectin EIA, Aplco), IGF-1 (Human IGF-I Quantikine ELISA, R&D Systems), IGFBP-3 (Human IGFBP-3 Quantikine ELISA, R&D Systems), and IL-6 (Human IL-6 Quantikine HS ELISA, R&D Systems). CRP was measured using CRP (3)-Wide Range reagent (Kamiya Biomedical Company) on Roche Cobas Mira chemistry analyzer with a high sensitivity protocol. The intra-assay CVs were 0.7%, 7.8%, 1.3%, 1.5%, 1.8%, 2.3%, and 3.3% for glucose, insulin, adiponectin, IGF-1, IGFBP-3, IL-6, and CRP, respectively. The details of specimen collection and analysis have been previously described (14). BODY.MATERIALS AND METHODS.GENETIC ANCESTRY ESTIMATION: DNA was extracted from baseline blood using the Qiagen whole blood kit. Genotypes were collected on the Illumina BeadExpress platform using standard protocols. We used a modified version of the AIMs selection algorithm developed by Galanter et al (16, 17), that allows for a 4-way population admixture model, to select a set of 220 AIMs (Supplemental Table 1). African, European, and Indigenous American (IA) ancestry estimates obtained with the AIMs panel were strongly correlated with genome-wide genotype-based estimates in reference samples (Correlation coefficients of 0.88, 0.95, and 0.96 respectively). It is well known that AIMs panels tend to overestimate the influence of minor ancestral components (16, 18). When evaluating the correlation between estimates for individuals who had more than 10% ancestry from a minor component (as estimated by the genome-wide panel), then the correlation between genome-wide estimates and panel estimates increased greatly (>0.90 correlation for African ancestry proportion). A panel of 214 of the 220 selected AIMs passed QC on the BeadExpress genotyping platform. In DNA samples passing QC, the call rate for the 214 AIMs analyzed averaged 99.7% +/− 0.3%, with a minimum of 97.5% of genotypes called. After Bonferroni correction for 214 tests, none of the AIMs showed significant departure from Hardy Weinberg Equilibrium in either reference ancestral population (669 reference European samples or 131 reference Native American samples). Ancestry was estimated using the ADMIXTURE software package v1.23 (19). The ADMIXTURE algorithm was primed with five EM steps at K=3 populations, and converged rapidly. Bootstrap replication using the default 200 bootstrap replicates yielded a standard error of less than 4% for each ancestry component within an individual. Reference populations were included in the analysis to anchor the inferred ancestries on nominally un-admixed individuals (K=3 ancestral populations). These reference populations included HapMap reference panels (YRI, ASW, HCB, JPT and MXC populations), and indigenous populations from the Americas (Nahua, Quechua, Aymara, Zapoteca, Tepehuano, and Maya) (16). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSES: The power analysis for the detection of the interaction of diet and ancestry was conducted using Quanto (version 1.2.4; 2009; University of Southern California, CA), and the coefficient of the interaction was on the scale of standard deviations of the biomarker measurements per unit increase in the ancestry measurement. Based on the calculation, we would have 80% power to detect changes of 2 standard deviations in the biomarkers. Indigenous American (IA) ancestry tertiles were created as: ≤45% IA ancestry (lowest), >45 to ≤62% IA ancestry (middle), and >62% IA ancestry (highest), respectively. Natural logarithmic transformation was applied to insulin, HOMAIR, adiponectin, hs-CR and IL-6 biomarker concentrations to achieve approximate normality. General linear models (unadjusted) were used to compare means of demographic and baseline characteristics across IA ancestry tertiles for continuous variables and chi-squared tests for categorical variables. General linear models adjusted for age, acculturation and BMI were used to compare baseline biomarker concentrations across IA ancestry tertiles, with the Duncan multiple range tests post hoc whenever the overall test indicated a statistically significant difference between IA ancestry tertiles. Linear mixed models were used to test the effect modification of ancestry on the metabolic response to the U.S. versus traditional Mexican diet, including participant as a random effect while treating diet sequence, feeding period, baseline and washout biomarker concentrations, age, acculturation, and BMI, ancestry (as continuous variable) and the interaction between ancestry and diet variables as fixed effects. Also, linear mixed effects models including participants as random effect and diet sequence, feeding period, baseline and washout biomarker concentrations, age, acculturation, and BMI as fixed effects were used to investigate the biomarkers responses to the diet intervention within each category of IA ancestry tertiles. We examined the presence of potential carry-over effects with the inclusion of the diet sequence variable (in addition to other variables including diet and feeding period) as a fixed effect in the mixed effects models. This variable (sequence) was not found to be significantly associated with the biomarkers responses to the diet intervention and hence, no carry-over effect was detected in our analysis. This might be related to the wash-out period of 28 days between each diet period being probably appropriate enough to minimize the carry-over effects. All analyses were performed using SAS (version 9.3; SAS Institute Inc., Cary, NC), all tests were two-sided and P values <0.05 were considered statistically significant. BODY.RESULTS: We first examined the overall distribution of African, European, and IA ancestry. In this sample of 58 healthy, first and second generation Mexican immigrant women, the overall distribution of ancestry was 56% IA, 38% European, and 6% African (Figure 1). We then examined the distribution of demographic characteristics and baseline measures for the women who completed the trial (n=53) across IA ancestry tertiles: 45% IA ancestry (lowest), >45 to 62% IA ancestry (middle), and >62% IA ancestry (highest), respectively (Table 1). Women in the highest IA ancestry tertile (>62%) were shorter in height (P<0.05), less educated (P<0.05), and less acculturated to the U.S. lifestyle (P<0.05), and tended to have higher waist-to-hip ratio (P=0.07) compared to women in the middle and lowest IA ancestry tertiles, respectively. Table 2 shows the baseline (pre-intervention) serum fasting concentrations of biomarkers of metabolic disease risk across IA ancestry tertiles. Women in the highest IA ancestry tertile (>62%) tended to have lower circulating concentrations of IGF-1 (P=0.09), and significantly lower circulating concentrations of IGFBP-3 compared to women in the middle and lowest IA ancestry tertiles, respectively (P<0.05). There was no association of ancestry with the IGF-1/IGFBP-3 ratio and baseline serum concentrations of adiponectin, CRP and IL-6. Results testing whether IA ancestry modified of the exploratory analysis on the metabolic response to the controlled intervention diets (U.S. versus traditional Mexican diet) stratified by IA ancestry tertile are shown in Table 3. Overall, the effect modification associations observed were modest. Compared to the U.S. diet, the traditional Mexican diet tended to reduce glucose (P=0.08), and insulin concentrations (P<0.05) among women in the middle IA ancestry tertile, and tended to reduce insulin concentrations among women in the lowest IA ancestry tertile (P=0.06). Similarly, compared to the U.S. diet, the traditional Mexican diet significantly reduced HOMAIR (P<0.05) and circulating concentrations of IGFBP-3 (P<0.01) and, while tended to reduce IGF-1 (P=0.06) among women in the middle IA ancestry tertile. We found no significant effect of effect modification of ancestry in the response to the diet intervention diets for IGF-1/IGFBP-3 ratio, and serum concentrations of CRP and IL-6 in any of the IA ancestry strata. Lastly, compared to the U.S. diet, the traditional Mexican diet tended to increase adiponectin concentrations among women in the middle IA ancestry tertile (P=0.07). There was no statistically significant difference in the biomarkers response to the diet intervention among the IA strata. In additional analyses, a cross-product interaction term of IA ancestry (as continuous variable) and diet treatment (U.S. vs. Mexican) were tested in adjusted linear mixed models that also included the main effect variables but we found no statistically significant interaction for any of the biomarkers examined (data not shown). BODY.DISCUSSION: In this randomized, crossover feeding trial among first and second generation, healthy Mexican immigrant women, we found a modest effect modification by IA ancestry in the metabolic response to the U.S. versus traditional Mexican diet among women in the middle IA ancestry tertile (>45 to ≤62%). Further, IA ancestry was associated with several baseline demographic and anthropometric characteristics, as well as baseline circulating concentrations of IGF-1 and IGFBP-3, independent of age, BMI, and acculturation status. In cross-sectional analyses of baseline measures (pre-intervention), we found that women with greater IA ancestry tended to have higher adiposity (waist-to-hip ratio), were more likely to be less educated and less acculturated to the U.S. lifestyle compared to women with greater European ancestry, consistent with previous findings (12, 13, 20). Greater IA ancestry tended to be associated with baseline waist-to-hip ratio, but not with BMI, as previously reported among Hispanic women (12, 13, 21) suggesting a greater contribution of visceral fat to the risk of metabolic disease in this ethnic group. Of particular interest is our finding of an inverse association between IA ancestry and baseline circulating concentrations of IGF-1 and IGFBP-3. Insulin-like growth factors (IGFs) are peptides known to promote cellular proliferation of normal breast cells, and therefore, high circulating concentrations of IGF-1 and IGFBP-3 are associated with increased risk of breast cancer (22–24). This is in agreement with studies by Fejerman et al, in which greater European versus IA ancestry among women of Mexican descent was associated with increased risk of breast cancer (25, 26). We previously showed that compared to the U.S. diet, the traditional Mexican diet improved insulin sensitivity and reduced circulating concentrations of IGF-1 and IGFBP-3 (14). Building upon these findings, in the present study we found a modest effect modification of IA ancestry in relation to the metabolic response to the intervention diets. This interaction seemed strongest for insulin sensitivity biomarkers and IGFs and only among women in the middle IA ancestry tertile. These findings suggest that genetic ancestral background may play a role in the metabolic response to specific dietary patterns kept or adopted by Mexican immigrants that can lead to future risk of diabetes. Consistent with our results, in a large observational study evaluating the association between genetic ancestry and risk of diabetes in a multi-ethnic cohort of postmenopausal women (n=16,476) who participated in the Women's Health Initiative, it was found that among Hispanic women, greater IA ancestry was associated with increased risk of diabetes (13). However, since our diet-ancestry findings in this report are limited to women in the middle IA ancestry tertile, it is possible that other factors underlie the observed associations. For example, women in the middle IA ancestry tertile had lower waist-to-hip ratio (non-statistically significant), and were more likely to have more education (P=0.02) and to be more acculturated to the U.S. lifestyle (P=0.04) compared to women in the lowest (≤45%) or highest IA ancestry tertiles (>62%), respectively. It is possible that these differences in SES (higher education status) influence the ancestry-metabolic response association in a manner that could not be captured using these standard methods. Similar results for differences in SES have been shown to be strongly associated with admixture proportions and disease risk in Hispanic populations. For example, in several studies among Hispanics evaluating the association between ancestry and disease risk, adjustments for SES significantly attenuated these associations (13, 27, 28), and in some cases the association become non-significant after adjustments for SES (29). We found no effect modification of IA ancestry in response to the intervention diets for the inflammatory biomarkers examined, including CRP and IL-6. Although, there was a non-statistically significant increase in adiponectin levels (P=0.07) in the Mexican vs US diet but only among women in the middle IA ancestry group. It is possible that the conditions of weight stability in our study played an important role in the null results. Similar to our results and under conditions of weight stability, others have found no diet-induced effect on inflammatory biomarkers, including CRP and adiponectin (30, 31). On the other hand, others have being able to demonstrate a diet-induced inflammatory response in dietary interventions that were coupled with weight loss. These findings suggests that diet-related changes in inflammatory profiles are greater when coupled with weight loss, in part, due to the strong association between adiposity and inflammation (32, 33). Our study is not without limitations. The modest sample size may have precluded us from finding a more robust effect modification of genetic ancestry in relation to the metabolic response to the intervention diets. Despite this limitation, a novel contribution of the present study is the use of AIMs to better understand the interplay between genetic ancestral background and diet-related risk of metabolic disease in a population that displays a great degree of genetic admixture. In conclusion, we observed a modest interaction between IA ancestry and the metabolic response to a U.S. versus traditional Mexican diet among women in the middle IA ancestry tertile, who were also more educated and acculturated to the U.S. when compared to their counterparts. Future experimental and longitudinal studies evaluating the extent by which IA ancestry plays a role in diet-related risk of metabolic disease will be necessary to confirm these results. BODY.SUPPLEMENTARY MATERIAL: 1

TITLE: A randomized, controlled trial to test the effectiveness of a glaucoma patient navigator to improve appointment adherence ABSTRACT.PURPOSE: Patients with glaucoma who do not keep their follow-up eye care appointments are at risk for developing more severe ocular disease. The primary aim of the current study was to evaluate whether the use of a patient navigator altered adherence to follow-up eye care appointments in community-versus office-based settings. ABSTRACT.PATIENTS AND METHODS: Patients diagnosed with a glaucoma-related condition following a comprehensive eye examination at 43 community sites in Philadelphia, PA, USA, were enrolled in this prospective, randomized, controlled trial. Patients were randomized into three groups for a 1-year period: Group 1 (G1) received follow-up eye care in a community-based setting with assistance from a patient navigator; Group 2 (G2) received follow-up eye care in an office-based setting with assistance from a patient navigator; and Group 3 (G3) received follow-up eye care in an office-based setting without a patient navigator (usual care). Adherence rates were compared among these three groups using a chi-squared test at a significance level of 0.05. ABSTRACT.RESULTS: A total of 155 patients with glaucoma-related diagnoses were enrolled. The mean age (±standard deviation) was 71.2 (±10.0) years. Patients were predominantly female (65.8%, n=102/155) and African-American (71.6%, n=111/155). The mean (±standard deviation) number of follow-up visits during the 1-year study period was 1.3 (±1.3) for G1, 1.6 (±1.3) for G2, and 1.3 (±1.1) for G3 (P=0.48). Appointment adherence, defined as attendance of ≥1 follow-up visit, was 69.8% (n=37/53) for G1, 82.5% (n=47/57) for G2, and 73.3% (n=33/45) for G3, (P=0.28). Sub-analysis of adherence rates for patients who attended ≥2 follow-up visits were 91.3% (n=21/23) for G1, 74.3% (n=26/35) for G2, and 66.7% (n=18/27) for G3, (P=0.11). ABSTRACT.CONCLUSION: Help from a patient navigator did not increase the likelihood of keeping ≥1 follow-up appointment in an office-based setting. Adherence rates for follow-up appointments reached close to 70% or above in a self-selected patient population. BODY.INTRODUCTION: Glaucoma, a progressive optic neuropathy, is the leading cause of irreversible blindness worldwide and affects over 65 million Americans.1,2 Most people are unaware they have glaucoma in early stages.3,4 Appropriate treatment and follow-up eye care are crucial in preventing vision loss due to glaucoma, as treatment can prevent blindness.3–7 While patient compliance is often difficult to identify precisely, studies have estimated nonadherence to follow-up eye appointments as high as 43% among those diagnosed with glaucoma who were prescribed intraocular pressure-lowering eye drops.8,9 A wide range of risk factors for low adherence to follow-up eye examination appointments has been identified, including advanced age, African-American or Latino ethnicity, and diagnosis of depression.10–13 Other factors associated with poor follow-up adherence, especially among the underserved, include poor health literacy and difficulty navigating the complex healthcare system.11,12,14,15 In previous studies, patients self-reported that a lack of transportation, difficulty affording copayment, uncertainty of where to find an ophthalmologist, a busy schedule, fear of an ocular diagnosis, and fear of treatment were all barriers to obtaining regular glaucoma-related eye examinations.3,8,9,11,12,14–16 Research has suggested many strategies to improve patient adherence, but one of the most promising options involves the addition of a dedicated healthcare worker to help and encourage patients to attend eye examination appointments.2–4,17–19 The introduction of this third party, often referred to as a patient navigator, is an intervention directly aimed at reducing the most frequently reported barriers preventing patients from receiving optimal care.20 The patient navigator concept was first introduced in the 1990s by Dr Harold Freeman to help females obtain breast cancer screening.20,21 Patient navigation programs have been shown in several randomized, controlled trials to improve appointment adherence, initiation of treatment, and patients' quality of life.21,22 Patient navigators have been utilized in a variety of medical fields with the navigators serving to help patients schedule doctor's appointments, determine insurance requirements, arrange transportation, make appointment reminder calls, and accompany patients to their appointments.20–24 However, there is limited data on the use of a patient navigator in an ophthalmic patient population and its efficacy in improving follow-up adherence and preventing vision loss, particularly among patients diagnosed with glaucomatous diseases. To determine the impact of a patient navigator on glaucoma eye care follow-up adherence, we initiated a 1-year prospective, randomized, controlled trial in an urban community setting versus office-based setting. BODY.PATIENTS AND METHODS.INSTITUTIONAL REVIEW BOARD APPROVAL: This trial was approved by the Institutional Review Board of Wills Eye Hospital and conducted in accordance to the Declaration of Helsinki. BODY.PATIENTS AND METHODS.PATIENT RECRUITMENT: This study enrolled 155 patients who were diagnosed with suspected glaucomatous disease, anatomically narrow angle, or glaucoma. All patients were recruited from a prior community outreach initiative (Philadelphia Glaucoma Detection and Treatment Project), funded by the US Centers for Disease Control and Prevention.25,26 During this program, a total of 1,649 individuals at high-risk for eye disease were examined, diagnosed, and given treatment recommendations by an ophthalmologist at 43 community sites throughout the Philadelphia region. Of this group, 645 patients were diagnosed with glaucoma-related conditions and required additional follow-up; they were eligible to participate in the present study (Figure 1). Beginning in August 2014, these eligible patients were sent three recruitment letters and called up to six times over the course of 4 months by study coordinators. This communication also included educational messages about the importance of ongoing follow-up eye examinations once diagnosed with a glaucoma-related condition. Eligible patients did not participate for a number of reasons. They were unreachable, because of either outdated information or unanswered voice mails. The remaining patients either opted out on their own or were unable to participate due to health or living situation. A small portion of patients were visiting other eye care providers or believed they did not have glaucoma. BODY.PATIENTS AND METHODS.STUDY DESIGN: Subjects were equally randomized into one of three groups for a 1-year period (Figure 1): Group 1 (G1) received follow-up eye care in a community-based setting with assistance from a patient navigator; Group 2 (G2) received follow-up eye care in an office-based setting with assistance from a patient navigator; and Group 3 (G3) received follow-up eye care in an office-based setting without a patient navigator (usual care). Of the prior 43 community sites in the Philadelphia Glaucoma Detection and Treatment Project, four sites were selected for subjects randomized to G1 for follow-up eye care in a community-based setting with assistance from a patient navigator. In order to maximize patient accessibility, these four sites were chosen in different regions of Philadelphia. Wills Eye Hospital was used as the office-based site for patients randomized to G2 and G3. Over the course of 1-year, subjects from all groups were offered a baseline eye examination and one or more follow-up visits depending on their diagnosis. Follow-up recommendations were based on the Practice Pattern Guidelines for Glaucoma from the American Academy of Ophthalmology (Table 1), but varied depending on the glaucoma specialists' clinical judgments.27 BODY.PATIENTS AND METHODS.BASELINE VISIT AND ASSESSMENT MEASURES: Baseline eye examinations were conducted at all locations by an intervention team, consisting of an ophthalmologist, a mobile unit coordinator, two ophthalmic technicians, a study coordinator, a community health educator, and a patient navigator. A mobile unit was used to transport the entire team and all screening equipment to the community sites. All subjects were formally enrolled in the study after obtaining written informed consent. All subjects received a comprehensive ophthalmic examination including: 1) ocular and medical history, 2) best corrected visual acuity measurement using Snellen eye charts (Precision Vision, La Salle, IL, USA), 3) slit-lamp biomicroscopy (Haag-Streit, Koeniz, Switzerland), 4) gonioscopy, 5) intraocular pressure measurement using Goldmann applanation tonometer (Haag-Streit), 6) undilated optic nerve evaluation, and 7) visual field test using the Octopus visual field analyzer (Haag-Streit). Optic disc color photography and central corneal thickness measurements were documented if not available from prior records. An ophthalmologist reviewed the test results with each subject and provided a diagnosis, recommended treatment, and follow-up plan within 1-year based on recommended guidelines (Table 1). During the baseline visit, the National Eye Institute-Visual Function Questionnaire-25 (NEI VFQ-25) was administered to all subjects to access barriers to eye care.28–30 Subjects also were given the Geriatric Depression Scale-15 (GDS-15), a validated 15-question assessment of depression in older adults that correlates with the severity of glaucoma.31,32 Complete results for both NEI VFQ-25 and GDS-15 are being published elsewhere. BODY.PATIENTS AND METHODS.FOLLOW-UP VISITS/USUAL CARE: Subjects who completed the baseline assessment and examination visit were scheduled for follow-up visits at one of the community sites or at Wills Eye Hospital, depending on their randomization. All subjects received a reminder phone call the day before their follow-up appointment. If the subject did not answer, a message was left on his or her phone. There was no charge or copay for any of the study visits. Follow-up visits consisted of an ocular and medical history review, an assessment of medication adherence, and a complete eye examination. Patients were given a brochure about glaucoma explaining the importance of regular eye examinations and treatment options. Subjects required a minimum of one follow-up visit within 1-year of their baseline visit regardless of their diagnosis or treatment plan. All subjects received instructions on proper eyedrop administration and assistance with medication refill and adherence, if needed. For subjects without prescription drug coverage that could not afford their medications, study coordinators helped them to receive either discounted medications through GoodRx.com or free medication through the pharmaceutical companies' patient assistance programs. All subjects were called to confirm that they filled their prescription following study visits. Glaucoma laser treatment (selective laser trabeculoplasty or laser peripheral iridotomy) was free of charge for all patients and performed by the ophthalmologist at all sites. If operating-room, nonlaser glaucoma surgery was recommended and a subject did not have insurance, a study coordinator would assist him or her to obtain the necessary health insurance. If a subject had health insurance that was not accepted at Wills Eye Hospital, a staff member would locate an ophthalmologist who accepted his or her insurance. In addition, staff members helped subjects with referrals to other nonglaucoma ophthalmology services, as needed. Of the subjects who were non-English speakers, interpreters (who spoke primarily Spanish or Mandarin Chinese, but also occasionally French or Arabic) were provided to assist with recommended follow-up visits, medication refills, and laser therapy. BODY.PATIENTS AND METHODS.PATIENT NAVIGATOR/INTERVENTION: Following the baseline visit, subjects in G1 (examined at community site) and G2 (examined at office-based site) were assisted by a patient navigator to identify barriers and improve appointment attendance. Two patient navigators were trained at the Wills Eye Hospital's Glaucoma Research Center on the potential barriers faced by subjects and the methods and resources to address these needs. Patient navigators employed the "teach-back" method to ensure that subjects understood what was explained to them by having them repeat what they had just heard. Navigators resolved subjects' transportation barriers by helping subjects with their access to public transportation or door-to-door transportation provided by the Philadelphia regional transportation services, if needed. In addition, navigators provided parking reimbursements for subjects seen at the office-based Wills Eye Hospital site. Subjects in G1 and G2 also received a reminder letter before their scheduled appointment and a retention letter for any missed appointments, along with the appointment reminder phone call that all subject groups received (Table 2). BODY.PATIENTS AND METHODS.PATIENT REMUNERATION: All subjects received a US$20.00 gift card from a local pharmacy for study enrollment and an additional US$10.00 gift card after completion of the final visit. BODY.PATIENTS AND METHODS.DATA COLLECTION: FileMaker Pro 11 (FileMaker Inc., Santa Clara, CA, USA), which automatically generates unique participant identifications, was used to track follow-up appointment adherence and scheduling. Specifically, the number of follow-up visits that each subject scheduled, attended, rescheduled, cancelled, and missed were recorded. Additionally, all attempts to contact each subject and reasons for cancellations were documented. Information within FileMaker Pro was exported into Statistical Package for Social Science (SPSS, version 19, IBM Corporation, Armonk, NY, USA) for subject data analysis. The results of NEI VFQ-25 and GDS-15 were also exported into SPSS by the data manager. BODY.PATIENTS AND METHODS.RANDOMIZATION AND STATISTICAL ANALYSIS: Subjects were randomized by a biostatistician who had no involvement in delivering any intervention using cluster randomization so that sites were randomized to one of the three groups in a 1:1:1 ratio. Due to differences in sample sizes by site, the number of subjects in each group was not the same. The original sample size goal of 345 randomized subjects provided 90% power to detect a difference in follow-up rates between any two groups of 25% assuming an alpha of 0.017, an intracluster correlation of 0.01, and an overall rate of adherence of 50%. Our final sample size of 155 provides ~55% power to detect the same difference under the same assumptions. Appointment adherence was defined as attendance of one or more of the recommended follow-up visits. Subjects who did not attend any appointments after the baseline appointment were consequently classified as nonadherent. The appointment adherence rates were compared among the three groups using chi-square test at a significance level of 0.05. Poisson regression models were also applied to estimate the relative ratio of the patient navigators' interventions and the baseline individual characteristics believed to be associated with follow-up appointment adherence, including: age, gender, race, ethnicity, glaucoma diagnosis, and GDS-15 and NEI VFQ-25 scores. BODY.PATIENTS AND METHODS.RANDOMIZATION AND STATISTICAL ANALYSIS.SECONDARY ANALYSES: We evaluated how community- and office-based interventions affected disease progression by assessing changes in clinical diagnoses. Subjects were considered to have disease progression if their diagnosis was glaucoma suspect at baseline and changed to glaucoma by their last follow-up appointment. Subjects who did not attend at least one follow-up appointment or were diagnosed with anatomically narrow angle were excluded from this analysis. BODY.RESULTS: A total of 155 subjects with glaucoma-related diagnoses were enrolled. The mean age (±standard deviation [SD]) was 71.2 (±10) years. Table 3 summarizes subjects' baseline demographic and clinical characteristic data by groups. Subjects were predominantly female (65.8%, n=102/155) and African-American (71.6%, n=111/155). There were more African-Americans in G1 (P<0.001), and more nonmarried subjects in G2 (P=0.005). The mean (±SD) number of follow-up visits during the 1-year study period was 1.3 (±1.3) for G1, 1.6 (±1.3) for G2, and 1.3 (±1.1) for G3 (P=0.48). Appointment adherence, defined as attendance of at least one or more follow-up visits, was 69.8% (n=37/53) for G1, 82.5% (n=47/57) for G2, and 73.3% (n=33/45) for G3, (P=0.28). A subanalysis was conducted to analyze whether subjects attended ≥ follow-up visits. The results are: 91.3% (n=21/23) of G1, 74.3% (n=26/35) of G2, and 66.7% (n=18/27) of G3 attended ≥2 follow-up visits (P=0.11). Overall, 73.3% (n=113/154) of the subjects were either single, separated, divorced, or widowed, while 54.5% (n=84/154) reported that they lived alone at the time of their baseline visit. Regarding education completed, 77.8% (n=119/153) had received at least 12 or more years of education. In all groups, 41.2% (n=63/153) of subjects felt that it was somewhat or very difficult to pay for basic necessities, such as food, housing, heating, and medical care; of them 88.5% (n=52/63) were African-Americans, 11.1% (n=7/63) Caucasians, and 6.3% (n=4/63) Asians. The mean (±SD) NEI VFQ-25 overall scores (on a scale of 1–100) were 83.98 (±14.75), 80.97 (±15.85), and 77.29 (±20.92) for G1, G2, and G3, respectively. The mean (±SD) GDS-15 total scores were 2.11 (±3.02), 2.18 (±2.43), and 3.28 (±3.25) for G1, G2, and G3, respectively. Table 4 shows the factors contributing to subjects' adherence to follow-up appointments, of which age was the only statistically significant variable. In terms of change in diagnosis from glaucoma suspect to glaucoma for subjects who attended at least one follow-up appointment at least 5 months after the baseline visit, 12 (80%, 95% confidence interval [59.8–100]) out of the 15 glaucoma suspect subjects remained glaucoma suspects in G1, while 19 (79.2%, 95% confidence interval [62.9–95.4]) out of 24 patients in G2, and ten (76.9%, 95% confidence interval [54.0–99.8]) out of 13 subjects in G3 remained glaucoma suspects and did not show disease progression. When disease severity was compared with appointment adherence, 57 (77%) out of a total of 74 glaucoma suspect subjects (less severe diagnosis) attended at least one scheduled follow-up appointment, while 32 (71.1%) out 45 glaucoma subjects (more severe diagnosis) attended their follow-up appointment(s). BODY.DISCUSSION: This study was a prospective, randomized, control trial to determine if the addition of a patient navigator in high-risk subjects diagnosed with glaucoma or a glaucoma-related condition (including glaucoma suspect and anatomically narrow angle) improved follow-up appointment adherence. Over the course of 1 year, 155 subjects were enrolled in this study, all of whom were diagnosed with or were suspected of a glaucoma-related eye condition in a prior community screening project.25,26 The primary outcome was adherence to follow-up eye appointments after a baseline assessment and eye examination visit. The three randomized study groups were demographically and clinically comparable, and results showed no statistically significant difference in appointment adherence rates between community- and office-based settings with and without a patient navigator at the first follow-up visit. In the intervention groups, patient navigators addressed transportation needs and sent additional reminder (before appointment) and retention (after missed appointment) letters. It is possible that the mitigation of transportation barriers and subject forgetfulness diminished the difference between community- and office-based care. However, while the intervention groups did receive additional support, the lack of differences between the intervention and usual care groups as well as the relatively high rates of follow-up appointment adherence in all groups, particularly among the females and African-Americans, could be due to the patient-centered study design. These patient-centered approaches included: all subjects received a reminder phone call prior to their scheduled appointment, per the standard of care protocol at Wills Eye Hospital. There were no fees for eye examinations or laser therapy, and medication adherence education and assistance. Medication financial support was provided to all subjects, as needed. Staff members also helped all subjects with obtaining health insurance, made nonglaucoma ophthalmic referrals, and provided interpreters as needed. All subjects also received financial compensations in the form of gift cards after randomization and at the final study visit. Several studies have indicated the need to address the lack of follow-up appointment attendance in an office-based setting among subjects diagnosed with glaucoma.14,15,33 Among these, poor follow-up adherence rates were highest among African-Americans, subjects aged 50–80 years, and those diagnosed with advanced stages of glaucoma.10–12,15,16 One of these studies included: a community-based eye screening program in Baltimore, supported by Hoffberger Family Philanthropies, which targeted a high-risk population similar to that found in our present study.9 The Hoffberger Program conducted community-based glaucoma screening examinations with a 59% rate of nonadherence to their follow-up visit. Among the most common reported reasons for lack of attendance were: no reminder letter received, forgot to come to appointment, lack of transportation, and fear of cost. Our study design addressed these barriers by providing reminder phone calls and giving free study visits to all subjects, which may explain the relative high rates of follow-up appointment adherence across all groups. The extra reminder and retention letters, along with the transportation assistance provided by patient navigators, did not appear to have made any additional impact on appointment adherence. One secondary outcome analyzed in our study was change in clinical diagnosis from baseline to the last follow-up appointment. Glaucoma diagnosis changes (glaucoma suspect to glaucoma) were comparable among the three groups, showing no differences between patient navigator and usual care groups, or community- versus office-based sites. Moreover, despite most of the glaucoma suspect subjects maintaining the same diagnoses within 5 months of their baseline visits, eleven (21.2%) out of 52 total glaucoma suspect subjects were diagnosed with glaucoma. This demonstrated that follow-up appointment adherence is particularly important for glaucoma suspect subjects, as early detection and treatment is vital to slowdown disease progression and prevent blindness.3–7,15 As a whole, disease severity did not correlate with an increase in follow-up eye examination appointment adherence, as suggested in previous published papers.9 Our results show that appointment adherence rates were relatively high and similar between all subjects diagnosed as glaucoma suspects (77%, n=57/74) and with glaucoma (71.1%, n=32/45). This high rate of follow-up is unexpected since our study patients had overall high NEI VFQ-25 scores, which indicated that the self-reported, subjective visual function was relatively high among our study population. A possible explanation for this unexpected result may be due to the effect of aforementioned patient-centered study design across all groups. Based on the relative ratios provided in Table 4, age >65 years may be associated with lower appointment adherence rates. This statistically significant result is contrary to previous reports that indicate younger subjects, male, minorities (especially African-Americans or Latinos), those with a less severe diagnosis or those who live far away from eye care providers are less likely to adhere to scheduled eye examination appointments.10 BODY.DISCUSSION.LIMITATIONS: In addition to the previously noted patient-centered study design, there are several limitations that may have contributed to our results and warrant discussion. All of our study subjects were recruited from and had completed the Philadelphia Glaucoma Detection and Treatment Project. Appointment adherence patterns were not analyzed in the previous study. Subjects in the current study were therefore self-selected and may not reflect the general patient population; however, they still represent a high-risk, targeted group. In addition, all the subjects recruited were familiar with the research staff, which likely increased appointment adherence across all groups. It is well known that a robust patient–provider relationship improves eye care and follow-up appointment adherence.10 Accordingly, it is possible that a sufficiently strong research team–patient relationship had developed after the 2-year Philadelphia Glaucoma Detection and Treatment Project. Lastly, the definition of appointment adherence has not been consistent or universally agreed upon by most researchers. We based our definition of appointment adherence, which was attendance of at least one or more scheduled follow-up appointments, on the fact that the glaucomatous conditions of our study subject population varied greatly. Actual appointment follow-up recommendations hence were often made based on clinical needs and not on study protocol guideline of returning in 6 months or 1 year. For example, some subjects were recommended to follow-up once a year, while others may have been asked to return in 1 month, especially if they had consented to receive laser therapy. This resulted in some subjects with more scheduled appointments (the most was 6 appointments) in 1 year. Undoubtedly, if the appointment adherence definition were to change, the result could potentially vary. Moreover, adherence was only measured in regard to appointment adherence, which is different from the common use of the term "adherence" to refer to taking prescribed medications. Adherence to glaucoma treatment (ie, prescribed eye drops) was not tracked throughout the course of this trial. According to Ung et al, patients prescribed glaucoma medications were less likely to attend their follow-up appointments.16 Glaucoma severity range of these subjects was not included in the current investigation. Further study is needed to evaluate the role of a patient navigator in subjects with varying degrees of glaucoma. Furthermore, our 1-year study period may be insufficient time to judge follow-up appointment adherence rates for certain subjects, since ongoing follow-up appointments are often required for those diagnosed with glaucoma. BODY.CONCLUSION: In summary, this prospective, randomized, controlled study provides useful information about strategies to increase follow-up appointment adherence rates in a high-risk, targeted population at high-risk for glaucoma through patient navigators at community-based locations. Subjects at high-risk for glaucoma with poor access to eye care due to accessibility and personal issues, such as finances, transportation, or forgetfulness, were enrolled, monitored, and treated. The results of this study suggest a comprehensive, patient-centered approach can improve access to eye care and follow-up appointment adherence for glaucoma care in a high-risk population. Although adherence rates to follow-up recommendations were similar between community- and office-based settings, overall adherence rates in the study were relatively high among this high-risk population, reaching close to 70% or above across all groups. We believe that this is due primarily to the fact that subjects self-selected to participate in the study, and all subjects received phone call reminders, no charge for examinations, and gift cards for participating.

TITLE: Comparison of sensory attributes and immediate efficacy of intranasal ciclesonide and fluticasone propionate in allergic rhinitis: A randomized controlled trial ABSTRACT.OBJECTIVES:: Intranasal corticosteroids (INCs) are the most effective modality for treating allergic rhinitis and their sensory attributes are important in patient compliance. This study aimed to compare the sensory attributes (scent, immediate taste, aftertaste, run down to throat, nose run off, soothing feel, nasal irritation, and urge to sneeze) and immediate response to the new intranasal steroid, ciclesonide (CIC), with fluticasone propionate (FLP) in allergic rhinitis. ABSTRACT.MATERIALS AND METHODS:: A randomized, double blind, single dose, crossover study was done with 74 patients presenting with acute allergic rhinitis. Eligible subjects were randomized in 1:1 ratio to one of the two treatment sequences – CIC followed by FLP or vice versa. Sensory attributes were assessed using a questionnaire to score each item on a seven-point Likert scale, immediately and 2 min after dosing. Total nasal symptom score (TNSS) was calculated to evaluate immediate efficacy 10 min after first drug administration. Overall preference was recorded 10 min after the second drug administration. Patients were queried about treatment emergent adverse events following study drug administration and also 24 h later over the phone. ABSTRACT.RESULTS:: Patients (58% males; pooled median age 32 years [Interquartile range, IQR, 25–41]; pooled median symptom duration 24 months [IQR 12–72]) preferred FLP over CIC nasal spray overall (55.41% vs. 25.68%, P = 0.007) and also with respect to attributes of scent, soothing feel, and nasal irritation. There was no statistically significant difference in immediate efficacy. Two patients reported mild headache following CIC first, while three felt mild headache, one dizziness, and one nasal congestion following FLP first administration. There were no delayed adverse events. ABSTRACT.CONCLUSIONS:: There was no difference in immediate outcome following use of either of the two INCs. FLP was preferred over CIC with respect to scent, soothing feel and nasal irritation, and also overall. There were no significant adverse events. BODY.INTRODUCTION: Allergic rhinitis is an inflammatory disorder of the nasal mucosa induced by allergen exposure triggering IgE-mediated inflammation. Clinically, it is characterized by four major symptoms – rhinorrhea, sneezing, nasal itching, and nasal congestion. Allergic rhinitis is a global health problem. A large section of the Indian population suffers from allergic rhinitis and symptoms can occur in up to 80% of asthmatic adults.[12] Intranasal corticosteroids (INCs) are recognized to be the most effective treatment modality for allergic rhinitis.[3] However, patient compliance is central to their successful use. Patient preference for a particular INC is largely influenced by the sensory attributes of the nasal spray, which in turn is largely dependent on formulation factors. For instance, preservatives in the spray formulation may produce nasal irritation, reducing tolerability. Other excipients may impart unacceptable taste or odor to the formulation. Further, the relative osmotic pressure or tonicity of the formulation can modulate nasal retention and absorption, thereby potentially influencing clinical efficacy. Intranasal ciclesonide (CIC) was introduced in the Indian market in 2008. Since sensory attributes of INCs play an important role in their acceptance and therapeutic compliance,[4] we aimed to assess the sensory attributes along with the immediate efficacy of ciclesonide in comparison to intranasal fluticasone propionate (FLP) that is being used for a longer time in India. BODY.MATERIALS AND METHODS: We conducted a randomized, double blind, single dose, crossover study at the Otorhinolaryngology out-patient department of Institute of Postgraduate Medical Education and Research (IPGME&R) and SSKM Hospital, Kolkata, India, between December 2009 and May 2010. Prior to study initiation, the protocol and informed consent documents were approved by the institutional Ethics Committee. All participants (12 years or above in age) had symptoms of allergic rhinitis for at least 1 year and presented with a total nasal symptom score (TNSS) of six or more plus an individual score of 2 or more for rhinorrhea or nasal congestion. Patients were excluded if they provided history of other nasal pathology (including polyps or respiratory tract malformations), recent nasal biopsy, nasal trauma or surgery, atrophic rhinitis, or rhinitis medicamentosa within last 60 days prior to screening; history of respiratory infections within last 14 days; and use of intranasal/oral corticosteroids, antihistamines, nasal decongestants, or other drugs used for rhinitis within last 7 days. Also excluded were expectant and nursing mothers and subjects with a history of smoking and/or substance abuse. Written informed consent was obtained from each study participant. In the case of enrolled adolescent subjects (between 12 – 18 years), consent was provided by the legal guardian while they themselves provided assent. The trial comprised a pretreatment screening period to verify inclusion and exclusion criteria, two single-dose treatments separated by 30 min, and a final follow-up over phone after another 24 h. Eligible patients were randomized in 1:1 ratio (simple randomization using a computer generated list) to one of two treatment sequences - aqueous suspension of FLP for intranasal administration (trade name: FLUTICONE, marketed by German Remedies, Mumbai, India) administered as two sprays in each nostril, total dose 200 mcg; followed by aqueous suspension of CIC (trade name: CINASE, marketed by German Remedies, Mumbai, India) administered as two sprays in each nostril, total dose 200 mcg; or the reverse sequence. A 30 min interval was kept between the two treatments. The dosing scheme is shown in Figure 1. Figure 1Sequence of study drug administration and related assessments. (CIC – ciclesonide, FLP – fluticasone) Ten minutes before receiving the study drug, participants cleansed their mouth by eating one unsalted biscuit and taking several swallows of water at room temperature. To cleanse the nasal passages of any secretions, they were required to sniff a wool swatch. This washout protocol was repeated before the cross-over drug administration. To ensure blinding, patients were blindfolded during product administration. In addition, the spray formulation labels were masked, they were coded as A or B, and administered under the supervision of an investigator who was not involved in generation of the randomization sequence. The code to be used first (A or B) was concealed in opaque serially numbered envelopes and revealed just prior to drug use. Thus, both patients and interviewing investigators remained unaware of study drug identity during administration as well as evaluation. Following each treatment, patients rated seven sensory attributes – scent, immediate taste, aftertaste, run down into the throat, run off from nose, soothing feel, sneezing urge, and nasal irritation – immediately and after 2 min. These were recorded through 13 questions, response to each was rated on a 7-point Likert scale (score 0–6, where 0 denoted absence or most positive response, 6 denoted most negative response, and 3 represented a neutral, i.e., neither positive nor negative response). This questionnaire [Table 1] was adapted from previous studies comparing sensory attributes of INCs.[4–7] Immediate efficacy of treatment was assessed by TNSS that rates each of five items – rhinorrhea, nasal congestion, sneezing, nasal itching, and ocular itching – on a scale of 0 to 3 [0: absent, 1: mild, 2: moderate, and 3: severe]. This was recorded before and 10 min after first drug-administration. Lastly patients were asked about overall preference, 10 min after the second drug administration. Table 1 The sensory attributes questionnaire For safety assessment, physical examination was conducted and vital signs (pulse, blood pressure, and breathing rate) were measured at screening and 30 min following study drug administration. Treatment emergent adverse events were captured by careful history taking and clinical examination. A follow-up assessment was conducted 24 h later over phone for delayed adverse events. The safety population was defined as all patients who received at least one actuation of the study spray formulations. BODY.MATERIALS AND METHODS.SAMPLE SIZE AND STATISTICAL ANALYSIS: It was calculated that 37 subjects would be required per group for the study to achieve 80% power, at 5% probability of type I error, in detecting a difference of 1 in TNSS following study drug administration. This calculation assumed a pooled standard deviation of 1.5 for TNSS. Numerical variables were compared between the two arms by Mann–Whitney U test. Individual sensory attributes were summarized as the proportion of subjects experiencing or expressing satisfaction with respect to that attribute. Independent proportions were compared between groups by Chi-Square test or Fisher's exact test as appropriate. Paired proportions were compared by McNemar's Chi-square test. Two tailed P-value <0.01 was considered to be statistically significant. Statistica version 6 (Tulsa, Oklahoma: Stat Soft Inc., 2001) software was used for analysis. BODY.RESULTS: Ninety subjects were screened, of whom 74 (82.22%) satisfied the selection criteria and consented to participate. There were no dropouts. The median (interquartile range) age of the participants who received CIC first was 32 (23–42) years while the median symptom duration was 24 (12–72) months. The corresponding figures for the arm receiving FLP first was 32 (28–39) years for age, and 24 (12–84) months for symptom duration. There were 22 (59.46%) and 21 (56.76%) males, respectively, in the two arms. These differences were not statistically significant. The pooled figures for the two arms were 58% males; median age 32 (25–41) years and median symptom duration 24 (12–72) months. The predominant presenting symptoms were rhinorrhea, nasal congestion, and sneezing. Regarding sensory attributes, patient ratings favored FLP over CIC with respect to satisfying scent, soothing feel and nasal irritation. Thirty seven (50%) patients preferred FLP compared to 6 (8.11%) preferring CIC with regards to satisfying scent (P < 0.001); 42 (56.76%) reported FLP to provide a more soothing feel compared to 15 (20.27%) preferring CIC in this regard (P < 0.001). Only 1 (1.35%) patient reported nasal irritation following FLP administration, in contrast to 21 (28.38%) following ciclesonide use (P = 0.002). Although, only one patient (1.35%) had urge to sneeze following FLP use, compared to 10 following CIC (13.51%), this difference was not statistically significant. The number reporting immediate taste, aftertaste, run down to throat and run off from nose were less with CIC compared to FLP, but these differences were also insignificant statistically. Figure 2 depicts the paired comparisons with regards to the individual sensory attributes. Figure 2Comparison of sensory attributes irrespective of the sequence of administration. No. of subjects have been shown (n = 74). Difference with respect to scent, soothing feel and nasal irritation are statistically significant (McNemar's Chi-square test) Regarding immediate efficacy, both CIC and FLP decreased TNSS compared to baseline, as well as individual symptom scores in majority of the subjects, within 10 min of administration. The median (interquartile range) TNSS declined from 8 (7–9) at baseline to 3 (2–4) after drug administration in subjects receiving ciclesonide first. In the fluticasone first arm, the corresponding decline was from 8 (6–10) to 2 (2–4). This difference was not statistically significant. Differences were also not significant when the proportions reporting decrease in individual symptom scores, rather than total score, were compared [Figure 3]. Figure 3Comparison of number of subjects (n = 37 for each treatment sequence group) reporting reduction in intensity of symptoms compared to baseline. The differences are not signifi cant statistically (Fisher's exact test) Overall, out of 74 subjects, irrespective of sequence of administration, 41 (55.41%) preferred FLP, while 19 (25.68%) preferred CIC. The rest had no selective preference. This difference was statistically significant (P = 0.007). The overall incidence of adverse events during treatment was 9.46% (7/74). This count excludes events like nasal irritation that are already covered under sensory attributes assessment. Two patients reported minor headache following CIC first, while three felt minor headache, one dizziness, and one nasal congestion following FLP first administration. No delayed adverse events were reported at the 24 h follow-up interview conducted over the phone. There were no adverse events that inconvenienced a subject to more than a mild degree. BODY.DISCUSSION: Allergic rhinitis is a common atopic condition of the nasal mucosa affecting a large number of Indians. The effect of rhinitis on the quality of life is remarkable. Standardized quality of life measures indicate that over 60% of allergic rhinitis patients are perturbed while symptomatic. Allergic rhinitis typically presents at a younger age[8] – the pooled median age in our study was 32 years. In a large survey of allergic rhinitis sufferers, participants reported nasal congestion as the most bothersome complaint.[9] This was also applicable and true in our study subjects. Intranasal corticosteroids are now the mainstay in treatment of allergic rhinitis.[10] This study was designed to assess overall preference based on patients' perceptions of the sensory attributes related to intranasal steroid use and the immediate efficacy of the steroid. We have not found any study comparing sensory attributes and immediate efficacy of intranasal ciclesonide and fluticasone propionate. In our study both drugs were delivered as aqueous nasal spray formulation that allows better dispersal and is considered to be less irritating to the nasal mucosa. Although the full effects of INCs take several hours to develop, their role in the inflammatory process suggests that onset of action would be rapid and possibly within minutes. It is reported that histamine levels in nasal secretion lavage fluid of allergic rhinitis patients can be counteracted within 30 min by intranasal administration of mometasone furoate.[8] This prompted us to undertake assessment of immediate efficacy of the study drugs, although this will not be the full or maximum effect on symptom scores. The results suggest that subjects favor fluticasone over ciclesonide with respect to scent, soothing feel, and less nasal irritation. There were no statistically significant differences between the two drugs for other sensory attributes either immediately or 2 min after dosing. Previous studies with nasal steroids have reported that patients usually prefer scentless and tasteless preparations but in our study patients preferred fluticasone that has a mild fruity odor, while ciclesonide has no odor but evokes a more irritating sensation in the nasal mucosa. Although the immediate efficacy of the two drugs was comparable, the differences in sensory attributes probably led to the greater preference for fluticasone over ciclesonide. The tolerability of both preparations was good and therefore this probably has not contributed to the difference in preference. Fluticasone did, however, produce taste, aftertaste, run down into throat, and run out from nose with greater frequency than ciclesonide, although differences were not statistically significant. In a previous study, comparing sensory attributes of fluticasone with budesonide in allergic rhinitis patients, a significantly greater number of subjects associated scent, taste, aftertaste and run down into throat with fluticasone.[11] Ciclesonide is used as a hypotonic solution that allows more diffusion in the nasal mucosa resulting in less nose run-off and less dripping down the throat.[12] Fluticasone, on the other hand, is used as an isotonic solution that produces more nose runoff and more dripping down the throat due to less absorption through nasal mucosa.[13] Formulation composition is important for intranasal corticosteroid preparations.[1314] Nasal sprays contain preservatives like benzalkonium chloride (BKC). The effects of such preservatives on the nasal mucosa have not been clearly defined, although benzalkonium chloride (0.05%) has been reported to produce a consistent and significant increase in nasal irritation, burning sensation, and nasal hypersecretion immediately after administration.[15] In our study, the fluticasone nasal spray, despite having 0.02% benzalkonium chloride, did not produce an appreciable nasal irritation in majority of the patients. It should be pointed out that compliance cannot be postulated on the basis of a single-dose study.[5] However, previous studies have reported that sensory attributes have important role in deciding patient compliance with INC therapy. When drugs within a given treatment class have similar efficacy and safety profiles, other characteristics (e.g., sensory attributes for INCs) can play a central role in product acceptance. Optimization of product formulation to meet the sensory preferences may further increase the patient adherence to INCs.[16] The study had some limitations. Exposure to any prohibited medication prior to recruitment was assessed only through the information volunteered by the subjects without insisting on detailed medical records. The sensory attributes questionnaire was adopted from those used in previous studies without separate validation. Patient preference seen in this single dose study, potentially may not match that following repeated use. Finally, the categorical rating with respect to both this questionnaire and the nasal symptom scoring is open to some subjective bias. BODY.CONCLUSIONS: Sensory attributes are potentially important in evaluation of intranasal products for allergic rhinitis. As newer intranasal corticosteroids are introduced, product attributes and immediate efficacy may decide the patient preference and adherence to therapy and thereby reflect on treatment outcome. In this study, although their immediate efficacy and tolerability were comparable, an overall patient preference was for fluticasone propionate formulation rather than ciclesonide. The former was also preferred over ciclesonide with respect to the sensory attributes of scent, soothing feel, and less nasal irritation. Comparing efficacy, tolerability, and acceptability with respect to various sensory attributes on continued or repeated use deserves further exploration.

TITLE: Beneficial but not sufficient: effects of condom packaging instructions on condom use skills ABSTRACT: Among those who are sexually active, condom use is the only method of protection against HIV/AIDS. Poor condom skills may lead to condom use failures, which can lead to risk of exposure. Despite the wide availability of condom use instructional leaflets, it is unclear whether these instructions sufficiently teach condom use skills. Ninety-two male and 113 female undergraduates were randomly assigned to a control condition (read non-condom instructions) or a treatment condition (read condom instructions). Participants completed self-report measures related to condom use and performed a condom demonstration task. Participants who read the condom instructions did not perform significantly better on the demonstration task, F (1, 203) = 2.90, P = 0.09, η2 = 0.014. At the item level, those who read the condom instructions better performed two of the seven condom use steps correctly. These data suggest that condom packaging instructions do not effectively teach condom use skills. BODY.INTRODUCTION: With over 50,000 new cases of HIV in the US each year1 and approximately 2.6 million worldwide,2 researchers continue their efforts to identify best practices for risk prevention. Because condom use is the only method of protection against HIV among those who are sexually active, many prevention programs are designed to increase consistent and correct condom use. Unfortunately, many condom users may be unwilling to attend an instructional program or may not have access to one. And thus, in the absence of formal condom use instruction or training, users may rely on alternative methods to learn correct condom use. The most available means through which users may learn correct condom usage is via instructional leaflets included in condom packaging. Despite the availability of these instructions, it is unclear to what extent users read and follow the instructions or if these leaflets effectively instruct correct condom usage. In fact, a review of the literature yielded no such evaluations. There is also evidence that condom users are experiencing failures at a much higher rate than would be expected (ie, relative to condom laboratory tests). Norris and Ford3 found that 60% of their sample reported having experienced a condom breaking, and Crosby and colleagues4 reported that over one-third of adolescents experienced a failure in the past 3 months. Similarly, Lindemann5 found that 47% of college students reported experiencing at least one condom use failure in the past 6 months, and 13% experienced at least one failure in the past 30 days. Other researchers have estimated that approximately 13% of all condom uses result in failure.6 When one considers that each instance of condom failure places both partners at risk, the importance of minimizing such failures becomes second only to encouraging consistent condom use in the first place. Condoms are highly effective, but only when used correctly. The importance of correct condom use is well recognized by condom manufacturers; for example, Trojan® brand latex condoms come with the following statement: "For maximum benefits, it is important to follow the instructions for use ... Failure to do so may result in the loss of the benefits of a condom." The high rates of condom failure reported may be attributed at least in part due to condom use error,7,8 and one's personal ability to use a condom correctly may be a consideration when weighing the benefits and costs of condom use.9 In addition, having negative experiences with condoms, such as a condom failure, may result in negative beliefs toward condom use, which in turn may decrease intentions to use condoms in the future.3 Likewise, condom use has been associated with high condom use self-efficacy and pro-condom norms.10 The importance of increasing correct condom use skills is fundamental to ongoing efforts to reduce transmission of HIV. The purpose of this research was to assess the efficacy of written condom packaging instructions for teaching correct condom use skills. More specifically, we compared condom use skills between college students who read condom packaging instructions immediately prior to performing a condom demonstration task to those who did not. BODY.METHODS.PARTICIPANTS: Participants were 92 male and 113 female undergraduate students ranging in age from 18 to 42 years (M = 19.36, SD = 2.63). Participants were predominately white (81%) and attending their freshmen or sophomore year of college (80%). Among the participants, 91% reported ever having sexual intercourse, and among those, 89% reported ever using a condom (see Table 1). Participants were recruited using a departmental human subjects pool, where students signed up for research participation opportunities in a central, public location, and for which participation earned credit toward meeting an introductory psychology research experience requirement. The use of human participants in this research was approved by the university's Institutional Review Board. BODY.METHODS.MEASURES AND MATERIALS: Condom use skills were assessed using the Measure of Observed Condom Use Skills (MOCUS).11,12 The MOCUS assesses seven singular, directly observable behaviors toward correct condom usage (see Table 2 for individual items). Items are scored as performed correctly ("yes") or not ("no"). All items on the MOCUS are behaviors that prevent condom breakage, slippage, or leakage of fluids. The MOCUS has high Guttman scalability (Reproducability = 0.93; Plus Percentage Ratio = 0.75). The MOCUS was administered individually by one of four trained observers (two male and two female). Inter-observer agreement was 93% from 18 pilot participants as part of the training, and the average chance-adjusted agreement across the seven MOCUS items was Cohen's κ = 0.78. In addition to the MOCUS, participants responded to self-report measures related to condom use. The Reported Condom Use and Failures scale (RCUF, unpublished measure) was used to assess the frequency and types of condom use failures experienced in the recent past, the Sexual Behavior Survey13 was used to assess current sexual practices, such as the number of recent sexual partners, and the Condom Use Self-Efficacy Scale (CUSES)14 was used as an indirect measure of skill, communication, and confidence with condoms. The CUSES was scored using the four self-efficacy subscales identified with acceptable internal consistency (α ≈ 0.80): (1) condom mechanics, (2) partner's disapproval, (3) assertiveness, and (4) intoxicants. 15 Internal consistency estimates for the present sample were similar, α > 0.70. Instructional pamphlets showing correct condom application and removal steps (Trojan brand condoms) and correct yoga-ball exercises20 (from http://www.about.com) were used. The pamphlets were similar in that they both contained small illustrations alongside step-by-step instructions for completing a physical procedure. Written instructions were provided in both English and Spanish for both condom application and removal, and yoga-ball exercises. In addition, lubricated condoms and a wooden penile model were used during administration of the MOCUS. The Trojan brand condom use leaflet included three application illustrations, showing the package being torn open, the condom placed on the tip of the penis, and the condom being pinched at the tip while being unrolled to the pubic hair line. There was also a fourth illustration showing that the condom should be held at the base of the penis during removal from the partner. The images and written instructions on the pamphlet clearly coincided with six of the seven items on the MOCUS. For the remaining item (Item 7, referring to holding condom at both tip and base while carefully sliding the condom off the penis) the pamphlet included a related, but more general instruction to hold the condom on the penis during the entire removal process. BODY.METHODS.DESIGN AND PROCEDURE: A between-subjects design was employed where participants in the Control Group read a non-condom related instructional pamphlet (ie, correct yoga-ball exercises) and participants in the Treatment Group read the condom packaging instructional pamphlet. Participants were randomly assigned to the group based on the research session and sex of the MOCUS observer. Random assignment based on the research session was used to prevent diffusion of treatment, and random assignment by MOCUS observer sex was used to ensure that the number of same sex and different sex participant-observer interactions were equivalent for both treatment groups. Upon arrival to the research session, participants provided written informed consent. At that time, participants were given the packet of self-report measures and instructed to generate a unique continuity code,16,17 which was written on the packet and a white file folder label (to be placed on the MOCUS at a later time). This continuity code procedure has been used to maintain anonymity of responses for sensitive data and allows researchers to link together multiple, independent data records for a particular participant. After completing the self-report measures, participants were given either the condom packaging or yoga-ball instructions and asked to carefully read the content. A researcher observed each participant as he or she read the assigned instructions. After reading the instructions, participants were escorted to a private room by a trained observer where the MOCUS was administered. All observers were blind to which instructions the participant had read. Participants were asked to respond to four self-report questions related to the MOCUS and were provided a written debriefing form that included the correct steps to condom usage. Before leaving, the researcher offered to answer questions and thanked each participant for their time. BODY.METHODS.EFFICACY MEASURES AND DATA ANALYSIS: Three efficacy measures were derived to evaluate the impact of condom packaging instructions on condom use skills. First, mean MOCUS scores were used as an aggregate efficacy measure to compare those who read the condom packaging instructions (ie, Packaging Instructions Group) and those who read the yoga-ball instructions (ie, Control). Second, the proportion of participants who correctly performed each item on the MOCUS was compared between groups to identify which, if any, aspects of condom use were best learned from reading the packaging instructions. A third efficacy measure was created by comparing the proportion of packaging instructions and control participants who performed all seven MOCUS items correctly. As each item on the MOCUS is designed to prevent a specific type of condom use failure,11 this third efficacy measure was important because only those who correctly perform all aspects of condom application and removal minimize the risk of condom use failures (eg, breakage, leakage, slippage). Data analysis proceeded in two phases. Initially, the three efficacy measures were compared between the Packaging Instructions and Control Groups. The same measures were then reexamined separately for men and women to account for potential gender differences in MOCUS scores that have been reported previously.11,12 ANOVA was used to compare the mean MOCUS scores between groups and to test for potential gender differences in MOCUS scores and differential effects of reading condom packaging instructions (ie, Gender X Group Interaction). Proportion data were compared using risk ratios (RRs). RRs represent the proportion of packaging instructions participants who successfully performed each item (or all MOCUS items) relative to the proportion of control participants. The RR was used because it summarizes two proportions with a readily interpretable ratio (eg, an RR of 1.50 indicates that packaging instructions participants were 50% more likely to successfully perform that particular MOCUS item) and associated confidence intervals could be compared to test for potential gender differences. RRs with non-overlapping confidence intervals would indicate differential effects of condom packaging instructions between genders. BODY.RESULTS: Table 1 displays the demographic composition, reported sexual experiences, and CUSES for the Packaging Instructions and Control Groups. Participants in both groups were similar in age, gender, ethnic composition, mean CUSES scores, sexual and condom use experiences, with one exception. Among those who reported ever using condoms, a greater proportion of control participants reported experiencing at least one condom use failure (62.2%) than those who read the packaging instructions (43.6%) (χ2 [1, n = 160] = 5.56, P = 0.02). No other differences between groups were significant. BODY.RESULTS.EFFICACY OF CONDOM PACKAGING INSTRUCTIONS: As can be seen in Figure 1, the mean MOCUS score among those in the Packaging Instructions Group was higher than that in the Control Group. This difference was modest and amounted to an average of approximately one-third of a MOCUS item better performance for the Packaging Instructions Group (ie, approximately one-quarter of a standard deviation) and was non-significant (F[1, 203] = 2.90, P = 0.09, η2 = 0.014). The proportion of participants who correctly performed each MOCUS item provides greater detail regarding this mean difference. As can be seen in Table 2, those who read the packaging instructions performed significantly better on two of the seven MOCUS items. Participants who read the packaging instructions were nearly 1.5 times more likely to successfully complete Application Step 3 (ie, "Pinch reservoir tip with two fingers") and Removal Step 5 (ie, "Hold condom at base and remove penis from the partner"), χ2's were 11.43 (P = 0.001) for Item 3 and 6.72 (P = 0.01) for Item 5. No significant differences were observed between groups on the remaining MOCUS items. Overall, 16.1% of participants performed all seven MOCUS items correctly. Although this proportion was slightly higher among the Packaging Instruction (18.6%) than the Control Group (13.6%), the difference was non-significant (χ2 [1, N = 205] = 0.96, P = 0.33) and small on a relative basis (RR: 1.37, 95% CI: 0.72–2.58). BODY.RESULTS.GENDER DIFFERENCES IN THE EFFICACY OF CONDOM PACKAGING INSTRUCTIONS: A two-way ANOVA was used to compare mean MOCUS scores between men and women and to test for differential impact of the condom packaging instructions between genders. On average, men performed approximately one-half of a MOCUS item (M = 4.98, SD = 1.46) better than women (M = 4.54, SD = 1.69). This difference amounted to approximately one-quarter of a standard deviation (Cohen's d = 0.28) and was non-significant (F[1, 201] = 3.81, P = 0.052, η2 = 0.019). As described above and displayed in Figure 1, the Packaging Instructions Group performed slightly better than the Control Group; however, the instructions were equally effective for men and women, Group × Gender interaction of F (1, 201) = 0.54 (P = 0.47, η2 = 0.003). Reading the packaging instructions had a similar impact for men and women when viewed item-by-item as well. Figure 2 displays the RRs comparing the proportion of participants in the Packaging Instructions Group who successfully performed each item relative to control participants for both men and women. As can be seen in Figure 2, the RRs are similar for all seven MOCUS items, and the 95% confidence intervals display considerable overlap between genders. The overall percentage of men (19.6%) and women (13.3%) who correctly performed all seven MOCUS items was also similar (χ2[1, N = 205] = 1.49, P = 0.22). The relative proportion of the Packaging Instructions (men: 25.0%; women: 13.0%) and Control Groups (men: 13.4%; women: 13.6%) showed that men in the Packaging Instructions Group were nearly twice as likely to score perfectly on the MOCUS (RR: 1.84, 95% CI: 0.75–4.46) than men in the Control Group. Women in the Packaging Instructions Group, on the other hand, were no more likely to score perfectly on the MOCUS than those in the Control Group (RR: 0.96, 95% CI: 0.37–2.46). It should be noted, however, that due to the small number of participants who scored perfectly on the MOCUS, irrespective of gender, the confidence intervals around the RR were wide and overlapped substantially. BODY.DISCUSSION: Participants who read a name-brand condom packaging instructional leaflet did not demonstrate significantly better condom use skills and were more likely to correctly perform only two of seven individual condom use steps on the MOCUS. In addition, the effect of reading the condom packaging instructions were similar for men and women on both an aggregate and an item-by-item basis. Lastly, only a small percentage of participants demonstrated errorless condom use (ie, perfect score on the MOCUS), irrespective of their group assignment. As such, these data suggest that condom packaging instructions alone do not provide sufficient information to teach errorless condom use. Because users may rely on these packaging instructions, it is important to ensure that they include the necessary information in a way that leads to correct condom usage. Although many of the differences between groups failed to reach statistical significance, there was a promising trend toward slightly better condom use skills among those who read condom packaging instructions. More specifically, the one-third of a MOCUS step difference observed for the aggregate efficacy measure can be attributed to the increased proportion of packaging instructions participants who correctly performed two of the seven condom use steps. As these two steps were among the least correctly performed by control participants, the greater than 40% improvement for those who read the packaging instructions becomes especially notable. Moreover, with Item 3 (ie, "Pinch reservoir tip with two fingers") designed to minimize condom breakage and Item 5 ("Hold condom at base as condom is removed from partner") designed to minimize slippage, correctly performing these steps reduces the risk of two different types of condom use failure. Despite this promising improvement, only two-thirds of participants correctly performed these steps immediately after reading the packaging instructions and these two steps alone are not sufficient to prevent condom use failures. The importance of this later issue is further echoed by the fraction of participants (16%) who completed all seven condom application and removal steps correctly. This may be, at least in part, due to characteristics of the written instructions, and researchers and condom companies should consider research outcomes on effective written instructions for procedural tasks when developing new leaflets.18 Similarly, it would be beneficial to identify how written condom instructions can be improved. For example, leaflets may be improved by using larger font, more images, or clearer and more detailed images. These features may be most important to users who have difficulty reading, or difficulty reading small font, or for those whose native language is other than that used in the leaflet. Potentially meaningful improvements to the content of condom packaging instructions may also be ascertained from the present study. More specifically, of the seven total MOCUS items only four items were performed correctly by approximately one-half of control participants (ie, Items 3, 5, 6, and 7; see Table 1). In contrast, approximately 85% of control and condom packaging participants successfully completed the remaining three items (ie, Items 1, 2, and 4). These data suggest that areas with the greatest potential for improvement would be (a) pinching the reservoir tip with two fingers prior to application (Item 3), (b) holding the condom at the base of the penis while removing the penis from the partner (Item 5), (c) pinching the tip of the condom to secure ejaculate in the reservoir (Item 6), and (d) holding the condom at both the tip and base while removing the condom (Item 7). Performance on two of these four items with the greatest potential for improvement was, in fact, better among those who read the condom packaging instructions (ie, pinching the reservoir tip prior to application and holding the condom at the base while removing the penis from the partner). Moreover, if only these four items with the greatest for potential improvement are considered, the significant performance difference on Items 3 and 5 translates to a significant mean difference between the Condom Packaging and Control Groups (F[1, 203] = 5.43, P = 0.02, η2 = 0.026). As such, the overall finding of a non-significant difference between groups on the aggregate MOCUS score (based on all seven items) should be considered in light of key differences between groups on two of the four items with the lowest performance overall and, perhaps more importantly, the greatest potential for improvement in the first place. The low percentage of participants who correctly applied and removed the condom is particularly surprising given our sample of college students, most of whom were sexually active and endorsed past condom use and intentions to use condoms in the future. Because every item on the MOCUS assesses a behavior related to a condom use failure, errorless performance is ideal for risk prevention. These data suggest that condom users who rely on similar written instructional materials as those tested here may be increasing their risk of experiencing a condom use failure. As further evidence of this problem, approximately one-half of the sample reported experiencing at least one condom use failure. This finding is consistent with other reports of condom use failures3–5 and the small number of participants who correctly applied and removed the condom in the present study offers a potential explanation for the generally high number of reported condom use failures. In considering the implications of these findings, the reader should also be aware of some limitations. First, only one set of condom use instructions were used in the present study. Although Trojan brand condoms comprise approximately 75% of condom sales19 and that competing brands include similar instructions as part of their packaging, it is possible that a different set of instructions may have yielded different results. Second, despite being equivalent on a number of key demographic and sexual history variables, the proportion of participants who reported experiencing a condom use failure was slightly higher among the randomly assigned Packaging Instructions Group. It should be noted, however, that this difference did not translate to corresponding differences in self-efficacy for a number of condom use factors, including the mechanics of condom application and removal, nor to differences in intentions to use condoms in the future. Similarly, prior and recent exposure to condom use instruction was not assessed in this study, which may present a potential alternative explanation for these findings if differences in exposure to condom use instructions existed between groups prior to the study. However, as noted above, random assignment was employed to minimize the possibility of such preexisting differences and the groups were similar on a variety of relevant demographic and sexual history variables (see Table 1). Also, the single-shot, cross-sectional nature of the current design permits conclusions regarding group differences. An alternative design, such as a pre-post design, may permit stronger conclusions regarding learning or skill improvements from written condom packaging instructions. Lastly, this study assessed the effect on condom packaging instructions on the behavior of college students, and it is unclear if the instructions would be more or less effective with other populations, such as men who have sex with men, injection drug users, or sexually active adolescents. Including others in future research would provide a more complete understanding of the efficacy of condom packaging instructions across populations. This research served as an initial evaluation as to whether or not college students can demonstrate correct condom use skills after reading published condom packaging instructional leaflets. Considerations for future research are many. There exists a need to evaluate the effectiveness of instructional leaflets published by other brands, as their content may better inform correct condom usage. It also remains unclear whether other populations effectively learn condom use skills from these pamphlets. If these findings are replicated using different instructional leaflets or among other populations, then efforts should be focused on improving packaging instructions. Also, with the continued increase and availability of technology, consideration should be given to assessing the effectiveness and feasibility of alternative types of instructions, such as video demonstrations that can be downloaded to mobile phones. This research strengthens the literature documenting the high frequency on condom use failures, in this case, among college students. Clearly, there is need to reduce the frequency of these failures, and this study provides evidence that condom use skills are lacking, even among condom using college students, and also that current instructional leaflets are not adequate for those looking to learn correct condom use skills. Nonetheless, these data show that instructional leaflets do help with two specific condom use steps, and provide promise that if improved, instructional leaflets may become an effective, low-cost, and easily distributed approach to reducing condom use failures – an accomplishment that may go far toward our efforts to reduce HIV risk.

TITLE: High sensitivity C reactive protein as a prognostic marker in patients with mild to moderate aortic valve stenosis during lipid-lowering treatment: an SEAS substudy ABSTRACT.AIMS: To assess the prognostic importance of high-sensitive C reactive protein (hsCRP) in patients with mild to moderate aortic valve stenosis during placebo or simvastatin/ezetimibe treatment in Simvastatin and Ezetimibe in Aortic Stenosis (SEAS). ABSTRACT.METHODS AND RESULTS: In 1620 SEAS patients, we measured lipids and hsCRP at baseline and after 1 year of treatment and registered during 4 years of follow-up major cardiovascular events (MCE) composed of ischaemic cardiovascular events (ICE) and aortic valve-related events (AVE). Simvastatin/ezetimibe reduced low-density lipoprotein cholesterol (3.49 (2.94 to 4.15) to 1.32 (1.02 to 1.69) vs 3.46 (2.92 to 4.08) to 3.34 (2.81 to 3.92) mmol/L) and hsCRP (2.1 (0.9 to 4.1) to 1.2 (0.6 to 2.4) vs 2.2 (0.9 to 4.9) to 1.8 (0.85 to 4.35) mg/L, all p<0.05) during the first year of treatment. In multivariable Cox regression analysis adjusting for traditional risk factors and baseline hsCRP, ICE was associated with a 1-year increase of hsCRP (HR=1.19 (95% CI 1.12 to 1.25), p<0.001) but not with active treatment (HRTreatment=0.86 (0.67 to 1.13), p=0.28). Patients in the top quartile of baseline hsCRP versus the rest were associated with a higher risk of MCE (HR=1.34(1.09 to 1.64), p=0.02). The prognostic benefit of reduction in hsCRP after 1 year was significantly larger (p<0.01 for interaction) in patients with high versus low baseline hsCRP; hence, a reduction in hsCRP abolished the difference in incidence of MCE between high versus low baseline hsCRP in patients with reduced hsCRP (31.1 vs 31.9%, NS) in contrast to patients with increased hsCRP. ABSTRACT.CONCLUSIONS: The treatment-associated reduction in ICE was in part related to a reduction in hsCRP but not in lipids. hsCRP reduction was associated with less MCE, especially in patients with high baseline hsCRP. ABSTRACT.TRIAL REGISTRATION: NCT00092677. BODY: Key questionsWhat is already known about this subject?Little is known concerning this subject. The present study is, to the best of our knowledge, the first study to test the prognostic importance of in-treatment changes in high-sensitive C reactive protein (hsCRP). In the smaller ASTRONOMER study, hsCRP was measured at year 1 as well, but these data have not been utilised.What does this study add?In our study, alterations in hsCRP are a stronger predictor of outcome than baseline hsCRP. We demonstrate that a reduction in hsCRP is associated with an improved prognosis, resembling that of patients with low baseline and low 1-year hsCRP.In the Simvastatin and Ezetimibe in Aortic Stenosis (SEAS) study, lipid-lowering treatment did not reduce the primary, combined end point, but a 22% reduction in ischaemic events was observed. This reduction in ischaemic events was associated with a reduction in hsCRP, but not in lipids. This is in line with findings from the JUPITER trial, but has not previously been shown in patients with aortic stenosis.How might this impact on clinical practice?We believe that better knowledge about the importance of inflammation in patients with aortic stenosis may improve risk stratification, making disease monitoring more individualised and better targeted and preventing morbidity and mortality in patients with aortic stenosis. BODY.INTRODUCTION: The role of inflammation in the pathogenesis of aortic valve disease remains unclear. Therefore, we investigated the prognostic importance of baseline high-sensitive C reactive protein (hsCRP) and changes in hsCRP during the first year of lipid lowering with simvastatin 40 mg and ezetimibe 10 mg versus placebo in the Simvastatin and Ezetimibe in Aortic Stenosis (SEAS) study. The SEAS study tested if simvastatin and ezetimibe in combination could reduce the need for aortic valve replacement (AVR). The study was neutral in reducing major cardiovascular events (MCE) consisting of both aortic valve-related events (AVE) and ischaemic cardiovascular events (ICE), but a 22% reduction in ICE in the treatment group was shown.1 This reduction primarily consisted of a reduction in coronary artery bypass grafting (CABG).2 Cholesterol and inflammation are involved in the development of atherosclerosis and subsequent cardiovascular disease.3 4 4a 4b 4c Histology of stenotic aortic valves shows subendothelial plaque-like lesions on the aortic side of the leaflets that resemble those seen in atherosclerosis and 'atherogenic' lipoproteins are accumulated in the lesions.5 6 hsCRP has shown its potential as a predictor of cardiovascular disease in several studies.7 8 8a 8b 8c Therefore, we hypothesised that a reduction in hsCRP would be associated with fewer ischaemic events, in line with findings from the JUPITER trial,9 and we also wanted to explore if a reduction of hsCRP would be associated with fewer MCE in this aortic stenosis (AS) population. BODY.METHODS.STUDY POPULATION: The SEAS study was a multicentre, randomised, double-blind, placebo-controlled study investigating whether intensive lipid lowering with simvastatin/ezetimibe combination versus placebo could reduce the need for AVR and risk of cardiovascular morbidity and mortality in 1873 patients aged 45–85 years with asymptomatic AS (defined as echocardiographic aortic valve thickening accompanied by a Doppler-measured aortic peak flow velocity >2.5 and <4 m/s and normal left ventricular (LV) systolic function). For this substudy, we included 1620 patients with hsCRP analysed at baseline. In total, 253 patients were excluded for missing baseline values of hsCRP. There were no differences between the study population and the 253 excluded patients with regard to age, gender, treatment and tobacco use. The complete study protocol, study design, organisation, clinical measures, exclusion criteria (most importantly, systolic heart failure, diabetes mellitus and known ischaemic heart disease) and baseline characteristics have been published previously.1 10 All patients gave written informed consent, and ethics committees in all participating countries approved the study (the SEAS trial is registered at http://ClinicalTrials.gov, identifier NCT00092677). BODY.METHODS.HIGH-SENSITIVE C REACTIVE PROTEIN: hsCRP was measured at baseline and at 1 year after inclusion. Serum hsCRP concentration was determined using a particle-enhanced immunoturbidimetry assay (Roche/Hitachi) range 0.1–20 mg/L and lowest detection limit 0.03 mg/L. A 1 year increase in hsCRP was defined as a rise from baseline to year 1. BODY.METHODS.WHITE CELL COUNT AND LIPIDS: White cell counts (WCCs) were measured only at baseline and lipids were measured at baseline, and then every 6 months during follow-up. All blood samples were analysed by the central laboratory, PPD Global Central Labs, in Belgium. Study investigators were blinded to lipid values throughout the entire study. BODY.METHODS.HYPERTENSION AND TOBACCO: Hypertension was defined in the SEAS study as a history of hypertension or the use of antihypertensive medication.11 This was reported by an attending physician or an elevated blood pressure at the baseline clinical visit (systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg), in accordance with the European Society of Hypertension Guidelines.12 Tobacco status was self-evaluated at study entrance. BODY.METHODS.ECG: The specifics about protocol, reading and reproducibility have been published previously.13 In short, ECGs were recorded at baseline and then annually at the local study centres, after which they were sent to the central ECG core laboratory at The Heart Center, Rigshospitalet, Denmark. A physician blinded to the randomisation and clinical data read and transferred all ECGs directly to a database by use of the Minnesota codes.13 BODY.METHODS.ECHOCARDIOGRAGRAPHY: The study protocol has been published earlier.14 In short, echocardiography was performed at baseline, once a year and before valve surgery if needed. Echocardiography was performed according to a standardised echocardiographic protocol. All images were read at the SEAS Echocardiography Core Laboratory at Haukeland University Hospital in Bergen, Norway. All readings were performed according to the American Society of Echocardiography guidelines. The readers were unaware of the sequence and site in order to minimise bias. BODY.METHODS.EFFICACY OUTCOMES: The SEAS study was event driven and was designed to continue until 464 events had occurred or all patients had been followed for at least 4 years. The primary outcome was MCE, a composite consisting of death from cardiovascular causes, AVR, congestive heart failure because of progression of AS, non-fatal myocardial infarction, hospitalisation for unstable angina, CABG, percutaneous coronary intervention (PCI) or non-haemorrhagic stroke. MCE included AVE and ICE. ICE was defined as death from cardiovascular causes, non-fatal myocardial infarction, hospitalisation for unstable angina, CABG, PCI or non-haemorrhagic stroke. All outcomes were classified by an independent End-point Classification Committee unaware of study group assignment.1 BODY.METHODS.STATISTICAL ANALYSES: Baseline characteristics are presented as mean±SD for continuous variables and proportions for categorical variables. Non-Gaussian distributed variables are presented as median values and IQR. hsCRP was log2 transformed in order to fulfil model assumptions in statistical analyses. Log2 was used to evaluate the risk associated with doubling of the concentration. Changes (1 year) in hsCRP are reported as the 1-year value minus baseline hsCRP. Unpaired Student t test was used to determine differences between treatment groups and paired Student t test was used to determine changes during treatment within each treatment group for normally distributed variables. For non-Gaussian distributed variables, Mann-Whitney U test and Wilcoxon signed-rank test, respectively, were used. Differences in continuous and categorical baseline characteristics were analysed using the one-way analysis of variance. Correlation between baseline characteristics of interest was explored using Spearman correlation analysis. For outcome prediction and comparison between groups, Kaplan-Meier plots and the log rank test were used. HRs were assessed by multiple Cox proportional hazard analyses and presented with 95% CI. hsCRP as a variable of interest along with gender, age and treatment allocation were forced into the models. The other variables of interest: blood pressure, body mass index (BMI), heart rate, glucose, WCC, total and low-density lipoprotein (LDL) cholesterol, 1-year changes in total and LDL-cholesterol, estimated glomerular filtration rate, tobacco, aspartate aminotransferase (AST) and ejection fraction were added to the models by backward selection. The performance of the Cox models was summarised using C-statistics to allow for right censoring of data and variable follow-up time.15 The two models compared were the basic model (the best statistical performing model predicting outcome without hsCRP and treatment forced into the model age, sex, heart rate, WCC, tobacco and treatment group) versus the new model (basic model plus baseline hsCRP and 1-year changes in hsCRP). Net reclassification improvement (NRI) was estimated as continuous NRI and presented with its CI and upward and downward reclassification rates.16 The bias-corrected 95% CIs for the C-statistics and NRI were assessed by the usage of bootstrapping. Finally, the integrated discrimination improvement (IDI) was calculated.17 This measure can be described as the difference in improved sensitivity and potential increase in 'one minus specificity', with the new model including the marker of interest versus the basic model.17 For all models, a two-tailed p<0.05 was required for statistical significance. Statistical analyses were performed using IBM SPSS V.20.0.0 (SPSS, Chicago, Illinois, USA) and R V.2.15.2 (R Foundation for Statistical Computing, Vienna, Austria). BODY.RESULTS: At baseline, the median age was 69 years (IQR 61–75; table 1). Table 1 Baseline characteristics according to quartiles of baseline hsCRP Variable hsCRP (mg/L) Quartiles of baseline hsCRP p ANOVA 1st quartile 0.00–0.90 2nd quartile 0.90–2.15 3rd quartile 2.15–4.50 4th quartile 4.50–15.80 Age (year) 66±10 67±10 69±9 68±9 <0.001 Female sex (n, %) 144 (34.1) 142 (34.5) 162 (41.8) 178 (44.9) 0.002 Smoking (n, %)  Current 62 (14.7) 77 (18.7) 74 (19.1) 91 (23) 0.03  Former 150 (35.5) 147 (35.7) 144 (37.1) 143 (36.1) 0.97  Never 210 (49.8) 188 (45.6) 170 (43.8) 162 (40.9) 0.08 Hypertension 342 (81) 355(86.2) 338(87.1) 362(91.4) <0.001 Randomised treatment (n, %) 207 (49.1) 219 (53.2) 214 (55.2) 180 (45.5) 0.03 Systolic BP (mm Hg) 143±0 146±20 144±20 146±20 0.045 Diastolic BP (mm Hg) 81±10 83±10 82±11 82±10 0.36 Body mass index (kg/m 2 ) 25±4 27±4 28±4 29±5 <0.001 Left ventricular mass index (g/m 2 ) 99.26±30.3 102.07±30.72 102.6±32.89 104.6±32.62 0.12 Left ventricular ejection fraction (%) 66.41±8.21 65.71±8.54 65.33±7.76 65.43±8.53 0.25 Aortic valve area index (cm 2 /m 2 ) 0.73±0.27 0.69±0.28 0.68±0.26 0.68±0.25 0.046 Aortic valve peak velocity (m/s) 3.25±0.64 3.24±0.65 3.27±0.65 3.25±0.64 0.96 Concomitant treatment  ACE inhibitor 80 (19) 126 (30.6) 108 (27.8) 134 (33.8) <0.001  Angiotensin II receptor blocker 72 (17.1) 82 (19.9) 67 (17.3) 86 (21.7) 0.272  Calcium antagonist 83 (19.7) 111 (26.9) 111 (28.6) 152 (38.4) <0.001  Aspirin or other platelet inhibitor 182 (43.1) 182 (44.2) 164 (42.3) 191 (48.2) 0.34  β-Blocker 173 (41) 212 (51.5) 202 (52.1) 208 (52.5) 0.002 ANOVA, analysis of variance; BP, blood pressure; hsCRP, high-sensitive C reactive protein. With ascending quartiles of hsCRP, most of the traditional cardiovascular risk factors increased significantly. The proportion of men proportion fell with increasing quartiles of baseline hsCRP. With increasing quartiles of hsCRP, LV mass indexed by body surface area (baseline mean=102 g/m2) and aortic valve peak velocity (baseline mean=3.05 m/s) did not increase, whereas aortic valve area index (baseline mean=0.63 cm2/m2) did decrease significantly (table 1). At baseline, hsCRP was associated with WCC (r=0.33, p<0.001), but not with total cholesterol (r=−0.041, p=0.10) or LDL-cholesterol (r=0.002, p=0.94). Baseline and 1-year values of lipids, liver marker AST and inflammatory markers are shown in table 2. Table 2 Effect of treatment on lipids and markers of inflammation Variable Allocation Baseline Year 1 Paired p value One-year changes hsCRP (mg/L) Treatment 2.1 (0.9–4.1)* 1.2 (0.6–2.4)* <0.001 1.76 (0.0–4.4)** Placebo 2.2 (0.9–4.9) 1.8 (0.85–4.35) 0.003 0.00 (−1.5–2.1) Total cholesterol (mmol/L) Treatment 5.72 (5.05–6.42) 3.39 (2.95–3.91)** <0.001 2.25 (1.7–2.8)** Placebo 5.67 (4.97–6.37) 5.5 (4.92–6.2) <0.001 0.13 (−0.3–0.5) LDL-cholesterol (mmol/L) Treatment 3.49 (2.94–4.15) 1.32 (1.02–1.69)** <0.001 2.13 (1.6–2.6)** Placebo 3.46 (2.92–4.08) 3.34 (2.81–3.92) <0.001 0.12 (−0.2–0.4) White cell count (10 9 /L) Treatment 5.72 (5.05–6.42) Placebo 5.8 (4.9–6.9) AST (U/L) Treatment 16 (14–19) 19 (16–23)** <0.001 −3.20 (−5.0–0.0)** Placebo 16 (14–19) 16 (14–19) 0.59 0.00 (−2.0–2.0) Between groups (allocation): *p<0.05; **p<0.001. Data are shown as median values and IQR. One-year changes are shown at baseline minus year 1 values. AST, aspartate aminotransferase; hsCRP, high-sensitive C reactive protein; LDL, low-density lipoproteins. At 1 year, hsCRP, total and LDL-cholesterol were lower in the treatment group, but unchanged in the placebo group. AST was similar in the two groups at baseline, but increased in the treatment group. In univariate Cox regression analyses, higher baseline hsCRP as well as an increase in hsCRP predicted MCE as well as ICE (table 3). Table 3 Cox regression models predicting major cardiovascular and ischaemic events Variable Cox regression model predicting major cardiovascular events p Value Cox regression model predicting ischaemic events p Value Baseline hsCRP (mg/L) 1.1 (1.04–1.17) 0.002 1.1 (1.00–1.20) 0.043 Increase in hsCRP (mg/L) 1.17 (1.1–1.24) <0.001 1.19 (1.12–1.25) <0.001 Hypertension 2.12 (1.23–3.67) 0.007 Treatment allocation 0.91 (0.75–1.1) 0.99 0.87 (0.67–1.13) 0.283 Gender 1.01 (1–1.02) 0.33 0.68 (0.52–0.89) 0.005 Age 1.01 (1–1.02) 0.12 1.05 (1.03–1.07) <0.001 Heart rate (beats/min) 1.01 (1–1.02) 0.01 WCC (10 9 /L) 1.12 (1.03–1.22) 0.010 Univariate (unadjusted)  Baseline hsCRP (mg/L) 1.06 (1.01–1.12)* 0.021 1.13 (1.06–1.21)** <0.001  Increase in hsCRP (mg/L) 1.12 (1.06–1.19)** <0.001 1.19 (1.1–1.29)** <0.001 *p<0.05; **p<0.001. hsCRP, high-sensitivity C reactive protein; WCC, white cell count. In multivariate Cox regression analyses, hsCRP values predicted both MCE and ICE independently of each other as well as the traditional cardiovascular risk factors (table 3). The highest event-free survival was observed in the two groups with reduced hsCRP values (figure 1A). Figure 1Event-free survival according to baseline and 1-year changes in high-sensitive C reactive protein (hsCRP). Survival free of (A) major cardiovascular event (MCE) and (B) ischaemic cardiovascular event (ICE) according to baseline and 1-year changes in hsCRP. More events (MCE) occurred with increasing hsCRP at baseline (p=0.014). However, an interaction was observed indicating that risk was primarily confined to those with increasing hsCRP values, in contrast to those with a decreasing hsCRP (table 4; p<0.01 for interaction). Table 4 Major cardiovascular event rates and HRs according to quartiles of baseline hsCRP in the whole cohort as well as in patients with increasing or decreasing hsCRP Baseline hsCRP One-year changes in hsCRP from baseline   Increase Decrease Quartiles of baseline hsCRP N HR Events per 1000 patient-years N HR Events per 1000 patient-years N HR Events per 1000 patient-years 1st quartile 422 1 83 171 1 92 231 1 75 2nd quartile 412 1.1 (0.87 to 1.39) 95 143 1.2 (0.84 to 1.71) 119 232 1.03 (0.73 to 1.44) 78 3rd quartile 388 1.28 (1.02 to 1.62)* 116 85 1.31 (0.87 to 1.96) 134 258 1.28 (0.94 to 1.75) 102 4th quartile 396 1.19 (0.94 to 1.51) 106 74 1.62 (1.09 to 2.4)* 168 280 1.07 (0.78 to 1.47) 82 1st–3rd quartile 1222 1.06 (0.88 to 1.28) 399 1.06 (0.88 to 1.28)* 721 0.96 (0.75 to 1.23) *p<0.05. hsCRP, high-sensitivity C reactive protein. Actually, in patients with a decrease in hsCRP during the first year, there was no significant difference in event rates in patients with high versus low baseline hsCRP (31.1% vs 31.9%, NS). However, in patients with an increase in hsCRP, a higher MCE rate was observed (56.8 vs 42.5%, p<0.01; table 4). The same pattern was observed for ICE (table 5 and figure 1B), except that the p value for interaction did not reach significance. Table 5 Ischaemic cardiovascular event rates and HRs according to quartiles of baseline hsCRP in the whole cohort as well as in patients with increasing or decreasing hsCRP Baseline hsCRP One-year changes in hsCRP from baseline   Increase Decrease Quartiles of baseline hsCRP N HR Events per 1000 patient-years N HR Events per 1000 patient-years N HR Events per 1000 patient-years 1st quartile 422 1 29 171 1 37 231 1 21 2nd quartile 412 1.64 (1.15 to 2.33)* 50 143 1.61 (0.97 to 2.67) 64 232 1.74 (1.01 to 3.02)* 37 3rd quartile 388 1.54 (1.08 to 2.22)* 48 85 1.47 (0.81 to 2.66) 58 258 1.75 (1.02 to 2.99)* 38 4th quartile 396 1.73 (1.21 to 2.46)* 52 74 2.13 (1.22 to 3.71)* 85 280 1.77 (1.04 to 3.01)* 37 1st–3rd quartile 1222 1.24 (0.96 to 1.61) 399 1.62 (1.03 to 2.55) 721 1.18 (0.82 to 1.69) *p<0.05. hsCRP, high-sensitivity C reactive protein. The risk for MCE rose significantly between the second and the third hsCRP quartile (HR 1.28, p<0.05) and remained elevated (table 4), while the risk for ICE increased between the first and the second quartile (HR 1.64, p<0.05; table 5). The addition of hsCRP (baseline and 1-year changes) to the basic model improved C-statistics from 0.57 to 0.60 (p<0.05) (table 6). Table 6 Performance of MCE risk models including traditional risk factors with and without hsCRP C-index (95% CI) p Value NRI (95% CI) IDI (95% CI) p Value Basic model 0.57 (0.43 to 0.71) Basic model+baseline hsCRP and 1-year changes 0.60 (0.41 to 0.79) <0.05 0.24 (0.13 to 0.38) 0.03 (0 to 0.07) 0.07 Basic model: age, sex, heart rate, white cell count, tobacco and treatment group. hsCRP, high-sensitivity C reactive protein; IDI, integrated discrimination improvement; MCE, major cardiovascular event; NRI, net reclassification improvement. The addition of hsCRP (baseline and 1-year changes) to the basic model resulted in an improvement in continuous NRI of 0.24 (95% CI 0.13 to 0.38), continuous NRI for events 0.03 and continuous NRI for non-events 0.20. The IDI was 0.03 (p=0.07). For ICE (table 7), the C-statistics improved from 0.65 to 0.68 (p<0.05), and continuous NRI improved by 0.26 (95% CI 0.02 to 0.39), when adding hsCRP (baseline and 1-year changes) to the basic model. Baseline hsCRP alone did not improve prediction of MCE or ICE significantly. Table 7 Performance of ICE risk models including traditional risk factors with and without hsCRP C-index (95% CI) p Value NRI (95% CI) IDI (95% CI) p Value Basic model 0.65 (0.34 to 0.96) Basic model+baseline hsCRP and 1-year changes 0.68 (0.33 to 1.03) <0.05 0.26 (0.02 to 0.39) 0.01 (−0.01 to 0.04) 0.4 Basic model: age, sex, heart rate, white cell count, tobacco and treatment group. hsCRP, high-sensitivity C reactive protein; ICE, ischaemic cardiovascular event; IDI, integrated discrimination improvement; NRI, net reclassification improvement. BODY.DISCUSSION: This study has two new observations relevant for the role of inflammation in aortic valve stenosis. First, elevated baseline hsCRP as well as a 1-year increase in hsCRP predicted MCE independently of traditional cardiovascular risk factors because an increase in hsCRP was strongly associated with more MCE in patients with elevated baseline hsCRP. Second, the beneficial effect of simvastatin and ezetemibe on ICE seemed to be related to a reduction in low-grade inflammation rather than a reduction in lipids. BODY.DISCUSSION.ELEVATED BASELINE HSCRP AS WELL AS A 1-YEAR INCREASE IN HSCRP PREDICTED OUTCOME INDEPENDENTLY OF TRADITIONAL CARDIOVASCULAR RISK FACTORS: To the best of our knowledge, this is the largest study investigating the prognostic importance of hsCRP in patients with asymptomatic mild to moderate AS to date. In the present study, baseline hsCRP as well as a 1-year increase in hsCRP predicted the combined end point independently of the traditional risk factors. In a study performed by Solberg et al,18 hsCRP predicted cardiovascular events in patients with severe AS significantly in univariate analysis, but not when included in a multivariate analysis. In a small study by Dichtl et al,19 CRP remained significant in multivariate analysis, with an HR of 1.6. The study by Dichtl et al19 resembled the SEAS study with regard to inclusion criteria and baseline characteristics, while the population in the study by Solberg et al18 had more advanced disease, an incidence of coronary artery disease of 50% and all were referred for AVR. However, in the ASTRONOMER trial, the only other study with in-treatment CRP values, although not to the best of our knowledge reported, CRP did not predict outcome and there was no difference in survival according to baseline CRP.20 The baseline characteristics in ASTRONOMER were different from those in SEAS.21 In total, 48.9% had congenital bicuspid aortic valves, they were younger and only fewer were currently smoking at inclusion. Our findings complement these studies. In a previous report from the SEAS study22 evaluating hsCRP as part of developing a risk score for predicting mortality in patients with asymptomatic mild to moderate AS, hsCRP also significantly predicted mortality. Furthermore, reduction in hsCRP during the first study year was associated with better outcome independently of baseline hsCRP and traditional cardiovascular risk factors. This is the first time that an association between treatment-induced changes in an inflammatory marker and MCE has been demonstrated in a population of patients with AS. The beneficial effect of a decrease in hsCRP during the first year of treatment was largest for those with a baseline hsCRP in the top quartile. Interestingly, the beneficial effect of a decrease in hsCRP seemed to obliterate the prognostic importance of baseline hsCRP (tables 4 and 5). These observations have also been reported in other populations, that is, in the general population,23 in patients with chronic heart failure,24 and in patients with non-ST elevation acute coronary syndrome.25 Furthermore, we found that hsCRP added significantly to the model predicting MCE assessed by C-index and continuous NRI. This suggests that low-grade inflammation assessed by hsCRP might be an important marker of progression of AS. However, our results need confirmation in other studies before hsCRP can be used clinically for estimation of cardiovascular risk in participants with AS. BODY.DISCUSSION.THE BENEFICIAL EFFECT OF SIMVASTATIN AND EZETIMIBE ON ICE WAS PARTLY DUE TO REDUCED HSCRP: Not surprisingly, baseline hsCRP as a marker of low-grade inflammation significantly predicted ICE as previously demonstrated in many other cohorts.7 26 27 When 1-year changes in hsCRP were added to the model, treatment allocation was no longer significant in the prediction of ICE. As expected, simvastatin and ezetimibe treatment reduced the lipid levels, but in Cox regression analysis changes in neither total nor LDL-cholesterol were significantly associated with ICE. Therefore, our study indicates that the reduction in ICE was related to the reduction in inflammation and not to changes in lipids. This corresponds well with previous findings from the JUPITER trial.9 However, neither studies were able to show conclusively whether it was the lipid reduction, reduced inflammation or a combination of the two, which accounted for the beneficial effect on ICE. Therefore, further studies are needed to answer this question. A possible confounder could be the inflammatory effects of LDL-cholesterol itself and its contribution to increased hsCRP. BODY.DISCUSSION.THE BENEFICIAL EFFECT OF SIMVASTATIN AND EZETIMIBE ON ICE WAS PARTLY DUE TO REDUCED HSCRP.LIMITATIONS: The inclusion and exclusion criteria for the SEAS study induce some limitations on this study. As this is a non-prespecified explorative substudy no causual relationship can be established. Additionally preconsisting inflammatory diseases or conditions were not considered in planning the original study design. The participants had to be asymptomatic, as well as jet velocity between 2.5 and 4 m/s. In addition, participants with known ischaemic heart disease and diabetes were excluded due to the placebo comparison of the study. However, the study population was otherwise not at low cardiovascular risk due to the relative high age, hyperlipidaemia, obesity, smoking and high prevalence of hypertension10 and did reflect patients seen in the daily clinic. As the findings were generated from post hoc analysis, any causal relationship cannot be inferred from these results. BODY.CONCLUSION: Although treatment with simvastatin 40 mg and ezetimibe 10 mg reduced both cholesterol and hsCRP, the treatment-associated reduction in ICE seemed to be related to the reduction in hsCRP rather than in cholesterol. hsCRP reduction was associated with fewer MCE and the prognostic benefit of a 1-year reduction in hsCRP was larger in patients with high baseline hsCRP.

TITLE: Effects of ABSTRACT: Bitter orange (Citrus aurantium) extract is widely used in dietary supplements for weight management and sports performance. Its primary protoalkaloid is p-synephrine. Most studies involving bitter orange extract and p-synephrine have used products with multiple ingredients. The current study assessed the thermogenic effects of p-synephrine alone and in conjunction with the flavonoids naringin and hesperidin in a double-blinded, randomized, placebo-controlled protocol with 10 subjects per treatment group. Resting metabolic rates (RMR), blood pressure, heart rates and a self-reported rating scale were determined at baseline and 75 min after oral ingestion of the test products in V-8 juice. A decrease of 30 kcal occurred in the placebo control relative to baseline. The group receiving p-synephrine (50 mg) alone exhibited a 65 kcal increase in RMR as compared to the placebo group. The consumption of 600 mg naringin with 50 mg p-synephrine resulted in a 129 kcal increase in RMR relative to the placebo group. In the group receiving 100 mg hesperidin in addition to the 50 mg p-synephrine plus 600 mg naringin, the RMR increased by 183 kcal, an increase that was statistically significant with respect to the placebo control (p<0.02). However, consuming 1000 mg hesperidin with 50 mg p-synephrine plus 600 mg naringin resulted in a RMR that was only 79 kcal greater than the placebo group. None of the treatment groups exhibited changes in heart rate or blood pressure relative to the control group, nor there were no differences in self-reported ratings of 10 symptoms between the treatment groups and the control group. This unusual finding of a thermogenic combination of ingredients that elevated metabolic rates without corresponding elevations in blood pressure and heart-rates warrants longer term studies to assess its value as a weight control agent. BODY.INTRODUCTION: The bioflavonoids hesperetin and naringenin are widely distributed in citrus fruits and juices as their glycosides, hesperidin and naringin, respectively. They are present in Citrus aurantium as well as a wide variety of other Citrus species 1. The sugar moieties for both flavonoids consist of rhamnose plus glucose, which must be removed in the intestinal tract before the flavonoids can be absorbed 2, 3. The absorption of these flavonoids in human subjects has been extensively studied and depends in part upon the forms in which they are ingested (juice or fruit, glycoside or aglycone, capsule, tablet, etc.) 4-8. Experiments involving human subjects, animals and cell culture systems have shown that the two flavonoids exhibit a wide range of potentially beneficial physiological and biochemical effects. Naringen and/or hesperidin or their aglycones have been shown to improve insulin sensitivity and glucose tolerance 9, prevent accumulation of triglycerides 9, inhibit cholesterol biosynthesis 10, 11, and serve as antioxidants and anti-inflammatory agents 12, 13 as well as hepatoprotectants 14 and neuroprotectants 15. However, the effects of naringin and hesperidin on metabolic rate and energy utilization have not been previously assessed. Studies have suggested that p-synephrine has thermogenic and lipolytic activities 16-18, increasing energy metabolism 19, 20 as well as athletic performance 21. p-Synephrine is the primary protoalkaloid in bitter orange extract which is derived from the immature fruits of Citrus aurantium 22-25, and is widely distributed in other Citrus species 22, 23, 26. The safety of p-synephrine and bitter orange extract has recently been reviewed 27, and based on the available studies in animals, humans and cell culture systems, it has been concluded that when taken orally and in recommended amounts both are safe. p-Synephrine is widely used in combination with caffeine and other ingredients in products designed to support weight management 19, 28. However, no studies have been reported comparing the thermogenic effects of p-synephrine when combined with the flavonoids hesperidin and naringin. This pilot study was designed to examine these effects using a comparative effectiveness research (CER) study design 29 in a double-blinded, randomized, placebo-controlled protocol to compare the effects of p-synephrine alone and in combination with different amounts of hesperidin and naringin on: (1) resting metabolism, (2) blood pressure, (3) resting heart-rate, and (4) self-reported mood/energy levels in healthy human subjects. BODY.MATERIAL AND METHODS: A total of 50 participants gave written informed consent in compliance with the Helsinki Declaration and approved by Integrative Health Technologies' Ethics Committee. Participants fasted for 8-10 hours without consuming caffeinated beverages, nicotine, exercising or participating in vigorous physical activities. Upon reporting to the research center, participants completed the 10-item self report in Table 1. After remaining seated in an isolated area for 10-15 min., participants completed measurements of their blood pressure, resting heart-rate and resting metabolism using Micro Life's MedGem® Indirect Calorimeter (Microlife Medical Home Solutions, Golden, CO. 80401). The MedGem® is a hand-held, self-calibrating calorimeter that measures oxygen consumption (VO2) to determine resting metabolic rate (RMR). In conjunction with the study, test-retest reliabilities of the instrument were measured on 41 subjects from test-retest periods ranging from 1-17 days between tests. The average reliability coefficient was 90.2 %, a coefficient that was consistent over the four test-retest periods. p-Synephrine was administered in the form of the patented bitter orange extract Advantra Z® which contained 60 % of this active material. Naringin and hesperidin were 96 % pure. All test materials were obtained from Nutratech Inc. (West Caldwell, NJ). Subsequent to RMR measurements, participants were randomly assigned to one of the five double-blinded treatment conditions in which they consumed one ounce of V-8 juice containing the following ingredient variations: Group 1: Placebo (V-8 juice only) Group 2: Advantra Z® (50 mg of p-synephrine) Group 3: Advantra Z® with 0 mg hesperidin and 600 mg naringin Group 4: Advantra Z® with 100 mg hesperidin and 600 mg naringin Group 5: Advantra Z® with 1,000 mg hesperidin and 600 mg naringin After remaining seated and resting for 45 min., participants completed a second self report rating scale. After 75 min., a third and final self report rating scale was completed, and measurements of blood pressure, heart-rate and RMR were determined. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSES: ANOVAs were calculated between the groups' baseline, ending and change scores for each treatment group in addition to Dunnett's t-test between each of the treatment groups and the placebo group. BODY.RESULTS: Figure 1 shows the changes in resting metabolic rates (RMRs) from baseline expressed as kcals for each of the treatment groups (N=10 per group). As expected, a small decrease occurred in the RMR of the placebo group since these participants continued their 8-10 hour fast (Group1). Increases occurred in the RMR of all treatment groups. As compared to placebo, 50 mg p-synephrine (Advantra Z®) alone (Group 2) more than doubled consumption by 65 kcals over placebo. In Group 3, adding 600 mg naringin to the 50 mg p-synephrine further increased calorie consumption more than three-fold to 122 kcals over placebo. The addition of 100 mg hesperidin to the p-synephrine plus naringin in Group 4 resulted in a further increase in calorie consumption by 5-fold to 183 kcal over placebo. However, in Group 5, when the amount of hesperidin consumed in conjunction with the 50 mg p-synephrine plus 600 mg naringin was increased to 1000 mg, the increase in calorie consumption over placebo was only 79 kcal, an increase between calorie consumption in the absence of hesperidin and in the presence of 100 mg hesperidin. In spite of the low levels of statistical power in these groups of only 10 subjects, the increases in RMR in Group 4 was significantly greater than placebo (P=0.039) and Advantra Z alone (Group 2). It is also worth noting the a repeated-measures Students' t-test revealed that the changes from baseline in Group 3 and Group 4 were both statistically significant (P=0.001 and 0.030, respectively). Table 2 shows changes in blood pressures and resting heart-rates from baseline to the ending test for each of the five study groups. The average baseline blood pressures and resting heart-rates for the study cohort were well within the normal ranges of 121/74 mm Hg and 70 BPM. At the conclusion of the study (75 min.), no increases in either systolic or diastolic blood pressures or heart rate were observed. None of the baseline-ending changes approached statistical significance nor were there any statistically significant differences between any of the five study groups. Table 3 depicts the results of the self-report ratings for the 10 symptoms. As compared to the placebo group, none of the changes in these 10 self-reported symptoms were statistically significant either 45 or 75 min. from baseline. No significant effects relative to the placebo group were observed by the participants in the four treatment groups for symptoms including anxiety, hunger, tension, sleepiness, energy, nervousness, headache, upset stomach, concentration or general discomfort. BODY.DISCUSSION: Using a double-blinded, randomized, placebo-controlled protocol, this study compared the effects of 50 mg p-synephrine (as Advantra Z®) alone with three different amounts of hesperidin and naringin on: (1) resting metabolism rate, (2) blood pressure, (3) resting heart-rate, and (4) self-reported mood/energy levels in healthy human subjects. As expected, the RMR of the placebo group decreased over the 75 min. test-retest period since subjects continued the 8-10 hour fast they began prior to the start of the study (Figure 1). The addition of p-synephrine, hesperidin, and naringin led to increases in RMR over placebo. However, while adding 100 mg of hesperidin in Group 4 led to an increase in RMR over placebo, an unexpected reduction was observed in the RMR in Group 5 which received 1000 mg hesperidin as compared to Group 4. These data indicate that increasing the hesperidin from 100 mg to 1,000 mg reversed some of the thermogenic effects of 100 mg hesperidin in combination with 50 mg p-synephrine plus 600 mg naringin. It may be that in this case, more is not better, and increasing the amount of hesperidin constitutes an example of hormesis 30, 31. The increase in the metabolic rate between p-synephrine alone and the placebo control was approximately 6.9 %. This increase in thermogenesis is similar to the increases reported in previous studies involving p-synephrine 17, 18. The increase in RMR between Group 4, the combination of p-synephrine with 600 mg naringin and 100 mg hesperidin, and the placebo control is approximately 17.7 %. If one assumes that the product was taken twice a day for one year, the theoretical increase in calorie consumption would amount to over 31 pounds. However, the actual extent of weight loss if the product was consumed under these conditions remains to be determined. The mechanism by which naringin and hesperidin increase the thermogenic effect of p-synephrine may involve enhancing the expression of adiponectin which is known to play a role in lipid and glucose metabolism. Liu et al. 32 have shown that naringenin and hesperetin up-regulate adiponectin expression, and both activate the peroxisome proliferator-activated receptor-γ (PPARγ) which has been recognized as the master regulator of adipocyte differentiation. In addition, naringenin also possessed significant ability to activate PPARα 32, a major regulator of lipid metabolism in the liver. Products with thermogenic properties are frequently associated with elevated blood pressures and heart-rates. p-Synephrine is a phenylethylamine derivative with some structural similarities to ephedrine, and is thus assumed to exert cardiovascular effects by many authors [see for example, 20, 21, 33-35]. However, no effects on heart rate or blood pressure were observed in response to p-synephrine or p-synephrine in combination with naringin and hesperidin (Table 2). p-Synephrine differs from ephedrine in that it has a hydroxyl group on the para position of the benzene ring and lacks the methyl group on the side chain of the molecule. These structural differences greatly alter the pharmacokinetic and receptor binding properties of p-synephrine, resulting in little or no cardiovascular effects 27, as confirmed by this study. No adverse events have been directly attributable to bitter orange extract and p-synephrine 36, 37. However, caffeine is commonly present in products that contain p-synephrine 19, 28, and is well known to produce cardiovascular effects 38, particularly in caffeine-sensitive individuals 39. The absence of changes in blood pressure, resting heart-rate and self-ratings in the four treatment groups involving p-synephrine, naringin and hesperidin relative to the placebo group is very positive, and in conjunction with the increased thermogenesis may result in the development of beneficial products with respect to weight loss and weight management. The findings were further substantiated by consistency of the results across all four treatment groups. However, longer term studies are required to assess the amount of weight that is actually lost in response to these products, and to provide information concerning safety when used over an extended period of time. A problem faced in studies of thermogenic effects is that noticeable changes in the mood states listed in Table 1 often make subjects aware of whether or not they are receiving a placebo, thus undermining blinding of the study. The lack of significant differences between the treatment groups and the control group with respect to the self-report ratings of symptoms increases confidence in the safety of the combination as well as in the blinding procedure used in this study. Strengths of the study are the use of randomized placebo-controlled double-blinded study design, the absence of thermogenic changes in the placebo group, the absence of adverse effects on blood pressure, heart-rate and self-reports in the four treatment groups, the consistency of the effects of p-synephrine on metabolism in all treatment groups, and the increases in RMR in the treatment groups. The weakness of this study is the small number (10) of subjects in the placebo and in each of the treatment groups. The study also reveals an interesting effect with respect to hesperidin where an increased amount may lead to a masking of the thermogenic effects found with lesser amounts. BODY.CONCLUSIONS: This randomized double-blinded placebo control pilot study supports the safety and thermogenic effects of p-synephrine, particularly when combined with 600 mg of naringin and 100 mg of hesperidin. No increase in heart rate or blood pressure was observed over the 75 min. of the study nor were there any differences in a self-reported rating of 10 common symptoms between the treated and placebo groups. The data suggests the combination of the flavonoids naringin and hesperidin with p-synephrine may assist weight management. Further studies are required to determine optimal doses as well as safety and efficacy associated with long term use.

TITLE: Smoking cessation intervention during pregnancy in a Polish urban community – what is the target population? ABSTRACT: The aim of this project was to evaluate the effect of intensive individual anti-smoking counselling among pregnant women from a Polish urban community with a large representation of socially underprivileged women. The study was conducted between 1 December 2000 and 31 December 2001. Out of 204 women who were asked to take part in a midwives-assisted program of educational counselling to stop smoking, 152 (74.5%) agreed to participate. The intervention program included four visits of a midwife trained in smoking cessation techniques to the home of a smoking pregnant woman. The control group were 145 pregnant women who on the first visit to a maternity unit received only a standard written information on the health risk from maternal smoking to the foetus. The percentage of pregnant women who quitted smoking during the project was 46.1% in the intervention group and 23.4% among the controls (p < 0.001). After combining the intervention group with the women who refused to participate in the project, the rate of quitting was 36.3%, still significantly higher than in controls (p = 0.01). The strongest influence of the intervention was found among women smoking more than 5 cigarettes/day. Women covered by the intervention programme, who reported smoking in previous pregnancies, were found to quit smoking to a much higher extent than the controls with a similar background. Such pattern was also observed for women whose husbands were smokers. The benefits of the intervention, especially for the socially underprivileged women, seem to result from an increased proportion of subjects who undertook a quitting attempt, rather than the effectiveness of these attempts. In the intervention group, among the subjects who did not manage to quit smoking during pregnancy, the number of women who at least slightly reduced their smoking rate was twice as high as in the controls. BODY.INTRODUCTION: From the late fifties, when the first report was published on the possible influence of maternal smoking on birth weight [1], hundreds of studies have demonstrated conclusively that smoking during pregnancy increases the risk of small-for-gestational-age infants and preterm delivery and, consequently, the perinatal mortality [2-4]. It was also observed that smokers who had changed their habit during pregnancy had a lower risk of these pathologies. A strong dose-response relationship has been found between the rate of smoking and the birth weight of their children [5,6]. More recent investigations imply that the health consequences of low birth weight can be seen in adult life e.g. as an elevated risk of cardiovascular diseases [7]. Butler and co-workers found that the infants of women who quitted smoking had the birth weights essentially the same as those of non-smokers [8]. However, the evidence that smoking cessation is effective has been provided by studies using the clinical trial design [9] often with an inclusion of objective biochemical measurements to confirm the smoking status of the examined groups [10-12]. A meta-analysis of the controlled trials of smoking cessation interventions during pregnancy concluded that overall there was a significant increase in the smoking cessation rates [13]. The benefits of these interventions, measured by the differences in the rate of quitters and average infant birth weight in the intervention and control populations, were varying and it was postulated that only the cognitive behavioural strategies are really effective [14]. The relative gain of the smoking cessation interventions depends not only on the type of activities undertaken but also on the characteristics of pregnant women. Despite the fact that the recognition of smoking as a hazard to reproductive health is fairly universal, one may hypothesize that for some groups of women it will be more difficult to quit smoking during pregnancy than for others. The decision-makers who are in charge of preventive health activities would like to be convinced that investment in the intervention programs in those specific target groups is really cost-effective. What are these groups? There are only a few reports on the results of smoking cessation programs that would consider the social characteristics of smoking pregnant women [15,16]. This paper reports the results of a randomised controlled trial measuring the effect of intensive individual anti-smoking counselling in the population of Polish women from an urban community, with a large representation of socially underprivileged women. BODY.METHODS.STUDY POPULATIONS: The cluster randomised trial was undertaken in the public district maternity centres in Łódź, Poland. The null hypothesis being tested was that the smoking cessation program conducted by midwives in the homes of pregnant women does not affect the quitting rate Basing on data for 1999, we found that 15 maternity units in the Łódź district provide prenatal care for about 1600 pregnant women a year. It was estimated from previous surveys that the smoking rate in I trimester of pregnancy is about 30%. Thus, it could be expected that annually approx. 32 smoking pregnant women book for prenatal care in each maternity unit. We calculated that in order to find a significant difference between the quitting rates of 35% and 20%, assuming α = 0.05 and 80% power, the compared populations should include at least 137 subjects. Assuming the 30% refusal rate in the intervention group and about 5% among the controls, the former group should comprise at least 200 subjects and the latter one 144. We found that to accumulate populations of this size, six maternity units should participate in the intervention and four in the control procedures. 204 women in total were asked to take part in a midwives-assisted program of intensive educational counselling to stop smoking. Out of them, 152 (74.5%) agreed to participate in such activities. At the same period of time, 145 (100%) smoking pregnant women who booked for maternity care in four control units agreed to respond to an inquiry about their smoking status and to update this data in 20th month of pregnancy and after delivery. The study was conducted between 1 December 2000 and 31 December 2001. We decided to use as controls a female population from a different administrative part of the city rather than to randomly allocate women to the intervention and control group from the same maternity units. We expected that the women participating in the program might have some influence on the latter group. BODY.METHODS.DESCRIPTION OF THE INTERVENTION AND CONTROL ACTIVITIES: On their first visit to one of six public maternity units participating in the smoking cessation program, all smoking pregnant women were informed by their physicians about the programme and asked whether they wanted to join it. According to the study protocol, a midwife trained in smoking cessation techniques was to visit the smoking pregnant women in their homes. The women received written materials prepared by Community Health Research Unit in Ottawa that were translated and adapted to the Polish conditions [17]. The programme included four midwife visits. The first visit included a diagnosis of the level of smoking addiction, using the Fagerström method, then discussing the benefits of smoking cessation, and encouraging the woman to take up a decision to quit smoking. The Fagerström Test for Nicotine Dependence measures physical dependence on nicotine [18]. This test includes 6 questions about the smoking habits. The total score above 6 points indicated that the woman examined may have been physically dependent on nicotine. On the second visit, about 1–2 weeks later, the pregnant woman who decided to give up smoking determined when this was to be done and signed the "Declaration to quit smoking". On the third visit, scheduled 1–2 days after the expected quitting day, the midwife inquired whether the woman actually acted as she promised. On the fourth visit, one month after the quitting day, the midwife informed the woman how to avoid smoking and keep smoking abstinence. During all the four visits it was stressed that the program is supposed to provide support only to those women who attempt to change their smoking status. The counselling emphasised the potential benefits to the foetus but the decision to quit or not was left to the pregnant woman herself. Once she made a step in this direction, all her further efforts were repeatedly reinforced. When the woman covered by the program did not manage to quit smoking during the four midwife's visits, she was offered a possibility to continue the intervention activities during another five visits. Women from the control group, on the first visit to a maternity unit, received a standard written information about the health risk from maternal smoking to the foetus. At the same time, the physician who passed this information encouraged them to stop smoking. BODY.METHODS.SCOPE OF INFORMATION OBTAINED: During the initial contact with the maternity unit the subjects filled in the questionnaire that contained information about their smoking profile (number of cigarettes smoked, years of smoking, partner's smoking, other household members' smoking, smoking in previous pregnancies if any, previous smoking cessation attempts). Such information was obtained from all women participating in the smoking cessation program. Similar data were also collected from those who refused to participate and the control group. In the control group, the data on smoking habit were updated in the 20th month of pregnancy whereas in the intervention group any changes in the smoking profile were recorded during each visit. As regards the group of women who refused to participate, it was possible to elicit information about their social characteristics and smoking habit. These were collected during their first visit to maternity unit. A few days after the delivery the midwives visited the women both from the intervention and control group in their homes. As for the former, they inquired whether anything had changed in their smoking status since the last meeting (maintaining abstinence, smoking relapses, quitting after the period of intervention). In the control group, the smoking status during the whole pregnancy was examined retrospectively. BODY.METHODS.DATA ANALYSIS: The data were analysed with the use of the SPSS package. The statistical measures of relationship included the t-test and the chi-square test or Fisher exact test as appropriate. The level of significance for acceptance of relationship between the variables was the conventional 0.05. BODY.RESULTS.COMPARISON OF SOCIAL CHARACTERISTICS OF THE EXAMINED GROUPS: The intervention and control subjects had comparable demographic profiles (Table 1). The average age of women in both groups was 25.8 years. The intervention group was more frequently found to be unmarried and primigravida and to have only the primary or vocational education. None of these differences was statistically significant. No differences were noted between the two groups with respect to the employment status. Table 1 Social characteristics of the intervention, control and refusal groups Variable Intervention (N = 152) Control (N = 145) Refusal (N = 52) n % n % n % Education  Primary or vocational 125 82.2 110 75.9 40 76.9  (8 or 11 years of education)  College or university 27 17.8 35 24.1 12 23.1  (12 or 17 years of education) Marital status  Married 73 48.0 86 59.3 18 34.6  Unmarried 79 52.0 59 40.7 34 65.4 Number of children  0 81 53.3 61 42.1 29 55.8  1 35 23.0 41 28.3 12 23.1  ≥ 2 36 23.7 43 29.6 11 21.1 Employment status  Employed 55 36.2 51 35.2 19 36.5  Unemployed 97 63.8 94 64.8 33 63.5 Years of smoking  <5 45 29.6 45 31.0 19 36.5  5–10 73 48.0 79 54.5 21 40.4  >10 34 22.4 21 14.5 12 23.1 No of cigarettes smoked/day  <5 10 6.6* 13 9.0 4 7.7  5–10 65 42.8 82 56.5 27 51.9  >10 77 50.6 50 34.5 21 40.4 Fagerström test  0–6 138 90.8* 143 98.6 46 88.5  7–9 14 9.2 2 1.4 6 11.5 Husband smoking  Yes 130 85.5 111 76.6 46 88.5  No 22 14.5 34 23.4 6 11.5 Any other household member smoking?  Yes 89 58.6* 63 43.4 33 63.5  No 63 41.4 82 56.6 19 36.5 Smoking in previous pregnancies  Primigravidas 76 - 58 - 25 -  Yes 68 89.5 74 85.1 23 85.2  No 8 10.5 13 14.9 4 14.8 * statistically significant (p < 0.05) difference in distributions of groups intervention and control. In the intervention group, the husbands/partners of the subjects were found to be smokers as often (85.5%) as those of the controls (76.6, p = 0.05). More pronounced differences were noted when other smokers in the household were taken into account (58.6% vs. 43.4%, p = 0.009). The women from the intervention group who were pregnant in the past, admitted smoking in previous pregnancies slightly more frequently than the controls did (89.5 vs. 85.1%, p = 0.4). The intervention group smoked more cigarettes per day on average (12.3 ± 5.9 vs. 10.9 ± 5.7, p = 0.04). The level of smoking addiction measured by the Fagerström test was significantly higher in the intervention group (3.6 ± 2.1 vs. 2.2 ± 1.8, p < 0.001). There were no statistically significant differences between the intervention and refusal groups. Women from the refusal group were more often unmarried than those from the intervention group (65.4 vs. 52.0, p = 0.09). In this group, the average period of smoking was 7.4 ± 5.9 and the number of cigarettes/day 12.3 ± 6.7. BODY.RESULTS.RATE OF QUITTING SMOKING: The percentage of pregnant women who quitted smoking was 46.1% in the intervention group and 23.4% among the controls (p < 0.001) (Table 2). In the refusal group (N = 52) four women quitted smoking, while the smoking status of others was unknown. After combining the intervention group with the group of women who refused to participate in the project, the rate of quitting was 36.3%, thus still significantly higher (p = 0.01) than in the control group. Table 2 Quitting smoking and social characteristics of the examined subjects (% calculated as a fractions of all subjects in the given subgroup) Variable Intervention Control Intervention and Refusal N n % N n % N n % All groups 152 70 46.1* 145 34 23.4 204 74 36.3** Education Primary or vocational 125 52 41.6* 110 19 17.3 165 54 32.7** College or university 27 18 66.7 35 15 42.9 39 20 51.3 Number of cigarettes/day smoked before the attempt <5 10 9 90.0* 13 5 38.5 14 9 64.3 5–10 65 35 53.8* 82 22 26.8 92 36 39.1 >10 77 26 33.8* 50 7 14.0 98 29 29.6** Fagerström test 0–6 138 63 45.7* 143 34 23.8 184 67 36.4** 7–9 14 7 50.0 2 0 0 20 7 35.0 Smoking in previous pregnancies Primigravidas 76 43 56.6* 58 20 34.5 101 45 44.6 Yes 68 20 29.4* 74 7 9.5 91 21 23.1** No 8 7 87.5 13 7 53.8 12 8 66.7 Smoking husband Yes 130 58 44.6* 111 20 18.0 176 61 34.7** No 22 12 54.5 34 14 41.2 28 13 46.4 * statistically significant (p < 0.05) difference in distributions of groups intervention and control; ** statistically significant (p < 0.05) difference in distributions of groups intervention and refusal combined and control. The effect of the intervention was much more evident in the group with only primary or vocational education (41.6% vs. 17.3%, p < 0.001) than with a college or university degree (66.7% vs. 42.9%, p = 0.06). A similar pattern was observed in the combined intervention and refusal groups versus the control group. When the number of cigarettes/day reported on the first visit was considered, the strongest influence of the intervention was found in the group smoking 5–10 cigarettes/day (53.8% vs. 26.8%, p < 0.001) and more than 10 cigarettes/day (33.8% vs. 14.0%, p = 0.01). Out of the subjects from the intervention group and the combined intervention and refusal group who scored above six on the Fagerström test, respectively fifty percent and thirty-five percent were found to quit smoking. Both findings were significantly higher than those obtained in the control group with similar test results. The women covered by the intervention program who reported smoking in previous pregnancies were noted to quit smoking to a much higher extent than the controls with a similar background (29.4% vs. 9.5%, p = 0.002). A similar pattern was observed in the combined intervention and refusal groups versus the control group (23.1% vs. 9.5%, p = 0.02). Such pattern was also observed in the group of women whose husbands were smokers (44.6% vs. 18.0%, p < 0.001). After combining the intervention group with the group of women who refused to participate in the project, the rate of quitting was still significantly higher than in the control group (34.7 vs. 18.0, p = 0.02). BODY.RESULTS.ATTEMPTS TO QUIT SMOKING: The intervention group almost twice as often as the controls undertook at least one attempt to quit smoking (78.9% vs. 40.0%, p < 0.001) (Table 3). This pattern could best be seen among the subjects with primary or vocational education. In the intervention group with such level of education 76.0% made such challenge, compared to 31.8% from the control group (p < 0.001). This finding was to a much lesser extent observed among women who had a college or university degree. It is worth noting that women from the intervention group who had primary or vocational education were trying to quit smoking even harder than those with a college or university degree in the control group (76.0% vs. 65.7%). As regards the percentage of women who had undertaken at least one quitting attempt, significant differences between the intervention and control subjects were found only in the subgroups smoking five or more cigarettes/day. Table 3 Quitting attempts among the intervention and control subjects (% calculated as a fraction of all subjects in the given subgroup) Variable Intervention Control N n % N n % All groups 152 120 78.9* 145 58 40.0 Education Primary or vocational 125 95 76.0* 110 35 31.8 College or university 27 25 92.6* 35 23 65.7 Number of cigarettes/day smoked before the attempt < 5 10 10 100 13 11 84.6 5–10 65 53 81.5* 82 35 42.7 >10 77 57 74.0* 50 12 24.0 Fagerström test 0–6 138 111 80.4* 143 58 40.6 7–9 14 9 64.3 2 0 0 Smoking in previous pregnancies Primigravidas 76 67 88.2* 58 31 53.4 Yes 68 45 66.2* 74 19 25.7 No 8 8 100 13 8 61.5 Smoking husband Yes 130 100 76.9* 111 38 34.2 No 22 20 90.9* 34 20 58.8 * statistically significant (p < 0.05) difference in distributions of groups intervention and control. Sixty four percent of the intervention subjects who scored above six on the Fagerström test, compared to none of two controls with similar test results, undertook an attempt to quit smoking (p = 0.2). The percentage of subjects from the intervention group who smoked in previous pregnancies and undertook a quitting attempt during the project was twice as large as that of the controls (66.2% vs. 25.7%, p < 0.001). A similar trend could be seen for the variable of living with smoking husband/partner (76.9% vs. 34.2%, p < 0.001). BODY.RESULTS.EFFECTIVENESS OF SMOKING CESSATION ATTEMPTS: The effectiveness of smoking cessation attempts (success rate), measured by the ratio of successful quitters to all who attempted to quit smoking, was 58.3% and 58.6%, respectively in the intervention and control groups (p = 1.0) (Table 4). No differences were found either in the success rate calculated for education subgroups. With respect to the number of cigarettes smoked, the effectiveness of quitting attempts was found to decrease with an increasing number of cigarettes/day in the intervention group. An opposite trend was observed in the control group. However, in view of the small number of subjects in each subgroup, the statistical significance of these patterns was not proved. Neither the results of the Fagerström test, nor the smoking in previous pregnancies or living with a non-smoking husband/partner enhanced the effectiveness of the quitting attempts. Table 4 Effectiveness of quitting attempts by social variables (% calculated as a fraction of all subjects with quitting attempts in given subgroup) Variable Intervention Control N n % N n % All groups 120 70 58.3 58 34 58.6 Education Primary or vocational 95 52 54.7 35 19 54.3 College or university 25 18 72.0 23 15 65.2 Number of cigarettes/day smoked before the attempt < 5 10 9 90.0 11 5 45.5 5–10 53 35 66.0 35 22 62.9 >10 57 26 45.6 12 7 58.3 Fagerström test 0–4 111 63 56.8 58 34 58.6 7–9 9 7 77.8 0 0 - Smoking in previous pregnancies Primigravidas 67 43 64.2 31 20 64.5 Yes 45 20 44.4 19 7 36.8 No 8 7 87.5 8 7 87.5 Smoking husband Yes 100 58 58.0 38 20 52.6 No 20 12 60.0 20 14 70.0 BODY.RESULTS.SMOKING REDUCTION AMONG NON-QUITTERS: The two groups examined were compared with respect to the smoking reduction rate in the subgroups of women who did not manage to quit smoking during pregnancy (Table 5). In the intervention group, the number of subjects who were able to at least slightly reduce their smoking rate was twice as large as in the controls (91.5% vs. 50.5%, p < 0.001). Both groups reduced the number of cigarettes smoked by more than 50%, which corresponded to about 7–8 cigarettes/day on average. Table 5 Changes in the smoking profile in the intervention and control groups (quitters excluded) Examined group Reduced number of cigarettes No change Mean daily reduction in cigarette consumption Mean difference in the daily consumption of cigarettes n % N % Av. % SD Av. SD Intervention (n = 82) 75 91.5 7 8.5 60.8 16.0 8.5 4.1 Control (n = 111) 56 50.5 55 49.5 57.1 14.5 7.5 3.9 p value <0.001 0.1 0.09 BODY.DISCUSSION: We found that the midwife-assisted smoking cessation intervention was effective when compared with the results obtained for controls covered by routine procedures. The quitting effect referred mainly to the subjects with primary education and who smoked more than 5 cigarettes/day at booking to the maternity unit. The rates of quitting smoking we noted were much higher than those reported by other investigators [3,9,19-24]. The latter were usually at the level of 2–17% in the control group and 6–27% in the intervention one. The high percentage of quitters in the Polish population may be explained by the fact that in this country the population of smoking women to a much higher extent includes the occasional smokers who may have less problems with quitting smoking during pregnancy than those strongly addicted to tobacco. The quitting rate of 23,4% in the control group is very close to those reported in other Polish surveys of pregnant women [25,26]. The large proportion of quitters in the intervention group may result from a rather low participation rate of women to whom the intervention was offered. We may speculate that women who agreed to take part in the programme were more inclined to quit smoking than those who refused. No such selection bias was found in the case of controls. We did not verify the self-reported smoking status by using biomarkers of exposure to tobacco smoke. From our previous investigations we know that about 20% of smokers may not admit that they smoke [26]. It may be possible that a similar proportion of smokers was not identified in our study and consequently was not assigned either to the intervention or control group. We cannot predict whether the inclusion of representatives of this group would result in an increase in the quitting rate (as they are more conscious of the smoking hazard) or a decrease (as they are not willing to accept any counselling). Our position is that whatever the direction of this bias might be, it should refer to both the groups compared, hence the final findings should not be affected. One of the consequences of not using any validation of the smoking status may be an overestimation of the quitting rate in the intervention group, since they may have reported false results just to please their counsellors. However, as a consequence, the effectiveness of such attempts must be lower in the intervention group (those claiming false attempts would not report abstinence for a long period of time and their attempt would be registered as unsuccessful), which was not the case in our study. Almost all the non-quitters in the intervention group and half of them in the controls reported some reduction in the amount of cigarettes smoked. These rates are twice as high as in the study of Hartman et al. [10] in which resident physicians provided self-helped materials to intervention subjects. The explanation may be what was already mentioned that the smoking pregnant women in Poland derive mainly from the population of occasional smokers, and those of them who do not manage to quit smoking can at least reduce the number of cigarettes smoked during pregnancy. It is also possible that the women willing to stop smoking may have been over-represented in the intervention group. Both the maternity units participating in the intervention program and the control ones were selected randomly. However, due to a substantial rate of non-participants the two examined groups were not exactly comparable. To eliminate the possible bias resulting from this situation, two major variables were controlled that affected the decision to cease smoking i.e. education and cigarette consumption prior to intervention. A similar approach was used for other characteristics well known to hinder the quitting attempts: smoking husbands/partners, other smoking household member, smoking in previous pregnancies. BODY.CONCLUSION: The midwife-assisted smoking cessation intervention seems to be an effective tool in the activities to help pregnant smokers from urban communities, where the smoking rates are relatively high, to make a decision to quit smoking in view of the potential hazard to their pregnancies and the children to be born. This kind of preventive program is well-suited for women with low education, who are heavy smokers and who do not receive support from their families while making efforts to give up smoking. More information about the potential benefits of the proposed intervention will be gathered when the analysis of pregnancy outcomes has been completed. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests.

TITLE: Comparative Evaluation of Root Canal Transportation by Three NiTi Single-File Systems in Curved Canals: A Cone Beam Computed Tomography Study ABSTRACT.INTRODUCTION: This study is aimed at evaluating root canal transportation in the mesiobuccal canal of mandibular first molars prepared with One Shape, Reciproc, and M-One nickel titanium (NiTi) single-file rotary systems using cone beam computed tomography (CBCT). ABSTRACT.MATERIALS AND METHODS: In this ex vivo study, CBCT scans of 45 extracted human mandibular first molars with 20–40° curvature were obtained. The teeth were randomly divided into three groups (n=15) for preparation of the mesiobuccal canal with One Shape, Reciproc, and M-One rotary systems according to the manufacturers' instructions. CBCT scans were obtained again after canal preparation. Changes caused by preparation in the coronal, middle, and apical thirds were determined on CBCT scans and analyzed using the Kruskal–Wallis test at P ≤ 0.05 level of significance. ABSTRACT.RESULTS: No significant difference was noted in the amount of canal transportation among the three groups (P > 0.05). M-One caused greater transportation in the apical third compared with Reciproc and One Shape, and One Shape caused greater transportation in the coronal third compared with other groups, although its magnitude was less than 0.3 mm. ABSTRACT.CONCLUSION: Reciproc, One Shape, and M-One are not significantly different in terms of canal transportation. BODY.1. INTRODUCTION: The main objective of endodontic treatment is to eliminate or minimize microorganisms in the root canal system while maintaining the original shape and path of the root canal [1]. However, no instrument/technique can predictably eliminate all the microorganisms from the root canal system. The cleaning efficacy of endodontic instruments significantly decreases at the apical third of root canals [2]. Many root canals have curvatures, and endodontic instruments tend to return to their original straight position during instrumentation of curved canals [3, 4]. Dentinal wall thickness in root canals has a direct relationship with root resistance to lateral forces [5, 6]. In the recent years, several nickel titanium (NiTi) instruments capable of faster and more efficient root canal preparation were introduced in the market. These systems have differences in some features such as cleaning efficacy, stress applied to dentinal walls, and ability to prepare oval-shaped root canals [7]. One Shape file (MicroMega, France) operates with continuous rotational movement compared with the other single-file systems. One Shape instruments have higher cutting efficacy in the root, which is probably attributed to electropolishing, flexibility, and variable cross section along its blade [8]. Antibreakage control in this file increases its fracture strength. This system has a sterile file with a tip size of ISO 25, 0.06 taper, variable pitch and a noncutting (safety) tip for cleaning and shaping of root canals [5]. Reciproc file (VDW, Munich, Germany) is made of M-Wire, which increases its strength and flexibility [9]. This file has an S-shaped cross section and a noncutting (safety) tip operating with reciprocal motion. Reciprocal movement was introduced in 1985 and is composed of two rotations, namely, 150° counterclockwise and 30° clockwise motions. This file is available in three sizes and tapers: R25 (25/0.08), R40 (40/0.06), and R50 (50/0.05) [10, 11]. M-One (Park, China) is a single-file system with a tip size of ISO 25, 0.06 taper, and full rotational motion. The alloys used in its composition are CM-Wire and Neo NiTi, and it has a regular triangular cross section. Cone beam computed tomography (CBCT) can be used to assess the amount of dentin removed from the root canal walls during root canal preparation. This imaging modality enables assessment of volume, surface area, cross-sectional shape, and taper of canal with no damage to tooth structure [8]. A previous study showed that WaveOne with reciprocating motion caused less canal transport than One Shape and ProTaper files [12]. Another study showed that Reciproc and WaveOne instruments caused significantly less canal transport than One Shape [13], while Cimilli and Kartal [14] indicated that continuous rotation had higher centering ability than reciprocating motion. No previous study has compared canal transportation caused by three NiTi single-file systems, namely, One Shape and M-One with continuous rotational movement and Reciproc with reciprocal movement. Thus, this study aimed at comparing canal transportation in the mesiobuccal canal of mandibular first molars prepared with One Shape, Reciproc, and M-One NiTi single-file rotary systems using CBCT. BODY.2. MATERIALS AND METHODS: The study was approved by the Ethics Committee of Zahedan University of Medical Sciences (IR.ZAUMS.REC.1395.83), and written informed consent was obtained from all patients. The study was conducted on 45 extracted mandibular first molars of patients presented to the Oral and Maxillofacial Surgery Department of Zahedan University of Medical Sciences. The teeth had been extracted due to periodontal or orthodontic reasons and had closed apices and mesial root curvature of 20–40° measured according to Schneider's method [15]. Root curvature measured 5–9 mm distance from the apex, and the mean length of the root was 19–22 mm. After collection of teeth, tissue residues and calcified debris were eliminated, and the teeth were disinfected in 0.1% thymol solution at 9°C for 24 hours. The teeth were rinsed with tap water to eliminate thymol residues, and they were then immersed in saline at 4°C. Primary radiographs of the mesial root were obtained to determine the degree of root curvature. Teeth with mesial root canals with one apical foramen and no sign of calcification or internal resorption were included in the study. Teeth with S- or C-shaped canals were excluded. All roots were evaluated under a stereomicroscope at ×12 magnification to ensure absence of craze lines, cracks, or fractures. Teeth with such defects were excluded from the study and replaced with sound teeth. Three-dimensional CBCT scans were obtained with the Vatech 3D system (Ez3D Plus, Korea) with the exposure settings of 89 kVp, 5.4 mA, 50 × 50 mm field of view, 0.08 mm voxel size, and 10 s time prior to preparation of root canals. Access cavity was prepared by a diamond bur and high-speed handpiece under air and water spray to negotiate mesiobuccal canal orifice. To determine the mesiobuccal canal working length, a #10 K file (Mani, Tochigi, Japan) was introduced into the canal until its tip was visible at the apex. The working length was determined to be 1 mm shorter than this length. A silicone impression material (Oranwash; Zhermack spa, Rovigo, Italy) was used to cover the cementum to simulate periodontal ligament. To prevent the entry of the silicone material into the apical foramen, the apex was sealed with red dental wax. The teeth were then mounted in blocks measuring 5 × 5 mm filled with putty wash to the level of cementoenamel junction. The teeth were embedded in a mold in a parallel fashion to standardize the pre- and postinstrumentation images. A small piece of orthodontic wire was placed at the corner of silicone blocks as a guide to ensure correct direction of scanning. The teeth were randomly divided into three groups of 15. Reciproc rotary file was used in group 1, One Shape was used in group 2, and M-One was used in group 3. BODY.2. MATERIALS AND METHODS.2.1. ROOT CANAL PREPARATION: All mesiobuccal canals were instrumented to the working length using the crown-down technique with a handpiece (X-Smart, Dentsply Maillefer, Japan) at the speed and torque recommended by the manufacturers for each system. The root canals were irrigated with 2.5% sodium hypochlorite solution delivered with a 30-gauge needle between instruments. Also, 17% EDTA and 5.25% sodium hypochlorite were used for final rinse and elimination of smear layer. BODY.2. MATERIALS AND METHODS.2.2. ROOT CANAL PREPARATION IN GROUP 1: Reciproc file with a tip size of ISO 25 and 0.08 taper was used to reach the working length with gentle pecking motion and reciprocating rotation at the speed and torque recommended by the manufacturer. Recapitulation was done frequently using a #10 K file, and the mesiobuccal canals were rinsed with 2.5% sodium hypochlorite after using each instrument. Glyde was used as the lubricant. BODY.2. MATERIALS AND METHODS.2.3. ROOT CANAL PREPARATION IN GROUP 2: One Shape file with a tip size of ISO 25 and 0.06 taper was used with continuous rotation and gentle in-and-out movement at the speed and torque recommended by the manufacturer to reach the working length. Recapitulation was done repeatedly using a #10 K file. The mesiobuccal canals were rinsed with 2.5% sodium hypochlorite after using each instrument. Glyde was used as the lubricant. BODY.2. MATERIALS AND METHODS.2.4. ROOT CANAL PREPARATION IN GROUP 3: M-One file with a tip size of ISO 25 and 0.06 taper was used to reach the working length with continuous rotation and torque recommended by the manufacturer. Recapitulation was done repeatedly using a #10 K file. The mesiobuccal canals were rinsed with 2.5% sodium hypochlorite after using each instrument. Glyde was used as the lubricant. CBCT scans of the teeth were obtained with the same settings mentioned earlier. The root canal wall thickness in uninstrumented and instrumented root canals was measured at 3, 6, and 9 mm from the apex. The amount of canal transportation was calculated using the following formula: CT = (a1 − a2) − (b1 − b2), where a1 was the shortest distance from the lateral edge of the uninstrumented canal to the lateral edge of the root, b1 was the shortest distance from the medial edge of the uninstrumented canal to the medial edge of the root, a2 was the shortest distance from the lateral edge of the instrumented canal to the lateral edge of the root, and b2 was the shortest distance from the medial edge of the instrumented canal to the medial edge of the root (Figure 1). Positive values obtained from this formula indicate the occurrence of transportation lateral to the curvature, whereas negative values indicate transportation in a direction facing the furcation. Based on this formula, zero value indicates no transportation, negative values indicate transportation in the distal direction (furcation side), and positive values indicate transportation in the mesial direction. It should be noted that canal preparation was done by the same operator in all groups, and canal wall thickness was measured by another operator blinded to the group allocation of teeth. Data were analyzed using SPSS version 20 (SPSS Inc., IL, USA). The Kolmogorov–Smirnov test was used to assess the distribution of data, which showed that data were not normally distributed. Thus, the mean and standard deviation (SD) of root canal transportation were calculated and compared using nonparametric Kruskal–Wallis test. P ≤ 0.05 was considered statistically significant. BODY.3. RESULTS: Figures 1 and 2 show the schematic view and CT scan images before and after instrumentation in the coronal, middle, and apical cross sections, respectively. Table 1 and Figure 3 show the amount of canal transportation in the apical, middle, and coronal thirds in the three groups. No significant difference was noted in the amount of canal transportation among the three groups (P > 0.05). M-One caused greater transportation in the apical third compared with Reciproc and One Shape, and One Shape caused greater transportation in the coronal third compared with the other groups, although its magnitude was less than 0.3 mm. BODY.4. DISCUSSION: The present study compared the amount of canal transportation caused by three single-file systems in root canals using CBCT. Reciproc, One Shape, and M-One were not significantly different in terms of canal transportation. It appears that reciprocating motion causes less transportation compared with full rotation. Mandibular molars are among the most common teeth requiring endodontic treatment [16, 17]. Thus, quality of root canal preparation in these teeth is an interesting topic of research. Mesial canals of these teeth often have mesiodistal and/or buccolingual curvatures. Due to more severe curves in the mesiobuccal canal, this canal is highly susceptible to transportation during mechanical preparation by endodontic instruments. Canal transportation refers to complete removal of dentin from the external wall of the curvature in the apical half of the canal, which is due to the tendency of file to straighten up and return to its original straight shape during preparation of curved root canals; this may lead to ledge formation and possible perforation of canal. In addition, canal transportation in the coronal third may lead to strip perforation and reduction in residual dentin thickness [18]. Wu et al. [19] reported that apical transportation more than 0.3 mm negatively affects the sealing ability of root filling materials. In our study, canal transportation over 0.3 mm was not seen in any group, and the magnitude of canal transportation was between 0 and 0.08 mm. Our results showed no significant difference in magnitude of canal transportation among the three rotary systems tested. M-One caused greater transportation in the apical third compared with Reciproc and One Shape, and One Shape caused greater transportation in the coronal third compared with other groups, although its magnitude was less than 0.3 mm. However, One Shape showed maximum transportation in the internal wall of the curvature in the coronal third, which can weaken the canal wall and increase the risk of strip perforation and microcrack formation. Similar to our study, Dhingra et al. [8] showed that One Shape removed more dentin from the coronal third than Reciproc and WaveOne, which may be related to decreased torsional and flexural stresses in reciprocating motion, resulting in higher centering ability and less taper lock [20]. Another study showed that WaveOne with reciprocating motion caused less transportation than One Shape and ProTaper [12]. It appears that One Shape and M-One have higher tendency to remove dentin from the internal wall of the curvature while Reciproc operates in a safer way. This finding can be explained by the difference between the reciprocating and continuous rotational motions. The reciprocating motion consists of a clockwise motion and a counterclockwise motion and allows the file to be continuously free against the internal wall of the curvature; thus, it operates opposite to the balanced force preparation technique and maintains the central canal path while shaping it. Also, this file is made of M-Wire alloy and has a variable angle and helical pitch, which increase its flexibility. Another study also showed that reciprocating motion, in contrast to continuous rotation, did not increase apical transportation [21]. Saber et al. [13] showed that Reciproc and WaveOne instruments caused significantly less transportation than One Shape, which may be attributed to the use of M-Wire alloy in fabrication of Reciproc and WaveOne files and their reciprocating motion. However, Cimilli and Kartal [14] indicated that continuous rotational motion has higher centering ability compared with reciprocating motion. Moreover, Beurklein et al. [22] indicated that One Shape had higher canal centering ability than Reciproc. Attempts are ongoing to improve the efficacy of chemomechanical preparation of root canals by new instruments and disinfecting agents. It appears that by use of files with reciprocating motion, compared to those with full rotational movement, optimal shaping of root canals with minimal canal transportation can be achieved. BODY.5. CONCLUSION: The magnitude of canal transportation was not significantly different among different rotary systems in root canal preparation, except for the mean transportation in coronal sections created by M-One rotary file, which was significantly greater than that in the control group.

TITLE: Effect of wood smoke exposure on vascular function and thrombus formation in healthy fire fighters ABSTRACT.BACKGROUND: Myocardial infarction is the leading cause of death in fire fighters and has been linked with exposure to air pollution and fire suppression duties. We therefore investigated the effects of wood smoke exposure on vascular vasomotor and fibrinolytic function, and thrombus formation in healthy fire fighters. ABSTRACT.METHODS: In a double-blind randomized cross-over study, 16 healthy male fire fighters were exposed to wood smoke (~1 mg/m3 particulate matter concentration) or filtered air for one hour during intermittent exercise. Arterial pressure and stiffness were measured before and immediately after exposure, and forearm blood flow was measured during intra-brachial infusion of endothelium-dependent and -independent vasodilators 4–6 hours after exposure. Thrombus formation was assessed using the ex vivo Badimon chamber at 2 hours, and platelet activation was measured using flow cytometry for up to 24 hours after the exposure. ABSTRACT.RESULTS: Compared to filtered air, exposure to wood smoke increased blood carboxyhaemoglobin concentrations (1.3% versus 0.8%; P < 0.001), but had no effect on arterial pressure, augmentation index or pulse wave velocity (P > 0.05 for all). Whilst there was a dose-dependent increase in forearm blood flow with each vasodilator (P < 0.01 for all), there were no differences in blood flow responses to acetylcholine, sodium nitroprusside or verapamil between exposures (P > 0.05 for all). Following exposure to wood smoke, vasodilatation to bradykinin increased (P = 0.003), but there was no effect on bradykinin-induced tissue-plasminogen activator release, thrombus area or markers of platelet activation (P > 0.05 for all). ABSTRACT.CONCLUSIONS: Wood smoke exposure does not impair vascular vasomotor or fibrinolytic function, or increase thrombus formation in fire fighters. Acute cardiovascular events following fire suppression may be precipitated by exposure to other air pollutants or through other mechanisms, such as strenuous physical exertion and dehydration. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01495325. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12989-014-0062-4) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Cardiovascular events are the leading cause of occupational death amongst fire fighters and account for approximately 45% of fatalities per year [1]. Moreover, the risk of acute myocardial infarction is increased 12- to 136-fold during fire suppression duties as compared to non-emergency duties and is likely to reflect a combination of factors including extreme physical exertion, mental stress, and exposure to heat and air pollutants [2]. Firefighters, during active fire suppression, are usually protected from smoke exposure by self-contained breathing apparatus (SCBA), however this is often disregarded in potentially hazardous, but tolerable situations, such as wildland fires where the long duration and remote location of fire fighting often renders SCBA wearing impractical [3]. Respiratory protection, therefore, often takes the form of a cotton rag or bandana tied around the nose and mouth. Air pollution is an established risk factor for the development of both acute and chronic cardiovascular diseases [4-11] with exposure to particulate matter (PM) consistently associated with adverse cardiovascular health effects. The mechanisms through which specific air pollutants, and in particular traffic-derived air pollutants, influence the cardiovascular system have been intensively studied and an understanding of their effects on the pathophysiology of disease is emerging. In contrast, the health effects of wood smoke and biomass exposure have received little attention. Wood smoke contributes large quantities of ultrafine particles to our environment through the combustion of biomass for heating and cooking, and during major wildland fires. Firefighters have significant and often prolonged exposures during wildland fire fighting, an important duty of the fire service. We have previously demonstrated that exposure to diesel exhaust impairs endothelial vasomotor and fibrinolytic function and increased ex-vivo thrombosis in man [12,13]. We have also demonstrated that exposure to wood smoke causes transient increases in arterial stiffness in healthy volunteers [14]. We therefore hypothesised that exposure to wood smoke, rich in ultrafine particulate matter, would have similar adverse effects and may explain the association between fire suppression and excess cardiovascular death. We therefore assessed the effect of exposure to wood smoke on vascular vasomotor and fibrinolytic function, and thrombus formation in healthy fire fighters. BODY.RESULTS: Exposures were well tolerated with no adverse symptoms reported and all subjects completed both study visits. Within the chamber, particulate matter with an aerodynamic diameter <1 μm (PM1) concentrations were 1,115 ± 151 μg/m3, with nitrogen oxides (NOx) and carbon monoxide (CO) concentrations of 0.6 ± 0.1 ppm and 16.0 ± 1.1 ppm respectively (Table 1; Figure 1). Total PM mass was consistent with the target concentration for the study, as shown by both TEOM and filter measurements. The high EC/TC ratio illustrates the high soot content in wood smoke. The total PAH concentration in the exposure chamber was 4.3 ± 2.5 μg/m3, of which 90% was associated with wood smoke PM. The concentration of PM associated benzo[a]pyrene was 443 ± 302 ng/m3. The most abundant PAH compounds in the wood smoke PM fraction, accounting for 88 ± 1% of the total analyzed PAH (both PM associated and gas phase), were (in descending order): benzo[a]pyrene, chrysene, benzo[b]fluoranthene, benz[a]anthracene, benzo[e]pyrene, benzo[ghi]perylene, benzo[ghi]fluoranthene, indeno[1,2,3-cd]pyrene, pyrene, fluoranthene, benzo[k]fluoranthene and coronene (Additional file 1: Table S1).Table 1 Characterization of wood smoke exposure and particulate matter n Unit Mean SD Min Max PM 1 mass concentration (TEOM) 16μg/m3 1,1151519221,561 PM 1 mass concentration (filter) 16μg/m3 8991007261,105 Carbon monoxide 16ppm166825 Nitrogen oxides 16ppm0.60.30.31.0 Elemental carbon/total carbon 1 6ratio0.800.020.790.83 Organic fraction of total PM 1,2 6%23.14.716.928.8 Soot fraction of total PM 1 6%60.115.739.978.8 PAH – PM associated (filter) 1,3 6μg/m3 3.92.31.56.7 PAH – semi-volatile (PUF) 1,3 6μg/m3 0.40.30.10.9 1OC-EC and PAH analysis from selected samples throughout the campaign (n = 6). 2Estimated based on the OC-EC analysis (assuming a factor of 1.8 used to covert OC to total organic PM and a factor of 1.1 used to convert EC to total soot PM). 3Includes the PAHs: phenanthrene, anthracene, 4H-cyclopenta[def]phenanthrene, 2-phenylnaphthalene, fluoranthene, pyrene, 1-methylfluoranthene, benz[a]fluorene, benz[b]fluorene, 2-methylpyrene, 4-methylpyrene, 1-methylpyrene, benzo[c]phenanthrene, benzo[ghi]fluoranthene, benzo[b]naphtho[1,2-d]thiophene, benz[a]anthracene, chrysene, 3-methylchrysene, 2-methylchrysene, 6-methylchrysene, 1-methylchrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[e]pyrene, benzo[a]pyrene, perylene, indeno[1,2,3-cd]fluoranthene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene, picene, benzo[ghi]perylene, dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, coronene, dibenzo[a,i]pyrene and dibenzo[a,h]pyrene.Figure 1 Particulate matter concentration during exposures. (a) A typical time-series of particle mass concentrations (PM1) in the chamber during a single 1 hour exposure measured with TEOM with data points every 30 seconds. (b) Average mass concentrations (PM1) in the chamber during the 1 hour exposures measured with TEOM (n = 16) every 30 seconds (mean ± standard deviation). The mean primary particle size was 168 nm and the number size distribution (mobility diameter) was clearly bi-modal with a lower peak around 50–80 nm and an upper peak around 150–200 nm (Figure 2). Wood smoke particulate generated superoxide free radicals in physiological saline solutions in the absence of cells or tissue. Comparing equivalent masses of particulate, the superoxide generating capacity of wood smoke particulates was greater than standard reference material urban dust or pyrogallol controls (P < 0.001) (Figure 2).Figure 2 Characterization of wood smoke particulate matter. (a) Scanning electron microscopy (SEM) image of wood smoke particles. (b) Size distribution graph of the particle size as assessed by photon correlation spectroscopy with the mean primary particle size indicated by the red line. (c) Average particle number size distribution in the exposure chamber, measured by SMPS system. The plot displays the distribution as mean and standard deviation from all 16 exposures. Previous studies demonstrate the 50–80 nm peak consists of alkali salt particles (e.g. potassium sulphate and potassium chloride) and the 150–200 nm peak soot particles with more organic material [15,16]. (d) Electron paramagnetic resonance (EPR) signal intensity showing oxygen free radical generation from wood smoke particulates in the presence of the superoxide-selective spin-trap Tempone-H. Particulates from exposures collected on Teflon filters suspended in physiological saline solution at a concentration of 100 μg particles/mL. The standard reference material urban dust (100 μg particles/mL) and pyrogallol (100 μM) were used as controls. Data expressed as mean ± SEM (n = 4–5). BODY.RESULTS.HAEMATOLOGY: Blood carboxyhaemoglobin concentrations increased from 0.9 ± 0.04 to 1.3 ± 0.04% immediately following exposure to wood smoke (P < 0.001) (Table 2). Total leucocyte, lymphocyte, neutrophil and platelet counts, were unaffected for up to 24 h after the exposure.Table 2 Haematological effects of exposure to wood smoke and filtered air Filtered air Wood smoke P value 1 Carboxyhaemoglobin, % Baseline0.9 ± 0.00.9 ± 0.0<0.0012 hours0.8 ± 0.01.3 ± 0.0*24 hours0.8 ± 0.00.8 ± 0.0 Leucocytes × 10 9 cells/L Baseline5.5 ± 0.45.6 ± 0.40.222 hours6.1 ± 0.36.4 ± 0.46 hours6.5 ± 0.36.9 ± 0.424 hours5.4 ± 0.35.6 ± 0.4 Lymphocytes × 10 9 cells/L Baseline2.0 ± 0.22.0 ± 0.20.112 hours1.9 ± 0.11.8 ± 0.16 hours2.0 ± 0.12.1 ± 0.124 hours2.0 ± 0.22.0 ± 0.2 Neutrophils × 10 9 cells/L** Baseline2.8 ± 0.32.9 ± 0.30.202 hours3.6 ± 0.33.9 ± 0.46 hours3.8 ± 0.34.1 ± 0.324 hours2.8 ± 0.23.0 ± 0.3 Platelets × 10 9 cells/L** Baseline225 ± 6229 ± 70.072 hours212 ± 6219 ± 76 hours205 ± 5203 ± 924 hours225 ± 7234 ± 6Values are reported as mean ± SEM. 12-way ANOVA with repeated measures comparing filtered air and wood smoke exposures.*P <0.001 following Bonferroni correction comparing filtered air and wood smoke.at a given time point.**P < 0.05 for trend across time. BODY.RESULTS.ARTERIAL STIFFNESS AND VASCULAR FUNCTION: Resting blood pressure and heart rate were unchanged during either exposure or for up to 24 h after exposure (Table 3). Augmentation index, augmentation pressure and pulse wave velocity increased immediately after exposure (P > 0.05), but changes were similar following exposure to wood smoke and filtered air (P > 0.05 for all comparisons of wood smoke versus filtered air).Table 3 Haemodynamic effects of exposure to wood smoke and filtered air Baseline Post-exposure 0 mins 10 mins 20 mins 30 mins 40 mins 50 mins 1 hr 2 hrs 6 hrs 24 hr mean P-value 1 Systolic pressure, mmHgFiltered air135 ± 2134 ± 2133 ± 2130 ± 3131 ± 2131 ± 2130 ± 3133 ± 2132 ± 2130 ± 2124 ± 20.59Wood smoke132 ± 2135 ± 3131 ± 3130 ± 3131 ± 3130 ± 3130 ± 3135 ± 4134 ± 5131 ± 3127 ± 2Diastolic pressure, mmHgFiltered air75 ± 274 ± 375 ± 275 ± 273 ± 476 ± 276 ± 276 ± 276 ± 276 ± 2768 ± 20.89Wood smoke75 ± 274 ± 274 ± 273 ± 274 ± 274 ± 276 ± 277 ± 277 ± 275 ± 270 ± 2Heart rate, bpmFiltered air63 ± 263 ± 363 ± 361 ± 360 ± 259 ± 259 ± 258 ± 258 ± 259 ± 256 ± 20.12Wood smoke61 ± 363 ± 363 ± 362 ± 362 ± 361 ± 361 ± 360 ± 360 ± 358 ± 355 ± 2∆ Augmentation pressure, mmHgFiltered air-0.2 ± 0.5−0.4 ± 0.7−0.7 ± 0.6−0.1 ± 0.7−0.2 ± 0.70.1 ± 0.70.7 ± 0.7---0.90Wood smoke-1.0 ± 0.8−0.2 ± 0.3−0.5 ± 0.3−0.7 ± 0.4−0.3 ± 0.50.6 ± 0.50.2 ± 0.5---∆ Augmentation index @75 bpm, %Filtered air-0.01 ± 4.6−1.7 ± 6.7−3.9 ± 6.1−2.8 ± 7.6−3.0 ± 7.5−2.2 ± 7.0−1.2 ± 7.2---0.72Wood smoke-2.3 ± 7.6−1.1 ± 4.0- 2.0 ± 5.1−3.2 ± 5.0−3.0 ± 6.4−1.3 ± 6.7−1.6 ± 6.0---∆ Pulse wave velocity, m/sFiltered air-0.1 ± 0.10.0 ± 0.10.1 ± 0.10.0 ± 0.1−0.1 ± 0.10.0 ± 0.10.4 ± 0.5---0.98Wood smoke-0.0 ± 0.1−0.1 ± 0.10.0 ± 0.1−0.1 ± 0.10.0 ± 0.1−0.1 ± 0.10.1 ± 0.1---Values are reported as mean ± SEM. 12-way ANOVA with repeated measures comparing wood smoke and filtered air (baseline to 6 hrs); paired Students t-tests were performed for 24 hr means (P > 0.05 for all). There was a dose-dependent increase in forearm blood flow with each vasodilator (P < 0.01 for all). However there were no differences in blood flow responses to acetylcholine (P = 0.91), sodium nitroprusside (P = 0.52) or verapamil (P = 0.63) between exposures (Figure 3). In contrast, there was an increase in forearm blood flow with bradykinin infusion following exposure to wood smoke compared to filtered air (P = 0.003). Bradykinin caused a dose-dependent release of tissue plasminogen activator antigen (P < 0.01), which was similar after both exposures (P = 0.72) (Figure 4).Figure 3 Effect of wood smoke and filtered air on forearm blood flow. There was a dose-dependent increase in forearm blood flow with each vasodilator (2-way ANOVA with repeated measures, P < 0.01 for all), however there were no differences in blood flow response to acetylcholine (P = 0.91), sodium nitroprusside (P = 0.52) or verapamil (P = 0.63) between exposures. In contrast, there was an increase in the forearm blood flow to bradykinin following exposure to wood smoke compared to filtered air (P = 0.003). All data expressed as mean ± SEM. There are no differences in blood flow in the non-infused arms and therefore these data points are overlaid.Figure 4 Effect of wood smoke and filtered air on platelet activation, fibrinolysis and thrombus formation ex vivo. (a) Platelet-monocyte binding and (b) platelet expression of P-selectin were unchanged 2 and 24 hours following exposure to wood smoke or filtered air (ANOVA with repeated measures, P > 0.05, n = 11–16). (c) Bradykinin caused a dose-dependent release of tissue-plasminogen activator (t-PA) antigen (2-way ANOVA with repeated measures, P < 0.01), which was similar after both exposures (P > 0.05, n = 16). (d) Thrombus formation under high-sheer conditions in the Badimon chamber was similar 2 hours after exposure to wood smoke or filtered air (Student's t-test, P > 0.05, n = 13). All data expressed as mean ± SEM. BODY.RESULTS.PLATELET ACTIVATION AND THROMBOSIS: Platelet-monocyte binding, monocyte surface expression of CD40 and platelet surface expression of CD40L and P-selectin were similar following wood smoke and filtered air exposure at 2 and 24 h following exposure (P > 0.05 for all) (Figure 4). There was no difference in thrombus formation following exposure to wood smoke compared with filtered air (thrombus area 12,216 ± 3,237 versus 12,775 ± 3,831 μm2, P = 0.54) (Figure 4). BODY.DISCUSSION: Controlled exposure to wood smoke at high particulate concentrations does not impair endothelial-dependent or -independent vasodilation or increase thrombosis in firefighters. Using established methodology and a comprehensive assessment of cardiovascular health we found no adverse effects of wood smoke to explain the cardiovascular risk associated with fire suppression duties. Whilst there have been no prior controlled exposures to wood smoke in firefighters, the effect of wood smoke on vascular function has been studied in healthy volunteers [17] and systemic inflammatory effects have been observed in firefighters responding to forest fires [18-20]. We have previously demonstrated that exposure to wood smoke for 3 hours at a lower PM concentration of 300 μg/m3 caused a transient increases in arterial stiffness and heart rate [14]. In contrast, we found no effect on arterial stiffness or heart rate following exposure to wood smoke at three-fold higher concentrations in firefighters over one hour. The overall dose was similar in both studies. These discordant findings may be explained by differences in susceptibility to wood smoke between healthy volunteers and firefighters with the latter having had multiple previous exposures to wood smoke through their occupation. There is some evidence that repeated exposure to smoke upregulates anti-oxidant levels in the airways and may diminish the effects of an acute exposure [21]. However we restricted enrollment to those firefighters who had not attended a major structural or wildland fire for more than one week prior to study visits and the effects of previous exposure on anti-oxidant levels are likely to be transient. It is also interesting to note that the baseline heart rates in our previous study [14] were approximately 10 bpm higher than in this study, perhaps suggesting previous participants were more susceptible to any effects of exposure on the autonomic nervous system. The duration of exposure may also be important with effects of wood smoke on arterial tone emerging after longer exposure periods. However, Forchhammer et al. recently observed no effect of wood smoke on peripheral arterial tone assessed by finger plethysmography despite delivering PM at 350 μg/m3 for up to 3 hours [17]. Venous occlusion plethysmography with intra-arterial infusion of vasodilators is widely regarded as the 'gold-standard' assessment of vascular function. We found no detrimental effect of exposure to wood smoke or filtered air on either endothelium-dependent or -independent vasodilatation. In fact we demonstrate a small increase in blood flow in response to bradykinin infusion following wood smoke exposure. It is plausible this was due to the effects of carbon monoxide exposure at concentrations that were sufficient to increase carboxyhaemoglobin concentrations and indeed, carbon monoxide is emerging as an important mediator of the vasodilator effects of bradykinin in the vessel wall [22]. The levels of carbon monoxide in this study were 4-fold higher than previous exposures to dilute diesel exhaust [23] and therefore it is plausible that vasodilation as a consequence of higher gaseous pollutants (carbon monoxide or nitrogen oxides [24]), may be important here. Nevertheless, it is unlikely that this would offset any detrimental effects of wood smoke PM on forearm blood flow across other vasodilators. There were no other important or adverse effects of wood smoke on vascular function, including endogenous fibrinolysis, platelet activation and thrombosis. Taken together these findings suggest that exposure to wood smoke is unlikely to be the primary cause of acute adverse cardiovascular events in firefighters. Whilst traffic-related air pollution is an established trigger for acute myocardial infarction [4,6,9,25], there are few studies that have linked exposure to wood smoke or biomass with cardiovascular events. The risk in firefighters may be mediated by other factors, such as exposure to extreme heat, physical exertion and psychological stress. Heat stress results from both high ambient temperatures and exercise-induced metabolic activity, exacerbated by insulated protective clothing. In controlled studies, heat stress causes vasodilatation and fluid loss, resulting in a reduction in cardiac output and a hypercoagulable state [26-29]. Strenuous physical exertion is an independent trigger of sudden cardiovascular events, particularly in individuals unaccustomed to exercise [30]. Fire suppression often requires firefighters to work at the extremes of physical capability associated with heart rates in excess of age predicted maximums [26,27,31-33] and for long periods with shifts frequently lasting 12 to 24 h [34]. Whilst exposure to wood smoke may not in isolation cause vascular dysfunction or induce a prothrombotic state, it remains plausible that firefighters responding to wildland fires are at increased risk of an acute cardiovascular event through a combination of factors that could still include wood smoke. Furthermore, firefighters are also exposed to a heterogenous mix of air pollutants during other activities and although the use of breathing apparatus is employed in these situations, at the perimeter of such fires and in the aftermath when, breathing apparatus is removed important exposures may occur. It is perhaps surprising that exposure to fine wood smoke particles at concentrations in excess of 1,000 μg/m3 had no adverse effects given that exposure to diesel exhaust at 300 μg/m3 has previously been shown to impair vascular function and increase thrombus formation in healthy men [12,13,35]. Differences in particle properties such as size, composition and surface chemistry between these exposures are likely to be important. Although the majority of wood smoke and diesel exhaust particles are in the ultrafine size fraction, the diameter of primary wood smoke particles was 5-fold larger than diesel exhaust particles (primary particle size of NIST standard reference material 2975 is 31 nm). Wood smoke particles are therefore perhaps less likely to deposit in terminal bronchioles or alveolar space and therefore to translocate or deliver soluble components into the circulation where they could directly effect the cardiovascular system. If wood smoke particles are unable to translocate due to larger size then they could perhaps cause a systemic inflammatory response, exerting late effects that were missed by undertaking assessments early after exposure. Others have shown that controlled exposure to fine and coarse PM is associated with early autonomic imbalance: rapid elevation of blood pressure and heart rate, and decreased heart rate variability immediately following exposure [36-39]. Conversely, we may have missed any immediate effects mediated by autonomic imbalance that were not comprehensively assessed in this study, although blood pressure and heart rate were unaffected acutely and over the 24 hour study period. Differences in surface chemistry are also likely to be important. Whilst wood smoke particles were able to generate super-oxide radicals, there were major differences in the PAH profile between wood smoke particles, where high molecular weight PAHs (≥228 Da) dominated, as compared to diesel exhaust particles [40]. There are some limitations to our study that merit consideration. The time points chosen to conduct our assessments post-exposure were based on the results of previous studies [12-14,23,24,35,41-46]. However, it is possible that wood smoke particles either exert an immediate or late effect on the cardiovascular system and we may have missed such effects. Additionally, the duration of exposure is also likely to be important with cumulative exposures over many days or weeks difficult to model experimentally. We recruited early career firefighters to minimize potential for confounding due to pre-existing vascular disease. Firefighters are exposed to complex mixtures of air pollutants derived from different sources, many of which may be more toxic than our simulated wildland fire exposure. It is plausible that firefighters with risk factors or subclinical disease would be more susceptible to any adverse cardiovascular effects of wood smoke. According to the widely recognized "healthy worker effect" in occupational medicine, it is common that susceptible individuals leave the workplace early due to symptoms, discomfort or acute illness. This may lead to selection bias with the remaining workers less sensitive or resistant to these noxious factors. Furthermore, we prospectively powered the study based on the measurements of primary end points made during previous studies [12,47-49]. Although we are confident that we have not missed effects on endothelial function or ex vivo thrombosis, we acknowledge that we may have had insufficient power to detect modest changes in some of the secondary end points, and thus cannot exclude the possibility of false-negative findings confounding their assessment. Nevertheless, even allowing for these limitations, in a carefully designed and controlled study with a comprehensive assessment of cardiovascular function, we found no adverse effects of exposure to wood smoke. BODY.CONCLUSIONS: Isolated wood smoke exposure at concentrations occurring in the vicinity of major wildland fires did not impair vascular vasomotor or fibrinolytic function, or increase thrombus formation in firefighters. The acute cardiovascular events associated with fire suppression may not be directly related to wood smoke exposure, rather they may be precipitated by other pollutants or mechanisms such as strenuous physical exertion and dehydration. BODY.METHODS.SUBJECTS: Sixteen healthy non-smoking male volunteers (median age 26, range 21–26 years) were enrolled into the study. The study was performed with the approval of local research Ethics Committees, in accordance with the Declaration of Helsinki and the written informed consent of all volunteers. Firefighters were recruited using advertisements in local fire stations. Exclusion criteria were cigarette smoking or the use of snus (tobacco snuff), the use of regular medication (specifically non-steroidal anti-inflammatory drugs, vitamins or anti-oxidant supplements), known ischemic heart disease, arrhythmia, diabetes mellitus, hypertension, renal or hepatic impairment, asthma, or inter-current infection. Subjects had normal lung function and reported no respiratory symptoms in the 6-week period preceding the study. Subjects had no occupational fire exposure (wildland or structural) for a week preceding study visit. BODY.METHODS.STUDY DESIGN: Subjects attended on two occasions at least one week apart and were exposed to filtered air or wood smoke for one hour in a double-blind randomized crossover design. Subjects attended at 8 am on the morning of study for initial bloods and for the fitting of Holter and ambulatory blood pressure monitors. Exposures were performed at 10 am in a dedicated exposure facility by researchers and technical staff not involved in the subsequent clinical assessment. Subjects remained indoors following exposure to minimize any confounding effects from ambient air pollution. Vascular studies were carried out in a quiet, temperature-controlled room maintained at 22°C to 24°C with subjects lying supine. All subjects abstained from alcohol and caffeine for 24 h, and from food for at least 4 h before each vascular study. The primary endpoints were forearm blood flow, estimated t-PA release from the forearm circulation and ex-vivo thrombus formation. Secondary endpoints included arterial stiffness, platelet activation, and changes in haematological variables. Based on previous studies [12-14,23,24,35,41-46], pulse wave analysis and velocity were assessed immediately after exposure, a study of ex-vivo thrombus formation, Badimon study was performed at 2 h, and venous occlusion plethysmography undertaken 4 to 6 h after exposures to wood smoke and filtered air. Venous blood was sampled at baseline, 2, 6 and 24 h after each exposure for storage and quantification of carboxyhaemoglobin. Subjects were fitted with an ambulatory blood pressure monitor (Spacelabs 90217; Spacelabs, Healthcare Ltd, Hertford, UK) prior to each exposure and monitored for 24 h. BODY.METHODS.WOOD SMOKE EXPOSURE: Exposures were performed in a purpose-built exposure chamber in Umeå, Sweden. During each exposure, subjects performed moderate exercise (to generate an average minute ventilation of 20 L/min/m2) on a bicycle ergometer that was alternated with rest at 15-min intervals. Wood smoke was generated using a common Nordic wood stove using birch wood in an incomplete combustion firing procedure (partial air-starved conditions), generating a soot-rich aerosol emission. To generate relatively constant incomplete combustion conditions during the 1 hour exposures, small batches (0.5–1.0 kg) birch wood logs were inserted every 5–15 minutes to maintain a high burn rate with repeated air-starved conditions. This procedure was in accordance with our previous wood smoke exposure study [14]. The birch wood was stored outdoors under roof cover for approximately 2.5 years before use and had a moisture content of 16–17% at the time of this study. The wood smoke was diluted with HEPA filtered air in three steps and continuously fed into and through a controlled environment exposure chamber (17 m3) to achieve a steady state concentration. The atmosphere in the chamber was monitored for pollutants using continuous measurement of oxides of nitrogen (NOx) (chemiluminescence, CLD 700 Ecophysics, >0.001 ppm) and carbon monoxide (CO) (IR, UNOR6N Maihak). Fine (<1 μm) particulate matter (PM1) mass concentration was measured on-line using a tapered element oscillating microbalance (TEOM 1400, Thermo Scientific). Integrated with the TEOM a filter (Teflon) sampling line was used to determine the mass concentration gravimetrically. A CO alarm instrument (MC400, Monicon Technology) was used in the chamber during the exposures. The target PM1 concentration in the chamber was 1,000 μg/m3. The equivalent mobility diameter (in the range 10–600 nm) of the wood smoke particles was measured in the chamber using a scanning mobility particle sizer (SMPS) (TSI GmbH). Organic (OC) and elemental carbon (EC) were determined using thermal-optical carbon analysis (according to the EUSAAR_2 protocol). These concentrations are regularly encountered at the perimeter of forest fires [50] and indoors when cooking with solid fuels [51], and are below the UK workplace 8 hour average exposure limits (HSE EH40 Workplace Exposure Limits 2005). The temperature in the chamber maintained between 21–24°C with a relative humidity of 50%. BODY.METHODS.POLYCYCLIC AROMATIC HYDROCARBON ANALYSIS: The collected wood smoke PM, and polyurethane foam (PUF) plugs with sampled semi-volatile PAHs were extracted with pressurized fluid extraction using an ASE 200 Accelerated Solvent Extractor system (Dionex Corporation, Sunnyvale, CA, USA). Wood smoke PM was extracted with a solvent composition consisting of toluene and methanol 9:1 at 200°C and 3000 psi (20.7 MPa). PUFs were extracted with hexane at 110°C and 500 psi (3.45 MPa). Details on instrumental parameters are available elsewhere [52,53]. Solid phase extraction sample cleanup was performed to remove polar constituents from the samples according to Christensen et al. [54] followed by instrumental analysis using an automated high pressure liquid chromatography-gas chromatography–mass spectrometry system (HPLC-GC-MS) [40]. The HPLC part of the system was used for PAH fraction using the back flush technique, where PAHs with 3 and more rings were isolated and introduced into the GC-MS system for separation and detection. The MS was operated in selected ion monitoring mode, and the PAHs were identified using compound specific mass to charge ratio and relative retention time on the GC capillary column. In total 36 PAHs in the range of 178 – 302 Da were analyzed (Additional file 1: Table S1). BODY.METHODS.ELECTRON PARAMAGNETIC RESONANCE: To provide a measure of particle reactivity EPR was used to establish oxygen-centred free radical generation from particulates collected from exposures (Langrish et al. [7]). A 1.6 mm diameter section of Teflon filter from the carbon analysis filter line of the exposure chamber and suspended in physiological saline solution (Krebs buffer, composition in mM: 118.4 NaCl, 25 NaHCO3, 11 glucose, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2) at a particle concentration of 100 μg/mL. Samples were vortexed for 1 min, followed by 30 min sonication sonication (100% power; Fisherbrand FB11002; Fisher Scientific, Loughborough, UK). Suspensions were incubated with the spin-trap, Tempone-H (1 mM; Enzo Life Sciences, Exeter, UK), immediately before the initial measurement. Tempone-H is a highly sensitive spin-trap that shows selectivity for superoxide, forming a stable product that can be measured by EPR [55]. The standard reference material urban dust (SRM1649a; National Institute of Standards and Technology, Gaithersburg, USA) was used as positive control particulate (note, that the results cannot be directly compared to filter particulates, as the proportion of the mass of wood smoke particulate unbound to the filter cannot be determined in the present study). Pyrogallol (100 μM) was used as a second positive control which spontaneously generates superoxide radicals in this buffer [56]. Samples were kept at 37°C throughout and measurements were taken after 30 and 60 min by drawing 50 μL of sample into a capillary tube (VWR International, Lutterworth, UK) and sealing with a plug of soft sealant (Cristaseal, VWR International). An X-band EPR spectrometer (Magnettech MS-200, Berlin, Germany) was used with the following parameters: microwave frequency, 9.3–9.55 Hz; microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 1500 mG; center field, 3365 G; sweep width, 50 G; sweep time, 30 s; number of passes, 1. Baseline signals from blank (non-exposed) filters were subtracted from that of filters with particulate. BODY.METHODS.ARTERIAL STIFFNESS: All measurements of arterial stiffness were performed at baseline, and at 10-min intervals after the exposure for one hour as previously described [14]. Pulse rate and blood pressure were measured using a validated semi-automated oscillometric sphygmomanometer (Boso-Medicus, Boso, Jungingen, Germany). Central arterial stiffness measured by pulse wave analysis was determined with a high-fidelity handheld tonometer (Millar Instruments, Texas, USA) at the right radial artery using the SphygmoCorTM system (AtCor Medical, Sydney, Australia). Carotid-femoral pulse wave velocity measurements were made using the Vicorder system (Skidmore Medical, UK). BODY.METHODS.VASCULAR STUDIES: All subjects underwent brachial artery cannulation with a 27-standard wire gauge steel needle under controlled conditions. After a 30-min saline infusion, acetylcholine at 5, 10, and 20 μg/min (endothelium-dependent vasodilator that does not release tissue plasminogen activator [t-PA]; Merck Biosciences); bradykinin at 100, 300, and 1000 pmol/min (endothelium-dependent vasodilator that releases t-PA; Merck Biosciences); sodium nitroprusside at 2, 4, and 8 μg/min (endothelium-independent vasodilator that does not release t-PA; David Bull Laboratories) and verapamil at 10, 30, and 100 μg/min (endothelium- and NO-independent vasodilator that does not release t-PA) were infused for 6 min at each dose. Vasodilators were separated by 20-min saline infusions and given in a randomized order except from verapamil, which was always given last due to its longer duration of action [57]. Forearm blood flow was measured in infused and non-infused arms by venous occlusion plethysmography with a mercury-in-silicone elastomer strain gauges as described previously [58]. Venous cannulas (17 gauge) were inserted into large subcutaneous veins of the ante-cubital fossae of both arms. Blood (10 mL) was withdrawn simultaneously from each arm at baseline and during infusion of each dose of bradykinin and collected into acidified buffered citrate (Stabilyte tubes, Biopool International). Samples were kept on ice before being centrifuged at 2000 g for 30 min at 4°C. Platelet-free plasma was decanted and stored at −80°C before assay. Plasma t-PA antigen and activity concentrations were determined by enzyme-linked immunosorbant assay (TECHNOZYM® t-PA Combi Actibind®, Technoclone, Austria). Hematocrit was determined by capillary tube centrifugation at baseline and during infusion of bradykinin at 1000 pmol/min. BODY.METHODS.FLOW CYTOMETRY: Samples were obtained at baseline, at 2 h immediately prior to the thrombosis study and at 24 h post exposure, and processed according to previously described protocols [59]. In brief, blood was taken from an ante-cubital vein using a 21-gauge cannula and anti-coagulated with D-phenylalanyl-Lprolyl-L-arginine chloromethylketone (75 μL; Cambridge Biosciences, UK). Samples were not analysed unless venesection achieved rapid and uninterrupted blood flow. Five minutes after sample collection, samples were stained with the following conjugated monoclonal antibodies: phycoerythrin (PE)-conjugated CD14 (Dako, Denmark), PE-conjugated CD62P, and PE-conjugated CD154 (Becton-Dickinson, UK); PE-conjugated CD40, fluorescein isothiocyanate (FITC)-conjugated CD42a, and FITC-conjugated CD14 (Serotec, USA); and appropriate control isotypes. All antibodies were diluted 1:20. Once stained, samples were incubated for 20 min at room temperature to identify P-selectin and CD40L on the platelet surface and CD40 on the monocyte surface. Platelet–monocyte samples were fixed with FACS-Lyse (Becton-Dickinson). Platelet samples were fixed with 1% paraformaldehyde. Samples were analysed within 24 h using a FACScan flow cytometer (Becton-Dickinson). Platelet–monocyte aggregates were defined as monocytes positive for CD14. Data analysis was performed using FlowJo (Treestar, USA). BODY.METHODS.EX-VIVO THROMBOSIS STUDIES: Thrombus formation was measured using the Badimon chamber as previously described [13,23]. In brief, a pump was used to draw blood from an antecubital vein through a series of consecutive cylindrical perfusion chambers maintained at 37°C in a water bath. Carefully prepared strips of porcine aorta, from which the intima and a thin layer of media had been removed, acted as the thrombogenic substrate. Each study lasted for 5 min during which flow was maintained at a constant rate of 10 mL/min. Porcine strips with thrombus attached were removed and fixed in 4% paraformaldehyde, wax embedded, sectioned, and stained with Masson's Trichrome. Images were acquired at × 20 magnification, and the thrombus area was measured using an Ariol image acquisition system (Leica Microsystems GmbH, Germany) by a blinded operator. Results from at least six sections were averaged to determine thrombus area for each chamber, as described previously [13,23]. BODY.METHODS.DATA ANALYSIS AND STATISTICS: A sample size of 16 gives us >90% power to detect a 10% difference in thrombus area, 17% difference in mean t-PA release and 22% difference in forearm blood flow at a significance level of 5% [12,47-49]. Continuous variables are reported as mean ± standard error of the mean (SEM). Statistical analyses were performed with GraphPad Prism, version 5.0 (Graph Pad Software, USA) by 2-way analysis of variance (ANOVA) with repeated measures and 2-tailed Student t-test, where appropriate. Statistical significance was taken at two-sided P < 0.05.

TITLE: Red yeast rice induces less muscle fatigue symptom than simvastatin in dyslipidemic patients: a single center randomized pilot trial ABSTRACT.BACKGROUND: About 10–15% patients who take statins experience skeletal muscle problems. Red yeast rice has a good safety profile could provide a compromise therapeutic strategy. Therefore, the aim of this study was to evaluate the effects of red yeast rice, when compared to simvastatin, on the muscle fatigue symptom and the serum lipid level in dyslipidemic patients with low to moderate cardiovascular risk. ABSTRACT.METHODS: A total of 60 dyslipidemic patients with low to moderate cardiovascular risk were recruited and randomly assigned to receive either simvastatin (n = 33) or red yeast rice (n = 27) for 4 weeks. The muscle fatigue score, the physical activity, the serum lipid profile and the safety profile were then evaluated. ABSTRACT.RESULTS: At the end of study, the fatigue score was significantly increased in patients treated with simvastatin, whereas no significant change was observed in patients receiving red yeast rice. In addition, the physical activity level was significantly decreased in patients from simvastatin group when compared to those from red yeast rice group. Similar lipid-lowering effects were observed in two groups. The safety profile was not affected after the treatments. ABSTRACT.CONCLUSIONS: Among dyslipidemic patients with low to moderate cardiovascular risk, red yeast rice induced less fatigue side effect and exerted comparable lipid-lowering effects when compared to simvastatin in this pilot primary prevention study. ABSTRACT.TRIAL REGISTRATION: NCT01686451. BODY.BACKGROUND: Statins have been shown to be beneficial for both primary and secondary cardiovascular (CV) prevention [1–5]. However, despite the efficacies of these agents in terms of lowering lipid levels, the rate of CV events, and, in some samples, mortality, some patients are unable to tolerate the adverse effects [6]. Unfortunately, failure to adhere to statin therapy can result in adverse CV outcomes [7, 8]. Increased fatigue has been recognized as one of the adverse effects of statins [9–13]. In a recent large scale randomized controlled trial, the majority of the patients were reported experiencing fatigue [13, 14]. Red yeast rice, which is a traditional dietary seasoning from the MONASCUS purpureus mold that contains lovastatin (Monacolin K) and other active ingredients, has been shown to exert lipid-lowering effects and CV benefits in both primary and secondary CV prevention studies [15–17]. Additionally, Red yeast rice is associated with few adverse events [15–17]. Unexpectedly, red yeast rice has been reported to have antifatigue effects [18]. In the present study, we compared the efficacy and fatigue-causing effects of red yeast rice and simvastatin in patients with dyslipidemia and moderate to low CV risk. BODY.METHODS.PARTICIPANTS: Patients with low-density lipoprotein-cholesterol (LDL-C) levels between 3 and 5 mmol/L (115–190 mg/dL) were enrolled. Patients were excluded if they were at a very high to high risk of fatal CV disease within 10 years based on the risk estimation system described in ESC/ESA guidelines (2011) of dyslipidemia management, [19] or they had symptomatic atherosclerotic disease (including coronary artery disease, peripheral arterial disease, and cerebrovascular disease), kidney failure or insufficiency, diabetes, a systematic coronary risk estimation (SCORE) value ≥5%, if they were currently using of any lipid-lowering medications or other medication, such as cyclosporin, erythromycin, clarithromycin, nefazodone, or any "azole" antifungal, (including fluconazole, itraconazole, ketoconazole, mibefradil, or protease inhibitors), or if they had conditions including active liver disease or unexplained persistently elevated transaminase levels, cancer, human immunodeficiency virus infection, a medical or psychiatric condition that prevented full study participation or follow-up (e.g., active psychosis), major surgery or hospitalization in the 3 months prior to study entry, if they were a female of childbearing potential, or if they were currently participating in another clinical trial. The study was approved by the institutional review board, and all patients provided written informed consent. BODY.METHODS.RANDOMIZATION AND MASKING: This trial was designed as a single-center, parallel-group study that took place at the medical clinic of the Second Affiliated Hospital of Wenzhou Medical University, China. According to the principle of the minimum distribution imbalance index, [20] the patients were randomly assigned to receive either red yeast rice (4 pills each containing 300 mg, hence in total 1200 mg daily) or simvastatin (0.5 pills each containing 40 mg, hence in total 20 mg daily) for 4 weeks. Patients were reminded weekly by telephone to take the medication on schedule and were asked to revisit at 28 ± 1 days (week 4). BODY.METHODS.STUDY PROCEDURES: Fasting blood samples were collected at week 0 (randomization) and 4 (at end of study) for lipid profile analyses and clinical chemistry (including serum lipid concentrations, alanine transaminase (ALT), aspartate transaminase (AST), creatine phosphate kinase (CPK) and serum creatinine (Cr)). All clinical laboratory analyses were performed in the hospital's central laboratory. BODY.METHODS.OUTCOME MEASURES: The primary endpoint was the fatigue score at the end of the study. Physical activity levels were also estimated. The baseline value was defined as the mean of the measurements obtained 1 week before randomization and on the day of randomization. The end-point value was defined as the measurement acquired after 4 weeks of treatment. We estimated different aspects of fatigue levels with psychological and physical questionnaires. The fatigue scores were assessed with a fatigue questionnaire [21]. Changes in physical activity levels as evaluated with a short version of the international physical activity questionnaire [22] was also recorded. Because of drugs' preparation issue, the measurement of the questionnaire was conducted in a single-blinded method that the physician did not know the treatment that patients received. The predefined secondary efficacy endpoints included the percentage changes from baseline to the study endpoint in lipid parameters (triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), and LDL-C levels). Safety was assessed by recording the prevalence and severity of adverse events and abnormal laboratory data. The patients who reported adverse events were also included. Compliance was assessed at each visit by counting the number of returned tablets. BODY.METHODS.STATISTICAL ANALYSES: All analyses were performed based on the intention-to-treat principle using data from all of the randomized patients. There were no treatment crossovers in the 4-week study period. Depending on the distributions, the continuous data are presented as medians (25th to 75th percentiles) or as the mean ± SD. The categorical data are presented as counts or proportions. The differences between groups were assessed with χ2 tests or Fisher's exact tests for the categorical data and with the nonparametric Wilcoxon rank-sum test or Student's t test for the continuous data. A 2-tailed value of P < 0.05 was considered to indicate statistical significance. All statistical analyses were performed using SPSS 17.0 software for Windows (SPSS for Windows version 17.0, Chicago, IL, USA). BODY.RESULTS.PATIENT CHARACTERISTICS: From August 10, 2012 to September 15, 2013, 243 patients were screened, and 60 patients who met the inclusion criteria were enrolled and randomly assigned to either the simvastatin (33 patients) or red yeast rice (27 patients) group. The flow of participants through the study is presented in Fig. 1. Table 1 shows the baseline characteristics of the patients, which were well balanced between the two treatment groups.Fig. 1Trial flow chart Table 1Baseline characteristics of the patientsa CharacteristicSimvastatin (n = 33)Zuezhikang (n = 27) P valueFemale sex, no. (%)15 (45.5)13 (48.2)0.835Age, yr46.0 ± 7.047.0 ± 5.80.614Current cigarette smoker, no. (%)3 (9.1)2 (7.4)1.000Arterial hypertension, no. (%)26 (78.8)16 (59.3)0.101Arterial blood pressure, mmHgSystolic148 ± 19148 ± 170.971Diastolic84 ± 783 ± 90.564FBG, mmol/L5.10 ± 0.365.33 ± 0.520.048TC, mmol/L5.91 ± 0.715.82 ± 0.730.488TG, mmol/L1.76 ± 1.001.61 ± 0.650.633LDL-C, mmol/L3.72 ± 0.483.74 ± 0.550.509HDL-C, mmol/L1.27 ± 0.251.23 ± 0.250.906ALT, U/L28.6 ± 10.630. 3 ± 13.30.570AST, U/L24.1 ± 7.025.6 ± 7.50.425CPK, U/L80.80 ± 24.9091.7 ± 23.70.092Cr, μmol/L72.6 ± 14.475.6 ± 17.20.463Fatigue score19.6 ± 2.619.3 ± 1.90.592Physical activity level0.870Low, no. (%)14 (42.4)3 (11.1)Moderate, no. (%)10 (30.3)12 (44.4)High, no. (%)9 (27.3)12 (44.4) aThe plus-minus values are the means ± the SDs. The percentages do not sum to 100 due to rounding. ALT alanine transaminase, AST aspartate transaminase, CPK creatine phosphate kinase, Cr creatinine, FBG fasting blood glucose, HDL highdensity lipoprotein-cholesterol, LDL low-density lipoprotein-cholesterol, TC total cholesterol. TG triglyceride BODY.RESULTS.EFFICACY RESULTS: Medication adherence was assessed by pill counting. All patients completed the self-rated fatigue assessment scale and the international physical activity questionnaire (short version) at the time of randomization and at week 4. The baseline values were comparable between the simvastatin and red yeast rice groups (Table 1). At week 4, the fatigue scores were significantly increased in the simvastatin group (P < .001 vs. baseline) and were significantly greater than those of the red yeast rice group (P < .01; Fig. 2). The fatigue scores did not change in the red yeast rice group (P = .16 vs. baseline; Fig. 2). Similarly, the physical activity levels, which were significantly reduced in the simvastatin group (P < .001 vs. baseline), remained unchanged in the red yeast rice group (P = .19 vs. baseline; Table 2) and were significantly lower in the simvastatin group than in the red yeast rice group (P < .001) at week 4 (Table 3).Fig. 2Comparison of the fatigue scores of the simvastatin and red yeast rice groups (mean ± SD). * P < .001 vs. baseline in the simvastatin group. ** P < .01 between the simvastatin and red yeast rice groups at week 4 Table 2Comparison of the physical activity levels at baseline and at week 4Simvastatin (n = 33) P valueXuezhikang (n = 27) P valueBaselineWeek 4BaselineWeek 4Physical activity level<.0010.19Low, No. (%)14 (42.4)25 (75.8)3 (11.1)3 (11.1)Moderate, No. (%)10 (30.3)8 (24.2)12 (44.4)15 (55.6)High, No. (%)9 (27.3)0 (0.0)12 (44.4)9 (33.3) Table 3Physical activity levels of two treatment groups at week 4Simvastatin (n = 33)Red yeast rice (n = 27) P valuePhysical activity level<.001Low, No. (%)25 (75.8)3 (11.1)Moderate, No. (%)8 (24.2)15 (55.6)High, No. (%)0 (0.0)9 (33.3) Before lipid-lowering treatment, there is no significant difference neither between the two groups of baseline level of TC(5.91 ± 0.71 vs. 5.82 ± 0.73 mmol/L for simvastatin and red yeast rice groups respectively, ns), nor between the two groups of baseline level of LDL-C(3.72 ± 0.48vs. 3.74 ± 0.55 mmol/L for simvastatin and red yeast rice groups respectively, ns). The administration of both simvastatin (20 mg daily) or red yeast rice (1200 mg daily) resulted in significant reductions in TC (−19.6% vs. -18.5% of baseline level for simvastatin and red yeast rice groups respectively, P < .001 vs. baseline for both) and LDL-C (−30.9% vs.-33.4% of baseline level for simvastatin and red yeast rice groups respectively, P < .001 vs. baseline for both) after 4 weeks of treatment (Fig. 3a-b) that were not significantly different between the two groups (P = 0.84 for the comparison of the percentage drop in the TC level and P = 0.64 for the comparison of the percentage drop in the LDL-C level; Fig. 3c). The improvements in TG and HDL-C concentrations were not significant in either treatment group (Fig. 3a-b).Fig. 3Comparison of the lipid-lowering efficacies of simvastatin and red yeast rice; a & b Comparison of lipid levels at week 4 and baseline in the simvastatin and red yeast rice groups (mean ± SD). No significant changes in TG or HDL levels were observed in either arm; c Comparison of the percentage changes in the lipid (TC and LDL) levels from baseline to week 4 (mean ± SD). The decrease in both the TC and LDL levels were comparable between the two groups.* TC levels, P < .001 vs. baseline in simvastatin group. ** LDL levels, P < .001 vs. baseline in simvastatin group. # TC levels, P < .001 vs. baseline in red yeast rice group. ## LDL levels, P < .001 vs. baseline in red yeast rice group BODY.RESULTS.SAFETY RESULTS: No significant increases of in the concentrations of ALT, AST, Cr, or CPK from baseline were observed in either arm (Fig. 4). No patient reported any adverse events.Fig. 4Comparison of the safeties at week 4 and at baseline (mean ± SD). No significant changes in ALT, AST, CPK or Cr levels were observed in either group BODY.DISCUSSION: Dealing with dyslipidemia should always be considered to be an essential and integral part of cardiovascular disease (CVD) prevention, and the tailoring of interventions to the baseline level of CVD risk is rational [19, 23]. In this trial, we compared the safeties and efficacies of simvastatin (20 mg daily) and red yeast rice (1200 mg daily) for patients with dyslipidemia and a 10-year risk of fatal CVD between 1% and 5% as evaluated with the SCORE system [19]. The findings indicated that the two treatments were comparable in terms of the decreases in LDL-C and TC levels. However, unlike the patients in the simvastatin group, the patients in the red yeast rice group did not experience fatigue or decreased physical activity. Statins are generally well tolerated and rarely cause serious adverse events [1–5]. In the Cholesterol Treatment Trialist's (CTT) meta-analyses that incorporated 26 randomized controlled trials, no increases in the risks for any non-CV cause of death and a small non-significant increase in rhabdomyolysis were observed in patients who were receiving statins [3]. Subsequent CTT studies and other meta-analyses that have addressed the issues of primary CV prevention have reported similar conclusions [1, 2, 4, 5]. However, statin-related fatigue has anecdotally been reported and confirmed by several studies, [9–14] including one randomized controlled trial [13] that enrolled 1016 participants (692 men and 324 women) who did not have heart disease or diabetes. The participants were randomly assigned to receive one of two statins (simvastatin or pravastatin) or placebo daily for six months. After six months, the participants taking the statins exhibited greater increases in overall fatigue than those who were taking the placebo. This effect was particularly pronounced in the women. The underlying mechanisms of statin-associated fatigue remain unclear but might be categorized as a manifestation of myopathy due to mitochondrial dysfunction [24–27]. Doctors should take these issues into account when considering the prescription of statins. Although the potential benefits of prescribing statins to a patient who is at high risk of CV problems might outweigh the risks of side effects, such as increased levels of fatigue, the opposite might be true for a person who is at a low risk for CV problems. This balance should be decided on a patient-by-patient basis by the doctor and the patient. An alternative approach is the selection of medications other than statins. In the present study, we compared the safeties, specifically in terms of fatigue, and efficacies of simvastatin and red yeast rice in patients with dyslipidemia and moderate to low CVD risks. Red yeast rice is a Chinese herbal medication that has been approved by the China Food and Drug Administration for dyslipidemia. Red yeast rice contains a family of monacolin-related substances, one of which is a naturally occurring lovastatin. In a large-scale randomized, placebo-controlled trial, called the China Coronary Secondary Prevention Study, 4870 patients with prior myocardial infarctions and baseline cholesterol levels between 170 mg/dL-250 mg/dL (4.40–6.47 mmol/L) exhibited significantly reduced rates of CV events following red yeast rice treatment [16]. In a meta-analysis involving 9625 participants with primary hyperlipidemia, red yeast rice preparations appeared to be as effective as statins in lipid modification [15]. In these trials, [15] Xuezhikang (red yeast rice preparation) was used at dosage of 1.2 g/day (containing 10 mg of lovastatin), Zhibituo (red yeast rice preparation) at 3.15 g/day (containing 9 mg of lovastatin), simvastatin at 10–20 mg/day, pravastatin at 10 mg/day, lovastatin at 20 mg/day, atorvastatin 10 mg/day, and fluvastatin 20 mg/day. Moreover, in sharp contrast to simvastatin, we did not observe any significant effect of red yeast rice on fatigue scores or physical activity levels after 4 weeks of treatment. In the present study, we found that the lipid modification efficacies both of red yeast rice (at 1200 mg daily) and simvastatin (at 20 mg daily) were comparable. Although the beneficial changes were limited to the TC and LDL-C parameters, these changes were still highly important because the current guidelines recommend that LDL-C should be the primary target of therapy [19, 23]. There are some limitations to this research. First, the sample size was relatively small, and future studies with larger sample sizes are needed to validate our results. Secondly, we followed the participants for only 4 weeks; thus, the long-term outcomes remain to be determined. Thirdly, two possibilities might confound the effects of simvastatin and red yeast rice on fatigue score and physical activity level reported by the present pilot study: 1) small sample effects and potential baseline disparities in fatigue score, physical activity level, and hypertension; and 2) population characteristics that might place them at higher risk, e.g. Asian ethnicity coupled with a high fraction of hypertension. BODY.CONCLUSIONS: Red yeast rice had similar lipid lowering properties to simvastatin in this small, short-term primary prevention trial, and may cause less fatigue. Further study of red yeast rice's long-term safety and efficacy in this patient population is warranted.

TITLE: Blood Loss and Transcapillary Refill in Uncontrolled Treated Hemorrhage in Dogs ABSTRACT.OBJETIVE:: This study evaluated retroperitoneal hematomas produced by bilateral injury of iliac arteries (uncontrolled hemorrhage), blood volume loss, transcapillary refill, the effects of volume replacement on retroperitoneal bleeding and the hemodynamic changes with and without treatment. ABSTRACT.METHODS:: Initial blood volume was determined with Tc99m-labelled red cells, and bleeding was evaluated by means of a portable scintillation camera positioned over the abdomen. Previously splenectomized mongrel dogs (16.8 ± 2.2 kg) were submitted to hemorrhage for 30 minutes and randomized into three groups: I - no treatment (n=7); II - treatment with 32 mL/kg of Lactated Ringer's for three to five minutes (n=7); and III - treatment with 4 mL/kg of 7.5% NaCl plus 6.0% dextran 70 for three to five minutes (n=7). They were studied for an additional 45 minutes. ABSTRACT.RESULTS:: Volume replacement produced transitory recovery in hemodynamic variables, including mean pulmonary artery pressure, pulmonary capillary wedge pressure and cardiac index, with significant increase in dogs treated with 32 mL/kg of Lactated Ringer's and 7.5% NaCl plus 6.0% dextran 70 (p<0.001, against no treatment), along with a decrease (p<0.001) in the systemic vascular resistance index. Groups II and III had significant initial decreases in hematocrit and hemoglobin. The treated dogs (groups II and III) presented rebleeding, which was greater during treatment with 32 mL/kg of Lactated Ringer's (group II). ABSTRACT.CONCLUSIONS:: Despite the rebleeding observed in treated groups, the utilization of hypertonic saline solution with dextran proved to be effective in the initial reanimation, producing evident transcapillary refill, while the Lactated Ringer's solution produced capillary extravasation and was ineffective in the initial volume replacement in this model of uncontrolled hemorrhage. BODY.INTRODUCTION: Volume resuscitation has been the mainstay of treatment for hypotensive trauma victims.1 However, controversy remains because there are concerns that this approach, before the control of bleeding, may result in increased blood loss due to increased blood pressure, which would dislodge blood clots, and the dilution of clotting factors.2–5 Hypotensive resuscitation and no fluid regimens are some strategies for avoidance of the potential side effects of volume infusion in uncontrolled hemorrhage.2,3,5,6 On the other hand, studies using small volumes of hypertonic saline or hypertonic saline plus dextran (HSD) have shown hemodynamic benefits and no significant increase in blood loss in several models of uncontrolled hemorrhage.7–15 None of the clinical studies evaluating hypertonic saline or hypertonic saline plus dextran (HSD) as the first fluid volumes infused into hypotensive patients suggested increased bleeding. On the contrary, these studies systematically suggest that hemodynamic benefits are achieved with less fluid and blood products in comparison to what is required for standard isotonic resuscitation.15–24 In the present study, we promoted a retroperitoneal hemorrhage with class III shock by ATLS with a simulation of blunt abdominal injury1,25–27 by bilateral posterior iliac artery puncture to evaluate fluid shifts and blood loss after HSD or isotonic saline infusion, in comparison to no fluid replacement. Our hypothesis is that small volumes of HSD promote volume expansion and hemodynamic benefits, in spite of modest increases in blood loss. BODY.MATERIALS AND METHODS: This project was previously approved by the Ethics Committee of the Heart Institute and conforms to National Institutes of Health guidelines for the use of experimental animals. BODY.MATERIALS AND METHODS.SURGICAL PREPARATION: Experiments were performed on 21 male mongrel dogs weighing 16.8 ± 2.2 kg (STD DEV) that had been splenectomized under sterile conditions three days before the experiment, under 2% halothane anesthesia to avoid the effect of splenic contraction and release of red cells to the circulation during shock. Dogs were fed standard dog chow and water ad libitum in the divisional kennel. Food was removed 12 h prior to the experiment, and water was discontinued 1 h before intravenous anesthesia with 1.5 mg/kg of morphine and 25 mg/kg of pentobarbital sodium. Animals were endotracheally intubated and mechanically ventilated (FiO2=50%, TV=10 cc/kg, respiratory rate=12 rpm) during the whole experiment. An arterial blood sample was drawn before the experiments and used to adjust the ventilator settings. The following interventions were performed through cutdown incisions: 1) bilateral femoral artery dissection for the introduction of Veress needles positioned under radioscopy with their tips at the level of the 6th - 7th vertebral body to create a retroperitoneal hematoma (see description of the experimental model below); 2) A 5F triple lumen Swan-Ganz catheter (Baxter Health Care Corporation) was introduced through the right internal jugular. The tip of the catheter was positioned into the pulmonary artery under fluoroscopic guidance and pressure wave analysis. The catheter was connected to a pressure monitor (MP 100 WSW, Biopac System, USA), and all pressure measurements were stored in a computer system (Acknowledge III) for analysis as well as for the collection of mixed venous blood; 3) Large bore (P240) polyethylene cannulae were inserted into the right common carotid artery and connected to a strain gauge transducer coupled to a model 1290-CP recorder to control mean arterial pressure; 4) A short catheter number 14 was inserted into the left external jugular vein for Tc99m infusion and volume replacement; 5) A 4F Foley catheter was used for urinary output monitoring. BODY.MATERIALS AND METHODS.HEMORRHAGE MODEL: The hemorrhage model of a retroperitoneal hematoma utilized in the present study was initially described by Cruz Jr.28 Briefly, after dissection of both femoral arteries in the groin, a Veress needle was introduced into each artery, advanced towards the common iliac artery under fluoroscopic visualization and pushed against the posterior-lateral wall of the vessel to create an injury. At the end of the experiment, animals were sacrificed by an intravenous injection of pentobarbital (50 mg/kg), followed by 20 ml of 19.1% KCl. An autopsy was performed to document the severity and extent of the retroperitoneal hematoma and the size and location of the iliac artery injury. The iliac arteries were dissected free, and each perforation created by the Veress needle was measured. Exclusion criteria included: a) animals with initial hemoglobin levels < 10 g/dL, b) arterial injuries ≤ 5 mm, c) extension of the hematoma greater than the area of image acquisition of the gamma camera, d) peritoneal perforation with subsequent bleeding into the peritoneal cavity, and e) mean arterial pressure (MAP) 50% higher than initial MAP five minutes after arterial injury. The study was divided into two phases. BODY.MATERIALS AND METHODS.PHASE I: DEVELOPMENT OF A METHOD FOR THE QUANTIFICATION OF BLOOD LOSS.: Physical simulation was performed to verify the reliability of quantifying a known, pre-determined volume of fluid using sequential gamma camera image acquisitions. Initially, a plastic syringe filled with 10 mL of water, containing 99m Technetium (99mTc, Instituto de Pesquisas Energéticas e Nucleares/CNEN, Universidade de São Paulo, Brazil) with a known amount of radioactivity (2 mCi) was placed under the gamma camera and radioactivity was measured. A plastic bag was then filled with successive 50 mL aliquots of water containing 99mTc (2 mCi) up to a total volume of 1300 mL. Radioactivity in the syringe was compared with that in the plastic bag to establish a correction factor accounting for the geometry of the bag containing larger amounts of radiolabeled fluid. Measurements were performed in triplicate. The following equation describes the correction factor used by ROCHA (1976)29 for the measurement of radioactivity in the plastic bag containing 99mTc dissolved in water: RC=MC×e(0.15×D×CF)where RC is the radioactivity in the bag ("real" count); MC is the measured radioactivity; 0.15 is the attenuation factor for water; D is the thickness of the bag; and CF is the required correction factor. An operative value for CF was determined from averaged RC values within the expected range of blood loss in the retroperitoneal hematoma. BODY.MATERIALS AND METHODS.EXPERIMENT B – ARTIFICIALLY CREATED RETROPERITONEAL HEMATOMA: This set of experiments was performed to establish the correction factor due to photon attenuation caused by the presence of the abdominal wall and intra-abdominal contents in the quantification of an artificially created retroperitoneal hematoma of a known, pre-determined volume 29. Five male mongrel dogs weighing 16.8±2.6 kg were used. Blood (250 mL) was withdrawn and radiolabeled with 99m Tc (99m Tc – TCK-11, CIS Bio International, France, 99m Tc activity of 2 mCi, Instituto de Pesquisa de Energia Nuclear, Universidade de São Paulo, Brazil). A syringe containing 10 mL of labeled blood was kept aside, and an image was acquired at the end of each experiment for radioactivity measurements. Following a midline laparotomy, two polyethylene catheters (PE240) were positioned in the area where a retroperitoneal hematoma secondary to bilateral iliac artery injury would be located. The abdominal wall was then closed, and the catheters were subsequently used to inject aliquots of 50 mL of blood mixed with 99mTc-labeled red blood cells into the retroperitoneal space. Radioactivity measurements were performed successively, at five-minute intervals following blood injection. The volume of the retroperitoneal hematoma was calculated as described in Experiment A, accounting for the dorsoventral thickness of the abdominal wall of the animal. In this set of experiments, the injected volume was limited to a maximum of 200 mL because the sutures securing the catheters in place were not reliably tight for volumes greater than 200 mL. BODY.MATERIALS AND METHODS.EXPERIMENT C – IN VIVO RETROPERITONEAL HEMATOMA: This set of experiments was performed to determine the volume of blood present in a retroperitoneal hematoma (VRPH) in vivo. In one group of animals, a gamma camera and 99mTc-radiolabeled red blood cells were used to determine the blood volume present in the retroperitoneal hematoma [GC group (n=7)]. In another group, the dilution technique with 99mTc- and Chromium (51Cr, Instituto de Pesquisas Energéticas e Nucleares/CNEN, Universidade de São Paulo, Brazil, 200 μCi)-radiolabeled red blood cells was used to quantify the initial and final circulating blood volumes (CBV), respectively [DT group (n=5)]. BODY.MATERIALS AND METHODS.QUANTIFICATION OF BLEEDING VOLUME: A portable gamma camera (Elscint, model Apex 209M, Israel) with a parallel collimator was positioned 5cm above the abdomen, centered over the putative area of the retroperitoneal hematoma. Images were acquired over 5 min at 15-min intervals throughout the experiment. At the end of each experiment, the cumulative radioactivity for each measurement was analyzed. Because of the short half-life of 99mTc (six hours), all counts were corrected for activity decay in the standard fashion. BODY.MATERIALS AND METHODS.MEASUREMENT OF INITIAL CIRCULATING RED BLOOD CELL VOLUME AND TOTAL BLOOD VOLUME: Initial circulating red blood cell volume (CRCV) was determined by means of isotope dilution of the radioactive tracer technetium (99mTc), as applied by Kowalsky and Perry, 1987.30 Previously-labeled red blood cells (3 mL) containing a known total load of tracer (L) were injected into the systemic circulation and allowed to mix uniformly over a period of 15 min. A syringe containing 10 mL of labeled blood was kept aside, and a radioactivity measurement of the sample was obtained at the end of each experiment to establish the baseline count of radioactive tracer per mL. A separate blood sample was collected for determination of the initial hematocrit (Htc) and for determining tracer concentration (C) using a scintillation counter (Phillips Medical System Division, model XL 1100 and XL 1151, Eindhoven, Netherlands). CRCV was calculated according to the following equation: CRCV=LC The circulating blood volume (CBV) was calculated as demonstrated below: CBV=CRCV0.96×Htc Plasma volume (PV) was determined by subtracting CRCV from CBV. BODY.MATERIALS AND METHODS.PHASE II. RESUSCITATED UNCONTROLLED HEMORRHAGE: The experiments were initiated 15 minutes after the injection of 99mTc-labeled red cells, when the first radioactivity measurement was obtained using the gamma camera (baseline measurement value, Figure 3). The arterial lesions were subsequently created (T0). The experiment lasted a total of 75 minutes, during which six radioactivity measurements, 15 minutes apart, were obtained (T0, T15, T30, T45, T60, T75). Coinciding with the time of each radioactivity measurement, mean arterial pressure (MAP) and cardiac index (CI) were determined, and arterial blood samples were obtained for Htc measurements. BODY.MATERIALS AND METHODS.PHASE II. RESUSCITATED UNCONTROLLED HEMORRHAGE.STUDY GROUPS: Five minutes before T30, the animals were randomly assigned to one of three groups, according to the fluid resuscitation regimen: NT, untreated controls (n=7) in which the animals underwent bilateral iliac artery injury and were observed for 75 min without any fluid resuscitation; LR (n=7) in which the animals received 32 mL/kg of lactated Ringer's; or HSD (n=7), in which animals received 4 mL/kg of 7.5% NaCl, 6% Dextran 70 in five minutes, and followed up to 75 min. BODY.MATERIALS AND METHODS.PHASE II. RESUSCITATED UNCONTROLLED HEMORRHAGE.VOLUME OF RED CELLS IN THE HEMATOMA (VRCH): The VRCH was calculated considering the initial circulating red blood cell volume (CRCV) determined by the dilution technique and the VRPH, as described in Experiment C. Before fluid resuscitation (T30), the VRCH was calculated as follows 30: VRCH(Tx)=VRPH(Tx)Htc(T0) For all time points after fluid resuscitation, the VRCH was calculated according to the following equation30: VRCH(Tx)=VRCH(T30)+[VRPH(Tx)−VRPH(T30)Htc(T45)] where Tx is the time point of the actual measurement. BODY.MATERIALS AND METHODS.PHASE II. RESUSCITATED UNCONTROLLED HEMORRHAGE.VOLUME OF RETROPERITONEAL HEMATOMA (VRPH): Before fluid resuscitation (T30), the VRPH (mL/kg) was calculated according to the following equation 30: VRPH(Tx)=VRCH(Tx)Htc(T0) After fluid resuscitation, the VRPH was calculated as follows 30: VRPH(Tx)=VRPH(T30)+[VRCH(Tx)−VRCH(T30)Htc(T45)] BODY.MATERIALS AND METHODS.PHASE II. RESUSCITATED UNCONTROLLED HEMORRHAGE.CIRCULATING RED CELL VOLUME (CRCV): The CRCV (mL/kg) was determined by the difference between the initial circulating red cell volume (CRCV) and the VRPH. BODY.MATERIALS AND METHODS.PHASE II. RESUSCITATED UNCONTROLLED HEMORRHAGE.CIRCULATING BLOOD VOLUME (CBV): The CBV (mL/kg) at a specific time point (Tx) was calculated by dividing the CRVC (Tx) by the Htc (Tx). BODY.MATERIALS AND METHODS.PHASE II. RESUSCITATED UNCONTROLLED HEMORRHAGE.EXPECTED CIRCULATING BLOOD VOLUME (ECBV): The ECBV (mL/kg) at Tx was calculated as the difference between the initial CBV and the VRPH at Tx until T30. After volume resuscitation (T45 and beyond), ECBV for the LR group was calculated as follows: ECBV(Tx)=CBV(T0)−VRPH(Tx)+VI where VI is the volume of the resuscitation fluid used, i.e., 32 mL/kg for LR- and 4 mL/kg for HSD-resuscitated animals. BODY.MATERIALS AND METHODS.PHASE II. RESUSCITATED UNCONTROLLED HEMORRHAGE.TRANSCAPILLARY REFILL (TR): TR (mL/kg) was calculated as follows: TR(Tx)=CBV(Tx)−ECBV(Tx) Volume of Retroperitoneal Bleeding Before Fluid Resuscitation (VRB shock) The VRB shock (% of CRCV) caused by the arterial injuries was calculated from T0 to T30 as follows: TR(Tx)=CBV(Tx)−ECBV(Tx) Volume of Retroperitoneal Bleeding After Fluid Resuscitation (VRBresus) The VRBresus occurring after fluid resuscitation (from T30 to T75) was calculated as follows: VRBresus(%)=[CRVC(T30)−CRVC(T75)]×100CRVC(T30) BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Data are presented as the mean ± standard error of the mean. For comparisons of continuous variables, either the Student's t-test or two-way analysis of variance (ANOVA) with Turkey's B correction for multiple comparisons was used. A p value < 0.05 was considered statistically significant. BODY.RESULTS.PHASE I: DEVELOPMENT OF A METHOD FOR THE QUANTIFICATION OF BLOOD LOSS.: A final CF value of 0.015 was determined after averaging RC values obtained from multiple measurements (see methods). A comparison between a known volume in the plastic bag and the calculated volume based on radioactivity measurements by the gamma camera (Experiment A) was made. For a volume of 50 mL, the observed averaged error was 17%, and the error was greater than 8% for volumes larger than 800 mL. A significant correlation was observed between the expected and the measured volumes of fluid in the bag, within a range of 100 – 800 mL, with a calculated error of less than 3% (Figure 1). The range of variation for calculated values obtained from sequential radioactivity measurements in this phase was between 4% and 6% from the mean. Because results of measurements obtained during Experiment A were reliably linear within the 100 mL to 900 mL range, and because blood volumes in the retroperitoneum following induction of a hematoma ranged from 400 mL to 600 mL, the CF value obtained in Experiment B, which accounted for the thickness of the animal's abdominal wall, was extrapolated and used in Experiment C. BODY.RESULTS.EXPERIMENT C – IN VIVO RETROPERITONEAL HEMATOMA: Cardiac index (panel A), mean arterial pressure (panel B), and hematocrit (panel C) were measured before (time = 0 min) and after (time = 15 min and 30 min) the creation of bilateral iliac artery injuries (Figure 2). No differences were observed in these parameters between the GC and DT groups. Retroperitoneal hematoma formation led to a significant decrease in cardiac index and mean arterial pressure at 15 min and 30 min (p<0.001). Examples of gamma camera images acquired from one animal during Experiment C, taken before and 5, 10, 15, 30, and 75 minutes after the arterial injuries were created, are shown in Figure 3. Background images and radiation obtained from a syringe containing blood and a radiotracer (see methods) are shown for comparison. Every third image acquired by the gamma camera was used for quantification of the retroperitoneal bleeding volume. Figure 4 represents the volume of the retroperitoneal hematoma (VRPH) and volume of red cells in the hematoma (VRCH) over time measured at 5-min intervals. Bleeding was more pronounced within the first five minutes after the arterial injury was created, and it ended spontaneously by 15 minutes after the bilateral iliac artery injuries were created. Comparisons between the volume of the retroperitoneal hematoma (VRPH), volume of red cells in the hematoma (VRCH), and plasma volume of animals in the gamma camera (GC group) or the dilution technique (DT Group) are shown in Figure 5. No significant differences were found when comparing these two methods. In a comparison between the circulating blood volume (CBV) measured in the GC and DT groups, a significant decrease in the circulating blood volume in both groups was observed, which paralleled the formation of the hematoma (Figure 6). More importantly, the gamma camera technique was as accurate as the classic dilution technique in determining the total red blood cell volume in the hematoma (31.2 ± 1.4% for the GC group vs. 32.8 ± 3.9% for the DT group, p > 0.7). BODY.RESULTS.PHASE II-RESUSCITATED UNCONTROLLED HEMORRHAGE: A total of 25 animals were used in Experiment D. Four animals were excluded, one due to anemia before the beginning of the experiment and three because of bleeding from the retroperitoneum into the peritoneal cavity. Figure 7 presents the results of MAP, CI, and Htc measurements throughout the experiment. The mean arterial pressure (MAP) decreased significantly during the first fifteen min after the injury, and it increased equally in all groups (panel A). A reduction in cardiac index (CI) was observed 15 min after injury in all groups. After fluid resuscitation, a significant increase in CI was observed in LR- and HSD-treated animals (T45) when compared with the NT group (* p<0.05 NT vs. LR and HSD) (panel B). The mean pulmonary artery pressure and pulmonary capillary wedge pressure were similar to the cardiac index. The hematocrit (Htc) was significantly reduced during the first fifteen minutes after the injury. After fluid resuscitation occurred, there was an initial decrease in the Htc in treated groups (from T30 to T45), which was similar in all groups from T45 to T75 (panel C). The hemoglobin level was similar to the hematocrit. CBV, CRCV, VRPH, VRCH, and TR results are shown in Figure 8. CBV was equally reduced during the initial 15 minutes after injury in all groups. Fluid resuscitation with subsequent rebleeding led to a decrease in CBV in both treatment groups, although changes were more pronounced in LR-treated animals (p<0.05 NT vs. LR) (panel A). The circulating red cell volume (CRCV) decreased slightly during hemorrhage. Fluid resuscitation with subsequent rebleeding led to a decrease in CBV in both treatment groups, although changes were more pronounced in LR-treated animals (p<0.05 NT vs. LR; p<0.05 LR vs. HSD) (panel B). Blood volume in the retroperitoneal hematoma (VRPH) at 15-minute intervals is shown. Bleeding was more pronounced in the initial 15 minutes after the bilateral iliac artery injuries were created. Significantly higher VRPH was observed in LR-treated animals when compared to the HSD group, indicating more pronounced rebleeding (p<0.05 NT vs. LR and HSD; p<0.05 LR vs. HSD) (panel C). The volume of red cells in the retroperitoneal hematoma (VRCH) at 15-minute intervals is shown. Bleeding was more pronounced in the initial 15 minutes after the bilateral iliac artery injuries were created. Fluid resuscitation with subsequent rebleeding led to an increase in VRCH in both treated groups. No differences between LR- and HSD-treated animals were observed (p<0.05 NT vs. LR and HSD) (panel D). Transcapillary refill increased in all groups during hemorrhage. Fluid resuscitation determined plateaus in the NT group. It increased in HSD-treated animals and decreased significantly in the LR group (p<0.05 NT and HSD vs. LR) (panel E). In addition, LR-treated animals had negative TR values, indicating transcapillary leaks, while HSD-treated animals had significantly increased transcapillary refill. BODY.DISCUSSION: The ideal fluid resuscitation used in the treatment of hemorrhagic shock remains a subject of significant debate. Issues regarding infusion volume, sodium load, rebleeding, pulmonary complications, and ultimately mortality have not yet been solved. Recently, hypertonic resuscitation regained attention due to its immunomodulatory potential. Evidence points to the fact that hypertonic saline resuscitation decreases the amount of fluid required to restore tissue perfusion when compared to LR. More importantly, however, is the fact that hypertonic saline solutions draw fluid from the interstitium into the intravascular space. This effect is in contrast to isotonic or hypotonic saline solutions, which lead to leaks into the interstitial space of at least two-thirds of the total infusion volume, leading to significant decreases in the intravascular volume and formation of tissue edema, further compromising tissue perfusion. In addition, improvements in microcirculatory blood flow, decreased neutrophil adhesiveness, and reduced susceptibility to sepsis after hemorrhagic shock have been documented after hypertonic saline resuscitation.9,12,14–15,31–36 Several animal models have been used to simulate naturally occurring uncontrolled hemorrhage leading to shock by creating mesenteric injury,37 major vascular injury,38–39 solid organ injury,11,40 external hemorrhage,2,4 and retroperitoneal hematomas.28 Several studies quantified blood loss by weighing surgical sponges, some used the clearance technique with Evans' blue or green indocyanine, and others employed the dilution technique with radiolabeled albumin or red blood cells radiolabeled with 99mTc,41,42 or 51Cr. 43 The gamma camera was developed in 1964. With the development of short half-life radioisotopes and low energy photon emission, this apparatus is now widely employed in diagnostic procedures. It has been used in the evaluation of ventricular function44 and in the detection of bleeding sites secondary to upper or lower gastrointestinal pathology.45 This study is the first to report use of a gamma camera to reliably quantify major blood loss in an experimental setting. We selected the present animal model because it reflects the clinical scenario seen in pelvic fractures or vascular injuries contained by the retroperitoneum, allowing us to adequately quantify bleeding volumes before and after fluid resuscitation. Average blood loss in the present study was 31%, which corresponds to a class 3 hemorrhage according to the American College of Surgeons Committee on Trauma.1 We compared gamma camera measurements with the dilutional technique and found the gamma camera to be potentially useful for the follow-up of experimental uncontrolled hemorrhage models. When compared to previously described methods, this technique has many potential advantages. It allows for the detection of an area of hemorrhage, quantification of bleeding, and determination of the moment at which bleeding ceases. It is non-invasive and can result in values closer to reality when compared to previously described methods. It is, however, strictly limited to experimental conditions since it requires labeling of blood before hemorrhage begins. Another shortcoming is that the spleen must be removed to avoid distribution of red cells between two dynamically different compartments. Other important points should be emphasized. The circulating blood volume within the acquisition area of the gamma camera must not vary, although this restriction is of small significance within the abdominal cavity. The use of a second radioisotope is not feasible because it may interfere with image acquisition. Therefore, validation had to be performed in a separate group of experiments. Bleeding into a confined space, as occurs in the retroperitoneum, is very difficult to quantify. Direct measurements of blood loss are usually performed by draining blood from the abdominal cavity and weighing sponges; however, errors as large as 60% have been described with these and other less accurate methods.40,46 These methods were not applicable to our model because retroperitoneal hematomas are usually formed by clotted blood. Computed tomography scanning has been frequently used in the diagnosis of hemoperitoneum; however, the quantification of retroperitoneal bleeding volume by this method is inaccurate, and its use is contraindicated in patients who are in shock. The use of labeled plasma albumin to evaluate the circulating plasma volume can be misleading because of normal protein leakage into the extravascular compartment, resulting in calculated values higher than the actual values.47 Other devices developed to measure bleeding volume have proven to be costly and difficult to use.45 Even with the use of 99mTc or 51Cr-labeled red blood cells in the dilution technique, it is difficult to determine the moment at which bleeding stops or resumes.41,43 In addition, radiolabeled red cells must be injected at each time point of interest to determine the circulating blood volume after hemorrhage starts. A homogeneous distribution of the labeled red cells in the circulation is always required, which requires 15 minutes to occur. Therefore, it is not possible to determine the circulating volumes at shorter time intervals. The present method covers all of these shortcomings. Employing the two techniques (dilution and gamma camera) to quantify the initial circulating blood volume and the volume of blood present in the retroperitoneal space allows for attainment of important parameters. The circulating blood volume, red cell and plasma volumes after injury, as well as the transcapillary refill can be measured over short time intervals. The use of a portable gamma camera in the present study introduced a new application to an old method.48 This method of monitoring bleeding volumes has a number of positive aspects to be considered in future research: 1) it is possible to quantify blood loss at relatively short time intervals, 2) the natural interruption of bleeding is clearly demonstrated, and 3) the gamma camera is as reliable as the dilution technique in measuring several parameters, as demonstrated in the present study. However, there are a number of restrictions to this method. For each experiment, a value for the correction factor must be determined. Furthermore, the method is strictly for experimental use, and the spleen must be removed if large animals are to be used, to avoid sequestration of volume in the splenic parenchyma and the effects of splenic contraction with subsequent changes in the circulating blood volume. Several studies have reported increased rebleeding with aggressive fluid resuscitation. Strategies for controlling blood loss and therefore the use of blood components have also been reported.49 The volume of rebleeding has been related to the sodium load of the solution, amount of fluid resuscitation, and the resultant hemodilution. One research study,4 using an animal model of uncontrolled hemorrhage, reported higher rebleeding volumes in animals treated with hypertonic saline with or without dextran, compared to non-treated animals or with animals treated with conventional isotonic resuscitation regimens.4 However, the total sodium load was different between groups. Several studies suggested that a state of "controlled hypotension" is probably the best strategy before definitive control of the bleeding site is achieved.2–6 Hemodynamic parameters such as the MAP, CI, and Htc have been used to monitor the hemodynamic response to fluid resuscitation and to detect new or recurrent bleeding episodes. MAP decreased during bleeding and increased in all groups after T30. The increase in MAP in the NT group can be explained by the action of compensatory mechanisms, such as vasoconstriction and tachycardia, in an attempt to maintain adequate perfusion to the heart and brain. MAP increased in the HSD and LR groups to levels slightly higher than those in the NT group up to T45, most likely due to fluid infusion. However, MAP decreased again in the resuscitated groups, most likely because of rebleeding. The same observations were made with CI measurements. Hemodilution and rebleeding contributed to decreases in Htc after fluid resuscitation in both resuscitation groups. Changes observed in the hemodynamic parameters and in Htc levels were paralleled by those seen in VRPH, VRCH, CBV, CRCV, and TR. LR-treated animals had higher rebleeding volumes as indicated by increased VRPH compared to the HSD group. TR following hypertonic saline resuscitation has been demonstrated in animal models of controlled hemorrhage.9–10,12,50 In the present study, hemorrhage led to transcapillary refill during the initial fifteen minutes after hemorrhage started in all groups. No transcapillary refill was observed during the shock period (T15 to T30). After fluid resuscitation and despite the occurrence of rebleeding in both resuscitated groups, a clear loss of intravascular volume to the interstitial space (transcapillary leak) was observed in LR-resuscitated animals. In contrast, TR was clearly observed in HSD-treated animals. BODY.CONCLUSION: This model produces an effective bilateral retroperitoneal hematoma (red cell loss of nearly 31%). Fluid resuscitation with either HSD or LR increased MAP, but it also led to rebleeding. Higher rebleeding volumes were observed in LR-treated animals, despite similar sodium loads given to HSD-treated animals. Transcapillary refill was only present in HSD-treated animals. LR-treated animals developed capillary leaks. The combination of increased recruitment of fluid to the intravascular space associated with decreased rebreeding volumes makes HSD, in the present model of uncontrolled hemorrhage, superior to LR resuscitation.

TITLE: Comparing the Efficacy of Low Dose and Conventional Dose of Oral Isotretinoin in Treatment of Moderate and Severe Acne Vulgaris ABSTRACT.OBJECTIVE:: This study was conducted to compare the effect of low-dose isotretinoin with its conventional dose in patients with moderate and severe acne. ABSTRACT.METHODS:: This was a clinical trial conducted on 60 male and female patients with moderate and severe acne vulgaris. The patients were divided into two treatment groups: 0.5 mg/kg/day isotretinoin capsule and low-dose isotretinoin capsule (0.25 mg/kg/day). Patients in both groups received 6-month treatment. At the end of the 6th month and 12th month (6 months after the end of the treatment), they were examined again, and their improvement was determined and compared. ABSTRACT.FINDINGS:: The average severity of acne in the two treatment groups did not differ significantly within any of the study periods. The most common side effects were nose dryness in the low-dose group (17%) and hair thinning and loss in the conventional-dose group (33.2%), although all the patients had dry lips. ABSTRACT.CONCLUSION:: According to the same severity of the acne in two groups in different study periods, as well as fewer side effects and more patients' satisfaction, the low-dose isotretinoin can be considered in the treatment of acne. BODY.I: Acne vulgaris is a common disease during adolescence. It is the most common inflammatory diseases of the sebaceous unit and a chronic inflammatory disease of the sebaceous glands.[1] There are different common treatments for acne vulgaris based on the severity of lesions.[2] In that, topical solutions (e.g., clindamycin or erythromycin), topical ointment (e.g., tretinoin, benzoyl peroxide, and adapalene), and other keratolytic drugs (e.g., alpha-hydroxy acid), salicylic acid-containing medicine, and/or sulfur or azelaic acid-containing drugs are used to treat mild cases. In more severe cases, systemic treatments, such as tetracycline, doxycycline, azithromycin, minocycline, azithromycin, and cotrimoxazole are used. In cases with the risk of scar, oral isotretinoin is administered. In cases with associated hormonal abnormalities, androgen, estrogen, spironolactone, and dexamethasone are used.[34] Despite all traditional and modern treatments, retinoid compounds are key components in the treatment of acne.[5] In early 1980, the administration of oral isotretinoin was limited in patients with nodulocystic acne. Nevertheless, the use of oral isotretinoin was made wider with broadening relevant experience. It was also prescribed for patients with milder acne, who did not respond satisfactorily to common treatments, such as topical retinoids plus oral antibiotics. Patients with moderate acne, who exhibit the scar symptoms, are also a candidate for treatment with oral isotretinoin. Acne is mainly associated with physical and cosmetic morbidity. The psychological consequences of acne vary from depression and anxiety to work and interpersonal relationship disorders. Studies, used quality of life instruments, have shown that the treatment with isotretinoin results in a significant improvement of socialization and self-confidence.[6] There may be a delay of 1–3 months before the initiation of treatment effects. In many cases, improvement continues even after discontinuation of the treatment. Therefore, the continuation of the treatment until the complete healing of lesions is not necessary.[7] Although isotretinoin is a very effective medication for the treatment of acne, its association with several complications necessitates the precise selection of patients.[6] The recommended dose is 0.5–1 mg/kg/day for 4–6 months.[8] A study conducted in India (2014) on 50 patients with moderate to severe acne showed the effectiveness of low-dose isotretinoin with lesser complications and greater cost-effectiveness. In this study, patients received 0.3–0.4 mg/kg/day isotretinoin.[9] In addition, a study (2012) showed the effective treatment of severe acne with low-dose isotretinoin (0.1–0.3 mg/kg/day).[8] Since, side effects of this medication are dose-dependent, the administration of low-dose isotretinoin (0.25 mg/kg/day) for 6 months seems logical; however, there is no relevant, comprehensive study with follow-up period in Iran, and although the patient's compliance is dependent on side effect and cost of drug and its cost and economic status of patients has turned it into a significant problem.[10] Thus, the present study has been conducted to compare the low- and common-dose oral isotretinoin in patients with moderate and severe acne vulgaris. BODY.M: This study was a prospective randomized clinical trial conducted during 2014–2015. The population comprised patients (both male and female) with moderate to severe acne vulgaris referred for treatment to Alzahra Medical and Training Center, several clinics affiliated to Isfahan University of Medical Sciences and a privately-owned doctor's office. The inclusion criteria are as following: patient consent to participate in the study, no sensitivity to retinoids, no pregnancy, not willing to become pregnant, and absence of hormonal disorders in patients. Patients with the following criteria excluded from the study: Failure of the patient to attend follow-up sessions for any reason, and adoption of other supplementary therapies during the study. The required sample size was estimated to be 30 patients using the sample size formula, serving to compare the ratios given the confidence level of 95% (Z1-α/2 = 1.96) and test power of 80% (Z1-β = 0.84). The minimum significant difference between the two groups was considered to be 0.25. After obtaining a permit from the Ethics Committee of Isfahan University of Medical Sciences that Ethical code is 393894 and IRCT code is 2015061722780N1, the simple sampling was applied to patients (male and female) with moderate acne vulgaris and referred for treatment to one of the largest referral centers in isfahan (Iran's third largest city, located in the center of Iran). The patients were randomly treated by 0.5 and 0.25 mg/kg/day of oral isotretinoin (Roaccutane, F. HOFFMAN-LA ROCHE Switzerland) for 6 months. The effort was made every day to arrange equal numbers of patients in Groups 1 and 2 so as to fulfill the random sampling. The patients were advised to take the medication with meals, avoid fat-free diet, and yet refrain from the excessive fat intake. This process continued until the number of patients per group amounted to 30. The method of follow-up in two groups was an intention to treat. At the baseline and 6 months later, the patients were examined for the severity of acne by a dermatologist that was blinded to intervention group. The severity of acne was determined through the Global Acne Grading System.[11] This system was used in studies conducted by the British Association of Dermatologists and the American Society for Dermatologic Surgery (2013 and 2014).[12] In this system, the numbers of lesions including comedones, papules, pustules, and nodules were counted on the forehead, right cheek, left cheek, nose, chin, chest, and upper back torso. Each type of lesion is given a grade based on its severity as follows: 1 - comedones; 2 - papules; 3 - pustules; and 4 - nodules. Moreover, each zone is given a factor as follows: 1 - nose, chin; 2 - forehead, right cheek, left cheek; and 3 - chest, back and upper back torso. The severity of lesion for each zone (local score) is calculated as follows: local score = Factor × grade (0–4). Global score is obtained from the sum of local scores in different zones. The severity of acne is considered based on the global score as follows: mild: 1–18; moderate: 19–30; severe: 31–38; and very severe: Over 38. To prevent the complications of treatment, the female subjects in reproductive age were initially tested for pregnancy. They were included in the study if the results were negative. Then, the test was repeated every month. At baseline and at the end of 1st, 2nd, 4th, and 6th months, the levels of liver enzymes, blood cholesterol, and triglyceride were checked in all patients. The treatment discontinued in case the triglyceride level elevated over 400 mg/dl, cholesterol over 300 mg/dl, alkaline phosphatase over 246, alanine aminotransferase over 62, and aspartate aminotransferase over 80.[13] All patients were simultaneously treated with daily 250 mg of oral azithromycin in the first 2 weeks.[13] Moreover, 0.25 mg of prednisolone was prescribed to treat patients with isotretinoin over the first week. To prevent complications such as dry lips and skin, identical topical emollient was administered on all patients. Furthermore, they were advised to apply sunscreen regularly and avoid sunlight as much as possible. After the end of treatment with isotretinoin, the patients went through local treatment with 2% clindamycin during the follow-up phase. The patients were visited on a monthly basis. The cases with no new lesions were recorded in a data collection form. At the end, it was found out which group recovered from the lesions sooner than the other. The side effects of isotretinoin were examined and recorded at each visit by a dermatologist that was blinded to intervention group. The patient satisfaction level was assessed and recorded at the end of treatment by visual analogue scale (VAS) for satisfaction.[14] VAS was a horizontal line. The patient rated his satisfaction by making a vertical mark on the line. There were two descriptors representing extremes of satisfaction (i.e., no satisfaction [0 point] and extreme satisfaction [5 points]). We analyzed complications by the questionnaire, physical examination, and laboratory tests that were conducted by a dermatologist that was blinded to intervention group. The collected data were described through several tables, graphs and measures of central tendency and dispersion. Then, they were analyzed by Chi-square test, independent t-test, and we used two-way repeated measure ANCOVA to determine the significance difference between these two sets of observations within the same group and then to compare the significant difference between the low dose versus high-dose therapy. The SPSS 22 (released 2013, SPSS Inc, Chicago, IL) was also used as statistical software. BODY.R: Results of the frequency distribution of demographic variables (qualitative and quantitative) in each treatment group indicated that the average age of participants in the first treatment group (low dose) and second group (conventional dose) was 22.94 ± 6.25 and 23.1 ± 4.66 years, respectively. Therefore, there was no statistically significant difference between age results (P = 0.911). Similarly, the average age and duration of acne did not differ significantly in the two groups (P > 0.05). Frequency distributions of qualitative research variables including gender differed significantly in the two groups, because 5 participants in the first group (13.9%) and 8 participants in the second group (26.7%), which added up to 13 (19.7%), were male, whereas 31 participants in the first group (86.1%) and 22 (73.3%) in the second group (which added up to 53 or 80.3%) were female (P = 0.011). However, there was no significant difference between the two groups in terms of family history of acne and frequency of previous acne treatments (P > 0.05). Results of determining and comparing the average severity of acne vulgaris in the first group prior to treatment (mean = 54.6, standard deviation [SD] = 2.9), 6 months into treatment (mean = 2.5, SD = 0.8), and 6 months following the treatment (mean = 8.7, SD = 0.8), showed that there was a significant difference in the average severity of acne in the first group before treatment and 6 months into the treatment as well as before treatment and 6 months following the treatment in the 12th month (P < 0.001). According to the classification of severity of acne based on the global score, it was found that average severity of acne was extremely high in the first group before treatment (with average acne severity of 58.6), while 6 months into treatment and 6 months following the treatment severity of acne was mild (with respective average acne severity of 2.5 and 8.7). Figure 1 depicts the linear diagram of changes of average acne severity over the three periods under study. Frequency distributions of acne severity in the first and second groups before treatment, 6 months into treatment, and 6 months after treatment based on the gender and age variables revealed that there was no significant difference between the two groups in terms of severity of acne based on gender and age (P > 0.05). Figure 1Diagram of changes of acne average severity in the two treatment groups over three study periods Results of determining and comparing the average severity of acne vulgaris in the second group (which received 0.5 mg isotretinoin capsules) before treatment (mean = 58.8, SD = 3.1), 6 months into treatment (mean = 1.8, SD = 0.7), and 6 months following the treatment (mean = 9.7, SD = 1.1) showed there was a significant difference in the average severity of acne in the second group before treatment and 6 months into the treatment as well as before the treatment and 6 months following the treatment in the 12th month (P < 0.001). According to the classification of severity of acne based on the global score, it was found that average severity of acne was extremely high in the second group before treatment (with average acne severity of 58.87), while 6 months into treatment and 6 months following the treatment severity of acne was mild (with respective average acne severity of 1.87 and 9.73). Results of comparing the average severity of acne in the two study groups showed that the average severity of acne in the two treatment groups did not differ significantly within any of the study periods (P > 0.05). Figure 1 shows the linear diagram of changes of average acne severity in the two groups over different periods. Results of comparing the average severity of acne in the two treatment groups before treatment, 6 months into treatment, and 6 months after treatment based on the gender and age variables showed that there was no significance difference between the severity of acne in the two groups based on gender and age (P > 0.05). The frequency distribution of the severity of acne in the first and the second groups before treatment, 6 months after treatment, and finally 6 months after completion of treatment in terms of duration of acne showed that no significant difference existed between the severity and duration of acne vulgaris in the first and second groups separately in any of the studied times (P > 0.05). In addition, comparison of the mean severity of acne in both treatment groups before treatment, 6 months after treatment, and 6 months after completion of treatment in terms of duration of acne showed no significant difference between the mean severities of acne in terms of the duration of acne in both groups (P > 0.05). The frequency distribution of the severity of acne in the first and second groups separately, before treatment, 6 months after treatment, and finally 6 months after completion of treatment in terms of family history of acne showed that no significant difference existed between the severity of acne and family history of acne in none of the groups in any of the studied times (P > 0.05). In addition, comparison of the mean severity of acne in both treatment groups at different times in terms of family history of acne showed no significant difference between the mean severities of acne in terms of family history of acne (P > 0.05). Considering the significance level of 5%, a significant difference can be observed between patients' satisfaction in the two treatment groups (P < 0.05), so the average of patients' satisfaction score in the first group (mean = 4.78, SD = 0.4) was significantly higher than the second group (mean = 4.43, SD = 0.6) (P = 0.02), which shows that the satisfaction of patients treated with lower doses of medication (Group I) was higher than patients treated with higher doses of the drug (Group II). As shown in Table 1, considering the significance level of 5%, a significant difference can be seen in the frequency of side effects dry mouth, dry nose, repeated rhinorrhea, and hair thinning and loss in both groups (P < 0.05), so that the frequency of side effects in the group receiving a higher dose of the drug (the second group) was more than the group receiving the low dose of the drug (the first group). Table 1 shows the most common side effects were nose dryness in the first group (17%) and hair thinning and loss in the second group (33.2%) also all the patients had dry lips. Table 1 The frequency of adverse reactions in patients with acne in the study groups BODY.D: There are different studies about this study that we mentioned some of them here. Amichai et al. and Lee et al. believed that doses of isotretinoin lower than 0.5 mg/kg/day may be effective for the treatment of some patients with acne.[1516] In another study, Rasi et al. investigated the efficacy of low daily dose isotretinoin in moderate to severe acne patients. They found that low-dose isotretinoin was found to be a safe and effective choice for patients with moderate to severe scar prone acne vulgaris[17] so their results are consistent with our results. Ghalamkarpour and Nasiri studied about isotretinoin in the treatment of acne and this study was performed on patients with acne to examine the therapeutic effects, recurrence rate, and adverse effects of this drug. They concluded that oral isotretinoin appears to have favorable results and the least adverse effects in the treatment of carefully-selected patients with acne.[18] In addition, no significant difference was observed between the severity of acne and patients' sex and age at any time point in our results. Furthermore, in the study of Duman et al. there were no statistically significant differences between control and acne groups with respect to age, sex, and Hospital Anxiety and Depression Scale (HAD) score.[19] A significant difference was observed between the two groups in terms of the frequency of the side effects such as dry mouth, nasal dryness, frequent nosebleeds, thinning hair, and hair loss. The above-mentioned side effects were more frequent in the high-dose group (second group) than in the low-dose group ( first group). Therefore, prescribing lower doses of this drug is more appropriate and effective in such patients. Our results are supported by the findings of a study from India, a low-dose isotretinoin treatment (0.15–0.28 mg/kg/day) lead to clinically significant results in 87.54% of the participants, including 68.20% very good and 19.34% of good results.[20] Another study that is about safety and efficacy of low-dose isotretinoin in the treatment of moderate to severe acne vulgaris and conducted by Rao et al. In this study, they evaluated 50 participants diagnosed as having moderate to severe acne vulgaris and the participants were recruited over a period of 2 years and were followed up for 3 months to know the safety and efficacy of low-dose isotretinoin in the treatment of moderate to severe acne vulgaris. Hence, they recommended that judicious use of low-dose isotretinoin in patients with moderate to severe acne because acne not only scars the face, but also the mind and the heart.[9] Lee et al. evaluated the clinical efficacy and tolerability of low-dose and intermittent isotretinoin regimens and to compare them directly with conventional isotretinoin treatment. They suggested that when considering tolerability, efficacy and patient satisfaction, low-dose treatment is most suitable for patients with moderate acne[15] which is consistent with this study results. The results of this study are consistent with the results of the previous study. This study shows the results of determining and comparing the average severity of acne vulgaris in the group receiving isotretinoin (0.5 mg capsules). The results show that in this group, there was a significant difference between the average acne intensity before and 6 months after treatment. Moreover, there was a significant difference between the average severity of disease before and 6 months after treatment. The satisfaction level of the low-dose patients was higher than the high-dose patients. In addition, the frequency of complications was greater in the high-dose patients (the second group) than in the low-dose patients (the first group). On average, there was no significant between-groups different in terms of the severity of the acne in the aforementioned times. According to the same recurrence of patients with acne after follow-up at 6 months, more satisfied patients and fewer side effects can be considered low-dose isotretinoin in the treatment of acne. BODY.A: Gita Faghihi and Fatemeh Mokhtari contribute to Concept and design of study. Nasrin Motamedi Fard contribute to intervention and data gathering. Narges Motamedi contribute to Drafting the article and revising it. Sayed Mohsen Hosseini contribute to analysis and interpretation of data. BODY.A.FINANCIAL SUPPORT AND SPONSORSHIP: This study was supported by Isfahan University of Medical Sciences. BODY.A.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Does Nursery-Based Intensified Anticipatory Guidance Reduce Emergency Department Use for Nonurgent Conditions in the First Month of Life? A Randomized Controlled Trial ABSTRACT: Objective. We aimed to evaluate the impact of an intensified anticipatory guidance program in the nursery on Emergency Department (ED) use for nonurgent conditions (NUCs) in the neonatal period. Methods. Parturient mothers of healthy newborns were randomized to an intervention group or control group. Baseline and 1-month follow-up knowledge surveys regarding newborn care were conducted. The primary outcome was the proportion of neonates who used the ED for a NUC. Secondary outcome was change in caregivers' knowledge on NUC. Results. Of a total of 594 mothers, 323 (54%) agreed to participate and were randomized to intervention (n = 170) or control (n = 153) group. Most were Hispanic (68%), single (61%), primiparous (39%), and without high school diploma (44%). 35 (21%) neonates in the intervention group and 41 (27%) in the control group were brought at least once for a NUC to the ED (p = 0.12). There was no statistically significant difference in within subject change on knowledge scores between the two study arms. Conclusions. Neonatal ED visits for NUCs occur frequently. This nursery-based intensified anticipatory guidance program had no statistically significant impact on neonatal ED use for NUC, nor on neonatal care-relevant knowledge among parturient mothers. Alternative modalities and timing of parental educational intervention may need to be considered. This trial is registered with Clinical Trials Number NCT01859065 (Clinicaltrials.gov). BODY.1. BACKGROUND: Both pediatricians and parents agree that anticipatory guidance is an important component of the well-child visit. However, parents report unmet expectations related to parenting advice, education, or screening during their child's health supervision visit. Few available studies suggest that there may be large variations in the delivery of anticipatory guidance, depending on the population and clinical setting [1, 2]. Paradis et al. found that parents receiving a video intervention rated higher confidence with specific infant care skills and reported feeling better prepared to care for their baby, compared to parents receiving only handouts [3]. First-time parents have many questions about the care of their newborns and most of them are not addressed during the standard follow-up visit at two days or two weeks of age. These concerns bring them into the physician's office or the Emergency Department (ED) for unnecessary visits that could have been resolved at home. Utilization of the ED for nonurgent conditions (NUCs) has long been recognized to be problematic because of cost and lack of continuity of routine care [4]. It has been estimated that about 20 million children in the US are annually brought to the ED for medical care and that up to half of those visits are for NUCs [4]. ED visits for NUCs occur especially frequently in the immediate postnatal period and early infancy [5, 6]. In the literature, management of NUCs in the ED can be considered when a nonurgent triage code was assigned at the time of presentation, no laboratory or radiologic investigations were performed, no physician referral occurred, and the final disposition was to discharge the child home [6, 7]. Newborns obviously represent a unique group of pediatric patients as the increased risk of serious bacterial infection is paired with often only subtle signs of illness naturally leading to heightened parents' anxiety. Indeed, one study showed that more than a third of neonates who returned to the ED within 5 days after being discharged with NUC required subsequent hospitalization [8]. However, there might be an additional burden of newer parents with limited parenting experience or knowledge of their infants' primary pediatric medical home [9]. As this help seeking behavior characterized by unnecessary ED use during the newborn period may be the onset of a pattern of future frequent ED use, we asked whether it could be influenced through a focused educational intervention [7]. Specifically, we aimed to evaluate the impact of an intensified nursery-based anticipatory guidance program on ED use for newborns with NUC. BODY.2. METHODS.2.1. DESIGN: This study was a prospective randomized control trial. BODY.2. METHODS.2.2. STUDY PARTICIPANTS: Eligible for participation were English- and Spanish-speaking parturient mothers (aged ≥ 18 years) of healthy full-term newborns (≥36 weeks of gestational age) who received care during the study period at the neonatal service of the Bronx-Lebanon Hospital Center. Inability to converse in either English or Spanish and existing hearing/vision impairments were exclusion criteria. BODY.2. METHODS.2.3. RECRUITMENT AND RANDOMIZATION: A trained research assistant and the coinvestigators enrolled participants between December 1, 2011, and March 31, 2012. Parturient mothers were approached at the neonatal service on the day of discharge and the ones who agreed to participate in the study were asked to sign an informed consent. The participants were randomly assigned to a control arm receiving a routine anticipatory guidance program or to an intervention arm receiving an intensified anticipatory guidance program. Assignment to respective study arms was determined by month of birth. During months #1 and #3 of the study participant mothers were assigned to the intervention arm, while during months #2 and #4 of the study they were assigned to the control arm. Video-based and handout materials were in English and Spanish. BODY.2. METHODS.2.4. INTERVENTIONS.2.4.1. ROUTINE ANTICIPATORY GUIDANCE CONTROL GROUP: The routine anticipatory guidance program consisted of the already established educational materials (handout and videos) on breastfeeding, reducing the risk of sudden infant death syndrome (from the "Safe to Sleep Campaign") [10], and prevention of Shaken Baby Syndrome (from the "Portrait of Promise: Preventing Shaken Baby Syndrome") [11]. BODY.2. METHODS.2.4. INTERVENTIONS.2.4.2. INTENSIFIED ANTICIPATORY GUIDANCE INTERVENTION GROUP: The intensified anticipatory guidance program included in addition to the routine anticipatory guidance program a 30-minute video entitled "Newborn Care: A Guide to the First Six Weeks" with detailed information based on the latest American Academy of Pediatric safety guidelines on newborns' NUC (e.g., jaundice, well-baby visits, taking temperature, and when to call the doctor) [12]. This video was shown to the participants on the day of discharge by the coinvestigators and/or the research assistant in the nursery. BODY.2. METHODS.2.5. OUTCOME MEASURES.2.5.1. NONURGENT ED VISIT: Our primary outcome was to determine the proportion of neonates with at least one nonurgent ED visit during the first month of life. Nonurgent ED use was assessed at 1-month follow-up and by reviewing newborns' electronic medical record. A nonacute ED visit was defined as follows: the infant presented for an acute health concern but a nonurgent triage code at the time of presentation was assigned by the triage nurse, no laboratory or radiologic investigations were performed, and the infant was subsequently discharged home with no physician referral [6, 7]. BODY.2. METHODS.2.5. OUTCOME MEASURES.2.5.2. MATERNAL KNOWLEDGE ON NONURGENT CONDITIONS: Knowledge on nonurgent conditions was surveyed using a self-administered, anonymous questionnaire at baseline (before receiving the respective anticipatory guidance program) and at 1-month follow-up (phone interview). After reviewing the 30-minute video, a questionnaire (11 questions) was drawn up to assess maternal knowledge on nonurgent conditions in newborns at baseline and at 1-month follow-up as follows.During feeding, baby should be burped: (a) Never, (b) If breastfed, after changing breast, (c) If formula fed, after 2-3 ounces of formula, (d) (b) and (c), (e) I don't knowWhere is the best place to take the temperature in a newborn baby? (a) Under arm, (b) Mouth, (c) Buttocks, (d) I don't knowA newborn baby has fever when the temperature is above: (a) 98.5 F, (b) 99.4 F, (c) 100.4 F, (d) I don't knowThis question is to assess your knowledge regarding normal range of stool frequency in newborns: 1 soft stool after 3 days to multiple soft stools in one day: (a) True, (b) False, (c) I don't knowThis question is to assess your knowledge regarding normal range of wet diapers in newborns: 5–8 wet diapers in one day: (a) True, (b) False, (c) I don't knowHow should your baby sleep? (a) Sideways, (b) On back, (c) On belly, (c) I don't knowWhere do you think your baby should be sleeping? (a) Mother's lap, (b) Mother's bed, (c) In his own crib/playpen/bassinet, (d) I don't knowWhen should you call the doctor or take the baby to the Emergency room? (a) Constant/distressed crying, (b) Vomiting/choking, not feeding well, (c) Tightness or shaking or hands or legs, (d) Breathing fast for long time, turning blue, (e) All of the aboveWhich of these is normal for babies? (a) Sneezing, hiccups, (b) Spit up after feeding or during burping, (c) Irregular breathing with no change in skin color, (d) Startle on loud noise or stimulation, (e) All of the aboveWhich of these are normal for a newborn baby? (a) A bluish green or gray birthmark on the lower back or buttocks, (b) A newborn rash or red splotches on skin, (c) Tiny white bumps on the face, (d) Soft spot on the head, (e) All of the aboveWhat are the common reasons for crying of normal newborn babies? (a) Wet diaper, (b) Hungry, (c) In pain, (d) Feeling lonely or tired, (e) All of the above.A composite score (0–11) based on the correct answers on the knowledge questions was calculated. The questionnaire was pretested for clarity and timing in the first 2 weeks of the study period. BODY.2. METHODS.2.6. DATA ANALYSIS: Demographic characteristics were summarized using descriptive statistics. Proportion of newborns who presented for nonurgent ED use at least once during the first month of life in the two trial arms were compared using Fisher's exact test. The difference in within subject nonurgent conditions knowledge score change from baseline to follow-up between the two study groups was assessed using a paired t-test. Analyses were based on subjects with complete data on both assessments and on intention to treat with zero score change imputed to subjects absent for the follow-up assessment. We powered our trial for analysis on the primary outcome, nonurgent ED visit. With an anticipated event rate of 20% in the control group, we randomized at least 150 subjects (allowing for a 10% rate of loss to follow-up) to each trial arm to have 95% power with p < 0.05, two-sided, to detect a 15% difference between the two arms in the proportions of newborns making at least one nonurgent ED visit [13, 14]. Data were analyzed using SPSS version 19. The hospital's institutional review board approved the study. BODY.3. RESULTS.3.1. STUDY POPULATION: Of a total of 594 parturient mothers during the study period, 323 (54%) consented to participate in the study and were randomized to the control (n = 153) or the intervention (n = 170) group (Figure 1). Table 1 displays the demographic characteristics of enrolled subjects in each group. The overall mean age was 26.5 years; most were single (61%), of Hispanic ethnicity (68%), and with an incomplete high school education (44%). A third (35%) was new mothers who had just delivered their first child. There were no significant differences between groups regarding age, race/ethnicity, education, marital status, and number of children. 44% (n = 74) and 40% (n = 61) of the participants were unable to be contacted for the 1-month follow-up survey from the intervention group and the control group, respectively. BODY.3. RESULTS.3.2. NONURGENT ED VISIT: Overall, a quarter (76/323, 24%) of the subjects' newborns had at least one nonurgent ED visit reported during the first month of life. While such visits were reported in only 35/170 (21%) infants of the intervention group compared to 41/153 (27%) infants of the control group, this difference was not statistically significant (p = 0.12). Subgroup analysis (marital status, level of education, maternal age, ethnicity, and number of children) also did not reveal any statistically significant difference in reported nonurgent ED visits between intervention and control group (data not shown). BODY.3. RESULTS.3.3. MATERNAL KNOWLEDGE ON NONURGENT CONDITIONS: There was no significant difference in mean baseline scores between groups (control group: 7.0, SD 2.2; intervention group: 6.9, SD 2.4; p = 0.59). Likewise, the mean scores at the 1-month follow-up survey were comparable (control group: 8.5, SD 1.9; intervention group: 8.0, SD 2.2; p = 0.11). Further, there was no statistically significant difference in within subject change scores between the two study arms (p = 0.52 and p = 0.80 for subjects with complete data and intention to treat analysis, resp.). BODY.4. DISCUSSION: First and foremost this study documents that about a quarter of newborns in the Bronx are brought to the ED for NUC within the first month of life. Further, we showed that providing parturient mothers in the nursery with intensified anticipatory guidance about such NUC in neonates did not lead to a significant reduction of the rate of nonurgent ED use among the subjects' newborns, nor to superior knowledge gain regarding nonurgent conditions compared to mothers in the control group. Nationally, ED use overall has continued to rise, with children and especially infants being brought there by caretakers for concern about NUC [15]. A study conducted in Cincinnati, Ohio, more than 10 years ago described that about 20% of infants had an ED visit for a NUC in the first 3 months of life [7]. Our apparent higher rate of this help seeking behavior may be explained by the fact that our institution serves almost exclusively an indigent minority population with poor educational attainment while about half of the subjects in the study from Cincinnati were White [7]. Indeed, non-White race of the mother apart from younger maternal age and Medicaid insurance were identified as significant risk factors associated with ED visits for NUC in the study from Cincinnati [7]. Subgroup analysis in our study failed to find any relevant associations. With low caregiver health literacy found to be an independent predictor for higher ED use overall and for use of ED for NUC, interventions targeting health literacy skills in parents have been of great interest [16–20]. Preventive pediatric care guidelines by the American Academy of Pediatrics prescribe the discussion of many topics to be covered at each office visit to provide parent with anticipatory guidance [21]. Anticipatory guidance is a developmentally based counseling technique that focuses on the needs of a child at each stage of life [21]. These needs are discussed during well-child care visits to increase parental satisfaction and help them to become a more effective caregiver [1, 2]. Barriers to delivering better anticipatory guidance include limited time and lack of confidence in counseling techniques [22]. However, a recent trial of newborn anticipatory guidance delivered by a DVD during the infant's first visit to the pediatrician's office demonstrated increased parental confidence in specific infant care items that were emphasized in the video and most importantly succeeded in reducing additional health care utilization [13]. We were unable to demonstrate a similar benefit with our video-enhanced anticipatory guidance program on NUC in the nursery. This may be because our study cohort in the South Bronx consisted predominantly of single women who were of Hispanic ethnicity. Both characteristics have been previously identified as leading predictors for seeking care for NUC with children in the ED [23]. Other explanations for lack of impact may be that the added information about NUC was brief, presented only once, and competed with other preventive health messages to be discussed in the nursery (e.g., breastfeeding, SIDS prevention, and Shaken Baby Syndrome) therefore limiting likelihood of parental recall [20, 22]. Indeed, discussing more than 8 anticipatory guidance topics during a pediatric health maintenance visit has not been found to be helpful [2]. Our study has some potential limitations. First, our study may have been underpowered to detect smaller differences in ED use and gained knowledge due to a high attrition rate in both study groups. Second, the intensified anticipatory guidance program that we used in the nursery may not suffice and/or may not be at the right time to improve parental nonurgent conditions knowledge and reduce nonurgent ED visits. Further, during the phone follow-ups, mothers might not remember well or answer truthfully about ED visits of their newborns. BODY.5. CONCLUSIONS: A significant proportion of healthy newborns in the South Bronx are brought to the ED for a NUC during the first month of life. Incorporating an intensified anticipatory guidance program in the nursery did not have a significant impact on reducing the rate of such ED visits for NUCs nor did it result in an improved gain of NUC-relevant knowledge in parturient mothers. It is possible that the video-enhanced educational program we used in the nursery may not suffice and/or may not be at the right time to improve parental knowledge on NUC and reduce ED use for NUC in our patient population. Introduction of NUC-related topics to future parents using alternative educational modalities (e.g., parenting class) or alternative timing (e.g., pediatric prenatal visit) may be potentially more promising strategies in urban, low-income communities to effect reduced ED use [14].

TITLE: Effects of pressure support and pressure-controlled ventilation on lung damage in a model of mild extrapulmonary acute lung injury with intra-abdominal hypertension ABSTRACT: Intra-abdominal hypertension (IAH) may co-occur with the acute respiratory distress syndrome (ARDS), with significant impact on morbidity and mortality. Lung-protective controlled mechanical ventilation with low tidal volume and positive end-expiratory pressure (PEEP) has been recommended in ARDS. However, mechanical ventilation with spontaneous breathing activity may be beneficial to lung function and reduce lung damage in mild ARDS. We hypothesized that preserving spontaneous breathing activity during pressure support ventilation (PSV) would improve respiratory function and minimize ventilator-induced lung injury (VILI) compared to pressure-controlled ventilation (PCV) in mild extrapulmonary acute lung injury (ALI) with IAH. Thirty Wistar rats (334±55g) received Escherichia coli lipopolysaccharide intraperitoneally (1000μg) to induce mild extrapulmonary ALI. After 24h, animals were anesthetized and randomized to receive PCV or PSV. They were then further randomized into subgroups without or with IAH (15 mmHg) and ventilated with PCV or PSV (PEEP = 5cmH2O, driving pressure adjusted to achieve tidal volume = 6mL/kg) for 1h. Six of the 30 rats were used for molecular biology analysis and were not mechanically ventilated. The main outcome was the effect of PCV versus PSV on mRNA expression of interleukin (IL)-6 in lung tissue. Regardless of whether IAH was present, PSV resulted in lower mean airway pressure (with no differences in peak airway or peak and mean transpulmonary pressures) and less mRNA expression of biomarkers associated with lung inflammation (IL-6) and fibrogenesis (type III procollagen) than PCV. In the presence of IAH, PSV improved oxygenation; decreased alveolar collapse, interstitial edema, and diffuse alveolar damage; and increased expression of surfactant protein B as compared to PCV. In this experimental model of mild extrapulmonary ALI associated with IAH, PSV compared to PCV improved lung function and morphology and reduced type 2 epithelial cell damage. BODY.INTRODUCTION: Intra-abdominal hypertension (IAH) is a clinical condition characterized by intra-abdominal pressure (IAP) ≥ 12 mmHg. Among other causes, it may result from sepsis, intra-abdominal bleeding, or trauma, and is associated with worse outcomes in these conditions [1,2,3,4]. IAH is highly prevalent in critically ill patients, affecting up to 64% of this population, and has a major impact on the function of the lungs and peripheral organs [3,5]. In the presence of preexisting alveolar-capillary damage, IAH promotes lung injury [6,7,8], edema, and increased intra-thoracic pressures, leading to atelectasis, airway closure, and deterioration of gas exchange [6]. Controlled mechanical ventilation with low tidal volume and optimization of positive end-expiratory pressure (PEEP), combined with neuromuscular blockade, has been recommended as a strategy to minimize ventilator-induced lung injury (VILI) [4,9]. However, IAH has been shown to potentiate dorsal atelectasis formation [6], and the relaxation of the respiratory muscles during controlled mechanical ventilation allows further cephalad displacement of the diaphragm, predominately in the ventral regions. In experimental acute lung injury (ALI) [10,11], it has been demonstrated that pressure support ventilation (PSV) improves gas exchange and hemodynamics and prevents VILI as compared to controlled mechanical ventilation. On the other hand, PSV may lead to further lung injury if the inspiratory transpulmonary pressure and effort are excessively high [12,13,14]. So far, however, no study has compared the impact of PSV and pressure-controlled ventilation (PCV) on lung damage in experimental ALI with IAH. Within this context, the present study was designed to test the hypothesis that, when delivered at a protective tidal volume, PSV compared to PCV would improve respiratory function, reduce the amount of collapsed areas in the lung, and prevent VILI in experimental extrapulmonary mild ALI with IAH. Part of the results of this study were published previously as an abstract [15]. BODY.MATERIAL AND METHODS: 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 Committee on the Ethics of Animal Experiments of the Health Science Center, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil (CEUA: 019). All efforts were made to minimize suffering. BODY.MATERIAL AND METHODS.ANIMAL PREPARATION AND EXPERIMENTAL PROTOCOL: Thirty Wistar rats (weight 334±55 g) received Escherichia coli O55:B5 lipopolysaccharide (LPS) (Sigma Chemical Co., St. Louis, MO, USA) intraperitoneally (i.p.) at a dose of 1,000 μg, suspended in saline solution to a total volume of 1,000 μL [16,17], to induce mild extrapulmonary ALI. After 24 h, animals were premedicated with 10 mg/kg diazepam i.p. (Compaz, Cristália, Itapira, SP, Brazil), followed by 100 mg/kg ketamine i.p. (Ketamin-S+, Cristália, Itapira, SP, Brazil) and 2 mg/kg midazolam i.p. (Dormicum, União Química, São Paulo, SP, Brazil). Following local anesthesia with 2% lidocaine (0.4 mL), a midline neck incision and tracheostomy were performed. Six of the 30 rats were used for molecular biology analysis and were not mechanically ventilated (non-ventilated, NV). An intravenous (i.v.) catheter (Jelco 24G, Becton, Dickinson and Company, New Jersey, NJ, USA) was inserted into the tail vein, and anesthesia induced and maintained with midazolam (2 mg/kg/h) and ketamine (50 mg/kg/h). Additionally, 10 mL/kg/h Ringer's lactate (B. Braun, Crissier, Switzerland) was administered i.v. in all groups. A second catheter (PE-50, Becton, Dickinson and Company) was then placed in the right internal carotid artery for blood sampling and gas analysis (Radiometer ABL80 FLEX, Copenhagen NV, Denmark), as well as monitoring of mean arterial pressure (MAP) (Networked Multiparameter Veterinary Monitor LifeWindow 6000 V; Digicare Animal Health, Boynton Beach, FL, USA). A 30-cm-long water-filled catheter (PE-205, Becton, Dickinson and Company) with side holes at the tip, connected to a differential pressure transducer (UT-PL-400, SCIREQ, Montreal, QC, Canada), was used to measure the esophageal pressure (Pes). The catheter was passed into the stomach and then slowly returned into the esophagus; its proper positioning was assessed with the "occlusion test" [18]. Heart rate (HR), MAP, intra-abdominal pressure (IAP), and rectal temperature were continuously monitored (Networked Multiparameter Veterinary Monitor LifeWindow 6000V, Digicare Animal Health, Florida, USA). Body temperature was maintained at 37.5±1°C using a heating bed. Colloids (Gelafundin®, B. Braun, São Gonçalo, RJ, Brazil) was administered intravenously (i.v., in 0.5-mL increments) as needed to maintain MAP> 60 mmHg. Animals were mechanically ventilated (Servo-i, MAQUET, Solna, Sweden) in PCV or PSV (flow triggering) with PEEP = 5 cmH2O and FiO2 = 0.4. During PCV, animals were paralyzed with pancuronium bromide (2 mg/kg, i.v.). In both PCV and PSV, the driving pressure was adjusted to achieve VT = 6 mL/kg. Following this step (5 min), animals were randomized to normal intra-abdominal pressure (nIAP) or intra-abdominal hypertension (IAH) subgroups. To induce IAH, a midline laparotomy 3 cm in length was performed to expose the abdominal cavity, and 15-cm hydrophilic gauze packs (Cremer, Blumenau, SC, Brazil) were placed in its four quadrants (one pack per quadrant). A catheter (PE-240) was inserted into the peritoneum for continuous IAP measurement [7,8,19], and a 2–0 silk suture was used to tie the catheter in place and ensure there was no leak. Both layers of the abdominal cavity were closed with 3–0 monofilament nylon suture (Ethilon®, São Paulo, SP, Brazil), which was tightened to maintain an IAP of 15 mmHg [7,8]. IAP was kept at this level throughout the experiment. In the nIAP group, a sham surgery was performed with the same technique used in the IAH group and manipulation of the abdominal cavity for the same amount of time, but no packing. Animals were kept on the same ventilator settings described above (PSV or PCV). Arterial blood gases and respiratory system and lung mechanics were measured immediately after surgery and after 1 h of mechanical ventilation with PSV or PCV (End) (Fig 1). FiO2 was set at 1.0 and, after 5 min, arterial blood (300 μL) was drawn into a heparinized syringe to determine arterial oxygen partial pressure (PaO2), arterial carbon dioxide partial pressure (PaCO2), and arterial pH (pHa) (Radiometer ABL80 FLEX, Copenhagen NV, Denmark). Animals were then killed with sodium thiopental 60 mg/kg i.v., and their lungs extracted at PEEP = 5 cmH2O for lung histology and molecular biology analysis (Fig 1). 10.1371/journal.pone.0178207.g001Fig 1Timeline representation of the experimental protocol.First randomization: pressure-controlled ventilation (PCV) or pressure support ventilation (PSV). Second randomization: intra-abdominal hypertension (IAH) or normal intra-abdominal pressure (nIAP). Start: immediately after surgery (Sham) or IAH induction at PCV or PSV. VT, tidal volume; PEEP, positive-end expiratory pressure; FiO2, fraction of inspired oxygen. Mechanics and arterial blood gases were evaluated at Start and End (after 1 h of mechanical ventilation in PCV or PSV). Airflow, airway pressure (Paw), and esophageal pressure (Pes) were continuously recorded throughout the experiments on a computer running customer-made software written in LabVIEW (National Instruments, Austin, TX) [20,21]. VT was calculated by digital integration of the airflow signal. Transpulmonary pressure (P,L) was calculated during inspiration and expiration as the difference between Paw and Pes. Peak and mean airway pressures (Ppeak,aw and Pmean,aw), transpulmonary pressures (Ppeak,L and Pmean,L), and the esophageal pressure generated 100 ms after onset of inspiratory effort (P0.1) were calculated. The respiratory rate (RR) was calculated from the Pes swings as the frequency per minute of each type of breathing cycle. The pressure–time product (PTP) per breath was calculated as the integral of ΔPes over time [10,11,22,23]. The asynchrony index was calculated as the total number of asynchronous breaths divided by the total number of triggered and untriggered breaths, multiplied by 100 [22]. All signals were amplified in a four-channel signal conditioner (SC-24, SCIREQ, Montreal, QC, Canada), and sampled at 200 Hz with a 12-bit analog-to-digital converter (National Instruments; Austin, Texas, USA). All mechanical data were computed offline by a routine written in MATLAB (Version R2007a; The Mathworks Inc, Natick, Massachusetts, USA). BODY.MATERIAL AND METHODS.HISTOLOGY.LIGHT MICROSCOPY: A laparotomy was performed immediately after blood sampling at the end of experiments. Heparin (1,000 IU) was injected into the tail vein. The trachea was then clamped at end-expiration (PEEP = 5 cmH2O) and the abdominal aorta and vena cava were sectioned, yielding a massive hemorrhage that quickly killed the animals. The lungs were removed en bloc. The left lung was frozen in liquid nitrogen, immersed in Carnoy's solution, embedded in paraffin, cut longitudinally at the level of the central zone into slices of 4 μm thickness, and stained with hematoxylin-eosin for histological analysis [22]. Photomicrographs at magnifications of ×100, ×200 and ×400 were obtained from eight non-overlapping fields of view per section under a light microscope (Olympus BX51, Olympus Latin America-Inc., Brazil). Diffuse alveolar damage (DAD) was quantified using a weighted scoring system by a researcher blinded to the experimental protocol [24]. Briefly, scores of 0 to 4 were used to represent alveolar collapse, interstitial edema, and septal thickening with 0 standing for no effect and 4 for maximum severity. Additionally, the extent of each scored characteristic per field of view was determined on a scale of 0 to 4, with 0 standing for no visible evidence and 4 for complete involvement. Scores were calculated as the product of severity and extent of each feature, and thus ranged from 0 to 16. The cumulative DAD score was calculated as the sum of each score characteristic, and ranged from 0 to 48. Scoring was assessed independently by two co-authors (J.D.S. and C.S.S.) who are experts in lung pathology. Both assessors were blinded to group assignment. The scores of each expert were combined to yield a final score by arithmetic averaging. BODY.MATERIAL AND METHODS.BIOLOGICAL MARKERS OF INFLAMMATION, FIBROGENESIS, ALVEOLAR STRETCH, AND EPITHELIAL AND ENDOTHELIAL CELL DAMAGE: Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to measure biomarkers associated with inflammation (interleukin [IL]-6), fibrogenesis (type III procollagen), alveolar stretch (amphiregulin), type II alveolar cell mechanotransduction (surfactant protein [SP]-B), and endothelial cell injury (vascular cellular adhesion molecule [VCAM]-1). The primers used are described in the online supplement (Additional file 1: S1 Table). Central slices of the right lung were cut, collected in cryotubes, flash-frozen by immersion in liquid nitrogen, and stored at −80°C. Total RNA was extracted from frozen tissues using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), in accordance with the manufacturer's recommendations. RNA concentrations were measured by spectrophotometry in a Nanodrop ND-1000 system (ThermoScientific, Wilmington, DE, USA). First-strand cDNA was synthesized from total RNA using a Quantitec reverse transcription kit (Qiagen, Hilden, Germany). Relative mRNA levels were measured with a SYBR green detection system in an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, California, USA). Samples were run in triplicate. For each sample, the expression of each gene was normalized to the acidic ribosomal phosphoprotein P0 (36B4) housekeeping gene [25] and expressed as fold change relative to respective NV animals, using the 2–ΔΔCt method, where ΔCt = Ct (reference gene)–Ct (target gene) [26]. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: Sample size calculation was based on pilot studies and on previous studies in rodents using similar ventilator settings [22,27]. A sample size of six animals per group would provide the appropriate power (1 − β = 0.8) to identify significant (α = 0.05) differences in the percentage of IL-6 between PCV and PSV, during nIAP, taking into account an effect size d = 1.38, a two-sided test, and a sample size ratio = 1 (G*Power 3.1.9.2, University of Düsseldorf, Düsseldorf, Germany). Data were tested for normality using the Kolmogorov-Smirnov test with Lilliefors' correction, while the Levene median test was used to evaluate the homogeneity of variances. If both conditions were satisfied, the effects of different ventilatory strategies (PCV and PSV) in nIAP and IAH were analyzed by using two-way repeated measures ANOVA followed by Bonferroni's test. One-way ANOVA on ranks followed by Dunn's post-hoc test was employed to compare lung histology and molecular biology data. Parametric data were expressed as mean ± SD, while non-parametric data were expressed as median (interquartile range). The significance level was set at p = 0.05. All tests were performed in GraphPad Prism version 6.01 (GraphPad Software, San Diego, CA). BODY.RESULTS: Thirty animals were used, with 6 animals allocated to each group, including the NV group. No animals died during the experiments. MAP was maintained above 60 mmHg throughout the experiments (Additional file 2: S2 Table). At Start, MAP was higher in PSV than PCV, regardless of nIAP or IAH. At End, the amount of fluid administered was higher in PCV than PSV (p<0.05), both in nIAP and IAH animals (Additional file 2: S2 Table). Oxygenation improved significantly from Start to End in IAH animals ventilated with PSV. There were no significant differences in PaCO2 between PCV and PSV at Start and End, while pHa was lower in PCV than PSV animals (Table 1). 10.1371/journal.pone.0178207.t001 Table 1 Arterial blood gases at Start and End. nIAP IAH Time point PCV PSV PCV PSV PaO 2 /FiO 2 Start 304 ± 174 341 ± 155 280 ± 143 245 ± 146 End 334 ± 115 429 ± 132 329 ± 112 421 ± 92 * pHa Start 7.31 ± 0.08 7.31 ± 0.07 7.31 ± 0.04 7.35 ± 0.09 End 7.22 ± 0.05 * 7.31 ± 0.05 # 7.20 ± 0.06 * 7.30 ± 0.03 # PaCO 2 (mmHg) Start 44.9 ± 15.8 35.2 ± 14.0 46.6 ± 15.7 39.1 ± 12.4 End 43.5 ± 17.5 39.8 ± 11.3 50.5 ± 16.8 47.7 ± 8.5 IAH, intra-abdominal hypertension; nIAP, normal intra-abdominal pressure; PCV, pressure-controlled ventilation; PSV, pressure support ventilation; Start, after sham surgery (nIAP) or IAH induction; End, nIAP or IAH after 1 h mechanical ventilation with PCV or PSV; PaO 2 /FiO 2 , arterial oxygen partial pressure divided by fraction of inspired oxygen; pHa, arterial pH; PaCO 2 , arterial carbon dioxide partial pressure. Comparisons were performed by two-way repeated-measures ANOVA followed by Bonferroni’s post-hoc test. Values are given as mean ± standard deviation of 6 animals/group. *Significantly different from Start (p<0.05). #Significantly different from PCV group at the corresponding time point (p<0.05). At Start, mean VT and RR were similar in all groups. PSV yielded lower Pmean,aw in nIAP and IAH animals compared to PCV. No significant differences were observed in Ppeak,aw, Ppeak,L, Pmean,L, P0.1, PTP, or asynchrony index between PSV and PCV animals in the nIAP or IAH groups (Table 2). 10.1371/journal.pone.0178207.t002 Table 2 Respiratory parameters at Start and End. Parameters Time nIAP IAH PCV PSV PCV PSV V T (mL/kg) Start 6.3 ± 0.6 5.9 ± 0.5 6.0 ± 0.5 5.8 ± 0.4 End 5.9 ± 0.4 6.7 ± 1 5.6 ± 0.5 6.2 ± 0.5 RR (breaths/min) Start 80 ± 0 62 ± 20 79 ± 2 77 ± 20 End 80 ± 0 70 ± 20 80 ± 0 67 ± 30 P peak,aw (cmH 2 O) Start 13 ± 1 14 ± 4 16 ± 2 12 ± 4 End 13 ± 1 13 ± 4 16 ± 2 12 ± 2 P mean,aw (cmH 2 O) Start 8.3 ± 0.7 6.8 ± 0.5# 9.1 ± 1.0 6.4 ± 0.7# End 8.0 ± 0.7 6.8 ± 0.9# 9.2 ± 0.7 6.2 ± 1.0# P peak,L (cmH 2 O) Start 13 ± 1 16 ± 4 13 ± 3 14 ± 3 End 12 ± 0.9 16 ± 5 13 ± 1 14 ± 2 P mean,L (cmH 2 O) Start 7.9 ± 0.7 7.5 ± 0.6 7.4 ± 1 7.4 ± 0.3 End 7.4 ± 0.6 7.5 ± 2.0 7.4 ± 0.8 7.4 ± 1 P 0.1 (cmH 2 O) Start - 0.28 ± 0.6 - 0.81 ± 0.6 End - 0.66 ± 0.4 - 0.94 ± 0.9 PTP (cmH 2 O*s) Start - 2.2 ± 0.7 - 1.4 ± 0.7 End - 2.1 ± 0.9 - 1.5 ± 0.8 Asynchrony index (%) Start - 3.9 ± 2.0 - 3.5 ± 2.0 End - 2.8 ± 2.0 - 1.3 ± 1.0 nIAP, normal intra-abdominal pressure, IAH, intra-abdominal hypertension; PCV, pressure-controlled ventilation; PSV, pressure support ventilation; Start, after sham surgery (nIAP) or IAH induction; End, nIAP or IAH after 1 h mechanical ventilation with PCV or PSV; V T , tidal volume; RR, respiratory rate; Ppeak,aw, peak airway pressure; Pmean,aw, mean airway pressure; Ppeak,L, peak transpulmonary pressure; Pmean,L, mean transpulmonary pressure; P 0.1 , driving pressure; PTP, pressure-time product. Comparisons were performed by two-way repeated-measures ANOVA followed by Bonferroni’s post-hoc test (p < 0.05). Values are given as mean ± standard deviation of 6 animals/group. #Significantly different from PCV group at the corresponding time point (p<0.05). In nIAP animals, no significant differences were observed between PCV and PSV with regard to alveolar collapse, interstitial edema, septal thickening, or DAD score (Additional file 3: S1 Fig, Fig 2). However, in the IAH group, PSV was associated with less alveolar collapse, interstitial edema, and DAD compared to PCV (Fig 2). In the presence of IAH, alveolar collapse, interstitial edema, septal thickening, and DAD score were higher in PCV than NV. 10.1371/journal.pone.0178207.g002Fig 2Cumulative DAD score (scores arithmetically averaged from two independent investigators) representing injury from alveolar collapse, interstitial edema, and septal thickening in animals with normal intra-abdominal pressure (nIAP) or intra-abdominal hypertension (IAH) mechanically ventilated in pressure-controlled ventilation (PCV) or pressure support ventilation (PSV) mode.NV: non-ventilated animals. Values are given as medians, interquartile ranges, and minimum/maximum of 6 animals in each group. Statistical significance was accepted at p < 0.05. *Significantly different from NV. Gene expression of biological markers associated with inflammation (IL-6), fibrogenesis (PCIII), pulmonary stretch (amphiregulin), and type II epithelial cell and endothelial cell damage (SP-B and VCAM-1 respectively) is illustrated in Fig 3. In both nIAP and IAH, IL-6, PCIII, and VCAM-1 expressions were higher in groups ventilated with PCV compared to NV. In nIAP, PCV was associated with higher amphiregulin expression than NV. In IAH, SP-B expression was lower in PCV than NV and higher in PSV than PCV, whereas VCAM-1 expression was higher in PSV than NV. 10.1371/journal.pone.0178207.g003Fig 3Real-time polymerase chain reaction analysis of biological markers associated with inflammation (interleukin [IL]-6), fibrogenesis (type III procollagen [PCIII]), pulmonary stretch (amphiregulin), type II epithelial cell damage (surfactant protein [SP]-B), and endothelial cell damage (vascular cellular adhesion molecule [VCAM-1]) in animals with normal intra-abdominal pressure (nIAP) or intra-abdominal hypertension (IAH) mechanically ventilated in pressure-controlled ventilation (PCV) or pressure support ventilation (PSV) mode.Values are given as medians, interquartile ranges, and minimum/maximum of 6 animals in each group. Relative gene expression was calculated as a ratio of average gene expression compared with the reference gene (36B4) and expressed as fold change relative to non-ventilated (NV) animals. *Significantly different from NV (p<0.05). #Significantly different from PCV (p<0.05). BODY.DISCUSSION: In the rat model of mild extrapulmonary ALI used in this study, we found that, regardless of IAP, PSV resulted in lower Pmean,RS (with no differences in Ppeak,RS, Ppeak,L, or Pmean,L) and decreased mRNA expression of biomarkers associated with inflammation and fibrogenesis as compared with PCV. Specifically, in the presence of IAH, PSV improved oxygenation, increased expression of surfactant protein (SP)-B, and was associated with less alveolar collapse, interstitial edema, and alveolar damage than PCV. To the best of our knowledge, this is the first study investigating the impact of PSV versus PCV on VILI in experimental ALI with IAH. The model of extrapulmonary ALI induced by E. coli LPS was chosen for this study because, in the clinical setting, IAH is most often associated with abdominal sepsis. Accordingly, 24 h after LPS administration, changes in lung histology, alterations in the alveolar-capillary barrier, inflammation, and physiologic dysfunction were observed, reproducing several of the main features of human ARDS [28]. In addition, at this time point, IAH was induced to simulate the course of increased IAP in critically ill patients with a model of extrapulmonary ALI [17,29]. IAH was induced by inserting gauze packs into the abdominal cavity until an IAP of 15 mmHg was achieved [7,8,19]. According to WSACS recommendations [30], IAH is defined as an IAP higher than 12 mmHg, and the average IAP in patients undergoing mechanical ventilation is 15 mmHg. Other methods used to mimic IAH, such as CO2/air inflation [31,32,33] and intraperitoneal fluid infusion [34], may interfere with the pathophysiological response to IAH or represent an additional physiologic variable altering the body's response to IAH. The advantage of our IAH model was the maintenance of a high IAP without any side effects related to gas inflation of the peritoneal cavity. In both PCV and PSV mode, animals were ventilated with a protective VT (6 mL/kg) and PEEP = 5 cmH2O. In the presence of IAH, increased PEEP may have a deleterious impact on hemodynamics and may increase fluid and/or vasoactive drug requirements, with no effects on lung injury [8]. We chose to analyze mRNA expression of biomarkers associated with inflammation (IL-6), type II epithelial cell damage (SP-B), and endothelial cell damage (VCAM-1) in the lung because of the role of these substances as mediators in the pathogenesis of VILI [35,36]. The expression of PCIII mRNA in lung tissue was also evaluated because it this the first form of collagen to be remodeled in the course of lung fibrogenesis, and is an early marker of lung parenchymal remodeling [37]. Amphiregulin, in turn, was measured because its expression is positively modulated by hyperinflation, it activates chemokines, cytokines, and adhesion molecules, and represents a novel candidate gene in VILI [20,38]. IAH is a primary cause of organ dysfunction and abdominal compartment syndrome (ACS), a clinical entity which carries a high mortality rate and requires urgent, targeted intervention [39,40]. Over the years, several strategies have been developed to attempt to mitigate IAH and prevent its progression to ACS [41]. IAH does not only affect abdominal organs, but also has a great impact on respiratory function [2], with major practical consequences for mechanically ventilated patients [42]; high IAP makes it particularly difficult to satisfy the mandates of lung-protective mechanical ventilation while providing adequate oxygenation. The optimal mechanical ventilation settings for ARDS with IAH have yet to be determined. A protective ventilation strategy with a low VT (6 mL/kg ideal body weight) and an airway plateau pressure of < 30 cm H2O has been demonstrated to improve survival in patients with ARDS [9]. Optimization of mechanical ventilation and recruitment [4], combined with neuromuscular blockade, has been proposed as a strategy to reduce IAP in patients with IAH or ACS [43]. Research suggests the optimal ventilator management of patients with ARDS and IAH should include the following: (a) monitoring of IAP, Pes, and hemodynamic parameters; (b) ventilation with protective VT, recruitment maneuvers, and PEEP set according to the "best" compliance of the respiratory system or lung; (c) deep sedation (with or without neuromuscular blockade in severe ARDS); and (d) an open abdomen in selected patients with severe ACS [44]. Previous experimental studies investigating the optimization of mechanical ventilation during IAH were performed with controlled mechanical ventilation and mainly focused on respiratory mechanics, partitioned into its lung and chest wall components, and/or gas exchange in healthy and diseased animals [31,32]; others yet focused on hemodynamics [45,46]. Assisted mechanical ventilation has been proposed as a potential alternative to controlled mechanical ventilation, with advantages of better alveolar recruitment and gas exchange with less hemodynamic impairment, muscle atrophy, and lung injury in experimental ARDS [10,11,47,48,49]. However, in severe ARDS, high spontaneous inspiratory effort may also lead to increased lung injury due to abnormally high inspiratory activity and transpulmonary pressure [12,13]. Our data suggest that, in the presence of mild extrapulmonary ARDS with IAH, PSV with moderate PEEP reduced atelectasis, likely due to moderate activation of respiratory muscles (and, in particular, the diaphragm) [50,51,52]. This reduction in atelectasis was associated with oxygenation improvement from Start to End in the PSV group, probably due to increased blood flow distribution [53]. However, the increase in transpulmonary pressure associated with PSV may also result in further lung damage [14,54,55], as lungs with pre-existing damage are more susceptible to increased stress, and regional changes in transpulmonary pressure may be associated with pendelluft (movement of air from more recruited regions to less recruited regions during early inspiration without a gain in tidal volume) [56]. Additionally, negative pleural pressures have been shown to yield negative alveolar pressures and increased vascular pressure, thus worsening lung edema [55]. In the presence of IAH, we cannot rule out that the beneficial effects of PSV on lung histology, decreasing DAD, atelectasis, and interstitial edema were associated with the reduced amount of fluids administered during mechanical ventilation [57]. The reduction in atelectasis was not associated with a decrease in Ppeak,L, which suggests that transpulmonary pressure might not be the only parameter influencing alveolar recruitment. Our data suggest that, in the presence of IAH, transpulmonary pressure is more effective to reduce atelectasis when induced by decreased pleural pressure than by increased airway pressure [58]. Even though DAD score was similar in the PCV and PSV groups in nIAP, biological markers differed according to mechanical ventilation strategy. In both the IAH and the nIAP groups, PSV was also associated with decreased mRNA expression of biological lung tissue markers associated with inflammation and fibrogenesis compared to PCV, while in IAH specifically, SP-B expression was higher with PSV than with PCV, thus suggesting less type II epithelial cell damage. The main mechanisms leading to lung injury may be associated to [10]: 1) peak airway and transpulmonary pressure (i.e., stress); 2) respiratory rate and minute ventilation; 3) shear stress due to continuous opening and closing of collapsed alveoli during tidal breathing; 4) mean airway and transpulmonary pressures (i.e., static strain); 5) regional stress and strain (transpulmonary pressures); and 6) redistribution of perfusion from collapsed towards aerated lung regions. In the present study, total inspiratory stress, respiratory rate, and minute ventilation were comparable between PSV and PCV. However, different distributions of forces leading to the same transpulmonary stress pressure can play a role. PSV only reduced atelectasis in the IAH group; thus, shear stress may not fully explain the reduction in lung injury observed during assisted ventilation. Mean airway pressure was lower during PSV both with and without IAH. The present study thus suggests that reductions in static stress and strain may markedly affect lung injury in the context of PSV [59]. Finally, we cannot exclude that redistribution in perfusion from collapsed towards aerated lung regions, which likely occurred at least in the IAH group, might have contributed partly to reductions in lung injury. Although VCAM-1 mRNA expression was increased in animals ventilated with PCV (in both nIAP and IAH) and PSV (in IAH) compared to NV, interstitial edema was more pronounced in PCV than in either NV or PSV in IAH. This apparent dissociation between morphological and molecular data may be attributed to the fact that VCAM-1 was evaluated at the RNA level instead of the protein level, and it takes time to observe the consequences of endothelial dysfunction in lung morphology. Moreover, interstitial edema is associated not only with endothelial cell dysfunction but also with lung perfusion distribution, which may differ according to mechanical ventilation strategy [60] and to inspiratory effort during PSV [54]. PSV was associated with asynchrony indices of 3–4%, suggesting that, within a minimal threshold limit as recommended (10%) [23,61], asynchronies do not seem to play a relevant role in promotion of lung injury. In line with our results, previous experimental studies using mild ARDS models without IAH reported a reduction in lung injury when assisted ventilation was compared to PCV [10,11,49]. In contrast, a previous study in healthy pigs with IAH at 30 mmHg reported greater histopathological damage to the lungs with assisted ventilation than with controlled mechanical ventilation [62]. BODY.DISCUSSION.LIMITATIONS: This study has several limitations. First, we used a specific model of mild extrapulmonary ALI induced by intraperitoneal endotoxin injection. Thus, our results may not be extrapolated to other ALI models in small or large animals, nor to severe ALI. Second, we did not assess possible long-term effects of PSV, nor did we assess other types of assisted ventilation in the setting of IAH. Technical limitations include the fact that a specific level of IAH (15 mmHg) was used, and that mediators were measured in lung tissue, but not in blood. Third, higher IAP may lead to increased pulmonary artery pressure (PAP) [63,64]. Pulmonary arterial pressure was not measured due to technical difficulties faced when inserting catheter into pulmonary artery while keeping the animal alive without hindering our primary hypothesis (PSV versus PCV in ALI with IAH). Future studies are required to investigate the cardiopulmonary interaction during IAH using different ventilation strategies. Fourth, even though it is important to exclude procedure-related issues, we were unable to keep ALI animals with IAH alive during 1 hour spontaneous breathing. Lastly, since the observation time was relatively short (1 h mechanical ventilation), the expression of mediators was quantified using RT-PCR instead of ELISA. It is well known that 1 h is sufficient time to produce changes in mRNA expression, but not to significantly change levels of protein [7,8,10,11,20]. Keeping small ALI animals alive in the presence of increased IAH and mechanical ventilation (PCV or PSV) during longer periods of time would have required higher amounts of fluids, occasional use of vasoactive drugs to maintain MAP > 60 mmHg, and bicarbonate for metabolic acidosis. All these therapeutic strategies might have interfered with individual gene activation, thus hindering assessment of our primary hypothesis. BODY.CONCLUSION: In the model of mild ALI with IAH used in this study, PSV was associated with less atelectasis, interstitial edema, diffuse alveolar damage, and biological markers of inflammation, fibrogenesis, and type II epithelial cell damage than PCV. BODY.SUPPORTING INFORMATION: S1 TableForward and reverse oligonucleotide sequences of target gene primers.Primers used in experiments. IL-6, interleukin-6; PCIII, pro-collagen III; SP-B, surfactant protein B; VCAM-1, vascular cell adhesion molecule-1; 36B4, acidic ribosomal phosphoprotein P0.(DOCX)Click here for additional data file. S2 TableAmount of fluids administered and mean arterial pressure.(DOCX)Click here for additional data file. S1 FigPhotomicrographs of lung parenchyma stained with hematoxylin-eosin.Original magnification ×200. Arrows: alveolar collapse. AD: alveolar duct. nIAP: animals with normal intra-abdominal pressure. IAH: animals with intra-abdominal hypertension. PCV: mechanically ventilated in pressure-controlled ventilation. PSV: mechanically ventilated with pressure support ventilation. NV: non-ventilated animals.(DOCX)Click here for additional data file.

TITLE: Comparison of paracetamol, ibuprofen, and diclofenac potassium for pain relief following dental extractions and deep cavity preparations ABSTRACT.OBJECTIVES:: To compare the effectiveness of different oral analgesics for relieving pain and distress in adults following the extraction of teeth and deep cavity preparations under local anesthesia. ABSTRACT.METHODS:: This randomized controlled study was conducted between November 2015 and May 2016. One hundred and twenty patients were randomly allocated to 3 groups. Forty patients were in the paracetamol (1 gram) group, 40 in the ibuprofen (400 mg) group and 40 in the diclofenac potassium (50 mg) group. Evaluation of the post extraction and deep cavity preparations pain was made by patients immediately postoperatively, 2, 4 and 6 hours postoperatively on standard 100 mm visual analogue scales (VAS). Furthermore, each patient was observed preoperatively and immediately postoperatively for signs of distress by using a 5 point face scale. ABSTRACT.RESULTS:: There were significant decreases in mean pain VAS scores for diclofenac potassium group compared to paracetamol and ibuprofen groups at 4 hours postoperatively (one-way Analysis of Variance: p=0.0001, p=0.001) and 6 hours postoperatively (p=0.04, p=0.005). Changes in distress scores from the preoperative score to the postoperative score were made using the paired sample t-test. There were significant decreases in distress scores between the preoperative and postoperative scores (p=0.0001). ABSTRACT.CONCLUSIONS:: Diclofenac potassium was more effective than paracetamol or ibuprofen for reducing postoperative pain associated with tooth extraction and deep cavity preparation. Patients' distress levels can be alleviated by using preemptive analgesics. BODY: It was well documented that the postoperative pain associated with surgical tooth extractions can range in intensity from moderate to severe during the first 24 hours.1-4 Pain intensity reaches its maximum peak between 6 and 8 hours when a conventional local anesthetic is used.5 Surgical dental extraction and deep cavity preparation are usually associated with a trauma to both the soft and hard tissues. This trauma is often accompanied with pain and swelling.6,7 Nonsteroidal anti-inflammatory drugs are one of the treatment options to be used as pain relief for surgical teeth extraction and cavities preprations.4,8 By administering the pre-operative analgesics, the postoperative pain intensity can be subsided and delayed as a result of the reduction in the amount of pain triggers (prostaglandins) discharged into the site of the injuries.9-11 Accumulation of prostaglandins released from the injured tissues increased by the time leading to the amplification of the pain intensity.5,9-11 Diclofenac works by blocking the chemical substances called cyclo-oxygenase (COX) enzymes. These enzymes trigger the information of prostaglandins in the body. Sites of injury or harm are considered the normal place for production of the prostaglandins, which cause pain and inflammation. By obstructing the influence of COX enzymes, a smaller amount of prostaglandins are formed and as a consequence less pain and inflammation are felt.12 There are 2 formulas of diclofenac in the market; diclofenac sodium and diclofenac potassium. The absorption of diclofenac potassium to patient's blood stream is faster than diclofenac sodium.7 As a rule, the earlier the analgesic absorbed the quicker the onset of action will be. So, diclofenac potassium is a fast acting analgesic and useful for the patients who are required immediate relief from inflammatory pain.12 A study by Ferraiolo and Veitz-Keenan13 was conducted to compare the effectiveness of different types of analgesics following third molar extractions. Data regarding the level of pain relief and the need for additional analgesics were collected at 6 hours following the surgery. The outcome of this study revealed that the ibuprofen was more effective than paracetamol at doses of 200 mg to 512 mg and 600 mg to 1000 mg, respectively. Krishnan et al14 carried out a study on 40 healthy patients aged from 18-50 years with deeply carious, lower molars teeth indicated for extraction under local anesthesia (LA). Subjects were divided into 2 groups. First group received transdermal diclofenac patches whilst second group received oral diclofenac for control of post-extraction pain. The pilot study outcome revealed that the effectiveness of transdermal diclofenac was similar to oral diclofenac in reducing post-extraction pain. Bauer et al15 study concluded that the preemptive analgesia with only ibuprofen is insufficient to prevent pain in third molar surgery. However, the combination of ibuprofen with dexamethasone was more effective in inhibiting central sensitization. The aim of this study was to investigate the effectiveness of 50 mg diclofenac potassium compared with 400 mg ibuprofen, and 1 g paracetamol, on post extraction and deep cavity preparation pain in a double-blind randomized controlled trial. The null hypothesis was oral administrations of diclofenac potassium, ibuprofen and paracetamol are equally effective in reducing post-surgical teeth extraction and deep cavity preparation pain. BODY.METHODS: This randomized controlled study was conducted between November 2015 and May 2016. The design and performance of this clinical study was carried out in accordance with the principles of Helsinki Declaration. Taibah Dental School Research Ethics Committee had approved the study. Written consent had been obtained from 125 patients who attended the Oral and Maxillofacial Department. Inclusion criteria for enrolling patients in this study were American Society of Anesthesiologists (ASA) class I or II patients who were healthy, or with mild systemic disease and had no risk from administration of LA with adrenaline; aged 16-70 years; presenting for elective one simple tooth extraction or one deep cavity restoration with the maximum of 2 cartridges of LA. Exclusion criteria involved patients who were sensitive to ibuprofen, declofenac potassium, or paracetamol; having surgical or multiple teeth extractions; having teeth with reversible pulpitis; having history of active peptic ulcer, attack of asthma, angioedema, aurticaria or acute rhinitis and acute porphyria. Slips of paper were labeled with 1g paracetamol (control group), 400 mg ibuprofen, or 50 mg emifenac using computer generated random number and placed in sequentially numbered envelops. The secretary of the clinic who was not associated with the study did this work. When all the screening procedures had completed and the eligibility of the patient had confirmed; the patient was allocated the next numbered envelope. This was opened The dental assistant not associated with the study opened the envelope and named it analgesic on the slip of the paper and handed it over to the patient. The slip was placed back into the envelope, and put back into the patient's records. To ensure that both the patients and investigator were blinded to the study group assignment. Patients were pre-medicated with paracetamol (1 g), ibuprofen (400 mg) or diclofenac potassium (50 mg) at least 15 minutes before administering the local anaesthetic for extractions or deep cavity preparations (Figure 1). The paracetamol, diclofenac potassium and ibuprofen doses used for this study were chosen according to manufacturer's recommendation and set at such a level as to keep the minimum side effects.16-19 Local anesthesia (1.8 ml mepivacaine 2% with epinephrine 1:100,000) was administered to the patient after sitting on dental chair. For upper teeth, patients were given buccal infiltrations and for lower teeth, either buccal infiltrations or inferior alveolar nerve block (IANB). Standard deep cavity preparation diamond bur is used and for extraction elevators and dental forceps were employed. The researcher was just an observer. Each patient was observed for signs of distress and these were recorded using Faces Distress Scale. This scale was developed by the first author (Giath Gazal) who evolved the Modified Smiley Faces Scale.20 Figure 2 shows the evaluation of distress, which was measured immediately after pre-operative, and postoperative extraction or deep cavity preparation. The distress scores were rated as; score 0 representing "no distress" to score 4 representing "very severe distress" whilst the scores in between representing mild, moderate, and severe distress. Pain assessment was carried out postoperatively, 2 hours postoperatively, 4 hours postoperatively and 6 hours postoperatively by independent assessor. Pain scores were recorded by asking the patients directly after each treatment on standard 100 mm visual analogue scales (VAS), tagged at the endpoints with "no pain" (0 mm) and "unbearable pain" (100 mm). However, pain assessment after 2, 4, and 6 hours postoperatively, were carried out through the phone call. Any extra analgesic was reported during the first 6 hours following the extraction. The pain and distress observation was measured by a trained and completely independent of the whole process. Figure 1Description of 120 patients with 40 in the normal dose of 1 gram of paracetamol (control group), 40 in the normal dose of 400 mg of ibuprofen, and 40 in the normal dose of 50 mg of diclofenac potassium. Figure 2Evaluation of distress measured immediately after pre-operative, and postoperative extraction or deep cavity preparation using the Giath distress scale. BODY.METHODS.STATISTICAL ANALYSIS: Sample size calculation of this study was based on the study by Gazal et al.21 Forty patients in each group would have 90% power to detect a difference in means of 0.90 for both pain and distress scores. A total of 130 patients were recruited for this study. Statistical analysis was performed using a software package (SPSS; version 20, SPSS Inc., Chicago, IL). These statistical tests were independent sample t-test, one-way Analysis of Variance (ANOVA), and paired sample t-test. BODY.RESULTS: Of the 130 recruited, 10 patients were excluded by the dental surgeon as they were considered unsuitable for including in this study (5 had LA overdoses, 2 fainted after local anesthetic injection, 2 of their teeth extraction was carried out surgically and one refused extraction after local anesthetic injection). The final sample size therefore comprised 120 patients with 40 in the normal dose of 1 gram of paracetamol (control group), 40 in the normal dose of 400 mg of ibuprofen, and 40 in the normal dose of 50 mg of diclofenac potassium (Figure 1). It was considered appropriate to use parametric tests to analyze the data due to the distribution of the data and the large numbers in each group. The mean number of teeth extracted per patient was 1 and the mean age was 40.7 years. Immediately postoperatively, patients were still under local anesthetic effects for that reason their pain intensity was very mild or absent. There were no statistically significant differences between the mean pain scores for the paracetamol, ibuprofen, and diclofenac potassium groups post-operatively (p-values from one-way ANOVA: 1.00, 1.00, and 1.00). For both paracetamol and diclofenac potassium groups at 2 hours postoperatively, there were no significant decreases in mean pain scores compared with the ibuprofen group (p-values from one-way ANOVA: 1.000, 0.06, Table 1). However, for diclofenac potassium group there were statistically significant decreases in the mean pain scores at 2 hours postoperative comparing with the paracetamol (control group) (p-values from one-way ANOVA: 0.008, Table 1). At 4 and 6 hours postoperatively, it was found that patients in the diclofenac potassium group recorded lower pain scores than patients in the paracetamol and ibuprofen groups. These differences were statistically significant (Table 1). However, there were no significant differences between the mean pain scores for the paracetamol and ibuprofen groups 4 and 6 hours post-operatively (p-values from one-way ANOVA: 1.00, and 1.00, Table 1). Clinically, all groups showed reduction in maximum and medium pain intensity scores and pain frequency. However, the extent of reduction was greater in the diclofenac potassium group. Table 1 Comparison between mean pain scores for the paracetamol, ibuprofen, and diclofenac potassium groups 2, 4 and 6 hours post operatively. The number of patients who requested rescue analgesia was 32 (n=14 [35%] in the paracetamol group, n=14 [35%]) in the ibuprofen group, and 4 [10%] in the diclofenac potassium group, Table 2). Table 2 A summary of the number of patients who requested rescue analgesics in paracetamol, ibuprofen, and diclofenac potassium groups postoperatively. For all the groups, ibuprofen, diclofenac potassium and control [paracetamol] changes in distress scores from the preoperative score to the post-operative score were made using the paired sample t-test. There were significant decreases in distress scores between the pre-operative and post-operative scores (p=0.0001, Table 3). Patients' distress levels eased off automatically once the dental treatment has completely finished. Table 3 Comparisons between mean distress scores for the patients in the preoperative and postoperative groups. BODY.DISCUSSION: Clinically, the findings of this study revealed that all the 3 preemptive analgesics (paracetamol at dosage of 1 g, ibuprofen at dosage of 400 mg and diclofenac potassium at dosage of 50 mg) reduced the post extraction and deep cavity preparation pain intensity and the need for rescue analgesic. The mean pain scores recorded by patients in all the groups on standard 100 mm visual analogue scales (VAS) were respectively (10.8) at 2 hours postoperatively, (19.5) at 4 hours postoperatively and (21.2) at 6 hours postoperatively. However, the result of this study also showed that the diclofenac potassium was statistically stronger than paracetamol and ibuprofen in terms of reduction of pain intensity and frequency. Taking a single dose of 50 mg diclofenac potassium preoperatively is sufficient to achieve reliable pain control following exodontia and deep cavities preparations. In the present study, there were only 4 (10%) patients in diclofenac potassium group who requested additional analgesics postoperatively. In contrast, there were 14 (35%) patients with paracetamol and 14 (35%) patients with ibuprofen groups who required supplementary rescue analgesia. Interestingly, preemptive analgesics resulted in increased time to first rescue analgesic request. Out of 4 patients who request rescue analgesics in the diclofenac potassium group, 3 were at 6 hours postoperatively. However, there were 16 patients in both paracetamol and ibuprofen groups who asked for additional analgesics at 6 hours postoperatively. There are 2 possible explanations for recommending the use of diclofenac potassium over both the paracetamol and ibuprofen regimes. First, the diclofenac potassium (dissolvable tablets of Emifenac 50 mg) used in this study has rapid onset of action due to its rate of absorption into the body was fast.2,3,12 Early pain relief and function after teeth extraction and deep cavity preparations were achieved. Derry et al's22 study compare the effectiveness of 2 different formulations of a single dose of oral diclofenac used for acute postoperative pain management in adults. The findings of this study revealed that oral diclofenac potassium was significantly more effective than diclofenac sodium for relief moderate to severe postoperative pain. The number of patients who experienced 50% pain relief over 4-6 hours postoperatively in the diclofenac potassium group was more than those having the diclofenac sodium. A double blind placebo controlled trial conducted by Yue et al23 to investigate the efficacy and speed of action of different strengths of normal and fast-dissolving paracetamol on postsurgical dental pain. The outcome of this study demonstrated that the fast dissolving paracetamol was significantly more effective than placebo. Patients in the fast dissolving paracetamol 1000 mg group reported less postoperative pain and increased time to first rescue analgesics than patients in either the normal dose of paracetamol 500 mg or fast dissolving paracetamol 500 mg tablets groups. The current study result is similar to the findings from the study by Derry et al,24 who reported that the clinical advantages for using fast-dissolving and absorbed diclofenac potassium is including better pain relief than those that are absorbed slowly. They concluded that diclofenac potassium at 50 mg single dose is considered as a good pain relief for moderate postoperative pain in adults. A Cochrane overview was conducted by Moore et al25 to summarize the efficacy of a single dose of oral analgesics for postoperative acute pain relief in adults. Thirty-nine Cochrane reviews of randomized trials have examined the analgesic efficacy of individual drug interventions in acute postoperative pain. The results of this Cochrane study confirmed that the use of single dose of analgesics is sufficient to achieve good and long lasting pain relief at relatively low doses. This can be achieved by using fast acting formulations and fixed dose combinations of analgesics. The second possible account for the superiority of diclofenac potassium over ibuprofen and paracetamol could be as a result of the slight differences in their mechanism of actions. Both diclofenac potassium and ibuprofen have similarity in the mode of action because they exert their efficacy by blocking the effect of COX enzymes. Less prostaglandins are produced.12,26 Diclofenac potassium is derivative from acetic acid and ibuprofen is derivative from propionic acid. Peripherally, both diclofenac potassium and ibuprofen Inhibit the construction of the prostaglandin particularly PGE2 at peripheral pain receptor.27,28 The PGE2 enhances the pain receptor's sensitivity to algesic substances such as bradykinin and substance P.29 Centrally, prostaglandins act in the spinal cord and higher centers to promote the transmission of pain signals to the brain.30 A study by Silva et al,31 was carried out to investigate the effectiveness of ibuprofen and etodolac for controlling pain, swelling, and trismus following surgical removal of the lower third molars. The result of this study reported that the patients in the etodolac group recorded lower intensity of postoperative pain, swelling, and trismus than those having oral dose of ibuprofen. The great ability of Etodolac bound to plasma proteins (99%) justifies its potency over the ibuprofen.32 This study demonstrated the effectiveness of diclofenac potassium over ibuprofen which could be as a result of the differences in the chemical properties rather than the mode of action. Diclofenac potassium 50 mg tablets used in this study was faster in dissolving and absorbing than ibuprofen 400 mg tablets. So, a rapid onset and longer duration of action was achieved by using diclofenac potassium. Patients in diclofenac potassium group had less pain at 4 hours and 6 hours postoperatively, and required less rescue analgesic than patients in the paracetamol group. Differences between diclofenac potassium and paracetamol in reducing postoperative pain intensity due to their mode of actions. There is considerable evidence that the antipyretic effect of paracetamol is centrally by inhibiting of prostaglandin E synthesis within the hypothalamus.33 However, the analgesic effect of paracetamol is peripherally by blocking impulse generation within the bradykinin sensitive chemoreceptors.34,35 The findings of this study are consistent with the results of another 2 studies. El Batawi36 administered one hour preoperatively a paracetamol and diclofenac sodium for children with traumatic dental treatments under general anesthesia. El Batawi's36 reported that the diclofenac sodium was more effective than paracetamol for pain relief postoperatively. Another study carried out by Eslampour et al,37 who compared the effectiveness of 3 analgesic drugs, which were administrated preoperatively for reducing postoperative pain associated with photorefractive keratectomy. Their findings revealed that the patients in diclofenac group reported less pain than patients in paracetamol and ibuprofen groups. These results also exposed that patients who showed greater levels of distress preoperatively, showed significantly lower levels of distress after the dental extraction and deep cavity preparations. There was a very interesting point to show in this study, that distress scores were influenced by the lower levels of pain intensity which the patients recorded at the postoperative stage of the assessment. Literatures have provided evidences that the level of patient's distress is determined as a combination of 2 factors: pain and anxiety.38-41 In this study by using a single dose of pre-emptive analgesics, postoperative pain intensity was eased off and as a consequence a reduction in patient's level of distress was achieved. This study has highlighted a new area for further research. These include investigating if there are analgesics effects of paracetamol, ibuprofen, and diclofenac on the level of cortisone and adrenaline in the patients who showed high and lower scores of pain and distress. Do analgesics exert effect on the level of cortisone and adrenaline in the blood? In conclusion, this study has shown that diclofenac potassium was more effective than paracetamol or ibuprofen for postoperative analgesia in adults who are having teeth extracted and deep cavities prepared under local anesthesia. Patients' distress levels can be alleviated by using preemptive analgesics. Preemptive analgesics play an important role in reducing postoperative pain and distress associated with painful dental procedures under LA. Using diclofenac potassium could be of great help to patients who are in moderate to severe pain. So it is strongly recommended for all dental surgeons and practitioners in the Saudi Arabia to administer diclofenac potassium preoperatively for their patients with traumatic dental treatments. In this study, there were no female patients participation because in Taibah University College of Dentistry, only male patients are treated. Therefore, the sample size with male and female patients might have more valid conclusion.

TITLE: Improvement of postoperative cognitive dysfunction and attention network function of patients with ischemic cerebrovascular disease via dexmedetomidine ABSTRACT: The protective effect of dexmedetomidine on cognitive dysfunction and decreased attention network function of patients with ischemic cerebrovascular disease after stenting was investigated. Fifty-eight patients with ischemic cerebrovascular disease undergoing stenting in Guizhou Provincial People's Hospital were selected and randomly divided into control group (n=29) and dexmedetomidine group (n=29). The dexmedetomidine group was treated with dexmedetomidine before induced anesthesia, while the control group was given the same dose of normal saline; and the normal volunteers of the same age were selected as the normal group (n=29). At 3 days after operation, the levels of serum S100B and nerve growth factor (NGF) in each group were detected using the enzyme-linked immunosorbent assay, and the level of brain-derived neurotrophic factor (BDNF) was detected via western blotting. Montreal cognitive assessment (MoCA) and attention network test (ANT) were performed. Moreover, the cognitive function and attention network function, and the effects of dexmedetomidine on cognitive function and attention network function were evaluated. The concentrations of serum S100B and NGF in dexmedetomidine group was lower than those in control group (P<0.01). The results of western blotting showed that the levels of serum BDNF in control group and dexmedetomidine group were significantly lower than that in normal group (P<0.01), and it was higher in dexmedetomidine group than that in control group (P<0.01). Besides, both MoCA and ANT results revealed that the visual space and executive function scores, attention scores, delayed memory scores, targeted network efficiency and executive control network efficiency in dexmedetomidine group were obviously higher than those in control group (P<0.01). The cognitive function and attention network function of patients with ischemic cerebrovascular disease have a certain degree of damage, and the preoperative administration of dexmedetomidine can effectively improve the patient's cognitive dysfunction and attention network function after operation. BODY.INTRODUCTION: Ischemic cerebrovascular disease can lead to hemiplegia or even death. According to epidemiological data, ischemic cerebrovascular disease ranks third in the diseases causing the death of the elderly, seriously affecting the life quality and life health of patients (1,2). Clinically, the ischemic cerebrovascular disease is often treated with surgery supplemented with drug therapy, and a large number of studies have shown that patients with ischemic cerebrovascular disease will suffer from vascular cognitive dysfunction; reducing the thrombosis via early diagnosis can effectively prevent the occurrence of vascular cognitive dysfunction (3–5). Stenting can be effective in the treatment of ischemic cerebrovascular disease, which is widely used in clinic due to its definite efficacy and small trauma (6). Stenting for patients with ischemic cerebrovascular disease may lower the risk of vascular cognitive dysfunction of patients. However, El Hammi et al (7) found that the cognitive function of patients with ischemic cerebrovascular disease treated with stenting still has a certain degree of cognitive dysfunction and damage to attention network function compared with normal people. Li and Liu (8) found through detecting the levels of serum S100B, nerve growth factor (NGF) and brain-derived neotrophic factor (BDNF) that dexmedetomidine provides significant improvement on the recovery of cognitive function of patients receiving epilepsy foci resection, and S100B, NGF and BDNF are highly sensitive and can accurately predict the patient's brain damage. The effects of dexmedetomidine on recovery of cognitive function and attention network function of patients with ischemic cerebrovascular disease have not been studied. Therefore, in this study, the cognitive function and attention network function of patients with ischemic cerebrovascular disease after operation were studied, expecting to clarify the effects of dexmedetomidine on the cognitive dysfunction and attention network function of patients with ischemic cerebrovascular disease, and to provide a theoretical basis and related guidance for the recovery of patients with ischemic cerebrovascular disease. BODY.PATIENTS AND METHODS.PATIENTS OF THE STUDY: The samples in this study were from patients diagnosed as ischemic cerebrovascular disease by deputy director and above in Guizhou Provincial People's Hospital from March 2015 to September 2016. Inclusion criteria: 1) patients diagnosed as ischemic cerebrovascular disease via head CT or MRI; 2) patients without obvious hemiplegia, with normal language skills; 3) patients who could complete the cognitive and attention network function tests; 4) patients who agreed to receive stenting, signed the informed consent and were willing to participate in this experimental study. Fifty-eight patients meeting inclusion criteria were selected, and they were aged 48–73 years, including 31 males and 27 females. Other wasting diseases were excluded from patients enrolled, and the clinical and pathological data and complete treatment program of the above patients in the treatment process were retained. The study was approved by the Ethics Committee of Guizhou Provincial People's Hospital. BODY.PATIENTS AND METHODS.EXPERIMENTAL GROUPING: The above patients selected were randomly divided into control group (n=29) and dexmedetomidine group (n=29). The dexmedetomidine group was treated with intravenous administration of 1 μg/kg dexmedetomidine using a micro-injection pump before induced anesthesia, while the control group was given the same dose of normal saline. Other operative procedures and treatment regimens were the same, and the normal volunteers at the same age were selected in the same period as the normal group; the cognitive function and attention network function of patients were analyzed. BODY.PATIENTS AND METHODS.DETECTION OF SERUM S100B AND NGF LEVELS VIA ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): The peripheral venous blood was drawn at 3, 7 and 30 days after operation, and 2 ml blood was collected after separation of serum to detect the levels of serum S100B and NGF strictly according to the S100B and NGF ELISA kit (Boster Biological Technology Co. Ltd., Wuhan, China). The remaining serum was stored at −80°C for subsequent experiments. BODY.PATIENTS AND METHODS.SERUM BDNF LEVEL: Serum specimens stored at −80°C and collected from each group of patients were thawed and the total protein was extracted after addition of RIPA lysate (1:1; Beyotime Biotechnology Co., Ltd., Shanghai, China), the protein content of each group was detected using the BCA protein quantification kit (Thermo Fisher Scientific, Waltham, MA, USA), and the same concentration of loading sample was prepared, followed by preparation of 10% sodium dodecyl sulfate-polyacrylamide separation gel and spacer gel and electrophoresis separation. Then the protein was transferred onto the PVD membrane and sealed in 5% skimmed milk powder for 1 h. The target bands were cut and incubated with BDNF and GAPDH antibodies (diluted at 1:1,000; cat. nos. SAB1405514 and SAB1405848; Sigma) at 4°C overnight. The bands were washed with Tris-buffered saline Tween (TBST) 3 times (10 min each time), and the rabbit anti-mouse secondary polyclonal antibody (diluted at 1:5,000; cat. no. SAB3701038; Sigma) were incubated at room temperature for 2 h. After that, bands were washed with TBST again 3 times (10 min each time). The DAB coloring solution was added in the dark room for development and fixation, and the gray value of band was analyzed using the gel imaging system. The BDNF/GAPDH ratio indicated the expression level of BDNF protein in serum. BODY.PATIENTS AND METHODS.MONTREAL COGNITIVE ASSESSMENT (MOCA): The cognitive function was evaluated via MoCA in an independent and quiet environment, including visual space and executive function: 1 point, the patients can describe the profile of clock, record the number order and position, and identify the distinction between the hour hand and the minute hand; 0 point, the patients cannot identify as above. Attention score: 1 point, the patient can repeat a series of figures said by the experimental staff; 0 point, the patient cannot repeat; 1 point, the patients can quickly find the difference of figures between the first and second time; 0 point, the patients cannot recognize that. Continuous subtraction (100 minus 6) is performed for a total of 5 times; 3 points, one error or normal; 2 points, 2 or 3 errors; 1 point, 4 errors; 0 point, all errors. Delayed memory score: 1 point for each of 5 words recalled by the patients. BODY.PATIENTS AND METHODS.ATTENTION NETWORK TEST (ANT): The attention network function was evaluated via ANT in an independent and quiet environment, including the targeted network efficiency and executive control network efficiency. The patients were trained and tested in strict accordance with the training and testing process of ANT. The patients were stimulated using the E-Prime 2.0 program; the thumb was placed on the number key 1 and 3 to determine the direction of target arrow. The patient's attention network function was evaluated based on the targeted network efficiency and executive control network efficiency of patients. BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: The data in this study are presented as mean ± standard deviation, and SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used for data processing. The t-test was used for intergroup comparison, while the analysis of variance was used for comparison among groups. The homogeneity test of variance was performed; Bonferroni method was used for pairwise comparison if the variance was homogeneous; otherwise, Welch method was used for analysis. Dunnett's T3 method was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference. BODY.RESULTS.GENERAL DATA ANALYSIS: The general data of patients in control group and dexmedetomidine group are recorded in detail and analyzed (Table I). The age, sex, percentage of hypertension history, percentage of diabetes mellitus history, percentage of drinking history and percentage of smoking history had no statistically significant differences between control group and dexmedetomidine group (P>0.05). BODY.RESULTS.SERUM S100B AND NGF LEVELS: The levels of serum S100B and NGF in each group were detected using the ELISA kit (Figs. 1 and 2). Compared with those in normal group, the levels of serum S100B and NGF in control group and dexmedetomidine group were significantly decreased (P<0.01), and they were significantly higher in dexmedetomidine group than those in control group (P<0.01). BODY.RESULTS.SERUM BDNF LEVEL: The level of serum BDNF in each group was detected via western blotting (Fig. 3). The levels of serum BDNF in control group and dexmedetomidine group were significantly decreased compared with that in normal group (P<0.01), and it was higher in dexmedetomidine group than that in control group (P<0.01). BODY.RESULTS.MOCA SCORES OF PATIENTS IN EACH GROUP: MoCA was used to record the visual space and executive function scores, attention score and delayed memory score of each group in detail in the experiment, and the cognitive function of patients was evaluated (Fig. 4). The visual space and executive function scores, attention scores and delayed memory scores in control group and dexmedetomidine group were obviously decreased compared with those in normal group (P<0.01), and they were obviously higher in dexmedetomidine group than those in control group (P<0.01, P<0.05). BODY.RESULTS.ANT OF PATIENTS IN EACH GROUP: ANT was used to record the targeted network efficiency and control network efficiency of each group (Fig. 5). The targeted network efficiency and control network efficiency in control group and dexmedetomidine group were obviously decreased compared with those in normal group (P<0.01), and they were obviously higher in dexmedetomidine group than those in control group (P<0.01, P<0.05). BODY.DISCUSSION: Patients with ischemic cerebrovascular disease will suffer from dysfunction or deletions in memory, attention, perception and feeling, making it impossible for patients to participate in the normal social activities and daily work and life, seriously affecting the life quality of patients; the above symptoms are collectively referred to as cognitive dysfunction (9). Researchers proposed the attention network theory by analyzing a large number of research results of cognitive psychology and brain function imaging, and evaluated the network function with the targeted network efficiency and control network efficiency (10,11). Cognitive function evaluation and attention network function evaluation are applied in the research on patients with stroke, Parkinson's disease, epilepsy and Alzheimer's disease (12–14). The mechanism of cognitive dysfunction and attention network damage in patients with cerebrovascular disease is not clear, and some studies have reported that the possible mechanism may be the vascular inflammation, cerebrovascular injury, environmental factors and genetic factors (15,16). In recent years, clinicians and researchers have paid increased attention to the cognitive dysfunction and attention network deletion in patients with central nervous system diseases, which is helpful to recover the patients' cognitive function and attention network function. In this study, the cognitive function and attention network function of patients with ischemic cerebrovascular disease after stenting were evaluated using the classical MoCA and ANT. The results showed that patients with ischemic cerebrovascular disease will suffer from severe cognitive dysfunction and attention network damage after operation. Yang and Rosenberg (17) found that the cognitive dysfunction and attention network function damage will be recovered in patients with carotid atherosclerosis after stenting, but it was found in this study that the cognitive function and attention network function of patients with ischemic cerebrovascular disease still have obvious abnormality compared with normal people even after stenting. In this study, patients with ischemic cerebrovascular disease received intravenous administration of dexmedetomidine before treatment, and the surgical process and treatment protocol were the same as those of the control group. After operation, the serum S100B, NGF and BDNF expression levels in patients were detected, and the cognitive function and attention network function were evaluated. The results showed that the levels of serum S100B, NGF and BDNF in dexmedetomidine group were significantly higher than those in control group, and the cognitive function and attention network function were also significantly superior to those in control group. The above results indicated that dexmedetomidine has a significant protective effect on patients with ischemic cerebrovascular disease and contributes to the recovery of cognitive function and attention network function of patients with of ischemic cerebrovascular disease. Moreover, the cognitive function and attention network function are closely related to the brain tissue structure and brain blood flow distribution; if the brain tissues are in an ischemia and hypoxia state for a long time, there will be severe cognitive dysfunction and attention network function damage (18,19). Dexmedetomidine is a highly-selective excitatory center and peripheral α2 receptor drug, which can make the blood pressure change stable under the stimulation and the blood flow more stable. Substantial research evidence reveals that dexmedetomidine has a significant protective effect on brain, which can significantly reduce the coma time and promote the postoperative cognitive function recovery of patients with brain injury (20). Dexmedetomidine can contract the vessels and produce sedative and analgesic effects. The latest research data show that dexmedetomidine can affect the expression levels of inflammatory factors and reduce the inflammatory response in blood vessels through activating the corresponding signal pathway (21). In this study, the protective effect of dexmedetomidine on cerebral blood vessels may be also manifested as reducing the level of inflammatory factors in cerebral blood vessels, reducing the level of inflammation in brain tissues of patients with ischemic cerebrovascular disease and reducing the damage of inflammation to the brain tissues, thus improving the cognitive function and attention network function. In conclusion, patients with ischemic cerebrovascular disease will suffer from obvious cognitive dysfunction and attention network function damage after stenting, and the preoperative administration of dexmedetomidine can effectively protect the cerebral blood vessels, improve the cognitive dysfunction and attention network function damage, and increase the postoperative life quality of patients.

TITLE: Long-Term Oxygen Therapy 24 vs 15 h/day and Mortality in Chronic Obstructive Pulmonary Disease ABSTRACT: Long-term oxygen therapy (LTOT) ≥ 15 h/day improves survival in hypoxemic chronic obstructive pulmonary disease (COPD). LTOT 24 h/day is often recommended but may pose an unnecessary burden with no clear survival benefit compared with LTOT 15 h/day. The aim was to test the hypothesis that LTOT 24 h/day decreases all-cause, respiratory, and cardiovascular mortality compared to LTOT 15 h/day in hypoxemic COPD. This was a prospective, observational, population-based study of COPD patients starting LTOT between October 1, 2005 and June 30, 2009 in Sweden. Overall and cause-specific mortality was analyzed using Cox and Fine-Gray regression, controlling for age, sex, prescribed oxygen dose, PaO2 (air), PaCO2 (air), Forced Expiratory Volume in one second (FEV1), WHO performance status, body mass index, comorbidity, and oral glucocorticoids. A total of 2,249 included patients were included with a median follow-up of 1.1 years (interquartile range, 0.6–2.1). 1,129 (50%) patients died and no patient was lost to follow-up. Higher LTOT duration analyzed as a continuous variable was not associated with any change in mortality rate (hazard ratio [HR] 1.00; (95% confidence interval [CI], 0.98 to 1.02) per 1 h/day increase above 15 h/day. LTOT exactly 24 h/day was prescribed in 539 (24%) patients and LTOT 15–16 h/day in 1,231 (55%) patients. Mortality was similar between the groups for all-cause, respiratory and cardiovascular mortality. In hypoxemic COPD, LTOT 24 h/day was not associated with a survival benefit compared with treatment 15–16 h/day. A design for a registry-based randomized trial (R-RCT) is proposed. BODY.INTRODUCTION: Long-term oxygen therapy (LTOT) improves survival time in patients with hypoxemic chronic obstructive pulmonary disease (COPD) when given for 15 h/day or more.[1, 2] LTOT is common and is associated with considerable logistics and costs.[3, 4] More than one million people use LTOT in the USA alone,[3] and the incidence is projected to increase in coming decades.[5] Morbidity and mortality are high despite LTOT, with a median survival of less than 2 years after start of oxygen therapy.[6] Strategies to improve prognosis in hypoxemic COPD are needed. Although current guidelines state that LTOT should be provided continuously for 24 h/day,[7] data are limited on whether LTOT 24 h/day provides additional survival benefit compared with treatment 15 h/day.[1, 2, 8, 9] The recommendation of LTOT 24 h/day is based on an unadjusted comparison of the treatment arms of two randomized trials from the 1970s,[8] which showed that the mortality rate was lower for the 18 h/day group in one study than for the 15 h/day group in another study.[1, 2, 8] However, the effects of each LTOT arm compared to controls (12 h/day and no treatment respectively) were similar.[1, 2] The comparison was non-randomized and did not adjust for potential confounders.[8] Furthermore, most patients starting LTOT on therapeutic indication nowadays are women, elderly with multimorbidity and median survival less than two years, as compared to the original trials which might decrease the validity of the original RCT findings for current practice.[5, 9–12] Evidence from randomized trials whether continuous LTOT duration 24 h/day provides an additional survival benefit above 15 h/day is lacking.[9] LTOT 24 h/day might pose an unnecessary burden for many patients compared with treatment 15 h/day where they can be unconnected to the machine for 9 hours each day.[13, 14] Supplemental oxygen therapy has been consistently associated with feelings of dependence, anxiety, guilt and shame among patients and caregivers, which could contribute to increased social isolation and restrictions of activity and daily life.[13, 15–17] The aim was to test the hypothesis that LTOT 24 h/day compared with LTOT 15–16 h/day and adjusting for potential confounders decreases all-cause, respiratory, and cardiovascular mortality in hypoxemic COPD. BODY.MATERIALS AND METHODS.DESIGN AND POPULATION: This was a prospective national observational study of patients starting LTOT on therapeutic indication for physician-diagnosed COPD in the National Register for Respiratory Failure (Swedevox) between October 1, 2005 and June 30, 2009. Swedevox has a population-based coverage of approximately 85% of patients starting LTOT in Sweden since 1987.[18] Details of the register were described in a recently published study using the same database.[11] All clinics prescribing LTOT in Sweden have agreed to observe the guidelines from the Swedish Respiratory Society that LTOT should be given for 15 h/day or more.[19–21] Nationwide, 48 clinics are prescribing LTOT.[6] LTOT is mainly prescribed by a pulmonologist or an internist with special training in COPD and respiratory failure.[6] The indication criteria for LTOT are: COPD and resting hypoxemia for at least three weeks despite optimal management of the underlying disease(s). The required level of resting hypoxemia breathing ambient air is defined as an arterial blood gas tension of oxygen (PaO2) < 7.4 kPa; or PaO2 7.4 − 8.0 kPa together with signs of right-sided heart failure/pulmonary hypertension and/or secondary polycythemia (erythrocyte volume fraction, EVF > 0.54). LTOT is titrated with the goal to obtain a PaO2 > 8 kPa or oxygen saturation > 90% on oxygen.[1, 2, 22, 23] LTOT daily duration prescribed may differ due to local traditions and based on patient-physician agreements in order to increase patient compliance when taking in regards patients' special needs and preferences. For patients who started LTOT more than once (N = 62) during the period, only the most recent treatment episode was included in the analysis. Exclusion criterion was a diagnosis of lung cancer before starting LTOT.[11] BODY.MATERIALS AND METHODS.DATA: Swedevox contains data on resting arterial blood gas tension of oxygen (PaO2) and carbon dioxide (PaCO2) breathing air and during oxygen therapy, forced expiratory volume in one second (FEV1), measured body mass index (BMI), smoking history and WHO performance status registered at the start of LTOT.[6] The prescribed daily dose (l/min) and duration (h/day) of LTOT are registered by a responsible specialized oxygen nurse at the time of starting LTOT. Data on comorbidity during the four-year period before baseline were obtained from the National Patient Register for in- and outpatient care, which covers more than 99% of all admissions in the study period and about 80% of all hospital based outpatient care since 2001 in Sweden.[24] Diagnoses were coded according to the ninth (before 1997) [25] and tenth revisions of the International Classification of Disease (ICD),[26] as previously described.[27] Data on all dispensed drug prescriptions in outpatient care after July 1, 2005 were obtained from the Swedish Prescribed Drug Register.[28] Vital status and cause of death were obtained from the Swedish Causes of Death Register. Patients were prospectively followed until the first of LTOT withdrawal, death, or study end December 31, 2009. The primary endpoint was death from all causes. Secondary endpoints were mortality from respiratory disease (ICD-10, J00–J99) or cardiovascular disease (ICD-10, I00–I99) as underlying cause of death. BODY.MATERIALS AND METHODS.ETHICAL CONSIDERATIONS: All patients participating in the study were informed according to directives from the authorities. Participants provided their verbal consent when registered in Swedevox and the consent procedure and the study was approved by the Lund University research ethics committee (DNr 157/2007 and 350/2008), the Swedish National Board of Health and Welfare, and the Swedish Data Inspection Board. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSES: Baseline patient characteristics were summarized using mean with standard deviation (SD) and median with range or interquartile range (IQR) for continuous variables with normal and skewed distribution, respectively. Categorical variables were expressed as frequencies and percentages. All-cause mortality was analyzed using Cox regression and expressed as hazard ratios (HR). Cause-specific mortality was analyzed using Fine-Gray regression accounting for the competing risk of death from other causes,[29] and expressed as subdistribution hazard ratios (SHR).[29] All associations were calculated with 95% confidence intervals (CIs). The observation time was from the start date of LTOT until the date of death from all causes, with censoring at withdrawal of LTOT or 31 December 2009. Covariates to be included in the final model were selected using subject matter knowledge and prior mortality analyses.[11, 27, 30] Missing elements were imputed for PaO2 (air) (N = 289), PaCO2 (air) (N = 301), FEV1 (N = 849), body mass index (BMI) (N = 701) and WHO performance status (N = 199), as previously described.[11] The model estimates were robust to the imputations. The final models were adjusted for baseline age, sex, prescribed oxygen dose, PaO2 (air), PaCO2 (air), FEV1, WHO performance status, BMI categories, maintenance treatment with oral glucocorticoids and comorbid diseases in terms of anxiety, renal failure and number of cardiovascular diagnoses (cerebrovascular disease, heart failure, hypertension, ischemic heart disease, peripheral artery disease, pulmonary embolism, and other circulatory disease). The primary analysis was among all patients (N = 2,249) with the prescribed daily LTOT duration analyzed as a continuous variable (h/day). In a secondary analysis, a comparison of LTOT prescribed for 24 h/day versus 15–16 h/day was carried out including only patients with either LTOT prescription (N = 1,770). Statistical significance was defined as two-sided p-value < 0.05. The differences among the groups were tested with t tests for continuous and chi-square tests for categorical variables. Statistical analyses were conducted using the software packages Stata, version 13 (StataCorp LP; College Station, TX), and SAS, version 9.3 (SAS Institute, Inc., Cary, NC). BODY.RESULTS.PATIENT CHARACTERISTICS: A total of 2,249 patients (59% women) started LTOT for COPD during the study period and were included in the main analysis. During a median follow-up of 1.1 years (IQR, 0.6 to 2.1 years), 138 (6%) patients withdrew from LTOT mainly because of improved oxygenation, and 1,129 (50%) patients died. The median survival time was 1.9 years (IQR, 0.7 to 4.0 years). Main causes of death included respiratory disease (68%), cardiovascular disease (20%) and cancer (6%). In the cohort, 539 (24%) patients were prescribed LTOT 24 h/day, 1,231 (55%) were prescribed 15 h/day and 470 (21%) had other daily durations prescribed. In the LTOT 24 h/day group, 288 (53%) patients died and 629 (52%) patients died in the LTOT 15–16 h/day group. Compared with the LTOT 15–16 h/day group, patients with LTOT 24 h/day had worse functional status (Table 1). PaO2 on oxygen above 8 kPa was achieved in the majority of patients, with similar rates for LTOT 24 h/day (77%) and 15–16 h/day (80%). 10.1371/journal.pone.0163293.t001 Table 1 Baseline characteristics in oxygen-dependent chronic obstructive pulmonary disease patients. Characteristic All on LTOTN = 2,249 LTOT 24 h/dayN = 539 (24%) LTOT 15–16 h/dayN = 1,231 (55%) P-value Age, years 74.7 ± 8.2 75.0 ± 8.1 74.7 ± 8.2 0.57 Women, n (%) 1,328 (59) 283 (53) 767 (62) < 0.001 PaO 2 air, kPa 6.5 ± 0.9 6.3 ± 0.9 6.6 ± 0.8 < 0.001 PaO 2 oxygen, kPa 8.7 ± 1.1 8.6 ± 1.1 8.7 ± 1.1 0.006 PaCO 2 air, kPa 6.3 ± 1.2 6.2 ± 1.3 6.2 ± 1.2 0.90 PaCO 2 oxygen, kPa 6.5 ± 1.3 6.6 ± 1.3 6.5 ± 1.3 0.12 FEV 1 , L 0.84 ± 0.48 0.89 ± 0.54 0.83 ± 0.45 0.10 FEV 1 , % of predicted 33.6 ± 17.3 34.8 ± 19.5 33.8 ± 17.0 0.45 Prescribed Oxygen dose, L/min 1.6 ± 1.3 2.0 ± 1.3 1.5 ± 1.0 < 0.001 Ever smoking, n (%) 2,106 (94) 478 (89) 1110 (90) 0.35 Body mass index, kg/m 2 24.0 (6.3) 24.0 (6.4) 23.9 (6.0) 0.80 WHO performance status, n (%) 0 132 (6) 26 (5) 76 (6) 0.26 1 881 (39) 173 (32) 493 (40) 0.001 2 714 (32) 167 (31) 385 (31) 0.90 3 292 (13) 108 (20) 132 (11) < 0.001 4 31 (1) 11 (2) 14 (1) 0.14 Missing 199 (8) 54 (10) 131 (11) 0.70 Cardiovascular diagnoses, n (%) 0 755 (34) 158 (29) 428 (35) 0.03 1 823 (37) 203 (38) 433 (35) 0.32 2 449 (20) 113 (21) 242 (20) 0.53 ≥3 222 (10) 65 (12) 128 (10) 0.30 Depression, n (%) 207 (9) 52 (10) 114 (9) 0.80 Anxiety, n (%) 196 (9) 44 (8) 123 (10) 0.23 Diabetes mellitus, n (%) 291 (13) 73 (14) 154 (13) 0.55 Renal failure, n (%) 97 (4) 28 (5) 57 (5) 0.60 Oral glucocorticoids, n (%) 1375 (61) 327 (61) 731 (60) 0.61 Data presented as mean ± SD unless otherwise specified. Hospitalizations and diagnoses were assessed within the four-year period before the start of long-term oxygen therapy (LTOT). Abbreviations : FEV 1 , forced expiratory volume in one second; PaO 2 , arterial blood gas tension of oxygen; PaCO 2 , arterial blood gas tension of carbon dioxide on air; WHO, world health organization. BODY.RESULTS.LTOT 24 VS 15 H/DAY: In the primary analysis among all patients (N = 2,249), higher daily LTOT duration analyzed as a continuous variable was not associated with any change in mortality rate, HR 1.00 (95% CI, 0.98 to 1.02) per 1 h/day increase above 15 h/day (Table 2). WHO performance status was found to be an important confounder (Table 2). Findings were similar for adjusted respiratory deaths (SHR 0.99; 95% CI, 0.97 to 1.02) and cardiovascular deaths (SHR 1.00; 95% CI, 0.96 to 1.05). 10.1371/journal.pone.0163293.t002 Table 2 Cox regression of daily oxygen duration and adjusted mortality in 2,249 patients with COPD. Variable Hazard ratio 95% CI P-value Continuous LTOT (24 vs. 15 h/day) 1.00 0.98–1.02 0.88 Age (per year) 1.04 1.03–1.05 < 0.001 Men 1.29 1.08–1.46 < 0.001 PaO 2 air (per 1 kPa) 0.93 0.86–1.00 0.04 PaCO 2 air (per 1 kPa) - - 0.001 PaCO 2 air † - - < 0.001 FEV 1 (per liter) 0.96 0.80–1.15 0.65 Prescribed oxygen dose (per 1 l/min) 1.03 0.98–1.08 0.23 Body mass index, kg/m 2 < 18.5 1.37 1.16–1.63 < 0.001 18.5–24.9 Ref - - 25–29.9 0.73 0.60–0.87 0.001 ≥ 30 0.80 0.64–1.00 0.06 WHO performance status 0 Ref - - 1 1.03 0.75–1.40 0.88 2 1.51 1.10–2.07 0.01 3 2.45 1.76–3.42 < 0.001 4 3.19 1.93–5.28 < 0.001 Missing 1.35 0.94–1.93 0.10 Cardiovascular diagnoses 0 Ref - - 1 1.26 1.09–1.46 0.002 2 1.40 1.18–1.66 < 0.001 ≥3 1.35 1.08–1.67 0.007 Anxiety 1.28 1.05–1.58 0.01 Renal failure 1.33 1.03–1.73 0.03 Oral glucocorticoids 1.20 1.06–1.35 0.004 † PaCO 2 air was included as second degree polynomial (Wald P < 0.001), wherefore a linear hazard ratio is not reported. Abbreviations : CI, confidence interval; for others see Table 1 . In the secondary analysis (N = 1,770), LTOT 24 h/day was associated with a higher all-cause mortality rate than treatment 15–16 h/day, HR 1.17 (95% CI, 1.02 to 1.34) in unadjusted analysis. However, in the final model adjusted mortality was similar between LTOT 24 h/day and LTOT 15–16 h/day, HR 0.98 (95% CI, 0.85 to 1.14; P = 0.81). The adjusted hazard plots of the treatment groups closely overlapped at all time points (Fig 1). 10.1371/journal.pone.0163293.g001Fig 1Cumulative risk of death for LTOT 24 h/day (N = 539) versus 15–16 h/day (N = 1,231) in oxygen-dependent COPD.The hazard ratio was 0.98 (95% CI, 0.85 to 1.14); adjusted for baseline age, sex, oxygen dose, PaO2 (air), PaCO2 (air), FEV1, WHO performance status, body mass index, treatment with oral glucocorticoids, and comorbid diagnoses including anxiety, renal failure and number of cardiovascular diagnoses. In unadjusted analysis, there was a trend towards increased respiratory deaths (SHR 1.09; 95% CI, 0.89 to 1.34) and cardiovascular deaths (SHR 1.17; 95% CI, 0.81 to 1.71) with LTOT 24 h/day compared with LTOT 15–16 h/day. Adjusted cause-specific mortality was however similar between the two treatment groups for respiratory deaths (SHR 0.96; 95% CI, 0.76 to 1.20) and cardiovascular deaths (SHR 0.89; 95% CI, 0.60 to 1.32). BODY.DISCUSSION.MAIN FINDING: Longer daily duration of LTOT above 15 h/day was not associated with improved adjusted survival time in a large cohort of patients with oxygen-dependent COPD. Adjusted mortality rates were similar for deaths from all causes and from respiratory and cardiovascular disease. BODY.DISCUSSION.STRENGTHS AND LIMITATIONS: Strengths of the present study include its national, population-based design and large study population. This study included 2,249 patients with COPD and therapeutic LTOT compared to a total of 280 patients in the randomized trials in severe hypoxemia.[1, 2] Analyses were adjusted for a range of relevant confounders, including blood gases, lung function, performance status, and comorbid diseases. Cause-specific mortality was analyzed as respiratory and cardiovascular disease are leading causes of death among these patients and may be affected by hypoxemia and supplemental oxygen therapy.[12] A limitation is that we lacked data on actual daily oxygen utilization. Therefore, we cannot exclude the possibility that patients prescribed LTOT 24 h/day utilized oxygen fewer hours/day in real life and explain why similar rated of mortality was found in both groups. Our findings reflect effectiveness of prescribed oxygen durations in clinical practice. Adherence to LTOT may be insufficient but has been reported to be better in patients with significant hypoxemia like those in the present study.[16] A second limitation common to all observational designs is possible confounding by indication owing to the lack of randomization.[31] Patients with more severe illness may be prescribed higher oxygen flow rates and longer LTOT duration. In the present study, LTOT 24 h/day was associated with an increased mortality risk in the unadjusted analysis, but the survival difference disappeared when controlling for confounders including prescribed oxygen dose and WHO performance status. Our findings support our research hypothesis that a longer LTOT duration does not provide additional survival benefit above 15 h/day. However, absence of residual confounding needs to be evaluated through randomized controlled trials. BODY.DISCUSSION.WHAT THIS STUDY ADDS: This is the first large comparative study of LTOT prescribed 24 h/day versus treatment 15 h/day on survival in oxygen-dependent COPD. Our findings are made more representative and robust due to its population-based and multi-center design. The previous four studies of the effects of LTOT on mortality were small (in total 501 patients), conducted 20–40 years ago, totally un-blinded, and included selected patients at tertiary specialist centers.[1, 2] The included patients were younger, mostly men with no or limited comorbidity and did not receive modern treatments including for COPD and cardiovascular disease.[1–3, 32, 33] The dramatic survival benefit of LTOT seen in the 1970s was not evident in the studies from the 1990s.[1–3] The validity of the initial observations for today's patients is unclear as the majority of current patients are women, elderly with multiple comorbidities and limited expected survival, similar to our study population.[5, 10–12] Our findings are in line with a recent small study (N = 228) which reported no survival difference between intermittent LTOT (mean utilization 6.7 ± 3.4 h/day) and continuous LTOT (mean utilization 18.1 ± 2.5 h/day).[34] BODY.DISCUSSION.MECHANISMS: Whereas the survival benefit of LTOT 15 h/day or more is established,[1, 2] the mechanisms for the improvement in survival still remains unknown. There is limited evidence, but possible effects of LTOT might be mediated through prevention of hypoxemia-related cardiovascular disease and stabilization of pulmonary arterial pressure.[35] However, the effect on pulmonary arterial hypertension was found to be similar between for LTOT 15 h/day and 18 h/day.[36] LTOT prescribed 15 h/day might thus be sufficient which is in line with our findings of similar rates of cardiovascular mortality between LTOT prescribed 24 h/day compared with LTOT 15–16 h/day. BODY.DISCUSSION.IMPLICATIONS: The lack of association between LTOT 24 h/day and improved survival suggests that there is no advantage of LTOT prescribed 24 h/day compared with 15 h/day. On the contrary, LTOT 24 h/day may pose an unnecessary burden and limitation for many patients compared with treatment 15 h/day, where patients can be disconnected from the equipment for 9 hours each day. There is no consistent evidence that LTOT reduces breathlessness or health status in daily life.[37–40] LTOT has been associated with feelings of dependence and shame, which could contribute to a reluctance to leave the house, increased social isolation and deconditioning, especially with oxygen therapy given continuously 24 h/day.[13, 15, 17] Low-flow oxygen therapy has been associated with oxidative stress [41] and inflammation [42] which could contribute to increased morbidity and adverse health effects.[43, 44] LTOT 24 h/day is also associated with an increased electricity cost, which might be problematic for severely ill patients with already strained finances. Current guidelines recommending continuous LTOT (optimally 24 h/day) are based on an observational, unadjusted comparison of the treatment arms from two randomized trials from the 1970s. [1, 2] [8] This recommendation is not evidence-based as no randomized study has compared the effect of LTOT 24 h/day with 15 h/day. The validity of LTOT among today's patients is difficult to assess as comparisons with none or intermittent LTOT <15 h/day may be ethically unacceptable. In light of the present findings,[9] there is equipoise between LTOT prescribed 24 h/day and 15 h/day. We propose that a next step would be to validate the effect on survival of LTOT 24 h/day compared 15 h/day in a registry-randomized controlled trial (R-RCT).[45] BODY.DISCUSSION.REGISTER-BASED RANDOMIZED TRIAL (R-RCT): An R-RCT uses a health care registry to recruit, randomize, and follow-up patients in a clinical trial.[45] The R-RCT approach combines the features of a prospective randomized interventional trial with a large-scale clinical registry and thus enables evaluation of clinically important patient outcomes in the real world setting.[45] The strengths of an R-RCT include a simple design, fast enrolment, control of non-enrolled patients, being inexpensive and the possibility of very long-term follow up.[45] The method was recently pioneered in a randomized trial of thrombus aspiration during percutaneous coronary interventions, which included more than 60% of patients with acute ST-elevation myocardial infarction nationwide at low costs.[46] Hospitalization and mortality rate were assessed using national registries, with complete follow-up.[46] An R-RCT using the National Swedevox register would be optimally suited for assessment of the effectiveness of continuous (24 h/day) versus low duration LTOT 15 h/day in a large representative sample of patients with chronic respiratory failure. BODY.CONCLUSIONS: LTOT prescribed 24 h/day was not associated with improved survival compared with LTOT 15–16 h/day in hypoxemic COPD after adjusting for potential confounders. The novel design of a registry-based randomized controlled trial is proposed to drive forward evidence-based care in patients with respiratory failure.

TITLE: Effect of aclidinium bromide on cough and sputum symptoms in moderate-to-severe COPD in three phase III trials ABSTRACT.BACKGROUND: Cough and sputum are troublesome symptoms in chronic obstructive pulmonary disease (COPD) and are associated with adverse outcomes. The efficacy of aclidinium bromide 400 μg twice daily in patients with stable COPD has been established in two phase III studies (ACCORD COPD I and ATTAIN) and a phase IIIb active-comparator study. This analysis evaluated cough-related symptoms across these studies. ABSTRACT.METHOD: Patients were randomised to placebo, aclidinium 200 μg or 400 μg twice daily in ACCORD (12 weeks) and ATTAIN (24 weeks), or to placebo, aclidinium 400 μg twice daily or tiotropium 18 μg once daily (6-week active-comparator study). Analysed end points included changes from baseline in Evaluating Respiratory Symptoms (E-RS; formerly known as EXAcerbations of Chronic pulmonary disease Tool), total and cough/sputum scores and frequency/severity of morning and night-time cough and sputum symptoms. ABSTRACT.RESULTS: Data for 1792 patients were evaluated. E-RS cough/sputum domain scores were significantly reduced with aclidinium 400 μg versus placebo in ATTAIN (−0.7 vs −0.3, respectively; p<0.01) and the active-comparator study (−0.6 vs −0.2, respectively; p<0.01). In the active-comparator study, significantly greater improvements were observed with aclidinium versus placebo for severity of morning cough (−0.19 vs −0.02; p<0.01) and phlegm (−0.19 vs −0.02; p<0.05). In ACCORD, aclidinium reduced night-time cough frequency (−0.36 vs 0.1 for placebo; p<0.001) and severity (−0.24 vs −0.1 for placebo; p<0.05), and frequency of night-time sputum production (−0.37 vs 0.05 for placebo; p<0.001). ABSTRACT.CONCLUSIONS: Aclidinium 400 μg twice daily improves cough and sputum expectoration versus placebo in stable COPD. ABSTRACT.TRIAL REGISTRATION NUMBERS: NCT00891462; NCT01001494; NCT01462929. BODY: Key messagesCough and sputum in COPD have a substantial impact on patients' health status, yet there are relatively few studies that have investigated the effect of bronchodilators on these symptoms.In this paper, we analyse data from three Phase III studies to elucidate the effect of aclidinium bromide on cough and sputum. The results suggest that in addition to improving lung function, LAMAs, such as aclidinium, can improve cough and sputum expectoration compared with placebo in patients with COPD.As cough and sputum impact negatively on overall patient wellbeing, controlling these symptoms may represent an important additional therapeutic benefit of this class of drugs. BODY.INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is characterised by persistent and progressive airflow limitation and an enhanced inflammatory response to noxious stimuli.1 The resulting lung injury leads to breathlessness and other characteristic symptoms of COPD, including cough and sputum. In patients with COPD, chronic cough and sputum production are associated with lung-function decline,2 more frequent exacerbations and hospitalisations, and increased risk of death.3 4 Accumulation of mucus in small airways is also associated with disease progression,5 and a productive cough has been shown to be independently associated with increased mortality in smokers with mild-to-moderate airflow obstruction.6 Cough symptoms also impact adversely on the health status of patients with COPD to a similar degree to that observed in bronchiectasis, asthma and chronic cough.7 The importance of cough and sputum symptoms in defining a patient's overall well-being is reflected in the inclusion of these items in the COPD Assessment Test, a patient-reported outcomes tool designed to assess overall COPD-related health status.8 9 Between disease exacerbations, when COPD is considered stable, there may still be marked daily variability in patients' perceptions of symptom severity. In a pan-European cross-sectional study in patients with COPD, cough and phlegm were reported to be most troublesome in the morning.10 However, in a recent observational study of COPD symptoms, despite overall night-time symptoms being less prevalent than in the morning and during the day, cough was still the most common symptom at night.11 Despite the evidence of a clear association between cough and adverse clinical outcomes, its significance in patients with COPD is often underappreciated.12 13 In addition, almost nothing is known about the effect of current first-line COPD treatments on symptoms of cough and sputum, and the need for studies to address this has been highlighted.12 Aclidinium bromide is a long-acting muscarinic antagonist (LAMA) that inhibits the action of acetylcholine at M3 receptors in the lungs, indirectly leading to airway smooth muscle relaxation. Aclidinium is approved as a maintenance bronchodilator treatment in patients with COPD.14–16 Several phase III studies have shown that aclidinium 400 μg twice daily improves lung function and symptoms in patients with moderate-to-severe airflow limitation.17–23 In this manuscript, we report our analysis of the data from three of these studies, ACCORD COPD I, ATTAIN and a 6-week active-comparator study, which was undertaken to determine the effect of the approved dose of aclidinium (400 μg twice daily metered dose; equivalent to aclidinium 322 μg delivered dose) on cough and sputum symptoms in patients with stable moderate-to-severe COPD. The three phase III studies reported here were selected on the basis that they had similar inclusion/exclusion criteria and included end points that assessed the efficacy of aclidinium 400 μg twice daily on cough and sputum symptoms. Four additional phase III studies of aclidinium did not record cough data so could not be included in this analysis. BODY.METHODS: In these analyses, only data from patients randomised to placebo, aclidinium 400 μg twice daily (the dose approved for use in patients with COPD) or tiotropium 18 μg once daily (also the approved dose) were evaluated. The purpose of this additional analysis was to assess the impact of aclidinium on cough and sputum symptoms across three clinical studies, including the relationship between symptoms and time of day. All end points were preplanned, with the exception of post hoc analyses assessing the correlation between Evaluating Respiratory Symptoms (E-RS; formerly known as EXAcerbations of Chronic pulmonary disease Tool) cough and sputum domain score and cough severity score in the active-comparator study and change from baseline in E-RS total and cough and sputum domain scores in patients who had ≥1 exacerbation event in the ATTAIN study. BODY.METHODS.STUDY DESIGN: ACCORD COPD I (ClinicalTrials.gov identifier: NCT00891462) and ATTAIN (ClinicalTrials.gov identifier: NCT01001494) were multinational, randomised, double-blind, placebo-controlled phase III studies.17 18 Following screening and a 2-week run-in period, patients were randomised (1:1:1) to receive aclidinium 200 μg, aclidinium 400 μg (metered dose; equivalent to aclidinium 322 μg delivered dose) or placebo twice daily via the GenuairTM/Pressair®i inhaler for 12 weeks in ACCORD COPD I and 24 weeks in ATTAIN. The third study was a randomised, double-blind, double-dummy, placebo-controlled and active-controlled phase IIIb study (ClinicalTrials.gov identifier: NCT01462929).19 Following a 2–3-week run-in period, patients were randomised (2:2:1) to receive aclidinium 400 μg twice daily (metered dose; equivalent to aclidinium 322 μg delivered dose), tiotropium 18 μg once daily in the morning via HandiHaler® or placebo for 6 weeks. In all three studies, inhaled albuterol/salbutamol (108/100 μg/puff) was permitted as relief medication as long as it was discontinued 6 hours prior to study visits. Additional permitted medications included inhaled corticosteroids, oral or parenteral corticosteroids (≤10 mg/day of prednisone or 20 mg every other day), oral sustained-release theophyllines and oxygen therapy (<15 hours/day), provided that treatment was stable for ≥4 weeks before screening. Other long-acting bronchodilators and anticholinergic drugs were washed out prior to screening and were not allowed during the treatment periods. All studies were conducted in accordance with the Declaration of Helsinki, International Conference on Harmonisation/Good Clinical Practice Guidelines and local regulations. The protocols were approved by institutional review boards/independent ethics committees at each site, and all patients gave written informed consent. BODY.METHODS.STUDY POPULATIONS: Detailed inclusion/exclusion criteria for the three studies have been reported previously.17–19 Briefly, each study enrolled male and female patients (≥40 years old) with a diagnosis of stable COPD and moderate-to-severe airflow obstruction (postbronchodilator forced expiratory volume in 1 s (FEV1) ≥30% and <80% of the predicted value and FEV1/forced vital capacity ratio <70%)1 who were current or former smokers with a smoking history of ≥10 pack-years. The presence of cough or sputum symptoms at baseline was not a specific inclusion criterion in any of the studies. Exclusion criteria included any respiratory tract infection or COPD exacerbation within 6 weeks prior to screening (3 months if exacerbation resulted in hospitalisation), any clinically relevant respiratory conditions, including a history or current diagnosis of asthma and a history of hypersensitivity to inhaled anticholinergics or other inhaled medications. BODY.METHODS.STUDY ASSESSMENTS: A summary of patient-reported outcome measures used to capture symptoms in each clinical trial and corresponding end points are shown in online supplementary table S1. Baseline values for all end points were calculated as the average scores over the 2–3-week screening period prior to randomisation. 10.1136/bmjresp-2016-000148.supp1supplementary tables BODY.METHODS.STUDY ASSESSMENTS.DAILY SYMPTOMS: In ATTAIN and the active-comparator study, daily respiratory symptoms were assessed using the E-RS algorithm.24–26 The EXACT is a 14-item electronic daily diary used to quantify and measure exacerbations of COPD. It is completed by patients at night with a recall period of 'today' and captures symptoms of COPD including cough and sputum production. The E-RS total score (range 0–40) is a derivative tool which uses the 11 EXACT items that relate specifically to respiratory symptoms, with higher scores indicating more severe symptoms; the E-RS cough and sputum domain score is the sum of the three EXACT items that relate specifically to cough and sputum symptoms (range 0–11). Responder criteria for E-RS total score and E-RS cough and sputum domain scores have been proposed as a change of ≥−2.0 units in the E-RS total score and ≥−0.7 in the E-RS cough and sputum domain score.27 The E-RS was not used in the ACCORD study. BODY.METHODS.STUDY ASSESSMENTS.MORNING AND NIGHT-TIME COUGH AND SPUTUM SYMPTOMS: Cough and sputum symptoms during the morning and night-time were assessed in the three phase III studies using questionnaires developed by the study sponsors.28 29 In ATTAIN, a 6-item night-time and morning symptoms of COPD questionnaire, completed by patients at approximately the same time every morning using an electronic patient diary, was used to assess the number of days patients experienced a range of morning or night-time symptoms, including coughing and bringing up phlegm or mucus. The questionnaire included one item that asked patients if they experienced symptoms during the night and one item that asked about their symptoms since they got out of bed to start the day. In the active-comparator study, morning symptoms were assessed using a 9-item COPD symptom questionnaire, completed daily by patients between 7:00 am and 11:00 am using an electronic diary. Early morning was defined as the time from when patients got out of bed to start the day until they started their daily activities. One item of the questionnaire was related to the presence of a range of early-morning symptoms, including cough and phlegm, with five items related to the severity of these symptoms. Patients assessed the severity of their overall morning symptoms (5-point scale: 1='I did not experience any symptoms'; 2='mild'; 3='moderate'; 4='severe'; 5='very severe') and the severity of individual symptoms, including cough and difficulty bringing up phlegm (5-point scale: 0='no symptoms'; 1='mild'; 2='moderate'; 3='severe'; 4='very severe'). In ACCORD COPD I, night-time symptoms were assessed using an 11-item COPD night-time symptoms questionnaire, adapted from an existing COPD symptom questionnaire30 to include additional items assessing the frequency of COPD symptoms, such as night-time breathlessness, cough, sputum production and wheezing, during the previous night. Patients completed the questionnaire daily in the morning using an electronic patient diary (the recall period was ≤24 hours). The frequency of night-time symptoms was assessed on a 5-point scale: 0='never'; 1='1–2 times'; 2='3–4 times'; 3='5–6 times'; 4='7 or more times'. The severity and impact of night-time symptoms were assessed on a 5-point scale: 0='no symptoms'; 1='symptoms present but caused little/no discomfort'; 2='mild symptoms that were unpleasant but caused little/no discomfort'; 3='moderate symptoms that caused discomfort but did not affect daily activities'; 4='severe symptoms that interfered with normal daily activities'. BODY.METHODS.END POINTS: Predefined efficacy end points included: changes from baseline in E-RS total score and E-RS cough and sputum domain score over the study period (ATTAIN and active-comparator study); the percentage of days with morning or night-time symptoms over the study period (ATTAIN); changes from baseline in the percentage of days without morning symptoms and the severity of morning cough and difficulty bringing up phlegm over the study period (active-comparator study) and changes from baseline at week 12 in COPD night-time symptoms (ACCORD COPD I). To investigate the reliability of different measures of cough symptoms used in these analyses, a post hoc analysis assessed the correlation between changes from baseline in E-RS cough and sputum domain scores in those patients who had ≥1 exacerbation event in the ATTAIN study, and the severity of morning cough (based on the symptom questionnaires) at week 6 in the active-comparator study. These were selected as both measures assess the improvement from baseline in symptom severity. Safety and tolerability were assessed in all three studies by recording adverse events. Additional safety assessments included a physical examination, laboratory tests, vital signs and ECGs. BODY.METHODS.STATISTICAL ANALYSES: Demographic and baseline characteristics were assessed in the intent-to-treat (ITT) population (all treated patients who had baseline and at least one postbaseline FEV1 assessment) and are reported as mean (SD) or percentage, as appropriate. Efficacy analyses were performed in the ITT population. Changes from baseline in E-RS total and cough and sputum domain scores (ATTAIN and active-comparator study), percentage of days with morning or night-time cough symptoms (ATTAIN) and changes from baseline in the percentage of days without morning symptoms and the severity of morning symptoms (active-comparator study) were analysed using an analysis of covariance (ANCOVA) model, with treatment group and sex as factors and age and corresponding baseline as covariates. Changes from baseline in the frequency and severity of night-time symptoms (ACCORD COPD I) were analysed using an ANCOVA model with treatment as a factor and the corresponding baseline as a covariate. Data are reported as least squares mean (SEM), least squares mean differences (95% CIs) or percentages, as appropriate. For the post hoc analysis, Pearson coefficients were used to evaluate the correlation between improvements in E-RS cough and sputum domain score and the scores from the cough severity question in symptom questionnaires. Additional post hoc analyses assessed the change from baseline in E-RS total and cough and sputum domain scores in those patients who had ≥1 exacerbation event identified using the EXACT in the ATTAIN study. An EXACT-identified event was defined as a persistent increase from baseline in total EXACT score of ≥9 points for ≥3 days or ≥12 points for ≥2 days.26 31 BODY.RESULTS.PATIENT POPULATION: The ITT populations in ACCORD COPD I, ATTAIN and the active-comparator study included 559, 819 and 414 patients, respectively. Demographics and baseline clinical characteristics of the study populations have been reported previously;17–19 the demographics and baseline clinical characteristics in the placebo, aclidinium 400 μg and tiotropium arms are shown in table 1. E-RS scores and symptom questionnaire scores at baseline in the placebo, aclidinium 400 μg and tiotropium study arms are shown in online supplementary table S2. Table 1 Demographics and baseline clinical characteristics (ITT population) ACCORD COPD I ATTAIN Active-comparator study Characteristic Placebo (N=185) Aclidinium 400 µg twice daily (N=190) Placebo (N=273) Aclidinium 400 µg twice daily (N=269) Placebo (N=85) Aclidinium 400 µg twice daily (N=171) Tiotropium 18 µg once daily (N=158) Age (years), mean (SD) 65.0 (9.2) 64.9 (9.5) 62.0 (8.0) 62.9 (8.4) 62.2 (8.2) 61.8 (8.2) 62.8 (7.9) Gender (male), n (%) 95 (51.4) 100 (52.6) 189 (69.2) 182 (67.7) 48 (56.5) 114 (66.7) 116 (73.4) Current smoker, n (%) 87 (47.0) 80 (42.1) 144 (52.8) 148 (55.0) 47 (55.3) 93 (54.4) 84 (53.2) Smoking history (pack-years), mean (SD) 52.9 (28.1) 57.2 (28.5) 38.9 (18.3) 41.7 (21.1) 39.6 (15.4) 41.5 (22.4) 45.0 (21.8) Postbronchodilator FEV 1 ,* mean (SD), L 1.6 (0.6) 1.5 (0.5) 1.6 (0.5) 1.6 (0.5) 1.6 (0.5) 1.6 (0.5) 1.7 (0.5) Postbronchodilator FEV 1 % predicted,* mean (SD) 54.7 (13.4) 54.1 (12.9) 56.6 (12.8) 56.2 (12.2) 55.5 (11.8) 55.8 (13.3) 56.0 (13.2) Severity of airflow limitation, †, ‡ n (%)  Moderate 111 (60.0) 118 (62.1) 178 (65.9) 184 (68.7) 58 (68.2) 108 (63.2) 104 (66.2)  Severe 72 (38.9) 68 (35.8) 92 (34.1) 84 (31.3) 27 (31.8) 63 (36.8) 53 (33.8) ≥1 COPD exacerbation in previous year, ‡ n (%) 52 (28.1) 43 (22.6) 88 (32.6) 97 (36.2) 19 (22.4) 61 (35.7) 47 (29.7) Concomitant use of ICS, n (%) 70 (37.6) 81 (42.6) 145 (53.1) 128 (47.6) 36 (42.4) 82 (48.0) 67 (42.4) *At screening visit. † Moderate COPD: 50% ≤postbronchodilator FEV 1 <80% predicted and FEV 1 /FVC <0.70; severe COPD: 30% ≤postbronchodilator FEV 1 <50% predicted and FEV 1 /FVC <0.70. ‡ Patients with available data. COPD, chronic obstructive pulmonary disease; FEV 1 , forced expiratory volume in 1 s; FVC, forced vital capacity; ICS, inhaled corticosteroid; ITT, intent-to-treat. BODY.RESULTS.SAFETY AND TOLERABILITY: Safety and tolerability outcomes have previously been reported for each study.17–19 In summary, aclidinium is well tolerated with the most common adverse events being nasopharyngitis, headache, COPD exacerbation and cough. No clinically significant differences in other safety assessments were observed. No new safety and tolerability findings were anticipated based on these additional analyses. BODY.RESULTS.DAILY COPD SYMPTOMS: Treatment with aclidinium 400 μg significantly reduced total daily COPD symptoms compared with placebo, as assessed by E-RS total score over 24 weeks in ATTAIN (p<0.001; figure 1A) and 6 weeks in the active-comparator study (p<0.001; figure 1A).19 In the active-comparator study, E-RS total score was also significantly reduced with tiotropium compared with placebo (p<0.05; figure 1A). Figure 1Change from baseline in (A) E-RS total score and (B) E-RS cough and sputum domain score over the study period in ATTAIN and the active-comparator study. Data are reported as LS mean+SE. E-RS total score ranged from 0 to 40; E-RS cough and sputum domain score ranged from 0 to 11. Higher scores indicate more severe symptoms. *p<0.05, **p<0.01, ***p<0.001 vs placebo. E-RS, Evaluating Respiratory Symptoms, formerly known as EXAcerbations of Chronic pulmonary disease Tool; LS, least squares; MCID, minimum clinically important difference. Daily cough and sputum symptoms, assessed by E-RS cough and sputum domain score, were also significantly reduced with aclidinium 400 μg versus placebo in ATTAIN and the active-comparator study (both p<0.01; figure 1B). There was no significant difference between tiotropium and placebo treatments on cough and sputum symptoms in the active-comparator study (p=0.109; figure 1B). Post hoc analysis of a patient subpopulation with ≥1 exacerbation event identified by the EXACT (n=178) indicated that E-RS total score and E-RS cough and sputum scores were similar at baseline between aclidinium 400 μg and placebo in ATTAIN. After 24 weeks, treatment differences (95% CI) between aclidinium 400 μg and placebo in this group were significant for E-RS total score (−1.9 [−3.1 to −0.6]; p<0.01) and E-RS cough and sputum scores (−0.5 [−0.9 to −0.2]; p<0.01). BODY.RESULTS.MORNING AND NIGHT-TIME COUGH AND SPUTUM SYMPTOMS: In ATTAIN, the percentage of days with any morning or night-time symptoms over the study period was significantly lower in patients treated with aclidinium 400 μg compared with placebo (both p<0.001; figure 2). Aclidinium treatment also significantly reduced the percentage of days with morning or night-time cough symptoms compared with placebo (both p<0.01; figure 2). Similarly, the percentage of days with morning or night-time bringing up phlegm or mucus was also significantly lower over the study period in patients treated with aclidinium 400 μg compared with placebo (p<0.01; figure 2). Figure 2Percentage of days with (A) morning symptoms and (B) night-time symptoms over the study period in ATTAIN. Data are reported as least squares mean. **p<0.01, ***p<0.001 vs placebo. In the active-comparator study, both aclidinium and tiotropium significantly increased the change from baseline in the percentage of days without any morning symptoms over the study period versus placebo (treatment differences [95% CI] 8.9% [4.1% to 13.8%] with aclidinium and 5.6% [0.6% to 10.6%] with tiotropium; p<0.001 and p<0.05 vs placebo, respectively). Similarly, there was a significant increase in the percentage of days without morning cough symptoms in the aclidinium group compared with placebo (treatment difference [95% CI] 7.2% [1.1% to 13.4%]; p<0.05); there was no significant difference with tiotropium versus placebo (treatment difference [95% CI] 5.5% [−0.8% to 11.8%]; p=0.084). While the change from baseline in the percentage of days without difficulty bringing up phlegm was numerically higher with aclidinium (7.7%) and tiotropium (4.8%) compared with placebo (2.0%), the differences between the active treatments and placebo did not reach statistical significance (p=0.100 for aclidinium and p=0.425 for tiotropium). Patients' assessment of the overall severity of their morning symptoms over the study duration was significantly reduced with aclidinium (−0.22; p<0.001) and tiotropium (−0.12; p<0.05) compared with placebo in the active-comparator study.19 When the severity of morning cough and difficulty bringing up phlegm was assessed, there was a significant reduction in the severity of both symptoms with aclidinium versus placebo over 6 weeks (p<0.05; figure 3). There was no significant change from baseline in the severity of either cough or difficulty bringing up phlegm in patients treated with tiotropium compared with placebo. Figure 3Change from baseline in the severity of morning cough and severity of difficulty bringing up phlegm in the morning over the study period in the active-comparator study. Data are reported as LS mean+SE. Assessed on a 5-point scale: 0='no symptoms' to 4='very severe symptoms'. *p<0.05, **p<0.01 vs placebo. LS, least squares. ACCORD COPD I investigated the prevalence and severity of night-time cough and sputum symptoms.17 After 12 weeks of treatment, aclidinium 400 μg significantly reduced the frequency of night-time cough compared with placebo (p<0.001; figure 4). The severity and impact of night-time cough symptoms was also significantly reduced at week 12 with aclidinium 400 μg compared with placebo (p<0.05; figure 4). In addition, the frequency of night-time sputum production was significantly lower in patients treated with aclidinium 400 μg compared with placebo (p<0.001; figure 4). Figure 4Change from baseline in the severity of night-time cough and the frequency of night-time sputum production at week 12 in ACCORD COPD I.17 Data are reported as mean+SE. Symptom frequency assessed on a 5-point scale: 0='never' to 4='7 or more times'. Symptom severity assessed on a 5-point scale: 0='no symptoms' to 4='severe symptoms that interfered with normal daily activities'. *p<0.05, ***p<0.001 vs placebo. BODY.RESULTS.CORRELATION BETWEEN E-RS AND SYMPTOM QUESTIONNAIRES: When all treatment groups were combined, there was significant correlation between the improvement in E-RS cough and sputum domain score and the improvement in the severity of morning cough symptoms assessed using the symptom questionnaire (r=0.684; p<0.001). Similar results were observed when the correlation between scores was assessed in each active treatment group (data not shown). BODY.DISCUSSION: This analysis is the first to specifically investigate the impact of a LAMA, or indeed any bronchodilator, on cough and sputum symptoms in patients with stable moderate-to-severe COPD. The results of the ATTAIN, ACCORD COPD I and active-comparator studies provide evidence that aclidinium is effective at reducing the severity and frequency of cough and sputum symptoms in patients with COPD, with improvements in E-RS total and E-RS cough and sputum scores as well as in evaluations of morning and night-time symptoms. These improvements were seen throughout the day and irrespective of the assessment tools used. Furthermore, aclidinium has previously been shown to be well tolerated in patients with COPD.17–19 23 Chronic cough and mucus accumulation in the airways are strongly associated with disease progression, lung-function decline and risk of adverse outcomes in patients with COPD.2–4 However, most clinical trials designed to evaluate the efficacy of COPD treatments have focused on improvements in lung function and breathlessness and reductions in exacerbation risk as clinical outcomes. The few trials that have assessed the efficacy of a LAMA on cough and sputum symptoms to date have reported negative results. In phase III studies, there was no significant difference in physicians' assessment of cough symptoms between patients receiving tiotropium and those receiving placebo.32 33 Furthermore, no effect on mucociliary clearance was observed with ipratropium or tiotropium in patients with COPD.34 35 In contrast, patients with severe COPD treated with an inhaled corticosteroid (fluticasone) and a long-acting β2-agonist (salmeterol) have been shown to report significantly reduced cough symptoms versus placebo.36 Before the current analysis, only smoking cessation has consistently been shown to reduce cough and phlegm symptoms in patients with COPD.37 38 Patients with COPD report variability in the frequency and severity of cough and sputum symptoms throughout the day, with greatest impact first thing in the morning and at night-time.10 11 39 The prevalence and severity of cough symptoms at the start of the day may relate to periods of increased activity associated with getting washed and dressed, whereas the night-time cough and sputum symptoms may be a consequence of mucus hypersecretion or reduced ciliary activity. In the analyses reported here, treatment with aclidinium significantly reduced the percentage of days and nights with symptoms of coughing and bringing up phlegm or mucus. The severity of morning and night-time cough and sputum symptoms was also found to be reduced. Treatment approaches that impact on cough and sputum symptoms throughout the whole 24-hour day may provide clinical benefits to some patients in terms of their overall well-being, particularly in the morning and night-time when patients report that these symptoms are most troublesome. In the active-comparator study, while both LAMAs had an effect, the magnitude of improvement in overall symptoms and cough and sputum symptoms was greater with aclidinium compared with tiotropium. The reasons for this are unclear; however, the fact that both LAMAs improved symptoms of cough and sputum suggests that these may be class effects. Precisely how these compounds might exert an effect on cough and sputum is unclear, but there is an emerging body of preclinical evidence suggesting that multiple pathways may be involved. For example, there is evidence that muscarinic antagonists reduce experimental cough,40 and that tiotropium and ipratropium act on TRPV1 to reduce the cough response in preclinical models.41 In addition, a 2016 preclinical study in rabbits showed that, further to their anticholinergic activity and any action on TRPV1 receptors, aclidinium and tiotropium may also have antitussive actions involving mechanoreceptors and acid-sensing ion channels.42 These studies provide preclinical evidence of LAMA antitussive activity; however, it is not yet clear how this may translate into clinical practice. Studies of capsaicin responsiveness suggested an increased cough reflex in patients with COPD;43 however, a recent study which evaluated predictors of cough frequency found no significant relationship between cough frequency and capsaicin cough reflex sensitivity.39 In contrast, cough frequency was independently associated with being a current smoker, smoking history, sputum production and neutrophilic inflammation.39 A recent study has demonstrated that M3 receptors may play a proinflammatory role in cigarette smoke-induced inflammation in animal models of COPD, suggesting another potential mechanism by which LAMAs may improve cough in patients with COPD.44 This is further supported by preclinical studies that have shown LAMAs can reduce neutrophils and inflammatory mediators, such as interleukin-6, tumour necrosis factor-α and interferon-γ, in cigarette smoke-exposed animal models.45 46 The efficacy of LAMAs to improve cough and sputum symptoms requires further investigation to determine if the effects observed with aclidinium are also seen with other drugs in this class. The E-RS and night-time symptoms of COPD questionnaires are validated tools for assessing cough and sputum symptoms in patients with COPD.24 28 The observed improvements in E-RS cough and sputum symptoms with aclidinium 400 μg in ATTAIN (0.7 decrease from baseline in 24 weeks) and the active-comparator study (0.6 decrease from baseline in 6 weeks) compare well with the recently proposed minimum clinically important difference (MCID) of ≥0.7 decrease from baseline.47 The lack of a validated MCID in the other tools used in these studies may be considered to be a potential limitation of this analysis. However, the significant correlation between improvements from baseline in E-RS cough scores and the severity of morning cough symptoms assessed using symptom questionnaires in the active-comparator study supports the clinical utility of these tools to assess cough symptoms. This study has other potential limitations. It should be stated that none of the three phase III studies reported here was powered to detect differences in cough and sputum symptoms, and the studies were not specifically designed to assess these symptoms. Furthermore, there was no prespecified minimum level of symptoms in any of the studies, meaning the population was relatively heterogeneous in terms of symptoms. Clinical trials designed specifically to assess the effects of treatments on cough and sputum symptoms in patients with COPD, using a combination of patient-reported outcomes, cough-specific quality-of-life measures and objective measures of cough and sputum symptoms, are needed to fully understand the efficacy of novel treatments on these symptoms. BODY.CONCLUSIONS: While few studies have investigated the effect of bronchodilators on cough and sputum symptoms, the results reported here suggest that in addition to improving lung function, LAMAs, such as aclidinium, can improve cough and sputum expectoration compared with placebo in patients with COPD. As cough and sputum symptoms impact negatively on overall patient well-being, controlling these symptoms may represent an important additional therapeutic benefit of this class of drugs.

TITLE: The efficacy of flapless implant surgery on soft-tissue profile comparing immediate loading implants to delayed loading implants: A comparative clinical study ABSTRACT.AIMS AND OBJECTIVES: To assess the efficacy of flapless implant surgery on soft-tissue profile and to compare the clinical outcomes of flapless implant therapy on immediate loading (IL) implants to delayed loading (DL) implants. ABSTRACT.MATERIALS AND METHODS: The study sample consisted of 20 patients who were partially edentulous in the anterior maxillary region. They were divided into two groups. In group I (IL) 10 implants were placed and immediately provisionalized and restored with a metal ceramic crown on the 14th day. In group II (DL) 10 implants were placed and loaded after 4 months. Single-piece implants were used for the IL group and two-piece implants were used for the DL group. All soft tissue parameters i.e., modified plaque index (mPI), modified bleeding index (mBI), papillary index (PPI), marginal level of soft tissue (ML) and width of keratinized mucosa (WKM) were recorded at baseline, Day 60, Day 120 and Day 180. ABSTRACT.RESULTS:: The success rate in group I was found to be 80%, which was lower than the success rate in group II which was found to be 90%. On comparison, there is no statistically significant difference in success rate between the two study groups. There was no statistically significant difference between the groups over time in parameters like mPI, mBI, ML and WKM. The mean PPI score in group II showed a significant increase from when compared to group I. ABSTRACT.CONCLUSION:: The results of this study indicated that flapless implant surgery using either immediately loading implants or DL implants, demonstrate enhancement of implant esthetics. BODY.INTRODUCTION: Dental implants are devices, usually alloplastic in nature that are surgically inserted into or onto the jawbone, which support a single prosthetic tooth and serve either as abutments or as cosmetic replacements for missing teeth. Dental implant therapy has been demonstrated to be a highly successful and predictable treatment modality for replacement of missing teeth. Flapless or minimally invasive surgery offers clinicians the possibility of placing implants in less time, without extensive flaps, and with perceived less bleeding and postoperative discomfort for the patient. Medicine has implemented minimally invasive procedures for prostate, abdominal, orthopedic and other types of surgery. Minimally invasive surgery reduces bleeding and discomfort.[1] The favorable outcomes using the immediate loaded (IL) implants enabled the clinicians to broaden the arena of implant dentistry enhancing esthetic and functional outcomes in addition to osseointegration. The accelerated treatment time and less surgical intervention via IL implants and one-stage surgical approach can significantly enhance patient comfort, satisfaction and acceptance. Francetti et al. demonstrated high bone-to-implant contacts ranging from 78 to 85% using the IL implants in edentulous mandibles.[2] Chiapasco et al. compared the effect of IL with DL of implants and concluded that no significant difference was present between the two groups, suggesting that IL is not detrimental for osseointegration.[3] With the advancement of dental implant therapeutics, the current trend is more geared toward enhancing patient esthetics and patient comfort and satisfaction. Van der Zee reported postsurgical tissue loss following flap reflection in the two-stage procedure of implant placement, implying that flap surgery for implant placement may negatively influence implant esthetic outcomes especially in the maxillary anterior region.[4] William et al. suggested that implants placed without flap reflection remain stable and exhibit clinically relevant osseointegration similar to implants placed with flapped procedures.[1] The purpose of this study is to examine the soft-tissue profile changes of single-tooth implants in the premaxillary region after flapless implant surgery comparing IL implants to DL implants. BODY.MATERIALS AND METHODS: The present clinical study was carried out in the Department of Periodontology and Implantology, Meenakshi Ammal Dental College and Hospital, Chennai, after obtaining approval from the ethical committee. The study sample consisted of 20 patients with missing teeth in the premaxillary region. They were randomly selected and divided into two groups (group I and group II) of 10 each. In group I, single-piece implants and in group II two-piece implants were placed using flapless implant surgery. The criterion for inclusion was partially edentulous patients with missing teeth in the premaxillary region and patients with natural teeth present mesial and distal to the edentulous spaces. Patients with complicated medical history such as uncontrolled diabetes, bleeding disorders, osteoporosis, radiation therapy and smoking were excluded from the study. Also patients with untreated chronic periodontitis and patients with history of bruxism were omitted from the study. BODY.STUDY DESIGN.PRESURGICAL PROCEDURE: Selection of patients was followed by full-mouth scaling, root planing and oral hygiene instructions. Informed consent was obtained after explaining the proposed nature of the study. Orthopantomograms and intraoral periapical radiographs (IOPA) were taken to assess the quality and quantity of bone around the proposed implant sites. Casts and surgical stents were fabricated for all the patients. All clinical parameters i.e., modified plaque index (mPI), modified bleeding index (mBI), papillary index (PPI), marginal level of soft tissues (ML) and width of keratinized mucosa (WKM) were recorded on Day 0. A single-piece and two-piece implant system (LifeCare Devices Private Limited) was used for this study. These implants are well-suited for IL protocol and DL protocol. They are available in diameters ranging from 2.8 to 5.0 mm and from lengths 10 to 16 mm. BODY.STUDY DESIGN.SURGICAL PROCEDURE: The surgical field was prepared using betadine solution and the areas were anesthetized using 2% xylocaine hydrochloride with adrenaline (1:200000). In the recipient implant sites, guided by the surgical stent, implants were placed using flapless technique. A small round bur was used to enter the osteotomy site. Pilot drill was then used to establish the depth and align the long-axis of the implant recipient site. Then a series of drills were used sequentially, according to the implant site selected to widen the osteotomy site. Implants were then placed into the site. Single-piece implants were used for IL and two-piece implants were used for DL group. BODY.STUDY DESIGN.POSTSURGICAL PROCEDURE.GROUP I -IL: IOPA were taken. For the site to be IL (group I) an acrylic provisional restoration was given. The patient received amoxicillin 500mg, three times a day for 5 days and ibuprofen 400 mg thrice a day for three days. In this group metaloceramic restorations were given on the 14th day. On the Day 60, Day 120 and Day 180 all clinical parameters were checked and IOPAs were taken [Figure 1c–g]. Figure 1Group I (IL) - (a)Pre-operative; (b) Immediate post-operative; (c) Provisional prosthesis placed; (d) Final prosthesis placed on day 14; (e) Final prosthesis on day 60; (f) Final prosthesis on day 120 ;(g) Final prosthesis on day 180 BODY.STUDY DESIGN.POSTSURGICAL PROCEDURE.GROUP II -DL: IOPAs were taken. For the DL all the same procedures were carried out as group I and appropriate instructions were given, except for temporization of the implant site. Instead of a temporary crown, a healing cap was placed over the implant and permanent crowns were delivered 4 months after implantation. All clinical parameters were recorded and IOPAs were taken on the Day 60, Day 120 and Day 180 [Figure 2d–h). Figure 2Group II (DL) - (a) Pre-operative; (b) Immediate post-operative; (c) Healing cap placed; (d) Second stage surgery; (e) Gingival former placed; (f) Abutment placed; (g) Final prosthesis on day 120; (h) Final prosthesis on day 180 BODY.STUDY DESIGN.CLINICAL PARAMETERS.MPI[: 0: No plaque 1: Plaque detected by probe 2: Plaque visible to naked eye 3: Abundant plaque BODY.STUDY DESIGN.CLINICAL PARAMETERS.MBI[: 0: No bleeding 1: Isolated bleeding spots 2: Confluent blood line 3: Heavy bleeding BODY.STUDY DESIGN.CLINICAL PARAMETERS.PPI[: Measurements were made from the reference line connecting the highest gingival curvatures of implant crown restoration and adjacent tooth on buccal side. 0: No papilla 1: Less than half 2: More than half but not complete fill 3: Complete fill 4: Overfill BODY.STUDY DESIGN.CLINICAL PARAMETERS.ML[: It was measured from the reference line drawn fromthe free gingival margins of adjacent teeth. The tissue levelapical to the reference line was recorded as positive,whereas a negative value was given when the tissue levelwas coronal to the reference line (Figure 3]. Figure 3Measurement of marginal level (ML) of soft tissue BODY.STUDY DESIGN.CLINICAL PARAMETERS.WKM: The width of the peri-implant keratinized tissue calculated by painting the gingiva with Schiller's potassium iodide at the implant site [Figure 4a–c]. Figure 4Measurement of Width of Keratinised Mucosa (WKM) 0: > 2 mm 1: 1 mm 2: 0 mm BODY.STUDY DESIGN.STATISTICAL ANALYSIS: Mean and standard deviation were estimated from the sample for each study group. Mann-Whitney's U-test was used to calculate intergroup variations. Wilcoxon signed rank test was used to calculate intragroup variations Single piece implants were used for IL [Figure 1a–b] and two piece implants were used for DL group [Figure 2a–c]. BODY.RESULTS: The sample for the study included 20 subjects who were randomly divided into two groups (groups I and II). In group I, single-piece and in group II two-piece implants were placed using flapless implant surgery. Group I (IL) consistedof 10 patients where implants were placed and immediately loaded. Group II (DL) consisted of 10 patients where the implants were placed and loaded after 4 months. The success rate in group I was found to be 80%, which was lower than the success rate in group II which was found to be 90%. On comparison, there was no statistically significant difference in success rate between the two study groups [Table 1]. Table 1 Comparison of clinical parameters between groups It was also inferred that there is no significant difference in mean mPI, mean mBI, WKM and ML of soft tissue between group I and group II at different time point's viz., at Day 0, Day 60, Day 120 and Day 180 [Tables 1 and 2]. Table 2 Comparison of clinical parameters (ML) On comparison the difference in mean PPI score between the groups on Day 60 was statistically significant and the mean change in PPI score from Day 60 to Day 180 between the groups was statistically significant [Table 1]. BODY.DISCUSSION: Flapless implant surgery has been suggested as one possible treatment option for enhancement of implant esthetics in the anterior maxilla. Flapless implant surgery requires penetration of the alveolar mucosa and bone without the reflection of mucoperiosteal flaps.[8] Flapless or minimally invasive implant surgery offers the clinician, the possibility of placing implants in less time, without extensive flaps, less bleeding and less patient discomfort.[4] There have been reports of postsurgical tissue loss from flap reflection, implying that flap reflection may have a negative influence on soft tissue profile, especially in the anterior maxilla. Concerns in flapless implant surgery are that tissues might be forced into the osteotomy site, potentially compromising osseointegration, but conclusions of a histological study showed that flapless implant placement is as biologically successful as placement of implants following mucoperiosteal flap reflection and does not produce any deleterious effects.[1] However, the clinician should keep in mind that patients treated with flapless implant surgical approach must be carefully planned and have sufficient bone volume for implant placement. Earlier studies reported that implants must be in a load-free condition for some months because micromotion at the bone-implant interface produced a fibrous repair.[9] However, now it is believed that IL of dental implants will be of clinical interest and of great benefit to the patient. Immediate loading of a dental implant not only includes a non-submerged; one-stage surgery but also actually loads the implant with a provisional restoration at the same time or shortly thereafter.[3] Earlier reports with IL implants were unpredictable; however, recent studies have showed encouraging results. In our study in group I, two of the 10 IL implants failed to osseointegrate leading to an 80% success rate. Previous studies also reported a wide range of failure rates in IL single tooth implants ranging from 0 to 19%.[10] The failure in the IL implants can be attributed to poor quality of bone, which could not be assessed as flapless technique was used and compromised healing phase. Another reason for failure in the IL group maybe due to excessive occlusal forces applied by the patient, by not complying with the postoperative instructions after loading of the implant. In an earlier study with no implant failure permanent crowns were delivered 6 months after use of temporary crown, unlike this study where permanent restorations were delivered on the 14th day.[11] In group II, one of the 10 implants placed using DL protocol failed, leading to 90% survival rate. The failure to osseointegrate might be due to inability to assess bone quality as flapless technique was used or improper angulations during placement of the implant using flapless technique. This is in contrast with most of the studies which show high success rate with DL protocol. The mPI and mBI were recorded using the index described by Mombelli A et al.[5] When the mean score between the groups were compared at different time points, there was no statistically significant difference. This is in accordance with other studies by Buser et al.[12] and Oh et al.[7] In general, the patients performed good home care and maintained good oral hygiene. Patients were informed about the subsequent consequences of plaque accumulation around implants and they were motivated to maintain oral hygiene by demonstrating oral hygiene techniques at every visit. The PPI was evaluated using the index described by Jemt et al.[6] Generally, the cause for papilla reduction after implant placement could be due to elevation of adjacent papilla during implant surgery.[4] A clinical study by Gomez Roman G showed that the elevation of adjacent papilla caused more bone loss compared to a technique that does not include the papilla.[13] This was minimized by using a flapless approach in our study. The interdental papilla height increased 2 months after IL implant therapy, which might have resulted from tissue remodeling after surgery and reformation of biological width. This is similar to a previous randomized controlled clinical trial by Oh et al.[7] In the DL group, papilla growth was observed after 4 months. This can be attributed to the fact that the permanent restorations for DL group were delivered 4 months after implant surgery. This corresponds to an 18-month follow-up study by Jemt et al.[6] The MLs were measured from the reference line drawn from the free gingival margin of adjacent teeth. No significant changes occurred in ML from baseline to 6 months. These results are similar to the conclusions of a study by Oh et al.[7] The WKM was measured by painting the mucosa with Schiller's potassium iodide which stains the keratinized tissue pale yellow. When the mean width of keratinized mucosa between group I and group II were compared at different time points, there was no statistically significant difference. This is in accordance with other studies by Buser et al.[12] and Oh et al.[7] which showed similar results. The level of the facial mucosa is an important soft tissue parameter for the esthetic outcome, and it has gained increasing attention in recent years. To achieve a correct mucosa level on the facial aspect, two prerequisites need to be fulfilled according to Kan et al. which are that the implant has to be correctly positioned in the orofacial and corono-apical directions and the mucosa must be supported by a facial bone wall of sufficient height and thickness, because the peri-implant mucosa has a rather constant dimension of 3.5-4.5 mm on the facial aspect.[14] With the rapid advancement of dental implant therapeutics, the current trend is enhancing patient comfort and esthetics.[15] Therefore, flapless implant therapy can be used to preserve soft tissue profile and increase patient comfort and satisfaction. However, flapless implant surgery has its limitations such as inability for the clinician to directly assess bone quality during implant placement, which may lead to implant failure. The present study has several limitations. The study did not follow a split-mouth design which would have ensured minimizing the effects of interpatient variability. Other limitations include small sample size, short-term follow-up and failures in the IL group. More studies with larger sample size and longer follow-up periods are required to understand the effect of flapless implant surgery on soft-tissue profile. BODY.CONCLUSIONS: The results of this study indicate that flapless implant surgery using either IL or DL implants, demonstrate enhancement of implant esthetics. Long-term clinical trials with high level of evidence, adequate sample size and comparison group (i.e., implant surgery with flap surgery) are required to verify the conclusions of this study.

TITLE: Long-term effects of laparoscopic sleeve gastrectomy versus roux-en-Y gastric bypass for the treatment of Chinese type 2 diabetes mellitus patients with body mass index 28-35 kg/m ABSTRACT.BACKGROUND: To compare long term effects of two bariatric procedures for Chinese type 2 diabetes mellitus (T2DM) patients with a body mass index (BMI) of 28-35 kg/m2. ABSTRACT.METHODS: Sixty four T2DM patients with Glycated hemoglobin A1c (HbA1c) ≧ 7.0 % were randomly assigned to receive laparoscopic sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB) procedure. Weight, percentage of excess weight loss (%EWL), BMI, waist circumference, HbA1c, fasting blood glucose (FBG), and C-peptide were measured. Serum lipid levels were also measured during three-year postoperative follow-up visits. ABSTRACT.RESULTS: Fifty five patients completed the 36-month follow-up. Both groups had similar baseline anthropometric and biochemical measures. At the end point, 22 patients (78.6 %) in SG group and 23 patients (85.2 %) in RYGB group achieved complete remission of diabetes mellitus with HbA1c < 6.0 % (P = 0.525) and without taking diabetic medications, and 25 patients in each group (89.3 % vs. 92.6 %) gained successful treatment of diabetes with HbA1c≦6.5 % (P = 0.100). Change in HbA1c, FBG and C peptide were comparable in the two groups. The RYGB group had significantly greater weight loss than the SG group [percentage of total weight loss (%TWL) of 31.0 % vs. 27.1 % (P = 0.049), %EWL of 92.3 % vs. 81.9 % (P = 0.003), and change in BMI of 11.0 vs. 9.1 kg/m2(P = 0.017), respectively]. Serum lipids in each group were also greatly improved. ABSTRACT.CONCLUSION: In this three-year study, SG had similar positive effects on diabetes and dyslipidemia compared to RYGB in Chinese T2DM patients with BMI of 28-35 kg/m2. Longer term follow-ups and larger sample studies are needed to confirm these outcomes, however. BODY.BACKGROUND: Obesity and type 2 diabetes mellitus (T2DM) are two of the most common metabolic disorders in the world. Both have significantly increased during the last decades [1, 2]. In China, the prevalence of obesity and T2DM is similar to the worldwide statistics. In China it is estimated that the number of people with diabetes was 98.4 million 2013 and will reach 142.7 million by 2035 [2]. Bariatric procedures are superior to conservative therapies in managing T2DM [3, 4]. Roux-en-Y gastric bypass (RYGB) is the most commonly supported procedure that can cure most T2DM in morbidly obese patients [3, 5, 6]. Sleeve gastrectomy (SG), a novel technique, is highly effective in the treatment of severe or morbid obesity [7, 8]. It is still controversial, however, whether SG has the same positive outcomes on T2DM in mild obese patients compared to RYGB [9, 10]. Importantly, most of the Chinese T2DM patients that have been studied have BMI less than 35 kg/m2 and are newly detected diabetes cases with short disease durations [11]. Other relevant reports about long term effects of SG on Chinese diabetes with BMI of 28-35 kg/m2 are scarce. The aim of this study was to compare the long term efficacy of SG and RYGB in Chinese T2DM patients with BMI of 28-35 kg/m2 using a prospective randomized trial over 36 months post-operatively. BODY.METHODS: We designed a prospective randomized study to determine whether SG is as effective as RYGB for T2DM remission in Chinese patients with BMI of 28-35 kg/m2 and a short history of disease. The study was conducted in Department of Gastrointestinal Surgery of the 1st affiliated hospital and Jihua hospital of Jinan University, Guangzhou, China. The trial was conducted from July 1, 2009 through July 30, 2014. The human ethics committee of Jinan University approved and supervised the whole study. BODY.METHODS.PATIENTS: Sixty-four patients enrolled in this study. Inclusion criteria included: (a) diagnosis of poorly controlled T2DM after 6 months medicine therapy [glycated hemoglobin A1c (HbA1c) level ≥7.0 %], (b) measured BMI of ≥28 and ≤ 35 kg/m2, (c) aged 25 to 60 years old, (d) diabetes duration of less than ten years, and (e) patients were excluded if they had undergone previous bariatric surgery or other complex abdominal surgery or if they had poorly controlled medical problems. Patients were also excluded if they had C-peptide levels below 0.8 ng/ml. In addition to the assessments for inclusion, each patient was assessed for their general condition and mental status, complications of obesity and diabetes mellitus, risk factors, and motivations for surgery (Fig.1). A computer-generated variable block schedule was used for randomization. Allocation to treatments was not concealed and patients knew which procedure they were to undergo.Fig. 1Flow diagram of patients in the study BODY.METHODS.SURGICAL INTERVENTIONS: In order to reduce the differences in surgical techniques, the same team and the same physician (Cunchuan Wang) carried out the operations for both groups. Described briefly, the surgical methods were as follows: For laparoscopic SG, four trocars were placed and 38 Fr. Bougie was used to calibrate the sleeve. The greater curvature was cut out 4 cm from the pylorus using a linear stapler towards His angle to completely remove the fundus of the stomach. The remnant gastric cutting edge was 2 cm from the lesser curvature of stomach. Then, the cutting edge was continuously sutured with 3-0 absorbable sutures, which are good for hemostasis. For laparoscopic RYGB, 5 trocars were used. The volume of gastric pouch was approximate 10-20 ml. The length of the biliopancreatic limb was 25 cm, and the Roux limb was 125 cm. The anastomotic stoma between stomach and jejunum was 1.5 cm and 6 cm between jejuna. During the operation, no routine stomach and drainage tubes were placed. Patients fasted the first day post-operation and followed a complete liquid and a soft diet for one month. Subsequently, patients followed a half liquid diet for three months and gradually arrived at a general diet. The patients received follow-up examinations in an outpatient clinic, Patients took a proton pump inhibitor and gastric mucosa protective agent for six weeks post-operation. In addition, the patients routinely took multivitamin supplementation and calcium tablet for a long period. The vitamin status was not checked regularly. BODY.METHODS.FOLLOW UP AND DATA COLLECTION: In one year post operation, the patients attended the visit every three months, and half-yearly thereafter. We collected the patients' height, body weight, BMI, waist circumference, usage of medication and adverse events. The laboratory test included HbA1c, FBG, C-peptide, and serum lipid profiles. BODY.METHODS.STUDY END POINTS: The primary outcome was glycemic control with HbA1c values less than 6.0 % in addition to fasting plasma glucose levels less than 7.0 mmol/L without glycemic agents at the 36-month visit. Secondary outcome measures included the percentage of weight loss and improvement of dyslipidemia. Any adverse events were also recorded. BODY.METHODS.STATISTICAL ANALYSIS: As previous study has shown a remission rate in RYGB group of 80 % [12], we assumed that SG would lead to a lower remission rate of 40 % in the lower BMI patients. Using a sample size of 64 patients (32 per group), we would have had the power to detect this difference with an ɑ level of 0.05 and power of 90 %. All analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, Illinois). Chi-square and t-tests were used to compare differences between two groups. Continuous variables were reported as means with standard deviation. A 2-sided P value of <0.05 was considered statistically significant. BODY.RESULTS.PATIENT CHARACTERISTICS: Nine (14.1 %) patients failed to finish the whole 36 months follow-up, and this included four from SG group and five from RYGB group. The patients' characteristics at baseline are summarized in Table 1. Both groups had similar baseline anthropometric measurements, including age, gender, weight, height, BMI, waist circumference, duration of diabetes, and medication usage conditions (Table 1). Baseline values of HbA1c (8.5 % vs. 8.9 %, P = 0.321), FBG (10.2 vs. 10.4 mmol/L, P = 0.700), and C-peptide (2.2 vs. 2.6 ng/ml, P = 0.062) in the SG group were comparable to the RYGB group. The two groups also had similar baseline serum lipid levels that included cholesterol, triglyceride, HDL, and LDL.Table 1Baseline patients characteristicsCharacteristicSG (n = 32)RYGB (n = 32) P valueDemographic, mean (SD)Age (yrs)40.4 ± 9.441.4 ± 9.30.681Sex, female-no.(%)23 (71.9)19 (59.4)0.292Height (cm)166.8 ± 6.8170.3 ± 8.60.077Weight (kg)88.4 ± 6.894.3 ± 13.30.055Body mass index (kg/m2)31.8 ± 3.032.3 ± 2.40.374Waist circumference (cm)103.0 ± 7.7104.5 ± 6.80.404Duration of diabetes (yrs)4.0 ± 1.74.2 ± 1.90.710Glycemia, mean (SD)HbA1c (%)8.5 ± 1.28.9 ± 1.30.321FBG (mmol/L)10.2 ± 2.710.4 ± 2.20.700C-peptide (ng/ml)2.2 ± 0.72.6 ± 1.00.062Serum lipids, mean (SD)Cholesterol (mmol/L)5.0 ± 1.14.6 ± 0.90.092Triglyceride (mmol/L)3.2 ± 1.73.0 ± 2.00.545HDL (mmol/L)1.1 ± 0.21.0 ± 0.10.067LDL (mmol/L)3.8 ± 1.13.9 ± 0.90.702Medication usage-no.(%)Oral hypoglycemic31 (96.9)30 (93.8)0.554Insulin usage15 (46.9)18 (56.2)0.453Antihypertension10 (31.2)12 (37.5)0.599Lipid-lowering drug21 (65.6)18 (56.2)0.442 BODY.RESULTS.SURGICAL TREATMENTS AND COMPLICATIONS: All procedures were successfully performed by laparoscopic techniques. The surgical time was shorter for the SG group than the RYGB group (58.0 vs.103.8 mins, P = 0.000). The mean post-operative hospital stay was 5.2 days for the SG group and 6.6 days for the RYGB group (P = 0.000). There were no deaths or major complications in either group. Minor complications occurred in 3 of 55 patients (5.5 %), including 2 gastroesophageal reflux cases in the SG group and 1 case of anemia in the RYGB group. All cases with complications were resolved with medications. The case with anemia was cured with ferralia and vitamin B12 for a long term. BODY.RESULTS.TREATMENT EFFECTS: Primary and secondary outcomes at 36 months are shown in Table 2. 22 patients (78.6 %) in SG group and 23 patients (85.2 %) in RYGB group achieved complete remission of diabetes mellitus with HbA1c < 6.0 % (P = 0.525) and without taking antidiabetic medications, and 25 patients in each group (89.3 % vs. 92.6 %) gained successful treatment of diabetes with HbA1c ≤ 6.5 % (P = 0.100). Meanwhile, at study end point, 27 patients in SG group and 28 in RYGB group stopped receiving oral hypoglycemic agents, and 13 patients in the SG group and 18 patients in the RYGB group no longer needed insulin injections.Table 2Outcomes at 36 monthsVariableSG (28)RYGB (27) P ValuePrimary outcome-no.(%)HbA1c ≤ 6.5 % without medications25 (89.3)25 (92.6)1.000HbA1c ≤ 6.5 % with medication2 (7.1)1 (3.7)1.000HbA1c < 6.0 % without medications22 (78.6)23 (85.2)0.525HbA1c < 6.0 % with medications1 (3.6)0 (0)1.000Glycemia, mean (SD)HbA1c (%)5.9 ± 0.75.7 ± 0.70.334Change from baseline (%)2.7 ± 1.13.1 ± 1.30.175FBG (mmol/L)5.9 ± 0.75.8 ± 0.70.371Change from baseline (mmol/L)4.3 ± 2.74.8 ± 2.00.448C-peptide (ng/mL)1.7 ± 0.51.8 ± 0.60.285Change from baseline (ng/mL)0.5 ± 0.50.7 ± 0.40.060Weight, mean (SD)%TWL27.1 ± 7.131.0 ± 7.10.049%EWL81.9 ± 14.092.3 ± 10.50.003Weight (kg)63.3 ± 7.964.4 ± 8.90.610Change from baseline (kg)24.3 ± 6.529.5 ± 8.90.017BMI (kg/m2)22.8 ± 1.722.0 ± 1.10.032Change from baseline (kg/m2)9.1 ± 2.711.0 ± 3.20.017Waist circumference (cm)81.2 ± 3.679.2 ± 3.10.029Change from baseline (cm)21.6 ± 10.825.0 ± 6.30.166Serum lipids, mean (SD)Cholesterol (mmol/L)3.9 ± 0.73.8 ± 0.80.674Triglyceride (mmol/L)1.5 ± 0.61.4 ± 0.60.310HDL (mmol/L)1.5 ± 0.31.7 ± 0.40.105LDL (mmol/L)2.2 ± 0.71.9 ± 0.70.120Medication usage-no.(%)Oral hypoglycemic agents4 (14.3)2 (7.4)0.700Insulin usage2 (7.1)00.488Antihypertension agent5 (17.9)3 (11.1)0.744Lipid-lowering drug3 (10.7)1 (3.7)0.630 Each group had significant weight loss compared to baseline in the follow-up. At each visit time, percentage of total weight loss (%TWL), %EWL and change in BMI were greater in the RYGB group compared to the SG group. The most weight loss time point was two-year post operation in both groups, and after that maintained the weight reduction outcomes (Fig. 2).Fig. 2Percentage of excess weight loss are plotted for the 3, 6, 9, 12, 18, 24 and 36 month time points. Error bars indicate 95 % CIs; P values for differences are all <0.05Fig. 3Values of HbA1c% (a), changes of HbA1c% (b) and fasting blood glucose (c) are plotted for the 3, 6, 9, 12, 18, 24 and 36 month time points. Error bars indicate 95 % CIs; P values for differences are all < 0.05 At three-year post operation, HbA1c were similar in the two study groups (5.9 vs. 5.7 mmol/L, P = 0.334). At 3-month and 6-month visiting post operation, HbA1c values were much lower for RYGB group than SG group, and meanwhile reductions of HbA1c were more significant for RYGB group (P < 0.01). After that, the values of HbA1c and changes of HbA1c were similar in the two groups (P > 0.05) (Fig. 3ab). FBG levels were comparable for the SG and RYGB groups at all-time points (Fig. 3c ). In both groups, HbA1c and FBG levels were significantly improved after 3 months (P < 0.05), and the improvements were maintained through the 36-month evaluation. Compared to the baseline, post-operative serum lipid levels in each group were significantly improved. The serum levels of cholesterol, triglyceride, HDL, and LDL were similar at each time point for the SG group compared to the RYGB group. 35 patients (18 from SG group, and 17 from RYGB group) no longer needed lipid-lowering medications and 14 patients (5 from SG and 9 from RYGB group) no longer needed antihypertensive medications at the 36-month follow-up. BODY.DISCUSSION: Bariatric surgery has favorable effects on obesity and related metabolic problems. Of available procedures, Roux-en-Y gastric bypass is a common choice. For T2DM patients with severe obesity and BMI over 35 kg/m2, a large number of studies have shown that both sleeve gastrectomy and RYGB procedures have favorable effects [5, 6, 13, 14]. As for T2DM patients with mild obesity, gastric bypass surgery has also shown to be effective. However, it is still controversial whether sleeve gastrectomy has the same effect for the lower BMI patients [10, 15, 16]. In Asian and Chinese populations, obesity related health risks are observed in people with BMI as low as 22 to 23 [17]. Compared to Caucasians with the same BMI, Chinese populations have significantly higher levels of subcutaneous and visceral fat, which corresponds to higher risk of cardiovascular and metabolic diseases. Thus, BMI used for diagnosing obesity in Asian and Chinese populations should be lower than in western populations [18, 19]. The Asian branch of ASMBS suggests that when treating T2DM patients with bariatric surgery, BMI should be lowered appropriately, and T2DM patients with BMI over 28 kg/m2 should be enrolled in clinical studies [20]. To our knowledge, there are rare studies onT2DM patients with BMI of 28-35 kg/m2 in the Chinese mainland. At the same time, more studies have shown that early bariatric surgical intervention can enhance the remission rates of T2DM [21, 22]. Therefore, the subjects in this study are mildly obese T2DM patients with BMI of 28-35 kg/m2 and disease histories of less than 10 years. The results of this study show that three years after operation, both SG and RYGB procedures were effective in weight reduction and remission of T2DM. RYGB had significantly better effects on %TWL, %EWL, and BMI change when compared with SG, which is consistent with previous studies [23, 24]. Moreover, patients in both groups had normal BMI and achieved ideal weights one year after operation without major complications. Three years after operation, the complete T2DM remission rates (HbA1c < 6.0 % without taking anti-diabetic medicines) were 78.6 % in the SG group and 85.2 % in the RYGB group. The average HbA1c and FBG levels in both groups reached normal levels, indicating that the effects of SG were equivalent to RYGB in mildly obese T2DM patients. This is consistent with the previous prospective study from Andrei Keidar and the retrospective study from Sylvie Pham with the patients of BMI > 35 kg/m2 [13, 14]. However, a research outcomes from Lee et al. suggested that RYGB achieved better blood glucose control compared to SG at one and five years post-operation for the T2DM patients with BMI 25-35 kg/m2 [10, 16]. In Lee's study, BMI of the patients was relatively lower and the diabetic history was longer (RYGB 5.8 years vs. SG 6.9 years). These factors may have caused the patients to be more pancreatic insufficient than peripheral insulin resistance, and that may cause the lower remission rate of T2DM. Additionally, our study showed that in both groups, all blood lipid indexes were significantly decreased after operation in the patients with dyslipidemia. Three years after operation, the blood lipid indexes, including total cholesterol, triglyceride, LDL, and HDL, stayed at normal levels with similar degrees of decline. Meanwhile, the percentages of patients that stopped taking lipid-lowering drugs and antihypertensive drugs were the same, illustrating that both SG and RYGB have similar effects on obesity relevant metabolic disturbances. Even now, the mechanism through which bariatric surgery treats T2DM is unclear. This study investigates clinical effects but not the underling mechanism. We can see from this study, RYGB gained more significant HbA1c reduction than SG in the first 6 months after operation, and that implied RYGB improves more rapidly for T2DM. Because the RYGB operation bypasses the proximal intestine, hypotheses of its mechanism include the Ghrelin hypothesis, hindgut hypothesis, and foregut hypothesis [25, 26]. After the SG operation, insulin resistance was obviously alleviated, while the incretin hormones level was significantly increased [26–29]. Peterli et al. found that one year after surgery, RYGB ghrelin levels approached preoperative values while SG ghrelin levels were still markedly attenuated. Meanwhile cholecystokinin concentrations after test meals increased less in the RYGB group than in the SG group. They suggested that bypassing the foregut is not the only mechanism responsible for improved glucose homeostasis [30]. Schauer PR et al. concluded that weight loss and a shorter duration of diabetes were the main predictors of having a glycated hemoglobin level of 6.0 % or less after surgery [31]. Our study suggests that both groups obtained similar diabetic remission rate but different weight loss effects 3 years after operation. The relationship between glycemic control and weight loss needs to be further investigated. This comparative study on clinical effects has some limitations that include lack of data collection on insulin resistance alleviation degree and lack of gastrointestinal GLP-1, GIP, and PYY hormones data collection. These data would help to determine the surgical mechanism for T2DM resolution in Chinese patients with BMI of 28-35 kg/m2. In addition, three-year follow-up is not long enough to assure that RYGB or SG can completely alleviate T2DM. Therefore, a longer period of follow-up is required. BODY.CONCLUSION: Through three-year clinical data analysis, it can be concluded that for Chinese mildly obese T2DM patients with BMI of 28-35 kg/m2, SG had similar effects to RYGB in remission of T2DM and metabolic disorders, but a longer follow-up period is still required to confirm the long-term effects.

TITLE: Procalcitonin Guidance to Reduce Antibiotic Treatment of Lower Respiratory Tract Infection in Children and Adolescents (ProPAED): A Randomized Controlled Trial ABSTRACT.BACKGROUND: Antibiotics are overused in children and adolescents with lower respiratory tract infection (LRTI). Serum-procalcitonin (PCT) can be used to guide treatment when bacterial infection is suspected. Its role in pediatric LRTI is unclear. ABSTRACT.METHODS: Between 01/2009 and 02/2010 we randomized previously healthy patients 1 month to 18 years old presenting with LRTI to the emergency departments of two pediatric hospitals in Switzerland to receive antibiotics either according to a PCT guidance algorithm established for adult LRTI or standard care clinical guidelines. In intention-to-treat analyses, antibiotic prescribing rate, duration of antibiotic treatment, and number of days with impairment of daily activities within 14 days of randomization were compared between the two groups. ABSTRACT.RESULTS: In total 337 children, mean age 3.8 years (range 0.1–18), were included. Antibiotic prescribing rates were not significantly different in PCT guided patients compared to controls (OR 1.26; 95% CI 0.81, 1.95). Mean duration of antibiotic exposure was reduced from 6.3 to 4.5 days under PCT guidance (−1.8 days; 95% CI −3.1, −0.5; P = 0.039) for all LRTI and from 9.1 to 5.7 days for pneumonia (−3.4 days 95% CI −4.9, −1.7; P<0.001). There was no apparent difference in impairment of daily activities between PCT guided and control patients. ABSTRACT.CONCLUSION: PCT guidance reduced antibiotic exposure by reducing the duration of antibiotic treatment, while not affecting the antibiotic prescribing rate. The latter may be explained by the low baseline prescribing rate in Switzerland for pediatric LRTI and the choice of an inappropriately low PCT cut-off level for this population. ABSTRACT.TRIAL REGISTRATION: Controlled-Trials.com ISRCTN17057980 ISRCTN17057980 BODY.INTRODUCTION: Lower respiratory tract infection (LRTI) is a leading cause of morbidity and mortality in children and adolescents worldwide; pneumonia is the number one cause of childhood mortality worldwide, and in Europe accounts for 9% of deaths in children under 5 years of age. Depending on age and diagnostic methodology, a bacterial etiology has been shown to occur in 33 – 70% of pneumonia in children. The lack of clinical, radiological, and laboratory tests to safely rule out bacterial involvement in LRTI still drives antibiotic treatment today. A reduction of antibiotic exposure in children with LRTI could be expected to have an impact on antibiotic consumption and the development of antibiotic resistance worldwide [1]–[11]. Procalcitonin (PCT) guided treatment for respiratory tract infections has been shown to markedly reduce antibiotic exposure in adults [12]–[18]. Smaller, single center trials have suggested that PCT may be helpful in the pediatric patient population [19], [20]. The purpose of the ProPAED trial was to investigate whether PCT guided treatment can reduce the antibiotic prescribing rate and the duration of antibiotic treatment in children and adolescents with LRTI presenting to an emergency department using the cut-off ranges successfully established in adults. BODY.METHODS.TRIAL DESIGN AND PARTICIPANTS: The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Protocol S1 and Checklist S1, respectively. We included all children and adolescents, 1 month to 18 years of age, presenting with LRTI to the emergency departments of two pediatric hospitals in Switzerland (Basel, Aarau) between 01/2009 and 02/2010 regardless of antibiotic treatment history. Patients were excluded if they or their care-takers were unwilling to participate or were unable to give written informed consent due to language problems. Additional exclusion criteria were severe immune suppression (HIV infection with a CD4 count <15% of normal age-specific counts), immunosuppressive treatment, neutropenia (<1000×109/L), cystic fibrosis, acute croup, hospital stay within previous 14 days, or other severe infection. The trial was approved by both the ethics committee of the University Basel and Kanton Aargau and conducted according to the principles of good clinical practice, and supervised by a steering committee and an independent data safety monitoring board. The trial is registered with the International Standard Randomized Controlled Trial Number (ISRCTN) register (number 17057980). Acute LRTI was defined as the presence of fever (core body temperature ≥38.0° C measured in hospital or at home) and at least one symptom (cough, sputum production, pleuritic pain, poor feeding) and at least one sign (tachypnea, dyspnoea, wheezing, late inspiratory crackles, bronchial breathing, pleural rub) for less than 14 days. In the case of fever, poor feeding and tachypnea without other signs, persistence of tachypnea following effective antipyretic treatment was required. Community acquired pneumonia (CAP) was defined as acute febrile LRTI with a new or increasing alveolar infiltrate on chest radiograph as assessed by the attending pediatrician. Non-CAP LRTI (i.e. bronchitis, bronchiolitis) was defined as acute febrile LRTI presenting with hyperinflation or new or increased peribronchial infiltrates without alveolar infiltrates on chest radiograph. Fever was considered a necessary sign, because antibiotic treatment for potential bacterial LRTI would not usually be considered in a pediatric patient with an afebrile LRTI. To assess the potential of recruitment bias, all patients with LRTI seen in the emergency departments of the study hospitals during the trial were identified by retrospective chart review. BODY.METHODS.RANDOMIZATION: Eligible patients were randomly assigned to either PCT guided antibiotic treatment (PCT group) or to clinically guided standard care (control group) by a pre-specified computer-generated scheme (1:1 ratio). Patient allocation was concealed by use of web-based online patient registration. We used variable blockrandomization with stratification for the participating clinic and the type of LRTI. BODY.METHODS.PROCEDURES: After obtaining informed consent and performing randomization (day 1), blood samples for PCT, C-Reactive Protein (CRP) and full blood counts (FBC) plus nasopharyngeal aspirates were taken from all participants. Performance of chest X-ray was encouraged for all patients. Serum PCT was measured by B.R.A.H.M.S. PCT sensitive Kryptor® (B.R.A.H.M.S., Hennigsdorf, Germany), a rapid sensitive assay with a functional sensitivity of 0.06 μg/L and a lower detection limit of 0.02 μg/L with an assay time of less than 30 minutes. CRP was measured by an immunoturbidimetric assay, the Tina-Quant C-Reactive Protein Generation 3 assay (Roche Diagnostics, Mannheim, Germany) on Hitachi 912 Modular P analyzer. For this test, functional sensitivity is 0.6 mg/L and detection limit 0.3 mg/L. FBC was done by Sysmex xT-2000i and differentiation was performed manually. On days 3 and 5, patients were re-evaluated clinically and PCT measurements were repeated. BODY.METHODS.ANTIBIOTIC GUIDANCE AND ENDPOINT ASSESSMENT: In the PCT group, initiation, continuation or termination of antibiotic treatment was strictly guided by PCT cut-off levels used in previous trials in adults with LRTI [12], [14], [21]–[23]. The algorithm provides PCT based decision categories for the likelihood of requiring antibiotic treatment for bacterial LRTI: "definitely" (>0.5 μg/L), "probably" (0.26–0.5 μg/L), "probably not" (0.1–0.25 μg/L), and "definitely not" (<0.1 μg/L). The PCT algorithm could be overruled for patients with life threatening infections, defined as severe co-morbidity, emerging ICU need during initial follow-up, or hemodynamic or respiratory instability. For all patients, discontinuation of antibiotics was encouraged upon clinical stabilization and when PCT values fell below 0.25; for patients with initial PCT values >10 μg /L when levels decreased below 90% of the initial value. Continuation of treatment on day 5 was determined according to the following algorithm: >1 μg/L: 7 days, 0.51–1 μg/L: 5 days, 0.26–0.5 μg/L: 3 days, and ≤0.25 μg/L: no antibiotic. In the control group, antibiotic treatment was initiated based on physician assessment and clinical guidelines for a duration of 7–10 days for uncomplicated CAP and 14 or more days for complicated CAP, e.g., parapneumonic effusions, empyema, abscess [24]. The hospital outpatient services and responsible non-hospital based primary care pediatricians were informed about study procedures and given guidance concerning assessment of adverse events. Children 14 years of age or older, or care takers of children under 14 years of age, completed a diary from day 1 through 14 including items on antibiotic intake, consumption of other medication, hospitalization, and occurrence of standardized symptoms. A questionnaire and visual analogue scale (0 to 100%) for self-assessment of impairment of overall daily activity thought attributable to the LRTI was also distributed [25], [26]. For endpoint assessment each patient was contacted on day 14 by a study pediatrician blinded to treatment allocation of the child. Contact consisted of a structured telephone interview with the parents. Safety monitoring included assessment of complications of LRTI: occurrence of serious adverse events (SAE) or disease specific failure including hospital readmission, recurrent infection requiring antibiotics, any co-morbidity in need of antibiotics, or worsening of impairment of daily activity by ≥20% on the visual analogue scale according to parent interview and diary. BODY.METHODS.STATISTICAL ANALYSES: The primary endpoint of this trial was antibiotic prescibing rate within 14 days of randomization; secondary endpoints were (i) duration of antibiotic treatment, (ii) rate and duration of side effects of antibiotic treatment, (iii) rate and duration of hospitalization, (iv) occurrence of serious adverse events, complications of LRTI or disease specific failure, and (v) impairment of daily activity attributable to LRTI during the 14 days following randomization. For sample size calculation, we assumed that PCT guidance would reduce antibiotic prescribing from 90% to 60% and from 30% to 15% in children and adolescents with CAP and non-CAP LRTI, respectively. With a 2-sided type I error rate of α = 0.05, 64 and 242 patients with CAP and non-CAP LRTI, respectively, had to be included to attain a targeted power of 80%. Assuming that 20% of all randomized patients would have CAP, a total sample size of 320 patients was determined, giving a power of 93% to detect a decrease in antibiotic prescribing from 42% (control group) to 24% (PCT group) for all LRTI patients. In an intention-to-treat analysis, we used a two-sided chi-squared test to compare the primary endpoint (antibiotic prescribing within 14 days of randomization) between PCT and control groups. We performed this test in all LRTI patients and in the pre-specified subgroups of patients with CAP and non-CAP LRTI according to diagnosis at randomization. To compare the primary and secondary binary endpoints between PCT and control groups, we estimated the rate difference and the odds ratio by logistic regression. In this model, we additionally included an interaction term between the therapeutic group and diagnosis at randomization (CAP versus non-CAP LRTI) to obtain effect estimates of PCT guidance in the two pre-specified subgroups and to investigate differences in effect of PCT guidance between patients with CAP and non-CAP LRTI. For secondary continuous endpoints, we used the Wilcoxon rank sum test and report the estimated mean difference between PCT and control group. We used exploratory statistics to assess mean impairment of daily activity. Confidence intervals for rate differences were calculated using Newcombe's method [27], and for mean differences, using the bootstrap percentile method [28]. For our analyses and graphics, we used R version 2.14.0 (R Foundation for Statistical Computing, Vienna, Austria) and the R add-on packages epiR version 0.9–32 and boot version 1.3–3. BODY.RESULTS: Of 946 patients with LRTI, 470 were formally screened for eligibility and of those, 337 randomized patients were available for analysis (Figure 1). All eligible patients were formally screened for baseline characteristics and included patients did not differ from excluded patients. However, the study population was more likely to have CAP compared to the population not assessed for eligibility (Table S1). Follow-up was complete for 329 (98%) patients with a telephone interview after 14 days (median 14 days; interquartile range [IQR] 13–15). Clinical recovery could be confirmed for the two patients withdrawing consent and two further patients with incomplete follow-up, all in the control group. We received 208 (62%) complete and 59 (18%) incomplete diaries (median 14 missing values; IQR 10–58). Seventy diaries (21%) were not returned. 10.1371/journal.pone.0068419.g001Figure 1Trial profile. Baseline characteristics of randomized patients were similar in both groups (Table 1). Median age in the PCT and control group was 2.7 and 2.9 years respectively; 48% of children in both groups were hospitalized. In 215 (64%) children, the initial diagnosis was CAP. 10.1371/journal.pone.0068419.t001 Table 1 Patient baseline characteristics. PCT group (N = 168) * Control group (N = 169) * Demographics Age, years, Median (IQR) 2.7 (1.1–5.2) 2.9 (1.2–5.7) Male gender, N (%) 98 (58) 98 (58) Study centre, N (%) Basel 128 (76) 121 (72) Aarau 40 (24) 48 (28) Day care, N (%) At home 84 (52) (N = 162) 86 (52) (N = 167) Day care/nursery/school 78 (48) 81 (48) Siblings, N (%) Siblings 0 48 (30) (N = 158) 39 (23) (N = 168) Siblings ≥1 110 (70) 129(77) Vaccination Status, N. (%) Streptococcus pneumoniae (PCV7) 0–2x 115 (74) (N = 155) 112 (74) (N = 151) ≥3x 40 (26) 39 (26) Haemophilus influenzae type b 0–2x 28 (18) (N = 157) 27 (17) (N = 155) ≥3x 129 (82) 128 (83) Clinical history Antibiotic pre-treatment, N (%) 25 (15) 17 (10) Days of fever before presentation, Median (IQR) 2 (1–4) (N = 164) 3 (1–4) (N = 166) Fever, N (%) 168 (100) 169 (100) Cough, N (%) 167 (99) 169 (100) Sputum production, N (%) 62 (37) 79 (47) Poor feeding, N (%) 79 (47) 74 (44) Pleuritic pain, N (%) 42 (25) 53 (31) Clinical findings Body temperature, °C, Median (IQR) 38.5 (37.9–39.1) (N = 167) 38.3 (37.8–39.0) (N = 168) Respiratory rate, Median (IQR) 40 (30–48) (N = 156) 40 (28–48) (N = 164) Heart rate, Median (IQR) 144 (124–160) (N = 163) 141 (120–160) (N = 164) Tachypnea, N (%) 127 (76) 116 (69) Dyspnea, N (%) 110 (65) 107 (63) Wheezing, N (%) 53 (32) 48 (28) Late inspiratory crackles, N (%) 71 (42) 69 (41) Reduced breathing sounds, N (%) 60 (36) 49 (29) Laboratory findings, Median (IQR) PCT, ug/L 0.26 (0.14–1.06) 0.21 (0.12–2.24) CRP, mg/L 23 (8–88) (N = 162) 20 (7–55) (N = 165) Leukocyte count, cells/ul 11.9 (8.7–18.9) (N = 164) 11.3 (7.7–16.4) (N = 166) Diagnosis at randomization, N (%) Non-CAP LRTI 60 (36) 62 (37) Community-acquired pneumonia 108 (64) 107 (63) Abbreviations: PCT, procalcitonin; IQR, interquartile range; CRP, C-reactive protein; Non-CAP, non-community-acquired pneumonia; LRTI, lower respiratory tract infection. * N in this column indicate the number of individuals with information on a particular variable. In the PCT group 104 of 168 (62%) patients and in the control group 93 of 165 (56%) patients received antibiotics. The estimated difference in antibiotic prescribing rate between the PCT and the control group was 6% (95% CI −5%, 16%; P = 0.359) in all LRTI patients, 28% (95% CI 12%, 43%; P = 0.002) in the subgroup of 120 patients with non-CAP LRTI and −8% (95% CI −19%, 4%; P = 0.250) in the subgroup of 213 patients with CAP. The odds ratio (OR) of receiving antibiotic treatment within 14 days of randomization in the PCT compared to the control group was 1.26 (95% CI 0.81, 1.95) in all LRTI patients, 4.09 (95% CI 1.80, 9.93) in non-CAP LRTI patients and 0.66 (95% CI 0.35, 1.23) in CAP patients (Tables 2 and 3). The interaction term between therapeutic group (PCT versus control) and diagnosis at randomization (CAP versus non-CAP LRTI) indicated a statistically significant difference in the effect of PCT guidance on antibiotic prescribing rate between CAP and non-CAP LRTI patients (OR for interaction 0.16; 95% CI 0.06, 0.45). 10.1371/journal.pone.0068419.t002 Table 2 Efficacy and safety, primary and secondary endpoints. Outcome Measure PCT group (N = 168) Control group (N = 169) Rate difference, % (95% CI) Odds ratio (95% CI) Mean difference (95% CI) Primary endpoint * * Antibiotic prescription within 14 days of randomization N (%) 104 (62) 93 (56) (N = 165) 6 (−5, 16) 1.26 (0.81, 1.95) Secondary endpoints Duration of antibiotic treatment, days Mean (median [IQR]) 4.5 (4 [0–8]) (N = 167) 6.3 (6 [0–11]) (N = 164) −1.8 (−3.1, −0.5) Antibiotic side effects * N (%) 56 (39) (N = 144) 57 (38) (N = 149) 1 (−10, 12) 1.03 (0.64, 1.65) Duration of antibiotic side effects, days Mean (median [IQR]) 1.4 (0 [0–2]) (N = 144) 1.3 (0 [0–1]) (N = 149) 0.1 (–0.4, 0.7) Hospitalization N (%) 104 (62) 100 (60) (N = 167) 2 (−8, 12) 1.09 (0.70, 1.69) Duration of hospitalization, days Mean (median [IQR]) 2.6 (2 [0–4]) (N = 167) 2.7 (2 [0–5]) (N = 164) −0.1 (−0.8, 0.5) Safety † N (%) 38 (23) 33 (20) (N = 164) 2 (−6, 11) 1.16 (0.69, 1.97) * On days of antibiotic therapy patients showing an exanthema or vomiting or diarrhea as stated in the patient’s diary from day 1 up to day 14. † Occurrence of any of the following entities: complications from pneumonia or other LRTI (e.g., parapneumonic effusions in need of puncture, empyema, lung abscess, necrotizing pneumonitis, acute respiratory distress syndrome) or occurrence of SAEs (hospital readmission, admission to intensive care unit, unexpected life threatening condition, condition of compromising sequelae or death occurring in the 14 days following the inclusion of the patient) or disease specific failure , including hospital readmission, recurrent infection in need of antibiotics or development of any co-morbid condition in need of antibiotics irrespective of the primary LRTI diagnosis, worsening of ≥20% of daily restrictions from LRTI according to parent interview and diary, new onset of respiratory distress or worsening of pre-existing respiratory distress (i.e., tachypnea, and or dyspnea in spite of β 2 -mimetic treatment) or increasing or new onset of O 2 requirement or development of global respiratory insufficiency – increasing pCO 2 . * number of individuals with available data for a given endpoint. 10.1371/journal.pone.0068419.t003 Table 3 Subgroup analyses. Outcome Measure PCT group Control group Rate difference, % (95% CI) Odds ratio (95% CI) Mean difference (95% CI) Non-CAP LRTI (N = 60) * (N = 62) * Antibiotic prescription within 14 days of randomization N (%) 27 (45) 10 (17) (N = 60) 28 (12, 43) 4.09 (1.80, 9.93) Duration of antibiotic treatment, days Mean (median [IQR]) 2.4 (0 [0–5]) (N = 59) 1.6 (0 [0–0]) (N = 60) 0.8 (−0.5, 2.0) Antibiotic side effects N (%) 14 (26) (N = 54) 6 (10) (N = 58) 16 (1, 30) 3.03 (1.11, 9.22) Duration of antibiotic side effects, days Mean (median [IQR]) 1.0 (0 [0–0.8]) (N = 54) 0.5 (0 [0–0]) (N = 58) 0.5 (−0.2, 1.2) Hospitalization N (%) 37 (62) 32 (53) (N = 60) 8 (−9, 25) 1.41 (0.68, 2.93) Duration of hospitalization, days Mean (median [IQR]) 2.5 (2 [0–4]) 2.3 (1 [0–5]) (N = 60) 0.3 (−0.8, 1.2) Safety N (%) 15 (25) 13 (22) (N = 60) 3 (−12, 18) 1.21 (0.52, 2.85) Community-acquired pneumonia (N = 108) (N = 107) Antibiotic prescription within 14 days ofrandomization N (%) 77 (71) 83 (79) (N = 105) −8 (−19, 4) 0.66 (0.35, 1.23) Duration of antibiotic treatment, days Mean (median [IQR]) 5.7 (5 [0–9]) 9.1 (10 [4.5–12.3]) (N = 104) −3.4 (−4.9, −1.7) Antibiotic side effects N (%) 42 (47) (N = 90) 51 (56) (N = 91) −9 (−23, 5) 0.69 (0.38, 1.23) Duration of antibiotic side effects, days Mean (median [IQR]) 1.7 (0 [0–2]) (N = 90) 1.8 (1 [0–3]) (N = 91) −0.1 (−0.9, 0.6) Hospitalization N (%) 67 (62) 68 (64) −2 (−14, 11) 0.94 (0.54, 1.63) Duration of hospitalization, days Mean (median [IQR]) 2.6 (2 [0–4]) (N = 107) 2.9 (2 [0–5]) (N = 104) −0.3 (−1.1, 0.5) Safety N (%) 23 (21) 20 (19) (N = 104) 2 (−9, 13) 1.14 (0.58, 2.24) Abbreviations: PCT, procalcitonin; CI, confidence interval; Non-CAP, non-community-acquired pneumonia; LRTI, lower respiratory tract infection. * Number of individuals with available data for a given endpoint. For each diagnostic group (all LRTI, CAP, non-CAP LRTI), the proportions of patients receiving antibiotics in the PCT group compared to the control group between day 1 and 14 are shown in Figure 2. In comparison with clinical guidelines, PCT guidance reduced the duration of antibiotic treatment in LRTI patients and in the subgroup of CAP patients. The mean duration of antibiotic exposure was 4.5 and 6.3 days in the PCT and control group, respectively (mean difference −1.8 days; 95% CI −3.1, −0.5; P = 0.039) (Table 2 and Figure 3). In the subgroup of patients with non-CAP LRTI, the mean duration of antibiotic treatment was 2.4 and 1.6 days in the PCT and control group, respectively (mean difference 0.8 days; 95% CI −0.5, 2.0; P = 0.01). In patients with CAP, it was 5.7 and 9.1 days in the PCT and control group, respectively (mean difference −3.4 days; 95% CI −4.9, −1.7; P<0.001) (Table 3). 10.1371/journal.pone.0068419.g002Figure 2Antibiotic prescribing rate.Antibiotic treatment by day since randomization for all children and adolescents with lower respiratory tract infections (LRTI) and for pre-specified subgroups according to PCT guidance and control. (A) All lower respiratory tract infections; (B) Community-acquired pneumonia (CAP); (C) Bronchitis and Bronchiolitis (non-CAP LRTI). 10.1371/journal.pone.0068419.g003Figure 3Duration of antibiotic treatment.Box plots of the distribution of the duration of antibiotic (AB) treatment (in days) for children and adolescents with lower respiratory tract infection (LRTI) in the procalcitonin (PCT) and control group. Rates of side effects from antibiotic treatment, hospitalization, and the combined safety endpoint (including SAE, complications of LRTI, and disease specific failure) were similar in both study groups. The rate difference for the combined safety endpoint between PCT and control group was 2% (95% CI −6%, 11%), and the OR was 1.16 (95% CI 0.69, 1.97) (Table 2). In the subset of 267 patients who returned their diaries, mean impairment of daily activity attributable to LRTI declined during the 14 days following randomization in both PCT and control group patients, indicating no relevant difference between the two study groups (Figure 4). 10.1371/journal.pone.0068419.g004Figure 4Impairment of daily activities.Impairment of daily activities attributable to lower respiratory tract infection (LRTI) over time in 267 children and adolescents who returned diaries in the procalcitonin (PCT) and control group. The smooth curves are local averages calculated using the default loess smoother in R. BODY.DISCUSSION: Compared with clinical guidelines, PCT guidance did not reduce the antibiotic prescribing rate in children and adolescents with LRTI. However, antibiotic treatment duration was reduced. The failure to reduce the rate of antibiotic prescribing for children with LRTI may be due to several factors. First, pediatricians in Switzerland have a low rate of prescribing antibiotics in general. For example, in a preparatory study for this trial, the background antibiotic prescribing rates for LRTI, CAP, and non-CAP in children and adolescents in the greater metropolitan area of Basel were 72%, 93%, and 19%, respectively (Reppucci R, et al. unpublished data). In the present study antibiotics were prescribed for 79% of CAP and 17% of non-CAP patients in the control group, which is even lower than the 89% background rate. We interpret this observation as a Hawthorn effect introducing bias towards the null hypothesis. Second, the PCT cut-off values we used to guide decision-making on initiating antibiotic treatment, based on an algorithm successfully established in adults with LRTI, may have been too low for use in children with LRTI, especially those with non-CAP LRTI. The effect of low PCT cut-off levels would be more pronounced in patients expected to have low PCT levels close to the cut-off level, such as patients with non-CAP LRTI. In our study, although there was a trend for PCT guidance to reduce antibiotic prescribing in the CAP subgroup, there was an increased rate in the non-CAP LRTI subgroup. Regardless of subgroup, LRTI patients in the PCT group were treated with antibiotics for a shorter duration than controls. This reduction in antibiotic duration was most pronounced in the sub-group of patients with CAP. This is consistent with findings in adult patients with LRTI [12], [14], [22], [23] and in neonates treated for suspected sepsis [20]. Strengths of our study are the concealed allocation of patients and the excellent follow-up98% of patients79% return rate of diariesThe inclusion rate of CAP was higher than expectedadditional power to the most important clinical patient group. Previous studies suggested that short course antibiotic treatment in children with uncomplicated LRTI may be safe and effective [29]–[31]. Our trial indicates that PCT measurement identifies the children with complicated and uncomplicated LRTI in whom antibiotic treatment can be discontinued early even in the absence of known microbial etiology. This trial was not powered to assess safety by a non-inferiority design. However, there were favorable outcomes in all patients with no adverse effects attributable to early termination of antibiotic treatment. Although predictive determinants of the appropriate duration of antibiotic treatment for pediatric LRTI were lacking, previous studies suggest that short course antibiotic treatment in children with uncomplicated LRTI may be safe and effective [29]–[31]. PCT guidance in children with LRTI did not reduce the rate and duration of hospitalization. This is most likely due to the fact that determinants for admission were hypoxemia, failure to take oral fluids, or the need for intravenous antibiotic treatment. These factors are independent of PCT levels. In a recent single center trial in hospitalized children with CAP, which used the same PCT algorithm as in the present study, PCT guidance reduced the antibiotic prescribing rate by 14%, while 100% of patients in the control group received antibiotics (19). PCT guidance in this Italian study reduced antibiotic prescribing rates to levels comparable to our baseline rate in CAP control patients. There may have been several reasons for this. Our study population may have been more severely ill. For example, the mean PCT levels in hospitalized CAP patients in the Italian study were lower (PCT group: 1.8 ug/L; control: 1.8 ug/L) in comparison to the hospitalized CAP patients in our study (PCT group: 4.5 ug/L; control: 6.9 ug/L), in spite of using the same assay for PCT measurements. Also, in our study, all CAP patients had alveolar consolidation as assessed by the emergency care pediatrician, whereas 35 – 39% of patients in the Italian study only showed reticulo-nodular infiltrates based on the post hoc chest radiograph assessment of a single radiologist. In conclusion, our results suggest that PCT guidance of antibiotic treatment in children and adolescents with LRTI is feasible, and can contribute to a reduction in antibiotic exposure overall. Cut-off values derived from trials in adults with LRTI, however, may not be appropriate in pediatric patients with LRTI. Future research should focus on determining optimal PCT cut off values for children with LRTI to identify patients who require antibiotic treatment as well as those in whom antibiotic treatment can be withheld safely. As the baseline complication and mortality rate of pneumonia in Switzerland is low, it would be useful to demonstrate the safety of PCT guided short course treatment in pediatric populations at risk of higher rates of complications and mortality. Reducing antibiotic treatment in pediatric patients through PCT guidance could have an impact on overall antibiotic prescribing, as the burden of viral respiratory tract infections in this population is high, and there is a paucity of reliable tests to guide prudent antibiotic use [32]–[37]. BODY.SUPPORTING INFORMATION: Table S1 Characteristics of included, excluded, and missed patient populations. (DOC)Click here for additional data file. Checklist S1(DOC)Click here for additional data file. Protocol S1(DOC)Click here for additional data file.

TITLE: A comparison of the transillumination-assisted technique versus midline approach technique in novices: a prospective randomized controlled trial about the Bonfils intubation fiberscope ABSTRACT.BACKGROUND: The present study aimed to compare the safety and efficacy for novices to conduct intubation with the Bonfils intubation fiberscope (BIF) using the transillumination-assisted or midline approach technique in patients with normal airways. ABSTRACT.METHODS: In this prospective randomized control study, 10 trainees were assigned to the transillumination-assisted technique group (T group) or the midline approach technique group (R group). Each trainee was required to conduct intubation in 50 patients. The primary outcome was intubation time. The secondary outcomes were success rate (%), number of attempts, and complications. ABSTRACT.RESULTS: Among the cases of successful intubation, the intubation time was not significantly different between the two groups (P > 0.05). The overall success rate of intubation was not significantly different between the two groups (P > 0.05). The intubation success rates at the first, second, and third attempts as well as the average intubation times were similar between the two groups (P > 0.05), but in patients receiving successful intubation at the second attempt, the intubation time was longer in the T group (P = 0.0006). The incidences of dry throat, sore throat, and hoarseness were higher in the T group (all P < 0.05). ABSTRACT.CONCLUSIONS: For patients with a normal airway, the transillumination-assisted technique was unlikely to increase the success rate of intubation with the BIF compared with the midline approach technique, but led to more complications. ABSTRACT.TRIAL REGISTRATION: ChiCTR-INR-16009967, retrospectively registered on November 22, 2016 ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12871-017-0322-6) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Establishing a successful endotracheal intubation is important for pulmonary ventilation and gas exchange during anesthesia and critical care. Failed or difficult endotracheal intubation is an important cause of morbidity and mortality during anesthesia [1]. A variety of methods and tools (such as direct laryngoscope, video laryngoscopes, and fiber optic bronchoscope (FOB)) have been used for endotracheal intubation, but no single device or method can be applied to all airway scenarios. Therefore, anesthesiologists have to master a variety of airway devices. Among these devices, the Bonfils intubation fiberscope (BIF) is widely used. The BIF is a rigid, long, slender device with a curved tip. A tracheal tube is loaded onto the shaft of the BIF, inserted into the mouth, and advanced to the glottis aperture. After identifying the vocal cords, the tracheal tube is placed into the trachea [2, 3]. The BIF can be used for the endotracheal intubation normal airways, but is especially useful for difficult tracheal intubations for patients with limited cervical mobility or limited mouth opening [4, 5]. The most important disadvantage of the BIF is the long learning curve [2, 6]. Two methods are commonly used for targeting the glottis using BIF: the midline approach technique and the transillumination-assisted technique. The midline approach technique is one of the most common techniques for BIF; the operators directly observe through the eyepieces to look for the glottis under direct vision [6], intra-oral mist, secretions, or contamination impair the vision during intubation and are the main reasons for BIF intubation failure. Recent studies showed that the application of the transillumination-assisted technique can improve the success rate of intubation [7]. In the transillumination-assisted technique, an external light is used to locate the glottis through the cricothyroid membrane [8]. Since the transillumination-assisted technique does not entirely rely on the direct observation of the glottis through the eyepieces, it is less likely to be affected by blurred vision. Nevertheless, which technique is easier for trainees remain to be shown. Therefore, we hypothesized that the transillumination-assisted technique have a higher success rate for intubations performed by trainees. Therefore, this study compared the transillumination-assisted and midline approach techniques in terms of intubation time, success rate, and intubation-related complications, hence providing clinical data to further optimize the learning and training process of BIF-guided tracheal intubation. BODY.METHODS.STUDY DESIGN: This prospective randomized controlled trial was conducted at the Anesthesiology Department of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, the study was approved by the ethics committee of the hospital. The trial is registered with Clinicaltrials.gov (ChiCTR-INR-16009967), available online: http://www.chictr.org.cn/showproj.aspx?proj=15963. Between January 2013 and June 2014, 500 patients undergoing tracheal intubation were recruited. Tracheal intubation operations were conducted by 10 students (trainees) from the Anesthesiology Department of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. These trainees had ≤2 years of doctor's qualification, but did not have experience with the BIF. They were assigned to the transillumination-assisted technique group (T group) or the midline approach technique group (R group) using a simple randomization method based on a random number table. The trainees were provided standard training by a chief physician who was considered as the observer and was skilled in using the Bonfils optical stylet (>100 successful unsupervised intubation operations). The training included explaining the theory of the BIF technique, watching teaching videos, and guiding the trainees to conduct tracheal intubation using the BIF in manikins (Shanghai Xinman Co., Ltd., Shanghai, China) [9]. After the trainees had successfully conducted tracheal intubation in manikins for three times, they were randomized and performed intubation operations. BODY.METHODS.PATIENTS: The inclusion criteria were: 1) patients undergoing elective surgery under general anesthesia; 2) age 18–65 years; 3) ASA grade I or II; 4) Mallampati grade I–II; 5) body mass index (BMI) of 18–25 kg/m2; and 6) signed the written informed consent. The exclusion criteria were: 1) severe cardiopulmonary system diseases; 2) coagulation disorders; 3) missing teeth or removable denture; 4) obesity; or 5) difficult airway. BODY.METHODS.BLINDING: Since this test could not be blind to observers and operators, only the patients were blinded to the kind of intubation. BODY.METHODS.PREPARATION PRIOR TO INTUBATION: The BIF stylet (Karl Storz, Tuttlingen, Germany) was connected to the light source, the objective lens was cleaned with alcohol, and the focal length of the eyepiece was adjusted. The shaft was lubricated with paraffin oil and nested with a suitable type of tracheal tube whose front end was 0.5 cm beyond the front end of the lens shaft; 7.5- and 7-mm tracheal tubes were used for male and female patients, respectively. Prior to operation, the patients were fasted for 8 h and did not receive any medication. In the operation room, the patient was placed in a supine position, and the venous pathway was opened. Blood pressure, heart rate, electrocardiogram, oxygen saturation, and train-of-four stimulation (TOF) were routinely monitored. The bispectral index (BIS) was monitored using an ASPECT 2000 monitor (Aspect A-2000, MA, USA). The patient was given preoxygenation at 8–10 L/min for 3 min, followed by anesthetic induction with an intravenous injection of 0.05 mg/kg of midazolam, 2 μg/kg of fentanyl, 2 mg/kg of propofol, and 0.6 mg/kg of rocuronium, as well as tracheal intubation after the BIS value was 40–60 and TOF indicated the disappearance of T2. BODY.METHODS.TRACHEAL INTUBATION WITH TRANSILLUMINATION-ASSISTED TECHNIQUE: The head of the patient was maintained in a neutral position without flexion and extension movements. The operator stood near the left side of the patient's head. Then, the operator lifted the jaw of the patient with his left hand and inserted the optical laryngoscope into the mouth along the incisor midline with the right hand. The operator would look for the brightest transilluminated spot to identify the glottis (Fig. 1). The stylet position was identified under the eyepiece: if it was located at the superior glottis or entered the trachea, the tracheal tube was inserted into the trachea along the lens shaft. However, if the glottis or tracheae ring was invisible, the optical stylet position was adjusted again to continuously look for the brightest spot at the neck midline, and the aforementioned procedures were repeated [7] (Fig. 1).Fig. 1Transillumination-assisted technique and midline approach technique. Note: In intubation using the transillumination-assisted technique, the Bonfils optical stylet (a) with a preset tracheal tube was slowly inserted into the upper respiratory tract along the midline. If a centered bright spot was visible in the cricothyroid membrane region of the neck (b), it might indicate that the laryngoscope tip was located near the glottis, where the vocal cords, annulus tracheae, and other structures were visible (c). If the laryngoscope tip was deviated to one side, the spot was more focal and darker (d), where sinus piriformis, glossoepiglottic folds, and other structures were visible (e) BODY.METHODS.TRACHEAL INTUBATION VIA MIDLINE APPROACH TECHNIQUE: The operator directly observed through the eyepieces to adjust the depth and the direction of the lens body, thereby to look for the glottis and insert the tracheal tube into the trachea along the lens body under direct vision. After successful tracheal intubation, the optical stylet was retracted along the physiological curvature of the oropharynx. BODY.METHODS.OUTCOMES: The primary outcome was the intubation time. The secondary outcomes were success rate, number of attempts, and complications. All intubation operations were observed by the same observer, who was in charge of recording experimental results, to avoid interobserver bias. This observer also recorded the characteristics of the patients, information on operators, and complications. The data included intubation attempts, success rate of first intubation, overall success rate of three attempts of intubation, intubation time, and intubation-related complications. The tracheal intubation time was defined as the time elapsed from the insertion of the front end of the laryngoscope into the mouth to the retraction of the laryngoscope after tracheal intubation was completed. The total duration for intubating a patient could not be longer than 3 min, and the maximum number of attempts allowed was three. The cases with three unsuccessful attempts or total intubation time >3 min were considered intubation failure. In addition, patients with blood oxygenation falling to <92% before intubation could be completed were considered as failed. In all these cases, intubation was completed by the observer. BODY.METHODS.STATISTICAL ANALYSIS: In a preliminary study, the average intubation time was 30.0 ± 18.4 s using BIF with the midline approach technique. An intubation time difference of 50% was considered to be clinically significant. According to the calculation formula of sample size for two independent samples \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ N={\left[\raisebox{1ex}{$\left({Z}_{\raisebox{1ex}{$\alpha $}\!\left/ \!\raisebox{-1ex}{$2$}\right.}+{Z}_{\beta}\right)\times \sigma $}\!\left/ \!\raisebox{-1ex}{$\delta $}\right.\right]}^2\times 4\left({Q_1}^{-1}+{Q_2}^{-1}\right) $$\end{document}N=Zα2+Zβ×σδ2×4Q1−1+Q2−1, where type I error alpha value of 0.05 and power of 90%. Thus a sample size of about 500 patients is needed. Statistical analyses were performed using the SPSS 13.0 software (SPSS Inc., IL, USA). Categorical data was expressed as number or percentage. Continuous data was expressed as mean ± standard deviation. The categorical data was analyzed using the chi-square test, and the continuous data was analyzed using the Student t test. The linear regression analysis about the intubation time and patient's treatment order were performed. Two-sided P-values <0.05 were considered statistically significant. BODY.RESULTS.SUBJECTS CHARACTERISTICS: A total of 500 ASA grade I or II patients were recruited (Fig. 2). Ten trainees were selected to conduct tracheal intubation (five trainees/group). The general data of the trainees and patients are listed in Table 1. Additional file 1: Figures S1, S2, and S3 present the learning curves of the two groups.Fig. 2Patient flowchart Table 1Characteristics of trainees and airway data of patients undergoing tracheal intubation using the Bonfils optical styletT group (n = 250)R group (n = 250) P valueCharacteristics of patients Gender (male/female)137/113133/1170.720 Age (years)48.0 ± 12.647.1 ± 12.60.436 Height (m)1.68 ± 0.081.67 ± 0.080.207 Weight (kg)62.9 ± 9.063.1 ± 9.00.777 BMI (kg/m2)21.8 ± 2.622.2 ± 2.60.123 ASA 1/2/3105/145/0111/139/00.588 Mouth opening < 4 (cm)00- Mallampati score (1/2/3)165/85/0173/77/00.445 Limited neck extension00- Thyromental distance <4 cm00-Characteristics of trainees Number55- Age (year)29.6 ± 1.830.2 ± 2.40.003 Gender (male/female)3/23/2- Doctors' qualifications (years)22- BMI body mass index; data were expressed as mean ± standard deviation BODY.RESULTS.PRIMARY OUTCOME: The number of attempts was similar between the two groups (1.4 ± 0.7 vs. 1.3 ± 0.6, P = 0.056). In patients with successful intubation at the second attempt, the intubation time was longer in the T group (67.4 ± 23.7 vs. 52.7 ± 22.1 s, P = 0.006), without difference for successful intubation at the first or third attempt (46.7 ± 18.1 vs. 48.6 ± 19.9 s, P = 0.348; 80.7 ± 19.8 vs. 63.8 ± 25.0 s, P = 0.054). Among all patients with successful intubation, the intubation time was similar between the two groups (52.8 ± 22.2 vs. 49.9 ± 20.7 s, P = 0.137). No intubation was failed because of blood oxygenation <92%. BODY.RESULTS.SECONDARY OUTCOMES: The success rates of the first, second, and third attempts were similar between the two groups (71.6% vs. 78.0%, P = 0.100; 57.8% vs. 65.5%, P = 0.301; 58.1% vs. 57.9%, P = 0.991). In addition, the overall intubation success rate was not significantly different between the two groups (94.8% vs. 96.8%, P = 0.265). Among patients receiving intubation with the transillumination-assisted technique, intubation was successful at the first, second, and third attempts in 71.6% (179/250), 16.0% (40/250), and 7.2% (18/250) of patients, respectively. Intubation was unsuccessful after three attempts in 5.2% (13/250) of patients, mainly because the trainees failed to find the bright spot. Among patients receiving intubation with the midline approach technique, intubation was successful at the first, second, and third attempts in 78.0% (195/250), 14.4% (36/242), and 4.4% (11/242) of patients, respectively. Intubation was unsuccessful after three attempts in 3.2% (8/250) of patients, mainly because the trainees could not clearly observe the glottis. Compared to the midline approach technique, the frequencies of dry throat (52.2% vs. 40.4%, P = 0.020), sore throat (26.8% vs. 16.8%, P = 0.009) and hoarseness (14% vs. 8%, P = 0.045) were higher in the transillumination-assisted technique (Table 2).Table 2Main study results and complicationsT group (n = 250)R group (n = 250) P valueOverall intubation success rate (%)237 (94.8)242 (96.8)0.265Rate of Success at the first attempt (n, %)179 (71.6)195 (78)0.100Succeed at the second attempt (n, %)40 (16.0)36 (14.4)0.301Succeed at the third attempt (n, %)18 (7.2)11 (4.4)0.991Failed after three attempts(n,%)13 (5.2)8 (3.2)0.265Intubation frequency(n, mean ± SD)3521.4 ± 0.73241.3 ± 0.60.056Successful intubation time (s)52.8 ± 22.249.9 ± 20.70.137First attempt (s)46.7 ± 18.148.6 ± 19.90.348Second attempt (s)67.4 ± 23.752.7 ± 22.10.006* Third attempt (s)80.7 ± 19.863.8 ± 25.00.054ComplicationsDry throat (%)128 (51.2)101 (40.4)0.020* Sore throat (%)67 (26.8)42 (16.8)0.009* Hoarseness (%)35 (14)20 (8)0.045* Trauma (%)00- Note: For comparison of intubation time and successful intubation time, a difference with P less than 0.05 was considered statistically significant, and indicated with "*". The P value was calculated using the independent-sample Student t test or chi-square test. Data were expressed as mean ± standard deviation and number (percentage) BODY.DISCUSSION: This study compared the intubation time, success rate and complications of the BIF conducted by novices using the midline approach technique or the transillumination-assisted technique. It was found that the intubation time and intubation success rate were similar between the two techniques. The intubation time for patients receiving successful intubation at the second attempt was longer with the transillumination-assisted technique than with the midline approach technique. The complication frequency of the transillumination-assisted technique was significantly higher compared to the midline approach technique. In this study, the overall success rate of intubation with the BIF was 94.8–96.8%. Byhahn et al. reported a success rate of 81.6% in simulating patients with a difficult airway who received tracheal intubation with the BIF [10]. Piepho et al. conducted a study of novices using the BIF in a simulated difficult airway and showed that the success rate of the BIF intubation was 82%, while the success rate of BIF intubation was higher than that of Macintosh laryngoscopy for patients with tongue edema (84% vs. 76%) [3]. A number of studies reported a success rate of BIF intubation to be 82–100% [6, 11, 12]. In this study, the success rate of intubation with the transillumination-assisted technique was 97.3%, similar to that reported by Sui [13]. In this study, the average intubation time with the BIF was 50.0–52.8 s, which was close to the upper limit of the time range of 23–52 s reported previously [6, 12, 14]. Differences in approach such as left molar approach, para-tongue approach, or modified laryngoscopic structure [15] are possible factors affecting intubation time. Different methods can be used to judge successful intubation, such as the carbon dioxide test or lung auscultation, which may have different durations and lead to different time points, thereby exerting different effects on the results. Some scholars believe that compared to operators who are experienced in conventional laryngoscopy, novices would take shorter time to conduct BIF intubation [2]. When the operators are familiar with the new intubation tools after training, the trainees and experienced operators tended to achieve the same intubation success rate, even in difficult airways. Although the trainees received standard training before the test, they might have different learning abilities, which could lead to differences in intubation time. Thus, although the intubation time did not show any significant difference between the two groups, its clinical significance requires further investigation. Postoperative laryngeal complications, such as sore throat and hoarseness, are common anesthesia-related complications during tracheal intubation. The frequencies of sore throat and hoarseness were reported to be 11–48% and 18–53%, respectively [16–19]. In this study, no severe intubation-related complications were found, and the frequencies of sore throat and hoarseness in the two groups were consistent with previous studies. In addition, the frequencies of dry throat, sore throat, and hoarseness were found to be significantly higher in the T group than in the R group. The reason for this difference could be a lack of experience in using the intubation technique/instrument. Biro et al. revealed that differences in professional allocation and experience did not significantly affect the incidence and strength of intubation-induced laryngeal complications [20]. On the other hand, Tazeh-Kand et al. showed that the anesthesiologist experience was an independent factor influencing the frequencies of sore throat and hoarseness in male patients after intubation [18]. Recently, Inoue et al. conducted a retrospective propensity score study of 21,606 patients [21] and the frequencies of postoperative laryngeal complications were found not to be significantly different for novices and experienced anesthesiologists. Furthermore, it was believed that laryngeal complications commonly occurred after tracheal intubation, but the incidence was not associated with the experience of the operators [21]. Nevertheless, whether the incidence of complications after BIF intubation is associated with the operators' experience is still controversial and requires further investigation. In the transillumination-assisted technique, the operator positions the BIF by finding the bright spot. A direct contact of the tube and laryngoscope tip with the laryngeal tissues leads to intubation-related damage. Although the BIF optical stylet has been proved to be safe and feasible in intubation with transillumination-assisted technique, its light source does not have a penetrability as strong as that of a Trachlight, which may be the reason for more complications in the case of the Bonfils optical stylet [7]. However, in the midline approach technique, the operator adjusts the direction and depth of the laryngoscope by observing through the eyepieces, which minimizes the direct contact of the laryngoscope tip with the vocal cords or epiglottis and other fragile tissues, hence reducing direct damage. The lightwand intubation does not rely on observing through eyepieces, and is not affected by oral secretions, blood, mist, and other factors [22]; this may have more extensive indications compared with the midline approach technique. However, for novices and non-difficult airway, the transillumination-assisted technique may present a higher risk of complications. This study had the following limitations:Premedication: Application of anticholinergic agents before anesthesia can significantly inhibit respiratory tract secretions and create favorable conditions for fiberoptic intubation. However, the common adverse reactions caused by anticholinergic agents (e.g., dry mouth) may lead to postoperative discomfort and other complications. Therefore, anticholinergic agents were not used for medication before anesthesia. However, this may hinder exposing the laryngoscope during intubation. This study was not designed to assess whether anticholinergic agents were associated with increased intubation times.Applicability of the instrument: The difficulty in training on the use of the BIF directly leads to poor applicability of this instrument. Currently, many video laryngoscopes are available that are convenient and have a high success rate of intubation. Furthermore, many video laryngoscopes are provided with disposable laryngoscope blades, which can reduce the chance of cross-infection. Nevertheless, since the BIF has its unique scope of application and is difficult to be replaced by other laryngoscopes, it still has potential applications.Evaluating intubation safety: The intubation time is unlikely to fully reflect the quality of intubation, while the safety and effectiveness of the operational technique are crucial. In a study by Garcia et al. [23], two layers of pressure-sensitive adhesives were applied to the conventional laryngoscope to measure the intubation force, which was considered an indicator for intubation safety and trainee's skills. However, this method is not appropriate for evaluating the BIF. In addition, the sensitivity and specificity of methods used to evaluate intubation safety still require a large number of studies. Nevertheless, the transillumination-assisted technique could be more prone to complications, especially for trainees. Therefore, we still recommend the midline approach technique group method.Blinding: The two intubation techniques are obviously different in operation process and steps. Therefore, it is impossible to apply blinding to trachea intubation operators, observers, and data collectors. Only the patient could be blinded because they were unconscious during intubation.Generalizability: In this study, the patients were relatively young (around 48 years old) and lean (BMI of about 22 kg/m2). Therefore, caution should be taken when extrapolating these results to elderly, pediatric, or obese patients. BODY.CONCLUSION: For a normal airway, intubation with the transillumination-assisted technique conducted by novices is unlikely to increase the success rate of intubation, but tends to increase the risk of complications. Thus, intubation with the midline approach technique could be safer for novices. The safety and efficacy for novices to manage patients with a difficult airway using the BIF requires further confirmation.

TITLE: Successful long-term weight loss among participants with diabetes receiving an intervention promoting an adapted Mediterranean-style dietary pattern: the Heart Healthy Lenoir Project ABSTRACT.OBJECTIVE: To examine weight change by diabetes status among participants receiving a Mediterranean-style diet, physical activity, and weight loss intervention adapted for delivery in the southeastern USA, where rates of cardiovascular disease (CVD) are disproportionately high. ABSTRACT.RESEARCH DESIGN AND METHODS: The intervention included: Phase I (months 1–6), an individually tailored intervention promoting a Mediterranean-style dietary pattern and increased walking; Phase II (months 7–12), option of a 16-week weight loss intervention for those with BMI≥25 kg/m2 offered as 16 weekly group sessions or 5 group sessions and 10 phone calls, or a lifestyle maintenance intervention; and Phase III (months 13–24), weight loss maintenance intervention for those losing ≥8 pounds with all others receiving a lifestyle maintenance intervention. Weight change was assessed at 6, 12, and 24-month follow-up. ABSTRACT.RESULTS: Baseline characteristics (n=339): mean age 56, 77% female, 65% African-American, 124 (37%) with diabetes; mean weight 103 kg for those with diabetes and 95 kg for those without. Among participants with diabetes, average weight change was −1.2 kg (95% CI −2.1 to −0.4) at 6 months (n=92), −1.5 kg (95% CI −2.9 to −0.2) at 12 months (n=96), and −3.7 kg (95% CI −5.2 to −2.1) at 24 months (n=93). Among those without diabetes, weight change was −0.4 kg (95% CI −1.4 to 0.6) at 24 months (n=154). ABSTRACT.CONCLUSIONS: Participants with diabetes experienced sustained weight loss at 24-month follow-up. High-risk US populations with diabetes may experience clinically important weight loss from this type of lifestyle intervention. ABSTRACT.TRIAL REGISTRATION NUMBER: NCT01433484. BODY: Significance of this studyWhat is already known about this subject?Though not well studied in the USA, particularly among low-income and minority populations, a Mediterranean-style dietary pattern can reduce the risk for cardiovascular disease (CVD) among those with and without type 2 diabetes and has been shown in some studies to be associated with maintenance of weight loss.What are the new findings?We developed and evaluated a lifestyle and weight loss intervention for low-income and minority residents of eastern North Carolina, with a major focus on implementing a culturally adapted Mediterranean-style dietary pattern while also promoting physical activity. Among study participants with diabetes, the intervention was well received and associated with sustained weight loss at 24-month follow-up.How might these results change the focus of research or clinical practice?This study demonstrates high acceptability of a Mediterranean-style dietary pattern among low-income and minority populations residing in eastern North Carolina and appears to be promising as a weight loss intervention for participants with diabetes. This dietary pattern and type of intervention should be evaluated in randomized controlled trials to further assess the intervention's effectiveness at improving CVD risk factors, yielding sustained weight loss, and reducing CVD events. BODY.INTRODUCTION: Weight loss, defined by the American Diabetes Association as a sustained reduction of 5% of initial body weight,1 is recommended for overweight and obese patients with type 2 diabetes, while others have noted that weight loss as small as 2 kg appears to provide clinical benefit.2 However, weight loss is difficult to achieve and harder to sustain; and in general, individuals with diabetes have a harder time losing weight and maintaining weight loss, when compared with those without diabetes.3 This is especially true for high risk groups such as African-American women with type 2 diabetes.4 For example, in the Action for Health in Diabetes (Look AHEAD) trial,5 among female participants with diabetes, the year-1 weight loss was 6.8% for African-Americans compared with 9.1% for non-Hispanic whites. Thus, improved approaches for promoting weight loss are needed for patients with diabetes and especially for groups at higher risk for obesity and diabetes such as African-American women. Some have argued that a variety of weight loss dietary patterns are acceptable if they lead to weight loss.6 However, as patients with type 2 diabetes are at very high risk for cardiovascular disease (CVD), consideration should be given to advocating a weight loss dietary pattern that also reduces CVD risk. When a Mediterranean diet pattern, supplemented with olive oil or nuts, was evaluated in the Prevención con Dieta Mediterránea (PREDIMED) randomized trial,7 there was a 30% reduction in CVD risk among participants with and without diabetes. This contrasts with the lower fat weight loss diet evaluated in Look AHEAD,8 which led to significant weight loss but did not reduce CVD risk. Attention to dietary pattern as a critical component of weight loss diets may be even more important for populations at very high risk for CVD, such as minority populations and those residing in the 'stroke belt' of the southeastern USA, where CVD rates are substantially higher compared with national levels.9 Thus, we developed and evaluated a lifestyle and weight loss intervention for residents of eastern North Carolina, with a major focus on implementing a culturally adapted Mediterranean-style dietary pattern while also promoting physical activity. In this paper, we report the intervention's effect on diet and physical activity behaviors, CVD risk factors, and weight loss through 24 months of follow-up by participants' diabetes status. BODY.RESEARCH DESIGN AND METHODS.STUDY OVERVIEW: The Heart Healthy Lenoir (HHL) Project was a collaborative research effort designed to reduce CVD risk and disparities in CVD risk among Lenoir County, North Carolina residents, as previously described.10 11 It was funded by the National Heart, Lung, and Blood Institute (NHLBI) as part of an initiative with the National Cancer Institute 'to develop and test multilevel interventions to reduce health disparities.'12 This paper focuses on participants who took part in the HHL lifestyle study,11 one of three coordinated HHL studies which also included a study to improve high blood pressure (BP) management at local practices and a study examining associations between genetic markers and change in CVD risk factors. These studies were conducted in Lenoir County because of its location in the 'stroke belt'9 of eastern North Carolina, where rates of CVD are higher than state and national averages13 and because it has a large minority population (40% African-American) that experiences disproportionally higher rates of CVD.14 The HHL lifestyle study was designed and conducted with input from a local community advisory committee10 and was approved and monitored by the University of North Carolina's Institutional Review Board. Data were collected between 20 September, 2011 and 7 November, 2014. The lifestyle study consisted of three phases as shown in figure 1, which depicts the three sequential phases of the study and the number of participants with and without diabetes who took part in each component of the intervention. Phase I, which lasted 6 months and was the same for all study participants, focused on improving diet quality and increasing physical activity. In Phase II, which also lasted 6 months, participants with a body mass index (BMI) ≥25 kg/m2 were offered an intensive weight loss intervention while those with a BMI<25 kg/m2 and those who declined the weight loss intervention received a maintenance of lifestyle intervention. In Phase III, participants who lost ≥8 lbs (3. 6 kg) at the conclusion of Phase II were invited to take part in a year-long, randomized controlled trial (RCT), comparing a more intensive and less intensive maintenance of weight loss intervention; a year-long maintenance of lifestyle intervention was given to those who did not take part in the RCT. As prior RCTs conducted by our research team15–18 have shown that similar formats of lifestyle and weight loss interventions are effective in improving lifestyle and achieving weight loss among low socioeconomic status participants, we did not include a control group for Phases I and II of this study. Furthermore, our community advisory committee strongly encouraged a study design in which all participants received 'active treatment.' Figure 1Study flow diagram. BODY.RESEARCH DESIGN AND METHODS.PARTICIPANTS: The enrollment goal for this study, based on having an adequate sample for the Phase III maintenance of weight loss RCT, was 350 with about 150 participants recruited from the community and 200 from the high BP study, as previously described.11 19 Criteria for screening from the community were age ≥18 years and interest in improving lifestyle behaviors to reduce CVD risk. Screening criteria for the high BP study were age ≥18, being an established patient at a participating practice, and systolic BP≥150 mmHg when assessed during routine care within the prior 12 months. After obtaining verbal informed consent, research staff conducted phone interviews to screen potential participants.11 If eligibility criteria were met, participants were invited to an enrollment visit at a central research office or at participating clinics. Written informed consent was obtained prior to collecting baseline measures. Participants attending the enrollment visit for the high BP study were invited to also take part in the lifestyle study until 200 agreed to do so. BODY.RESEARCH DESIGN AND METHODS.INTERVENTION.PHASE I (MONTHS 1–6)—LIFESTYLE INTERVENTION: This intervention focused on dietary and physical activity behaviors and did not address other aspects of lifestyle relevant to CVD risk reduction, such as smoking cessation. Further, the same intervention was given to those with and without diagnosed diabetes. (All participants with diabetes had type 2 diabetes). The lifestyle intervention was originally developed by Ammerman et al20 21 and subsequently revised to emphasize carbohydrate quality as an important component of a heart-healthy diet.15 Consistent with the evolving literature suggesting frequent consumption of food with high-quality fats (polyunsaturated and monounsaturated fats primarily from plant sources and fish) is also important in reducing CVD risk in those with and without diabetes,7 22–26 this study's dietary intervention was further modified to include a major focus on improving dietary fat quality. These changes rendered the HHL lifestyle intervention dietary pattern very similar to the PREDIMED study's nut intervention arm diet;7 therefore, the dietary pattern was termed 'Med-South' because of its intended use in the southeastern USA. The intervention format and content have been previously published and described in detail.11 Phase I included four monthly sessions delivered by a trained counselor administered as hour-long individual counseling sessions or 90 min group sessions given at a centrally located research office or participating clinic. Participants chose their preferred counseling format. About 75% of counseling time was devoted to dietary behaviors with the rest focusing on physical activity. Dietary counseling included culturally relevant content to improve fat quality (such as increasing consumption of nuts, full-fat salad dressings and mayonnaise, and vegetable oils), increase fruit and vegetable consumption, promote fish and poultry intake while reducing red and processed meat intake, and minimize consumption of sugar-sweetened beverages, high-sugar-content desserts, and snacks. Physical activity counseling focused mostly on walking with a recommended goal of at least 7500 steps/day or ≥30 min/day of physical activity on ≥5 days/week. Spouses and friends were invited to attend the counseling intervention sessions. When participants could not physically attend counseling sessions, telephone counseling was offered. Participants also received a pedometer and activity logs to self-monitor physical activity as well as a listing of local community resources that promoted healthy eating (eg, farmers' markets) and physical activity (eg, community parks). Those participants who were co-enrolled in the high BP study received a home BP monitor and were instructed to measure their BP at least three times per week. They also received monthly phone calls for a year, primarily promoting BP medication adherence. During counseling sessions, participants worked with their counselor to create individually tailored action plans to improve dietary and physical activity behaviors. Dietary and physical activity tips were tailored to problematic lifestyle behaviors assessed on the baseline lifestyle questionnaire. Dietary tips included recipe suggestions from a southern-style cookbook that was given to all participants. At the beginning of sessions 2–4, the counselor and participants reviewed progress made towards previously stated goals. BODY.RESEARCH DESIGN AND METHODS.INTERVENTION.PHASE II (MONTHS 7 THROUGH 12)—WEIGHT LOSS AND MAINTENANCE OF LIFESTYLE INTERVENTIONS: Participants with a BMI≥25 kg/m2 could choose to take part in the weight loss intervention. Those who were not eligible for the weight loss intervention (BMI<25 kg/m2) and those who declined the intervention received a maintenance of lifestyle intervention consisting of three phone calls, as previously described.11 The weight loss intervention was offered in two formats over ∼16 weeks: 16 weekly group sessions as previously tested,16 18 27 or five group sessions plus 10 phone contacts (combination intervention), as recently described.11 The major modification from the previously tested weight loss intervention was the focus on the Med-South dietary pattern and addition of newer evidence-based behavioral components (eg, daily self-weighing).28 BODY.RESEARCH DESIGN AND METHODS.INTERVENTION.PHASE III (13–24 MONTHS)—WEIGHT LOSS AND LIFESTYLE MAINTENANCE INTERVENTIONS: Participants who took part in the Phase II weight loss intervention and lost ≥8 lbs (3.6 kg) were invited to take part in the maintenance of weight loss RCT. All other study participants received brief, quarterly maintenance of lifestyle intervention phone calls (similar to Phase II), as previously described.11 For the maintenance of weight loss RCT, participants were randomized 1:1 to receive either 36 phone contacts (24 weekly calls over 6 months followed by 12 biweekly calls over 6 months; more intensive intervention) or 18 phone contacts (12 biweekly calls over 6 months followed by 6 monthly calls over 6 months; less intensive intervention). BODY.RESEARCH DESIGN AND METHODS.MEASURES: Outcome measures were assessed at baseline, 6, 12, and 24 months. Previously validated questionnaires were administered to assess lifestyle change including the Dietary Risk Assessment (DRA) which assessed overall diet quality,20 29 the brief Block fruit and vegetable questionnaire,30 a dietary fat quality screener,31 and a questionnaire to assess walking and overall physical activity.32 33 In addition, the SF-12 (SF-12 instrument, Quality Metric, Lincoln, RI) was administered to assess quality of life. Weight was assessed by electronic scale (Seca 874, Seca, Hanover, MD) as the average of two measures to the closest tenth pound. Height was measured with a portable stadiometer at baseline only. BP was calculated as the average of three measurements recorded at 60 s intervals (Omron HEM-907XL, Omron Healthcare, Lake Forest, IL) after being seated for 5 min. Blood lipids and glycated hemoglobin (A1c) were assessed by a commercial laboratory (LabCorp, Burlington, North Carolina, USA). At follow-up measurement visits, questionnaires were administered to assess acceptability of the intervention and adverse outcomes. Participants received compensation for measurement visits: $40 for enrollment, $25 for 6 and 12-month visits, and $30 for the 24-month visit. BODY.RESEARCH DESIGN AND METHODS.SAMPLE SIZE AND STATISTICAL ANALYSIS: The enrollment goal of 350 participants was based on having a sufficient sample (N=100) for the embedded RCT of weight loss maintenance.11 In addition, the overall sample of 350 was considered sufficient to describe the primary objective of the lifestyle intervention to improve diet quality at 6-month follow-up, and the major secondary objectives of improved diet quality, physical activity, and weight loss at 12 and 24-month follow-up. Sample characteristics were summarized using descriptive statistics, with subgroups by diabetes status and race. Outcomes were assessed using pre–post changes by diabetes status and race with paired t-tests for continuous outcomes, McNemar's tests for binary outcomes, and χ2 tests for subgroup analysis when appropriate. Data are reported for returnees at follow-up without imputation for missing data. As described,11 among all study participants, weight loss was substantially less than anticipated and observed in our prior weight loss studies.16 18 Only 27 participants overall (eight with diabetes) took part in the maintenance of weight loss RCT. Given this small number, weight loss results are reported in the aggregate and not separately for those who took part in the RCT. Because age, race, sex, education, and baseline weight are potential confounders for weight loss, a linear regression analysis was conducted adjusting for these variables. Participants who became pregnant, had bariatric surgery, or were diagnosed with cancer (excluding non-melanoma skin cancer or localized breast or prostate cancer diagnosed by screening tests) were excluded from analysis (eight overall, four with diabetes). SAS V.9.3 was used for analysis. BODY.RESULTS.BASELINE CHARACTERISTICS: As outlined in detail elsewhere,11 of 642 individuals assessed as eligible for this study, 366 (57%) attended the enrollment visit and 339 (53%) completed all baseline measurements and comprised the study sample, including 134 recruited from the community and 205 from the clinic-based high BP study. Participants' baseline characteristics, by diabetes status then further categorized by race, are outlined in table 1. Table 1 Baseline characteristics: overall, by diabetes status then by race Characteristics Diabetes No Diabetes Overall All African-American White All African-American White n=339 n=124 n=89 n=34 n=215 n=130 n=83 Demographics Age, mean (SE) 56 (0.6) 59 (0.9) 59 (1.1) 61 (1.7) 54 (0.8) 51 (1.1) 57 (1.2) Female 260 (77) 93 (75) 70 (79) 22 (65) 167 (78) 111 (85) 54 (65) Race  African-American 219 (65) 89 (72) 130 (61)  White 117 (35) 34 (28) 83 (39) Education, years  ≤8 (middle school or less) 16 (5) 7 (6) 4 (5) 3 (9) 9 (4) 7 (5) 2 (2)  9–11 (some high school) 45 (13) 25 (20) 22 (25) 2 (6) 20 (9) 13 (10) 7 (8)  12 (high school graduate) 128 (38) 46 (37) 36 (40) 10 (29) 82 (38) 58 (45) 24 (29)  13–15 (some college) 79 (23) 24 (19) 16 (18) 8 (24) 55 (26) 29 (22) 25 (30)  16 (college graduate) 49 (14) 14 (11) 9 (10) 5 (15) 35 (16) 17 (13) 17 (21)  >16 (graduate school) 22 (7) 8 (7) 2 (2) 6 (18) 14 (7) 6 (5) 8 (10) Education: high school or less 189 (56) 78 (63) 62 (70) 15 (44) 111 (52) 78 (60) 33 (40) Marital status  Married or living with a partner 159 (47) 51 (41) 32 (36) 19 (56) 108 (50) 50 (39) 57 (69)  Other 180 (53) 73 (59) 57 (64) 15 (44) 107 (50) 80 (62) 26 (31) Currently have health insurance 251 (74) 94 (76) 68 (76) 25 (74) 157 (73) 88 (68) 67 (81) Current employment  Working full time 124 (37) 25 (20) 18 (20) 7 (21) 99 (46) 64 (49) 33 (40)  Working part time 42 (12) 17 (14) 14 (16) 2 (6) 25 (12) 17 (13) 8 (10)  Do not work due to health reasons 69 (20) 38 (31) 26 (29) 12 (35) 31 (14) 17 (13) 14 (17)  Retired 53 (16) 26 (21) 18 (20) 8 (24) 27 (13) 8 (6) 19 (23)  Other 51 (15) 18 (14) 13 (15) 5 (15) 33 (15) 24 (19) 9 (11) Annual household income  <$10 000 62 (20) 31 (29) 24 (32) 6 (18) 31 (16) 26 (22) 5 (7)  $10 000 to<$20 000 64 (21) 28 (26) 23 (31) 5 (15) 36 (18) 22 (19) 14 (18)  $20 000 to<$40 000 84 (28) 29 (27) 20 (27) 9 (27) 55 (28) 40 (34) 14 (18)  $40 000 to<$60 000 33 (11) 9 (8) 3 (4) 6 (18) 24 (12) 12 (10) 12 (16)  $60 000 to <$80 000 27 (9) 9 (8) 4 (5) 5 (15) 18 (9) 9 (8) 9 (12)  ≥$80 000 34 (11) 2 (2) 0 (0) 2 (6) 32 (15) 10 (8) 22 (29) CVD and risk factors for CVD Known coronary heart disease 49 (14) 28 (23) 20 (23) 8 (24) 21 (10) 10 (8) 11 (13) Known cardiovascular disease 62 (18) 33 (27) 23 (26) 10 (29) 29 (13) 14 (11) 15 (18) Hypertension 291 (86) 121 (98) 88 (99) 32 (94) 170 (79) 107 (82) 63 (76) Cholesterol  High (≥240 mg/dL) 187 (56) 82 (67) 57 (65) 24 (71) 105 (49) 53 (42) 52 (63)  Borderline (200–239 mg/dL) 46 (14) 8 (7) 7 (8) 1 (3) 38 (18) 26 (21) 11 (13)  Desirable (<200 mg/dL) 102 (30) 33 (27) 24 (27) 9 (27) 69 (33) 48 (38) 20 (24) Diabetes 124 (37) Current cigarette smoker 54 (16) 21 (17) 17 (19) 4 (12) 33 (15) 20 (15) 13 (16) Packs of cigarettes smoked per day, mean (SE) for current smokers 0.7 (0.1) 0.6 (0.1) 0.6 (0.1) 0.8 (0.2) 0.7 (0.1) 0.6 (0.1) 0.9 (0.2) Taking BP lowering medications 260 (77) 114 (92) 83 (93) 30 (88) 146 (68) 93 (72) 53 (64) Lifestyle DRA total score 27.8 (0.3) 28.5 (0.5) 28.3 (0.6) 28.9 (0.7) 27.4 (0.4) 27.2 (0.5) 27.7 (0.7) Fat quality screener score 15.5 (0.2) 15.5 (0.2) 15.4 (0.3) 15.7 (0.3) 15.4 (0.2) 15.3 (0.2) 15.7 (0.4) Fruit and vegetable servings per day 3.4 (0.1) 3.7 (0.2) 3.8 (0.2) 3.3 (0.3) 3.3 (0.1) 3.1 (0.2) 3.5 (0.2) Walking time (min/wk) 91 (11.3) 67 (12.8) 68 (15.6) 54 (20.5) 105 (16.1) 122 (24.9) 80 (14.3) Total physical activity time (min/wk) 149 (14.0) 112 (18.1) 112 (20.5) 99 (36.0) 171 (19.3) 176 (27.1) 161 (26.3) Physiological Weight, kg 98 (1.4) 103 (2.2) 103 (2.4) 104 (4.7) 95 (1.7) 99 (2.3) 90 (2.5) BMI, kg/m 2 36 (0.5) 38 (0.8) 38 (0.9) 38 (1.6) 35 (0.7) 37 (0.9) 32 (0.9) Systolic blood pressure, mm Hg 135 (1.2) 136 (2.0) 138 (2.4) 131 (3.6) 134 (1.5) 136 (2.1) 133 (2.2) Diastolic blood pressure, mm Hg 82 (0.7) 81 (1.1) 82 (1.4) 78 (1.9) 83 (0.8) 84 (1.1) 81 (1.2) HbA1c, % 6.6 (0.1) 7.9 (0.2) 8.0 (0.2) 7.6 (0.3) 5.8 (0.0) 5.8 (0.0) 5.7 (0.0) HbA1c, mmol/mol 49 (1.1) 63 (2.2) 64 (2.2) 60 (3.3) 40 (0.0) 40 (0.0) 39 (0.0) Total cholesterol, mg/dL 193 (2.3) 188 (4.1) 188 (5.0) 187 (7.2) 196 (2.7) 193 (3.2) 200 (4.8) HDL cholesterol, mg/dL 54 (0.8) 51 (1.4) 55 (1.6) 43 (2.1) 56 (1.0) 58 (1.3) 53 (1.6) Data are means (SE) or n (%). BMI, body mass index; BP, blood pressure; CVD, cardiovascular disease; DRA, dietary risk assessment; HDL, high-density lipoprotein. A total of 124 (37%) participants had diabetes. The overall average age was 56 years; participants with diabetes were older on average than those without diabetes (59 vs 54 years). Men, particularly African-American men, were underrepresented in the sample. More than half of participants did not have any college education. Those without diabetes and whites were more likely to be married or living with a partner compared with others. Most participants had health insurance (74%) and this did not vary by diabetes status or race. Those with diabetes were more likely to be unemployed due to health reasons (31% vs 14%) and less likely to be currently employed full-time or part-time (34% vs 58%). Median annual household income was <$40 000 overall and <$20 000 among participants with diabetes. In terms of CVD risk factors, reported rates of hypertension were very high in the study (86% overall), which may be largely attributed to how participants were selected. Participants with diabetes were more likely to report a history of hypertension (98% compared with 79%) and African-Americans reported higher rates of hypertension than whites within each subgroup. Participants with diabetes also reported higher rates of prior coronary heart disease and CVD. At baseline, overall diet quality and fat quality were similar across diabetes status and race, while participants with diabetes had slightly higher fruit and vegetable consumption (average 3.7 compared with 3.3 servings/day). Participants without diabetes reported much higher total walking and total activity time at baseline (mean 105 and 171 min per week, respectively, compared with 67 and 112 min per week, respectively, among those with diabetes). Participants with diabetes weighed more on average than those without diabetes (103 kg compared with 95 kg). Systolic BP was similar across all categories, with a somewhat higher average for African-Americans than whites (138 mmHg compared with 131 mmHg) among participants with diabetes. Among those with diabetes, African-American participants had higher hemoglobin A1c percentage at baseline than whites (8.0% compared with 7.6%). BODY.RESULTS.OUTCOMES: Figure 1 depicts the three sequential phases of the study and the number of participants with and without diabetes that took part in each component of the intervention and returned for follow-up. Follow-up rates at 6, 12, and 24 months were ∼75% and were similar between participants with and without diabetes. Lifestyle outcomes are shown in table 2. Table 2 Change in lifestyle outcomes by diabetes status and race from baseline to 6, 12, and 24 months Outcome N Phase 1 N Phase 2 N Phase 3 Baseline to 6 months Baseline to 12 months Baseline to 24 months Mean, 95% CI Mean, 95% CI Mean, 95% CI Dietary DRA total score 235 4.4 (3.7 to 5.0)*** 227 3.3 (2.5 to 4.0)*** 226 3.0 (2.3 to 3.6)*** Diabetes (all) 86 4.2 (3.1 to 5.3)*** 86 3.0 (1.8 to 4.2)*** 84 2.0 (1.0 to 3.1)***  African-American 60 4.4 (2.9 to 5.9)*** 63 3.8 (2.3 to 5.3)*** 62 2.5 (1.2 to 3.7)***  White 25 4.0 (2.6 to 5.4)*** 22 1.1 (−0.6 to 2.8) 21 0.7 (−1.3 to 2.7) No diabetes (all) 149 4.4 (3.6 to 5.3)*** 141 3.4 (2.5 to 4.4)*** 142 3.5 (2.7 to 4.3)***  African-American 95 4.7 (3.7 to 5.7)*** 93 4.1 (3.0 to 5.3)*** 96 3.3 (2.3 to 4.4)***  White 52 3.9 (2.5 to 5.4)*** 47 2.0 (0.3 to 3.7)* 45 3.7 (2.5 to 5.0)*** Fat quality screener score 229 1.4 (1.0 to 1.8)*** 225 1.0 (0.6 to 1.3)*** 224 0.7 (0.3 to 1.1)*** Diabetes (all) 84 1.3 (0.7 to 2.0)*** 85 1.2 (0.6 to 1.8)*** 83 0.3 (−0.3 to 1.0)  African-American 58 1.2 (0.4 to 2.0)** 62 1.5 (0.8 to 2.2)*** 61 0.4 (−0.3 to 1.1)  White 25 1.6 (0.4 to 2.7)** 22 0.4 (−0.3 to 1.2) 21 0.1 (−1.2 to 1.5) No Diabetes (all) 145 1.4 (1.0 to 1.9)*** 140 0.8 (0.4 to 1.3)*** 141 0.8 (0.4 to 1.3)***  African-American 92 1.5 (1.0 to 2.1)*** 92 1.1 (0.6 to 1.6)*** 95 0.8 (0.3 to 1.4)**  White 51 1.2 (0.5 to 2.0)** 47 0.4 (−0.4 to 1.2) 45 0.9 (−0.1 to 1.9) Fruit and vegetable servings per day 249 0.3 (0.1 to 0.5)* 253 0.5 (0.3 to 0.8)*** 250 0.4 (0.2 to 0.6)*** Diabetes (all) 93 −0.1 (−0.5 to 0.4) 98 0.2 (−0.3 to 0.7) 96 0.1 (−0.4 to 0.5)  African-American 67 −0.1 (−0.7 to 0.5) 75 0.2 (−0.4 to 0.8) 74 0.1 (−0.4 to 0.6)  White 25 0.1 (−0.4 to 0.7) 22 0.0 (−0.6 to 0.6) 21 0.1 (−0.6 to 0.7)  No Diabetes (all) 156 0.5 (0.3 to 0.7)*** 155 0.8 (0.5 to 1.1)*** 154 0.6 (0.3 to 0.9)***  African-American 101 0.5 (0.2 to 0.8)** 103 0.8 (0.4 to 1.1)*** 103 0.6 (0.2 to 0.9)**  White 53 0.5 (0.0 to 0.9)* 51 0.8 (0.3 to 1.2)*** 50 0.6 (0.2 to 1.1)** Summary score for drinks, desserts, snacks 236 1.1 (0.9 to 1.3)*** 229 1.3 (1.1 to 1.6)*** 228 1.1 (0.8 to 1.3)*** Diabetes (all) 87 1.1 (0.7 to 1.4)*** 87 1.2 (0.8 to 1.6)*** 85 0.9 (0.5 to 1.3)***  African-American 61 1.1 (0.7 to 1.6)*** 64 1.3 (0.8 to 1.7)*** 63 1.1 (0.7 to 1.6)***  White 25 1.0 (0.5 to 1.5)*** 22 1.1 (0.3 to 1.8)** 21 0.1 (−0.7 to 1.0) No diabetes (all) 149 1.1 (0.8 to 1.5)*** 142 1.4 (1.1 to 1.8)*** 143 1.2 (0.8 to 1.5)***   African-American 95 1.3 (0.9 to 1.8)*** 93 1.7 (1.2 to 2.1)*** 96 1.3 (0.9 to 1.8)***  White 52 0.7 (0.2 to 1.2)** 48 0.9 (0.4 to 1.4)*** 46 0.8 (0.3 to 1.3)** Physical activity Walking time, min/wk 249 64 (19 to 109)** 253 71 (28 to 113)** 250 22 (−13 to 56) Diabetes (all) 93 101 (17 to 184)* 98 126 (58 to 194)*** 96 62 (11 to 113)*  African-American 67 119 (18 to 221)* 75 136 (52 to 220)** 74 75 (13 to 137)*  White 25 71 (−81 to 223) 22 117 (14 to 219)* 21 38 (−36 to 112) No diabetes (all) 156 42 (−9 to 94) 155 36 (−17 to 89) 154 −3 (−49 to 42)   African-American 101 29 (−45 to 103) 103 19 (−53 to 91) 103 −17 (−82 to 47)  White 53 63 (8 to 118)* 51 68 (−3 to 138) 50 22 (−22 to 66) Total physical activity time, min/wk 249 97 (36 to 158)** 253 83 (30 to 136)** 250 48 (−7 to 103) Diabetes (all) 93 120 (19 to 221)* 98 109 (29 to 188)** 96 62 (−8 to 131)  African-American 67 136 (17 to 255)* 75 127 (32 to 223)** 74 89 (3 to 175)*  White 25 106 (−94 to 305) 22 74 (−63 to 212) 21 −5 (−91 to 81) No Diabetes (all) 156 83 (6 to 159)* 155 67 (−3 to 137) 154 40 (−39 to 118)  African-American 101 86 (−17 to 189) 103 37 (−42 to 116) 103 28 (−76 to 132)  White 53 79 (−33 to 192) 51 125 (−15 to 265) 50 58 (−55 to 171) Significance level: *p<0.05; **p<0.01; ***p<0.001. DRA, dietary risk assessment; min, minute; wk, week. Overall diet quality, as assessed by the DRA total score, improved by about 4 points at 6-month follow-up for all participants and each of the subgroups. Improvement in DRA total score was maintained at 12 and 24 months compared with baseline among all subgroups except white participants with diabetes. Fat quality score improved by 1.4 points on average at 6 months with no significant difference between subgroups by race or diabetes status, but this improvement was attenuated over time for participants with diabetes. Only participants without diabetes reported a statistically significant increase in fruit and vegetable servings per day at 6, 12, and 24-month follow-up. The improvement in the summary score for drinks, desserts, and snacks was higher among African-American participants with and without diabetes compared with whites. Increased walking time was sustained at 24 months among participants with diabetes and among African-Americans with diabetes. Physiological outcomes are shown in table 3. Table 3 Change in physiological outcomes by diabetes status and race from baseline to 6, 12, and 24 months Outcome n Phase 1 n Phase 2 n Phase 3 Baseline to 6 months Baseline to 12 months Baseline to 24 months Mean, 95% CI Mean, 95% CI Mean, 95% CI Systolic BP, mm Hg 249 −6.4 (−8.7 to −4.1)*** 251 −6.2 (−9.0 to −3.3)*** 250 −7.3 (−9.9 to −4.6)*** Diabetes (all) 93 −7.1 (−11.3 to −3.0)*** 97 −5.8 (−10.9 to −0.6)* 96 −7.4 (−11.9 to −3.0)**  African-American 67 −8.6 (−13.9 to −3.2)** 74 −6.4 (−12.7 to −0.1)* 74 −9.3 (−14.6 to −4.0)***  White 25 −3.8 (−9.6 to 2.0) 22 −4.1 (−13.2 to 5.0) 21 −0.4 (−8.7 to 8.0) No diabetes (all) 156 −5.9 (−8.7 to −3.2)*** 154 −6.4 (−9.7 to −3.1)*** 154 −7.1 (−10.5 to −3.8)***   African-American 101 −5.6 (−8.9 to −2.2)** 102 −7.3 (−11.6 to −3.1)*** 103 −7.9 (−12.2 to −3.5)***  White 53 −6.5 (−11.3 to −1.7)** 51 −4.3 (−9.5 to 0.8) 50 −5.6 (−10.7 to −0.5)* Diastolic BP, mm Hg 249 −3.7 (−4.9 to −2.5)*** 251 −5.0 (−6.4 to −3.6)*** 250 −6.7 (−8.3 to −5.2)*** Diabetes (all) 93 −4.4 (−6.4 to −2.3)*** 97 −5.6 (−8.2 to −3.0)*** 96 −7.2 (−10.1 to −4.3)***  African-American 67 −4.6 (−7.2 to −2.1)*** 74 −5.8 (−8.8 to −2.7)*** 74 −8.2 (−11.7 to −4.7)***  White 25 −4.1 (−7.6 to −0.7)* 22 −4.7 (−10.0 to 0.6) 21 −3.3 (−8.3 to 1.7) No diabetes (all) 156 −3.4 (−4.8 to −1.9)*** 154 −4.6 (−6.2 to −3.0)*** 154 −6.4 (−8.2 to −4.7)***  African-American 101 −2.8 (−4.8 to −0.9)** 102 −4.8 (−7.0 to −2.7)*** 103 −6.5 (−8.9 to −4.0)***  White 53 −4.3 (−6.5 to −2.1)*** 51 −3.9 (−6.2 to −1.6)*** 50 −6.3 (−8.5 to −4.1)*** Weight, kg 248 −0.7 (−1.2 to −0.3)** 250 −1.7 (−2.5 to −1.0)*** 247 −1.6 (−2.5 to −0.8)*** Diabetes (all) 92 −1.2 (−2.1 to −0.4)** 96 −1.5 (−2.9 to −0.2)* 93 −3.7 (−5.2 to −2.1)***  African-American 66 −1.1 (−2.0 to −0.3)** 73 −1.0 (−2.5 to 0.4) 71 −3.1 (−4.6 to −1.5)***  White 25 −1.6 (−4.0 to 0.7) 22 −3.3 (−6.9 to 0.4) 21 −5.5 (−9.9 to −1.2)* No diabetes (all) 156 −0.4 (−0.9 to 0.1) 154 −1.8 (−2.7 to −1.0)*** 154 −0.4 (−1.4 to 0.6)   African-American 101 −0.7 (−1.4 to 0.0) 102 −2.2 (−3.4 to −1.1)*** 103 −0.9 (−2.2 to 0.4)  White 53 0.2 (−0.5 to 0.8) 51 −1.0 (−2.3 to 0.4) 50 0.9 (−0.8 to 2.5) ≥5% weight loss, % 248 9.3 (5.6 to 12.9) 250 23.2 (17.9 to 28.5) 247 23.1 (17.8 to 28.4) Diabetes (all) 92 10.9 (4.5 to 17.3) 96 20.8 (12.7 to 29) 93 34.4 (24.7 to 44.1)   African-American 66 12.1 (4.1 to 20.2) 73 19.2 (10.0 to 28.4) 71 32.4 (21.3 to 43.5)  White 25 8.0 (0.0 to 18.8) 22 27.3 (8.3 to 46.2) 21 38.1 (16.9 to 59.3) No diabetes (all) 156 8.3 (4.0 to 12.7) 154 24.7 (17.8 to 31.5) 154 16.2 (10.4 to 22.1)   African-American 101 11.9 (5.5 to 18.3) 102 26.5 (17.8 to 35.1) 103 18.5 (10.9 to 26.0)  White 53 1.9 (0.0 to 5.6) 51 19.6 (8.6 to 30.6) 50 10.0 (1.6 to 18.4) HbA1c, %† 217 0.01 (−0.09 to 0.12) 220 −0.07 (−0.20 to 0.06) Diabetes (all) 80 −0.11 (−0.38 to 0.17) 84 −0.30 (−0.63 to 0.02)   African-American 60 −0.10 (−0.45 to 0.25) 66 −0.33 (−0.72 to 0.06)  White 19 −0.20 (−0.60 to 0.20) 17 −0.28 (−0.85 to 0.28) No diabetes (all) 137 0.08 (0.04 to 0.11)*** 136 0.07 (0.01 to 0.13)*   African-American 87 0.10 (0.05 to 0.14)*** 88 0.08 (0.01 to 0.16)*  White 48 0.05 (0.00 to 0.10) 47 0.05 (−0.03 to 0.13) HbA1c, mmol/mol† 217 0.1 (−1.0 to 1.3) 220 −0.8 (−2.2 to 0.7) Diabetes (all) 80 −1.2 (−4.2 to 1.9) 84 −3.3 (−6.9 to 0.2)   African-American 60 −1.1 (−4.9 to 2.7) 66 −3.6 (−7.9 to 0.7)  White 19 −2.2 (−6.6 to 2.2) 17 −3.1 (−9.3 to 3.1) No diabetes (all) 137 0.9 (0.4 to 1.2)*** 136 0.8 (0.1 to 1.4)*   African-American 87 1.1 (0.5 to 1.5)*** 88 0.9 (0.1 to 1.7)*  White 48 0.5 (0.0 to 1.1) 47 0.5 (−0.3 to 1.4) Total cholesterol, mg/dL‡ 221 −3.2 (−7.0 to 0.7) 211 −3.1 (−7.8 to 1.7) Diabetes (all) 84 0.4 (−6.7 to 7.6) 83 −4.2 (−13.1 to 4.6)   African-American 66 −0.4 (−8.3 to 7.6) 65 −7.1 (−17.5 to 3.4)  White 17 6.9 (−9.5 to 23.3) 17 9.1 (−6.6 to 24.8) No diabetes (all) 137 −5.4 (−9.8 to −1.1)* 128 −2.3 (−7.7 to 3.0)   African-American 89 −6.8 (−11.8 to −1.8)** 83 −2.4 (−8.9 to 4.2)  White 47 −2.5 (−11.0 to 6.0) 44 −2.2 (−11.6 to 7.2) HDL cholesterol, mg/dL‡ 220 −1.6 (−2.8 to −0.4)* 211 −1.0 (−2.3 to 0.3) Diabetes (all) 84 −2.0 (−4.0 to 0.0)* 83 −2.1 (−4.4 to 0.3)   African-American 66 −1.4 (−3.7 to 0.8) 65 −2.2 (−4.8 to 0.5)  White 17 −3.9 (−8.7 to 0.8) 17 −1.8 (−7.6 to 4.0) No diabetes (all) 136 −1.3 (−2.8 to 0.2) 128 −0.3 (−1.7 to 1.2)   African-American 88 −1.6 (−3.6 to 0.4) 83 −0.1 (−2.1 to 2.0)  White 47 −0.9 (−3.1 to 1.2) 44 −0.6 (−2.6 to 1.3) Significance level: *p<0.05; **p<0.01; ***p<0.001. †HbA1c was not measured at 24 months. ‡Lipids were not measured at 6 months. BP, blood pressure; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein. Among all participants, there was a statistically significant reduction in systolic BP of about 6 to 7 mmHg across all follow-up time points. The reduction was similar for those with diabetes and larger for African-Americans with diabetes compared with whites. Outcomes for diastolic BP were similar. Of note, there was very little change in participants' use of blood measure medication at follow-up (data not shown). For those with diabetes, the number reporting use of BP medication was unchanged at 6-month, one less at 12-month, and two less at 24-month follow-up. For those without diabetes, use increased by one at 6, two at 12, and two at 24-month follow-up. There was a trend toward significant improvement in hemoglobin A1c at 12 months among participants with diabetes (−0.30% (95% CI −0.63 to 0.02)/−3.3 mmol/mol (95% CI −6.9 to 0.2), p=0.07). Overall, there was a small decrease in total and HDL cholesterol. Weight loss was a major outcome of interest. Participants with diabetes had significant, sustained, and progressive weight loss compared with baseline at 6, 12, and 24 months (mean −1.2 kg, −1.5 kg, and −3.7 kg, respectively) across all intervention groups. White participants with diabetes had greater weight loss than African-American participants with diabetes. Weight change for participants with diabetes at 12-month follow-up (data not shown) by intervention group selected at the start of the HHL weight program (start of Phase II) was: −3.9 kg (95% CI −7.4 to −0.4, p=0.03) for the group weight loss program (n=17); −2.6 kg (95% CI −5.0 to −0.2, p=0.03) for the combination weight loss program (n=27); and –0.2 kg (95% CI −2.0 to 1.6, p=0.81) for the maintenance of lifestyle intervention program (n=52). At 24-month follow-up (data not shown), it was −5.2 kg (95% CI −9.6 to −0.8, p=0.2) for the group weight loss program (n=18); −2.2 kg (95% CI −4.6 to 0.1, p=0.06) for the combination weight loss program (n=25); and –3.8 kg (95% CI −5.9 to −1.8, p<0.001) for the maintenance of lifestyle intervention program (n=50). After adjusting for age, race, sex, education, and baseline weight, only participants with diabetes had significant sustained weight loss at 24 months (p<0.0001) and participants with diabetes had significantly more weight loss on average than participants without diabetes (p=0.01). Figure 2 depicts weight change (loss and gain) at follow-up by diabetes status with cut points for weight change from baseline of ≥2.5, ≥5, and ≥7.5%. Consistent with mean weight loss, a greater proportion of participants with diabetes demonstrated substantially greater weight loss than gain at these time points, especially at 24 months. At 24 months, 18 participants (20%) lost more than 7.5% body weight while none gained this amount. For those without diabetes, the percentages of those who lost and gained weight at 24 months were similar. Figure 2N and percentage of participants, by diabetes status, for weight change from baseline to follow-up at 6, 12, and 24 months. Weight change is shown for three cut points (≥2.5, ≥5, and ≥7.5% of baseline weight). N, number. From baseline to 24-month follow-up, 32 participants (34%) with diabetes lost at least 5% body weight compared with 25 participants (16%) without diabetes. Percentages for ≥5% weight loss were similar between African-Americans and whites with diabetes; however, more African-American participants without diabetes achieved ≥5% weight loss at 24 months than whites without diabetes (18% compared with 10%, respectively). Percentages of participants with diabetes who achieved ≥5% weight loss at 24 months, by Phase II intervention group, were (data not shown): 44% (95% CI 21 to 68) for the group weight loss program (n=18); 24% (95% CI 7 to 41) for the combination weight loss program (n=25); and 36% (95% CI 22 to 50) for the maintenance of lifestyle intervention program. At the conclusion of Phase I, 85 of 88 (97%) participants with diabetes and 151 of 154 (98%) without diabetes either strongly agreed or agreed that they would recommend the lifestyle program to others. After Phase II, among participants completing the acceptability survey with diabetes, all in the group weight loss program (n=12) and 16 of 19 (84%) in the combination weight loss program were satisfied or very satisfied with the intervention. Participants without diabetes were similarly satisfied. In addition, no adverse outcomes were attributed to the intervention. BODY.CONCLUSIONS: This study evaluated a lifestyle and weight loss intervention promoting a Mediterranean-style diet in a way that was designed to be appealing to residents of the southeastern USA residing in the 'stroke belt,' where the population's CVD risk is very high. Though this type of dietary intervention has been carefully evaluated in Europe,7 it has not been adapted for and assessed in low-income and minority US populations. In this study, the intervention was well received by participants and, compared with baseline, there was improvement in self-reported lifestyle behaviors, BP, and weight, with substantially greater sustained weight loss observed for participants with diabetes. Among participants with diabetes, our intervention did not achieve the same degree of weight loss documented in the Diabetes Prevention Program (DPP) study,34 which was > 5 kg at 24 months (20% of participants were African-American, average age 51). However, in the small subgroup of participants (n=25) who selected the group-based weight loss option, weight loss at 24 months was >5 kg, as it was for all of the white participants with diabetes. In essence, among participants selecting the group-based weight loss format, weight loss at 24 months in our low-income and high-minority population was similar to that observed in the DPP. Moreover, weight loss among all participants with diabetes was greater than in most weight loss studies enrolling disadvantaged population groups.4 Different from the PREDIMED study, which reported weight loss <0.5 kg at 24 months follow-up in all study arms (all participants, not stratified by diabetes status),35 participants in our study with diabetes who selected the lifestyle only option (which focused on diet quality and physical activity, but not weight loss) lost 3.8 kg at 24-month follow-up. A possible explanation for weight loss in this group of our study, as compared with PREDIMED, is difference in baseline diet, with more poor quality carbohydrates and processed food in the American diet compared with the European diet36 and particularly so in the southeastern USA.37–40 In this setting of excess intake of poor quality carbohydrates, a change to a Mediterranean-style, unrestricted fat diet, may have positive metabolic and appetite-suppressing effects, as recently outlined by Ludwig.41 Although the dietary pattern advocated in this study was similar to the PREDIMED intervention arms (especially the nut intervention arm),7 we recommended regular consumption of vegetable oils (high in polyunsaturated and monounsaturated fats) as opposed to four tablespoons of extra virgin olive oil per day as tested in the PREDIMED olive oil intervention arm. In this regard, it is worth noting a recent publication assessing outcomes in two large cohort studies which indicated diets higher in polyunsaturated and monounsaturated fats are associated with a reduction in CVD mortality and total mortality.26 As illustrated in figure 2, among all participants with diabetes, including the majority who did not elect to take part in the weight loss intervention offered in Phase II of the intervention, weight loss increased steadily over the 24-month study period. This weight loss began during Phase I, which focused on diet quality and physical activity, but not specifically on weight loss, and continued at about the same rate during Phases II and III. The observed pattern of weight loss in this study was different from that observed in most other weight loss studies, which is usually greatest weight loss in the short term (6–12 months) with attenuation of weight loss beyond 1 year.42 In a recent meta-analysis of weight loss among named weight loss diet programs, only one of 10 programs achieved greater weight loss at 12 months than at 6 months.6 Even in programs with intensive interventions that include maintenance of weight loss components such as the DPP43 and Look AHEAD,8 there was attenuation in weight loss at 18 and 24 months, respectively. A possible explanation for the observed sustained weight loss in our study was that all participants received some level of intervention over 24 months and unlike most weight loss studies, which try to achieve weight loss over 6 months, our approach was to focus on weight loss over a longer time frame. Additionally, the Mediterranean dietary pattern may have contributed to the sustained weight loss over 24 months, as this dietary pattern has previously been shown to be associated with maintenance of weight loss.44 This study has several limitations including a pre–post design without a control group. While the observed changes may be due to the intervention, they could also be due to other factors, including secular trends. However, sustained weight loss among adults is uncommon. For example, in the control groups of weight loss RCTs such as DPP and Look AHEAD, average weight loss at 24-month follow-up was <1kg.8 34 With regard to secular trends in North Carolina during the time frame of this study (2011 to 2014), there were no changes in the rates of overweight and obesity as assessed by the Centers for Disease Control and Prevention's behavioral risk factor surveillance system.45 Another major limitation is the sample size of this study, especially with regard to outcomes by intervention groups (group weight loss, combination weight loss, or maintenance of lifestyle intervention) and race. Further, because weight loss was less than expected at the end of Phase II, as discussed in detail elsewhere,11 the sample size for the 'embedded' RCT of weight loss maintenance was so small that we did not undertake a formal analysis of outcomes for this RCT. Another limitation is that lifestyle outcomes were self-reported and may have been exaggerated due to social desirability reporting bias. Finally, our findings may not be generalizable to populations different from the sample enrolled from one community in eastern North Carolina. The cost-effectiveness of an intervention is a very important consideration for community-based and clinic-based weight loss programs. Without a control group, we did not undertake a cost-effectiveness analysis, which is another limitation of this study. However, we have previously reported a cost-effectiveness analysis for the group-based intervention format that we tested in Phase II, comparing it with a delayed intervention control group.46 In that study, in which the weight loss intervention was considered cost-effective (assuming weight loss could be sustained over time), weight loss at 6-month follow-up was 3.7 kg in the intervention group, very similar to the weight loss of 3.9 kg observed at the completion of Phase II for our participants with diabetes for our participants with diabetes who received the group-based intervention. The study has several strengths. First, is the relatively unselected sample (few exclusion criteria were applied and no run-in period) which enhances its generalizability; second, it used a design that mimicked real-world situations, allowing participants to choose between two weight loss intervention formats or to focus on lifestyle change without weight loss as a goal; third, the follow-up was 74% at 24 months, which is a larger follow-up percentage and longer follow-up interval than reported in many weight loss studies; and fourth, physiological outcomes were obtained using standardized objective measures. In this study, with a relatively unselected sample, largely minority and of lower socioeconomic status, the tested Mediterranean-style dietary pattern, a pattern associated with substantial reduction in CVD risk7 was very well received. Among participants with diabetes, there was sustained improvement in self-reported lifestyle behaviors, BP, and weight change at 24-month follow-up. Though the study did not have a control group, our findings confirm the acceptability of a Mediterranean-style dietary pattern among this very high-risk population and suggest that this dietary pattern may be associated with sustained weight loss. Further study of interventions promoting this dietary pattern is warranted in high-risk US populations with diabetes, including RCTs that assess intermediate outcomes and CVD events.

TITLE: Induction of Labor Using Native (OXYTIP) in Comparison to Foreign Oxytocin (SYNTOCINON) ABSTRACT.OBJECTIVE: This study was conducted with the aim to investigate and compare Iranian produced and foreign oxytocin for use in induction of labor. ABSTRACT.MATERIALS AND METHODS: This random clinical trial was conducted on a population of 198 pregnant women with live fetus and cephalic presentation and conditions conducive to induction of labor, monitored by obstetricians and gynecologists. They were randomly divided into group A (n = 99) received 10 units of Syntocinon (Novartis Pharma Canada) in 500 cc Ringer lactate, and group B (n = 99) received 10 units of Oxytip (Caspian Tamin Company Iran) in 500 cc serum, who entered the study to commence induction, by signing written consent. Study variables such as induction indications (post-term, ruptured membranes, diabetes, and..), induction duration, duration of the 1st and the 2nd stages of labor, and delivery method; as well as labor outcomes like hyper-stimulation of uterine, postpartum bleeding, 5-minute Apgar score, and infant's birth weight; and neonatal outcomes (admission to NICU, oxygen and intubation) were assessed for the two groups by a trained midwife and registered in the patient's questionnaire. Data were analyzed in SPSS software using statistical tests: t-test, Chi-square, and Mann-Whitney. ABSTRACT.RESULTS: Two groups were similar in demographic variables such as; age, BMI, parity, education. There was no significant difference regarding to obstetric and gynecologic characteristics such as: gestational age, dilatation, effacement, and fetal positioning, as well as the indication for labor induction when the study began. After intervention, variables including: induction duration, duration of the 1st and the 2nd stages of labor, delivery method; and labor outcomes such as: hyper-stimulation of uterine, postpartum bleeding, 5-minute Apgar score, and infant's birth weight; and neonatal outcomes (admission to NICU, oxygen and intubation), in the two groups, were found to be the same (P < 0.05). Mean oxytip dosage needed was less than that of oxytocin to reach for appropriate pain (P = 0.042). ABSTRACT.CONCLUSION: The two drugs in terms of labor induction and neonatal complications had similar outcomes and the locally made drug with a lower dosage appears to produce the desired outcome. BODY.INTRODUCTION: Labor induction is a common intervention technique in childbirth, which is performed in 1 out of every 4 pregnant women in cases as: pre-eclampsia, diabetes, prolonged pregnancy, when cervix is ready or membranes are ruptured. It is administered intravenously as a labor induction strategy in term pregnancies (1). Shortening duration of labor by induction, especially when prolonging pregnancy, for whatever reason, poses a serious risk to mother and/or fetus, is a valuable successes in obstetrics (1). In addition to increased rate of maternal and fetal infections, prolonged labor has other consequences such as: increased hospitalization costs, increasing need for cesarean section. It is also an important factor in lowering Apgar score and infant's need for NICU (1, 2). Among standard indications for labor induction, gestational hypertension, and prolonged pregnancy are the most common criteria. In the U.S., labor induction from 9.5% in 1990 reached 22.1% by 2004 (2). Various labor induction methods including use of oxytocin, prostaglandins, and amniotomy are available. In the Parkland Hospital, oxytocin was used to induce labor in 30% of deliveries. At Alabama University, Birmingham Hospital, oxytocin was used in 20% of deliveries between 1996 and 1999 (3). Oxytocin is the most common medication used to induce labor in viable pregnancies, and it is the first synthetic polypeptide hormone made, which brought its creators the Noble Prize (1). This octa-peptide is secreted in pulse form and has a half-life of 10-12 minutes (4, 5). The level of oxytocin that causes effective uterine contractions varies in different people, mostly due to the difference in clearance speed and number of oxytocin receptors in the uterus (6). Its success in induction of labor depends on condition of cervix at the onset of induction. To stimulate the pregnant uterus, this drug is administered intravenously because it is possible to measure dosage of administered drug, and in case of a complication, it can quickly be discontinued. The initial dose of this drug varies from 0.5 ml/min to 2 ml/min, and the interval between increased dosage also varies from 15 to 40 minutes (low dosage regimen) (7). According to the new pharmacokinetics data, most obstetricians use a regimen in which oxytocin dosage is increased by 1-2 ml/min every 40 minutes (8, 9). Considering the importance of labor induction and the drugs used, and given the circumstances in the country, the global sanctions, and the rising cost of medication, an important and valuable strategy for social empowerment is to turn to, and support national products. Also, in the particular economic circumstance of the country, it is important to try and locally produce medications that require valuable foreign currency to import. Thus, considering local production of these drugs and implementation of different approval stages, this study was conducted to compare the effects of foreign (Novartis Pharma Canada) and Iranian-produced oxytocin (Oxitip, Caspian Tamin Company) in labor induction in term and post-term deliveries. If the Iranian drug is efficient, it will help self-sufficiency of the country in pharmaceutical production. Through comparison of the locally-produced and foreign produced oxytocin, we can determine its particular side-effects and report them to the manufacturer in order to improve this drug. With its improved quality, or similarity of studied effects, this drug can be marketed globally, thereby reducing unwanted import costs of the drug. BODY.MATERIALS AND METHODS: This study was a randomized double-blind clinical trial that was performed from 2012 till September 2013 in the Academic Medical Centre, the Vali-e-Asr Hospital, Tehran, Iran, with a target population of 220 pregnant women with live fetus and cephalic presentation and more than 37th week of gestational age (Fig. 1). According to gynecologists' and obstetricians' opinions, induction of labor was indicated for these women because their cervix did not conform to medical parameters and was not appropriate for childbirth without intervention. Study inclusion criteria were singleton, cephalic presentation, healthy fetal membranes, Bishop score less than 4, reactive NST, or BPS8/10 and uterine contractions less than 3 in every 10 minutes. Figure 1The consort flowchart Regular uterine contractions (more than 6 contractions per hour), fetal death, contraindication for amniotomy (like HIV positive), mother's age less than 18 years, history of cesarean section, multi-parity (>3), chorio-amniotitis, history of asthma, multiple pregnancy, placenta previa, non-cephalic presentation, glaucoma, vaginal bleeding, history of allergy to prostaglandins or beta-adrenergics, abnormal fetal heart rate, were exclusion criteria. 198 women was randomly and equally divided into group A and group B. Patient follow-up, in terms of delivery process to the end was carried out by obstetricians other than research team. Before performing induction protocol, Bishop score was calculated for each pregnant woman by a qualified person and dilatation less than 3 cm and effacement of 10-30% were considered. To that end, all patients were first examined for uterine contractions by the researcher patients with Bishop score of 4 or less and absence of uterine contractions were entered the treatment and intervention phase of the study. Before initiating induction, all patients underwent NST, and fetal heart rate was monitored. Patients that showed signs of fetal distress were excluded from the study. Written consent for participation in the study was obtained from all patients. This project was approved by the ethics committee of Tehran University of Medical Sciences, and was registered in the clinical trials site IRCT 201011275181N3. Induction of labor initially began with 10 unit oxytocin in 1000 cc Ringer solution (2.5 mu/min), then increased every 15 minutes at infusion rate of 4 drops of 2.5Cc per minute, to reach a maximum of 40 mu/min, and continued at this rate, or until number of drops reached a maximum of 64 drops/min. With the right rate of contractions (3 contractions in every 10 minutes, each lasting more than 40 seconds and maximum of 60 seconds) there was no need to increase dosage and injection was maintained at the same rate. Obviously, healthcare providers in the delivery ward were blinded to the nature of the intervention. With the onset of good contractions, vaginal examination was performed every hour with intact membranes, and every 2 hours with ruptured membranes, and uterine contractions were monitored every half hour. Before oxytocin injection, maternal vital signs were controlled, and then monitored every hour. Oxytocin was discontinued when fetal distress or uterine hyper-stimulation signs were observed. When uterine hyper-stimulation and fetal distress were improved, infusion re-started at half the previous dose, and continued as before. It was decided that, if within 6 hours of induction no change was observed, the patient was considered as an induction failure, and induction was discontinued. However, with change in status of cervix, induction continued. With non-spontaneous rupture of membranes, amniotomy was performed at dilatation of 5 cm. Furthermore, during induction, fetal heart rate was controlled every 15 minutes, and with any change in fetal heart rate, induction was stopped and permanent monitoring was performed. Also, during induction, uterine contractions were controlled, and with more than 4 contractions in every 10 minutes, or with each contraction lasting more than 90 seconds and contraction intervals less than 60-90 seconds, uterine hyper-stimulation was assumed and induction was discontinued. Also, to assess progress of labor, cervical dilatation and effacement were controlled every 30 minutes during the 1st active stage of labor, and every 15 minutes during the 2nd stage of labor. Data such as age, gestational age, number of pregnancies, induction indications, type of delivery, 5-minute Apgar score, birth weight, and primary and secondary outcomes were completed in the questionnaire. Data were analyzed with SPSS software using statistical tests: t-test, Chi-square, and Mann-Whitney. Primary outcomes such as induction interval and labor duration, and secondary outcomes like duration of the second stage, and abnormal fetal heart rate pattern forceps delivery rate, cesarean rate, 5-minute Apgar score, number of indications for neonatal counseling cases, NICU cases were measured and compared in two groups. BODY.RESULTS: Mean age of the foreign oxytocin (group A) was 25.7 years and mean age in the locally produced oxytocin (group B) was 26.3 years. Mean BMI was 28.55 kg/m2in group A, and 28.7 kg/m2in group B. There was no difference between the two groups in terms of age (P = 0.633), weight (P = 0.678), height (P = 0.257), or BMI (P = 0.846) (Table 1). Table 1 Pre-intervention qualitative and quantitative variables in two groups Oxytocin (A) n= 98 Oxitip (B) n= 98 p value Age (year) (mean± SD) 25.7± 4.56 26.30± 5.23 0.633 Weight (kg) (mean± SD) 75.92± 11.56 75.17± 14.30 0.687 BMI (kg/m2) (mean± SD) 28.55± 4.40 28.70± 5.91 0.846 Number of pregnancies (mean± SD) 1.71± 0.92 1.66± 0.96 0.706 Gestation age (weeks) (mean± SD) 39.10± 1.47 38.75± 1.33 0.080 Mean prenatal cervical dilatation (fingers) (mean± SD) 1.44± 0.63 1.46± 0.63 0.821 Education  Illiterate-primary school (n,%) 6 (6.1) 9 (9.2) 0.821  Junior-high school 33 (33.3) 35 (35.7)  High school Diploma (n,%) 57 (57.6) 51 (52.0)  Bachelor's degree (n,%) 3 (3.0) 3 (3.0) Labor induction reasons  Post-term fetus (n,%) 10 (10.1) 8 (8.1) 0.239  Intensifying labor pains (n,%) 39 (39.4) 40 (40.4)  Maternal diabetes (n,%) 1 (1.0) 3 (3.0)  Membrane rupture (n,%) 18 (18.2) 29 (29.3)  Pre-eclampsia (n,%) 3 (3.0) 4 (4.0)  Others (n,%) 27 (27.3) 16 (16.2) No significant difference was found between the two groups in the reasons of labor induction like post-term, maternal diabetes, membrane rupture, or pre-eclampsia (p= 0.239); uterine hyper-stimulation was not reported in either of the group. Mean drug dose was 28.98 drops in group A, and 24.29 drops in group B, and the difference between the two groups was statistically significant (p= 0.042). Also, duration of contractions was 42.96 seconds in the oxytocin group and 40.25 seconds in oxytocip group, which was less than that in foreign-made drug (P = 0.016) (Table 1). Study variables such as labor induction interval and onset of the first pain, duration of labor induction, duration of 1st and 2nd stages of labor and frequency of natural childbirth were the same in both oxytocin and oxytocip groups (Table 2). Table 2 Post-intervention childbirth variables in two groups Variables Cyntocinon (A) n= 98 Oxytip (B) n= 98 p value Drug dosage at onset of pain (drops) (mean ±SD) 11.27± 9.11 7.45± 3.38 0.003 Interval between labor induction and onset of first pain (min) (mean ±SD) 41.9± 49 32± 54.9 0.067 Interval between labor induction and main pains (min) (mean ±SD) 198.96± 279.50 160.95± 182.95 0.245 Duration of labor induction (hr) (mean ±SD) 11.59± 7.92 9.74± 6.06 0.067 Duration of 1st phase of labor (hr) (mean ±SD) 2.62 4.75 0.158 Duration of 2nd phase of labor (hr) (mean ±SD) 31.96± 17.21 37.77± 24.06 0.695 Drug dose at onset of proper contractions (drops) (mean ±SD) 28.98± 15.84 24.29± 15.83 0.042 Duration of contractions (seconds) (mean ±SD) 42.96± 10.28 40.25± 4 0.016 Interval between contractions (min) (mean ±SD) 2.34± 1.43 2.52± 1.87 0.454 Cesarean (n,%) 47 (48%) 41 (41.4%) 0.454 Postnatal hemorrhage 0 4 (4.1%) 0.059 Neonatal complications  5-minute Apgar score(mean 9.80± 0.51 9.84±0.40 0.261  Admission to NICU (n,%) 3 (3%) 6 (6.1%) 0.306  Nasal oxygen (n,%) 4 (4%) 5 (5.1%) 0.733  Oxygen mask (n,%) 3 (3%) 4 (4%) 0.700  Intubation (n,%) 1 (1%) 0 0.999 Neonatal outcomes (admission to NICU, receiving oxygen, and intubation) were also the same in both oxytocin and oxytocip using pregnant women groups. Postpartum hemorrhage was slightly more in group B compared to group A; however, the difference was insignificant (P = 0.59). Hyper-stimulation did not occur in any of the two groups. BODY.DISCUSSION: Use of oxytocin in labor induction or augmentation is common in childbirth and delivery (10). Oxytocin is a potent drug, and its improper and prolonged use at high doses can cause complications such as cardiovascular effects, hypotension, uterine over-stimulation, fetal distress, uterine rupture, and water intoxication (11, 12). Considering that an important and valuable strategy for social empowerment is to turn to and support national produce, the present study was conducted to examine and compare the effects of Iranian produced with foreign produced oxytocin, and to determine the specific side-effects of the drug to report to the producing company, so that steps could be taken to improve the drug. Through enhanced quality and similarity of effects studied, the company could then market the drug globally. In the present study, mean induction duration (induction onset to full opening of cervix) in the oxytocin group was 11.59± 7.92 and in the oxytocip group was 9.74±6.06 hours, which was nearly the same as the study conducted in the U.S. on 816 patients that used low dose of oxytocin, in which induction duration was 9.7 hours (13). Also, in a study by Aram in Isfahan, induction duration until completion of labor was nearly 771 minutes (12.5 hours) (14). In a study by Sohrabi et al., in which a low dose of oxytocin was administered, duration of induction until labor was 14.5 hours (412.8 minutes) (15). Another study by T Tam in 2013 conducted on 625 patients from population of women undergoing induction with oxytocin, duration of induction until labor was 11.917 hours, and cesarean rate of 16% and natural birth of 74% were also reported (16). It seems duration of induction until onset of labor in this study was almost similar to those of other studies, and within the normal range. Given that type of delivery, duration of induction, and duration of effective pains were the same in both groups, the main cause of cesarean and lack of progress of labor was CPD (Cephalo-Plevic Disproportion). In a study by Charoenboon, cesarean rate caused by CPD increased from 3.2% in 1992 to 7.8% in 2011, which was more common than other indication like breech presentation, fetal distress, and multiple pregnancy [13]. As for Vali-e-Asr Hospital is a referral and teaching hospital, perhaps reports of high number of cesarean cases may be due to reasons other than oxytocin use. Selection of subjects with no particular risk factors largely reduced this limitation. BODY.CONCLUSION: Both drugs had similar labor induction properties, and also similar effects on neonatal complications. However, the locally-made drug achieves the same results at low dosage. Considering the results obtained and lack of specific complications, the Iranian oxytocin (Oxytip, Caspian Company) produces similar labor induction results to its foreign counterpart and it can be used as an Iranian-made drug in current circumstances, benefiting from resistive economy in production and distribution of drugs, which will reduce current costs in the country.

TITLE: Polyethylene Glycol and Lactulose versus Lactulose Alone in the Treatment of Hepatic Encephalopathy in Patients with Cirrhosis: A Non-Inferiority Randomized Controlled Trial ABSTRACT: BACKGROUND In this clinical trial, polyethylene glycol (PEG) solution was compared with lactulose in the treatment of hepatic encephalopathy in patients with cirrhosis. METHODS This randomized controlled trial was performed on 40 patients in two groups. The patients in the lactulose group received either 20-30 grams of lactulose orally or by a nasogastric tube, or 200 grams of lactulose enema by a rectal tube. The patients in the PEG–lactulose group received the same amount of oral or rectal lactulose, plus 280 grams of PEG in 4 liters of water orally as a single dose in 30-120 minutes. Serial physical examinations, hepatic encephalopathy scoring algorithm (HESA), blood level of ammonia, and serum biochemical studies were used to evaluate the severity of hepatic encephalopathy. RESULTS In comparison with lactulose alone, PEG-lactulose could improve HESA score in 24 hours more effectively (p =0.04). Overall, PEG-lactulose regimen was associated with a decrease in length of hospital stay compared with lactulose treatment (p =0.03) but in subgroup analysis we found that PEG-lactulose regimen could only decrease the length of hospital stay in women significantly (p =0.01). CONCLUSION The use of PEG along with lactulose in comparison with lactulose alone is more effective in the treatment of hepatic encephalopathy in patients with cirrhosis and results in more rapid discharge from hospital. BODY.INTRODUCTION: Cirrhosis is one of the leading causes of death in the United States and results in a significant economic burden ranges from $14 million to $2 billion due to its underlying etiology.1,2 According to the global burden of disease (GBD) study in 2013, cirrhosis is the 6th cause of death in developed countries and among the 10 most common causes of death in different world areas.3 This burden is expected to rise in the forthcoming years due to the increasing prevalence of cirrhotic cases related to HCV infection and non-alcoholic steatohepatitis (NASH).4,5 Different studies show that most of patients with cirrhosis ultimately develop some degrees of hepatic encephalopathy and this condition is highly associated with increased mortality and health care costs.6 Hepatic encephalopathy, is further classified to overt hepatic encephalopathy (OHE) and minimal hepatic encephalopathy (MHE).7 It is estimated that 30-45% and 60-80% of patients with cirrhosis finally show OHE and MHE, respectively.6,8-10 There is a dearth of evidence backed by large, well controlled clinical trials in favor of specific treatment options currently in use for patients with MHE.11 However the existing evidence suggests some benefit of lactulose in the treatment of MHE.12,13 Treatment of patients with OHE is mainly based on the eliminating of underlying precipitating factors, nutritional supports, and reduction of blood ammonia level.11,14 Lactulose and rifaximin are the most widely used medications to reduce ammonia production; however their exact mechanism of action is still unclear.11 Rifaximin is a non-absorbable antibiotic that has been used for patients with OHE. It acts against coliforms like Escherichia coli, reduces the amount of serum ammonia and improves hepatic encephalopathy.15 A meta-analysis of 19 randomized controlled trials showed that rifaximin had beneficial effects on patients with OHE and may also reduce mortality in this population.16 However its cost remains a concern. A decision-analysis study found that rifaximin was not cost-effective as monotherapy in the treatment of patients with hepatic encephalopathy.17 Approximately 70% of patients with OHE improve on lactulose treatment.18 When used for secondary prophylaxis, lactulose reduces recurrent episodes of hepatic encephalopathy, but does not affect mortality.19 Lactulose is currently considered as the first line treatment of patients with OHE although there is no high-quality evidence to support it.20 In a Cochrane review, no significant differences in outcome was seen between the patients treated with or without lactulose.21 As a patient with OHE is admitted to hospital, predisposing factors often resolve along with the improvement in mental status; thus understanding the exact role of lactulose in this improvement is somehow difficult.22 Polyethylene glycol (PEG) is being studied for the treatment of hepatic encephalopathy in patients with cirrhosis and limited research has shown positive effects.23 In this clinical trial, PEG solution plus lactulose is compared with lactulose in the treatment of hepatic encephalopathy in patients with cirrhosis. BODY.MATERIALS AND METHODS: This randomized controlled trial (RCT) was reviewed and approved by Tehran University of Medical Sciences (TUMS) and the review board of Digestive Disease Research Institute (DDRI; approval letter number 416/984). All the studied patients provided an informed consent signed by themselves or their legally authorized representatives (LARs). LARs and the patients were free to quit this study anytime. This RCT was performed in Shariati Hospital, a referral center for gastroenterological and liver diseases, Tehran province, Iran, from September 2015 to January 2016. According to previous trials that had studied the effect of PEG and lactulose in the treatment of patients with cirrhosis and hepatic encephalopathy, we estimated the 24-hour improvement in hepatic encephalopathy scoring algorithm (HESA) score (see the next part) for PEG-lactulose group about 80% and for lactulose group about 50%.23-26 In addition, we assumed 2-sided first type error and second type error as 0.05 and 0.20, respectively and a drop-out rate of about 15%. Thus it is estimated that in order to have a statistically significant difference between two treatment groups, we need 24 patients in each group. All patients with a known history of cirrhosis who presented with episodic hepatic encephalopathy were eligible to participate. Hepatic encephalopathy was defined as the onset of disorientation or asterixis according to The International Society for Hepatic Encephalopathy and Nitrogen Metabolism consensus and was assessed using HESA score.8 Patients having all the following criteria were included: - Documented cirrhosis with any underlying etiology - Hepatic encephalopathy of any grade - 18 to 80 years of age Exclusion criteria were as follows: - Acute liver failure defined as severe acute liver injury with encephalopathy and international normalized ratio (INR)≥1.5 in a patient without pre-existing liver disease - Acute change in mental status due to a diagnosis other than hepatic encephalopathy - Hemodynamic instability obviating vasopressors for resuscitation - Pregnancy - Lack of LAR - Refusal of consent by the LAR - Patient's unwillingness to participate The patients were randomly assigned to two groups according to a series of block randomization numbers (8 blocks of 6 patients). The patients in the lactulose group received either 20-30 grams of lactulose (at least 3 doses in 24 hours) orally or by a nasogastric tube, or 200 grams of lactulose enema by a rectal tube. The patients in the PEG–lactulose group received the same amount of oral or rectal lactulose, plus 280 grams of PEG in 4 liters of water orally as a single dose during 30-120 minutes. Lactulose dose increased until at least two soft or loose bowel movements per day were produced. All the patients received otherwise routine care by the treating physician. Serial physical examinations were performed at presentation and 24 hours later. We used HESA score to evaluate the severity of hepatic encephalopathy. This scoring system is an objective instrument and is more widely adopted than West Haven criteria.27 In order to avoid interpersonal variations, HESA scores at baseline and 24 hours afterwards were calculated by the same investigator. Blood level of ammonia and serum biochemical studies were checked in all patients at baseline and 24 hours after starting treatment. All samples were collected in EDTA containing tubes and transported to the laboratory in ice in less than 15 minutes of collection. Child-Turcotte-Pugh (CTP) score and Model for End-Stage Liver Disease (MELD) scores were calculated for all the patients. Cause of cirrhosis was obtained from past medical records. Major outcome was the change in HESA score after 24 hours. The patients were also compared regarding the length of hospital stay and blood ammonia levels and biochemical studies. One of the team members was responsible for prescribing either PEG-lactulose or lactulose alone to eligible patients, while HESA scores at presentation and 24 hours later were calculated by another team member avoiding possible biases. The clinical evaluator had been experienced previously in the diagnosis of hepatic encephalopathy and in the performance of HESA. Also statistical analyses were done by another member who was totally masked to the treatment groups. Primary outcome of this trial was defined as at least one scale improvement in HESA score. Secondary outcomes were overall length of hospital stay and changes in blood ammonia level. IBM SPSS software version 22 for windows was used to analyze the data. We used Pearson Chi-square test and Fisher's exact test for qualitative analysis, Shapiro-Wilk test to assess normal distribution, independent sample Mann Whitney U test and Log Rank test for non-parametric analysis and independent sample t test for parametric analysis. A cut-off of 0.05 for statistical significance was presumed. BODY.RESULTS: Of the 48 eligible patients to enter the study, 2 patients in each group refused to consent. Three patients in lactulose group and one patient in PEG-lactulose group had received sedative drugs. Since the diagnosis of hepatic encephalopathy is based on the exclusion of other diagnoses, we excluded their data from the final analysis. Basic demographic data were similar between the groups (table 1). Hepatitis B, hepatitis C, and cryptogenic cirrhosis were the most common underlying causes of cirrhosis. There were no significant differences in the underlying etiology of cirrhosis. Gastrointestinal (GI) bleeding and constipation were the most common precipitating factors of hepatic encephalopathy. No significant difference was seen in the precipitating factors of encephalopathy between the treatment groups. Severity of cirrhosis according to MELD score and CTP score was also comparable between the two groups. Three patients in lactulose group and two patients in PEG-lactulose group received lactulose enema (p =0.63). Table 1 Basic demographic and clinical information Variables All Patients (n=40) Lactulose Group (n=19) PEG-lactulose Group (n=21) Significant Difference Age, mean (SD), y 56.45 (10.86) 59.63 (9.24) 53.57 (11.61) No Men, n 27 11 16 No Cirrhosis Cause, n No Alcoholic fatty liver disease 2 1 1 Autoimmune hepatitis 1 1 0 Cryptogenic 11 6 5 Hepatitis B 12 6 6 Hepatitis C 9 4 5 NASH 2 1 1 Portal thrombosis 2 0 2 Primary sclerosing cholangitis 1 0 1 Encephalopathy Cause, n No Constipation 15 9 6 Electrolyte disturbance 2 1 1 GI bleeding 18 7 11 HCC 1 0 1 Infection 4 2 2 MELD score, median (IQR) 17.5 (10) 17.5 (6) 17.5 (15) No CTP score, median (IQR) 9 (3) 9 (2) 9.5 (3) No Lactulose enema, n 5 3 2 No NASH: non-alcoholic steatohepatitis, HCC: hepatocellular carcinoma, CTP: Child-Turcotte-Pugh, MELD: Model of End-stage Liver Disease, PEG: polyethylene glycol 3350. In addition, baseline lab data including blood level of ammonia was similar between PEG-lactulose group and lactulose group (tables 2 and 3). Table 2 Laboratory data Variables All Patients (n=40) Lactulose Group (n=19) PEG-lactulose Group (n=21) Significant Difference WBC count, mean (SD), × 10 9 /L 8.6 (5.8) 9.8 (7.1) 7.6 (4.2) No Hemoglobin concentration, mean (SD), mg/dL 10.7 (2.5) 10.7 (2.0) 10.6 (2.9) No Platelet count, mean (SD), × 10 3 /uL 99.7 (59.5) 98.0 (65.3) 101.2 (55.3) No BUN, mean (SD), mg/dL 35.7 (25.8) 38.8 (25.5) 32.8 (26.4) No Creatinine, mean (SD), mg/dL 1.4 (0.8) 1.5 (0.8) 1.2 (0.6) No Total bilirubin, mean (SD), mg/dL 7.3 (11.6) 5.1 (7.9) 9.3 (14.0) No Direct bilirubin, mean (SD), mg/dL 3.7 (6.9) 2.2 (3.9) 5.1 (8.7) No Prothrombin time, mean (SD), sec 21.8 (10.4) 19.7 (7) 23.7 (12.7) No Partial thromboplastin time, mean (SD), sec 37.6 (17.4) 35.6 (11.5) 39.4 (21.6) No INR, mean (SD) 1.8 (1.0) 1.6 (0.6) 2.0 (1.3) No Total protein, mean (SD), g/dL 4.9 (0.8) 4.8 (0.8) 4.9 (0.7) No Serum albumin, mean (SD), g/dL 2.8 (0.5) 2.8 (0.5) 2.8 (0.6) No WBC, white blood cell; BUN, blood urea nitrogen; INR, international normalized ratio. Table 3 Study outcomes Variables All Patients (n=40) Lactulose Group (n=19) PEG-lactulose Group (n=21) Significant Difference Baseline HESA score 0.34 2 16 8 12 1 24 11 9 24-h HESA score change, mean (SD) 1 (0.57) 0.77 (0.53) 1.23 (0.53) *0.04 One or more score improvement in HESA score, n 34 14 20 *0.05 24-h ammonia level change, mean (SD), µmol/L Baseline level 77.6 (30.5) 72.5 (28.4) 82.1 (32.3) 0.30 After 24-h 41.2 (25.5) 37.0 (18.6) 45.1 (30.4) 0.55 Difference 36.3 (21.1) 35.6 (17.8) 37.0 (24.2) 0.81 Length of hospital stay, mean (SE), day 7.8 (0.4) 8.9 (0.7) 6.8 (0.5) *0.03 We used Mann Whitney U test to analyze “baseline HESA score”, “24-h HESA score change” and “24-h ammonia level change” due to lack of normal distribution. Fisher’s exact test was used to analyze “one or more score improvement in HESA score”. Data for “length of hospital stay” were analyzed using Log-rank test. In comparison with lactulose alone, PEG-lactulose could improve HESA score in 24 hours more effectively (p =0.04, table 3). 20 patients in PEG-lactulose group had a decrease of at least 1 HESA score after 24 hours, as compared with 14 patients in lactulose group and the difference was significant (p =0.05, figure 1). Fig. 124-hour HESA score changes in lactulose and PEG-lactulose groups HESA score at presentation and 24 hours after treatment have been shown with different bullet colors for each treatment group. Dark gray for the beginning and light gray for 24 hours after treatment. The patients whose HESA score did not show any changes after 24 hours have been shown with black bullets. Of the 21 patients in PEG-lactulose arm, 1 patient had no improvement in HESA score, 14 patients had improvement of one score and 6 patients improved by two scores. Of the 19 patients in lactulose arm, 5 patients had no improvement in HESA score, 13 patients had improvement as much as one score and 1 patient had improvement up to two scores (figure 1). There was no significant change in the blood level of ammonia after 24 hours between the treatment groups (p =0.858, table 3). But, PEG-lactulose regimen was associated with a decrease in length of hospital stay as compared with lactulose treatment (p =0.03, figure 2). Fig. 2Time to discharge in patients presented with hepatic encephalopathy(HE) In a Kaplan-Meier graph, depicting the proportion of hospitalized patients and length of hospital stay for patients presented with hepatic encephalopathy. The patients who received PEG in addition to lactulose more rapidly discharged from hospital (p=0.03). There were no notable adverse effects in either PEG-lactulose group or in lactulose group. Death did not occur in any of the groups up to the end of hospital discharge. There were not any significant changes in electrolyte levels and kidney functions as well. BODY.DISCUSSION: The results of this study show that use of PEG along with lactulose in comparison with lactulose alone, is more effective in the treatment of hepatic encephalopathy in patients with cirrhosis. By accelerating the treatment of hepatic encephalopathy, PEG can help patients to return to normal life more rapidly and decrease the direct and indirect cost of illness caused by hepatic encephalopathy. PEG, which resolves decreased level of consciousness more effectively and more rapidly in the first 24 hours, can also help physicians to identify the other causes of altered mental status more quickly and more accurately. The exact mechanism resulting in hepatic encephalopathy is poorly understood.28 Many studies have suggested the role of ammonia production in GI tract and impaired ammonia elimination by an imperfect liver 21,29; thus reduction in the amount of GI bacteria by the use of antibiotics and colon cleansing agents may play a therapeutic role in hepatic encephalopathy. This therapeutic strategy is dissociated with the underlying cause of hepatic encephalopathy i.e. whether GI bleeding or electrolyte disturbance was the precipitating factor, administration of cleansing agents might be helpful in resolving hepatic encephalopathy. PEG is a safe osmotic laxative most commonly used for colon preparation before colonoscopy and is an inexpensive, widely accessible laxative in many settings. Due to the past literature review, this randomized controlled trial could show the effectiveness of PEG in the treatment of hepatic encephalopathy for the second time. Compatible with a previous study 23, in this trial PEG-lactulose could improve hepatic encephalopathy more rapidly than lactulose alone, but there were no significant differences in blood level of ammonia between the two groups. Up to the best of our knowledge, ammonia or better to say NH3 (not NH4+) plays the major role in altered mental status in encephalopathic cirrhotic patients, because only NH3 can easily pass over blood brain barrier.30 If we measured the amount of free NH3 instead of total blood level of ammonia, we could probably find a difference between the two treatment groups. It is noteworthy that PEG causes a mild metabolic acidosis, resulting in increased NH4+ level and decreased NH3. These chemical changes might explain the effect of PEG in rapid improvement of altered mental status without any significant change in ammonia level.30,31 In addition, some other studies have challenged the use of blood ammonia as an indicator for severity of hepatic encephalopathy, especially in grade 0 to grade 2 encephalopathy.32-34 It is worth mentioning that because of GI bleeding, 7 patients in lactulose group and 11 patients in PEG-lactulose group (p =0.32) received ceftriaxone 1g daily for 3 days as a preventive strategy against spontaneous bacterial peritonitis. Since there is no significant difference in the number of these patients between the two groups, we can assume that prophylactic antibiotic therapy did not confound the results of this trial. There are some notable limitations in our study. This trial assessed the effect of PEG in only one hospital. In this trial, we used a unique dose of PEG, namely the same dose being used for preparing the colon for colonoscopy. More studies of escalating doses should be designed to reach the optimum dose of PEG with minimum adverse effects in the treatment of hepatic encephalopathy. Finally this was a non-inferiority trial; superiority trials that weigh PEG alone versus other conventional treatments like lactulose, help researchers to understand the effect of PEG much better. This randomized controlled trial showed that the use of PEG together with lactulose in comparison with lactulose alone is a more effective way in the treatment of hepatic encephalopathy in patients with cirrhosis and results in more rapid discharge from hospital. Larger and multicenter trials with different doses of PEG and different target population should be performed to make PEG as a routine treatment for patients with cirrhosis and encephalopathy.

TITLE: A pharmacoeconomic analysis to determine the relative cost-effectiveness of bimatoprost 0.03% eye drops and brimonidine 0.2% eye drops in patients of primary open-angle glaucoma/ocular hypertension ABSTRACT.AIMS:: The aim was to compare efficacy and cost-effectiveness of bimatoprost 0.03% and brimonidine 0.2% in primary open-angle glaucoma (POAG)/ocular hypertension (OHT). ABSTRACT.SETTINGS AND DESIGN:: Open, randomized, cross-over, comparative study. ABSTRACT.MATERIALS AND METHODS:: Forty patients of POAG or OHT with intraocular pressure (IOP) <30 mm Hg were included in the study after a written informed consent. The patients were divided randomly into two groups of 20 patients each. Patients of group A were administered bimatoprost 0.03% eye drops once daily, and those of group B brimonidine 0.2% eye drops twice daily for a period of 4 weeks. After a washout period of 4 weeks, the patients were crossed over that is, group A was administered brimonidine 0.2% and group B bimatoprost 0.03%. Fall in IOP at 4 weeks was recorded. The daily cost of each drug was calculated by maximum retail price and the average number of drops per bottle. The cost-effectiveness was then calculated as the cost of drug/mm Hg fall in IOP. ABSTRACT.STATISTICS:: Independent samples t-test was used to compare the efficacy of both drugs. ABSTRACT.RESULTS:: IOP lowering with bimatoprost (8.9 ± 1.598 mm Hg) was significantly (P < 0.0001) higher than brimonidine (6.55 ± 1.26 mm Hg). The number of drops/ml were 33.43 ± 0.52 and 25.49 ± 0.26, respectively, for bimatoprost and brimonidine. Treatment with bimatoprost was costlier than brimonidine with daily costs/eye Rs. 4.02 ± 0.06 and 3.14 ± 0.03, yearly costs/eye Rs. 1467.46 ± 20.74 and 1147.75 ± 11.15, respectively. Bimatoprost was more cost-effective than brimonidine with the cost-effectiveness ratio (CER) respectively Rs. 13.10 ± 2.61/mm Hg and Rs. 13.96 ± 2.86/mm Hg. Incremental CER Rs. 10.43/mm Hg implies lower costs/mm Hg extra IOP lowering by bimatoprost than Rs. 13.96 for brimonidine. ABSTRACT.CONCLUSION:: In spite of being costlier, bimatoprost is more efficacious and cost-effective than brimonidine. BODY: Glaucoma is a chronic debilitating disease requiring life-long treatment. Being the largest cause of bilateral blindness, second only to cataract; glaucoma is a major public health problem.[1] Prevalence of blindness in India, according to National Survey on Blindness 2001–2002, is 1.1%.[2] Leading cause of blindness in India is cataract accounting for 62.6%, whereas glaucoma accounts for 5.8%.[3] Worldwide, it is estimated that about 66 million people have visual impairment from glaucoma, with 6.7 million suffering from blindness due to it.[4] From 2010 to 2020, the most detectable change in glaucoma worldwide would be, its increase in India. The largest absolute number of glaucoma cases have been reported in China, followed by Europe and India.[5] Primary open-angle glaucoma (POAG) is a subset of the glaucomas defined by raised intraocular pressure (IOP) consistently above 21 mm Hg in at least one eye with typical glaucomatous visual field and/or optic nerve head damage and an open, normal appearing anterior chamber angle with no other underlying disease.[6] Ocular hypertensives are defined as a subset of patients with open angles, raised IOP but neither optic nerve head nor visual field changes.[7] Studies provide strong evidence that high IOP plays an important role in the neuropathy of POAG. It has been demonstrated that the reduction in the level of IOP lessens the risk of visual field progression in open-angle glaucoma.[8] Treatment strategies of glaucoma aim at lowering IOP, which helps to prevent optic nerve damage and glaucoma-related blindness. An even a single unit lowering of IOP has been associated with significant clinical improvements.[9] Pharmacotherapy is usually the first line of treatment for elevated IOP and open-angle glaucoma. Major drug classes for medical treatment of POAG include alpha-agonists (brimonidine), beta-blockers (timolol, betaxolol, levobunolol), topical carbonic anhydrase inhibitors (dorzolamide, brinzolamide), oral carbonic anhydrase inhibitors (acetazolamide), miotic agents (pilocarpine), prostaglandin (PG) analogs (travoprost, latanoprost), prostamides (bimatoprost), and sympathomimetic drugs (epinephrine, dipivefrine). PG analogs lower IOP by increasing the uveoscleral outflow of aqueous humor.[10] They are effective in reducing IOP and have the additional advantage of requiring only once a day administration. Bimatoprost, a synthetic prostamide analog, used as a 0.03% topical preparation once daily is efficacious in the treatment of open-angle glaucoma, ocular hypertension (OHT) and other forms of glaucoma. Brimonidine, a selective alpha-2-agonist, available as 0.2% ophthalmic solution to be administered twice daily, causes a suppressed aqueous humor production. With IOP lowering efficacy comparable to β-blockers, brimonidine confers neuroprotection in glaucoma by stimulating an ongoing neuronal survival pathway.[11] It is used as one of the first-line therapies in patients who have contraindications to β-blockers. The ophthalmologists have a wide range of choices for management of glaucoma, in terms of cost, efficacy, and adverse effects. There is an increased demand from society and health care payers that clinical medicine in particular when aimed at treatment of chronic life-long disease should justify its cost. Taking into consideration, the broadening gap between therapeutic possibilities and resources available, the choices have to be made by prioritizing (rationing) all treatment strategies.[12] Economic evaluation of glaucoma therapy needs to be targeted at assessment of efficiency, that is, health effects weighed against the sacrifices or costs incurred for attaining them.[13] Therapeutic decisions for glaucoma therapy should be taken with due consideration of cost of drug along with efficacy and safety. It is no longer enough that an intervention is clinically effective; it needs to be cost-effective as well if we are to make the most of our finite resources. Contemplating the importance of cost-effectiveness, the idea of our study is to update the ophthalmologists' knowledge regarding the cost-effectiveness along with daily and yearly cost of treating glaucoma with bimatoprost, an effective prostamide and brimonidine, an alpha-2-agonist. BODY.MATERIALS AND METHODS: In this open, randomized, cross-over, comparative study, 40 subjects of POAG or OHT attending the outpatient Department of Ophthalmology, were included after obtaining written informed consent. The inclusion criteria were a minimum age of 18 years and unilateral or bilateral POAG or OHT with IOP < 30 mm Hg. Patients with bilateral POAG were treated for both eyes, but only left eye of every patient was considered as the study eye. Patients with closed anterior chamber angle or acute angle closure glaucoma, any history of intraocular surgery within 6 months of study, ocular infection or inflammation, history of allergy to the drugs or other side effects with the drugs or with established diagnosis of secondary glaucoma were excluded from the study. Apart from this, the female patients who were pregnant, lactating or not employing adequate measures to prevent conception or patients with a history of chronic obstructive pulmonary disease/asthma were also excluded. BODY.MATERIALS AND METHODS.INTRAOCULAR PRESSURE LOWERING (EFFECTIVENESS) DETERMINATION: In this study conducted over 12 weeks, 40 eyes (left side) of these 40 patients were included with the main treatment outcome as the number of mm Hg fall in IOP. The patients were randomly divided into two groups of 20 each. Baseline IOP was recorded on 1st visit, day 0 at 9 am. Patients in group A were instructed to instill one drop of bimatoprost 0.03% (Lumigan) once a day at 9 pm daily for 4 weeks. Patients in group B were instructed to instill one drop of brimonidine 0.2% (Alphagan) twice a day at 9 am and 9 pm for 4 weeks. Measurement of IOP was done subsequently at 2 weeks and 4 weeks. The patients were counseled for regular instillation of eye drops at first visit and compliance to treatment ensured at subsequent visits at 2 weeks and 4 weeks by questioning the patient. The 4 weeks of therapy were followed by a 4 week washout period when no drug was administered to the patients in either of the groups. The 4-week washout period was decided after literature search. Most of the studies with bimatoprost and brimonidine were carried out with a washout period of 4–6 weeks.[141516] The drugs were then crossed over, that is, group A was instilled brimonidine 0.2% and group B was instilled bimatoprost 0.03% for a period of another 4 weeks after a baseline IOP measurement and subsequent measurements on 2nd week and 4th week. Effectiveness data used for this economic analysis were the number of mm Hg of IOP reduction at 4 weeks compared with the baseline. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The efficacy in terms of IOP lowering was subjected to statistical analysis using independent samples t-test. BODY.MATERIALS AND METHODS.COST ANALYSIS: To determinate the cost, five bottles each of bimatoprost (Lumigan) and brimonidine (Alphagan) were taken and number of drops per bottle calculated with bottles held at 135° (the angle at which the drops are instilled in the eyes) and drops collected in graduated measuring cylinder. The actual, not labeled volume was determined for each bottle. Thus, the number of drops/ml were determined by counting the number of drops in one bottle and dividing by the total actual volume measured. Daily cost of particular anti-glaucoma medication was calculated by dividing the cost of one bottle by total number of drops in a bottle and multiplying by number of drops required daily. Thereafter, 4 weekly and yearly costs of both drugs were calculated. BODY.MATERIALS AND METHODS.COST-EFFECTIVENESS ANALYSIS: Cost-effectiveness i.e. cost/mm Hg of IOP reduction was calculated as: Where one treatment strategy is both more expensive and more effective than its comparator, an incremental cost-effectiveness ratio (ICER) can be calculated that depicts the extra cost per unit of outcome obtained in comparing one treatment option to another.[17] BODY.MATERIALS AND METHODS.ETHICS: The study was approved by the institutional ethical committee. BODY.MATERIALS AND METHODS.OBSERVATIONS: The age- and gender-related characteristics of study groups were as shown by Table 1. The baseline IOP of group A and group B before recruitment was 26.45 ± 1.98 and 26 ± 1.89 mm Hg, respectively; and after washout was 26 ± 1.65 and 25.75 ± 1.74 mm Hg. Table 1 Age and gender distribution in both groups Efficacy of both drugs was compared by measuring IOP lowering effect of bimatoprost and brimonidine. Statistical analysis was done using independent samples t-test. As shown in Table 2, absolute fall in IOP with bimatoprost was by 8.9 ± 1.598 mm Hg by the final visit. This was significantly (P < 0.0001) higher than the reduction of 6.55 ± 1.26 mm Hg seen in brimonidine-treated patients. Among the patients on bimatoprost, 85% (34/40) showed IOPs ≤ 18 mm Hg at 4 weeks as against 25% (10/40) with brimonidine. Table 2 Statistical analysis of IOP reduction with bimatoprost and brimonidine using independent samples t -test Fig. 1 summarizes the mean IOP at visit 0, 1, 2 and Fig. 2 summarizes the IOP fall with a standard deviation. The number of drops per bottle, the number of drops/ml and overfilling/underfilling of bottles were as shown by Table 3. Thus, bimatoprost had more drops/ml than brimonidine. Figure 1IOP changes in patients on Bimatoprost and Brimonidine on subsequent visits Figure 24-weekly cost s of both drugs Table 3 Volumetric analysis In our study, we found treatment with bimatoprost to be costlier than brimonidine with daily costs for each eye, Rs. 4.02 ± 0.06 and 3.14 ± 0.03, respectively. The yearly costs for the drugs per eye were Rs. 1467.46 ± 20.74 and 1147.75 ± 11.15 for bimatoprost and brimonidine, respectively. The 4 weekly costs for both drugs used for calculation of CER were as shown by [Fig. 2]. Cost-effectiveness that is, cost/mm reduction of IOP was calculated. The costs and effectiveness included in the calculation were the 4 weekly costs (28 days) and IOP lowering at V3 that is, at 4 weeks. CER analysis as shown by Fig. 3 shows a lower cost incurred by bimatoprost than brimonidine per millimeter lowering of IOP. Thus, bimatoprost is superior to brimonidine in cost-effectiveness analysis. Figure 3Comparision of cost-effectiveness of bimatoprost and brimonidine Incremental cost-effectiveness ratio is the ratio of the difference in costs and the difference in IOP lowering by both drugs. It represents an additional cost for each unit of additional fall in IOP. The ICER was calculated as 10.43 which means extra Rs. 10.43 were required for each additional mm Hg IOP reduction given by bimatoprost for 4 weeks as compared with brimonidine. Selecting brimonidine, the less costly alternative, implies a willingness to pay Rs. 13.96/mm Hg (CER of brimonidine). An additional IOP reduction obtained with bimatoprost costs Rs. 10.43, which is below willingness to pay the amount for brimonidine. Therefore, on the basis of its incremental CER, bimatoprost could be considered a cost-effective strategy as compared with brimonidine. The adverse drug reactions reported by the patients were as shown in Table 4. No patient in either group had any serious adverse drug reactions warranting discontinuation of therapy or requirement of additional medication for the treatment of adverse effects. Adverse events were mostly mild in both groups, and the drugs were well-tolerated. Table 4 Adverse effects of drugs BODY.DISCUSSION: An ophthalmologist has a wide range of choices for the medical management of glaucoma. Hypotensive lipids (PG analogues/prostamides) have high efficacy, a favorable safety profile, ease of once daily regimen and are often reasonable on a cost per day basis. They have thus fast become a favorite among both physicians and patients despite their higher costs. Bimatoprost has been shown to be most cost-effective among PG analogues.[18] Alpha-agonists like brimonidine are available at lower maximum retail price (MRP's) than PG analogs and are being studied for additional advantages of neuroprotection.[19] Bimatoprost and brimonidine, both are commonly prescribed drugs in glaucoma, each with its own advantages. As the treatment is life-long, studies have been done to compare the efficacy and cost of various anti-glaucoma drugs previously. In our study, the efficacy of drugs as shown by IOP lowering of 8.9 ± 1.6 (34.10%) and 6.55 ± 1.26 (25.19%), respectively, for bimatoprost and brimonidine was comparable to studies by Thomas et al., 2003 and Whitcup et al., 2003 which showed IOP lowering 8 (32.4%) and 6 ± 3.3 (21%) for bimatoprost and brimonidine, respectively.[2021] Several ways have been to devised by the drug manufacturers to minimize the wastage of eye drops, like overfilling of the bottles, bottle design, medication dispensing mechanisms and administration techniques. Hence, a lot of preparations may be cheaper, but due to large drop size, they may end up being less cost-effective. The dispensing angles, and viscosity of the medication are the other factors that may influence drop size.[22] The MRP of an anti-glaucoma agent is just one of the multitudes of factors to consider when choosing a medication for a patient. The products with higher actual volume, smaller drop size; hence, the larger number of drops/ml may in reality cost less. In the present study, the average number of drops/ml was 33.43 ± 0.52 for bimatoprost (Lumigan) and 25.49 ± 0.26 for brimonidine (Alphagan) whereas, in study by Rylander an Vold, 2008 these were, respectively, 32.27 ± 0.45 and 24.83 ± 1.57.[23] The daily costs in the present study, Rs. 4.02 ± 0.06 and Rs. 3.14 ± 0.03, respectively, for bimatoprost and brimonidine were comparable with the cost analysis studies by Fiscella et al., 1999 and Fiscella et al., 2003; which showed daily costs to be $0.95 and $0.90 for bimatoprost and brimonidine, respectively.[2224] Thus, bimatoprost showed higher daily costs in all these studies. But, cost analysis alone is not a complete economic analysis as the treatment with higher costs may be more efficacious too. On the other hand, a comparatively cheaper therapy may have the disadvantage of lower efficacy and higher adverse effects. The CER for both drugs in the present study was calculated for the study period of 4 weeks and cost/mm Hg lowering found to be Rs. 13.10 ± 2.61/mm Hg and Rs. 13.96 ± 2.86/mm Hg for bimatoprost and brimonidine, respectively. Thus in the present study, bimatoprost, in spite of being costlier had a more favorable CER. Another study showing comparison of cost-effectiveness of bimatoprost and brimonidine by Galindo-Ferreiro et al. too shows similar results placing bimatoprost at a superior position to brimonidine with respect to CER, with CER being 9.1 for bimatoprost and 9.6 for brimonidine.[25] The gross difference in numerical values of CER in both of above studies is due to variability in monetary units, MRP's and cost of patient visits in different countries. In the present study, ICER of bimatoprost was Rs. 10.43/mm Hg which is lower than cost/mm Hg reduction incurred by using brimonidine Rs. 13.96 ± 2.86/mm Hg). Selecting brimonidine, the less costly alternative, implies "willingness to pay" Rs. 13.96 ± 2.86/mm Hg lowering of IOP. An additional reduction in IOP obtained with bimatoprost costs Rs. 10.43/mm Hg which is less than the "willingness to pay" amount for brimonidine. Therefore, on the basis of ICER, using bimatoprast over the brimonidine would be considered a cost-effective strategy. We have compared cost-effectiveness of bimatoprost 0.03% (Lumigan, Allergan) and brimonidine 0.2% (Alphagan, Allergan). The findings of the study thus may not be generalized to other brands and concentrations of these drugs. Similar studies could be carried out to prioritize the anti-glaucoma therapy based on cost-effectiveness analysis. BODY.CONCLUSION: Bimatoprost and brimonidine are efficacious drugs in the treatment of POAG/OHT, but in spite of daily costs being higher, bimatoprost is a better choice than brimonidine because of: Greater cost-effectivenessGreater reduction in IOPGreater ocular hypotensive efficacyBetter compliance due to once daily dosing. Physicians consider many factors when prescribing patients with glaucoma. Ultimately, the goal of eye care providers is to give the best, most cost-effective care to their patients taking into consideration efficacy, tolerability, medication response, compliance, and dosing regimens along with the cost of medication. Economic evaluation of glaucoma therapy needs to be targeted at assessment of efficiency, that is, health effects weighed against the sacrifices or costs incurred for attaining them. The deciding criterion should be cost-effectiveness of treatment strategy rather than efficacy or cost alone. As glaucoma management requires life-long therapy and the options available are many, future studies would be needed to update the rapidly changing economic information pertaining to the medical management of glaucoma.

TITLE: Clinical Trial of a Home Safety Toolkit for Alzheimer's Disease ABSTRACT: This randomized clinical trial tested a new self-directed educational intervention to improve caregiver competence to create a safer home environment for persons with dementia living in the community. The sample included 108 patient/caregiver dyads: the intervention group (n = 60) received the Home Safety Toolkit (HST), including a new booklet based on health literacy principles, and sample safety items to enhance self-efficacy to make home safety modifications. The control group (n = 48) received customary care. Participants completed measures at baseline and at twelve-week follow-up. Multivariate Analysis of Covariance (MANCOVA) was used to test for significant group differences. All caregiver outcome variables improved in the intervention group more than in the control. Home safety was significant at P ≤ 0.001, caregiver strain at P ≤ 0.001, and caregiver self-efficacy at P = 0.002. Similarly, the care receiver outcome of risky behaviors and accidents was lower in the intervention group (P ≤ 0.001). The self-directed use of this Home Safety Toolkit activated the primary family caregiver to make the home safer for the person with dementia of Alzheimer's type (DAT) or related disorder. Improving the competence of informal caregivers is especially important for patients with DAT in light of all stakeholders reliance on their unpaid care. BODY.1. INTRODUCTION: Dementia of the Alzheimer's type is a growing public health problem. An estimated 35.6 million people were living with dementia in 2010, and the number is expected to double every 20 years to 65.7 million in 2030 and 115.4 million in 2050 [1]. In the United States, an estimated 5.2 million people have dementia of the Alzheimer's type (DAT), a number that is projected to grow to 13.8 million by 2050 [2]. Other population studies estimate that 13% or 1 in 8 Americans aged 71 or older suffers from this disease [3], the sixth leading cause of death in the United States for which there is no treatment or cure [4]. A person with DAT will live an average of four to eight years and as long as 20 years after the onset of symptoms [5] during which 80% of their care is provided by family and friends [6]. These family caregivers absorb the largest costs of care in both dollars and emotional distress. In high-income countries, informal care accounts for 45% of the worldwide costs of dementia, $604 billion USD, and, in low-income and lower-middle-income countries, the costs of informal (unpaid) care by family caregivers account for the majority of costs [7]. Further, because of the demands of caring for a person with DAT, family caregivers have negative health consequences and increases in health care costs for themselves [8, 9]. The currently incurable nature of dementia of the Alzheimer's type, the duration of the illness, the suffering it can cause to patients and their families, and the cost of care make providing safe community care a priority. Health-related safety in the home and community is a public health concern for all individuals, especially older adults striving to "age in place" [10]. A review of empirical evidence regarding home safety of people across the age span and with or without disability revealed that home modifications increase functional ability outcomes although inconsistencies in study results and limitations in research designs exist [11]. The authors emphasized the importance of future studies to examine not only the physical environment but also the impact of intraindividual factors and the social environment of the home on functional ability. These authors note the relatively little home safety research that has been conducted with people who have dementia-related disorders. Older adults are at increased risk of accidents and injuries in the home compared to the general population. Among those over the age of 65 in the United States, accidental injuries are the ninth leading cause of death, in particular fatality due to falls and fire; common nonfatal injuries include falls, being struck by objects, motor-vehicle incidents, and lacerations [12]. Persons with dementia are at even greater risk than their peers because of the cognitive and functional impairments associated with the illness [13, 14]. Prior research has revealed risks for injury for persons with dementia living at home that can be categorized as either high frequency risks, high severity risks, or both: falling, cooking, wandering, driving, home fires, medication problems, and unsafe firearms and sharps [15–17]. For example, general wandering around the home may be considered a high frequency risk for injury (i.e., common but not necessarily dangerous), whereas nighttime wandering in inclement weather is a less frequent but more severe risk. Even in early stages of memory loss, if a person is unaware of memory loss, they are more likely to have difficulty with everyday decisions such as medication management [18]. In addition, patients' self-evaluations of their ability to conduct instrumental activities of daily living (IADL) such as medication management and ability to use the telephone are significantly higher than caregivers' objective evaluations [19]. Not surprisingly, home safety is an immediate concern for families when a member is diagnosed with DAT. In a recent descriptive study of 82 older adults with dementia living in the community, the authors identified the prevalence of home safety modifications as well as barriers to implementation [20]. Marquardt and colleagues found 44% of homes had outdoor steps with no railings; only 23% had modified bathrooms with at least three safety components: grab bars, walk-in shower, and shower seat; 12% of homes were rated as very cluttered, which did not allow for safe mobility inside the house. These authors also found caregivers attributed almost three times more home modifications to physical impairments as opposed to memory loss in their loved ones; this suggests that caregivers may lack insight into how cognitive deficits impact the way people with dementia navigate their environments [20]. Caregivers have identified several barriers to making the home safer; in particular, lack of knowledge and impractical options were identified; conversely, trained professionals can facilitate the implementation of home safety modifications by providing caregivers with practical information and advice about what to do [21, 22]. This randomized controlled trial (RCT) continues a program of research guided by the Home Safety/Injury Model [15, 21, 23] which aims at giving informal caregivers the knowledge and resources to prevent risky behaviors and accidents in the homes of persons with dementia of the Alzheimer's type (DAT) or a related dementia. Our initial studies revealed that family caregivers were overwhelmed by the potential for safety hazards in the home and did not know where or how to make the most important home modifications. Even in the presence of adequate skills, however, a person's perception of low self-efficacy and self-doubts about the ability to perform a task can interfere with achievement [24]. Thus, a Home Safety Toolkit was designed to focus on improving caregiver competence by increasing practical abilities through a health literacy tested booklet that focuses on the high frequency/high severity risks for accidents and injuries in the home (Figure 1). In addition, the Home Safety Toolkit (HST) includes sample items (Table 1) which allow the caregiver to immediately and easily make home safety modifications, increasing caregiver self-efficacy through enactive attainment—theorized to be the most powerful of the influences on self-efficacy [25]. Our initial studies used an intensive professional assessment/intervention by a registered nurse (RN) and an occupational therapist. In contrast, this current HST, informed by health literacy principles, is specifically designed to be a self-directed, self-paced intervention for the caregivers which allows them to decide when and how to make modifications in the environment. Experience from prior studies informed the generation of hypotheses for this RCT. For instance, we previously found correlations between caregiver self-efficacy and years of formal education and perceived social support which lead to the decision to include these variables as covariates in the hypothesis testing: Hypothesis 1. After controlling for the effects of caregiver years of formal education, baseline caregiver self-efficacy, baseline caregiver strain, and social support, caregivers who receive the Home Safety Toolkit intervention will have higher postintervention self-efficacy, lower postintervention caregiver strain, and improved environmental home safety than caregivers in the control group who receive standard patient education: Hypothesis 2. After controlling for the effects of caregiver years of formal education, baseline caregiver self-efficacy, baseline caregiver strain, and social support, care recipients who receive the Home Safety Toolkit intervention will have fewer risky behaviors and accidents when compared to the care recipients in the control group who receive standard patient education. BODY.2. METHODS.2.1. PARTICIPANTS: Study participants were recruited from the Bedford, Massachusetts Veterans Administration Medical Center (VAMC), VA Boston Healthcare System (VABHS), and Boston University Alzheimer's Disease Center (ADC). Participants were dyads of primary caregivers and persons with a progressive dementia of the Alzheimer's type who lived in the community, were willing to have home visits for home safety education, and read and spoke English. Inclusion criteria for care recipients were diagnosis of dementia of the Alzheimer's type or a related disorder, score of 24 or less on the Mini-Mental State Examination [26], expected to continue living in the community for the next 6 months, and having the ability to ambulate without help from the caregiver (assistive devices to self-ambulate were acceptable). Inclusion criteria for the primary informal caregiver were living in the home with the care recipient, provid a minimum of 4 hours of caregiving per day, and have no known or apparent cognitive impairment upon screening. Exclusion criteria were a previous home safety visit and admission to a long-term care facility. Persons with DAT who were living alone were excluded because their safety issues are more complex and there is no primary informal caregiver who can make consistent observations about risky behaviors and accidents. BODY.2. METHODS.2.2. PROCEDURES: The study was reviewed and approved by the Institutional Review Boards (IRB) of Bedford VAMC, VA Boston Healthcare System, and Boston Medical Center and monitored semiannually by a Data Safety Monitoring Board of VA Health Services Research and Development as required for multisite studies. Potential subject dyads were enrolled from geriatric specialty clinics. Two sites, the Bedford Dementia Clinic and the Boston University Alzheimer's Disease Center (ADC), maintain a registry of families who have agreed to participate in research. At the Boston University ADC, the recruitment coordinator sent a postcard to eligible caregivers who returned the card to indicate whether they agreed to be contacted by one of the researchers [27]. In addition, because this "Opt-In" strategy can lead to a more self-selected group of participants, the recruitment coordinator also telephoned eligible participants if they neglected to return the postcard. At Bedford VAMC, unless a family "opted out" of all research, they were sent an introductory flier (approved by the IRB) with a follow-up phone call by the project director or research assistant. At VA Boston Geriatric Consultation Clinics, the research staff was available onsite to give the flier to potential patient/caregiver dyads and requested a follow-up phone call to discuss the project in more detail. Thus, subject dyads were essentially self-referred rather than referred by a clinical provider, an approach that was favored by clinical providers who did not want to use their limited clinical time to discuss research. The randomization method was stratified by site, and a permuted-block randomization was used in order to balance the number of patients assigned to each group [28]. Computer-generated random numbers were used by the statistician to allocate group assignment by the sealed envelope method [29]. Home visits were used for the initial and final data collection because in our preliminary studies caregivers had requested home visits because it was the most convenient location for them and the home environment is the most comfortable for the care recipient. In order to be consistent throughout the study, all subject dyads in both groups had baseline and final data collection done in the home setting. After informed consent but before random assignment, the project director (PD) or research assistant (RA) collected demographic and baseline data on the outcome variables and covariates. Then, the subject/dyad was randomized to either the intervention group or the control group. Upon learning the group assignment of the subject dyad, the investigator returned to his/her car and brought either the HST (booklet and sample items) or a standard patient information worksheet on home safety. We kept the two forms of patient education, HST and standard worksheet, separate so subject dyads would not see what the other group was receiving (single blinded). Subject dyads were told that at the end of their 3-month participation in the study they would be offered the alternative home safety education. After randomization, the intervention group received the Home Safety Toolkit (HST), which has two components: (1) the booklet "Keep the Home Safe for a Person with Memory Loss" and (2) a canvas bag with low-cost sample items that were found in prior studies to be acceptable to families and effective to reduce risky behaviors and accidents. The HST booklet (Figure 1) was developed using well-established principles of reading comprehension and health literacy and was learner verified for attractiveness, comprehension, self-efficacy, and persuasiveness [30]. Caregivers in the intervention group had an opportunity to manipulate and practice using the home safety items. This was intended to increase self-efficacy for injury prevention and increase practical ability. Sample safety items included a motion sensor, slide bolt lock, stove knob covers, grab bar, rubber bathmat, medicine dispenser, smoke alarm, and general items such as a flashlight and nightlights (Table 1). The control group received the "Worksheet to Make the Home Safer," a patient information sheet that has been used in clinical practice since 2003 [31]. The worksheet was used to standardize "customary care" among the 3 study sites, which was a requirement of the Institutional Review Board that was concerned about the subject dyads in the control group being at higher risk during the study period. The worksheet has accurate and practical recommendations for home safety in dementia of the Alzheimer's type (DAT) with a reading level of 5th to 6th grade; however, it is in a conventional format using words only and does not conform fully to the principles of health literacy. In addition, there are no sample safety items to stimulate self-efficacy. Caregivers in both groups were given 15–20 minutes to look over the information and ask questions if they wished to clarify any of the provided information. Given that the intervention was designed to be self-directed and self-paced, there was no specific training provided by the research investigator to either the intervention or the control group, but the research investigator did answer questions initiated by the caregiver in both cohorts. During the study period of 3 months, the caregivers in both the intervention and control groups were called biweekly by the project director (PD) or research assistant (RA) to collect information on the Risky Behavior Questionnaire (RBQ). Interim (between the two home visits) data collection of risky behaviors and accidents was conducted to facilitate the caregivers' memory of "close calls" during the 3 months of the study. Close calls were behaviors by the person with dementia that worried the family caregiver and/or could have resulted in an injury, for example, lighting the stove or smoking. In our prior study, the use of a home safety log over the course of several months was difficult because risky behaviors were episodic and unpredictable. At 3 months after enrollment, a second home visit was conducted to collect Time 2 data. After this final data collection, caregivers were offered the alternative home safety education materials. Fidelity to the protocol was achieved through training by the principal investigator ((PI) Kathy J. Horvath). The PI first demonstrated the home safety protocol and then the PD and RA each did a simulated home visit for a potential subject in each of the groups: intervention and control. Any observed differences between the PD and RA were discussed and resolved to achieve a standardized approach to the home safety protocol. At regularly scheduled team meetings, the protocol was discussed to review procedures and any new issues. A change in the RA incumbent midway through the study led to a demonstration and return demonstration by the PD and RA, respectively. BODY.2. METHODS.2.3. MEASURES: Demographics. Baseline data included a conventional demographic sheet with care recipient's age, gender, and race and caregiver's age, gender, race, relationship to care recipient, and years of formal education. BODY.2. METHODS.2.3. MEASURES.2.3.1. FOLSTEIN MINI-MENTAL STATE EXAMINATION (MMSE) [: The MMSE is a commonly used and well-documented measure of global cognition. Although other brief cognitive assessments have shown better sensitivity and specificity for early cognitive impairment [32, 33], the MMSE was the most readily available instrument across the 3 study sites. Because the inclusion criteria required a confirmed diagnosis of Alzheimer's disease or a related disorder, the need to identify mild cognitive impairment was not required, and the MMSE was used as a measure of disease severity because of the availability of scores across study sites. For this study, the MMSE was used only to describe the sample and was not used for diagnostic purposes. Scores on the MMSE range from 0 to 30 with higher scores representing better cognition. Age and education level impact population-based norms for mean scores on the MMSE indicating that persons over the age of 60 with a minimum of 9–12 years of education have a mean score of 28 with a standard deviation of 1.7 [34]. Measuring MMSE in cohorts of older persons aged 75, 80, and 85 years or older indicates population norms as low as 22 in the oldest old with limited education [35]. BODY.2. METHODS.2.3. MEASURES.2.3.2. PHYSICAL SELF-MAINTENANCE SCALE (PSMS) [: This scale measures 6 basic activities of daily living: toileting, feeding, dressing, grooming, physical ambulation, and bathing. The scale was developed and tested with a diverse sample of 265 subjects aged 60 or older, living in both institutional and community settings. The scale has good interrater reliability (r = 0.87; r = 0.91), and construct validity was demonstrated when compared to measures of mental status and behavioral adjustment (r = 0.38 and r = 0.38, resp.). In our prior studies, Cronbach's alpha for the scale was 0.81 and 0.85. Items are rated on a scale of 1 (can perform the task without any help) to 5 (person is totally dependent). Scores range from 6 to 30 with higher scores representing greater disability. Internal consistency reliability for this study was a = 0.855. BODY.2. METHODS.2.3. MEASURES.2.3.3. FUNCTIONAL ACTIVITIES QUESTIONNAIRE (FAQ) [: The FAQ measures instrumental activities of daily living (IADL) that are commonly performed by people living in the community. Initial testing of the FAQ demonstrated good sensitivity (0.85) and specificity (0.81) for detecting impairment which was comparable to the MMSE. Convergent validity was demonstrated through correlation with an independent neurologist's estimate of global functioning (r = −0.83), and concurrent validity was demonstrated with another measure of IADL (r = 0.72). Item to total correlations on the FAQ ranged from 0.80 to 0.90 [37]. There are 10 items, such as preparing a balanced meal and paying attention to TV, which are rated on a scale from normal = 0 to dependent = 3. The scores range from 0 to 30 with higher scores representing greater degrees of functional impairment. Internal consistency reliability for this study was a = 0.746. BODY.2. METHODS.2.3. MEASURES.2.3.4. REVISED SCALE FOR CAREGIVING SELF-EFFICACY [: This instrument is composed of 3 discrete subscales that are measured and interpreted separately, which is consistent with self-efficacy (SE) theory as being domain specific. This study used the subscale: SE for obtaining respite. This subscale is conceptually congruent with the Home Safety/Injury Model [21, 23]. One of the most unsafe environments is when the care receiver is left alone unsupervised. Obtaining respite for times when the caregiver has to do errands or other activities is an important task in order to avoid gaps in supervision. Additional items reflecting behaviors specific to home environmental safety were added in procedures common to self-efficacy measurement [39]. The additional 12 items asked caregivers to rate their confidence to prevent the high frequency/high severity risks for safety in the home of a person with DAT [15], for example, preventing the care receiver from using the stove or sharp knives and preventing the care receiver from leaving the house alone. Each of the total 17 items is rated as 0% confidence to 100% confidence. For statistical purposes, the summed score for all items was used in the analysis with potential scores ranging from 0 to 1700 with higher scores representing increased caregiver self-efficacy for home safety. Understanding the scores from a clinical perspective is discussed under Section 3. Internal consistency reliability for this study was a = 0.837 Time 1 and a = 0.803 Time 2. BODY.2. METHODS.2.3. MEASURES.2.3.5. MBRC CAREGIVER STRAIN INSTRUMENT [: The Margaret Blenkner Research Center Caregiver Strain Instrument is composed of four subscales which are scored separately. For this study, the subscale for health strain was used because the items capture potential negative consequences for the caregiver, such as physical health deterioration, more nervousness, and having less energy. Construct validity was demonstrated through factor analysis and convergent validity with other psychosocial measures. The scale consists of 5 items, which are rated as strongly agree (3), agree (2), disagree (1), and strongly disagree (0). Scores for the scale range from 0 to 15 with higher scores representing more strain. Internal consistency reliabilities for this study were a = 0.855 Time 1 and a = 0.842 Time 2. BODY.2. METHODS.2.3. MEASURES.2.3.6. MEDICAL OUTCOMES STUDY SOCIAL SUPPORT SURVEY (MOS-SSS) [: This instrument was developed to measure functional support in contrast to structural support. Functional support is defined as the perception of the availability of interpersonal relationships for particular needs, such as preparing meals, helping with daily chores, and giving advice. The MOS-SSS consists of 19 items which are scored from 1 to 5 with 1 = "None of the Time" and 5 = "All of the Time." The scale consists of 4 subscales which can be scored separately or as a total overall social support index. The overall index was used for this study. Internal consistency reliability for the overall index was Cronbach's a = 0.97. Construct validity was tested with product moment correlations with other measures of emotional and physical health [41]. The highest correlation was with a measure of loneliness, and the lowest correlation was with a measure of pain severity (r = −0.67; r = −0.19; P < 0.01). Scale items are averaged and then transformed so the possible range of scores is 0–100, with higher scores representing higher perceived social support. Internal consistency reliabilities for this study were a = 0.939 Time 1 and a = 0.948 Time 2. BODY.2. METHODS.2.3. MEASURES.2.3.7. RISKY BEHAVIOR QUESTIONNAIRE [: This tool was developed for use in the first home safety project in order to capture the behaviors that often lead to accidents and injuries. Content validity is derived from empirical evidence demonstrating the high severity and high frequency behaviors in persons with dementia of the Alzheimer's type (DAT) that could lead to an injury in the home. The number of risky behaviors and accidents is recorded at baseline and biweekly for 3 months. The additional biweekly interim data points were added to ensure good memory of "close calls," the caregiver's perception of behavior that was risky but did not result in an accident or injury. For example, the care recipient tried to leave the house unsupervised but may not have exited. The baseline number of risky behaviors is summed for the month prior to the first home visit. The outcome measure for the variables of risky behaviors and accidents is the summed total of incidents that occurred during the 3-month duration of the study. Individual behaviors are not weighted because it is difficult to determine the severity of an incident if an actual injury does not occur. Based on our preliminary studies, we consider all of the risky behaviors as having an equal potential to cause harm, and thus the scores for risky behaviors and accidents are summed. Potential scores range from 0 to undetermined. The maximum score is undetermined because the measure represents the caregiver count of the number of times an incident occurred. In this study, postintervention sum scores ranged from 0 to 180 with a mean of 35.20 (SD 34.55). BODY.2. METHODS.2.3. MEASURES.2.3.8. HOME SAFETY CHECKLIST (HSC): The HSC is used to measure the variable overall home safety. This tool was developed and used in preliminary studies with interrater reliabilities of 0.80–0.85, internal consistency reliability a = 0.84) and was sensitive to changes in home safety between enrollment (Time 1) and 6 months later (Time 2) with a statistically significant change in scores (t = 9.402; P ≤ 0.001). This relatively new tool was used because there is no other instrument available to measure home safety specifically. Lach et al. used a Home Safety Inventory as a daily log for caregivers to record risky behaviors, but the Home Safety Inventory is not a measurement tool [42]. Gitlin and colleagues developed and tested the Home Environmental Assessment Protocol (HEAP); however it consists of 192 items that assess not only hazards but also adaptations and comfort in the home [43]. Consequently, we believe that the HSC is the best tool to measure specifically the variable of home safety. The 64 items on the HSC reflect the specific recommendations in the educational materials that were given to both the intervention and the control groups. Each item is scored as NA = not applicable (e.g., for issues not included in all homes such as sliding glass doors), 1 = no safety issue; 2 = Safety modification implemented, 3 = traditional unsafe but not immediately threatening (e.g., hand rails on one side of stairs), and 4 = safety modification needed. Scores are summed and range from 5 to 256 with lower scores reflecting better home safety. BODY.2. METHODS.2.4. DATA ANALYSIS: The statistical package SPSS-PC was used to run all analyses [44]. Descriptive statistics were first computed on all study variables for all data collection points to examine the data for the presence of marked skewness, outliers, and systematic missing data. Hypotheses were tested using Multivariate Analysis of Covariance (MANCOVA) in order to test all outcome variables and covariates simultaneously. Prior to testing the hypotheses with MANCOVA, assumptions of normality of sampling distribution, homogeneity of variance-covariance matrices, linearity, multicollinearity, and singularity were checked. Tests for univariate and multivariate outliers were computed separately for each cell of the design in each hypothesis, and appropriate transformations or deletion of outlying cases was performed when needed using techniques specified in Tabachnick and Fidell [45]. BODY.3. RESULTS: We identified an initial population of 165 care recipient/caregiver dyads for the study, of which 127 dyads were enrolled. A CONSORT [46] flow diagram is depicted in Figure 2. Among the 38 dyads that did not enroll, 20 did not meet inclusion criteria, and 18 refused participation. Randomization of the sample used a blocked design, stratified by setting, in order to achieve balanced representation of each site in both the intervention and the control groups. Of the 127 dyads that enrolled, 70 were assigned to the intervention group and 57 to the control group using the sealed envelope method. A final sample of 108 dyads (60 in intervention group; 48 in control group) completed the study. No participant chose to withdraw, but rather, withdrawal was required because of changes in the status of either the care receiver or the caregiver that no longer met the inclusion criteria. Attrition was relatively low (15%) and dispersed evenly across intervention and control groups. We examined baseline characteristics (age, gender, years of caregiving, race, marital status, level of education, care receiver MMSE score, PSMS score, FAQ score, MBRC score, and MOS-SSS score) between the dropouts and completers using the appropriate statistics (chi-square for nominal data and t-tests with adjustments for type 1 error for continuous data) revealing no significant differences between the groups. The final sample had power of 0.82 or greater for all proposed analyses. The sample is typical of care dyads for people with dementia of the Alzheimer's type (Table 2). The care recipients, people with DAT or a related disorder, are an older group with a wide range of disease severity. The caregivers are somewhat younger as a group, reflecting some primary caregivers who were adult children. Because two of the recruitment sites were veteran's administration facilities, the care recipients are more likely to be male with female caregivers; however there were no significant differences between the intervention and control groups on these demographic and disease severity measures. Internal consistency reliabilities were acceptable for all instruments with Cronbach's alpha ranging from 0.746 to 0.948. Statistical analysis of the demographic variables and the dependent measures revealed that the variable of caregiver age correlated with the caregiver outcome measures in Hypothesis 1 (self-efficacy and caregiver strain), and it was therefore added to the analysis as a covariate. Years of education and social support did not correlate with the outcome variables in either hypothesis and were dropped from the analysis as covariates. Based on this preliminary data analysis, all MANCOVA analyses included the demographic variable of caregiver age and all Time 1 baseline measures set as covariates. Table 3 reports the adjusted means for the outcome variables and the corrected MANCOVA model. Effect sizes were computed using Cohen's d and revealed effect sizes for home safety (0.21), caregiver self-efficacy (0.29), and caregiver strain (0.22). In relation to the significance of home safety as a problem area for persons with DAT living at home, these small effect sizes translate into clinically relevant findings. Making even modest improvements in home safety, caregiver self-efficacy and caregiver strain will make an impact on the often tentative circumstances of home caregiving for a person with DAT. Effect sizes must also be considered in the context of an educational intervention in the setting of a neurodegenerative chronic illness. The Institute of Educational Science suggests that effect sizes for educational interventions of 0.25 standard deviations or larger are considered substantively important [47]. Given that the analyses were adequately powered and statistical significance was found, the effect sizes in this study represent a reasonable magnitude for this intervention. The means for caregiver self-efficacy represent relatively high self-confidence on average. Converting the total sum to an average confidence in home safety, the Control group had 75% overall confidence in caregiving home safety, and the intervention group had 80% overall confidence in their ability to make a home safer (1305/1700 and 1350/1700, resp.). As discussed above, we believe that these small changes overall can have a significant clinical impact, but, more importantly, the use of the measurement instruments in a clinical setting is more relevant if the individual items are examined. An average score might represent high self-efficacy for preventing the person with DAT from using the stove or sharp knives but low self-efficacy for obtaining respite for the caregiver. Clinical interventions would be tailored to the caregiver's specific needs. Caregiver strain is lower in the intervention group than in the control group, which represents less perceived strain in caregiving. The developers of the MBRC Caregiver Strain Instrument suggest a score of greater than 10 to indicate heightened risk requiring clinical investigation. Also a score can be used to assess change in the care situation over time. The group means in our study, which differ significantly in statistical analyses, indicate caregivers that are not on average at high risk. As with the other assessment instruments, however, in a clinical setting, an individual score that changes over time is important to monitor. Overall home safety improved in the intervention group compared to the control group, with a lower score that represents fewer safety risks. With a range of potential scores on the Home Safety Checklist of 5–262, the means for the intervention group (133.58) and control group (129.32) reflect home environments that on average are at the midrange of environmental safety. Therefore, a small improvement (lower score) that is statistically significant still suggests that home safety modifications are an important part of caring for a person with DAT that requires ongoing monitoring. The means for risky behaviors and accidents for the intervention (37.44) and control (33.95) groups indicate a low-moderate number of incidents on average (range = 0–180). This study was a small randomized trial to test an evidence-based intervention for efficacy. Although actual accidents and injuries can be devastating to a patient and family, in a sample of 108 dyads, the number is likely to be small in the 3 months of study participation. Therefore, risky behaviors, known to be a source of anxiety for the caregiver, were summed with accidents in order to have a measure for the outcome variable that could be analyzed statistically. Epidemiologic studies, reported in the literature review, establish the higher rate of accidents and injuries in this population of persons with DAT or a related disorder [12–14, 18, 19]. Analysis Results for Hypothesis 1. After controlling for the effects of caregiver age, and baseline measures of caregiver self-efficacy, baseline caregiver strain, and baseline home safety, caregivers who receive the Home Safety Toolkit will have improved home environmental safety higher postintervention self-efficacy, and lower postintervention caregiver strain than the control group which receives standard patient education. Hypothesis 1 was confirmed. Caregivers in the intervention group had significantly improved home environmental safety (P ≤ 0.000) higher caregiver self-efficacy (P ≤ 0.002), and lower caregiver strain (P ≤ 0.000) than caregivers in the control group. Analysis Results for Hypothesis 2. After controlling for the effects of caregiver age, baseline measures of caregiver self-efficacy, baseline caregiver strain, and baseline risky behaviors and accidents, care recipients who receive the Home Safety Toolkit will have fewer risky behaviors and accidents when compared to the care recipients in the control group who received standard patient education. Hypothesis 2 was confirmed. Care recipients in the intervention group had significantly less risky behaviors and accidents (P ≤ 0.000) than care recipients in the control group. BODY.4. DISCUSSION: The Home Safety Toolkit is a practical intervention that significantly improved caregiver self-efficacy to prevent injury to the person with DAT living at home and reduced caregiver strain. The findings from this clinical trial support observations in clinical practice that, when caregivers are given an easy-to-read publication on making the home safer and an opportunity to practice home safety modifications, their competence and confidence as caregivers increase. The small effect sizes are a consideration regarding the value of implementing the Home Safety Toolkit intervention. We note, however, that our study actually tested two interventions: the Home Safety Toolkit with health literacy materials and sample items in contrast to a conventional patient information worksheet with written recommendations for making the home safer. Customary care in some dementia and geriatric clinics often does not include comprehensive home safety information, and the IRB at the participating sites were concerned about withholding safety information from a vulnerable group of patients and family caregivers. Thus, subject dyads in the control group received standardized home safety education that is unlikely in customary clinical care and decreased the size of the effects between the intervention and control groups. Nevertheless, the statistically significant results support the efficacy of the Home Safety Toolkit. The findings have health policy implications with regard to funding for the Home Safety Toolkit sample items that promote caregiver self-efficacy. In order to standardize the intervention, all families in the intervention group were given the same type and amount of sample items with the exception of some large items such as a tub transfer bench. A large, special order item such as this was ordered based on individual need. Without the cost of the occasional tub bench, the cost per family for the Home Safety Toolkit booklet and sample items equals $210 USD. Some items were used immediately and some were kept for future use as the behavior of the person with dementia changed. A formal cost/benefit analysis was not undertaken because the study timeline would have been too long to capture health care utilization costs such as emergency room visits and hospitalization. Rather the focus of this current study was efficacy of the intervention. Nevertheless, most of the staff time in the study was related to research procedures such as informed consent and data collection and was included in the study budget. We specifically designed this intervention to be self-directed by the family caregiver, with an easy-to-read publication that would require little if any clinical staff time. A subsequent implementation research study, under development, is needed to analyze costs related to the Home Safety Toolkit. Further, the Home Safety Toolkit may be most appropriate in North America and Western European countries where housing structures are more similar to the housing arrangements of the study population. Two of the three study sites served the diverse urban population of Boston, Massachusetts, but we did not obtain income data from the participants and did not design the study to test for the potential effects of socioeconomic status on the study outcomes. However, we were surprised that years of education and perceived social support did not correlate with the outcome variables and therefore were not entered into the adjusted MANCOVA model. When these variables, often associated with socioeconomic status, were successfully randomized between the two groups, the Home Safety Toolkit still was effective to increase caregiver self-efficacy and home safety and decrease caregiver strain and care recipient risky behaviors and accidents. Study Limitations. Two of the participating facilities are US Department of Veterans Affairs medical centers, and therefore the study sample has few female care recipients and few male caregivers. Therefore, the potential for gender differences in the outcome variables was not tested. In a recent publication, male caregivers of a person with dementia reported less caregiver burden than female caregivers, even in situations where the care recipient was more impaired [48]. Future studies of the potential effects of gender on interventions such as the Home Safety Toolkit should be undertaken. The study design was single blinded in that the subjects did not know which group they had been assigned to randomly, but the project director and research assistants were aware of group assignment. Double blinding, as is done in drug clinical trials, could not be done, because the Home Safety Toolkit could not be disguised; the sample home safety items were an obvious sign of group assignment. Thus, there may have been bias in the data collection following randomization. Baseline data were collected before group assignment, as one control on potential bias, and procedures were reviewed frequently between the PI and statistician (who were blinded to both groups) and the project staff who were collecting data. BODY.5. CONCLUSION: The Home Safety Toolkit (HST) utilized principles of health literacy and self-efficacy to activate the primary family caregiver to manage the high frequency and high severity home safety issues for a person with dementia of the Alzheimer's type (DAT). The intervention is consistent with new models of patient-centered care where the patient and family caregiver are full partners with professional providers. In DAT, in particular, with the amount of unpaid care provided by family caregivers, this partnership is indispensable to the well-being of the person with DAT. This relatively low-technology evidence-based intervention now requires the implementation of strategies to enable primary care providers to prescribe a Home Safety Toolkit for persons with DAT and their family caregivers. Addressing policy issues regarding how the sample safety items in the Home Safety Toolkit will be stocked and incorporated into operational budgets will also contribute to successful implementation and sustainability.

TITLE: Effects of Dietary n-3 Fatty Acids on Hepatic and Peripheral Insulin Sensitivity in Insulin-Resistant Humans ABSTRACT.OBJECTIVE: Dietary n-3 polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), prevent insulin resistance and stimulate mitochondrial biogenesis in rodents, but the findings of translational studies in humans are thus far ambiguous. The aim of this study was to evaluate the influence of EPA and DHA on insulin sensitivity, insulin secretion, and muscle mitochondrial function in insulin-resistant, nondiabetic humans using a robust study design and gold-standard measurements. ABSTRACT.RESEARCH DESIGN AND METHODS: Thirty-one insulin-resistant adults received 3.9 g/day EPA+DHA or placebo for 6 months in a randomized double-blind study. Hyperinsulinemic-euglycemic clamp with somatostatin was used to assess hepatic and peripheral insulin sensitivity. Postprandial glucose disposal and insulin secretion were measured after a meal. Measurements were performed at baseline and after 6 months of treatment. Abdominal fat distribution was evaluated by MRI. Muscle oxidative capacity was measured in isolated mitochondria using high-resolution respirometry and noninvasively by magnetic resonance spectroscopy. ABSTRACT.RESULTS: Compared with placebo, EPA+DHA did not alter peripheral insulin sensitivity, postprandial glucose disposal, or insulin secretion. Hepatic insulin sensitivity, determined from the suppression of endogenous glucose production by insulin, exhibited a small but significant improvement with EPA+DHA compared with placebo. Muscle mitochondrial function was unchanged by EPA+DHA or placebo. ABSTRACT.CONCLUSIONS: This study demonstrates that dietary EPA+DHA does not improve peripheral glucose disposal, insulin secretion, or skeletal muscle mitochondrial function in insulin-resistant nondiabetic humans. There was a modest improvement in hepatic insulin sensitivity with EPA+DHA, but this was not associated with any improvements in clinically meaningful outcomes. BODY.INTRODUCTION: Insulin resistance is an early metabolic abnormality in the progression of type 2 diabetes. Genetic predisposition (1) is often intertwined with excess calorie intake and lack of exercise (2). Positive energy balance is widely accepted as the major culprit in the development of insulin resistance and the metabolic derangements that affect insulin-sensitive tissues. Ectopic lipid accumulation in skeletal muscle (3) and liver (4), oxidative stress (5), and mitochondrial dysfunction (6) are implicated in insulin resistance. Chronic inflammation (7) and macrophage infiltration of adipose tissue, sarcopenia, as well as progressive decline in β-cell function (8), especially in the context of obesity and aging, have also been linked in the pathogenesis of insulin resistance. Although exercise is a highly effective countermeasure to insulin resistance, there is great interest in alternative or supplemental therapeutic strategies for individuals who are unable to participate in exercise at recommended levels. n-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have emerged as a promising therapeutic strategy for their pleiotropic effects in the arena of diabetes (9,10), cardiovascular disease (11), and aging (12). The insulin-sensitizing effects of n-3 PUFAs are well documented in animal models (13–15). Leading mechanisms include repression of macrophage-induced tissue inflammation (15), improved mitochondrial function (13,16), and activation of anabolic pathways including a peroxisome proliferator–activated receptor signaling, a target of the current antidiabetic drugs thiazolidinediones. However, data from human studies have been conflicting. Some studies indicate that n-3 PUFAs improve insulin sensitivity in humans (17–21), whereas others find that n-3 PUFAs have no insulin-sensitizing effects (22–25) or may even worsen long-term glycemic control (26). Interestingly, studies in humans with a high inflammatory status such as overweight women (17), cancer cachexia (21), or hemodialysis (19) showed a beneficial effect of n-3 fatty acids. These discrepancies are at least partially attributed to the variation in the population studied, the source of n-3 PUFAs, dose, duration of treatment, underlying level of insulin resistance, and diversity of methods used to evaluate insulin sensitivity. A paucity of placebo-controlled studies of adequate treatment dose and duration using gold-standard measures of insulin sensitivity precludes a clear consensus about the efficacy of dietary n-3 PUFAs as insulin sensitizers in humans. In this study, we determined the effects of dietary n-3 fatty acids (3.9 g/day) on insulin sensitivity in a 6-month randomized, placebo-controlled, double-blind study in insulin-resistant, nondiabetic humans. Our primary aim was to systematically evaluate whole-body insulin sensitivity using the pancreatic clamp technique and to differentiate between hepatic and peripheral insulin sensitivity using deuterated glucose. We hypothesized that n-3 PUFAs would improve insulin sensitivity and that this improvement would be associated with increased skeletal muscle mitochondrial function. The rationale for this hypothesis originates from our recent observations in mice that n-3 PUFAs protect insulin sensitivity in association with transcriptional evidence of mitochondrial biogenesis (13). Here, we used a combination of in vitro and in vivo techniques to comprehensively evaluate mitochondrial function in skeletal muscle. BODY.RESEARCH DESIGN AND METHODS.SUBJECTS: A total of 62 overweight (BMI >25 kg/m2) individuals gave written informed consent as approved by the Mayo Foundation Institutional Review Board. Participants underwent screening for eligibility by standard hematologic and biochemical blood tests, resting electrocardiogram, physical examination, and medical history. As a general screening to exclude overweight participants with normal insulin sensitivity, participants were excluded if their fasting HOMA of insulin resistance (HOMA-IR) was >2.6, a threshold that was identified based on normal reference values (27). Participants were excluded if they were diagnosed with diabetes or if their fasting glucose exceeded 7 mmol/L (126 mg/dL). Additional exclusion criteria included cardiovascular disease, uncontrolled hypothyroidism, smoking and alcohol abuse, and participation in structured exercise more than 2 days per week for 30 min per day. Participants who reported using n-3 nutritional supplements and medications known to affect muscle metabolism such as β-blockers, corticosteroids, and anticoagulants were also excluded. A total of 31 participants (8 men and 23 women) were randomized. BODY.RESEARCH DESIGN AND METHODS.STUDY DESIGN: This was a randomized, placebo-controlled, double-blind study of n-3 PUFAs (EPA+DHA) versus placebo (ethyl oleate). The Mayo Clinic Research Pharmacy maintained the double-blind status and randomly assigned individuals to groups based on a table prepared by a statistician. The n-3 PUFAs and placebo softgels were supplied by Sancilio & Company, Inc (Riviera Beach, FL). Each n-3 softgel contained 675 mg EPA and 300 mg DHA and an additional 75 mg of "non EPA/DHA" n-3 fatty acids. Placebo softgels contained 1,200 mg ethyl oleate. Participants were instructed to consume two softgels with their morning meal and two with their evening meal for a total of 3.9 g/day EPA+DHA for 6 months. Every 4 weeks, participants reported to the Clinical Research Unit (CRU) to pick up a new prescription and return remaining capsules, which were counted to determine compliance. Compliance was also assessed from plasma levels of EPA and DHA measured by mass spectrometry at the end of the study. Participants were monitored for liver function (alanine aminotransferase, aspartate aminotransferase, and bilirubin), coagulation (international normalized ratio and prothrombin time), blood lipid profile, glucose, insulin, and creatine. At baseline and 6 months, all participants underwent a series of tests to evaluate body composition, energy metabolism, insulin sensitivity, and mitochondrial function. BODY.RESEARCH DESIGN AND METHODS.BODY COMPOSITION: DEXA was used to determine whole-body fat mass, percent body fat, and fat-free mass (FFM) (Lunar DPX-L; Lunar Radiation, Madison, WI). BODY.RESEARCH DESIGN AND METHODS.BODY COMPOSITION.MRI AND SPECTROSCOPY: Participants were positioned supine in the bore of a 3.0 Tesla GE Signa MRI scanner. Serial T1-weighted axial images were acquired through the abdominal region, between the pelvis and the upper end of the diaphragm, during a breath-hold phase. Abdominal subcutaneous and visceral fat areas were measured using Analyze Software System (Mayo Clinic Biomedical Imaging Resource) by a single trained analyst. Participants then performed in-magnet exercise to measure muscle oxidative capacity using phosphorous (31P)-MRS as previously described (28). A 31P-tuned transmission/receive coil was placed over the tibialis anterior muscle. After a habituation session, participants were asked to maximally dorsiflex for 30 s. Phosphorous metabolites were measured for 60 s of rest, throughout the 30-s muscle contraction, and during a 10-min recovery period. Phosphocreatine, inorganic phosphate, and ATP were analyzed using NUTS software (Acorn NMR, Livermore, CA). Oxidative capacity was measured from the rate constant of phosphocreatine recovery from a single exponential fit of the recovery data as previously described (28). BODY.RESEARCH DESIGN AND METHODS.HYPERINSULINEMIC-EUGLYCEMIC CLAMP: Subjects consumed a weight-maintaining diet (20% protein, 50% carbohydrate, 30% fat, and 10 kcal/kg body wt) for 3 days provided by the CRU metabolic kitchen after a consultation with a dietitian. Participants were admitted to the CRU on the evening of the 3rd day of the weight-maintenance phase. Participants consumed only water after 1900 h. Indirect calorimetry (TrueOne 2400; Parvo Medics) was used for measuring energy expenditure and respiratory quotient at 0600 h (baseline fasting) and 1230 h (insulin infusion). A retrograde catheter was inserted into a dorsal hand vein, and the hand was kept in a heated box (130°F) for collection of arterialized blood. Venous catheters in the contralateral arm were used for infusion of glucose and hormones. A two-stage hyperinsulinemic-euglycemic clamp was performed for 6 h. Three hours prior to the beginning of hormone infusion, a primed (6 mg/kg FFM), continuous (4 mg/kg FFM/h) infusion of [6,6-2H2]-d-glucose was initiated. Regular insulin was infused at a low dose (0.62 mU/kg FFM/min) for 3 h and then at a high dose (2.3 mU/kg FFM/min) for 3 h. Somatostatin (60 ng/kg total body wt/min), glucagon (0.65 ng/kg total body wt/min), and human growth hormone (3 ng/kg total body wt/min) were infused for the entire period to achieve a pancreatic clamp. Blood samples were taken at 10-min intervals for plasma glucose concentration measurement by glucose oxidase (Analox Instruments). Euglycemia (∼5.0 mmol/L [90 mg/dL]) was maintained with titrated infusion of 40% dextrose solution containing 2% [6,6-2H2]-d-glucose (29). Peripheral insulin sensitivity was evaluated from the average glucose infusion rates required to maintain euglycemia during the last 60 min of each stage of the clamp. Hepatic insulin sensitivity was evaluated from the suppression of endogenous glucose production (EGP) during the third hour of the low-insulin stage of the clamp compared with baseline EGP (29). BODY.RESEARCH DESIGN AND METHODS.HYPERINSULINEMIC-EUGLYCEMIC CLAMP.MIXED-MEAL TEST AND MUSCLE MITOCHONDRIAL FUNCTION: At least 1 week after the pancreatic clamp, participants were readmitted to the CRU after 3 days of standardized meals. At 0700 h, a biopsy was taken from vastus lateralis muscle under local anesthesia (2% lidocaine) using a Bergstrom needle as previously described (30). Muscle tissue was immediately prepared for measurements of mitochondrial function as described in detail previously (31). At 1230 h, participants consumed a mixed liquid meal containing 15% protein, 55% carbohydrate, and 30% fat, approximately equal to 35% of resting energy expenditure (REE) for evaluating meal glucose tolerance. Arterialized blood samples were taken from a heated hand vein for glucose and insulin measurements at intervals over 6 h after the test meal, which was consumed over 10 min. BODY.RESEARCH DESIGN AND METHODS.ANALYTICAL METHODS: Glucose was measured in plasma samples by glucose oxidase method (Analox Instruments). [6,6-2H2]-d-glucose enrichment of blood samples and infusions was measured by gas chromatography–mass spectrometry (32). Insulin was measured using a two-site immunometric assay using electrochemiluminescence immunoassay detection (Roche Diagnostics, Indianapolis, IN). C-peptide was measured using a two-site immunometric (sandwich) assay using electrochemiluminescence detection (Roche Diagnostics, Indianapolis, IN). Interleukin (IL)-6 was measured using the Quantikine HS IL-6 Immunoassay, a solid-phase ELISA designed to measure human IL-6 in serum, plasma, and urine. Leptin and adiponectin were measured by radioimmunoassay, and C-reactive protein was measured by particle-enhanced immune-turbidimetric assay. With the exception of D2 glucose, all blood measurements were done in the Mayo Clinic Immunochemical Core Laboratory. Plasma EPA and DHA free fatty acids were measured by high-performance liquid chromatography–mass spectrometry as previously described (33). BODY.RESEARCH DESIGN AND METHODS.STATISTICAL ANALYSIS: All outcomes were checked for normal distribution. Variables with normal distributions are presented as means ± SEM. Two-way (treatment group and time) repeated-measures ANOVA was used to compare outcomes from baseline to follow-up in placebo compared with n-3–treated groups. For variables that were not normally distributed, data are presented as medians and interquartile range with group comparisons made using Wilcoxon rank-sum test on the Δ from baseline to follow-up. Statistical analyses were performed using JMP Software (SAS Institute, Cart, NC). BODY.RESULTS: Thirty-one subjects underwent randomization with 16 participants assigned in the n-3 arm and 15 in the placebo arm. Four participants withdrew from the study for personal reasons. One participant was withdrawn by investigators for noncompliance with the protocol. In total, 14 participants completed the study in the n-3 group and 11 in the placebo group. Four subjects have missing insulin sensitivity data because of failed intravenous access during the clamp procedure. Four participants have missing meal tolerance test data because of failed intravenous access. Four participants did not have mitochondrial capacity measured in muscle biopsies because of technical difficulties. BODY.RESULTS.ANTHROPOMETRIC CHARACTERISTICS: Main clinical characteristics at baseline and postintervention are shown in Table 1. At baseline, the two groups were similar in age and body composition. Both groups exhibited similar increases in body weight (P = 0.036) and % body fat (P = 0.008) over the 6 months of the study. The distribution of abdominal fat between subcutaneous and visceral cavities was similar in both groups at baseline and postintervention, although there was a significant interaction (P = 0.048) reflecting a subtle increase in visceral fat in the n-3 group and subtle decrease in the placebo group. Table 1 Subject characteristics Placebo ( n = 11) n-3 ( n = 14) P Baseline Follow-up Baseline Follow-up Physical characteristics Age (years) 32.6 ± 2.5 33.2 ± 2.5 35.3 ± 2.9 35.8 ± 3.0 0.604 Height (cm) 168.1 ± 2.3 168.0 ± 2.0 172.3 ± 2.2 172.7 ± 2.3 0.174 Weight (kg) 99.6 ± 4.5 101.7 ± 4.4 105.3 ± 4.2 107.3 ± 5.1 0.724 BMI (kg/m 2 ) 35.2 ± 1.4 36.0 ± 1.4 35.5 ± 1.2 36.0 ± 1.3 0.412 FFM (kg) 51.8 ± 2.6 52.5 ± 2.5 56.6 ± 2.7 56.4 ± 2.8 0.191 Body fat (%) 45.1 ± 2.1 45.8 ± 2.1 43.9 ± 1.9 44.9 ± 1.6 0.791 Abdominal fat (cm 2 ) 46.5 (32.2–59.5) 50.2 (36.8–62.1) 46.7 (32.7–58.8) 47.1 (34.0–57.6) 0.271 Visceral fat (cm 2 ) 9.69 ± 1.18 8.55 ± 0.89 9.92 ± 1.04 10.50 ± 1.15 0.048 Lipids  Total cholesterol (mg/dL) 193 ± 7 196 ± 7 170 ± 11 171 ± 9 0.810  Triglyceride (mg/dL) 120 ± 19 153 ± 35 175 ± 18 141 ± 13 0.012  HDL cholesterol (mg/dL) 48 ± 3 46 ± 3 51 ± 3 50 ± 4 0.087  LDL cholesterol (mg/dL) 99 ± 8 95 ± 9 110 ± 6 113 ± 7 0.254  Non-HDL cholesterol (mg/dL) 124 ± 9 125 ± 11 145 ± 8 141 ± 8 0.414  EPA (μmol/L) 1.61 ± 0.37 2.34 ± 0.73 1.54 ± 0.40 11.47 ± 4.30 0.076  EPA (mg/dL) 0.049 ± 0.011 0.071 ± 0.022 0.047 ± 0.012 0.347 ± 0.130 0.076  DHA (μmol/L) 1.47 ± 0.20 2.22 ± 0.61 1.62 ± 0.35 4.49 ± 0.82 0.068  DHA (mg/dL) 0.048 ± 0.006 0.073 ± 0.020 0.053 ± 0.012 0.148 ± 0.027 0.068 Glucose and hormones  Glucose (mg/dL) 89.9 ± 1.6 95.2 ± 4.9 92.4 ± 2.8 93.4 ± 3.1 0.321  Insulin (μIU/mL) 17.3 ± 2.3 18.8 ± 2.0 16.4 ± 0.9 17.1 ± 1.8 0.934  HOMA-IR 3.83 ± 0.50 4.52 ± 0.54 3.76 ± 0.28 3.94 ± 0.52 0.715 Inflammatory markers  Leptin (ng/mL) 41.7 ± 5.6 41.2 ± 5.6 35.2 ± 5.8 35.2 ± 4.9 0.878  Adiponectin (μg/mL) 6.37 ± 0.68 6.80 ± 0.88 5.47 ± 0.49 5.70 ± 0.53 0.672  C-reactive protein (mg/dL) 0.11 (0.06–0.26) 0.21 (0.14–0.31) 0.34 (0.09–0.46) 0.32 (0.05–0.68) 0.487  Interleukin-6 (pg/mL) 1.81 ± 0.37 1.87 ± 0.29 1.73 ± 0.20 1.67 ± 0.22 0.688 Energy expenditure  RQ (fasting) 0.87 ± 0.01 0.86 ± 0.01 0.87 ± 0.02 0.86 ± 0.02 0.659  RQ (steady-state insulin) 0.93 ± 0.02 0.95 ± 0.01 0.92 ± 0.01 0.92 ± 0.02 0.268  REE (fasting) 1,701 ± 83 1,770 ± 89 1,862 ± 66 1,910 ± 70 0.763  REE (steady-state insulin) 1,777 ± 95 1,760 ± 51 1,846 ± 51 1,898 ± 66 0.306 Patients were randomly assigned to receive placebo (ethyl oleate) or n-3 (EPA+DHA) for 6 months. Measurements were made prior to randomization (baseline) and again after 6 months (follow-up). For normally distributed data, means ± SEM are given, and two-way (group and time) repeated-measures ANOVA was used to compare outcomes across groups. Precise P values are given for the group-by-time interaction. For data that were not normally distributed (abdominal fat and C-reactive protein), data are given as median (interquartile range), and Wilcoxon rank-sum test was used to compare the Δ from baseline to follow-up between placebo and n-3 groups. BODY.RESULTS.LIPIDS, GLUCOSE, INSULIN, AND INFLAMMATION PROFILES: Fasting blood lipids, glucose, insulin, and inflammatory markers are given in Table 1. Compliance measured from return pill counts was 90% in the placebo group and 92% in the n-3 group, with marked increases in fasting plasma EPA and DHA free fatty acid levels in the n-3 group (Table 1). Total cholesterol, HDL and LDL, did not change in either group. As expected, serum triglyceride levels decreased in the n-3 group and increased in the placebo group (P = 0.012). Fasting plasma glucose and insulin concentrations were not altered by the intervention, and likewise there was no change in HOMA-IR. Baseline levels of leptin and adiponectin were in the higher and lower normal range, respectively, and did not change over time. Likewise, the inflammation markers C-reactive protein and IL-6 were not altered in either group. BODY.RESULTS.HEPATIC AND PERIPHERAL INSULIN SENSITIVITY: Plasma glucose and insulin were similar during baseline and follow-up studies in both groups during the 6-h clamp procedure (Supplementary Fig. 1). The glucose infusion rates required to maintain euglycemia were also similar from baseline to follow-up studies in both groups (Fig. 1A and B). There were no differences between groups in the average glucose infusion rates measured during the last 60 min of each 3-h stage of the clamp (Fig. 1C and D), indicating that peripheral insulin sensitivity was unaffected by EPA+DHA supplementation. EGP was modestly suppressed during the low-dose insulin infusion (Fig. 1E and F), but the percent suppression decreased by 12% on average in the placebo group and increased by 15% on average in the n-3 group (P = 0.024) (Fig. 1G), indicating that hepatic insulin sensitivity improved in response to 6 months of EPA+DHA supplementation. Indirect calorimetry measurements at baseline and during steady-state high insulin infusion showed an increase in respiratory quotient (RQ) and REE in response to insulin in both groups (Table 1), with no changes in either group after the intervention. Figure 1Hyperinsulinemic-euglycemic clamp. The glucose infusion rates (GIRs) required to maintain euglycemia in 10-min intervals during the 6-h insulin infusion in placebo (A) and n-3 (B) groups at baseline and follow-up. The average glucose infusion rate during the final 60 min of each stage of the clamp (low insulin and high insulin) in placebo (C) and n-3 (D) groups at baseline and follow-up. EGP measured in the basal fasting state and during low-dose insulin (E and F). EGP suppression was greater with n-3 (G). Data bars are means ± SEM. Data points represent individual subject responses with dotted lines linking baseline with follow-up observations. *P < 0.05 for the group × time interaction. BODY.RESULTS.MEAL GLUCOSE TOLERANCE AND INSULIN SECRETION: Postprandial glycemia during the 6 h after a mixed meal was similar in placebo and n-3 groups at baseline and after 6 months (Fig. 2A and B). Although there was a trend for increased peak postprandial glucose after 6 months in the placebo group (Fig. 2A), the glucose areas above baseline were similar in both groups before and after the intervention (Supplementary Fig. 2). These data are in agreement with the measurements of peripheral insulin sensitivity assessed during the pancreatic clamp. Insulin secretion determined from C-peptide concentrations over 6 h after the mixed meal was also similar in both groups at both time points (Figs. 2E and F). The area above baseline for C-peptide was similar in placebo and n-3 groups and did not change with either intervention (Supplementary Fig. 2), indicating that insulin secretion did not change in response to high-dose EPA+DHA. Figure 2Mixed-meal tolerance test. Plasma glucose (A and B), insulin (C and D), and C-peptide (E and F) concentrations measured 6 h after ingestion of a liquid meal were similar in both groups and unchanged with either intervention. Data are means ± SEM. BODY.RESULTS.SKELETAL MUSCLE MITOCHONDRIAL FUNCTION: Skeletal muscle oxidative capacity measured in mitochondria isolated from muscle biopsy tissue was unchanged by n-3 fatty acids or placebo in response to carbohydrate-based mitochondrial substrates (Fig. 3A) or lipid-based substrates (Fig. 3B). Mitochondrial efficiency as evaluated by the respiratory control ratio, the index of mitochondrial proton leak, and ADP:O ratio, the index of phosphorylation efficiency, was also similar across groups (Fig. 3C and D). The absence of any effect of EPA+DHA on mitochondrial capacity was corroborated with in vivo measurements of muscle oxidative capacity using 31P-MRS, which also demonstrated no effect of n-3 or placebo (Fig. 3E). Figure 3Mitochondrial function in skeletal muscle. Mitochondrial oxygen consumption rates (JO2) were measured under state 3 respiration conditions with carbohydrate-based mitochondrial substrates (A) and lipid substrates (B). Mitochondrial coupling was assessed from the respiratory control ratio (RCR) (C) and ADP:O ratio (D). Oxidative capacity measured in vivo by magnetic resonance spectroscopy (E). Data bars are means ± SEM. Data points represent individual subject responses with dotted lines linking baseline (●) with follow-up (○) observations. BODY.CONCLUSIONS: This study demonstrates that 6 months of dietary EPA and DHA at 3.9 g/day does not alter insulin-stimulated peripheral glucose disposal, postprandial glucose disposal, or insulin secretion in overweight insulin-resistant nondiabetic individuals. We found that EPA+DHA treatment resulted in a small but significant increase in the suppression of EGP, although this was not accompanied by any changes in clinically meaningful outcomes related to hepatic insulin sensitivity. Furthermore, skeletal muscle mitochondrial function was unaffected by EPA+DHA. The results from this study provide some resolution to the current ambiguity concerning the therapeutic potential of dietary n-3 PUFAs in the context of human insulin resistance. Precedent literature consistently shows that n-3 PUFAs prevent insulin resistance in rodents (13,14,34). The leading mechanisms include activation of peroxisome proliferator–activated receptor-α (34), suppression of inflammation (15), and attenuation of ectopic lipid accumulation in insulin-sensitive tissues (13,34). Furthermore, there is early evidence that n-3 PUFAs may stimulate mitochondrial biogenesis (13) and improve skeletal muscle mitochondrial function (35). Since mitochondrial dysfunction has been implicated in the etiology of insulin resistance (1), it is possible that n-3 PUFAs may attenuate insulin resistance through their influence on mitochondria in insulin-sensitive tissues. In spite of promising evidence from animal studies, there is currently no clear consensus on the effects of n-3 PUFAs on insulin sensitivity in humans. Intervention studies in humans have shown some biological effects of n-3 PUFAs similar to what is observed in rodents such as repression of macrophage-induced inflammatory response (25), improved mitochondrial function (35), and decreased circulating triglycerides and small dense LDL particles (23). Although these factors are all believed to be important determinants of insulin resistance, none of the studies found that insulin sensitivity was affected by n-3 PUFAs. A recent meta-analysis including 11 randomized clinical trials concluded that there is no overall association between intake of n-3 PUFAs and insulin sensitivity (36). A closer examination of these studies revealed substantial variability in the daily dose of n-3 PUFAs (0.14–11 g/day), duration of the intervention (2–6 months), and subject characteristics or disease states (lean, obese, diabetes, fatty liver disease, and hypertension). The vast majority of these studies evaluated insulin sensitivity from fasting glucose and insulin or oral/intravenous glucose tolerance tests, which may lack sensitivity needed to distinguish between hepatic and peripheral insulin sensitivity. We are unaware of any randomized, placebo-controlled, double-blind studies of n-3 PUFAs at pharmaceutical doses and durations in insulin-resistant nondiabetic individuals where insulin sensitivity was evaluated using gold-standard techniques. In the absence of such studies, it is possible that real therapeutic potential of n-3 PUFAs in insulin-resistant humans may be overlooked. The current study was designed to fill this gap. We recruited overweight men and women who were insulin resistant but otherwise healthy. A pharmaceutical grade EPA+DHA preparation was given at the highest dose that the FDA generally regards as safe (4 g/day) for 6 months. All of the participants demonstrated low DHA and EPA levels in the fasting plasma free fatty acid pool (Table 1), indicating low n-3 consumption in their background diets based on values reported based on tertiles of fatty fish consumption (37). The n-3 group exhibited approximately a sevenfold increase in fasting EPA levels and a fourfold increase in DHA levels in plasma free fatty acids after 6 months, indicating significant enrichment of adipose tissue n-3 content. Thus, absorption and bioavailability of n-3 fatty acids were high. An appropriate placebo group was included in a randomized double-blind fashion. Participants were carefully characterized for body composition and clinical blood parameters, and insulin sensitivity was evaluated using the pancreatic clamp technique, which is the gold standard for evaluating insulin sensitivity and offers the additional advantage of distinguishing between hepatic and peripheral insulin sensitivity. With this robust study design, we find that dietary EPA+DHA at 3.9 g/day for 6 months did not improve peripheral insulin sensitivity or postprandial glucose disposal. Although this conclusion corroborates previous meta-analyses, it does so with a high level of confidence because many of the methodological shortcomings of previous negative reports have been addressed. In addition to the pancreatic clamp, we also followed postprandial glycemia for 6 h after a mixed meal as a physiologically relevant parameter of peripheral glucose disposal. Similar to the clamp, the mixed-meal glucose disposal was comparable in both groups and although a slight worsening was observed in the placebo group, this was not statistically significant. Postprandial insulin concentrations and insulin secretion were also similar in both groups and were unchanged with either intervention. Although our data clearly show that 6 months of EPA+DHA supplementation has no measurable effect on peripheral glucose disposal, we found a small but significant improvement in hepatic insulin sensitivity measured by the suppression of EGP during insulin infusion. By using somatostatin to suppress endogenous insulin secretion, we are confident that portal insulin levels are the same in all participants, thereby eliminating a source of variability in prevailing endogenous insulin levels that could confound EGP measurements. To our knowledge, this is the first study where hepatic and peripheral insulin sensitivities were evaluated separately in response to n-3 PUFA supplementation. It is interesting to note that a meta-analysis from 2011 concluded that n-3 PUFAs do not affect insulin sensitivity; however, subgroup analysis of studies using HOMA-IR, which primarily reflects hepatic insulin sensitivity, found improvements in insulin sensitivity with n-3 PUFAs (36). The notion that n-3 PUFAs may specifically act in liver is supported by evidence that n-3 PUFAs may improve hepatic steatosis, inflammation, and fibrosis in patients with nonalcoholic fatty liver disease (38). In contrast, others found that EPA at 2.7 g/day for 1 year did not improve HOMA-IR or histological features of nonalcoholic steatohepatitis (39). Nevertheless, the data from the current study suggest that the effects of EPA+DHA may be specific to liver. It is important to consider that although the effects of EPA+DHA on hepatic insulin sensitivity were statistically significant compared with the placebo group, the improvement was subtle and was not accompanied by any changes in fasting plasma glucose, HOMA-IR, or any other clinically relevant outcomes. The current study also examined the effect of EPA+DHA on skeletal muscle mitochondrial function, since these organelles have been linked to insulin sensitivity (1). We recently found that the insulin-sensitizing effects of n-3 PUFAs were associated with transcriptional evidence of mitochondrial biogenesis (13). Since mitochondrial dysfunction is a hallmark of insulin resistance and potentially involved in the etiology and progression of type 2 diabetes (1), we determined whether muscle mitochondrial function could be improved by n-3 PUFAs in insulin-resistant individuals. Measurements of oxidative capacity and efficiency in mitochondria isolated from muscle biopsy material showed no change with n-3 PUFAs. These in vitro findings were also verified in vivo by 31P-MRS. Recent work by Herbst et al. (35) found that fish oil supplementation in healthy young men increased EPA and DHA content of mitochondrial membranes but did not change mitochondrial capacity. These investigators did, however, report an increase in ADP sensitivity of mitochondria. Although we did not specifically evaluate ADP sensitivity, we did measure phosphorylation efficiency, which was unchanged. There are two important clinical implications of the mitochondrial measurements in skeletal muscle. There is widespread interest in strategies to improve mitochondrial function in skeletal muscle of insulin-resistant individuals because these organelles are responsible for lipid oxidation and sustaining the metabolic demands of skeletal muscle activity. In contrast to this widespread belief, we find that skeletal muscle mitochondrial dysfunction is not always a necessary feature of insulin resistance, since the participants in this study exhibited normal mitochondrial function in spite of marked insulin resistance. Nevertheless, although EPA+DHA had no effect on mitochondrial function in this population of insulin-resistant participants, it will be important for future studies to determine whether n-3 PUFAs influence mitochondrial physiology in patient populations where mitochondrial dysfunction is evident. In summary, the current study provides strong evidence against the notion that n-3 PUFAs improve peripheral insulin sensitivity in insulin-resistant nondiabetic individuals. It is difficult to reconcile these data with rodent studies where n-3 PUFAs clearly protect against insulin resistance. A key consideration is that the majority of the rodent studies have been preventive studies, while human studies are designed to determine whether n-3 PUFAs can reverse prevalent insulin resistance. Some insight into this issue comes from a recent study where n-3 PUFAs were found to prevent insulin resistance during an acute infusion of a lipid emulsion in healthy young men (40). Thus, although dietary n-3 PUFAs do not appear to be effective in reversing peripheral insulin resistance, additional studies are required to determine whether these bioactive lipids can be part of long-term preventive strategies. Although the subtle improvements in hepatic insulin sensitivity with EPA+DHA do not appear to be clinically meaningful in the current study, there is impetus for similar investigations in other insulin-resistant populations such as patients with fatty liver disease.

TITLE: Racemic ketamine in adult head injury patients: use in endotracheal suctioning ABSTRACT.INTRODUCTION: Endotracheal suctioning (ETS) is essential for patient care in an ICU but may represent a cause of cerebral secondary injury. Ketamine has been historically contraindicated for its use in head injury patients, since an increase of intracranial pressure (ICP) was reported; nevertheless, its use was recently suggested in neurosurgical patients. In this prospective observational study we investigated the effect of ETS on ICP, cerebral perfusion pressure (CPP), jugular oxygen saturation (SjO2) and cerebral blood flow velocity (mVMCA) before and after the administration of ketamine. ABSTRACT.METHODS: In the control phase, ETS was performed on patients sedated with propofol and remifentanil in continuous infusion. If a cough was present, patients were assigned to the intervention phase, and 100 γ/kg/min of racemic ketamine for 10 minutes was added before ETS. ABSTRACT.RESULTS: In the control group ETS stimulated the cough reflex, with a median cough score of 2 (interquartile range (IQR) 1 to 2). Furthermore, it caused an increase in mean arterial pressure (MAP) (from 89.0 ± 11.6 to 96.4 ± 13.1 mmHg; P <0.001), ICP (from 11.0 ± 6.7 to 18.5 ± 8.9 mmHg; P <0.001), SjO2 (from 82.3 ± 7.5 to 89.1 ± 5.4; P = 0.01) and mVMCA (from 76.8 ± 20.4 to 90.2 ± 30.2 cm/sec; P = 0.04). CPP did not vary with ETS. In the intervention group, no significant variation of MAP, CPP, mVMCA, and SjO2 were observed in any step; after ETS, ICP increased if compared with baseline (15.1 ± 9.4 vs. 11.0 ± 6.4 mmHg; P <0.05). Cough score was significantly reduced in comparison with controls (P <0.0001). ABSTRACT.CONCLUSIONS: Ketamine did not induce any significant variation in cerebral and systemic parameters. After ETS, it maintained cerebral hemodynamics without changes in CPP, mVMCA and SjO2, and prevented cough reflex. Nevertheless, ketamine was not completely effective when used to control ICP increase after administration of 100 γ/kg/min for 10 minutes. BODY.INTRODUCTION: Endotracheal suctioning (ETS) is essential for patient care in an intensive care unit, but may represent a cause of cerebral secondary injury. In the last few years, several authors found that it can have considerable negative impact on cerebral physiologic variables, causing coughing, increasing intracranial pressure (ICP) and altering cerebral perfusion pressure (CPP) in head-injured patients [1-5]. With the aim of controlling cerebral and systemic effects related to ETS, a lot of studies have been conducted to test different drugs or procedures, such as hyperventilation [6], administration of opioids [7-10], barbiturates [8], local anesthetics [8,11-13], and paralyzing agents [7,8]. Unfortunately, in many cases single-center nonrandomized clinical studies were performed, based on small samples, thus leading to conclusions with low level of scientific evidence. Even if recent recommendation for ETS in mechanically ventilated patients are available [14], so far no consensus exists about which strategy should be used to reduce intracranial effects of this procedure. Ketamine is an N-methyl-D-aspartate receptor antagonist recommended during minor procedures, for the induction of anesthesia prior to the administration of other agents, and to supplement low-potency anesthetics. It has unique properties as a dissociative anesthetic, analgesic, amnestic and anxiolytic; it exerts its effect by 'disconnecting' the thalamocortical and limbic systems, effectively dissociating the central nervous system from outside stimuli [15,16]. The result is a 'dissociative anesthesia', that is characterized by analgesia, amnesia and anxiolysis, while maintaining cardiovascular stability and preserving spontaneous respirations and protective airway reflexes [15,16]. It has been historically contraindicated for its use in patients with head injury because of a concern that it may increase ICP [17-21]. However, several recent studies suggested ketamine as a sedative in the setting of a neurosurgical intensive care unit, since no association was observed between ketamine and ICP in mechanically ventilated head-injured patients during continuous analgosedation [22-29]. Thus, it has officially been recommended for use in analgesia and sedation in European countries. Furthermore, several animal studies observed its neuroprotective activity against hypoxic, ischemic or mechanical neuronal insults [29]. We hypothesized that ketamine could be safe and effective in blunting ICP increase after ETS in these patients. Thus, we investigated the effect of ETS before and after ketamine on ICP, CPP, jugular oxygen saturation (SjO2) and cerebral blood flow velocity in head-injured patients during continuous infusion of propofol and remifentanil. BODY.MATERIAL AND METHODS.PATIENTS AND DATA COLLECTION: After approval by the Ethics Committee of the Catholic University School of Medicine (P/997/CE/2010), informed written consent was obtained from patients' next of kin. Inclusion criteria were: ● Age between 18 and 75 years ● Severe closed head-injured patients (GCS ≤8) within 72 hours after trauma ● ICP monitoring ● Analgesia and sedation with propofol (3 to 5 mg/kg/h) and remifentanil (0.05 to 2 γ/kg/min) in continuous infusion to obtain a Ramsey score of 5 to 6 Exclusion criteria were: ● Severe hemodynamic instability ● Paralyzed patients ● Known allergy to the treatment drugs or pregnancy Patients were managed according to Brain Trauma Foundation Guidelines [30]. Multimodal monitoring, including ICP and SjO2 was performed (SC7000 Monitor, Siemens, Erlangen, Germany). Patients were mechanically ventilated in volume-controlled ventilation, thus to maintain partial pressure of carbon dioxide in the blood (paCO2) between 32 and 35 mmHg and partial pressure of oxygen in the blood (paO2) >70 mmHg. Jugular bulb catheters were inserted in the larger internal jugular vein, under ultrasonographic control; correct position was verified by X-rays. Patients were nursed supine with a 30° head-up tilt. Blood flow velocity in the middle cerebral artery (mV MCA) was measured by 2 MHz pulsed Doppler ultrasound device (transcranial Doppler (TCD) H21-Hitachi Medical Systems Europe, Zug, Switzerland). BODY.MATERIAL AND METHODS.STUDY PROTOCOL: The study was structured in two phases: control and intervention. In the control phase, patients were sedated with propofol (3 to 5 mg/kg/h) and remifentanil (0.05 to 0.2 γ/kg/min) in continuous infusion, and ETS was performed according to recent guidelines through an orotracheal tube [14]. If cough was present, patients were assigned to the intervention phase, and an infusion of a racemic mixture of ketamine 100 γ/kg/min for 10 minutes was added before ETS. In both phases, each patient underwent the protocol as described in Table 1. At each step, a standard set of parameters was recorded: heart rate (HR), mean arterial pressure (MAP), ICP, CPP. In addition, at step 1, 3 and 5, arterial blood gas analysis and SjO2 were carried out; at these steps, mV MCA was bilaterally measured by TCD, and mean value was reported. In the control phase, step 2 was not carried out. Table 1 Study protocol Study protocol T1 Baseline T2 Immediately before ETS T3 Immediately after ETS T4 5 minutes after ETS T5 10 minutes after ETS An evaluation of cough strength was performed after ETS by a semi-quantitative scale (Harris Scale; component 'Response to endotracheal suctioning') [31]. It ranged between 1 and 4 (Table 2). Table 2 Scoring of cough strength [ 31 ] Cough strength 1 Agitation, distress, prolonged coughing 2 Coughs, distressed, rapid recovery 3 Coughs, not distressed 4 No cough BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: Repeated measures analysis of variance was used in order to study physiologic parameters before, during and after ETS. Cough reflex after ETS was evaluated by the Friedman test for repeated measures; in cases of multiple comparisons, level of significance was adjusted using the Bonferroni correction, and corrected P value was reported. Statistical calculations were performed using the Statistical Package for Social Sciences (Windows version 14.0, Microsoft Corp, Redwood, WA, USA). Results are reported as mean ± standard deviation, or median and interquartile range (IQR), as appropriated; a value of P <0.05 was considered statistically significant. BODY.RESULTS: Twenty-one head-injured patients consecutively admitted to 18-bed general ICU of 'A. Gemelli' Hospital between 1 January 2011 and 1 November 2011 were enrolled in the study. 'A. Gemelli' Hospital is a 1,200-bed university hospital located in Rome, Italy, that is a referral center for severely injured patients and serves an urban area of 1 million people. During baseline analgosedation (control group), a cough reflex was present in all patients, and all were included in the study. The sample was predominantly male (57%), with nine females (43%). The mean age was 54.6 ± 21.2, ranging from 19 to 74 yrs. The patients' severity of illness, as measured by the Glasgow Coma Scale (5.8 ± 3.8) and the Simplified Acute Physiology Score II (SAPS II) score (49.7 ± 9.2), showed severe injuries and significant comorbidities requiring high levels of medical and nursing care. At baseline, ICP was always below 25 mmHg. During the study, hyperventilation was not applied, and five patients were treated with hyperosmolar therapy. BODY.RESULTS.CONTROL: Despite deep sedation, ETS (T3) caused an increase of HR (from 74.3 ± 14.0 to 82.1 ± 17.6 min-1; P <0.001), MAP (from 89.0 ± 11.6 to 96.4 ± 13.1 mmHg; P <0.001), ICP (from 11.0 ± 6.7 to 18.5 ± 8.9 mmHg; P <0.001), SjO2 (from 82.3 ± 7.5 to 89.1 ± 5.4%; P = 0.01) and mV MCA (from 76.8 ± 20.4 to 90.2 ± 30.2 cm/sec; P =0.04) if compared with T1. CPP did not vary with ETS (Figures 1, 2, 3, 4 and 5). Furthermore, ETS stimulated a strong cough reflex, with a median cough score of 2 (IQR 1 to 2) (Figure 6). Figure 1Ketamine prevented any significant increase of mean arterial pressure (MAP) after endotracheal suctioning. Statistical significance in comparison with T1 is shown. *P <0.05; **P <0.01. Figure 2No significant difference in cerebral perfusion pressure (CPP) was observed during the study in both groups. Figure 3Differences between control and intervention in intracranial pressure (ICP) during the study are shown. Statistical significance in comparison with T1 is shown. *P <0.05; **P <0.01. Figure 4A significant increase of blood flow velocity in the middle cerebral artery (mV MCA) was observed in the control group. Statistical significance in comparison with T1 is shown. *P <0.05. Figure 5SjO2 increased after endotracheal suctioning (ETS) in both groups. In the control group a significant difference was observed. Statistical significance in comparison with T1 is shown. *P <0.05. Figure 6Cough score evaluated after endotracheal suctioning (ETS) is shown [31]. Each bar corresponds to the cough score of each patient. These changes were not sustained over time. As compared with T1, 10 minutes after ETS (T5) HR (from 74.3 ± 14.0 to 78.0 ± 20.4 min-1; P = 0.17), MAP (from 89.0 ± 11.6 to 88.8 ± 11.0 mmHg; P = 0.92), ICP (from 11.0 ± 6.7 to 11.4 ± 7.4 mmHg; P = 0.61) and SjO2 (from 82.3 ± 7.5 to 85.1 ± 6.0%; P = 0.13) were not modified; mV MCA increased (from 76.8 ± 20.4 to 84.6 ± 24.2; P = 0.03) (Figures 1, 2, 3, 4 and 5). BODY.RESULTS.INTERVENTION: No significant variation of HR, MAP, CPP, mV MCA, and SjO2 were observed in any step during the study. At baseline (T1) ICP was 11.0 ± 6.4 mmHg; at this step, in two cases it was higher than 20 mmHg. Following ketamine administration (T2), ICP did not modify (11.7 ± 7.3 vs. 11.0 ± 6.4 mmHg; P = 0.28); after ETS (T3) cough reflex was significantly reduced in comparison with controls (cough score 4 (IQR 3 to 4) vs. 2 (IQR1 to 2); P <0.0001) (Figure 6). ICP increased after ETS if compared with T1 (15.1 ± 9.4 vs. 11.0 ± 6.4 mmHg; P <0.05). (Figures 1, 2, 3, 4 and 5). Interestingly, in contrast with controls, ETS did not induce any significant change of mV MCA and SjO2. BODY.DISCUSSION: The main result of this study is that the administration of a racemic mixture of ketamine did not induce any significant variation of ICP, CPP, MAP, mV MCA and SjO2 in mechanically ventilated head-injured patients during continuous analgosedation. If administered before ETS, racemic ketamine reduced cough reflex, and prevented any significant change of MAP, CPP, mV MCA and SjO2 in comparison with controls. Nevertheless, its use before ETS was not sufficient to completely blunt ICP increases at the drug dose studied. Ketamine has been historically contraindicated for its use in patients with head injury, since an association with increased ICP was reported [17-21]. This concept originated from a few case reports and small case–control studies from the 1970s conducted on patients with abnormal cerebrospinal fluid pathways. The increase of ICP was observed in patients who were breathing spontaneously and who had received ketamine as a sole anesthetic agent. It was thought to be related to an increase in cerebral metabolic rate and to a corresponding increase of cerebral blood flow. However, several articles are challenging this message [22-29]. Mayberg et al. investigated cerebral hemodynamics in 20 patients undergoing craniotomy after induction of isoflurane/nitrous oxide anesthesia [23]. They found that an intravenous bolus of 1 mg/kg ketamine did not modify MAP, CPP and arterojugular difference of oxygen, while ICP and mV MCA were significantly reduced. Albanese et al. confirmed these data [24]. In patients with severe head injury who were sedated with propofol, they found that ICP decreased after increasing doses of intravenous ketamine boluses, and no significant differences in MAP, CPP, SjO2, and mV MCA were observed. Recently, Bar-Joseph found that a single ketamine dose decreased ICP by 30% (from 25.8 ± 8.4 to 18.0 ± 8.5 mm Hg; P <0.001) and increased CPP from 54.4 ± 11.7 to 58.3 ± 13.4 mm Hg (P <0.005) during analgosedation in pediatric mechanically ventilated head-injured patients [22]. Reasons for these conflicting results are not completely known. In particular, effects of ketamine on cerebral blood flow (CBF) and cerebral metabolic rate (CMR) are equivocal, since they varied in different brain regions, according to the type of ketamine used (racemic, S-, or R- enantiomers) and the dose administered. According to positron emission tomography (PET) studies by Vollenweider, subanesthetic doses (0.2 to 0.3 mg/kg) of S-ketamine increased CMR, whereas R-ketamine decreased it [32]. Schmidt demonstrated a dramatic decrease in CBF following the administration of a large (10 mg/kg) dose of racemic ketamine [33]. Also, the observed increase in CBF may be partly mediated by a direct effect of the drug on arterial pressure, and partly by a concomitant increase in PaCO2 in spontaneously breathing patients. Simultaneous administration of propofol or benzodiazepines, and mechanical ventilation may blunt these changes in CBF, and explain the results of recent studies. At present, the use of ketamine in neurosurgical patients is not considered completely safe. Even if it is recommended in some countries for analgesia and sedation in head-injured patients, the Federal Drug Administration (FDA) suggests its use with extreme caution in patients with preanesthetic elevated cerebrospinal fluid pressure. The Italian Authority for Drugs (AIFA) contraindicate its use in patients with head injury [34]. Recent guidelines on dealing with sedation and anesthesia for traumatic brain injury do not mention it [30]. Our work provides support not only for the absence of any significant variation of ICP after ketamine, but also for the stability of mV MCA and SjO2. In addition, we observed that the sedative effects of ketamine might be useful as an adjunct to continuous analgosedation for blunting cerebral and systemic response after ETS. Intracranial effects of ETS have been extensively studied. Several authors suggested that vasodilation occurs during ETS, with a resulting increase in CBF that is partially responsible for the increase in ICP [2,35,36]. Cruz found that ETS increased MAP and ICP, with a concomitant increase in SjO2 and mV MCA, suggesting a systemic and cerebral response to painful stimulus [37]. In addition, coughing may induce an increase of intrathoracic pressure and central venous pressure that may contribute to ICP elevation [38]. Our data confirm these results. Despite analgosedation, ETS caused an increase of MAP associated with an elevation of mV MCA and SjO2, as in the presence of an increase of CBF. ICP significantly increased, but came back to baseline after 10 minutes. This transient increase in ICP after ETS may be due to the fact that intracranial hypertension was not common in our patients. Actually, mV MCA was significantly increased, and it remained high during all the study. Unfortunately, we did not measure CBF, but it is reasonable that in patients with an altered intracranial compliance, the increase of CBF may induce a severe increase of ICP. After ketamine, cough reflex was significantly reduced with respect to controls. In the same way, we observed only an increase of ICP, in absence of any significant variation of systemic and cerebral parameters. During ICP increase, MAP did not modify, and CPP showed a slight and nonsignificant reduction. In contrast with what we observed in controls, we did not find any significant variation of mV MCA and SjO2, suggesting that ketamine could have prevented the increase of CBF induced by ETS. These data were in accord with Bar-Joseph, who observed that ketamine reduced ICP in 88% of cases during a potentially distressing intervention such as respiratory physiotherapy, endotracheal suctioning or bed linen change [22]. They did not study a standardized noxious stimulus, as we did, and probably cerebral effects were related to a less painful procedure. Nevertheless, their results refute the notion that ketamine could increase ICP, suggesting that ketamine could induce an additional anesthetic effect without decrease of CPP. We are aware that this study has some limitations. In fact, it was observational, monocentric and conducted on a small number of patients. Furthermore intracranial hypertension was not common in our cases and we did not know in how many cases autoregulation was impaired. Probably, cerebral effects of ETS after ketamine could be different in those subset of patients. We used ketamine at a dose of 100 γ/kg/min for 10 minutes before ETS to minimize hemodynamic variation due to rapid infusion. Actually, this dose can be low for clinical effects; moreover, because of its brief half-life, this slow infusion rate may lead to a less effective action of the drug. BODY.CONCLUSIONS: In conclusion, our data showed that racemic ketamine did not induce any significant variation in cerebral and systemic parameters in mechanically ventilated head-injured patients during continuous analgosedation. After ETS, ketamine maintained cerebral hemodynamics without changes in CPP, mV MCA and SjO2, and prevented cough reflex. Nevertheless, it was not effective enough to control ICP increase. Further studies should be encouraged to confirm these results, in order to update indications of ketamine in neurosurgical patients, and to recommend the best strategy to control cerebrovascular effects after ETS. BODY.KEY MESSAGES: ● Ketamine did not induce any significant variation of ICP, CPP, MAP, mV MCA and SjO2 in mechanically ventilated head-injured patients during continuous analgosedation ● Despite continuous infusion of analgosedation, endotracheal suctioning stimulated cough reflex and caused an increase of MAP, SjO2, mV MCA and ICP. ● If added to continuous analgosedation before endotracheal suctioning, ketamine reduced cough reflex, and prevented any significant change of MAP, CPP, mV MCA and SjO2 ● Ketamine was not sufficient to completely blunt ICP increases after endotracheal suctioning. BODY.ABBREVIATIONS: AIFA: Italian authority for drugs; CBF: Cerebral blood flow; CMR: Cerebral metabolic rate; CPP: Cerebral perfusion pressure; ETS: Endotracheal suctioning; FDA: Federal Drug Administration; GCS: Glasgow coma scale; HR: Heart rate; ICP: Intracranial pressure; ICU: Intensive care unit; IQR: Interquartile range; MAP: Mean arterial pressure; mV MCA: Mean velocity in the middle cerebral artery; PET: Positron emission tomography; SAPS II: Simplified acute physiology score II; SjO2: Jugular oxygen saturation. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: AC had full access to all the data in the study and takes responsibility for the integrity and the accuracy of the data analysis. AC conceived the study, and participated in its design and coordination and helped to draft the manuscript. SP participated in analysis and interpretation of data, helped to draft the manuscript, and critically revised the manuscript for important intellectual content. SP, AT, and MGB collected the data for the study and participated in statistically analysis. CS, MGA, MAP and MA participated in the conception, design and development of the database, helped in analysis and interpretation of data, helped in drafting of the manuscript and critically revised the manuscript for important intellectual content. CDW was in charge of the statistical analysis, and critically revised the manuscript. All authors read and approved the final manuscript.

TITLE: The Serotonin Transporter Gene Alters Sensitivity to Attention Bias Modification: Evidence for a Plasticity Gene ABSTRACT.BACKGROUND: Attention bias modification (ABM) procedures have been shown to modify biased attention with important implications for emotional vulnerability and resilience. The use of ABM to reduce potentially toxic biases, for instance, is a newly emerging therapy for anxiety disorders. A separate line of gene-by-environment interaction research proposes that many so-called vulnerability genes or risk alleles are better seen as plasticity genes, as they seem to make individuals more susceptible to environmental influences for better and for worse. ABSTRACT.METHODS: A standard ABM procedure was used with a sample of 116 healthy adults. Participants were randomly assigned to one of two training groups. One received an ABM procedure designed to induce a bias in attention toward negative material, while the other was trained toward positive pictures. Individuals with low- and high-expressing forms of the serotonin transporter gene (5-HTTLPR) were compared. ABSTRACT.RESULTS: Those with a low-expression form (S/S, S/Lg, or Lg/Lg) of the 5-HTTLPR gene developed stronger biases for both negative and positive affective pictures relative to those with the high-expression (La/La) form of the gene. ABSTRACT.CONCLUSIONS: Here, we report the first evidence that allelic variation in the promotor region of the 5-HTTLPR gene predicts different degrees of sensitivity to ABM. These results suggest a potential cognitive mechanism for the gene-by-environment interactions that have been found in relation to the serotonin transporter gene. Variation on this genotype may therefore determine who will benefit most (and least) from therapeutic interventions, adversity, and supportive environments. BODY: The search for vulnerability genes or risk alleles has been central to the field of psychiatric genetics. It now seems that while specific genes are unlikely to be linked in a direct way to psychopathology, they do moderate the impact that the environment has on stress sensitivity (1–3). Evidence for such gene-by-environment (GxE) interactions, in spite of ongoing controversy, has been gaining momentum. Central to this debate is the burgeoning number of studies examining a repeat length polymorphism in the promotor region of the human serotonin transporter gene (5-HTT, SLC6A4), which has become the most widely studied genetic variant in psychiatry, psychology, and neuroscience (4–9). The short (s) allelic form of the serotonin transporter-linked polymorphic region (5-HTTLPR) is associated with reduced activity of the serotonin transporter, resulting in higher levels of intrasynaptic serotonin (low expression) compared with the long (l) form, which leads to reduced levels of intrasynaptic serotonin (high expression) (3,8). In 1996, it was reported that the s allele was associated with increased self-reports of trait-anxiety or neuroticism, a personality construct known to be linked with increased risk of depression (10). Then, in 2003, an influential longitudinal study found that carriers of the s allele were indeed at increased risk of depression and suicidality but only if they had experienced serious stressful life events or childhood abuse (11). This classic GxE interaction led to a burgeoning of research that remains controversial (4–6,12). While some meta-analyses find that the GxE effects do not hold up across studies (12), others find that as long as a detailed and comprehensive analysis of stressful life events is documented, the 5-HTTLPR short variant does moderate the impact of life stress on psychopathology (6,7). Thus, when extensive details are taken with regard to life events, such as relationship breakups, etc. in one-to-one interviews, GxE effects are strong, while they are often not detected when such specifics are not obtained (4). Another factor that may contribute to the difficulty of replication in this field is the possibility that the s allele actually increases sensitivity to the environment in a more general way so that adverse environments will lead to bad outcomes, while positive and supportive environments will lead to benefits. In other words, the s allele may not be a vulnerability genotype so much as a plasticity genotype (13–15). Uher (2) has argued that one explanation as to why so-called risk alleles have been conserved throughout evolution might be because the social context shapes the outcome of these essentially neutral genetic factors. In other words, more malleable neural circuits can lead to negative outcomes under adversity but also hold the potential for positive gains when the environment is supportive. This means that the neural circuits relating to the processing of affective significance, which are controlled to some extent by the serotonergic system, may be sensitized in s-allele carriers (16). The 5-HTTLPR short variant may, therefore, act as a plasticity gene that renders individuals more susceptible to environmental influences for better and for worse (13–15). It is worth noting, however, that negative material has a stronger draw on attention than does positive material (17). This means that attentional biases to negative, especially threat-related, material is generally stronger than biases toward positive information when compared with a neutral baseline. Thus, while plasticity may operate to both negative and positive information, attention will generally be more responsive to the negative. A separate line of research shows that biases to selectively process threat-related, relative to positive or benign, information is a risk factor for psychopathology. For example, automatic selective biases to direct attention toward negative material better predicts stress reactivity 4 months later, as measured by cortisol response, relative to standardized measures of neuroticism and trait-anxiety (18). It is, therefore, unsurprising that s-allele carriers usually demonstrate increased attentional bias for threat (19–26), which has been confirmed in a recent meta-analysis, and increased amygdala reactivity to threat-related images (27,28). Of particular interest, a recent study shows that s-allele carriers are faster than l homozygotes to pick up fear responses in a fear-conditioning paradigm (29), supporting the notion that people with this genotype are more sensitive to fear-related cues in the environment. Because fear learning is a primary mechanism through which attentional biases for threat develop (30,31), we can speculate that this may be one mechanism through which s-allele carriers acquire a bias toward the more negative aspects of the environment. New techniques to actively induce or modify attentional biases provide a unique methodology to test the hypothesis that s-allele carriers' heightened sensitivity to threat results in the development of potentially toxic biases that leave them more susceptible to psychopathology. MacLeod et al. (32) first demonstrated that selective biases in attention could be modified by a simple computerized technique and that induction of a threat bias leads to increased stress reactivity, whereas the induction of a benign bias leads to a reduction in emotional vulnerability. These findings are important, as they provide evidence for the causal nature of biased attention in stress vulnerability; an experimentally induced bias changes stress reactivity. Their attention bias modification (ABM) technique involved participants being required to identify a nonemotional probe, such as a letter or a symbol, that could appear in one of two locations on the computer screen immediately following the presentation of two words, one of which was negative (e.g., failure, humiliation) and one of which was neutral (e.g., carpet). To train attention toward negative words, the critical probe always appeared in the location previously occupied by a negative word, whereas to induce a benign bias, the probe always appeared in the location previously occupied by a neutral word. Variants of this ABM task have been tested in a range of anxiety disorders and have been shown to reduce threat-related biases and produce marked improvements in clinical symptoms (33–35). Attention bias modification techniques demonstrate that attentional biases are highly plastic and might provide novel treatment strategies for anxiety disorders (35). The present study presents the first investigation of the hypothesis that carriers of the short variant of the 5-HTTLPR will be more responsive to ABM interventions. We used a novel form of the ABM task that presented only positive and negative pictures, rather than comparing each with a neutral item. The main reason for this was because the wider literature on ABM conflates valence and arousal. Because we wanted to isolate the effects of valence (negative and positive material), we used well-validated pictorial stimuli that were matched for arousal level. This would not have been possible if a neutral control had been included on each trial. Based on previous findings with fear conditioning (29), we expected s-allele carriers to develop stronger biases for threat in an ABM task when compared with those homozygous for the l allele. Moreover, if the s allele really does confer greater sensitivity to the environment for better and for worse, then we would also expect stronger development of a positive bias for pleasant images in people with this genotype. In contrast, if the s allele is better characterized as a vulnerability gene primarily responsive to fear-relevant information, then sensitivity to environmental contingencies should occur only with threat-related stimuli. BODY.METHODS AND MATERIALS.PARTICIPANTS: Participants were recruited from a pre-existing database at the University of Essex if they carried either the low-expression (i.e., S/S, S/Lg, or Lg/Lg) or the high-expression (La/La) variant of the 5-HTTLPR gene. Sixty-two participants with the low-expression and 54 with the high-expression genotype were recruited. None had a prior or current psychiatric diagnosis and all reported taking no medication that might affect their mental ability. All had normal or corrected-to-normal vision and gave written informed consent to participate in the study. For the low-expression group, 31 participants were randomly assigned to an attention training procedure to induce attentional bias toward negative images (negative ABM), while 31 were assigned to a training condition designed to induce bias toward positive images (positive ABM). For the high-expression genotype group, 26 participants were assigned to the negative ABM, while 26 were assigned to positive ABM. Participants were either paid £6 or awarded course credit for taking part in the experiment. BODY.METHODS AND MATERIALS.GENOTYPING OF SEROTONIN TRANSPORTER POLYMORPHISM: For DNA collection, participants provided three to four eyebrow hairs with their root ball intact, which were placed into a labeled 1.5 mL tube and centrifuged. DNA was extracted using the Qiagen (Qiagen GmbH, Hilden, Germany) DNeasy blood and tissue kit according to the manufacturer's instructions, using 180 uL of ATL buffer plus 20 uL of proteinase K for the extraction (both, Qiagen). DNA was eluted in 200 uL AE buffer from the Qiagen columns and stored at −20°C until analyzed. The samples were assayed with a combined polymerase chain reaction (PCR)/restriction digest procedure that enabled the distinguishing of three alleles of the serotonin transporter, a length polymorphism (long and short alleles), and a single nucleotide polymorphism (SNP) within the long allele of the locus. The following two primers were used for the PCR (the forward primer carries a 6-FAM label at the 5′ end):IDna5HTTP1FF Fam-CCCAGCAACTCCCTGTACCCCTCCTAIDna5HTTPA4R CGCAAGGTGGGCGGGAGGCTQiagen Type-It microsatellite PCR mix was used for the PCR amplification, using a final volume of 10 uL. Each PCR contained 2.5 uL DNA (or water for control subjects), 5 uL of 2 × PCR mix, 1 uL Q reagent, 1 uL of primers, and .5 uL of water. The final primer concentration was 200 nmol/L each primer. The PCR mixes were cycled using the following scheme: hot start at 95°C for 5 minutes; 40 cycles of 94°C for 30 seconds, 68°C for 90 seconds, and 72°C for 90 seconds; then a final extension at 60°C for 30 minutes. An aliquot of the PCR products was diluted 1:40 with water, then 1 uL mixed with 9 uL of formamide containing Rox500 GeneScan molecular weight markers (Applied Biosystems, Foster City, California). Samples were analyzed by capillary electrophoresis in an Applied Biosystems 3730 instrument, enabling the distinguishing of the long allele (351 bases) from the short allele (307 bases). A second aliquot of 2 uL of the PCR products was digested with the HpaII restriction enzyme in a reaction volume of 20 uL, with 1 unit of enzyme, at 37°C for >90 minutes (Invitrogen, Carlsbad, California). Digest products were diluted 1:40 as before, mixed with formamide plus markers, and separated as above. The sizes of bands generated were 259 bases (long allele plus A SNP base), 217 (short allele), and 86 bases (long allele plus G SNP base). BODY.METHODS AND MATERIALS.MATERIALS: Trait anxiety and depression were measured with the Spielberger Trait-State Anxiety Inventory (STAI) (36) and the Beck Depression Inventory-II (37), in addition to a standardized Attentional Control Scale (38).The State-Anxiety scale of the STAI and two 100-mm visual analogue scales (VAS) were also used to assess anxiety and depression both before and after the ABM procedure. Participants indicated their feelings on the dimensions of anxiety and depression by simply placing an X on a 100 mm VAS ranging from 0 (not at all) to 100 (extremely). BODY.METHODS AND MATERIALS.ATTENTION BIAS MODIFICATION: Stimuli for ABM were presented on a 24-inch Apple Mac OS X Leopard computer (Apple Inc., Cupertino, California) using Superlab software version 4 (Cedrus Corporation, San Pedro, California) and reaction time and error rate responses were recorded from a standard keyboard. Thirty pictures were selected from the International Affective Picture System (IAPS) (39) and gray-scaled using Adobe Photoshop (Adobe, San Jose, California). Fifteen pictures were negatively valenced (picture numbers: 1111, 1112, 1270, 1275, 1280, 1300, 2205, 2490, 2590, 2691, 2692, 2750, 2800, 9180, and 9253) and 15 pictures were positive images (picture numbers: 1440, 1460, 1463, 1500, 1510, 1540, 1590, 1604, 1610, 1710, 2040, 2660, 4641, 4643, and 4660). The two sets differed on the valence of pictures but were matched on arousal. From this set, four positive and four negative pictures were used in phase 1—pretraining—consisting of 128 trials. Each trial started with a fixation cross at center of the screen for 500 milliseconds. Then, a pair of positive and negative pictures appeared one on either side of the fixation point for 500 milliseconds. Immediately after this display, a target—either horizontal (..) or vertical (:) dots—replaced one of the pictures and remained on the screen until response. Participants had to press the "A" button if the dots were vertical and the "L" button if the dots were horizontal. Errors were indicated by a 50-MHz tone. Following the participant's response, a blank screen was presented for 750 milliseconds before a new trial began. There was a 50:50 chance for the target to replace the negative or the positive picture so that any underlying bias toward either type of image could be assessed. Phase 2 consisted of 648 training trials with a different set of nine positive and nine negative pictures. Everything was identical to phase 1 except that the stimulus valence to target location contingency was 100% rather than 50%. In the negative ABM, the target always replaced the negative picture (100% contingency), while in the positive ABM, the target always replaced the positive picture (100% contingency). Finally, in phase 3 (posttraining), the contingency reverted to 50:50, and four images (two positive and two negative) from phase 1, along with four new images (two positive and two negative), were presented. Otherwise, everything was identical to phase 1 to assess whether there was any change in attentional bias from before to after ABM training. BODY.METHODS AND MATERIALS.TEST PROCEDURE: Following written informed consent, each participant filled out the trait questionnaires followed by the pretraining state-anxiety questionnaire and the two VAS ratings. The nature of the computer task was explained. Following 20 practice trials, participants were then left to complete all three phases of the computerized ABM procedure. When finished, they again completed the state-anxiety questionnaire and the two VAS scales. BODY.METHODS AND MATERIALS.DATA ANALYSIS: For each participant, the mean correct reaction time (RT) was computed for each cell of the design by first removing any trials with errors and then removing RTs that were less than 200 milliseconds or greater than 2000 milliseconds (3.1% of data). Preliminary analysis revealed that overall RTs did not differ between the low- and high-expression genotype groups (mean = 750 milliseconds for both). To simply the analysis and presentation of results, an attentional bias index (AB index) was computed by subtracting the mean correct RT on trials where the target appeared in the location of a positive image from the mean correct RT on trials where the target appeared in the location of a negative image. Thus, a numerically positive AB index (e.g., +40 milliseconds) indicates vigilance for affectively positive images (or avoidance of negative images), whereas a numerically negative score (e.g., −52 milliseconds) indicates vigilance for negatively valenced IAPS pictures (or avoidance of positive images). Three separate index scores were computed: AB-pre was attentional bias before training, while AB-old and AB-new were the attentional bias indices found after training for old IAPS images (i.e., those that had been presented in phase 1) and for new IAPS images (i.e., those that had not been presented earlier in the study). Data were analyzed by means of a 2 (genotype group: low expression, high expression) × 2 (ABM group: negative ABM, positive ABM) × 3 (bias index: AB-pre, AB-old, AB-new) analysis of variance with the AB index as the dependent variable. The impact of ABM on mood was assessed by a series of 2 (genotype group: low expression, high expression) × 2 (ABM group: negative ABM, positive ABM) × 2 (session: before training, after training) analyses of variance with state-anxiety and VAS ratings of anxiety and depression as dependent variables. BODY.RESULTS: Male and female subjects were distributed equally between the low-expression (male subjects = 55%, n = 34) and the high-expression (male subjects = 56%, n = 30) genotype groups, which were comparable on the STAI trait-anxiety [mean for low expression = 40.08, SD = 10.6; mean for high expression = 39.3, SD = 8.6, t(114) < 1], Beck Depression Inventory-II [mean for low expression = 7.7, SD = 8.1; mean for high expression = 5.8, SD = 4.1, t(114) = 1.5, p < .126, two-tailed], and Attentional Control Scale [mean for low expression = 51.6, SD = 9.3; mean for high expression = 51.5, SD = 9.0, t(114) < .1]. The genotyping groups did not differ on any of the pretraining questionnaire measures for either the positive ABM (all ts < 1.2) or the negative ABM (all ts < 1.3) group. The attentional bias scores before and after ABM training is shown in Figure 1. The three-way interaction between genotype group, ABM group, and bias index was significant, F(2,224) = 14.8, p < .001, Cohen's f = .36. For the low-expression group, there was a significant ABM group × bias index interaction, F(2,120) = 13.7, p < .001, Cohen's f = .48, such that the positive bias induced by positive ABM training was larger than at pretraining for both old [t(30) = −3.1, p < .004, two-tailed, Cohen's d = .56] and new [t(30) = −2.9, p < .006, two-tailed, Cohen's d = .52] items, while negative ABM induced larger negative biases for both old [t(30) = 2.5, p < .018, two-tailed, Cohen's d = .45] and new [t(30) = 3.4, p < .002, two-tailed, Cohen's d = .60] items. For the high-expression group, while the ABM group × bias index interaction did reach significance, F(2,104) = 3.9, p < .05, Cohen's f = .27, follow-up comparisons revealed only trends for larger biases with new items following positive [t(27) = 1.9, p < .07, two-tailed, Cohen's d = .36] and negative [t(27) = −2.0, p < .06, two-tailed, Cohen's d = .40] ABM training. The interaction was also examined for each ABM group separately and this showed that the attentional bias of the low-expression group was altered significantly by positive ABM for both new [t(31) = −2.9, p < .006, two-tailed, Cohen's d = .82] and old [t(31) = −3.1, p < .004, two-tailed, Cohen's d = .76] items. For this group, negative ABM also changed bias significantly in the expected direction for both new [t(30) = 3.4, p < .002, two-tailed, Cohen's d = .53] and old [t(30) = 2.5, p < .018, two-tailed, Cohen's d = .53] items. In marked contrast, the bias of the high-expression group was not modified by either positive or negative ABM interventions. Figure 2 shows the mean state-anxiety reported for each ABM group. The three-way interaction was not significant, but there was an ABM group × session interaction, F(1,112) = 9.3, p < .003, Cohen's f = .29, due to a larger increase in state-anxiety following training for negative ABM [t(58) = −2.1, p < .04, Cohen's f = .29] compared with positive ABM [t(56) = −5.9, p < .000, Cohen's f = .78]. The mean ratings on the VAS scales also revealed ABM group × session interactions for anxiety [Figure 3: F(1,112) = 8.0, p < .005, Cohen's f = .27] and depression [Figure 4: F(1,112) = 5.5, p < .021, Cohen's f = .22], which were not qualified by genotype group. For the anxiety ratings, the increase following training was greater for negative ABM [t(56) = −7.1, p < .001, Cohen's d = .94] than for positive ABM [t(58) = −2.4, p < .018, Cohen's d = .32]. Depression ratings also increased more following negative ABM [t(56) = −7.1, p < .001, Cohen's d = .95] than positive ABM [t(58) = −3.6, p < .001, Cohen's d = .47]. Finally, a genotype group × session interaction for the depression ratings, F(1,112) = 17.1, p < .000, Cohen's f = .39, was due to larger increases in depression following ABM for the low-expression [t(61) = −7.4, p < .000, Cohen's d = .94] relative to the high-expression [t(61) = −2.9, p < .005, Cohen's d = .39] genotyping group. BODY.DISCUSSION: Changes in attentional bias following ABM were greater in people with low-expression relative to high-expression forms of the serotonin transporter gene. This cognitive malleability to environmental contingencies explains why s-allele carriers (i.e., low expression) are faster to learn fear and develop neural circuits that are more sensitive to threat (27,29). However, the attentional systems of s-allele carriers were also more responsive to positive ABM training, relative to long-allele carriers, providing direct support for the view that the low-expression form of the serotonin transporter gene is best conceived of as a plasticity gene rather than a vulnerability gene. The low-expressing form tunes people into the affective significance of their surroundings—whether negative or positive—moving us beyond the notion that the s allele is a risky genotype, whereas the l allele is protective. Instead, it seems that there's a cost to protective genotypes, such as a reduced ability to maximize the potential of favorable situations (2,13,15). The impact of ABM on mood and anxiety was relatively modest. Depression and anxiety both increased following ABM, with the negative ABM generally leading to larger increases than the positive ABM. This pattern is understandable given that every trial on our ABM procedure contained a negative image. Even though participants' attention was directed away from the negative images during the positive ABM, these threat-related images are powerful cues to attention (17) and therefore are likely to be noticed over several hundred trials. For clinical interventions, ABM procedures with positive and neutral images are unlikely to have such negative effects on mood. Genotype group strongly influenced the degree of change on attentional bias, while the effects on mood were less clear-cut. This is likely due to less sensitivity of the self-report scales when compared with measures of cognitive bias (18), in combination with just a single session of ABM training. Future research should include a wider range of more sensitive outcome measures. One implication of our results is that s-allele carriers should gain most from therapeutic interventions such as ABM. However, a recent study reported that posttraumatic stress disorder patients with the short allele showed a poorer response to cognitive-behavior therapy than did those with two long alleles (40). Thus, at 6-month follow-up, fewer patients in the l-allele group met diagnostic criteria for posttraumatic stress disorder (n = 2, 15%) than those in the s-allele group (n = 14, 48%). One possibility is that even though the cognitive systems of the short-allele carriers are more malleable, this malleability, in turn, results in a much more deeply ingrained set of biases over a lifetime. In other words, the heightened sensitivity to environmental input that occurs in s-allele carriers makes it difficult for them to overcome pre-existing biases and deeply engrained neural circuits that are particularly responsive to danger cues. By the same token, this group should, however, be ultimately more responsive to interventions such as CBT and ABM, and this is an important focus of future research. The use of procedures, like ABM, that specifically target low-level biases may prove particularly effective for this genotype. Several limitations to our study need to be addressed in future research. Our sample size was relatively small and we examined just one genetic polymorphism. These findings should be replicated with a larger sample and a greater range of potential genetic predictors (e.g., the catechol-O-methyltransferase Valine158Methionine polymorphism that is also linked to fear learning). A direct and sensitive measure of stress reactivity after the intervention should also be included in future studies. It would also be useful for future research to include more conventional ABM paradigms that include neutral, in addition to positive and negative, material. Even though this does confound arousal and valence, the absence of negative images in positive training is likely to prevent the negative effect on mood we saw here. Our results present the first evidence that variation on the serotonin transporter polymorphism leads to different degrees of sensitivity to short-term interventions that modify fundamental biases in cognitive processing. This provides a potential mechanism through which those with the low-expression form of this gene are more susceptible to environmental events for better and for worse. Our results imply that those with the low-expression form may benefit most from interventions aimed at reducing toxic biases in attention, even though they may have more deeply ingrained biases.

TITLE: Beetroot Juice Supplementation Improves High-Intensity Intermittent Type Exercise Performance in Trained Soccer Players ABSTRACT: It has been shown that nitrate supplementation can enhance endurance exercise performance. Recent work suggests that nitrate ingestion can also increase intermittent type exercise performance in recreational athletes. We hypothesized that six days of nitrate supplementation can improve high-intensity intermittent type exercise performance in trained soccer players. Thirty-two male soccer players (age: 23 ± 1 years, height: 181 ± 1 m, weight: 77 ± 1 kg, playing experience: 15.2 ± 0.5 years, playing in the first team of a 2nd or 3rd Dutch amateur league club) participated in this randomized, double-blind cross-over study. All subjects participated in two test days in which high-intensity intermittent running performance was assessed using the Yo-Yo IR1 test. Subjects ingested nitrate-rich (140 mL; ~800 mg nitrate/day; BR) or a nitrate-depleted beetroot juice (PLA) for six subsequent days, with at least eight days of wash-out between trials. The distance covered during the Yo-Yo IR1 was the primary outcome measure, while heart rate (HR) was measured continuously throughout the test, and a single blood and saliva sample were collected just prior to the test. Six days of BR ingestion increased plasma and salivary nitrate and nitrite concentrations in comparison to PLA (p < 0.001), and enhanced Yo-Yo IR1 test performance by 3.4 ± 1.3% (from 1574 ± 47 to 1623 ± 48 m; p = 0.027). Mean HR was lower in the BR (172 ± 2) vs. PLA trial (175 ± 2; p = 0.014). Six days of BR ingestion effectively improves high-intensity intermittent type exercise performance in trained soccer players. BODY.1. INTRODUCTION: While nitrate and nitrite were previously considered inert byproducts of the nitric oxide (NO) metabolism, recent insights suggest that (dietary) nitrate can also serve as a precursor for NO through the nitrate -> nitrite -> NO-pathway [1]. Different studies have shown that both plasma nitrate and nitrite concentrations increase following dietary nitrate supplementation in a dose-dependent manner [2,3]. These elevations in plasma concentrations have in turn been associated with improvements in exercise performance, suggesting ergogenic benefits from activation of the nitrate to NO pathway [4,5,6]. Multiple studies from different laboratories have shown that dietary nitrate ingestion can decrease the oxygen cost of submaximal exercise and increase high-intensity exercise tolerance in recreational athletes [4,7,8]. Furthermore, we have previously shown that nitrate-rich beetroot juice ingestion can not only increase oxygen efficiency during submaximal cycling exercise, but that it can also improve time trial performance in moderately trained cyclists and triathletes [5]. As such, this work, in line with others [6,9], has established a functional benefit of dietary nitrate supplementation on exercise performance. Most of the earlier work on the ergogenic effects of nitrate supplementation was focused on endurance type sports, while little attention has been given to high-intensity and/or intermittent type exercise performance. However, recent findings suggest that nitrate might largely convey its effects on exercise performance through type II muscle fibers [10,11]. Ferguson et al. [10] used a rat model to assess the effects of dietary nitrate supplementation on blood flow in vivo during submaximal exercise. The increases in blood flow and vascular conductance in the exercising limbs were primarily observed in fast twitch muscle fibers. In line with these observations, Hernandez et al. [11] reported that dietary nitrate supplementation improves intracellular calcium handling in fast-twitch muscles of mice, which resulted in increased force production. Based on these findings in rodents, it could be suggested that the ergogenic effects of nitrate might be most profound for activities that recruit type II muscle fibers [10,11], i.e., (very) high-intensity exercise bouts of short duration. Soccer is one of the world's most widely performed team sports and is characterized by players performing multiple bouts of high-intensity running and sprinting throughout the 90 min of a match, during which there is heavy reliance on the contribution of type II muscle fibers [12]. These periods of high-intensity activity are alternated with periods of relative recovery, resulting in an intermittent type intensity profile [12,13,14]. The Yo-Yo Intermittent recovery test level 1 (Yo-Yo IR1) is an often used measurement tool to simulate these soccer specific activities in a controlled setting, thereby allowing the reliable and feasible assessment of physical performance in soccer players [15]. Indeed, the Yo-Yo IR1 test has been shown to cover aspects of both aerobic as well as anaerobic performance in soccer players, with a strong link towards the ability to perform high-intensity intermittent type exercise throughout a match [12,15]. Using the Yo-Yo IR1, two previous studies described improved high-intensity intermittent type exercise performance following nitrate-rich beetroot juice ingestion in recreationally active team sport players [16,17]. These observations were the first evidence of ergogenic benefits that team sport players (such as soccer players) could have from nitrate ingestion. The earlier of the two studies observed these effects after ingestion of a high nitrate dose (1780 mg, 28.7 mmol) in the 30 h prior to the high-intensity intermittent running test [16]. Although effective, the dosing strategy that was applied in the study strongly deviates from that of current multiday supplementation protocols that have proven effective in endurance athletes [4,5,7]. More in line with current nitrate supplementation regimens, Thompson et al. recently concluded that a five-day nitrate supplementation protocol with a lower daily dose of nitrate was also effective in improving high-intensity intermittent running performance in recreational athletes [17]. Extending on this finding in recreational athletes, we hypothesized that a homogenous group of trained soccer players performing intermittent type exercise would also benefit from nitrate ingestion. Therefore, we assessed the effects of a six-day nitrate-rich beetroot juice supplementation protocol on high-intensity intermittent running performance in a group of trained soccer players. BODY.2. MATERIALS AND METHODS.2.1. SUBJECTS: A total of 40, first team, male soccer players competing in the 2nd and 3rd Dutch amateur league were recruited to participate in the study. After being informed about the purpose and potential risks of the study, all subjects provided written informed consent. The experimental protocol and procedures were approved by the medical ethical committee of the Maastricht University Medical Centre, the Netherlands (METC 153006; ClinicalTrials.gov: NCT02436629). Eight subjects failed to complete the study because of injury (n = 3), failure to comply with the protocol (dietary/activity standardization procedures; n = 4), or due to personal time constraints (n = 1). Data of the remaining 32 subjects (age: 23 ± 1 years, height: 181 ± 1 cm, weight: 77 ± 1 kg, BMI: 23.4 ± 0.4 kg/m2, playing experience: 15.2 ± 0.5 y) was used in the analysis. BODY.2. MATERIALS AND METHODS.2.2. STUDY DESIGN: This double blind, randomized, placebo-controlled, cross-over study was designed to investigate whether six days of nitrate-rich beetroot juice (BR) supplementation improves intermittent type exercise performance in trained soccer players. Subjects were required to report to the research facility on four occasions, spread over a three-week period. Following a screening session (visit one), subjects visited the research facility ~1 week prior to the first experimental trial to get familiarized with the Yo-Yo intermittent Recovery test level 1 (Yo-Yo IR1) and to receive their supplemental beverages (visit two). No blood or saliva samples were collected during familiarization. The experimental trial days (visits three and four) that followed were each on day six of the nitrate-rich or nitrate-depleted beetroot juice supplementation period, with the last supplemental bolus being ingested 3 h prior to performing the Yo-Yo IR1. Wash-out between the two supplementation periods was at least eight days. BODY.2. MATERIALS AND METHODS.2.3. SUPPLEMENTATION PROTOCOL AND STANDARDIZATION OF PHYSICAL ACTIVITY AND DIET: During the two 6-day supplementation periods, subjects ingested 2 × 70 mL/day of beetroot juice. The choice for beetroot juice was largely based on previous observations by us [18], and by others [19], that suggest more pronounced benefits from nitrate ingestion through plant based sources than following sodium nitrate ingestion. The daily 140 mL bolus of nitrate-rich beetroot juice (BR) provided ~800 mg of nitrate (~12.9 mmol), while the beetroot juice placebo (PLA) was similar in taste and appearance but instead was depleted of nitrate (both supplied by Beet It, James White Drinks Ltd., Ipswich, UK). Subjects were instructed to ingest the 2 × 70 mL shots around the same time each day (~5 pm), which was based on the time the final bolus was ingested on day six of supplementation; i.e., 3 h prior to the exercise test. In addition, subjects recorded their activities and dietary intake in the 36 h prior to the first experimental trial, which were then replicated in the 36 h prior to the second trial. Subjects refrained from strenuous physical exercise or labor in the 48 h leading up to the trial days, and did not consume caffeine or alcohol in the 12 h and 24 h prior to each trial, respectively. To prevent any attenuation in the reduction of nitrate to nitrite by commensal bacteria in the oral cavity, subjects refrained from using antibacterial mouthwash/toothpaste and chewing gum during the six-day supplementation periods [20]. No restriction was set for the consumption of nitrate-rich foods. This was done to allow for the determination of the additional effect of dietary nitrate on performance, on top of the normal diet. As has also been done previously [21], on test days, all subjects were provided with a standardized dinner that was consumed ~3.5 h prior to the exercise test. After consumption of this meal and the final supplemental bolus, subjects were only allowed to consume an ad libitum amount of water in the hours that followed. The amount of water consumed before and during the first trial was replicated during the second trial. BODY.2. MATERIALS AND METHODS.2.4. EXPERIMENTAL PROTOCOL: On the last day of each supplementation period, subjects reported to the research facility ~2 h after ingesting the last 140 mL bolus of beetroot juice. The trials started with collection of a single antecubital venous blood sample and collection of a saliva sample for determination of pre-exercise nitrate and nitrite concentrations (2.5 h after ingesting the last supplemental bolus). Subjects then filled out a gastrointestinal (GI) tolerance questionnaire to assess GI complaints as a result of supplement ingestion. A heart rate monitor (Zephyr Technology Corporation, Annapolis, MD, USA) was then fitted before subjects performed a standardized 10-min warm-up, after which the Yo-Yo IR1 was performed. Heart rate was monitored continuously (1 Hz) to calculate mean heart rate throughout the test, as well as peak heart rate reached near the end of the Yo-Yo IR1 (30-s peak heart rate). The warm-up and the Yo-Yo IR1 were performed indoors in a sports hall, on a 2 by 20 m running lane that was marked by cones, as described previously by Krustrup et al. [15]. The test consisted of repeated 2 × 20 m sprints between a starting, turning, and finishing line at a progressively increasing speed controlled by audio bleeps from an audio system. Between each 2 × 20 min run, subjects had a 10-s active recovery period in an area of 5 × 2 m that was marked by cones behind the start/finishing line. When a subject failed to cross the finish line before the final bleep, a warning was given. When a subject failed to cross the finish line before the bleep for a second time, the final distance covered was registered and represented the end result [15]. Immediately after completing the Yo-Yo IR1, subjects rated their perceived exertion on a Borg 6–20 scale [22]. BODY.2. MATERIALS AND METHODS.2.5. PLASMA AND SALIVA ANALYSIS: Blood samples were collected in Lithium-Heparin containing tubes and immediately centrifuged at 1000× g for 5 min, at 4 °C. Aliquots of plasma were frozen in dry-ice after centrifugation, and were stored at −80 °C for subsequent analysis of plasma nitrate and nitrite concentrations. Saliva samples were collected in 2 mL Eppendorf cups and stored at −80 °C until nitrate and nitrite concentrations were determined in both saliva and plasma using chemiluminescence, as described previously [18]. BODY.2. MATERIALS AND METHODS.2.6. STATISTICAL ANALYSIS: A sample size of 40 subjects, including a 20% dropout, was calculated with a power of 80% and an alpha of 0.05 (two-sided) to detect a 4.2% difference in the distance covered during the Yo-Yo IR1 between BR and PLA. Performance data from the Yo-Yo IR1, heart rate, and plasma and saliva data were analyzed with a paired samples t-test (BR vs. PLA). Effect size of Yo-Yo IR1 performance was determined using Cohen's dz statistical calculation for paired samples. Heart rate data of 7 subjects was incomplete (due to technical problems and/or shifting of the chest bands) and was therefore not included in the analysis. Pearson correlation coefficients were calculated to assess whether differences in plasma or saliva nitrate and nitrite concentrations between trials were associated with the difference in Yo-Yo IR1 performance or heart rate variables between BR and PLA. Statistical significance was set at p < 0.05, and all data were analyzed using SPSS 21.0 (version 21.0, IBM Corp., Armonk, NY, USA), and are presented as means ± SEM. BODY.3. RESULTS.3.1. PLASMA AND SALIVA NITRATE AND NITRITE CONCENTRATIONS: Ingestion of BR for six subsequent days resulted in elevated nitrate concentrations when compared to PLA, in both plasma (Figure 1A) and saliva (Figure 1C) (both p < 0.001). Similarly, nitrite concentrations were higher following BR vs. PLA supplementation in both plasma (632 ± 66 nM vs. 186 ± 13 nM; p < 0.001; Figure 1B) and saliva (2882 ± 519 μM vs. 375 ± 54 μM; p < 0.001; Figure 1D). BODY.3. RESULTS.3.2. YO-YO IR1 TEST: High-intensity intermittent running performance as assessed by the Yo-Yo IR1 significantly improved following BR ingestion (1623 ± 48 m) when compared to PLA (1574 ± 47 m; p = 0.027; Figure 2A). The average improvement in distance covered during the test was 3.4 ± 1.3%, with a Cohen's dz of 0.41. Of the 32 subjects assessed, 18 showed an improved performance during the BR trial vs. the PLA trial (+9 ± 5%), 10 had a slightly worse performance (−5 ± 3%) and 4 showed no difference between trials. Although peak heart rate did not differ between trials (p = 0.16; Table 1), average heart rate during the Yo-Yo IR 1 test was lower in the BR trial when compared to PLA (p = 0.014; Table 1). BODY.3. RESULTS.3.3. GI AND BORG SCORE: Subjects tolerated the interventional drinks well and GI discomfort did not differ between interventions. Only two participants reported a bloated stomach during the PLA trial, and one during the BR trial, while flatulence was reported by two participants during the PLA and two participants during the BR trial. Ratings of perceived exertion as determined with the Borg scale were also not different between interventions (p = 0.23; Table 1). BODY.3. RESULTS.3.4. CORRELATION ANALYSES: Despite the substantial elevations in plasma and saliva concentrations following BR ingestion, no significant correlations were found between plasma and saliva nitrate (r = 0.076, p = 0.697) or plasma and saliva nitrite (r = 0.264, p = 0.144) concentrations. In addition, no associations were observed between (differences in) plasma or saliva concentrations on the one hand, and the (differences in) distance covered, or heart rate variables on the other hand (all r ≤ 0.296; all p ≥ 0.092). BODY.4. DISCUSSION: The current study demonstrates that six days of nitrate-rich beetroot juice supplementation improves high-intensity intermittent type exercise performance in trained soccer players. The improvements in intermittent type exercise performance were accompanied by a lower mean heart rate during the high-intensity intermittent running test, and were preceded by increases in both plasma and saliva nitrate and nitrite concentrations. Nitrate related research in the past years has primarily focused on establishing the effects of nitrate supplementation on endurance type exercise performance. While improvements in exercise capacity [4,8,23] and exercise performance [5,6] have indeed been observed in endurance athletes, recent literature suggests possible performance benefits of nitrate ingestion in more high-intensity and intermittent type sports and activities [16,24]. Extending on previous observations in recreationally active team-sport players [17], the present study specifically assessed the effects of a multiday supplementation protocol with nitrate-rich beetroot juice on high-intensity intermittent type exercise performance in a large sample of trained soccer players. We found that six days of BR supplementation elevated nitrate and nitrite concentrations in both plasma and saliva (Figure 1). The observed 11-fold increase in plasma nitrate and 3-fold increase in plasma nitrite concentrations are in line with previous observations where a similar nitrate dose was administered [18,24]. In addition to the changes in plasma concentrations, the current findings suggest that saliva samples might represent a (less invasive) alternative to assess the postprandial response to beetroot juice ingestion. Salivary nitrate and nitrite concentrations were respectively 13-fold and 7-fold higher following BR ingestion when compared to PLA (Figure 1C,D). However, no correlations were observed between plasma concentrations and saliva concentrations. As such, it seems that saliva samples may be used as a means to assess compliance to nitrate supplementation and to confirm the endogenous reduction of nitrate into nitrite. Nevertheless, analysis of salivary nitrate and nitrite does not seem to represent a valid surrogate for quantitative changes in plasma nitrate or nitrite concentrations. In addition to changes in nitrate and nitrite concentrations, the six-day BR supplementation protocol also resulted in quantifiable improvements in high-intensity intermittent running performance in the soccer players. We observed a 3.4% increase in intermittent type exercise performance on the Yo-Yo IR1 test (Cohen's dz: 0.41; Figure 2A). This is in line with a previous report of improvements in high speed running performance in recreationally active team sport players following a multiday BR supplementation regimen [17]. Although the exact mode of action explaining this effect is still unclear, animal studies have shown that nitrate supplementation can increase blood flow [10], and enhance contractile function in type II muscle fibers [11]. There is some suggestion that these adaptations might be responsible for the improved performance observed during high intensity/intermittent type exercise in which type II fibers are heavily recruited [25]. Interestingly, while such cellular changes have been proposed to only occur following a multiday supplementation regimen [10,11,26], two studies from the same laboratory observed improvements in high-intensity intermittent type exercise performance following both an acute high dose BR supplementation protocol (~29 mmol within 36 h; 4.2% improvement) [16], as well as following a five-day BR supplementation approach with a lower daily dose of nitrate (6.4 mmol/day; 3.9% improvement) [17]. The use of a multiday protocol would seem preferred as it likely allows sufficient time for (some of) the suggested cellular adaptations to occur, that might drive the ergogenic effects of nitrate [10,11]. Furthermore, it is believed that trained subjects may require a different nitrate supplementation strategy (i.e., higher dose and/or for a longer period) to elicit beneficial performance effects in comparison to recreational athletes [9,27,28]. The current study therefore assessed the ergogenic effect of a conventional six-day supplementation protocol with BR (12.9 mmol/day nitrate) in a homogenous sample of trained soccer players. Performance on the Yo-Yo IR1 test was on average ~15% higher when compared to the recreational subjects included in the recent study from Thompson et al. [17]. Nonetheless, we observed a 3.4% improvement in high-intensity intermittent running performance, suggesting that a six-day BR supplementation protocol represents a practical and effective regimen for trained soccer players to improve their performance. Clearly, such a performance benefit should be attained without any major negative side effects. In line with previous work [18], only very mild GI discomfort was reported in a few subjects during the current study, supporting the non-adverse use of beetroot juice in relative short-term interventions. Furthermore, as recently reviewed by Bryan and Ivy [29], there is currently no clear indication of adverse health risks accompanying high nitrate intakes for a prolonged period of time. At present, though any potential risks always need to be carefully considered, the established benefits of nitrate, which may be even more pronounced when consuming nitrate through 'natural' nitrate-rich vegetable sources [18,19], seem to outweigh the potential risks [29]. Intriguingly, and in contrast to previous studies in team sport players, we observed that ingestion of BR for six consecutive days also had an effect on heart rate during the high-intensity intermittent running test (Table 1). While no changes were observed in peak heart rate, mean heart rate during the Yo-Yo IR1 was lower following BR ingestion than following PLA ingestion. To the best of our knowledge, the current findings are the first evidence of changes in heart rate following nitrate ingestion in young healthy athletes. Whether the decrease in mean heart rate is related to the improved exercise performance is unclear, as the only available literature describing effects of inorganic nitrate-nitrite on heart rate are from heart failure patients [30,31]. Borlaug and colleagues showed that a nitrite infusion protocol in heart failure patients increased cardiac output during exercise [30]. The observed increase in stroke volume was suggested to be explained by improved contractility of the left ventricle. While it is currently unclear whether nitrate and/or nitrite ingestion can similarly increase cardiac contractility in healthy individuals, such an effect could explain the decrease in heart rate observed in our study; i.e., allowing the same cardiac output with increased stroke volume, but lower heart rate. Interestingly, a recent study in rodents also showed increased cardiac contractility following nitrate ingestion, most likely as a result of enhanced expression of calcium handling proteins [32]. As nitrate ingestion has also been shown to enhance expression of calcium handling proteins in type II skeletal muscle fibers [11], such an explanation would fit with both the observed increase in intermittent type exercise performance, and the lower mean heart rate in the current study. Although nitrate supplementation increased plasma and saliva nitrate and nitrite concentrations, improved exercise performance, and reduced heart rate, no correlations were observed between any of these parameters. Only a limited number of studies have been able to show correlations between plasma concentrations and subsequent performance benefits [2,17,24,27]. In the present study, only a single sample of saliva and plasma was collected ~30 min prior to the exercise test. It could be suggested that a time point closer to, or even during the exercise test may have revealed a relation between plasma concentrations and changes in performance. Despite the fact that all subjects showed substantially increased plasma and saliva nitrate and nitrite concentrations, not all subjects showed improvements in performance (Figure 2B). It is unclear what the exact explanation is for this lack of effect, although it seems likely that the large day-to-day variability inherent to the Yo-Yo test played a role [33] (Figure 2B). Taking this variability into account, the inclusion of a large sample of trained soccer players allowed us to show a significant and relevant improvement in Yo-Yo IR1 test performance following BR ingestion. Importantly, Yo-Yo IR1 performance has been described to strongly correlate with the ability to perform high speed running and sprinting activities throughout a soccer match [15]. As such, our findings suggest that nitrate supplementation could represent an effective nutritional strategy to improve exercise performance in soccer players, especially towards the end of the match when sprint intensity/frequency has been shown to decrease significantly due to fatigue [34]. Even though in general, day-to-day variation in exercise performance tests combined with small sample sizes make it difficult to study potential ergogenic benefits in highly-trained athletes, future work should be undertaken to establish whether these performance improvements in high-intensity intermittent-type exercise in trained soccer players can also be translated toward the elite level. BODY.5. CONCLUSIONS: Based on the present findings in a large sample of trained soccer players, we conclude that six days of nitrate-rich beetroot juice ingestion improves high-intensity intermittent type exercise performance.

TITLE: Medication intensification in diabetes in rural primary care: a cluster-randomised effectiveness trial ABSTRACT.OBJECTIVE: To determine the effectiveness of a provider-based intervention to improve medication intensification among patients with diabetes. ABSTRACT.DESIGN: Effectiveness cluster-randomised trial. Baseline and follow-up cross-sections of diabetes physicians' patients. ABSTRACT.SETTING: Eleven U.S. Southeastern states, 2006–2008. ABSTRACT.PARTICIPANTS: 205 Rural primary care physicians, 95 completed the study. ABSTRACT.INTERVENTION: Multicomponent interactive intervention including web-based continuing medical education (CME), performance feedback and quality improvement tools. ABSTRACT.PRIMARY OUTCOME MEASURES: Medication intensification, a dose increase of an existing medication or the addition of a new class of medication for glucose, blood pressure and lipids control on any of the three most recent office visits. ABSTRACT.RESULTS: Of 364 physicians attempting to register, 102 were randomised to the intervention and 103 to the control arms; 95 physicians (intervention, n=48; control, n=47) provided data on their 1182 of their patients at baseline (intervention, n=715; control, n=467) and 945 patients at follow-up (intervention, n=479; control, n=466). For A1c control, medication intensification increased in both groups (intervention, pre 26.4% vs post 32.6%, p=0.022; control, pre 24.8% vs post 31.1%, p=0.033) (intervention, adjusted OR (AOR) 1.37; 95% CI 1.06 to 1.76; control, AOR 1.41 (95% CI 1.06 to 1.89)); however, we observed no incremental benefit solely due to the intervention (group-by-time interaction, p=0.948). Among patients with the worst glucose control (A1c >9%), intensification increased in both groups (intervention, pre 34.8% vs post 62.5%, p=0.002; control, pre 35.7% vs post 61.4%, p=0.008). ABSTRACT.CONCLUSIONS: A wide-reach, low-intensity, web-based interactive multicomponent intervention had no significant incremental effect on medication intensification for control of glucose, blood pressure or lipids for patients with diabetes of physicians practising in the rural Southeastern USA. ABSTRACT.TRIAL REGISTRATION: NCT00403091. BODY: Article summaryArticle focusTo determine the effectiveness of a wide-reach and low-intensity intervention aimed at providers to improve medication intensification among patients with diabetes in rural areas.Key messagesIn an effectiveness cluster-randomised trial among 205 rural physicians in 11 US states (Southeastern states; 2006–2008; 24 months), a wide-reach, low-intensity, web-based interactive multicomponent intervention had no significant incremental effect on medication intensification for control of glucose, blood pressure or lipids for patients with diabetes.Medication intensification to control glucose increased in both the intervention and control groups and it was most significant at very poor levels of glucose control.Strengths and limitations of this studyStrenghts include a designed cluster-randomised trial in a diverse rural area.Limitations include the self-selection of charts for data abstraction. A high attrition rate may have introduced bias. BODY.INTRODUCTION: Diabetes mellitus is highly prevalent in the USA.1 Diabetes research has produced clear evidence that blood glucose, lipid and blood pressure (BP) control forestall the vascular complications witnessed with uncontrolled diabetes. Thus, control of such diabetes care measures are well-accepted indicators—as intermediate process outcomes of high quality of care,2 3 However, diabetes care measures of their appropriate control lag behind; only 7% of diabetes patients in USA meet the American Diabetes Association recommendations for glycated haemoglobin (A1c), BP and low-density lipoprotein (LDL) control.4 Furthermore, diabetes is more endemic in the Southern USA and individuals with diabetes in this area have worse control.5–8 Hence, public health interventions, which consider distance barriers in rural setting and aimed at improving diabetes control in patients living in rural areas, are warranted.9–11 The Rural Diabetes Online Care (R-DOC) study was a rigorously tested cluster-randomised clinical trial comparing a multicomponent physician intervention including web-based continuous medical education (CME), performance feedback12–14 and quality improvement tools13 to a concurrent control website in rural Southeast USA.15 The primary outcomes were measures of acceptable and optimal control for A1c, BP and LDL; the secondary outcomes were process measures (rate of assessment and group means of A1c, BP and LDL). Despite the non-significant effect attributable to the intervention for any of the primary outcomes in the R-DOC study, we observed an absolute increase in the rates of assessment of A1c and LDL in both study arms (A1c, intervention 30%, control 29%, p<0.001; LDL, intervention 35%, control 38%, p=0.05).15 However, whether physicians in rural areas adjust medications to control A1c, BP or LDL has not been examined in wide-reach, low-intensity interventions9–11 such as the one utilised in the R-DOC study. Thus, we now report the medication intensification outcomes of the R-DOC study—specifically, medication intensification to improve overall control of haemoglobin A1c, BP and LDL in the rural Southeastern USA. Our findings from the main R-DOC study and this report have important implications for distant CME programmes, quality improvement initiatives and implementation research. BODY.METHODS.STUDY DESIGN AND SETTING: The R-DOC study was a cluster-randomised trial (ClinicalTrials.gov identifier: NCT00403091). The main study results—effect of the intervention on diabetes control—have been published elsewhere.15 Further details of the study design, web-content, recruitment and retention processes and patient characteristics have been published elsewhere.15–18 Briefly, participants were family, general and internal medicine physicians located in rural areas of 11 Southeastern US states (population size <25 000 habitants). Physicians were enrolled in the study sequentially from September 2006 to September 2008, provided informed consent online, and were randomised to the intervention or control arms using block randomisation which was concealed to the investigators and statisticians. The unit of randomisation was the physician (and not a practice); however, for physicians working in a group practice, only one physician per practice could enrol in the study. The protocol was approved by the local institutional review boards at The University of Alabama at Birmingham and Tuscaloosa medical campuses. BODY.METHODS.INTERVENTION ARM DESCRIPTION: The website focused on helping physicians to achieve A1c, BP and LDL control in their diabetic patients.15 18 Specifically, the intervention site contained: (1) challenging cases; (2) individualised diabetes performance feedback reports based on the physician's own panel of patients; (3) practice timesavers; (4) practical goals and guidelines, including guidance for quality improvement and systems redesign;13 (5) patient resources; and (6) an area to track and view CME credit. Intervention arm physicians also received tailored email reminders about website updated and uncompleted sections of the website.15 Individualised performance feedback reports were based on physician's patients with diabetes. The feedback reports compared each intervention arm participant's personal performance compared to the performance for the top 10% of other intervention arm participants.12 The feedback reports consisted of diabetes control (A1c <7%, systolic BP <130 mm Hg, LDL <100 mg/dl), counselling on diet or exercise and medication intensification.15 BODY.METHODS.CONTROL ARM DESCRIPTION: The control website contained: (1) links to diabetes practice guidelines and patient education materials; (2) a list of educational conferences on general medical topics (updated monthly); (3) an area to track and view their CME credit and (4) a link to an external medical blog. Physicians in the control group did not receive performance feedback reports or electronic communications. BODY.METHODS.DATA SOURCES: All participating physicians provided copies of records of 15 (intervention arm physicians) or 10 (control arm physicians) of their own consecutively seen patients with diabetes at baseline and again at follow-up (representing two cross-sectional views of each physician's panel of patients). Since the focus of the intervention was the physician, we were less interested in specific patients but rather each physician's panel on average. Therefore, two samples as serial cross-sections would better represent any change in the physician's own panel on average. This method is similar to practice feedback for quality improvement purposes.19 20 The number of records was selected to balance rigour and cost (see also sample size description in the statistical section below). Patient inclusion criteria were having at least two office visits during the past year and no dialysis, dementia, organ transplantation, HIV/AIDS, terminal illness or malignancy (except for skin and prostate cancer). Data abstraction was performed by trained personnel on blinded records sent to the study centre (or abstracted on site). Details of data abstracted, quality controls and physician compensation are published elsewhere.15 BODY.METHODS.OUTCOMES: In our main study,15 the main outcomes were measures of acceptable and optimal diabetes control.2 3 The main outcome for this report is medication intensification, defined as a dose increase of an existing medication or the addition of a new class of medication for glucose, blood pressure and lipids control on any of the three most recent office visits. Medication intensification equalled the proportion of patients who had a glucose-lowering medication, antihypertensive medication or cholesterol-lowering medication added or increased over the three most recent visits divided by the total number of patients assessed at baseline or follow-up. As a secondary outcome, we also explored medication intensification by level of glucose, BP or lipid control. BODY.METHODS.STATISTICAL APPROACH: We calculated the sample size for the main study results.15 On the basis of clinically important differences, the study required 100 physicians per trial arm to detect a minimum of 0.4% difference in A1c, 6 mm Hg in BP and 6 mg/dl in LDL for the main outcomes (power 80%, α=0.05, up to 20% dropout) (10 patients per physician at baseline and at follow-up).15 However, intervention arm physicians were asked to provide 15 patient records at baseline (and not at follow-up) because of the need to construct audit and feedback reports as part of the intervention19 20 for A1c, BP and lipids, reflecting the fact that not all diabetes patients also have hypertension and hyperlipidaemia. The initial study was not powered to perform adjusted analysis for the intensification outcomes of this report. All analyses followed the intention-to-treat principle. The relationship between the main and secondary outcomes and the effect of the intervention were examined with generalied linear mixed models (GLMM), accounting for clustering of patients within physicians. ORs for follow-up versus baseline within the two groups were calculated, adjusting for covariates that differed between baseline and follow-up patient populations; thus, the covariates included in these adjusted analyses included race, and clinical diagnosis of hypertension or depression. GLMM was implemented by PROC GLIMMIX in SAS and was adjusted for race, and clinical diagnosis of hypertension or depression. Thus, the group coefficient represented differences between the intervention and control groups at baseline; the time of coefficient represented changes in the control group over time, thus capturing temporal trends. Finally, the group-by-time interaction coefficient represented the difference-of-differences for over-time change in the intervention versus control group, and was a direct comparison of over-time change for the intervention versus the control group. A positive OR meant that the odds of a patient being in control increased more over time for the intervention versus control group. All analyses were performed in SAS V.9.2 (Cary, North Carolina, USA). BODY.RESULTS.RECRUITMENT SCHEME, PATIENT CHARACTERISTICS AND WEB UTILISATION: The Consolidated Standards of Reporting Trials diagram and patients characteristics at baseline and at follow-up (modified from a prior publication15) are included in this report for completeness (figure 1 and table 1). We obtained baseline and follow-up data on 95 physicians (intervention, n=48; control, n=47) and 1182 of their patients at baseline (intervention, n=715; control, n=467) and 945 diabetes patients at follow-up (intervention, n=479; control, n=466). Table 1 Characteristics of diabetes patients of 95 physicians randomised to intervention (n=48) or control (n=47) who completed follow-up Patient characteristic Baseline Follow-up Intervention (n=715) Control (n=467) Total (n=1182) Intervention (n=479) Control (n=466) Total (n=945) Age (years) 58.7 (13.59) 60.6 (13.79) 59.4 (13.70) 61.3 (13.42) 60.5 (12.71) 60.9 (13.09) Gender, female 360 (51.1%) 230 (49.5%) 590 (50.5%) 260 (54.5%) 252 (54.4%) 512 (54.5%) Race, African American 97 (13.9%) 99 (21.3%) 196 (16.9%) 102 (21.4%) 143 (30.9%) 245 (26.1%) Obesity* 274 (38.3%) 193 (41.3%) 467 (39.5%) 97 (20.3%) 75 (16.1%) 172 (18.2%) Smoker, current 85 (12.5%) 55 (12.3%) 140 (12.4%) 63 (13.8%) 53 (13.0%) 116 (13.4%) No self-testing 168 (33.6%) 114 (36.3%) 282 (34.6%) 189 (41.4%) 183 (44.7%) 372 (43.0%) Non-adherence to appointments 75 (11.0%) 54 (12.0%) 129 (11.4%) 10 (2.2%) 23 (5.6%) 33 (3.8%) Insurance, Medicaid 65 (9.1%) 51 (10.9%) 116 (9.8%) 66 (13.8%) 58 (12.4%) 124 (13.1%) Comorbidities  Diabetes complications† 147 (20.6%) 110 (23.6%) 257 (21.7%) 156 (32.6%) 134 (28.8%) 290 (30.7%)  Insulin use 103 (14.4%) 66 (14.1%) 169 (14.3%) 84 (17.5%) 68 (14.6%) 152 (16.1%)  Hypertension 473 (66.2%) 304 (65.1%) 777 (65.7%) 327 (68.3%) 283 (60.7%) 610 (64.6%)  Hyperlipidaemia 110 (15.4%) 85 (18.2%) 195 (16.5%) 11 (2.30%) 17 (3.7%) 28 (3.0%)  Peripheral vascular disease 47 (6.6%) 31 (6.6%) 78 (6.6%) 41 (8.6%) 35 (7.5%) 76 (8.0%)  Coronary artery disease 135 (18.9%) 83 (17.8%) 218 (18.4%) 111 (23.2%) 95 (20.4%) 206 (21.8%)  Vascular intervention‡ 60 (8.4%) 47 (10.1%) 107 (9.1%) 36 (7.5%) 40 (8.6%) 76 (8.0%)  Depression 105 (14.7%) 61 (13.1%) 166 (14.0%) 84 (17.5%) 60 (12.9%) 144 (15.2%) A prior version of this table has been reported (reproduced with permission). 15 Values are mean (SD) or n (%). *Obesity: body mass index ≥  30 kg/m 2 or clinical diagnosis. †Diabetes complications: retinopathy, neuropathy or nephropathy. ‡Vascular intervention: coronary artery bypass grafting, stent, percutaneous coronary angioplasty. Figure 1Consolidated Standards of Reporting Trials (CONSORT) diagram (a prior version of this figure has been reported, reproduced with permission15). At baseline, intervention group physicians provided records for fewer African American patients and more patients with hypertension and depression compared with control group physicians. Of the 95 physicians, 90 (94.7%) had access to the Internet in the office. BODY.RESULTS.MAIN OUTCOMES: MEDICATION INTENSIFICATION: In the adjusted analysis, we saw no significant effect attributable to the intervention in the group-by-time interaction term for any of the medication intensification measures (A1c, BP and LDL; p=0.948, 0.216 and 0.995; respectively). In the unadjusted analysis, intensification of medications to control A1c increased in both trial arms when baseline and follow-up data were compared (intervention, 26.4% vs 32.6%, p=0.022; control, 24.8% vs 31.1%, p=0.033; figure 2). This was confirmed in the adjusted analysis (intervention, adjusted OR (AOR) 1.37; 95% CI 1.06 to 1.76; control, AOR 1.41 (95% CI 1.06 to 1.89)). Figure 2Medication intensification for haemoglobin A1c (%), blood pressure (BP, mm Hg) and cholesterol (low-density lipoprotein (LDL), mg/dl) control for intervention (n=48) or control (n=47) physicians, at baseline (pre, n=1182 patients) and follow-up (post, n=945 patients). See text for definitions of medication intensification. In the unadjusted analysis, intensification of medications to control BP did not differ for patients cared for by physicians in either trial arm when comparing baseline and follow-up data (intervention, 12.3% vs 15.9%, p=0.080; control, 16.5% vs16.3%, p=0.941; figure 2). This finding was consistent with the adjusted analysis (intervention, AOR 1.37 (95% CI 0.99 to 1.88); control, AOR 1.00 (95% CI 0.72 to 1.41)). In the unadjusted analysis, intensification of medications to control LDL did not differ for patients cared for by physicians in either trial arm when comparing baseline and follow-up data (intervention, 10.6% vs 11.3%, p=0.726; control, 8.1% vs 8.4%, p=0.898; figure 2). This finding was consistent with the adjusted analysis (intervention, AOR 1.05 (95% CI 0.73 to 1.50); control, AOR 1.00, (95% CI 0.65 to 1.53)). BODY.RESULTS.SECONDARY OUTCOME: MEDICATION INTENSIFICATION BY STRATA: In the unadjusted analysis and among patients with worse glucose control (A1c >9%), intensification of medications to control A1c increased in both trial arms between baseline and follow-up (intervention, pre 34.8% vs post 62.5%, p=0.002; control, pre 35.7% vs post 61.4%, p=0.008; figure 3 top panel). For patients whose last A1c ≥7 to ≤9, we noted an increase in intensification between the baseline and follow-up for the intervention group (p=0.005) but no difference in the control group (p=0.164; figure 3 top panel). Among patients with best glucose control (A1c <7%), medication intensification between baseline and follow-up was similar for both trial arms (intervention, p=0.673; control, p=0.543; figure 3 top panel). Figure 3Medication intensification for strata of haemogloblin A1c (%), blood pressure (BP, mm Hg) and cholesterol (low-density lipoprotein (LDL), mg/dl) control. See text for definitions of medication intensification. In the unadjusted analysis, intensification of BP medication between baseline and follow-up was similar at all levels of BP control for both trial arms (figure 3 middle panel). Similarly, in the unadjusted analysis, intensification of lipid-lowering medications between baseline and follow-up was similar at all levels of LDL control for both trial arms (figure 3 bottom panel). BODY.DISCUSSION: In a rigorously designed cluster-randomised trial, a wide-reach, low-intensity web-based multicomponent intervention for primary care physicians in the rural Southern USA had no significant incremental effect on medication intensification for their diabetes patients' A1c, BP or LDL control as compared to a concurrent control website focusing on diabetes care. However, the absolute rate of intensification of medications to control A1c increased in both study arms at the end of the study by a modest amount (∼6%). Although the study was not designed to perform subgroup analysis, the rate of intensification was greatest among the worst-controlled patients (A1c >9%) in both study arms (∼25–28%). Plausible explanations for our findings include the low engagement with the intervention, higher-than anticipated attrition rate, Hawthorne's effect or no effect of the intervention15 18 Medication intensification is emerging as a process measure for quality improvement efforts among patients with diabetes mellitus; pharmacotherapy intensification is strongly linked to improved A1c, BP and LDL. For example, in a study of over one million members with hypertension, hyperlipidaemia and diabetes mellitus at Kaiser Permanente Northern California, Selby et al21 found that a 5% improvement in treatment intensification led to a 1.0–1.9% improvement in control for the entire population. However, the association of treatment intensification rates to improved risk factor control in rural populations has not been examined. Further evidence and reviews of the importance of medication intensification among patients with diabetes are available elsewhere22–27. BODY.DISCUSSION.TRIALS USING MEDICATION INTENSIFICATION OUTCOMES: Relatively few studies involving physicians have examined medication intensification as an outcome, none in a rural setting. Studies have tested the impact of a simulated case-based education with opinion leader feedback,28 customised feedback29 and real-time feedback to patients and their physicians using cell phone software;30 these studies are summarised below. In a group randomised controlled trial28 of 57 primary care physicians and 2020 patients in an urban setting, physicians were randomised to no intervention, a simulated case-based physician learning intervention, or the same case-based and leader feedback. Glucose control, mean A1c value, worsened in the control group by 0.06, improved in the simulated case-based group by 0.01, and worsened in the case-based and leader feedback by 0.18. Among the 907 patients with A1c>7%, medication intensification was similar between groups (31.5%, control; 32.6%, case-based; 36.8%, case-based and leader feedback; p=0.41). Similarly, among the 701 patients with LDL-C >99 mg/dl, medication intensification was similar between groups (20.8%, control; 24.2%, case-based; 21.8%, case-based and leader feedback; p=0.66). Finally, among the 949 patients with BP values >130/mm Hg, medication intensification was similar between groups (27.3%, control; 24.7%, case-based; 28.2%, case-based and leader feedback; p=0.61). Medication intensification was defined by the initiation or titration in 12 months after the intervention for all patients with goals above target (A1c>=7%, LDL-C >=100 mg/dl, BP>130/80 mm Hg); insulin titration was excluded from the outcome. The attrition rate was 33% (38/59 physicians). Our case scenarios took less than 10 min to complete as compared with the ones in this study (over 60 min each).28 Perhaps striking a balance between more 'intense' cases to increase physician engagement and providing feedback information may lead to the improvement in medication intensification. Finally, the medication intensification rates definitions were different from ones used in our study; hence, they cannot be compared. In a randomised trial of 3703 patients with diabetes and their 123 physicians, participants were randomised to receive customised feedback of clinical information in four groups: patient only, physician only, both the patient and physician, or neither one.29 At 12 months, interventions had no effect on A1c test ordering, medication intensification or improvement in A1c or LDL control. Medication intensification was defined as in the above study.28 In our study, we included customised feedback as one of the many components of the intervention. In a pilot-controlled trial,30 30 patients with diabetes were randomised to an intervention group consisting of cell phone-based software which provided real-time feedback on glucose levels, medication regimens and suggested treatment algorithms or a control group. Intervention providers received patients' logbooks and suggested treatment plans to reach A1c<6.5%. During the 3 months of the study, the A1c levels decreased among patients in the intervention as compared with the control group (by 2.0% vs 0.7%; respectively); medications were changed or intensified in 84% in the intervention group as compared with 23% patients in the control group (p<0.002). The operational definition for medication intensification in this study was not reported. Cell phone technology provides the opportunity for wide reach approach. In our study, physicians and not their patients received the customised feedback on their group of patients. BODY.DISCUSSION.LIMITATIONS: Our study had limitations.15 First, physicians themselves provided the records of consecutively seen patients; although selection bias was a possibility, the wide range of A1c, BP and LDL values suggests that not only well-controlled patients were selected. Second, the high attrition, 95 of the 205 randomised physicians provided baseline and follow-up data, may have introduced biases. By design, to test a purely online intervention, we chose not to enhance retention activities as typically seen in randomised clinical trials and focused on several aspects of provider-based implementation tools.13 Although a comprehensive analysis would have been enlightening, we did not perform a systematic examination of this negative trial, or how such intervention might work it were effective. We worried that studying the study while being conducted would have introduced another variable. BODY.DISCUSSION.IMPLICATIONS: A framework has been proposed to evaluate the public health impact of interventions.9–11 The components of the framework include reach ('How many participate?'), effectiveness ('Does it work in usual settings?'), adoption ('How many use it?'), implementation ('Is it used as intended?') and maintenance ('Is it sustained over time?') (RE-AIM). In this study, using a wide-reach approach, we examined the effectiveness of a potential public health intervention. Effective interventions may be limited by their low-reach, specificity to fewer settings, expense and limited sustainability—thus limiting their generalisability. Testing less intensive interventions with wider reach in rural settings has been advocated in public health interventions.11 Finding effective strategies to accomplish continuing professional development for physicians practising in remote locations is also an important objective.31 In contrast, higher-intensity interventions in rural and community settings have shown promising results.32–36 BODY.CONCLUSION: In conclusion, a wide-reach, low-intensity, web-based interactive multicomponent intervention had no significant incremental effect on medication intensification for control of glucose, BP or lipids for patients with diabetes of physicians practising in the rural Southeastern USA. Despite low engagement in the web-based programme, intensification measures improved in both arms of the study. Our study raises important issues for wide-reach, low-intensity CME programmes that seek to improve patient outcomes. BODY.SUPPLEMENTARY MATERIAL: Author's manuscript Reviewer comments

TITLE: To condition or not condition? Analysing ‘change’ in longitudinal randomised controlled trials ABSTRACT.OBJECTIVE: The statistical analysis for a 2-arm randomised controlled trial (RCT) with a baseline outcome followed by a few assessments at fixed follow-up times typically invokes traditional analytic methods (eg, analysis of covariance (ANCOVA), longitudinal data analysis (LDA)). 'Constrained' longitudinal data analysis (cLDA) is a well-established unconditional technique that constrains means of baseline to be equal between arms. We use an analysis of fasting lipid profiles from the Group Medical Clinics (GMC) longitudinal RCT on patients with diabetes to illustrate applications of ANCOVA, LDA and cLDA to demonstrate theoretical concepts of these methods including the impact of missing data. ABSTRACT.METHODS: For the analysis of the illustrated example, all models were fit using linear mixed models to participants with only complete data and to participants using all available data. ABSTRACT.RESULTS: With complete data (n=195), 95% CI coverage are equivalent for ANCOVA and cLDA with an estimated 11.2 mg/dL (95% CI −19.2 to −3.3; p=0.006) lower mean low-density lipoprotein (LDL) cholesterol in GMC compared with usual care. With all available data (n=233), applying the cLDA model yielded an LDL improvement of 8.9 mg/dL (95% CI −16.7 to −1.0; p=0.03) for GMC compared with usual care. The less efficient, LDA analysis yielded an LDL improvement of 7.2 mg/dL (95% CI −17.2 to 2.8; p=0.15) for GMC compared with usual care. ABSTRACT.CONCLUSIONS: Under reasonable missing data assumptions, cLDA will yield efficient treatment effect estimates and robust inferential statistics. It may be regarded as the method of choice over ANCOVA and LDA. BODY: Strengths and limitations of this studyClarification of the statistical methods available for longitudinal randomised controlled trials (RCTs) as well as analysis recommendations is warranted.In many longitudinal RCTs, participants are measured at baseline, then at the same follow-up occasions with a small number of follow-ups, for example, 2–4. In this design, how should baseline values be handled?In practical applications, constrained longitudinal data analysis, an appropriate generalisation of analysis of covariance, is the most straightforward to implement and under reasonable missing data assumptions will yield robust estimates of treatment effect differences and valid inferential statistics. BODY.INTRODUCTION: In a recent longitudinal randomised controlled trial (RCT) designed to examine the effect of Group Medical Clinics (GMC) on cardiovascular outcomes in patients with diabetes, the statistical inference on the effect of the GMC intervention on low-density lipoprotein (LDL) levels was dependent on the method of analysis applied.1 Estimates of mean difference in LDL between treatment arms ranged from 6.9 to 11.2 mg/dL depending on the analysis method used. Reviewers questioned the primary analysis method which yielded an LDL improvement of 8.9 mg/dL for the GMC arm compared with usual care (p=0.03), and requested an alternative, and seemingly plausible but less powerful analysis that yielded an LDL improvement of 7.2 mg/dL for the GMC arm compared with usual care (p=0.15). Thus, the interpretation of the intervention effect varied significantly depending on the analytic technique used. Based on this experience and others of a similar nature, clarification of methods available as well as analysis recommendations is warranted and may be useful, not only to statistical analysts, but clinical researchers evaluating longitudinal RCTs. BODY.INTRODUCTION.LONGITUDINAL STUDY DESIGNS: In many longitudinal RCTs, research participants are measured at the same follow-up measurement occasions and the number of follow-up occasions is small, for example, 2–4. Typically, baseline outcomes are measured prior to randomisation. In analyses, baseline outcomes can be ignored, used to calculate change scores, used conditionally as covariates or can be part of the outcome response vector. In this design, how should baseline values be handled? Is the question of interest conditional, given one's baseline what is the difference in outcome between treatment arms, or is the question unconditional, what is the treatment difference in change? Or does it matter? These fundamental questions and their implications on the method of analysis were introduced over 50 years ago in a sentinel paper by Lord2 subsequently leading to the term 'Lord's paradox. In Lord's hypothetical example, the difference between the conditional and unconditional questions and how analyses are applied yielded different answers and statistical inference when applied to the same data set. In spite of extensive literature3–8 for this type of data, there remains a lack of clarity in biomedical research applications regarding longitudinal statistical analysis. As the issue is addressed here, a major complication requiring consideration in the analysis strategy of a longitudinal RCT is missing data. Attrition (dropout) or intermittent missingness occurs even in studies with few measurement occasions as we are addressing. Reducing the quantity of missing data in an RCT enhances the reliability of results;9 however, rarely is it possible to eliminate missing data completely? The assumptions and methods applied for handling missing data when analysing 'change' in a longitudinal RCT can greatly affect the robustness, validity and power of results and need to be clearly understood. The general consensus in the statistical literature for a longitudinal RCT is that the conditional approach, that is, analysis of covariance (ANCOVA), is the most powerful and robust method4 8 to address the fundamental questions of interest. We take this view, and elaborate on its implementation, taking into account the additional complication of missing data, and enumerate the benefits of unconditional constrained longitudinal data analysis (cLDA) models, illustrating that they generalise the ANCOVA approach in the longitudinal trials' setting. BODY.INTRODUCTION.ANALYSIS STRATEGIES: In a longitudinal RCT of the type addressed here, the outcome of interest is measured at each of the defined assessment periods of the trial (each participant in each arm of the trial). In this setting define the outcome as , the assessed value at each time point, t for each individual i (. In its simplest form, a longitudinal RCT is a pre/post-type of study where there are assessments at two time points, one pre-treatment baseline, , and one post-treatment, . In the more general longitudinal RCT study design with multiple but few follow-up time points, response profile modelling6 also known as mixed model repeated measures10 is generally used as it allows for arbitrary patterns in the mean responses over time (ie, time is dummy coded) as well as for the covariance of responses and can be used for fitting ANCOVA, cLDA or longitudinal data analysis (LDA) models. From a modelling perspective, the pre-treatment baseline assessment can be viewed as either a fixed predictor of post-treatment outcome or as an outcome assessment. For the former, the conditional ANCOVA approach yields estimates of treatment differences over time given the observed baseline values where post-treatment outcome measurements are the response variables. For the latter, an unconditional model (LDA or cLDA) yields estimates of treatment differences unconditionally, and in the context of a longitudinal RCT, both the pre-treatment and post-treatment outcome measurements are response variables. BODY.INTRODUCTION.ANALYSIS STRATEGIES.CONDITIONAL VERSUS UNCONDITIONAL MODELS: A fundamental difference between a conditional or unconditional analysis is in the modelling of the pre-treatment assessment. In unconditional analysis, baseline is part of the response vector requiring additional assumptions for modelling baseline. In an LDA, there are no modelling constraints on the baseline; separate baseline means are assumed and fit for each randomised group. The general test used for treatment difference over time in LDA is equivalent to a change score type of analysis; comparing change from baseline to follow-up between randomised groups. In contrast for cLDA baseline means are constrained to be equal between the randomised groups; a common baseline mean is assumed and fit across randomised groups. In an RCT, baseline precedes any treatment deliverance and under expectation the baseline means are equal. The test for treatment difference over time in the cLDA is essentially equivalent to a test for treatment difference in an ANCOVA when no outcome data are missing.5 11–13 BODY.INTRODUCTION.ANALYSIS STRATEGIES.CONDITIONAL MODEL: An ANCOVA model in a two-arm (j=1, 2) RCT with a pre/post design will have study outcome measures at baseline and one follow-up time (t=0,1). However, there will only be one response variable per participant (i=1,...,nj) as the baseline (Y0) is a covariate in the model. The model is written as:1 The marginal mean at follow-up time (t=1) is conditional on the baseline . The parameter is the slope for the baseline, and is the effect of treatment (j=2) at time (t=1) compared with treatment (j=1) adjusted for the baseline effect. BODY.INTRODUCTION.ANALYSIS STRATEGIES.UNCONDITIONAL MODELS.LDA MODEL: An LDA model in a two-arm (j=1, 2) RCT with a pre/post design will have study outcome measures at both the baseline and follow-up time (t=0,1). The model is written as:2 The parameter γ is the difference in baseline means at time (t=0) between arms, τ is the mean difference in change from baseline to follow-up for arm (j=1), and δ is the difference in change from baseline to follow-up between arms. BODY.INTRODUCTION.ANALYSIS STRATEGIES.CLDA MODEL: A cLDA model in a two-arm (j=1, 2) RCT with a pre/post design will have study outcome measures at both the baseline and follow-up time (t=0,1). The model is written as:3 The parameter is the mean change in outcome from baseline to follow-up time in arm (j=1), and δ′ is the mean difference in change from baseline to follow-up between arms, however, since baseline means are assumed equivalent this is the mean difference between arms at the follow-up time. BODY.INTRODUCTION.ANALYSIS STRATEGIES.CONDITIONAL AND UNCONDITIONAL MODEL COMPARISON: No missing baseline or follow-up data: When there is no missing baseline or follow-up data, Liang and Zeger5 and Liu et al11 have shown that ANCOVA and cLDA models produce identical point estimates for treatment differences. Using our model notation we can state under the case of no missing baseline or follow-up data:4 Similarly, for reasonably sized trials the variance of the treatment effect differences will be approximately equivalent between ANCOVA and cLDA models (see online supplementary appendix A). 10.1136/bmjopen-2016-013096.supp1supplementary appendix Frison and Pocock3 have shown that estimated treatment differences from ANCOVA or of POST-treatment means or CHANGE from baseline from an LDA model have the same expected value. However, they demonstrate the general superiority of inferential power for ANCOVA over both POST and CHANGE from baseline analysis. We can see from plotting equations of estimates of variance in figure 1 that ANCOVA and cLDA are superior to LDA-POST and LDA-CHANGE over the range of correlations between pre and post measurements (see online supplementary appendix A for variance estimates). For high correlations between pre-measurements and post-measurements the variances of LDA-CHANGE and ANCOVA/cLDA are similar. Figure 1Comparison of variance of treatment difference estimates over the range of correlations between pre and post measurements for LDA, cLDA, ANCOVA and SPO methods; plot generated from variance estimate formulas given in online supplementary appendix A. ANCOVA, analysis of covariance; cLDA, constrained longitudinal data analysis; LDA, longitudinal data analysis; SPO, simple post only. The conditional and unconditional models described above are easily extendible from the pre/post type design with only one follow-up time point to multiple follow-up time points by additional dummy variables to represent follow-up times. Similarly, comparison of models and estimates from the pre/post design apply to the follow-up time points. In the longitudinal RCTs with few measurement occasions, the comparison of interest is generally the treatment difference at the last follow-up time point (T)—the theory as described above for the post-time measurement in a pre/post design applies to analysing the differences between treatment arms at any specific follow-up time. BODY.INTRODUCTION.MOTIVATING EXAMPLE: GROUP MEDICAL CLINICS STUDY: We present an analysis of fasting lipid profiles from the GMC longitudinal RCT on patients with diabetes to illustrate applications of ANCOVA, LDA and cLDA models and demonstrate theoretical concepts described above including the impact of missing data.1 This study randomised 239 participants at a 3:2 ratio to the GMC arm (n=133) and usual care arm (n=106). Fasting lipid profiles are secondary outcomes from the GMC study measured at baseline, midpoint (median follow-up 6.8 months) and end of study (median follow-up 12.8 months).14 For illustration, we focus analysis on the baseline and 12-month LDL cholesterol (LDL-C) measurements. For notation, the two arms will be denoted with j=G for GMC or j=U for usual care and time with t=0 for baseline and t=12 for 12 months. All analyses were conducted using SAS V.9.2 (SAS Institute, Cary, North Carolina, USA). BODY.METHODS: For completeness of analyses approaches, in addition to the ANCOVA, LDA and cLDA models, we also conducted simple post only (SPO) and simple change score analysis (SACS). For SPO, we compared mean 12-month LDL-C () for GMC (j=G) to mean usual care (j=U) 12-month LDL-C () and for SACS, we compared mean LDL-C change from baseline () for GMC (j=G) to the mean change from baseline LDL-C () for usual care (j=U) using two-sample t-tests. We then applied the ANCOVA, LDA-CHANGE and cLDA models to, and , the assessed value of LDL-C. All three types of models were fit using linear mixed models15 and the response profile modelling approach. Models were fit using PROC MIXED with restricted maximum likelihood estimation and unstructured covariance. BODY.METHODS.COMPLETERS ANALYSIS: The first set of analyses was applied to participants that had both baseline and 12-month measurements (completers) to demonstrate theoretical comparisons of models as described without the added complication of missing data. BODY.RESULTS: Among the 239 patients randomised in the study, 195 participants had LDL-C measurements at both baseline and 12 months. All methods yield statistically significant differences in LDL-C between arms (table 1) with change score analysis methods (SACS and LDA) right at p=0.05. SPO had the largest estimated difference in mean LDL-C between GMC and usual care. As expected, estimated mean treatment differences at 12 months and 95% CI coverage are equivalent for the ANCOVA and cLDA with an estimated 11.2 mg/dL lower mean LDL-C in GMC compared with usual care. The SACS and LDA estimated differences and 95% CI coverage are equivalent with an estimated 10.1 mg/dL lowering between baseline and 12 months of mean LDL-C for GMC compared with usual care. LDA and SACS analyses yield a wider 95% CI than ANCOVA and cLDA methods. For completers, the ANCOVA and cLDA yield the most robust results and illustrate their superiority (as shown in figure 1) over SPO and change score methods (both SACS and LDA), as the correlation between baseline and 12-month LDL-C values was estimated in the 0.50 range. Table 1 Completers only (n=195 participants)—pre/post analyses Model Outcome (Y t and/or C t ) GMC (n=117) Usual care (n=78) GMC vs usual care (95% CI) p Value Post-only 12 months (Y 12 ) 81.9 94.1 −12.1 (−21.5 to −2.7) 0.01 SACS 12 months − baseline (C 12 ) −12.9 −2.8 −10.1 (−20.2 to 0.0) 0.05 ANCOVA 12 months (Y 12 ) 82.3 93.5 −11.2 (−19.2 to −3.3) 0.006 LDA Baseline (Y 0 ) 94.8 96.9 12 months (Y 12 ) 81.9 94.1 −10.1 (−20.2 to 0.0) 0.05 cLDA Baseline (Y 0 ) 95.7 95.7 12 months (Y 12 ) 82.3 93.5 −11.2 (−19.2 to −3.3) 0.006 ANCOVA, analysis of covariance; cLDA, constrained longitudinal data analysis; GMC, Group Medical Clinics; LDA, longitudinal data analysis; SACS, simple change score analysis. Among the 239 patients randomised, 6 participants had no baseline or 12-month LDL-C, so they are excluded yielding 233 patients for the all participants analysis. The estimated treatment differences diverge across analysis methods as well as statistical significance using a α=0.05 (table 2). ANCOVA and SACS are equivalent to completers analyses, as the 38 cases with either a missing baseline or 12-month LDL-C are deleted. ANCOVA and cLDA methods no longer have equivalent estimates of treatment differences. All 233 participants contribute at least one measurement to cLDA analysis so that participants missing either a baseline or 12-month LDL-C contribute to estimated treatment difference of an 8.9 mg/dL lower mean LDL-C at 12 months in GMC compared with usual care (see table 2). The discrepancy in the estimated treatment difference between ANCOVA and cLDA is due to missing data and assumptions that are made about missing data. As discussed previously, mixed-effect models yield unbiased estimates of treatment effects under the assumption that the missing data are conditional on observed quantities. Although this assumption cannot be specifically tested, the discrepancy in estimates between ANCOVA and cLDA model yields some evidence that data are missing conditional on observed quantities. If data were missing completely at random (not conditional on observed quantities) then estimated treatment differences between ANCOVA with case deletion and cLDA should be similar. Similarly, differences in estimates from the POST-only as well as LDA models between completers only and participants using all available data provide evidence that participants with missing observations may not be a completely random sample from the study population. Therefore, any analyses based on completers only (including the ANCOVA model) may be biased. Table 2 All available data (n=233 participants)—pre/post analyses Model Outcome (Y t and/or C t ) N GMC Usual care GMC vs usual care (95% CI) p Value Post-only 12 months (Y 12 ) 204 89.7 96.7 −6.9 (−14.2 to 0.4) 0.07 SACS 12 months − baseline (C 12 ) 195 −12.9 −2.8 −10.1 (−22.0 to −0.8) 0.05 ANCOVA 12 months (Y 12 ) 195 83.4 94.6 −11.2 (−19.2 to −3.3) 0.006 LDA* Baseline (Y 0 ) 233 96.7 99.6 12 months (Y 12 ) 83.5 93.6 −7.2 (−17.2 to 2.8) 0.15 cLDA* Baseline (Y 0 ) 233 98.0 98.0 12 months (Y 12 ) 84.0 92.9 −8.9 (−16.7 to −1.0) 0.03 *Baseline LDL-C is missing for 9 participants and 12-month LDL-C is missing for 29 participants. ANCOVA, analysis of covariance; cLDA, constrained longitudinal data analysis; GMC, Group Medical Clinics; LDA, longitudinal data analysis; LDL-C, low-density lipoprotein cholesterol; SACS, simple change score analysis. As shown for ANCOVA and cLDA, LDA and SACS methods estimated differences are discrepant due to missing data and assumptions that are made about missing data. LDA is using all available data and fit using mixed-effects model, whereas in SACS participants with missing data are deleted. Similar to completers analysis, LDA methods yield wider 95% CI than both cLDA and ANCOVA. BODY.RESULTS.ALL PARTICIPANTS ANALYSIS: The second set of analyses was applied to all participants (ie, including those with either missing baseline or 12-month measurements) to compare methods and illustrate the impact of missing data. SPO participants with missing 12-month LDL-C are deleted and SACS participants with either missing baseline or end of study measurements are deleted. Similarly, for ANCOVA with only two time points, participants with missing baseline or 12-month measurements are deleted. For LDA and cLDA, all available data were used; no participants were deleted due to missing data. The estimation procedure used in the mixed model framework for longitudinal analysis yields unbiased estimates of parameters when missing outcomes are assumed to be ignorable, that is, when missing values are related to either observed covariates or response variables but not to unobserved variables.16 17 BODY.DISCUSSION.MISSING DATA IMPLICATIONS: THE CASE FOR CLDA: The illustrative example presented above clearly demonstrates the statistical theory described for the analysis methods and the impact of missing data3 4 16 on the inference and interpretations of results. In general, the implications of how missing data are handled in a trial range from sample size and statistical power loss to potentially biased estimates of treatment effects. Olsen et al18 discuss principled methods for handling missing data in longitudinal sleep disorder trials that are relevant to the types of longitudinal RCTs we are discussing. The default method for handling missing data in many software packages is case deletion; if any variable or outcome is missing for a participant, then the participant is deleted from analysis. In order for estimates of treatment effect differences to be unbiased when case deletion occurs, we have to assume that the subset of participants with complete data is a random sample of the entire study sample. Therefore, for ANCOVA, SACS and SPO analysis above, we are making this assumption. For many longitudinal RCTs it is likely that the probability of dropout is related to some observable characteristic or quantity (eg, treatment arm assignment) and therefore the subset of participants with complete data is not a random sample of the entire study. Under this assumption unbiased estimates of treatment effects can be achieved with mixed-effect models or by performing multiple imputation.18 Methods for handling missing data when we cannot assume that missing data can be characterised by observable quantities are beyond the scope of this paper. The choice of whether to use multiple imputation is up to the analyst and based on the untestable assumption that all predictors of missing data can be included in the mixed-effect model. In many longitudinal RCTs it is a reasonable assumption that treatment group assignment, time of assessment and available outcome assessments along with potential baseline stratification variables would be the predictors of missing outcome data. If the number of potential predictor variables of missing data is expansive and unreasonable to include in the mixed-effects model then it may be necessary to perform a multiple imputation analysis. Multiple imputation is a more complicated process for handling missing data that is also based on the untestable assumption that all predictors of missing data are included in the imputation model.17 In the multiple imputation framework, comparisons of conditional and unconditional models would be equivalent to what was discussed above for no missing baseline or follow-up data. Once imputed data sets are created, there are no missing outcome data; the uncertainty from estimating imputed values is accounted for in the SE estimates. For ease of implementation and robustness of results15 16 mixed-effect models with maximum likelihood estimates are a good first-line choice for handling missing data. Even when applying mixed-effects models, the treatment of baseline values can impact results because of differences in case deletion. In the pre/post design if the baseline is a covariate (ANCOVA), participants with the missing covariate will be deleted. Similarly, with only one follow-up time, if the follow-up time is missing then there is no outcome data for the participant to be included. However, with unconditional models since both baseline and follow-up are part of the outcome vector, if a participant is missing either measurement, their available data will be included and participant is not deleted. When missing data occur and mixed-effect models are used, cLDA models are the optimal choice for providing the most precise estimate of treatment differences under a reasonable assumption that missing data are related to observable characteristics. Finally, it should also be noted that these methods will not diverge for every data set to which they may be applied to—however as certain missing data assumptions cannot specifically be tested, cLDA models are the optimal choice across a range of conditions and data sets. BODY.SUMMARY: It does not appear widely appreciated that cLDA can often be regarded as the method of choice for the analysis of a longitudinal RCT with few measurement occasions. Except for small samples it is equivalent to ANCOVA when there are no missing data, and cLDA is an appropriate generalisation of ANCOVA under reasonable missing data assumptions. Without question, potential baseline imbalance between treatment arms has implications for the analysis of longitudinal RCTs and is a source of confusion. In an RCT, baseline differences can be attributed to random chance assuming there were no problems or issues with the randomisation process and no significant measurement error issues. LDA or CHANGE score analysis is sometimes viewed as a more intuitive analysis; however, for an RCT most often the best method is an ANCOVA as far as bias, precision and power.19 In a longitudinal RCT, when within-participant correlations are not high, change score or LDA (an unconditional analysis) is not the most powerful analyses. Frison and Pocock3 found that ANCOVA treatment difference estimates have small bias under the assumption that measurement error is a small proportion of the between-participant variance; however, bias in a change score analysis may be somewhat larger especially if correlations between pre- and post-measurements are small. cLDA is sometimes erroneously viewed as more problematic when there is baseline imbalance in outcomes between treatment arms. However, cLDA and ANCOVA are equivalent when analysing complete data. cLDA generalises the ANCOVA approach and both are superior to an LDA in many cases. Therefore, the primary analytic issue is not necessarily whether or not to perform conditional analysis. ANCOVA is a conditional analysis and cLDA is an unconditional analysis, yet both are powerful methods that can be applied to examine treatment differences over time in a longitudinal RCT. In practical applications, cLDA is the most straightforward to implement and under reasonable missing data assumptions will yield robust estimates of treatment effect differences and inferential statistics. In most cases, it is the method of choice.

TITLE: The self-adjusting file instrumentation results in less debris extrusion apically when compared to WaveOne and ProTaper NEXT ABSTRACT.AIM:: The present ex vivo study aimed to evaluate the debris extrusion after instrumenting the root canals by three different files systems. ABSTRACT.MATERIALS AND METHODS:: Sixty extracted human mandibular premolars with single canals were selected and randomly divided into three groups (n = 20) for instrumentation with three different files. Group 1: WaveOne (primary) single reciprocating file (WO; Dentsply Maillefer, Ballaigues, Switzerland) (25/08), Group 2: Self-adjusting file (SAF; ReDent-Nova, Ra'anana, Israel) (1.5 mm), and Group 3: ProTaper NEXT X1 and X2 (PTN; Dentsply Tulsa Dental, Tulsa, OK) (25/06). Debris extruding by instrumentation were collected into pre-weighed Eppendorf tubes. These tubes were then stored in an incubator at 70°C for 5 days. The tubes were then weighed to obtain the final weight, with the extruded debris. Statistical analysis for the debris extruded apically was performed using one-way analysis of variance and post hoc Tukey's test. ABSTRACT.RESULTS:: The statistical analysis showed a significant difference between all the three groups tested (P < 0.01). The following post hoc Tukey's test confirmed that Group 2 (SAF) exhibited significantly least (P < 0.01) debris extrusion between the three groups tested. ABSTRACT.CONCLUSIONS:: The SAF resulted in significantly less extrusion of debris when compared to reciprocating WO and rotary PTN. BODY.INTRODUCTION: Biologically, all irritants must be removed from the root canal space (RCS), while at the same time mechanically create an adequate space and shape for flushing and debridement.[1] Complete debridement of the RCS using recently developed rotary/reciprocating files and newer irrigation systems can prove as an aide predicting endodontic success. However, the instrumentation, irrigation solutions, and the formed debris due to instrumentation containing necrotic tissue, micro-organisms, pulpal fragments, and dentin particles may be extruded from the RCS into the periapical region, resulting in post-operative inflammation and treatment failure.[2] The extrusion of debris using different protocols for instrumentation and irrigation has been reported.[345678910] The amount of debris extruded differs according to the preparation techniques because of the various available designs of the file systems and irrigation devices.[3] All the instrumentation techniques used for biomechanical preparation result in apical extrusion of debris, even after the root canal preparation is maintained short of the apical terminus. Most nickel-titanium (NiTi) instrument systems work in a crown-down manner with push-pull rotation filing movements. Various advances in instrument design and the use of different operational principles can successfully influence the amount of debris extrusion. An evolved generation in NiTi instrumentation files is marked by the introduction of single files systems for shaping and cleaning. The WaveOne (WO; Dentsply Maillefer, Ballaigues, Switzerland) single reciprocating file system simulates a reversed balanced force and a linear motion. The Self-Adjusting File (SAF; ReDent-Nova, Ra'anana, Israel) is also a single file system, which is a hollow compressible design. The SAF is used with vibrating movement accompanied by continuous irrigation with any desired solution.[4] The most recent generation of shaping files ProTaper NEXT (PTN; Dentsply Tulsa Dental, Tulsa, OK) presents uniqueness with the center of mass and the center of rotation offset design. These files produce a mechanical wave of motion that travels along the active length of the file. This unique design is advantageous in minimizing the engagement between the file and dentin, may also enhance removal of debris out of a canal and improves flexibility of the files.[4] Less apical extrusion associated with SAF and higher extrusion with WO have been reported in previous studies.[56] To the best of our knowledge, no report has compared apical debris extrusion of debris by WO, SAF, and PTN files; hence, the present study was designed to compare the apically extruded debris when the root canals were instrumented with these files. The null hypothesis was that the three different files (WO, SAF, and PTN) that used different kinematic motions (reciprocating, vibratory, and rotary) would have no effect on the apically extruding debris during instrumentation. BODY.MATERIALS AND METHODS: Sixty extracted human mandibular premolars with single canals confirmed radiographically (buccal and proximal) were collected from random collection. The soft-tissue remnants and calculi on the external root surface were removed using ultrasonics. The buccal cusp edge of each tooth was flattened aiding as a reference point, and coronal access was prepared using an access cavity kit (Endo Z Access Kit, Dentsply Tulsa). The canal patency was achieved and controlled with a size 15 K-file. The working length (WL) of each canal was determined as 1 mm short of the length of a size 15 K-file that was visible at the major diameter of the apical foramen. The teeth were then randomly divided into three experimental groups for different instrumentation techniques. The present study used the experimental model described by Myers and Montgomery[7] for assessing the debris extruded periapically. An analytical balance (Single Pan K-15, K Roy Instruments Pvt. Ltd., Varanasi, India) with an accuracy of 10-4 g was used to measure the initial weights of the tubes. Three consecutive weights were obtained for each tube, and the mean was calculated. Each tooth was inserted up to the cemento-enamel junction, and a 27-G needle was placed alongside the stopper for use as a drainage cannula and to balance the air pressure inside and outside the tubes. Then, each stopper with the tooth and the needle was attached to its Eppendorf tube, and the tubes were fitted into vials. The same operator trained to use all the file systems efficiently carried out the instrumentation. The Eppendorf tube was covered with a silver foil to blind the operator from seeing the apex during instrumentation. BODY.MATERIALS AND METHODS.INSTRUMENTATION: The glide path for all the 60 samples was created till a # 20 K-file for all the groups and a new instrument/file (WO, SAF, and PTN) was used for every sample. BODY.MATERIALS AND METHODS.GROUP 1 WAVEONE (WO): Twenty teeth were instrumented by single reciprocating file WO (primary) instrument, used in a pecking motion. The root canal orifice was flared using Sx file from the universal ProTaper (Dentsply Tulsa Dental, Tulsa, OK). A pre-programmed reciprocating endomotor (X-Smart Plus, DentsplyMaillefer) possessing the program for WO (small, primary, and large) files instrumentation was used. The flutes of the instrument were cleaned after three pecks. On meeting obstruction, the file was removed, the canal was irrigated, recapitulated, and the file was re-introduced into the canal again. The instrumentation was done till the WO (primary) file reached the apex. BODY.MATERIALS AND METHODS.GROUP 2 THE SELF-ADJUSTING FILE (SAF): Twenty teeth were prepared with the SAF system. A prior glide path established accommodated the 1.5 mm SAF. The SAF file was operated by using in-and-out manual motion for 4 minutes in the canal, with continuous irrigation by using bi-distilled (0.4-mm amplitude and 5,000 vibrations per minute), using VATEA peristaltic pump (ReDent-Nova) at a rate of 4 mL/min. A total of 3 mL bi-distilled water was then used for 3 minutes as a final rinse. BODY.MATERIALS AND METHODS.GROUP 3 PROTAPER NEXT (PTN): Twenty teeth were instrumented by PTN instruments, used according to the manufacturer's instructions using a gentle in-and-out motion with a torque-controlled endodontic motor (X-Smart Plus, Dentsply Maillefer) at 300 rpm and a torque of 2.6 Ncm. The root canal orifice was flared using Sx file from the universal ProTaper (Dentsply Tulsa Dental, Tulsa, OK). This was followed by the use of X1 and X2 files. On meeting obstruction the file was removed, the canal was irrigated, recapitulated, and the file was re-introduced into the canal again. The instrumentation was continued till the X1 and X2 both reached the working length. BODY.MATERIALS AND METHODS.IRRIGATION FOR THE ROTARY/RECIPROCATING FILES: After each instrument (rotary) or after three pecks (reciprocating) 2 mL of bi-distilled water was used as irrigant. The irrigation needle (NaviTip 31ga; Ultradent, South Jordan, UT) was placed as deep as possible into the canal but not deeper as the predetermined WL minus 1 mm. Ethylenediaminetetraacetic acid (EDTA) gel was used as a lubricant throughout the instrumentation procedures for all the groups. BODY.MATERIALS AND METHODS.DEBRIS COLLECTION: Following instrumentation, the teeth were removed from the tube and the debris adhering to the root surface was collected by washing off the apical area of the tooth with 1 ml of distilled water into the centrifuge tube. The centrifuge tube was stored in an incubator at 70°C for 5 days, to allow the moisture to evaporate, before weighing the dry debris, using an electronic balance. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Theraw pooled data of the weights were statistically analyzed using D'Agostino-Pearson test for normal distribution and test for homogeneity. This was followed by application of one-way analysis of variance (ANOVA) and post hoc test to determine the significant group (statistical package of social sciences (SPSS) 16, SPSS Inc, Chicago, IL). The alpha-type error was set at 0.05. BODY.RESULTS: The weights obtained were analyzed using D'Agostino-Pearson test for normal distribution, which was not significant (P > 0.05). Also, the test for homogeneity was satisfied (P > 0.05) aiding in successful application of parametric test one-way ANOVA to find out the significant difference. Statistical analysis exhibited a significant difference between all the three groups (P < 0.001) (one-way ANOVA; SPSS software v16). Post hoc tukey's test revealed Group 1 (WO) resulted in higher debris extrusion (P < 0.001) and Group 2 (SAF) resulted in least debris extrusion (P < 0.001) in the three groups tested (SPSS software v16). Whereas, PTN group presented with an intermediate debris extrusion when compared to SAF it was higher (P < 0.01) and with WO it was lesser (P < 0.01). The mean, minimum, maximum, and standard deviation of the three experimental groups are shown in Table 1. The mean extrusion of debris extruded apically is plotted in Figure 1. Table 1 Mean, minimum, maximum, and standard deviation of the debris extruded for all the three groups. (*Statistically significant differences between the three groups) Figure 1Meanwise distribution of the apical extrusion of debris for all the three groups. (A Highest and B Least debris extrusion among the groups) BODY.DISCUSSION: According to the results obtained from the current study, the resultant debris from instrumentation of root canals was extruded periapically regardless of the file design (convex triangular WO, hollow SAF, and rectangular PTN) and different kinematic motions (reciprocating, vibratory, and rotary) used. The reciprocating file WO resulted in maximum debris extrusion (P < 0.01), whereas the vibratory file SAF resulted in least debris extrusion in the three groups tested (P < 0.01). Thus, the null hypothesis was rejected. Previous studies have reported less debris extrusion associated with SAF and more with rotary (Protaper) and reciprocating files;[56] however to the best of authors' knowledge the apical extrusion of debris resulting from the recently evolved ProTaper NEXT rotary file is rarely reported.[8] The present study used method of Myers and Montgomery;[7] to simulate clinical conditions, only the buccal cusp edge of each tooth was flattened aiding as a reference point and the shaping procedures were conducted without de-coronation. Distilled water was used as an irrigation solution to avoid any possible crystallization of sodium hypochlorite. The amount of periapically extruded debris being extremely low, the contact of moist or greasy fingertips alters the weight of the extruded debris. Another important issue is the absence of a physical backpressure provided by periapical tissues, an imminent shortcoming of in vitro studies.[79] Also, the residual pulp tissue, condition of the pulp, and normal or pathological periapical tissues may act as barriers and inhibit the apical extrusion in vivo conditions.[91011] Some suggestions to simulate the periodontal ligament have been made, by the use of floral foam,[11] but they lead to absorption of the irrigant as well as the debris.[12] The present study compared three different instrumentation protocols, a reciprocating file (WO), a vibratory file (SAF), and a rotary file (PTN), a comparison yet to be reported in literature. The results obtained from the current study may be explained by differences in the instrument design and movement kinematics between the WO, SAF, and PTN systems. The WO file was associated with the maximum debris extrusion apically in the present study, which is accordance with other studies.[61314] The WO files are characterized by a modified triangular cross-section, which results in decreased cutting efficacy and smaller chip space resulting in auguring the formed debris after instrumentation, periapically.[613] The WO files also exhibit a larger taper of 0.08 at the apical 3 mm, which can be attributed for excessive debris formation apically, and extrusion periapically.[8] SAF a single-file system, devoid of a central metal core and any cutting edge or flutes, instead has an abrasive surface.[15] The SAF is operated with a transline in-and-out vibratory motion and associated continuous simultaneous irrigation. This continuous flow of irrigant does not build up any pressure in the canal as the metal meshwork allows the free escape of irrigant. In the narrowest apical part of a canal prepared up to a # 20 K-file, the SAF is effective, leaving more than 38% of the canal cross-section free for backflow of fluid and dentinal debris.[15] deMeloRibeiro et al.,[16] stated that in the apical third, the SAF system created cleaner inner canal walls when compared to rotary system. The PTN is a novel rotary file system and very rare reports on apical extrusion of debris resulting from its instrumentation. Capar et al., reported less debris extrusion associated with PTN files when it was compared to the universal ProTaper file systems.[8] The PTN possess a unique design, an offset center of mass and rotation. This design provides more cross-sectional space for enhanced cutting, loading and successfully allowing the debris to travel out of a canal (coronally), compared to a file with a centered mass and axis of rotation. It may also decrease the chances for the file packing the debris laterally, aiding in reducing the chances of blockage of the root canal system.[4] This can be the main advantage of the file and may lead to least debris extrusion; hence, it was used as one of the instrumentation techniques for the present study. The samples in the group instrumented by PTN resulted in less extrusion of the debris when compared to the WO group. With rotary and reciprocal systems a predictable and circularly standardized apical preparation can be expected but incase of the SAF group, the apical enlargement depends upon the apical anatomy of the samples. In the present study, SAF group resulted in less extrusion of debris when compared to the other two groups tested. In a study by Dietrich et al.,[17] comparing reciprocal movement to the SAF, reciprocal instrumentation resulted in more debris in the apical part of the root canal than the SAF or a rotary system. The amount of debris formed in the apical third may also cause its extrusion periapically. BODY.CONCLUSION: In the present ex vivo study, the extrusion of debris associated with three different files possessing different designs that used different kinematic motions was assessed. Within its limitations, it can be concluded that the three file systems used for instrumentation resulted in extrusion of debris even though the working length was maintained 1mm short of the apex. The SAF that used a vibratory motion with continuous irrigation resulted in significantly less debris extrusion when compared to PTN and WO files systems. The results of the current study are favorable to the SAF system, but further studies clinically evaluating the incidence of post-instrumentation pain with these instrumentation systems can provide a better understanding of these file systems.

TITLE: Restrictive intraoperative fluid optimisation algorithm improves outcomes in patients undergoing pancreaticoduodenectomy: A prospective multicentre randomized controlled trial ABSTRACT: We aimed to evaluate perioperative outcomes in patients undergoing pancreaticoduodenectomy with or without a cardiac output goal directed therapy (GDT) algorithm. We conducted a multicentre randomised controlled trial in four high volume hepatobiliary-pancreatic surgery centres. We evaluated whether the additional impact of a intraoperative fluid optimisation algorithm would influence the amount of fluid delivered, reduce fluid related complications, and improve length of hospital stay. Fifty-two consecutive adult patients were recruited. The median (IQR) duration of surgery was 8.6 hours (7.1:9.6) in the GDT group vs. 7.8 hours (6.8:9.0) in the usual care group (p = 0.2). Intraoperative fluid balance was 1005mL (475:1873) in the GDT group vs. 3300mL (2474:3874) in the usual care group (p<0.0001). Total volume of fluid administered intraoperatively was also lower in the GDT group: 2050mL (1313:2700) vs. 4088mL (3400:4525), p<0.0001 and vasoactive medications were used more frequently. There were no significant differences in proportions of patients experiencing overall complications (p = 0.179); however, fewer complications occurred in the GDT group: 44 vs. 92 (Incidence Rate Ratio: 0.41; 95%CI 0.24 to 0.69, p = 0.001). Median (IQR) length of hospital stay was 9.5 days (IQR: 7.0, 14.3) in the GDT vs. 12.5 days in the usual care group (IQR: 9.0, 22.3) for an Incidence Rate Ratio 0.64 (95% CI 0.48 to 0.85, p = 0.002). In conclusion, using a surgery-specific, patient-specific goal directed restrictive fluid therapy algorithm in this cohort of patients, can justify using enough fluid without causing oedema, yet as little fluid as possible without causing hypovolaemia i.e. "precision" fluid therapy. Our findings support the use of a perioperative haemodynamic optimization plan that prioritizes preservation of cardiac output and organ perfusion pressure by judicious use of fluid therapy, rational use of vasoactive drugs and timely application of inotropic drugs. They also suggest the need for further larger studies to confirm its findings. BODY.INTRODUCTION: Pancreaticoduodenectomy (PD) remains the primary treatment strategy for strategy peri-ampullary malignancies including pancreatic adenocarcinoma and a variety of benign conditions. It is a complex operative procedure associated with a significant physiological impact on patients requiring a long recovery period. Despite achieving relatively low peri-operative mortality in specialised centres [1,2] it continues to be associated with high peri-operative complication rates ranging between 17–50% [3,4]. With projected increases in the rates of pancreatic cancer [5], there is an increasing need to reduce complication rates associated with PD. Inappropriate perioperative fluid intervention and likely tissue hypoperfusion and/or oedema are strongly associated with the development of postoperative complications for patients undergoing major abdominal surgery including PD [4,6–8]. Enhanced Recovery After Surgery programmes (ERAS) for patients undergoing PD have advocated a more judicious use of intravenous (IV) fluid administration [9,10]. ERAS programs for PD have been widely adopted impacting positively on length of stay, while not increasing rates of peri-operative morbidity, mortality or readmission [11]. While ERAS has been shown to be beneficial to patient outcomes when compared to usual care in patients receiving PD, no trials have examined the additional impact of an intraoperative fluid optimisation algorithm. We hypothesised that for patients undergoing PD using ERAS protocols, the additional impact of an intraoperative fluid optimisation algorithm would influence the amount of fluid delivered, reduce fluid related complications, and improve length of hospital stay. We conducted a prospective multicentre randomized controlled trial to test our hypothesis. BODY.METHODS: The Austin Health Research and Ethics Committee approved this study (Approval number: HREC/13/Austin/30) and written informed consent was obtained from all participants. The study was retrospectively registered with the Australian New Zealand Clinical Trials Registry (ACTRN: 12616000538448). The study was conducted from September 2013 to December 2015 at four metropolitan hospitals with a dedicated hepatobiliary service. BODY.METHODS.KEY DATES: Trial design and final Protocol lock: 23 Apr 2013 Submitted for Research Ethics Committee approval: 23 Apr 2013 Research Ethics Committee Approval date: 2 Sept 2013 First participant enrolled: 9 Sept 2013 Last participant enrolled: 31 Dec 2015 Project completion date: 2 Feb 2016 Participants were identified from elective surgery waiting lists and included adult patients undergoing elective PD. We excluded the following patients: age less than 18 years, pregnancy, pre-operative coagulopathy, renal impairment (creatinine >250umol/L), chronic liver disease (Child Pugh classification), American Society Anaesthesiology physical status > class IV, and patients undergoing distal, central or total pancreatectomy or pancreatic enucleation. Prior to randomisation, in keeping with standard hospital practices at all centres, all patients regardless of their diagnosis being considered for PD underwent preoperative multidisciplinary team assessment where surgeon, anaesthetist, radiologist, oncologist, nutritionist and allied health professionals ensured patients were optimized for surgery. This included a haematology led multimodal perioperative haemoglobin optimization program based on the National Blood Authority of Australia's patient blood management initiatives to optimize preoperative red cell mass, minimize perioperative blood loss and tolerate postoperative anaemia [12]. Patients randomized to the ERAS group received standard ERAS care (usual care group). Patients randomized to the GDT group received standard ERAS care in addition to a surgery-specific fluid optimisation algorithm. BODY.METHODS.STANDARDISED ERAS PROTOCOL: Participants were fasted for six hours for solids and two hours for clear fluids. Intravenous (IV) fluid loading prior to induction of anaesthesia was prohibited. Unless contraindicated, intrathecal morphine analgesia (300-400ug) was inserted at a lumbar spinal level prior to induction of anaesthesia. Patients with morphine allergy received epidural analgesia via a low thoracic needle inserted at T8/9 or T9/10 level. Anaesthesia was induced with propofol (1-3mg/kg IV), fentanyl (1-3ug/kg IV) and a non-depolarizing neuromuscular blocker. Intraoperatively, participants received dexamethasone (8mg IV), clexane (40mg subcutaneously) and paracetamol (1g IV). Antibiotic prophylaxis included ceftriaxone (1g IV), ampicillin (1g IV) and metronidazole (500mg IV). Intraoperative monitoring included continuous electrocardiography, pulse oximetry, capnography, invasive blood pressure, central venous pressure, pulse pressure variation, urine output and core body temperature. Maintenance of anaesthesia was achieved using sevoflurane or desflurane in 50% oxygen: 50% air ratio titrated to a bispectral index (BIS) of 40 to 60. Remifentanil (0.1–0.3ug/kg/min IV) was started after induction of anaesthesia and ceased during surgical closure of the wound. Thirty minutes prior to wound closure, participants receiving epidural analgesia were loaded with ropivicane 0.2% (10mL via the epidural catheter) followed by an epidural infusion at 10mL/hr. All other participants received fentanyl (20-100ug/mL) at the discretion of the attending anaesthetist. BODY.METHODS.INTRAOPERATIVE FLUID MANAGEMENT AND USE OF VASOACTIVE MEDICATIONS: All participants had blood pressure measured directly from a 20G arterial line catheter inserted prior to induction of anaesthesia. Central venous pressure was recorded directly and continuously from a central venous catheter. The FloTracTM catheter (FloTrac System 4.0, Edwards Lifesciences, Irvine, CA, USA) was connected directly to the arterial line and then connected to the EV1000 haemodynamic monitor. The monitor displayed stroke volume index and cardiac index, which are derived variables calculated by the Frotrac Catheter using the arterial pressure wave form. The monitor also displayed the stroke volume variation (derived variable calculated from the maximum, minimum and mean stroke volume over a respiratory cycle), and the systemic vascular resistance that was calculated from mean arterial pressure, central venous pressure and cardiac output using standard physiologic formulae. The arterial line pressure bag was maintained at 300 mmHg, with the sensor stopcock kept level to the phlebostatic axis, located at the fourth intercostal space at the mid-anterior-posterior diameter of the chest wall (corresponding to the right atrium). Patients in the usual care group had the EV1000 monitor concealed with an opaque screen with all alarms silenced. Fluid management and use of any vasoactive medication was guided only by routine cardiovascular monitoring at the discretion of the attending anaesthetist. For participants in the GDT group, fluid intervention and use of vasoactive medications were guided by an intraoperative fluid optimisation algorithm (Fig 1) and based on physiological parameters from the EV1000 haemodynamic monitor in addition to conventional haemodynamic monitoring. A stroke volume variation of >20% was used as a target for fluid intervention. At the end of surgery, all haemodynamic information from the EV1000 monitor was downloaded from all participants. For all participants, the only available crystalloids solutions were Hartmann's solution or Plasma-Lyte 148 (Baxter Healthcare, Toongabie, New South Wales, Australia). The only colloid solutions were 4% or 20% Albumex (CSL Behring, Broadmeadows, Victoria, Australia). 10.1371/journal.pone.0183313.g001Fig 1Surgery-specific cardiac output algorithm. Postoperatively, all participants were admitted to the intensive care unit for at least 24 hours, and then discharged to a dedicated hepatobiliary surgical ward under a multidisciplinary team of surgeon, perioperative physician, and pain clinician. Postoperative analgesia was optimized twice daily by a dedicated acute pain service. Fentanyl patient controlled analgesia was initiated in the ICU (20ug bolus, five-minute lock out), until oral intake resumed, after which participants receive oral oxycodone (10-20mg every 4 hours). All participants received a low dose ketamine infusion (0.05–0.1mg/kg/hr) for 24 postoperative hours. Epidural analgesia was ceased at 48–72 hours postoperatively. All participants received strict paracetamol (1g QID) for 48 hours and proton pump inhibitor continued for two weeks post discharge. Rescue analgesia consisted of non-steroidal anti-inflammatory drugs (ketorolac 30mg IV 8 hourly) or tramadol (50-100mg IV 6 hourly). Physiotherapy was delivery twice daily and antibiotic prophylaxis continued for 24 hours. Nasogastric tubes were removed the day following surgery, unless there was a greater than 300mL drainage in a 6-hour period. Clear fluids were encouraged immediately postoperatively, and liquid diet was commenced on postoperatively Day 2. The surgical drains were removed when there was no evidence of pancreatic or biliary leakage. Pancreatic enzyme supplements commenced once a soft diet was tolerated. Strict serum glucose control (target of 6 to 10 mmol/L) was maintained by use of an insulin sliding scale. The indwelling urinary catheters was removed by Day 3. Laxatives (docusate sodium 200mg) every twelve hours were commenced from Day 4 post surgery to achieve regular bowel motions. BODY.METHODS.OUTCOMES AND DATA COLLECTED: The primary outcome was length of hospital stay (defined as discharge from theatre to formal discharge from the acute hospital ward). The criteria for discharge were unassisted mobilisation, eating and drinking without nausea or vomiting, defaecation, satisfactory oral analgesia, and no evidence of medical or surgical complications, particularly infection. Other outcomes collected were the amount of fluid administered perioperatively, use of vasoactive medications, and development of complications. Complications were recorded by two independent clinicians as unexpected events occurring in the postoperative period until hospital discharge, and graded according to Clavien-Dindo Classification [13]. Pancreatic leaks and delayed gastric emptying were graded and classified according to the International Study Group of Pancreatic Surgery [14–16]. All other complications were defined and classified according to the European Perioperative Clinical Outcome (EPCO) definitions, based on a statement from the European Society of Anaesthesiology and the European Society of Intensive Care Medicine joint taskforce on peri-operative outcome measures [17]. Other data collected included preoperative patient characteristics, body mass index, American Society of Anesthesiologists (ASA) class, comorbidities and preoperative biochemical and haematological laboratory test results. Operative details collected included anaesthetic technique, fluid balances, intraoperative blood transfusion requirements, duration of surgery, and use of vasoactive medications (type and amount). Fluid balances were calculated by subtracting total output (urine output, blood loss, loss from drains and vomitus) from total input (all intravenous fluid intervention, parental medications or feeding, oral water intake). Third space losses were not included, as they were considered negligible. Postoperative details included detailed fluid intervention for postoperative Days 1 and 2 (type and amount), detailed fluid balances for postoperative Days 1 and 2, blood transfusion requirements, daily body weight, routine biochemistry and haematology, and drain output. BODY.METHODS.SAMPLE SIZE ESTIMATION AND STATISTICAL METHODOLOGY: We powered the study to observe a large treatment effect (Cohen's d = 0.8). In order to observe such effect 52 patients in total (26 per group) would provide a power of 0.8, assuming the type 1 error threshold of 0.05. Based on previous pilot data from our institution, the expected LOS in the control group was 16 days (SD = 4 days). Effects size d = 0.8 would correspond to the difference of 3.2 days, thus the expected LOS in intervention group would be 12.8 days. An independent statistician generated a computerised sequence of 52 allocation codes, 26 for each group. An independent research nurse sealed the allocation codes into sequentially numbered opaque envelopes. Randomisation was done by a central unit immediately prior to induction of anaesthesia. Statistics were done by a statistician blinded to allocation and the code was broken after analysis was completed. Statistical analysis was performed using commercial statistical software STATA/IC v.13. Figures were constructed using Prism 7.0 GraphPad software (La Jolla, CA, USA). Results were expressed as either a median (range) or in the form of frequencies unless otherwise stated. Comparisons between categorical variables were determined by chi-square and Fisher's exact test as appropriate. Non-categorical variables were assessed by the Mann-Whitney U test. Associations between GDT and individual outcomes were investigated using appropriate regression models: negative binomial regression models for LOS (treated as the count of days) and for total per patient count of postoperative complications, linear regression models with robust standard error estimation for fluid outcomes, and logistic regression models for the use of individual vasoactive drugs. Duration of surgery was used as a marker of complexity for surgery and added as a covariate in all analyses performed. Corresponding associations are summarized as appropriate effect sizes with 95% confidence intervals (CIs). The difference between groups with highest grade of complications was estimated using Wilcoxon-Mann-Whitney Generalised odds ratio and corresponding 95%CI, A p value of 0.05 was chosen as the threshold to indicate statistical significance. In order to preserve Type I error, a p value of less than 0.01 was considered statistically significant for IV fluid and vasoactive drugs where multiple outcomes were being tested. The study was reported in accordance with the CONSORT Guidelines for reporting randomised trials [18]. BODY.RESULTS: Sixty-eight participants were screened for eligibility, 16 patients had changes to planned operative interventions and were excluded (distal pancreatectomy with splenectomy: 4, total pancreatectomy: 4, surgery aborted due to unresectable disease: 5, and palliative gastric/biliary bypass: 3). Twenty-six participants were randomized to GDT and twenty-six participants to usual care (Fig 2). There were no violations or breaches of the study or ERAS protocols. Baseline characteristics, co-morbidities, and preoperative biochemical and haematological results are summarised in Table 1. The median (IQR) age was 61 years (53,72) in the GDT group and 68 years (54,75) in the usual care group respectively. Nineteen patients (73%) in the GDT group received intrathecal morphine vs. 18 patients (69%) in the usual care group, the remaining patients received epidural anaesthesia. Median (IQR) duration of surgery was 8.6 (7.1,9.6) in the GDT group vs. 7.8 hours (6.8,9.0) in the Usual care group (p = 0.2). Cancer was the most common indication for surgery in both groups. 10.1371/journal.pone.0183313.t001 Table 1 Characteristics of patients undergoing pancreaticoduodenectomy with and without goal directed therapy. Data presented as median (interquartile range) or number (proportion). GDT group (n = 26) Usual care group (n = 26) Characteristics Age (years) 61 (53:72) 68 (54:75) Male:Female 15:11 14:12 BMI (kg/m 2 ) 27 (23:31) 28 (24:31) ASA Class I-II 7 (27%) 7 (27%) ASA Class ≥ III 19 (73%) 19 (73%) Diabetes 7 (27%) 11 (42%) COPD 4 (15%) 2 (8%) Hypertension 4 (15%) 3 (12%) Ischemic Heart Disease 1 (4%) 2 (8%) PVD 2 (8%) 1 (4%) Malignancy 25 (96%) 25 (96%) Preoperative bloods Hb (g/L) 141 (130:148) 135 (125:145) WCC (x10^ 9 /L) 7.1 (5.7:8.6) 7.2 (5.8:9.9) Platelets (x10^ 9 /L) 234 (185:296) 223 (199:303) Albumin (g/L) 40 (37:45) 37 (31:41) Bilirubin (μmol/L) 10 (7:15) 12 (8:42) Urea (mmol/L) 5.9 (4.4:6.7) 6.2 (4.7:7.6) Creatinine (μmol/L) 69 (59:86) 73 (59:98) eGFR (mL/min/1.73m 2 ) 90 (80:90) 82 (66:91) ASA–American society of anesthesiologists; BMI–body mass index; WCC–white cell count, COPD–Chronic obstructive pulmonary disease, PVD–Peripheral vascular disease. Missing values; Hb 1, WCC 1, Platelets 1, Albumin 1, Bilirubin 3, Urea 1, eGFR 1. 10.1371/journal.pone.0183313.g002Fig 2Consort diagram. BODY.RESULTS.PRIMARY ENDPOINT: LENGTH OF STAY: Median (IQR) length of hospital stay was significantly shorter in the GDT group: 9.5 days (IQR: 7.0, 14.3) compared to the Usual care group: 12.5 days (IQR: 9.0, 22.3) with an Incidence Rate Ratio (IRR) of 0.64 (95% CI 0.48, 0.85), p = 0.002. The median (IQR) for length of stay for each of the four recruiting hospitals was 12 days (9:16), 9 days (7.5:15), 11 days (6:23), and 10.5 days respectively (7.25:16.25). There were no differences between the length of stay between the four hospitals (p = 0.465: Kruskal-Wallis test ANOVA). BODY.RESULTS.SECONDARY ENDPOINTS.INTRAOPERATIVE FLUIDS AND VASOACTIVE MEDICATIONS: Detailed intraoperative fluid intervention, vasoactive medications and hospital length of stay are summarized in Table 2. Median (IQR) intraoperative fluid balance was lower in the GDT group compared to the usual care group (p<0.0001). Crystalloid use was significantly lower in the GDT group (p<0.0001). There were no significant differences in the use of intraoperative colloid or blood products. Three patients (12%) in the GDT group received intraoperative metaraminol vs. 23 patients (88%) in the usual care group (p<0.0001). Eleven patients (42%) received intraoperative ephedrine in the GDT group vs. 7 patients (27%) in the usual care (p = 0.125). Twenty-four patients (92%) in the GDT group received intraoperative noradrenaline vs. 8 patients (31%) in the usual care group (p<0.0001). Similarly, the use of dopamine and/or dobutamine was significantly higher in the GDT group compared to the usual care group: 12 patients (46%) vs. 1 patient (4%) (p = 0.007). Use of intraoperative beta-blockers was similar between groups. 10.1371/journal.pone.0183313.t002 Table 2 Intraoperative fluid intervention, vasoactive drug administration, operative factors, regional anaesthesia and length of stay in patients undergoing pancreaticoduodenectomy with and without goal directed therapy (GDT). Data presented as median (interquartile range) or number (proportion). GDT group (n = 26) Usual care group (n = 26) Effect size (95% CI) p value Fluid intervention Crystalloid (mL) 1750 (1000:2100) 4000 (2313:4206) -1787 (-2453:-1121) a <0.0001 Colloid (mL) 200 (0:500) 200 (0:500) -94 (-369:182) a 0.499 Blood products 0 1 (4%) Not estimable b >0.999 Total fluid (mL) 2050 (1313:2700) 4088 (3400:4525) -1881 (-2490:-1271) a <0.0001 Total fluid (mL/kg/hr) 3.2 (2.2:3.9) 6.8 (5.4:8.6) -3.40 (-4.37:-2.44) a <0.0001 Urine output (mL) 605 (310:1128) 669 (273:948) 25 (-313:364) a 0.880 Blood loss (mL) 200 (138:363) 400 (200:550) -135 (-284:15) a 0.076 Fluid balance (mL) 1005 (475:1873) 3300 (2474:3874) -1808 (-2469:-1148) a <0.0001 Vasoactive drugs Any vasoactive drug given 26 (100%) 25 (96%) Not estimable b >0.999 Metaraminol 3 (12%) 23 (88%) 0.02 (0.00:0.10) b <0.0001 Ephedrine 11 (42%) 7 (27%) 2.69 (0.76:9.49) b 0.125 Phenylephrine 0 2 (8%) 0.20 (0:2.42) b 0.2 Noradrenaline 24 (92%) 8 (31%) 28.07 (4.90:160.71) b <0.0001 Beta-blockers 5 (19%) 4 (15%) 1.37 (0.32:5.97) b 0.672 Dopamine/Dobutamine 12 (46%) 1 (4%) 26.17 (2.46:277.96) b 0.007 Operative factors Duration of surgery (hours) 8.6 (7.1:9.6) 7.8 (6.8:9.0) 0.56 (-0.31:1.43) a 0.2 Length of hospital stay Hospital stay (days) 9.5 (7.0:14.3) 12.5 (9.0:22.3) 0.64 (0.48:0.85) c 0.002 a Effect size reported as average difference with robust 95%CI b Effect size reported as odds ratio c Effect size reported as incidence rate ratio Bonferonni corrected threshold for statistical significance: p = 0.00625 for fluids, and 0.0071 for vasoactive drugs BODY.RESULTS.SECONDARY ENDPOINTS.POSTOPERATIVE FLUIDS: Details of postoperative fluid interventions are summarized in Table 3. Median (IQR) Postoperative Day 1 fluid balance was 1661mL (1253, 2041) in the GDT group vs. 1177mL (704, 1725) in the usual care group (p = 0.178). Postoperative Day 2 fluid balances were also similar between groups: 334mL (-426, 884) in the GDT group vs. 212mL (-767, 636) in the usual care group (p = 0.239). No statistically significant differences in the volumes of crystalloid or colloid fluids administered between the groups on both postoperative Day 1 and Day 2 were observed. 10.1371/journal.pone.0183313.t003 Table 3 Postoperative fluid intervention in patients undergoing pancreaticoduodenectomy with and without goal directed therapy (GDT). Data presented as median (interquartile range) or number (proportion). GDT group (n = 26) Usual care group (n = 26) Effect size (CI) p value Day 1 Crystalloids (mL) 2330 (2030:3119) 2627 (1908:3072) -59 (-552:435) a 0.813 Colloids (mL) 0 (0:350) 0 (0:750) -203 (-430:24) a 0.078 Blood products 0 0 Total IV fluid (mL) 2466 (2045:3323) 2946 (2199:3481) -262 (-819:296) a 0.35 Fluid balance (mL) 1661 (1253:2041) 1177 (704:1725) 331 (-156:818) a 0.178 Day 2 Crystalloids (mL) 1544 (1376:2151) 1900 (1544:2259) -226 (-511:59) a 0.118 Colloids (mL) 0 (0:0) 0 (0:0) 22 (-23:67) a 0.326 Blood products 0 2 (8%) 0.32 (0:4.50) b 0.4 Total IV fluid (mL) 1570 (1376:2151) 1900 (1594:2259) -247 (-544:51) a 0.102 Fluid balance (mL) 334 (-426:884) 212 (-767:636) 351 (-241:944) a 0.239 a Effect size reported as average difference with robust 95%CI b Effect size reported as odds ratio BODY.RESULTS.SECONDARY ENDPOINTS.POSTOPERATIVE COMPLICATIONS: Details of postoperative complications are summarized in Table 4. Postoperative complications were common and occurred at similar frequencies amongst the GDT (73%) and usual care (81%) groups (p = 0.179). Total number of complications per patient were significantly lower in GDT group (44) than with usual care (92): IRR: 0.41 (95%CI 0.24, 0.69) p = 0.001 (Fig 3). The majority of complications were graded as Clavien-Dindo Class 1 and 2. Assessment of most severe complications demonstrated no significant differences between the GDT group and usual care group (p = 0.414). Postoperative pancreatic fistula occurred in 2 patients (8%) in the GDT group vs. 5 patients (19%) in the usual care group (p = 0.191). Three patients (12%) in the GDT group developed delayed gastric emptying vs. 6 patients (23%) in the usual care group (p = 0.213). Patients in the usual care group were significantly more likely to receive blood transfusion: GDT (nil) vs. usual care (35%) (p = 0.0005). Patients in the usual care group were significantly more likely to develop electrolyte derangements: GDT (27%) vs. usual care (62%) (p = 0.012). A difference between the proportions of cardiorespiratory complications in the usual care group (54%) compared to GDT (27%) was noticed, which did not achieve statistical significance (p = 0.066). The rest of the complications were similar between the groups. No significant differences between the groups in return to theatre (p = 0.521), or return to ICU (p>0.999) were observed. 10.1371/journal.pone.0183313.g003Fig 3Modified Rankin scale showing the proportion of participants in the usual care and goal directed therapy (GDT) groups with complications. 10.1371/journal.pone.0183313.t004 Table 4 Summary of complications in patients undergoing pancreaticoduodenectomy with and without goal directed therapy. Data presented as number (proportion). GDT group (n = 26) Usual care group (n = 26) Effect size (CI) p value Patients with complications 19 (73%) 21 (81%) 0.34 (0.07–1.64) e 0.179 Number of complications (total) 44 (1.69) 92 (3.54) 0.41 (0.24:0.69) f 0.001 Clavien-Dindo Classification (most severe) 1.28 (0.70:2.33) g 0.414 I 6 (23%) 5 (19%) II 10 (38%) 13 (50%) III 3 (12%) 0 IV 0 3 (12%) V 0 0 Complication types Wound infection 5 (19%) 6 (23%) 0.76 (0.19:2.95) e 0.687 Superficial surgical site infection 4 (15%) 4 (15%) 0.83 (0.17:3.95) e 0.812 Deep surgical site infection 1 (4%) 2 (8%) 0.31 (0.02:4.33) e 0.387 Sepsis 2 (8%) 3 (12%) 0.61 (0.09:4.14) e 0.613 Postoperative pancreatic fistula a 2 (8%) 5 (19%) 0.30 (0.05:1.82) e 0.191 Grade A 1 (4%) 2 (8%) 0.39 (0.03:4.92) e 0.463 Grade B 1 (4%) 2 (8%) 0.51 (0.04:6.24) e 0.601 Grade C 0 1 (4%) Not estimable e - Delayed gastric emptying 3 (12%) 6 (23%) 0.37 (0.08:1.77) e 0.213 Bile leak b 1 (4%) 0 Not estimable - Cardiorespiratory complications 7 (27%) 14 (54%) 0.33 (0.10:1.07) e 0.066 Acute respiratory distress syndrome 0 1 (4%) 1 (0:39.00) e >0.999 Pneumonia 1 (4%) 1 (4%) 0.62 (0.03:12.56) e 0.758 Pulmonary atelectasis 1 (4%) 3 (12%) 0.20 (0.02:2.45) e 0.210 Pulmonary congestion 3 (12%) 3 (12%) 0.80 (0.14:4.70) e 0.805 Cardiogenic pulmonary oedema 0 3 (12%) 0.07 (0:1.08) e 0.057 Arrhythmia 2 (8%) 3 (12%) 0.56 (0.08:3.87) e 0.558 Acute pancreatitis c 1 (4%) 0 Not estimable e >0.999 Gastrointestinal bleed 0 2 (8%) 1 (0:39.00) e >0.999 Acute kidney injury 4 (15%) 4 (15%) 0.83 (0.17:3.96) e 0.813 Delirium 2 (8%) 7 (27%) 0.17 (0.03:1.03) e 0.054 Ischaemic hepatitis 0 1 (4%) 1 (0:39.00) e >0.99 Nausea and vomiting 2 (8%) 1 (4%) 1.23 (0.08:18.02) e 0.878 Electrolyte disturbances 7 (27%) 16 (62%) 0.21 (0.06:0.71) e 0.012 Hypokalaemia 4 (15%) 7 (27%) 0.50 (0.12:2.02) e 0.330 Hyponatremia 0 3 (12%) 0.29 (0:2.73) e 0.289 Hypomagnesemia 1 (4%) 1 (4%) 1.04 (0.06:18.37) e 0.979 Hypophosphatemia 0 2 (8%) 0.66 (0:6.39) e 0.714 Hyperkalaemia 1 (4%) 2 (8%) 0.45 (0.04:5.58) e 0.537 Hypernatremia 1 (4%) 1 (4%) 1.04 (0.06:18.37) e 0.979 Endocrine abnormalities 3 (12%) 4 (15%) 0.55 (0.10:2.99) e 0.486 Drug reaction 2 (8%) 0 0.71 (0.11:N/A) e >0.999 Refractory analgesia 1 (4%) 4 (15%) 0.21 (0.02:2.09) e 0.182 Other d 1 (4%) 4 (15%) 0.15 (0.01:1.69) e 0.126 Required blood transfusion 0 9 (35%) 0.04 (0:0.29) e 0.0005 Return to theatre 1 (4%) 4 (15%) 0.44 (0.04:5.44) e 0.521 Return to ICU 0 2 (8%) 1 (-0:39.00) e >0.999 All complications defined by European Perioperative Clinical Outcome definitions 17 except: a International Study Group of Pancreatic Fistula (ISGPF) b Presence of bile in the drainage fluid that persisted on postoperative day 4 c Elevations in serum lipase > 3× normal laboratory reference range d Urinary tract infection 2, Foot drop 1, Fluid overload 1, Fall 1 e Effect size reported as odds ratio f Effect size reported as incidence rate ratio g Effect size reported as generalised odds ratio BODY.DISCUSSION.KEY FINDINGS: This is a multicentre randomised controlled trial in patients undergoing PD to compare usual care based on ERAS principles or GDT using a cardiac output guided haemodynamic algorithm in addition to ERAS principles. We found that GDT-guided intra-operative treatment was associated with a less positive fluid balance, decreased administration of intraoperative fluids, greater use of intraoperative vasoactive drug infusions, a decreased number of complications, decreased administration of red cells, and shorter length of hospital stay. BODY.DISCUSSION.RELATIONSHIP WITH PREVIOUS STUDIES: The demographic and clinical features of patients in this study are consistent with other studies of this operation [19,20]. Moreover, complications in the usual care arm are similar to those reported in other high volume tertiary centres [21,22]. The beneficial role of ERAS after pancreatic surgery has been established in a recent systematic review [10], and has led to the implementation of specific ERAS guidelines [23]; however, there have been inconsistent findings when assessing the optimal intraoperative fluid regime for PD [19,24,25]. In this regard, despite ERAS protocols [9,10], we observed that usual care patients received almost twice the volume of fluid intraoperatively, when compared to their GDT group counterparts. Moreover, while it has been established that ERAS protocols are able to reduce length of stay in patients undergoing uncomplicated PD [26], our findings highlight the additional benefit of GDT in improving patient outcomes when combined with a standardized ERAS programme. Our findings also concur with other studies demonstrating an association between higher postoperative fluid balances in high-risk surgery and increased requirements for blood transfusion [27]. In this regard, patients in our study did not differ at baseline in terms of age, gender or preoperative haemoglobin, all of which are considered independent predictors of blood transfusion [28,29]. Thus, our observations further emphasise the additional impact a more liberal fluid therapy can have on haemodilution and blood transfusion requirements [30]. An increased prevalence of electrolyte disturbances, notably hypokalaemia and hyponatremia was also observed in the usual care group where fluid administration was liberal [31], a finding supported by other studies comparing fluid liberal to fluid restrictive regimens [32–34]. In patients receiving PD, we recently reported that restrictive perioperative fluid intervention and negative cumulative fluid balance were associated with fewer complications and shorter length of hospital stay [8]. Similar to these findings, fluid practices in the usual care group in the present study contradict several clinical guidelines, ERAS recommendations and reviews, which reinforce and endorse the benefits associated with a restrictive or "net-even" approach to fluid therapy [35–39]. Finally, our findings support those reported in the "OPTIMISE" Trial, where GDT was associated with a clinical benefit for patients undergoing small bowel surgery with or without pancreas surgery [40]. They are also consistent with a recent meta-analysis that concluded that goal-directed therapy reduces length of hospital stay and complications [40]. BODY.DISCUSSION.STUDY IMPLICATIONS: Our findings imply that, in patients undergoing PD, the benefits of GDT may be not only related to the volume of fluid infused intraoperatively, but to how and when fluid therapy is administered, and how and when vasoactive medications are introduced. The effects of fluid therapy combined with adrenergic and vasoactive therapy has not been formally evaluated in human clinical trials. In an animal model of fluid kinetics it was reported that adrenergic alpha1-receptors with vasoactive drugs accelerated, while beta1-receptors retarded the distribution and elimination of fluid [41]. Other kinetic models in animals have shown than low dose phenylephrine therapy may slow down the distribution of fluid from the plasma to the interstitial fluid space, thereby preventing hypovolemia [42,43]. It's plausible that both the higher use of noradrenaline and dopamine used in the GDT group in our study preserved plasma volume, offsetting the requirements for fluid intervention. Volume expansion with fluids in combination with vasoactive therapy is poorly understood due to the confounding effects of anaesthesia, surgery and patient positioning on vaso- and venodilatation, arterial pressure, cardiac contractility, and activation of the renin-aldosterone hormonal axis. Finally, the decreased administration of red cells seen with GDT may have long-term benefits as perioperative blood transfusion has been associated with reduced survival in patients with pancreatic cancer undergoing surgical resection [44]. BODY.DISCUSSION.STRENGTHS AND LIMITATIONS: There are several strengths to this study. First, it is the largest multicentre randomised trial of a haemodynamic management in PD patients receiving an ERAS protocol. All haemodynamic variables measured from the Flotrac device were assessed invasively and were not amenable to ascertainment bias or derivation. Moreover, the cardiac output haemodynamic algorithm used was pragmatic, and non-prescriptive with regards to type of fluid or specific class of vasoactive drug. This flexibility allowed anaesthetists to prescribe therapies they were most familiar with, whilst taking into consideration the patient's baseline physiological state, and targeting appropriate haemodynamic goals according to age and co-morbidity. In addition, and in contrast to conventional recommendations, we considered a SVV of 20% as a clear cut off value for fluid intervention. We chose such value because SVV may be inconclusive between 9% and 13% in approximately 25% of patients during general anaesthesia [45,46]. To our knowledge, ours is the highest SVV target that used for a major surgery related GDT protocol, and is more fluid restrictive than most previous conventional GDT protocols [47–53]. Finally, the outcomes used to assess the intervention were robust, quantitative, clearly different and not amenable to interpretation or ascertainment bias. Our study also has some limitations. We did not collect information on pancreatic duct size, texture of the pancreas, number of lymph nodes retrieved or surgical complexity. However, the focus of this study was on the association of non-surgical factors with patient outcomes. Moreover, we used multivariable statistical analysis to adjust for duration of surgery as a marker of complexity, a confounder that could impact on fluid intervention and postoperative outcomes. As all hepatobiliary surgeons and anaesthetists across all centres were part of a dedicated hepatobiliary-anaesthesia service, we did not collect outcomes of individual clinicians. Finally, this study was powered to measure differences in hospital length of stay, not postoperative complications. Whilst we showed that there were fewer total complications in the GDT group, there were no significant differences in proportion of patients with complications between groups. Clearly a much larger study is required to comprehensively answer this question. As this study was performed across four hospitals, the external validity of the study appears reasonably robust. However, larger confirmatory studies are desirable. Finally, as this study focused on patients undergoing PD, we cannot extrapolate our surgery-specific cardiac output guided algorithm to other types of complex surgeries, other scheduled (or emergency) types of operations, or to older, sicker or morbidly obese patients. BODY.DISCUSSION.CONCLUSIONS: The findings of this study indicate that GDT using a cardiac output guided algorithm can reduce positive fluid balance, the rate of complications, the requirement for blood transfusions and length of hospital stay after PD. Using a surgery-specific, patient-specific goal directed fluid therapy algorithm in this cohort of patients, can justify using enough fluid without causing oedema, yet as little fluid as possible without causing hypovolaemia i.e. "precision" fluid therapy. These findings support the use of a perioperative haemodynamic optimization plan that prioritizes preservation of cardiac output and organ perfusion pressure by judicious use of fluid therapy, rational use of vasoactive drugs and timely application of inotropic drugs. They also suggest the need for further larger studies to confirm its findings. BODY.SUPPORTING INFORMATION: S1 FileOriginal dataset.(XLSX)Click here for additional data file. S2 FileConsort checklist.(PDF)Click here for additional data file. S3 FileTrial protocol.(PDF)Click here for additional data file.

TITLE: The Effect of Four Different Gonadotropin Protocols on Oocyte and Embryo Quality and Pregnancy Outcomes in IVF/ICSI Cycles; A Randomized Controlled Trial ABSTRACT.BACKGROUND:: Despite the large number of papers published on the efficiency of different exogenous gonadotropins, no confirmed protocol exists. Therefore, the aim of the present study was to compare the efficacy of 4 exogenous gonadotropins in IVF/ICSI cycles. ABSTRACT.METHODS:: This study, performed from January 2014 to May 2014, recruited 160 women referred to Ghadir Mother and Child Hospital and Dena Hospital, Shiraz, Iran. The patients underwent standard downregulation and were randomly divided into 4 groups of A, B, C, and D and were administered hMG, hFSH, rFSH, and combined sequential hFSH/rFSH, respectively. Then, the duration of stimulation, number of oocytes and embryos as well as their quality, implantation rate, biochemical and clinical pregnancy rate, and live birth rate in each group were evaluated. ABSTRACT.RESULTS:: Group D patients required significantly fewer ampoules of FSH than did the women in groups A, B, and C (P=0.004). The duration of stimulation was significantly longer in group C than in groups A and D (P=0.030). The serum estradiol level was significantly higher in group D than in groups B and C (P=0.005). A significantly higher number of large-sized follicles was observed in group D than in group B (P=0.036). ABSTRACT.CONCLUSION:: Our data revealed no statistically significant differences in the mean oocyte number, embryo quality, clinical pregnancy rate, or live birth rate between the hMG, hFSH, rFSH, and sequential hFSH/rFSH protocols. However, several differences in the duration of stimulation, serum estradiol levels, and number of large-sized follicles were detected between the groups. Trial Registration Number: IRCT201408116541N7 BODY: What's Known Many studies have compared different exogenous gonadotropins for controlled ovarian stimulation to show which kind is more suitable and leads to greater IVF success.Which gonadotropin has more efficacy for controlled ovarian stimulation is not confirmed and it remains a controversial issue. What's New There are no significant differences in the mean oocyte number, embryo quality, clinical pregnancy rate, or live birth rate between hMG, hFSH, rFSH and sequential hFSH/rFSH.Several differences in the duration of stimulation, serum estradiol levels, and the number of large size were detected among the groups. BODY.INTRODUCTION: Today, assisted reproductive technology (ART) has become a well-established and highly efficient therapy for infertility. In ART, it is well understood that the most important factors for maximizing the success rate of in vitro fertilization (IVF) are retrieving greater numbers of high-quality oocytes using controlled ovarian hyperstimulation (COH) and establishing a receptive endometrium.1-4 Therefore, COH plays a principal role in achieving a high ART success rate. Nowadays, the use of long protocols using the gonadotropin-releasing hormone (GnRH) analog plus gonadotropins for COH has gained widespread popularity.5-7 Various gonadotropin preparations are commercially available and used for COH such as human menopausal gonadotropin (hMG), human-derived follicle-stimulating hormone (hFSH), and recombinant FSH (rFSH). hMG contains FSH and luteinizing hormone activity, while rFSH comprises only FSH, and in comparison to hFSH, rFSH includes a high proportion of fewer acidic isoforms with high purity and high in vitro bioactivity.8 There are many controversies surrounding which kind of exogenous gonadotropin is more suitable and leads to greater IVF success. Some studies have demonstrated that a better outcome in terms of oocyte and embryo quality, subsequent pregnancy rates, and live birth rate is obtained when hMG is used for ovarian stimulation, as compared with rFSH.9-11 However, other studies have shown that rFSH is as effective as urinary FSH or hMG in terms of the number of oocytes and embryos obtained and the total gonadotropin dose needed.7,9,12 Studies that compared hFSH with rFSH noted increased ovarian recruitment of follicles in the rFSH group.13,14 Daya12 showed that rFSH was better than hFSH in terms of the pregnancy rate, while van Wely et al. illustrated a borderline significant difference of 5% higher clinical pregnancy rate in women stimulated with hFSH compared with rFSH.7 Selman et al.8 demonstrated that the combination of hFSH/rFSH for ovarian stimulation had a positive effect on follicular development, oocyte quality, embryo development, and clinical outcome in patients with repeated IVF failures. Therefore, despite the large number of papers published on COH protocols comparing the efficiency of different exogenous gonadotropins, no confirmed protocol exists, and it is not quite clear which is superior to the others. Thus, the objective of the current study was to compare the efficacy of 4 different ovarian stimulation protocols, comprising hFSH, rFSH, hMG, and sequential use of hFSH and rFSH, on oocyte and embryo quality and IVF treatment outcome in patients undergoing IVF or intracytoplasmic sperm injection (ICSI). BODY.PATIENTS AND METHODS: This double-blinded, randomized, clinical trial study was registered in the Iranian Registry of Clinical Trials (code: IRCT201408116541N7) and approved by the Institutional Review Board and the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (code: CT-P-92-7249). The study was performed from January 2014 to May 2014. Written informed consent was obtained from each participant. A flow chart of the study design is depicted in figure 1. The CONSORT flow diagram is depicted in figure 2. The sample size used in this study was determined based on the criteria established by Kutner et al.15 using the following formula: type I error (α) =0.05, power of analysis (1-β=0.95), Effect Size and number of groups=4. Figure 1Study flow chart of the evaluation of the efficacy of different ovarian stimulation protocols, consisting of hFSH, rFSH, hMG, and sequential use of hFSH and rFSH, on oocyte and embryo quality and IVF treatment outcome in patients undergoing IVF or ICSI. Figure 2CONSORT flow diagram shows the sampling procedure. The study group consisted of 160 women referred to 2 hospital-affiliated IVF centers for infertility treatment in Shiraz, Iran (Ghadir Mother and Child Hospital and Dena Hospital). Patients with unexplained or male factor infertility were included in the study if they met the following criteria: 1) age between 20 and 38 years; 2) body mass index (body weight divided by the square of body height) between 19 and 29 kg/m2; 3) history of regular menstrual cycles, ranging from 25–35 days; 4) no relevant systemic disease, severe endometriosis, or uterine or ovarian abnormalities; 5) no more than 3 previous IVF cycles; and 6) no previous IVF cycle with a poor response or the ovarian hyperstimulation syndrome. Additionally, patients with FSH >10 IU/mL, with <5 follicles in antral follicle count, and anti-Müllerian hormone <1 ng/mL were excluded from the study. After the patients were assessed for eligibility according to the mentioned criteria, a standard downregulation protocol was performed for all of them via a subcutaneous injection of GnRH agonists (0.5 mg of buserelin, Suprefact, Serono), on day 21 of their menstrual cycle (1 wk before the expected menses). Subsequently, on day 2 of the next menstrual cycle, after confirming desensitization (estradiol serum concentration <50 pg/mL, the absence of follicles ≥10 mm in diameter, and endometrial thickness <5 mm), the patients were randomized by a person independent of the research team using a computer-generated random-number list. Thereafter, ovarian stimulation was commenced for the study population as follows: group A: 40 patients who received hMG (Menogon®, Ferring Pharmaceuticals A/S, Copenhagen, Denmark); group B: 40 patients who received hFSH (Fostimon®, IBSA Institut Biochimique SA, Geneva, Switzerland); group C: 40 patients who received rFSH (Gonal-F®, Merck, Serono, Rome, Italy); and group D: 40 patients who received hFSH (FostimonX®, IBSA Institut Biochimique SA, Geneva, Switzerland) for the first 6 days, followed by rFSH (Gonal-F®, Merck, Serono, Rome, Italy). In all the 4 groups, the gonadotropin administration was continued up to the day of human chorionic gonadotropin injection (hCG) (Gonasi® HP, IBSA Italia, Rome, Italy). It should be mentioned that both the subjects of the study and the investigators performing the study were blind to the type of the gonadotropin each patient received for ovarian stimulation. The monitoring of ovarian responses to gonadotropin stimulation during the treatment cycle began from day 6, using transvaginal sonography and the measurement of the plasma E2 level every 3 days. Each change in the gonadotropin dose was performed according to the follicle size and the plasma E2 level. The treatment was continued until the observation of at least 2 follicles having reached 17–18 mm in diameter (leading follicles) and some other follicles 14–16 mm in diameter. When the leading follicle was 18–20 mm, and there were at least 3 follicles of 16–17 mm, gonadotropin administration was stopped, and an intramuscular injection of 10,000 IU of hCG was administered for final oocyte maturation. Finally, 34–36 hours after hCG injection, transvaginal ultrasound-guided oocyte retrieval was performed. Oocyte maturity was tested according to the presence or absence of a germinal vesicle and first polar body and was graded as GV, MI, or MII according to the criteria established by Veeck et al.16,17 Subsequently, IVF or ICSI, based on indications, was performed. ICSI was used in the cases with male factor infertility. After fertilization, embryo scoring was carried out on the day of embryo transfer (3 d after oocyte retrieval).16,17 The embryos were graded as I, II, or III, where I indicates the best-quality embryo and III indicates the lowest-quality embryo. The luteal phase was supported by an intramuscular injection of 2 vials of progesterone (50 mg, Iran Hormone, Tehran, Iran) daily, from the day of oocyte retrieval for 3 days and continued with intravaginal progesterone (400 mg, Cyclogest®, Actavis UK Ltd., Barnstaple, UK) twice per day. Two weeks after embryo transfer, the chemical pregnancy test was carried out by evaluation of serum β-hCG. In addition, clinical pregnancy was evaluated by observing the pregnancy sac 6 weeks after embryo transfer, and the implantation rate was determined by the number of gestational sacs divided by the number of embryos transferred. The primary end points were oocyte and embryo quality and pregnancy outcomes. The secondary endpoints were the duration of stimulation, plasma E2 level on the day of hCG administration, number of used ampoules or vials of gonadotropin, number of large-sized follicles, total number of collected oocytes and transferred embryos, and implantation and miscarriage rates. Statistical analysis was performed using SPSS, version 16 (IBM, Armonk, USA). For the analysis of the data, the one-w ay ANOVA test was used followed by the Tukey test to compare the means. A P value <0.05 was considered statistically significant. BODY.RESULTS: According to table 1, age, body mass index, duration of infertility, and endometrial thickness at baseline were similar in all the groups. Table 1 Demographic characteristics of the women receiving hMG, hFSH, rFSH, and sequential hFSH/rFSH Characteristics Group A hMG Group B hFSH Group C rFSH Group D Sequential hFSH/rFSH P value Age 31.90±5.0 31.32±5.01 30.25±3.45 32.35±4.53 0.382 Body mass index (kg/m 2 ) 23.98±2.70 24.4±2.91 24.69±2.26 24.69±2.26 0.923 Duration of infertility (y) 6.60±3.68 6.50±3.62 7.60±6.43 5.40±3.71 0.757 Endometrial thickness at first day (mm±SD) 2.90±1.44 3.30±1.33 3.10±0.99 2.70±1.41 0.763 Endometrial thickness on hCG day (mm±SD) 7.60±1.42 7.70±1.41 7.90±1.96 8.30±1.70 0.792 Number of ampoules or vials of gonadotropin 18.8±6.52 18.7±5.91 19.8±6.09 15.8±3.85 ▲ , ■ , • 0.004 Duration of stimulation (day±SD) 8.90±2.90 9.1±2.1 10±2.4 ٭ , • 8.80±1.71 0.030 17ß-estradiol level on the day of triggering 1940.0±479.1 1925.9±675.3 2223.6±1026.1 2531.07±1087 ▲ , ■ 0.005 The significance was considered at P≤0.05. ▲ : Statistically significant differences between groups A and D; ■ : Statistically significant differences between groups B and D; • : Statistically significant differences between groups C and D; ٭ : Statistically significant differences between groups A and C The number of ampoules or vials of gonadotropin administered was lower in group D than in the other groups; this difference was statistically significant compared to groups A, B, and C. The duration of stimulation was longer in group C than in the other 3 groups, and the difference in group C in comparison to groups A and D was statistically significant. Endometrial thickness and the estradiol level on the day of hCG administration were higher in group D than in the other groups. Apropos the estradiol level, this difference was significant in group D in comparison to groups A and B. As is shown in table 2, the number of large-sized follicles was high in group D and then in group C, compared to groups A and B. This difference between groups B and D was statistically significant. Table 2 Ovarian response and oocyte maturity in the patients receiving hMG, hFSH, rFSH, or sequential hFSH/rFSH Characteristics Group A hMG Group B hFSH Group C rFSH Group D Sequential hFSH/rFSH P value Number of large follicles 9.9±4.7 8.9±4.8 10.8±7.0 12.45±5.4 ■ 0.036 Number of all retrieved oocytes 388 328 448 433 0.068 Number of retrieved oocytes/patient 9.5±4.83 8.2±4.7 11.2±6. 7 10.8±5.5 0.067 Number of degenerated oocytes (%) 8 (2.1) 8 (2.44) 12 (2.7) 20 (4.6) 0.178 Number of GV oocytes (%) 24 (6.3) 21 (6.40) 22 (5.0) 18 (4.2) 0.906 Number of MI oocytes (%) 32 (8.4) 30 (9.15) 35 (7.9) 27 (6.2) 0.923 Number of MII oocytes (%) 316 (83.2) 269 (82.01) 375 (84.5) 368 (85.0) 0.069 Significance was considered at P≤0.05. ■ : Statistically significant differences between groups B and D; NS: Nonsignificant The number of retrieved oocytes was higher in groups C and D than in groups A and B, but the difference did not constitute statistical significance. The number of degenerated oocytes was higher in group D than in groups A, B, and C; the difference, however, was not statistically significant. No statistically significant differences were observed in the number of GV and MI oocytes between the studied groups, but the number of mature oocytes (MII) was higher in group C and then in group D than in groups A and B; nevertheless, the difference was not statistically significant. The lowest number of MII oocytes was observed in group B. According to table 3, the number of transferred embryos was not different between the groups. The highest proportion of grade-I embryos and the lowest proportion of grade-II and grade-III embryos were in group D, followed by groups C, B, and A. Table 3 Embryo score of the patients after treatment with hMG, hFSH, rFSH, or sequential hFSH/rFSH Characteristics Group A hMG Group B hFSH Group C rFSH Group D Sequential hFSH/rFSH P value Number of embryos transferred/patient (mean±SD) 2.9±0.7 2.6±0.9 2.8±0.7 2.8±0.7 0.530 Grade-I embryos (%) 33 (28.9) 41 (39.42) 55 (50.0) 65 (59.09) 0.054 Grade-II embryos (%) 52 (45.6) 37 (35.9) 36 (32.7) 35 (31.83) 0.688 Grade-III embryos (%) 29 (25.4) 26 (25) 19 (17.3) 10 (9.09) 0.106 Significance was considered at P≤0.05 As is shown in table 4, the chemical and clinical pregnancy rate, implantation rate, and live birth rate were high in group D, followed by group C, in comparison to the other groups; nonetheless, the difference was not statistically significant. In addition, the abortion rate was highest in group D. Table 4 Clinical outcome of the patients after treatment with hMG, hFSH, rFSH, or sequential hFSH/rFSH Characteristics Group A hMG Group B hFSH Group C rFSH Group D SequentialhFSH/rFSH P value Biochemical pregnancy rate (%) 22 (55) 22 (55) 22 (55) 28 (70) 0.432 Clinical pregnancy rate (%) (n) 18 (45) 15 (37.5) 20 (50) 23 (57.5) 0.296 Implantation rate per embryo transferred (%) 15.6±17.9 15.55±25.69 22.7±27.6 25.2±24.6 0.176 Live birth rate per clinical pregnancy (n) (%) 11 (61.11) 9 (60) 16 (80) 19 (82) 0.614 Abortion rate per clinical pregnancy (%) 2 (11.11) 2 (13.13) 3 (15) 8 (34.87) 0.862 Significance was considered at P≤0.05 BODY.DISCUSSION: Among the different protocols for COH, the use of the GnRH analog plus gonadotropins (long protocol or standard protocol) is popular, owing to its more favorable results. The literature abounds with studies comparing exogenous gonadotropins for COH, but the issue still remains controversial. Exogenous ovarian stimulation increases oocyte yield but may compromise the developmental competence of the oocytes in stimulated cycles.18 In this study, we evaluated the efficacy of 4 different ovarian stimulation protocols using different gonadotropins in women undergoing IVF or ICSI programs. According to our results, the number of ampoules used was significantly lower in the sequential protocol than that in the other 3 protocols and the duration of stimulation in the rFSH-alone protocol was significantly longer than that in the hFSH and hMG protocols. Gerli et al.19 demonstrated that stimulation with the sequential protocol, compared with the rFSH protocol, necessitated a low gonadotropin dose and short duration of stimulation for the stimulation of ovaries. Other studies have shown no significant differences between the use of rFSH and hFSH or the sequential protocol in the duration of stimulation and the dose of gonadotropin used.6,13,14,20,21 These contradictory results may have originated from diversity not only among the products of pharmaceutical companies but also among patients' race and physiological status. The effect of serum estradiol level on the day of hCG on ART outcome is controversial. It is said that although the estradiol level increases endometrial proliferation, uterine perfusion, oocyte development and maturation, number of embryos transferred, implantation, delivery, and pregnancy rate, the supraphysiological level of estradiol may not only cause endometrial damage and disrupt the implantation but also exert negative effects on IVF-ICSI outcome. Nevertheless, this hypothesis has yet to be confirmed.22,23 COH leads to the development of groups of follicles of differing sizes. Gonadotropin stimulation changes in the steroid profile result in modifying the microenvironment of the developing follicle and its oocyte. Precise evaluation of follicle size is highly important, and it has been shown that larger follicles at the time of retrieval have consistently mature oocytes with a higher rate of fertilization.24 We observed that the level of estradiol was significantly higher in the sequential protocol than in the hFSH and hMG protocols, resulting in more large-sized follicles, retrieved oocytes, and MII oocytes in this protocol. Nonetheless, it did not lead to a clear increase in the endometrial thickness of these groups compared to the other 2 groups. The sequential use of hFSH/rFSH is the same as the natural physiologic cycle, where more acidic isoforms of FSH are produced in the follicular phase, when the estradiol level is low, and fewer acidic isoforms are produced in the late follicular and periovulatory phase, when estradiol is high.21 The significantly high number of large-sized follicles in the sequential protocol in comparison to the rFSH protocol may be related to the combined used of acidic (hFSH) and less acidic isoforms (rFSH) of FSH, which mimics the physiology of the normal menstrual cycle and is an important mechanism for the regulation of the final stages of follicle and oocyte maturation.25 Furthermore, in the rFSH protocol, the level of estradiol was nonsignificantly higher than that with the hFSH and hMG protocols. Gholami et al.13 showed a significantly high level of estradiol in the rFSH protocol compared with the hFSH protocol. Other studies have shown no differences in the estradiol level and endometrial thickness between the sequential, rFSH, hFSH, and hMG protocols.8,19,21,26,27 Although not significant, the number of retrieved oocytes and MII oocytes was high in the sequential and rFSH protocols compared with the hFSH and hMG protocols. However, the number of degenerated oocytes was nonsignificantly high in the sequential protocol compared to the other 3 groups. In other studies, no significant differences in the number of retrieved oocytes have been seen between the different protocols.8,19,27,28 We observed no significant differences in the number of retrieved oocytes and mature oocytes between the hFSH and rFSH patients, chiming in with other studies.6,21 Gerli et al.19 observed that the number of MII oocytes was significantly higher in the patients who received the sequential protocol than in the patients who received rFSH alone. Therefore, using a sequential protocol, our patients reached higher estradiol levels and sufficient numbers of suitable follicles with fewer ampoules and lower durations of stimulation. Furthermore, in the rFSH protocol, despite the need for more ampoules and a longer duration of stimulation than in the other groups, more retrieved oocytes and higher numbers of MI and MII oocytes were produced than with the hMG and hFSH protocols, although these differences were not significant. We observed that the use of the sequential and rFSH protocols, by comparison with the hMG or hFSH protocol, nonsignificantly led to more good-quality (grade I) embryos. In addition, the number of low-quality embryos was lowest in the sequential protocol, followed by the rFSH, hFSH, and hMG protocols. Selman et al.8 and Gerli et al.19 showed that the number of good-quality embryos was significantly high in the sequential protocol in comparison to the hFSH and rFSH protocols. In other studies, the number of good-quality embryos is similar in the rFSH, hMG, and hFSH protocols.13,24,27 Although the total number of transferred embryos was not different between the groups, the implantation and pregnancy and live birth rates were higher in the sequential protocol. This may be related to the higher number of good-quality embryos produced in the patients who received the sequential protocol. Selman et al.8,21 and Gerli et al.19 showed that the implantation rate and pregnancy and delivery rates were significantly high using the sequential protocol in comparison to the hFSH and rFSH protocols. In the rFSH protocol, these parameters were slightly higher than in the hFSH and hMG protocols. Gholami et al.13 and Selman et al.26 reported that the implantation rate and pregnancy rate were similar between the rFSH and hFSH protocols. In contrast, Daya12 demonstrated that rFSH was better than hFSH in terms of the pregnancy rate, while van Wely et al.7 showed a significantly high clinical pregnancy rate with the hFSH protocol compared with rFSH. Ludwig et al.27 and Turhan et al.5 showed that the pregnancy and live birth rates were similar between the rFSH and hMG protocols. These differences may be due to the heterogeneity of patients in the analysis, their age, type of GnRH analog suppressions, gonadotropin doses, etc. Nonetheless, the results of the study by Selman et al.8 and our results showed that the sequential protocol was better than the other protocols in terms of clinical pregnancy and the live birth rate. Our results regarding the superiority of rFSH over hFSH differed from their results. In our study, the pregnancy rate was higher in the sequential and rFSH protocols, although the abortion rate was higher in these protocols as well; however, the overall outcome (the live birth rate) stood higher in these 2 groups (not significantly). Accordingly, we concluded that the rFSH and sequential hFSH/rFSH protocols yielded more mature oocytes, but the sequential protocol was more valuable in terms of embryo quality, as was seen in implantation, pregnancies, and live birth rate. Still, there was no clear difference between the hMG and hFSH protocols. The sequential use of hFSH/rFSH is the same as the natural physiologic cycle, where more acidic isoforms of FSH are produced in the follicular phase, when the estradiol level is low, and fewer acidic isoforms are produced in the late follicular and periovulatory phase, when estradiol is high. This may be an important mechanism for the regulation of the final stages of follicle and oocyte maturation.21,25 Therefore, the difference and distribution of exogenously applied gonadotropins should be determined and used for ovarian stimulation. Obviously, these differences in the effect of FSH isoforms on follicular development patterns strongly suggest that oocyte development is also likely to be influenced, that normal follicle development and ultimately normal oocyte function depend on an appropriate balance of sequential differentiation, and that this balance is strongly influenced by FSH isoform distribution.28 BODY.CONCLUSION: In conclusion, the sequential protocol was able to improve the success rate of ART and could, as such, be deemed a valuable protocol in IVF programs. Further large randomized trials are needed to yield a precise estimation of any difference between the above-mentioned protocols.

TITLE: A controlled trial of the Litebook light-emitting diode (LED) light therapy device for treatment of Seasonal Affective Disorder (SAD) ABSTRACT.BACKGROUND: Recent research has emphasized that the human circadian rhythm system is differentially sensitive to short wavelength light. Light treatment devices using efficient light-emitting diodes (LEDs) whose output is relatively concentrated in short wavelengths may enable a more convenient effective therapy for Seasonal Affective Disorder (SAD). ABSTRACT.METHODS: The efficacy of a LED light therapy device in the treatment of SAD was tested in a randomized, double-blind, placebo-controlled, multi-center trial. Participants aged 18 to 65 with SAD (DSM-IV major depression with seasonal pattern) were seen at Baseline and Randomization visits separated by 1 week, and after 1, 2, 3 and 4 weeks of treatment. Hamilton Depression Rating Scale scores (SIGH-SAD) were obtained at each visit. Participants with SIGH-SAD of 20 or greater at Baseline and Randomization visits were randomized to active or control treatment: exposure to the Litebook LED treatment device (The Litebook Company Ltd., Alberta, Canada) which delivers 1,350 lux white light (with spectral emission peaks at 464 nm and 564 nm) at a distance of 20 inches or to an inactivated negative ion generator at a distance of 20 inches, for 30 minutes a day upon awakening and prior to 8 A.M. ABSTRACT.RESULTS: Of the 26 participants randomized, 23 completed the trial. Mean group SIGH-SAD scores did not differ significantly at randomization. At trial end, the proportions of participants in remission (SIGH-SAD less than 9) were significantly greater (Fisher's exact test), and SIGH-SAD scores, as percent individual score at randomization, were significantly lower (t-test), with active treatment than with control, both in an intent-to-treat analysis and an observed cases analysis. A longitudinal repeated measures ANOVA analysis of SIGH-SAD scores also indicated a significant interaction of time and treatment, showing superiority of the Litebook over the placebo condition. ABSTRACT.CONCLUSION: The results of this pilot study support the hypothesis that light therapy with the Litebook is an effective treatment for SAD. ABSTRACT.TRIAL REGISTRATION: Clinicaltrials.gov: NCT00139997 BODY.BACKGROUND: Seasonal Affective Disorder (SAD, winter depression) is a well-recognized form of recurrent depressive disorder, characterized by typical and atypical (increased appetite, weight, sleep and fatigue) depressive symptomatology and a distinct seasonal nature [1,2]. SAD is thought to be related to natural seasonal variations in light levels. Bright light therapy – exposure of the patient each morning to bouts of artificially produced high intensity light – has been shown to produce amelioration of depressive symptoms. Over 70 trials addressing the efficacy of light therapy have now been conducted, including 2 large controlled trials [3,4] which demonstrated clear efficacy. Light therapy was found to be similar in efficacy to treatment with fluoxetine in a large controlled trial [5]. Several meta-analyses have found that light treatment is effective for SAD [6-8]. While light therapy appears to be an efficacious form of treatment, the traditional mode of delivery via a relatively large and bulky light box can be cumbersome for patients. Finding easier and briefer forms of treatment has been a major goal of the field. Light therapy using light-emitting diodes (LEDs) may offer advantages over conventional light boxes based on fluorescent or incandescent sources. First, recent data indicate that the human circadian rhythm system is most sensitive to light with wavelength in the range 450 – 480 nm [9-11]. LEDs can be selected to emit light with energy concentrated in this range, while fluorescent and incandescent sources emit across the visible spectrum. Although the role of the circadian rhythm system in the pathophysiology of SAD is unclear [12], one study has shown that LED-generated blue light (398 lux, peak energy output around 468 nm) was more effective than LED-generated red light (23 lux, peak output around 654 nm) [13]. Secondly, LEDs are more efficient and lighter than traditionally used fluorescent tubes, and may permit significantly smaller and lighter treatment devices. The aim of the present study was to conduct a randomized placebo-controlled trial to test the efficacy of a white LED device whose light emission was relatively concentrated in shorter wavelengths (the "Litebook", The Litebook Company Ltd., Alberta, Canada). Since negative ion generators have been reported to be effective in treatment of SAD [14], a "credible placebo" design similar to that of Eastman and colleagues [4], in which an inactivated negative ion generator was used as a "no light" control condition, was employed. The results suggest that treatment with the Litebook LED device is an effective treatment for SAD. BODY.METHODS.STUDY PROTOCOL: This is a multi-center, randomized, double-blind, parallel-group clinical trial of light therapy for participants with SAD (winter type). Participants were seen at a Baseline Visit, a Randomization Visit, and after 1, 2, 3 and 4 weeks of treatment. Participants who appeared to meet the inclusion criteria and not meet exclusion criteria at the Baseline Visit were invited to return in 1 week for a Randomization Visit. At this visit participants who continued to meet study criteria were issued either an active light treatment device or a placebo inactivated ion generator. Participants were seen at weekly intervals during 4 weeks of treatment. Participants were enrolled between October 1 and March 1 to reduce confounding effects of natural remission as expected in the spring. Severity of depressive symptoms was rated at each visit using a 24-item SIGH-SAD, a scripted version of the Hamilton Depression Rating Scale [15] modified to reflect better the atypical symptomatology of SAD [16]. This version of the SIGH-SAD consists of the HDRS 17-item scale plus the first 7 atypical items (i.e., excluding Reverse Diurnal Variation). At the Randomization Visit and the subsequent 4 visits, SIGH-SAD ratings were carried out by a clinician blinded to the assigned treatment device. The blinded clinician also completed a systematic inquiry about any adverse events. A separate unblinded clinician dispensed and demonstrated the treatment device at the Randomization Visit, and was available at subsequent visits if required. The study was conducted at 5 sites, in New Haven (USA), Vancouver, Montreal and Ottawa (Canada), and Groningen (The Netherlands). The research protocol was approved by applicable institutional review boards and met standards established by the Helsinki Declaration, and participants signed appropriate consent forms. The trial was registered at the U.S. National Institutes of Health clinical trials database [17]. BODY.METHODS.PARTICIPANTS: Participants were recruited through media advertisements or professional referrals, screened by experienced interviewers by telephone, and if appropriate invited for a Baseline Visit. At this visit, participants received a full psychiatric evaluation, physical exam, urine toxicology for commonly abused substances, and urinary beta-HCG for female participants. Participants were required to be between ages 18 and 65, to have a DSM-IV diagnosis of SAD (major depressive episode, with seasonal pattern, winter type [18] and to have a SIGH-SAD score of 20 or greater. Diagnosis was established with the Structured Clinical Interview for DSM-IV (SCID) [19]. Participants also completed the Morningness-Eveningness Questionnaire (MEQ), a measure of preference for activity in the early or late part of the day [20]. Participants were told that the study involved treatment with either a new light treatment device or a negative ion generator, that both types of treatment were experimental, and that the study was placebo-controlled. In particular, participants were told that one half of the devices in the study were modified in such a way that the investigators did not expect the device to be efficacious. In order to demonstrate informed consent, participants had to demonstrate understanding that if they participate they have a one in two chance of being assigned to treatment expected to be inactive for 4 weeks. Exclusion criteria were: significant medical illness, any retinal disease or medical disorder associated with retinal disease; pregnancy; use of photosensitizing medications, mood-altering medications, light therapy or other treatment for SAD within 1 week of the Baseline Visit (except within 4 weeks in the case of pharmacological antidepressant agents); initiation of psychotherapy within 3 months of the Baseline Visit, except where terminated by the participant prior to this visit; current organic mental disorder, panic disorder, anorexia or bulimia nervosa, obsessive-compulsive disorder or posttraumatic stress disorder; a history of any psychotic disorder or bipolar I disorder (history of manic episode); a history of substance use disorder not in full remission for at least one year; unstable sleep or mood patterns (such as severe premenstrual syndrome); previous unsuccessful trial of light therapy with an accepted device for at least 2 weeks; inability to provide informed consent; poor likelihood of complying reliably with study requirements; suicidal risk or other factor making trial participation clinically inappropriate. Participants were required to have a habitual sleep onset time before 1 A.M., and a habitual sleep end time before 9 A.M., prior to entry in the trial. Participants were required to agree to avoid other treatments for SAD or excluded medications, alteration of daily schedule to change light exposure, or travel to sunny destinations, to maintain a stable sleep schedule, and if female and potentially fertile to use an appropriate form of contraception during the trial. BODY.METHODS.TREATMENT DEVICES: At the Randomization Visit, eligible participants were issued an active or control treatment device by the unblinded clinician. Assignment to active or control group was determined by telephone call by the unblinded clinician to the trial sponsor, and was balanced in blocks of 4 for each site and gender. The proper use of the device was demonstrated to the participant by the unblinded clinician. After experiencing the assigned device in operation, the participant completed a brief questionnaire about expectations [21]. Participants were given a tape 20 inches in length to indicate the correct distance from the device. The active treatment consisted of a Litebook treatment device with 60 LEDs (The Litebook Company Ltd., Alberta, Canada). The 60 LEDs employed in this Litebook model contain emitters which have a spectral emission peak at approximately 464 nm and fluorescent phosphors which provide a broader, secondary spectral peak near 564 nm: of the energy emitted over the range 400 to 700 nm, about 48% is emitted over the range 420 to 508, and 37% is emitted over the range 512 to 616 nm. Collectively the emitted light appears white. This device produces approximately 1,350 lux light at 20 inches. Participants assigned to this device were carefully instructed on aligning the device to illuminate maximally the eyes. An evaluation by an independent consultant physicist confirmed that the Litebook device meets the relevant sets of standards for light exposure safety [22-24]. Control treatment consisted of a negative ion generator, modified to emit no negative ions (SphereOne, Inc., Silver Plume, CO) and to generate a faint high-pitched whine, used at the same distance. Participants using the ion generator were instructed to wear a wrist strap connected to the device to maximize the transfer of negative ions, as this intervention has been found to increase expectations regarding efficacy for the device [3]. Participants were instructed to use the device for 30 minutes each morning, as soon as possible upon arising, and to complete treatment before 8 A.M. Participants were asked to maintain as stable a schedule of sleep and treatment as possible during the trial, and were asked to complete a log of the times of the beginning and end of sleep and of treatment. Participants were asked not to disclose to the blinded clinician which treatment device they were assigned. The blinded study clinician was permitted to reduce the duration of treatment to 15 minutes per day until the next study visit in the event of jitteriness or over stimulation, but this reduction was not required for any participant during the trial. BODY.METHODS.STATISTICAL ANALYSIS: SIGH-SAD scores were analyzed in both a last observation carried forward (LOCF) analysis, including all 26 participants who were randomized, and an observed cases (OC) analysis, including all 23 participants who completed the trial. Remission was defined as a SIGH-SAD score less than 9. The a priori endpoint hypothesis was whether the proportion of participants in remission differed between the active and control treatment groups in the LOCF analysis using the Fisher's exact test. In a secondary analysis, end trial SIGH-SAD scores, as %SIGH-SAD scores consisting of final score as percentage of individual score at randomization, were compared between active and control groups by t test. Post hoc comparisons of the proportion of participants in remission and mean %SIGH-SAD score were made at Weeks 1, 2 and 3. Secondary analysis also included a repeated measures ANOVA mixed model with SIGH-SAD as dependent variable and time, treatment, and interaction of time and treatment as fixed effects, including all randomized participants. A variety of models were considered, including participant intercept and slope as random effects, autoregressive time-dependent, compound symmetry or unstructured correlation structures, and possible transformation of time by the log of one plus the week of treatment. The best model was selected by Schwartz Bayesian criterion, but all models indicated a significant interaction of time by treatment. The final model included linear time trend, no random effects, transformed time, and autoregressive correlation structure (SAS PROC MIX procedure, Kenward-Rogers method for degrees of freedom). Statistical assumptions were verified by examination of residuals. Comparisons between the groups at baseline were made with t-tests in the case of continuous variables and Fisher exact tests in the case of dichotomous variables. Changes in time of sleep or treatment were analyzed with paired t-tests for participants who completed the trial, excluding one participant with incomplete sleep log data. End trial %SIGH-SAD scores were used to assess any relationship between therapeutic response and times of sleep or treatment or other covariate. Statistical analysis was performed with STATVIEW version 5.0.1 and SAS version 9.1.3 (both from SAS Institute, Cary, NC). All results are reported as means ± standard deviations. BODY.RESULTS: Twenty six participants were randomized into the study, 15 in the active treatment group and 11 in the control treatment group. In the active treatment group, 1 participant withdrew after the visit Week 1 for unclear reasons, possibly related to adverse effects of jitteriness and headache or to travel plans. In the control treatment group, 1 participant was withdrawn after Week 1 due to lack of improvement, and 1 participant was withdrawn after Week 1 due to missed treatments related to a motor vehicle accident. Thus, 23 participants completed the Week 4 visit, 14 in the active treatment group and 9 in the control treatment group. There were no instances of accidental unblinding of the depression rating clinicians during the trial. Mean SIGH-SAD scores for the active and control treatment groups did not differ significantly at randomization (28.0 ± 5.35 versus 25.1 ± 3.22, respectively; as shown in Table 1). There were no significant differences between the active and control groups in age (44.7 ± 12.3 years versus 47.6 ± 10.8 years), fraction of female participants (64.3% versus 88.9%), fraction of Caucasian participants (85.7% versus 100%; in the active treatment group, 1 participant was Black and 1 participant Hispanic), number of previous episodes of SAD (11.1 ± 9.9 versus 10.6 ± 9.0), age of first SAD episode (30.3 ± 11.6 versus 35.4 ± 13.4), weight (78.4 ± 18.0 kg versus 71.1 ± 14.1 kg), BMI (28.9 ± 6.5 versus 26.1 ± 5.1), expectation scores (3.88 ± 0.70 versus 3.37 ± 0.86), or MEQ scores (51.5 ± 10.2 versus 55.2 ± 6.3). Table 1 SIGH-SAD Outcome Measures at Randomization and after 1, 2, 3 and 4 weeks of treatment. Mean SIGH SAD score* Mean SIGH SAD score As % of randomization score % Participants in remission (SIGH SAD <9) Active Control Active Control p value Active Control p value Randomization 28.0 ± 5.3 25.1 ± 3.2 - - - - - - Week 1 18.6 ± 7.9 19.0 ± 7.2 67.1 ± 26.7 74.0 ± 22.5 0.527 7.1 11.1 0.999 Week 2 14.9 ± 10.0 17.9 ± 4.9 54.5 ± 36.4 71.7 ± 19.5 0.208 28.6 0.0 0.127 Week 3 11.1 ± 10.1 14.9 ± 4.2 39.0 ± 30.7 54.4 ± 17.9 0.080 42.9 11.1 0.176 Week 4 8.7 ± 8.4 13.4 ± 5.4 29.9 ± 25.4 54.4 ± 21.8 0.027** 57.1 11.1 0.040*** Notes: Observed cases analysis: for active treatment n = 14 and for placebo treatment n = 9. * interaction of time and treatment significant in repeated measures ANOVA as noted in text. ** comparison significant at p ≤ 0.05 by t test. *** comparison significant at p ≤ 0.05 by Fisher's exact test. SIGH-SAD scores improved in both groups over the 4 weeks of treatment, with active treatment participants showing greater improvement (Table 1, Figure 1). The proportion of participants achieving remission was significantly greater in the intent-to-treat LOCF analysis: 53.3% versus 9.1%, p = 0.036 (the a priori endpoint hypothesis of the trial). The proportion of participants achieving remission was also significantly greater with active than control treatment in the OC analysis of all randomized participants: 57.1% versus 11.1%, p = 0.040 (Fisher's exact test; remission defined as SIGH-SAD score <9). There were no significant differences in proportion of participants in remission in pairwise post hoc comparisons prior to Week 4. Figure 1Mean %SIGH-SAD Score after 1, 2, 3 and 4 weeks of treatment. The mean SIGH-SAD score, as percent of individual participant value at the Randomization Visit, is shown for participants receiving active (n = 14) and placebo (n = 9) treatment in the observed cases analysis, at the Randomization Visit ("R") and after 1, 2, 3 and 4 weeks of treatment. Error bars indicate standard error of the mean. Mean %SIGH-SAD scores (final SIGH-SAD score as percent of the individual participant score at randomization) were significantly different between the active and control groups at trial end, in both the intent-to-treat LOCF analysis, 34.5% ± 30.47% versus 60.4% ± 23.61%, p = 0.028, and the OC analysis, 29.9% ± 25.4% versus 54.4% ± 21.8%, p = 0.027. Mean %SIGH-SAD scores did not differ significantly in pairwise post hoc comparisons prior to Week 4. Analysis of SIGH-SAD scores with a repeated measures ANOVA indicated a significant effect of time (F (1,115) = 71.2, p < 0.0001) and a significant interaction of treatment and time (F(1,115) = 5.30, p = 0.023). These results indicate that the active light treatment condition was significantly superior to the placebo control condition. There was no significant correlation between expectation scores and therapeutic response measured as final %SIGH-SAD scores: for all participants, r2 = 0.00 (p = 0.86), for participants on active treatment, r2 = 0.07 (p = 0.35), and for participants on control treatment, r2 = 0.03 (p = 0.63). There was no significant correlation between pre-treatment MEQ scores and therapeutic response measured as %SIGH-SAD scores for all participants, r2 = 0.03 (p = 0.45), for participants on active treatment, r2 = 0.00 (p = 0.79), or for participants on control treatment, r2 = 0.00 (p = 0.84). Times of self-reported sleep start, sleep midpoint, sleep end, and treatment start are shown in Table 2 for all participants who completed the trial, for the week before treatment, the first week of treatment and the last week of treatment in Table 2. There was no significant difference between active and control groups in any of these variables in any week. There were no significant changes in time of sleep start between the baseline week and first week of treatment, or between the first and last week of treatment in either group. The time of sleep end was earlier in the first week of treatment than in the baseline week in both active and control groups (within-group difference significant at p < 0.0001 and p = 0.047, respectively, paired t test), presumably reflecting the need to complete treatment by 8 A.M. as required by the protocol. Between the first and last week of treatment the time of sleep end shifted somewhat later in the active group (p = 0.011, paired t test). Times of sleep midpoint and of treatment start showed a similar pattern to time of sleep end, as might be expected. Table 2 Mean times of sleep and treatment during baseline week and during first and last weeks of treatment. Sleep onset time Sleep midpoint time Sleep end time Active Placebo Active Placebo Active Placebo Baseline week 23:04 ± 1:01 23:08 ± 0:50 3:06 ± 0:44 1 3:17 ± 0:59 7:07 ± 0:51 2 7:26 ± 1:22 First week treatment 23:11 ± 0:45 22:58 ± 0:30 2:48 ± 0:32 1 2:57 ± 0:46 6:25 ± 0:40 2,3 6:56 ± 1:14 Last week treatment 23:05 ± 0:49 22:58 ± 0:60 2:52 ± 0:38 2:55 ± 0:55 6:39 ± 0:44 3 6:52 ± 0:60 Time of treatment Time to treatment after midpoint of sleep Active Placebo Active Placebo First week treatment 6:42 ± 0:44 4 7:03 ± 0:45 3:54 ± 0:29 5 4:03 ± 0:34 Last week treatment 6:55 ± 0:45 4 7:07 ± 0:50 4:01 ± 0:28 5 4:02 ± 0:35 Notes: Data is included for all participants who completed the trial, n = 14 for active and n = 9 for placebo treatment. No comparison between active and placebo group was statistically significant for any of the variables shown. Comparisons between the first week treatment and the baseline week or last week of treatment that were significant at p ≤ 0.05 in paired t test are indicated by shared superscripts. There was no significant statistical correlation or apparent relationship between end trial %SIGH-SAD and time of treatment (r = 0.01, p = 0.74), in participants on active treatment who completed the trial. Most participants (11 of 15) received treatment between 6:10 A.M. and 7:40 A.M., and all of these showed a response with final score less than 50% of pre-treatment. There was no significant statistical correlation or apparent relationship between end trial %SIGH-SAD and the interval between time of treatment and sleep midpoint (r = 0.03, p = 0.53), in completing participants on active treatment. Most participants (11 of 15) received treatment beginning between 3:20 and 4:30 hours after sleep midpoint and all but 1 had a final %SIGH-SAD score less than 50% of pre-treatment. Few treatment-related adverse effects were reported during the trial. In the active treatment group, jitteriness and headache were reported by 1 participant at Week 1, dry mouth and difficulty falling asleep by another participant at Week 1. In the control treatment group, jitteriness was reported by 1 participant at Week 1. BODY.DISCUSSION: The results of this pilot study suggest that 30 minutes of daily light exposure to the Litebook LED device is efficacious in the treatment of SAD: the a priori hypothesis of a difference in remission rate between active and control treatment was supported. The rate of remission in the active group, 57%, was comparable to the remission rate observed by Eastman et al [4] with 1 hour daily use of a 5,000 lux light box (61%). Recent studies have indicated that the human circadian rhythm system is most sensitive to short wavelength light. For example, melatonin secretion is most powerfully inhibited by light with wavelength in the range 450 – 480 nm [9,11], and melatonin rhythms are best shifted by such wavelengths [10]. One study found light with wavelengths around 468 nm more effective in the treatment of SAD than light with wavelengths around 654 nm [13]. The spectral energy distribution of light emitted by the Litebook LED device peaks at about 464 nm, and 48% of its energy is in the range of 420 nm to 508 nm. It is reasonable to hypothesize that the LED device is therapeutically similar to the brighter light box due to this concentration in the short wavelengths. Demonstration that the therapeutic effect of the Litebook device is similar to that of a standard light box would require direct comparison trials. Therapeutic response with the Litebook device appeared to be gradual, with separation of the active and control groups increasing between 1 and 4 weeks. Our results are similar to those of Eastman et al [4], who observed a significant difference in response rate at Week 3 and 4. Most studies of light therapy for SAD have been 1 or 2 weeks in duration, but gradual onset of response was observed in the 4 week trial by Bauer et al [25], and in the 8 week trial by Lam et al [5]. It is possible that an 8 week trial would have shown a further increased therapeutic response. There is some evidence that trial length may affect speed of therapeutic effect in light therapy, with participants randomized to shorter treatment having a faster response than those randomized to longer treatment [26]. Selection of an appropriate control has been problematic in light treatment research, since, as in the case of some other medical devices, the modality of treatment cannot be "blind". Most such research has used treatment with dim red light as a placebo intervention. There are 2 problems with this approach. First, as the use of bright light is increasingly recognized by the public as a treatment for SAD, participants may become more likely to perceive dim red light as the placebo condition, while participants exposed to bright light may be more likely to conclude they are receiving the active condition. Second, even dim light can affect the circadian rhythm system and may have some positive therapeutic effect [27]. The present study used a "credible placebo" design. A no-light device with a plausible therapeutic mechanism served to control for non-specific behavioral effects of light therapy (e.g., sitting for 30 minutes, waking before 8:00 A.M.). Expectations for the light device were not significantly higher than for the ion generator, and there was no significant correlation between expectation score and therapeutic response. The relationships between therapeutic response and times of sleep and of treatment are important for theoretical and practical issues. It has been proposed that SAD is related to a phase delay of circadian rhythms, and that light treatment in SAD is effective by advancing circadian rhythms [28]. Terman and colleagues [29] observed a shift to earlier time of sleep midpoint during light treatment for SAD, suggesting a phase advance of circadian rhythm. In the present trial we observed only a shift towards later time of sleep end and midpoint during the 4 weeks of treatment. In the present trial, participants in both active and control groups appeared to move their times of awakening earlier between the pre-treatment week and the first week of treatment, likely because they were required to complete treatment by 8 A.M. Light treatment then appeared to be associated with a small delay in time of awakening during the treatment period. The protocol of the trial may have obscured the ability to observe shifts in circadian phase. The observations of Terman et al [29] would suggest that response to treatment ought to be strongest about 1.5 – 2 hours after sleep midpoint in a participant with typical 11 P.M to 7 A.M. sleep cycle. The present results do suggest that treatment between 6:10 and 7:40 A.M. in clock time, or between 3:20 and 4:30 hours after sleep midpoint, was effective in alleviating SAD. With the limited number of participants in this trial it is not possible to draw detailed conclusions about the dependence of therapeutic response on time of treatment. Studies with other methodologies, such as that of Murray et al [30], have not observed relationships like those observed by Terman et al [29]. A more detailed version of the phase shift hypothesis suggests that the relationship between times of sleep and time of temperature minimum is critical in the pathogenesis and treatment of SAD [31]. We were unable to measure any such shift in phase angle difference as there was no measure of physiological rhythms in our participants (nor did we measure end trial MEQ, which might serve as a surrogate measure). There was no discernable relationship between MEQ and therapeutic response to light treatment. A study of this size might not be adequate to detect such an association. Reports of adverse events were rare in the trial and the light treatment was well tolerated by participants. Jitteriness was observed in 1 participant each in the active and control treatment groups. This trial is too small to permit accurate assessment of a difference in the occurrence of this symptom between active and control treatment. No ocular adverse events were observed. Studies with ophthalmological examination before and after treatment have disclosed no harmful effect of treatment with conventional light boxes [32], but such studies have not been conducted with the Litebook device. In summary, this pilot randomized controlled trial supported the hypothesis that the Litebook LED device is significantly superior to a credible placebo control condition for the treatment of SAD. However, the results of a small-sample clinical trial must be interpreted with caution. A trial with a larger sample size would provide more definitive information about the efficacy and safety of this LED device. A more convenient form of light therapy might lead to increased use of light for SAD and other biological rhythm disturbances. BODY.CONCLUSION: At the end of this 4 week randomized, double-blind, placebo-controlled trial, the proportions of participants in remission (SIGH-SAD < 9) were significantly greater, and SIGH-SAD scores (as percent of individual score at randomization) were significantly lower with treatment with the Litebook LED light therapy device than with placebo treatment. A longitudinal repeated measures ANOVA analysis of SIGH-SAD scores also indicated a significant interaction of time and treatment. These results are consistent with the hypothesis that the Litebook device is an effective therapy for SAD. There was no significant correlation between therapeutic response and expectation scores, MEQ scores, or time of treatment expressed as clock time, or as time since the midpoint of sleep. Treatment was well-tolerated, with only transient minor adverse effects. BODY.COMPETING INTERESTS: PHD and RWL have received research funding and served as consultants for manufacturers of pharmaceutical antidepressants which could be considered alternative therapies for SAD. RWL has received honoraria as a member of the professional advisory board of The Litebook Company Ltd., and holds stock options in the company: he did not participate in the clinical conduct of the trial or the analysis of unblinded data. DBB reports the gift of light treatment devices for other research studies from The Litebook Company Ltd, but has no other potential conflicts of interest. AJE, EEM, EMT, YM, MJR, EH, JE and HI reported no potential conflicts of interest. BODY.AUTHORS' CONTRIBUTIONS: The original version of the experimental protocol was written by PHD and EEM, with important input from RWL in the initial design and conduct of the study. PHD served as the overall project principal investigator, and AJW served as the overall research coordinator. EMT, EH, DBB, PHD and YM served as principal investigators at the individual sites. MJR, HI, and JT participated in the clinical conduct of the trial. The final manuscript was written by PHD, with comments from all co-authors, all of whom read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here:

TITLE: Peri-operative chemotherapy with or without bevacizumab in operable oesophagogastric adenocarcinoma (UK Medical Research Council ST03): primary analysis results of a multicentre, open-label, randomised phase 2–3 trial ABSTRACT.SUMMARY.BACKGROUND: Peri-operative chemotherapy and surgery is a standard of care for patients with resectable oesophagogastric adenocarcinoma. Bevacizumab, a monoclonal antibody against VEGF, improves the proportion of patients responding to treatment in advanced gastric cancer. We aimed to assess the safety and efficacy of adding bevacizumab to peri-operative chemotherapy in patients with resectable gastric, oesophagogastric junction, or lower oesophageal adenocarcinoma. ABSTRACT.SUMMARY.METHODS: In this multicentre, randomised, open-label phase 2–3 trial, we recruited patients aged 18 years and older with histologically proven, resectable oesophagogastric adenocarcinoma from 87 UK hospitals and cancer centres. We randomly assigned patients 1:1 to receive peri-operative epirubicin, cisplatin, and capecitabine chemotherapy or chemotherapy plus bevacizumab, in addition to surgery. Patients in the control group (chemotherapy alone) received three pre-operative and three post-operative cycles of epirubicin, cisplatin, and capecitabine chemotherapy: 50 mg/m2 epirubicin and 60 mg/m2 cisplatin on day 1 and 1250 mg/m2 oral capecitabine on days 1–21. Patients in the investigational group received the same treatment as the control group plus 7·5 mg/kg intravenous bevacizumab on day 1 of every cycle of chemotherapy and for six further doses once every 21 days following chemotherapy, as maintenance treatment. Randomisation was done by means of a telephone call to the Medical Research Council Clinical Trials Unit, where staff used a computer programme that implemented a minimisation algorithm with a random element to establish the allocation for the patient at the point of randomisation. Patients were stratified by chemotherapy centre, site of tumour, and tumour stage. The primary outcome for the phase 3 stage of the trial was overall survival (defined as the time from randomisation until death from any cause), analysed in the intention-to-treat population. Here, we report the primary analysis results of the trial; all patients have completed treatment and the required number of primary outcome events has been reached. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN 46020948, and with ClinicalTrials.gov, number NCT00450203. ABSTRACT.SUMMARY.FINDINGS: Between Oct 31, 2007, and March 25, 2014, 1063 patients were enrolled and randomly assigned to receive chemotherapy alone (n=533) or chemotherapy plus bevacizumab (n=530). At the time of analysis, 508 deaths were recorded (248 in the chemotherapy alone group and 260 in the chemotherapy plus bevacizumab group). 3-year overall survival was 50·3% (95% CI 45·5–54·9) in the chemotherapy alone group and 48·1% (43·2–52·7) in the chemotherapy plus bevacizumab group (hazard ratio [HR] 1·08, 95% CI 0·91–1·29; p=0·36). Apart from neutropenia no other toxic effects were reported at grade 3 or worse severity in more than 10% of patients in either group. Wound healing complications were more prevalent in the bevacizumab group, occurring in 53 (12%) patients in this group compared with 33 (7%) patients in the chemotherapy alone group. In patients who underwent oesophagogastrectomy, post-operative anastomotic leak rates were higher in the chemotherapy plus bevacizumab group (23 [10%] of 233 in the chemotherapy alone group vs 52 [24%] of 220 in the chemotherapy plus bevacizumab group); therefore, recruitment of patients with lower oesophageal or junctional tumours planned for an oesophagogastric resection was stopped towards the end of the trial. Serious adverse events for all patients included anastomotic leaks (30 events in chemotherapy alone group vs 69 in the chemotherapy plus bevacizumab group), and infections with normal neutrophil count (42 events vs 53). ABSTRACT.SUMMARY.INTERPRETATION: The results of this trial do not provide any evidence for the use of bevacizumab in combination with peri-operative epiribicin, cisplatin, and capecitabine chemotherapy for patients with resectable gastric, oesophagogastric junction, or lower oesophageal adenocarcinoma. Bevacizumab might also be associated with impaired wound healing. ABSTRACT.SUMMARY.FUNDING: Cancer Research UK, MRC Clinical Trials Unit at University College London, and F Hoffmann-La Roche Limited. BODY.INTRODUCTION: Randomised controlled trials1, 2 have shown that the addition of peri-operative chemotherapy to surgery improves survival for patients with resectable oesophagogastric adenocarcinoma compared with surgery alone. Despite this increase in survival, mortality in patients with this disease remains high, with 5-year overall survival for localised disease at diagnosis of only about 40%.3, 4 Bevacizumab, a monoclonal antibody that targets VEGF, improves responses to chemotherapy and progression-free survival, but not overall survival, in patients with advanced gastric cancer.5, 6 In oesophagogastric cancer, a complete surgical resection (R0 resection) is an important predictor of long-term survival.7 We postulated that a higher proportion of patients responding to pre-operative chemotherapy would increase the likelihood of an R0 resection and lead to improved survival outcomes.8 Therefore, we designed ST03 as a phase 2–3 trial to assess the safety and efficacy of adding bevacizumab to peri-operative chemotherapy for patients with resectable oesophagogastric cancer. The initial phase 2 stage of the trial focused on safety and feasibility in the first 200 patients; these results have been reported previously.9 The addition of bevacizumab was feasible and did not seem to significantly increase toxic effects or the likelihood or severity of surgical complications. Therefore, the study proceeded to the phase 3 stage. BODY.METHODS.STUDY DESIGN AND PARTICIPANTS: We recruited patients aged 18 years and older with previously untreated, histologically proven, resectable adenocarcinoma of the lower oesophagus, oesophagogastric junction, or stomach from 87 UK hospitals and cancer centres. The original design (from January, 2007) included patients with gastric or Siewert type III oesophagogastric junction tumours, with eligibility widened in July, 2009, to include type II oesophagogastric junction tumours (in response to centres reporting diagnostic difficulty in distinguishing between type II and III oesophagogastric junction tumours), and further widened in March, 2011, to also include type I oesophagogastric junction and lower oesophageal tumours (after the closure of the Medical Research Council [MRC] OE05 trial on Oct 31, 2011), which recruited patients with such tumours). Staging investigations included CT scans for all patients and endoscopic ultrasound for all lower oesophageal and junctional tumours, or according to local practice for gastric tumours. Laparoscopy was mandated for gastric and type II and III oesophagogastric junction tumours, and according to local practice for type I oesophagogastric junction and lower oesophageal cancers. PET scans, MRI, or bone scans were used when clinically indicated according to local practice. To be eligible, patients were required to have a WHO performance status of 0 or 1 and adequate cardiac, liver, renal, and bone marrow function to be eligible. Patients with lower oesophageal or oesophagogastric junction tumours also had to have adequate respiratory function (FEV1 ≥1·5 L). Blood pressure of a maximum of 140/90 mmHg, a left ventricular ejection fraction of at least 50%, and the absence of proteinuria were also required. Research in contextEvidence before this studyWe searched PubMed in April, 2005, for publications relating to bevacizumab in oesophagogastric cancer and bevacizumab in cancer. In 2006, the MAGIC trial showed an improvement in progression-free survival and overall survival when peri-operative chemotherapy was given in addition to surgery, compared with surgery alone, in patients with resectable oesophagogastric adenocarcinoma. In 2011, the AVAGAST trial in advanced gastric cancer reported an improvement in tumour response and progression-free survival, but not overall survival, when bevacizumab was combined with chemotherapy.Added value of this studyTo the best of our knowledge, this trial is the first study in which bevacizumab was given to patients with resectable oesophagogastric adenocarcinoma in the peri-operative setting; the results provide no evidence of a benefit of bevacizumab administration in combination with peri-operative chemotherapy in these patients. Moreover, the safety results indicate that bevacizumab administration might also be associated with impaired wound healing.Implications of all the available evidenceThe results of this trial suggest that there is unlikely to be a role for bevacizumab in the treatment of localised, operable oesophagogastric cancer. The implication of the results is that patients given standard perioperative chemotherapy are unlikely to benefit from receiving bevacizumab. Future research should consider alternative new treatments in combination with standard therapy in this patient population. Patients were excluded if they had a medically significant co-existing or previous medical condition, defined as cerebrovascular disease (transient ischaemic attack or stroke), myocardial infarction or angina requiring nitrate therapy within the preceding year, uncontrolled hypertension, a recent history of any gastrointestinal inflammatory disorder or any history of uncontrolled hypertension, congestive heart failure (New York Heart Association grade 2 or worse), or serious cardiac arrhythmia. Those taking corticosteroids or undergoing thrombolytic therapy within 10 days before starting chemotherapy were also ineligible. All patients gave written informed consent before randomisation. The trial protocol was approved by a national ethics committee and the UK Medicines and Healthcare products Regulatory Agency. Every participating centre obtained local approvals. BODY.METHODS.RANDOMISATION AND MASKING: Eligible patients were randomly assigned (1:1) to receive either peri-operative epiribicin, cisplatin, and capecitabine chemotherapy or epiribicin, cisplatin, and capecitabine plus bevacizumab, in addition to surgery. Treatment allocation was done via a telephone call to the Medical Research Council Clinical Trials Unit at University College London (normally by the research nurse at the site who was responsible for following up the patient), where trial management staff used a computer programme that implemented a minimisation algorithm with a random element and stratification by chemotherapy centre, site of tumour (lower oesophagus vs oesophagogastric junction type I vs type II vs type III vs stomach), and tumour stage (according to TNM 6th edition). The algorithm established every patient's treatment group allocation at the point of entry (rather than through the use of a pre-determined allocation list). Patients and investigators were not masked to treatment allocation. BODY.METHODS.PROCEDURES: Epirubicin, cisplatin, and capecitabine chemotherapy was given as three pre-operative and three post-operative 21-day cycles, consisting of 50 mg/m2 intravenous epirubicin and 60 mg/m2 cisplatin on day 1 and 1250 mg/m2 oral capecitabine on days 1–21. Patients in the chemotherapy plus bevacizumab group were given 7·5 mg/kg bevacizumab as a continuous intravenous infusion on day 1 of each of the chemotherapy cycles (either before or after the chemotherapy was given). To maximise any potential treatment effect with an acceptable toxicity profile, patients in the bevacizumab group also received six further infusions of bevacizumab alone (7·5 mg/kg intravenously alone every 21 days) as maintenance treatment after post-operative chemotherapy. No bevacizumab dose reductions were allowed. Bevacizumab was discontinued in the event of any new case of gastrointestinal perforation, arterial thromboembolic events (including transient ischaemic attack, stroke, myocardial infarction, or new diagnosis of ischaemic heart disease), grade 3 or 4 haemorrhage, grade 3 or 4 congestive heart failure or left ventricular dysfunction, grade 4 hypertension, grade 4 proteinuria, tracheoesophageal fistula at any grade or any other fistula deemed to be possibly related to bevacizumab. These events were classified as notable, and subject to expedited reporting together with all other toxic effects meeting the standard definitions of serious adverse events. Dose reductions and interruptions in chemotherapy were permitted according to guidance in the trial protocol. All serious adverse events were reviewed for categorisation and severity by the chief investigator (DC) or trial physicians (ECS, AFO). Surgery was scheduled 5–6 weeks after the last day of the final pre-operative chemotherapy cycle; therefore, there were at least 8 weeks between the last pre-operative bevacizumab administration and surgery. Surgical procedures were specified as follows; for gastric or Siewert type III oesophagogastric junction tumours either proximal, total, or distal subtotal gastrectomy was recommended with a lymphadenectomy to include as a minimum lymph node stations 1–7 to ensure at least 15 nodes were excised; for Siewert type II oesophagogastric junction tumours, either extended gastrectomy or two-phase oesophago-gastrectomy with a two-field lymphadenectomy; for Siewert type I oesophagogastric junction or lower oesophageal tumours, oesophagogastrectomy with either a two phase right thoraco-abdominal approach or a left thoracoabdominal approach with a two field lymphadenectomy. Minimal access procedures were allowed only in centres that had sufficient experience (at least 20 such procedures done) after review of outcomes and complication rates by surgeons from the Trial Management Group. Pathological evaluation of resected tumour specimens followed guidance that was compliant with the Royal College of Pathologists' dataset for oesophagogastric cancer resections. We assessed tumour response to pre-operative chemotherapy with Response Evaluation Criteria In Solid Tumors (RECIST) criteria (version 1.0; assessed by CT scan, with laparoscopy, endoscopic ultrasound, or PET scans if clinically indicated) and post-operatively at each centre by pathologists who assessed the resected tumour specimen to establish the extent of resection, margin involvement, extent of lymph node dissection, and Mandard tumour regression grade. Resections were judged to be curative (R0) if the pathologist considered a radical resection had been undertaken and there was no evidence of microscopic residual disease with longitudinal margins (proximal and distal) microscopically clear and there were no viable tumour cells present within 1 mm of the oesophageal circumferential resection margins. Post-operative chemotherapy was started 6–10 weeks after surgery. Patients were followed up every 6 months post-surgery for the first 3 years and every year thereafter until death, or at comparable timepoints if treatment was discontinued early. Cause of death and disease progression events were reported according to local investigator assessment. We assessed quality-of-life data with the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 and STO22 questionnaires, administered before and after post-operative chemotherapy and twice during the maintenance phase, then every 6 months post-surgery for 3 years and annually thereafter. For patients who were not fit for surgery, quality-of-life questionnaires were carried out at similar timepoints. Analysis of quality of life will be presented in a separate publication. We assessed cardiac function by echocardiogram or multiple gated acquisition scan at baseline and after pre-operative and post-operative chemotherapy. After completion of the phase 2 stage, left ventricular ejection fraction measurements were done only at baseline. Before every chemotherapy cycle, a full blood count and blood pressure measurements were taken and patients were tested for proteinuria. In February, 2010, a protocol amendment mandated a nadir neutrophil count on day 10 of the first pre-operative chemotherapy cycle with granulocyte colony stimulating factor (GCSF) recommended for all cases of grade 4 neutropenia, and at the investigator's discretion for grade 3 cases, during all subsequent chemotherapy cycles. BODY.METHODS.OUTCOMES: For the phase 3 analysis, the primary outcome measure was overall survival defined as the time from randomisation until death. Secondary outcomes were macroscopic disease-free survival, progression-free survival, response rates to pre-operative chemotherapy and curative (R0) resection rates. All efficacy analyses were done on an intention-to-treat basis. Disease-free survival was measured from a landmark point, taken to be 6 months from randomisation to allow for any difference in timing of surgery across all patients, to the first occurrence of disease recurrence or death. Patients who had an event before the landmark point and those who had a macroscopically incomplete (R2) resection or no resection were deemed to have had a disease-free survival event at time zero. Progression-free survival was measured from randomisation to the first occurrence of disease recurrence or death; unlike disease-free survival, an R2 resection was not considered an event for this outcome measure. In all survival analyses, patients who had not had the event of interest by the time of analysis were censored at the time they were last followed up. A RECIST response to pre-operative chemotherapy was defined for this trial as a partial or complete response. Those with stable disease, progressive disease, or who had died before the RECIST assessment were regarded as non-responders. For the analysis of pathological tumour response, a Mandard tumour regression grade of 1, 2, or 3 was considered a response and in the intention-to-treat comparison those who did not undergo a resection were included as non-responders. Sensitivity analysis of overall survival was repeated on the following pre-defined baseline subgroups: age (<60 years; 60–70 years; >70 years); sex; WHO performance status; baseline tumour site; baseline tumour stage (separately for gastric/type III oesophagogastric junction tumours and type I/II oesophagogastric junction/lower oesophageal tumours). We did not do an analysis by the type of surgery because this was not known at baseline; instead we analysed tumour site as a baseline surrogate for this variable. BODY.METHODS.STATISTICAL ANALYSIS: 5-year overall survival in the epiribicin, cisplatin, and capecitabine chemotherapy alone group was estimated to be 40%. This estimate was based on the proportion of patients in the peri-operative chemotherapy group of the MAGIC trial who were alive at 5 years (36%),1 taking into consideration the possible effect of improvements in surgical technique, staging, and supportive care over time. A 10% improvement in survival would have been consistent with the benefit seen when adding bevacizumab in other settings at the time the trial was designed.10 To detect an absolute 10% improvement in 5-year survival (corresponding hazard ratio [HR] 0·76), with 80% power and a two-sided 5% significance level, 420 deaths were required. On the assumption that the trial would take 3–4 years to complete recruitment, with 18–24 months' follow-up, the target sample size was estimated to be between 900 and 1100 patients. The trial database was frozen for analysis on Sept 30, 2015, after the target number of deaths had occurred. The accumulating data were monitored by an Independent Data Monitoring Committee (IDMC), which met 13 times between May, 2008, and November, 2014, to review safety data and efficacy analyses. Analyses of survival data were done with the log-rank test. Analyses of overall survival, disease-free survival, and progression-free survival were based on all randomly assigned patients, whereas analysis of overall survival by resection status and Mandard tumour regression grade were based on all randomly assigned patients with available pathological resection status and Mandard tumour regression grade, respectively. We assessed consistency of treatment effect across pre-defined subgroups with tests for heterogeneity that were based on all randomly assigned patients within each subgroup. To compare the two groups in terms of the proportions of patients responding to chemotherapy and the proportions in whom curative resection was achieved, we used the χ2 test based on all randomly assigned patients who had available data from the relevant assessment. Comparison of curative resections and pathological responses were repeated on only those patients with available data who underwent a resection. All analyses were unadjusted for covariates and done at a two-sided 5% significance level, with no adjustment for multiple comparisons.11 All statistical analyses were done with STATA (version 13.0). This trial is registered as an International Standard Randomised Controlled Trial, number 46020948, and with ClinicalTrials.gov, number NCT00450203. BODY.METHODS.ROLE OF THE FUNDING SOURCE: Cancer Research UK reviewed and approved the study design. F Hoffmann-La Roche did a factual accuracy check on the final article but any decision to incorporate comments was made solely at the discretion of the authors. Neither funder had any role in the collection, analysis, or interpretation of the data. DC, SPS, ECS, SR, and REL had access to the raw data. The corresponding author had full access to all the data and the final responsibility to submit for publication. BODY.RESULTS: Between Oct 31, 2007, and March 25, 2014, 1063 patients were enrolled and randomly assigned to receive peri-operative epiribicin, cisplatin, and capecitabine chemotherapy (n=533) or peri-operative chemotherapy plus bevacizumab (n=530). The baseline characteristics were well balanced between the two groups (table 1). The median age of all enrolled patients was 63 years (IQR 56–68), 859 (81%) of 1063 patients were men, and 646 (61%) had stage 3 or 4 disease (according to TNM 6th edition). 144 (14%) had lower oesophageal tumours, 128 (12%) Siewert type I, 199 (19%) Siewert type II, 209 (20%) Siewert type III, and 383 (36%) gastric. The proportion of patients undergoing PET scanning as part of staging has increased steadily during the course of the trial (table 1), but did not appear to differ between the groups. 1054 (99%) of 1063 patients (529 in the chemotherapy alone group and 525 in the chemotherapy plus bevacizumab group) started chemotherapy after randomisation (figure 1). 472 (89%) of 529 patients in the chemotherapy group and 463 (88%) of 525 in the chemotherapy plus bevacizumab group who started chemotherapy received all three pre-operative cycles. 895 (84%) of 1063 randomly assigned patients (457 [86%] of 533 in the chemotherapy alone group vs 438 [83%] of 530 in the chemotherapy plus bevacizumab group) underwent a resection in the trial. Figure 1 provides reasons why the remaining patients did not undergo surgical resection. The median time from the start of the last pre-operative cycle to surgery was 62 days (IQR 56–68) in the chemotherapy alone group and 62 days (57–69) in the chemotherapy plus bevacizumab group. 843 (95%) of 895 patients had at least 7 weeks between the start of their final pre-operative cycle and surgery. 293 (55%) of 533 patients randomly assigned in the chemotherapy group and 257 (48%) of 530 randomly assigned in the chemotherapy plus bevacizumab group re-commenced chemotherapy post-operatively; 215 (73%) of 293 patients in the chemotherapy group and 197 (77%) of 257 patients in the chemotherapy and bevacizumab group received all three post-operative cycles (figure 1). Of all randomly assigned patients, 212 (40%) of 533 in the chemotherapy alone group and 195 (37%) of 530 in the chemotherapy and bevacizumab group received all six scheduled cycles of chemotherapy. Bevacizumab was given in 1407 (94%) of 1492 cycles administered pre-operatively and 605 (89%) of 679 administered post-operatively. The appendix provides further details about pre-operative and post-operative chemotherapy, including numbers of patients who discontinued treatment or had dose reductions (appendix pp 4, 5). At the time of analysis, we used a reverse Kaplan-Meier method to calculate median follow-up, which was 38·4 months (IQR 27·5–50·8) in the whole population and 36·2 months (27·4–51·4) in the chemotherapy alone group and 39·1 months (27·6–50·5) in the chemotherapy and bevacizumab group. 508 patients died (248 in the chemotherapy group and 260 in the chemotherapy and bevacizumab group) and 85% of patients in each group (451 of 533 given chemotherapy alone and 452 of 530 given chemotherapy and bevacizumab) had either died or been followed up for at least 2 years. 3-year overall survival was similar in the two groups: 50·3% (95% CI 45·5–54·9) in the chemotherapy alone group and 48·1% (43·2–52·7) in the chemotherapy and bevacizumab group (HR 1·09 in favour of the chemotherapy alone group, 95% CI 0·91–1·29; log-rank p=0·36; figure 2). Insufficient patients (n=56) had reached the 5-year timepoint at the time of analysis to give a reliable estimate of 5-year overall survival. We recorded 300 disease-free survival events in the chemotherapy alone group and 303 in the chemotherapy plus bevacizumab group. Disease recurrence was confirmed in 210 and 192 patients, respectively (170 and 159 of whom subsequently died), with the remaining events attributable to death before reported recurrence (78 in the chemotherapy group and 101 in the chemotherapy plus bevacizumab group) and a macroscopically incomplete resection (12 vs 10). For progression-free survival, a macroscopically incomplete resection was not considered an event of interest, so the total number of events was therefore 288 in the chemotherapy alone group and 293 in the chemotherapy plus bevacizumab group. There was no evidence of a treatment effect of bevacizumab on either disease-free survival (HR 1·04, 95% CI 0·89–1·22; p=0·62) or progression-free survival (HR 1·05, 95% CI 0·89–1·23; p=0·56). Analysis of RECIST responses to pre-operative chemotherapy (partial or complete response vs stable disease, progressive disease, or death before the tumour assessment) is based on 875 patients (438 in the chemotherapy alone group and 437 in the chemotherapy plus bevacizumab group); we excluded 188 patients (95 in the chemotherapy alone group and 93 in the chemotherapy plus bevacizumab group) with missing response data from the pre-operative tumour assessment. The proportion of patients responding to treatment according to RECIST were similar in the two groups (183 [42%] of 438 patients in the chemotherapy group vs 177 [41%] of 437 in the chemotherapy and bevacizumab group; p=0·70). Analysis of pathological tumour responses based on Mandard tumour regression grade (grade 1–3 vs grade 4–5 or no resection) includes 895 patients (452 chemotherapy alone, 443 chemotherapy plus bevacizumab); those excluded are those who underwent a resection but had unavailable pathological tumour assessment data. The proportion of patients achieving pathological tumour responses were also similar between the groups (147 [33%] of 452 patients in the chemotherapy alone group vs 135 [30%] of 443 in the chemotherapy plus bevacizumab group; p=0·51). A repeat of this comparison including only the 727 patients with available data who underwent a resection (376 in the chemotherapy alone group and 351 in the chemotherapy plus bevacizumab group) also yielded a similar result (147 [39%] of 376 patients in the chemotherapy alone group vs 135 [38%] of 351 in the chemotherapy plus bevacizumab group; p=0·86). Comparison of the proportion of resections that achieved R0 is based on 1002 patients (505 in the chemotherapy alone group and 497 in the chemotherapy and bevacizumab group); we excluded 61 patients (28 in the chemotherapy alone group and 33 in the chemotherapy plus bevacizumab group) who underwent a resection but had unavailable pathological assessment data. Resections were judged to be R0 by local pathologists in 321 (64%) of 505 patients in the chemotherapy alone group and 305 (61%) of 497 in the chemotherapy plus bevacizumab group (p=0·47). When the comparison was repeated including only patients who underwent a resection (R0 vs R1), the proportions of R0 resections were again similar between the groups (321 [75%] of 429 patients in the chemotherapy alone group vs 305 [75%] of 405 in the chemotherapy plus bevacizumab group). Post-hoc, the proportion of R0 resections varied by baseline tumour site; gastric tumours had the highest proportion of R0 resections (265 [87%] of 304 resections), compared with type III oesophagogastric junction (117 [75%] of 157), type II oesophagogastric junction (106 [72%] of 148), type I oesophagogastric junction (62 [61%] of 102), and lower oesophageal (76 [66%] of 116). Of 208 R1 resections, 42 (20%) had a positive proximal margin and 33 (16%) had a positive distal margin (table 2). Circumferential margin involvement was only reported routinely by the pathologist for oesophagogastrectomies; of 146 R1 oesophago-gastrectomies, 72 (49%) had a positive circumferential margin and 132 (90%) were either at or within 1 mm of the circumferential margin. Figure 3 shows the results of pre-defined baseline subgroup analyses for overall survival. The results of these analyses were generally consistent with the main result. Although we did not note any heterogeneity overall (I2=0%, p=0·78) or in most subgroups (sex I2=0%, p=0·99; WHO performance status I2=0%, p=0·77; tumour site I2=0%, p=0·44; gastric tumour stage I2=0%, p=0·63; oesophageal tumour stage I2=12%, p=0·29), we did note a trend toward heterogeneity across age groups (I2=66%; p=0·053). In particular, in patients aged 70 years and older, significantly fewer patients who received chemotherapy alone died compared with those given chemotherapy plus bevacizumab (figure 3). However, in this subgroup, the proportion of deaths that were reported to be non-disease related was higher in the chemotherapy plus bevacizumab group than in the chemotherapy alone group (61 [30%] patients given chemotherapy and bevacixumab vs seven [19%] of 36 patients given chemotherapy alone). Survival beyond the scheduled surgery timepoint assesed post-hoc was significantly longer in patients who had an R0 resection than in those with an R1 resection or no resection (HR 0·23, 95% CI 0·19–0·28; p<0·0001; figure 4). Our earlier comparison of the proportion of patients achieving a pathological tumour response categorised patients with a Mandard tumour regression grade of 1, 2, or 3 as responders. However, our data suggest that those patients with a Mandard tumour regression grade of 1 or 2 might represent a group with improved post-operative survival (figure 5). When such patients were compared with those with a Mandard tumour regression grade of 3, 4, or 5, or no resection post-hoc, post-operative survival was significantly better (HR 0·30, 95% CI 0·21–0·44; p<0·0001; figure 5). The frequency and severity of adverse events occurring during either pre-operative or post-operative chemotherapy were similar between the groups (table 3, table 4). Neutropenia was the most common grade 3 or worse adverse event, both pre-operatively (occurring in 145 [27%] of 529 patients in the chemotherapy alone group vs 139 [26%] of 525 patients in the chemotherapy plus bevacizumab group) and post-operatively (95 [33%] of 292 vs 81 [32%] of 254). This includes two fatal cases of infection with neutropenia, one in the chemotherapy alone group and one in the chemotherapy plus bevacizumab group. Of the 257 patients in the chemotherapy plus bevacizumab group who began post-operative chemotherapy, 179 (69%) went on to receive maintenance bevacizumab and 16 (9%) of these 179 patients reported a grade 3 or 4 toxicity during maintenance treatment, the most common of which were neutropenia (four patients), anorexia (3 patients) and lethargy (3 patients). The most commonly reported serious adverse events were gastrointestinal (60 events in the chemotherapy alone group vs 63 in the chemotherapy plus bevacizumab group), anastomotic leaks (30 events vs 69), and infections with normal neutrophil count (42 events vs 53). 248 patients in the chemotherapy group and 260 in the chemotherapy plus bevacizumab group had died at the time of analysis. Causes of death were reported to be mainly disease-related (204 [82%] of 248 in the chemotherapy alone group vs 204 [78%] of 260 in the chemotherapy plus bevacizimab group); other deaths were due to chemotherapy-related toxic effects (six [2%] vs five [2%]), or related to resection or reoperations (13 [5%] in each group). Other reasons were given for 58 patients (23 [9%] in the chemotherapy group vs 35 [13%] in the chemotherapy plus bevacizumab group; appendix) and the cause of death was unavailable for the remaining five patients (two [<1%] vs three [<1%]). 30-day post-operative mortality was similar in the two groups (14 [3%] of 457 patients who underwent resection in the chemotherapy alone group vs 11 [3%] of 438 who underwent resection in the chemotherapy and bevacizumab group). 21 (5%) of 457 patients in the chemotherapy alone group and 22 (5%) of 438 in the chemotherapy and bevacizumab group died within 90 days of surgery. Of the patents who underwent a resection, data from the post-operative assessment were available for 446 patients in the chemotherapy alone group and 427 in the chemotherapy plus bevacizumab group. The overall incidence of post-operative complications was slightly higher in the chemotherapy plus bevacizumab group, with 215 (48%) of 446 patients in the chemotherapy alone group reporting complications compared with 243 (57%) of 427 patients in the chemotherapy plus bevacizumab group. Wound healing complications in particular were more prevalent in the bevacizumab group, occurring in 53 (12%) patients in this group compared with 33 (7%) patients in the chemotherapy alone group. However, the overall incidence of complications that were deemed to be life-threatening was similar in both groups, at 8% (37 of 446 patients in the chemotherapy alone group and 34 of 427 in the chemotherapy plus bevacizumab group). Appendix p 6 provides full details of the post-operative complications. An increased incidence of post-operative anastomotic leak in the chemotherapy plus bevacizumab group became apparent towards the end of the trial. At a planned IDMC review in June, 2013, leaks were recorded in 30 (10%) of 312 patients in the chemotherapy group and 48 (16%) of 297 in the chemotherapy plus bevacizumab group (compared with 14 [8%] of 179 and 19 [11%] of 170, respectively, at the previous IDMC review in July, 2012). Further investigation showed that the increased leak rate in the bevacizumab group was restricted to those patients who underwent oesophagogastrectomy. In June, 2013, 12 (9%) of 132 patients in this subgroup who received chemotherapy alone had post-operative anastomotic leak versus 29 (24%) of 123 who received bevacizumab, compared with 18 (10%) of 180 versus 19 (11%) of 174, respectively, in all other patients. No other relevant clinical characteristics were identified that might explain the increased frequency of anastomotic leak, nor was there any evidence of a centre effect (data not shown). Consequently, with 1057 patients randomly assigned, recruitment was closed to patients with lower oesophageal or junctional tumours planned for an oesophagogastric resection, and pre-operative bevacizumab was discontinued in such patients who had already been recruited. At the time of the final analysis (Sept 30, 2015), in patients undergoing oesophago-gastrectomy, we recorded post-operative anastomotic leaks in 23 (10%) of 233 patients in the chemotherapy alone group versus 52 (24%) of 220 in the chemotherapy plus bevacizumab group compared with 20 (9%) of 213 and 23 (11%) of 207, respectively, in all other patients. Overall, most of the 103 cases in which onset dates were available occurred during the period immediately after surgery (40 [39%] within 5 days of surgery and 80 [78%] within 10 days). Leak onset dates were provided on serious adverse event reports and were therefore not available for 15 cases in which the event did not satisfy the criteria for a serious adverse event. In those who had an anastomotic leak, three (7%) of 43 patients in the chemotherapy alone group died within 30 days of the operation versus seven (9%) of 75 in the chemotherapy plus bevacizumab group and revisional operations (appendix) were required in 22 (51%) of 43 patients in the chemotherapy alone group compared with 24 (32%) of 75 in the chemotherapy plus bevacizumab group. BODY.DISCUSSION: The results of our trial show that the addition of bevacizumab to peri-operative epiribicin, cisplatin, and capecitabine chemotherapy did not improve overall survival in patients with potentially resectable oesophagogastric adenocarcinoma. There was no clinical evidence of a differential biological effect on tumour growth; the proportions of patients responding to pre-operative chemotherapy assessed by both radiological RECIST criteria and Mandard tumour regression grade from the resected specimen, as well as R0 resection rates, were similar in both groups. This finding is in contrast to those of one small study (n=80), which reported that bevacizumab in combination with docetaxel, oxaliplatin, and fluorouracil increased the proportion of R0 resections achieved in patients with locally advanced gastric cancer,12 and results from studies in advanced (unresectable and metastatic) gastric cancer in which bevacizumab in combination with cisplatin and capecitabine given as first-line treatment increased the proportion of patients achieving a response (37·4% vs 46·0%; p=0·032) and progression-free survival (HR 0·80; p=0·037) but not overall survival.5 Additionally, ramucirumab, a monoclonal antibody against VEGF receptor 2, increases median overall survival compared with placebo from 3·8 months to 5·2 months as second-line treatment in advanced disease13 and from 7·4 to 9·6 months compared with placebo, in combination with paclitaxel in the same setting.14 Although an insufficient number of patients had reached the 5-year timepoint to give a reliable estimate of 5-year overall survival, the scarcity of evidence for an effect on tumour growth and overall survival to this point suggest that it is unlikely a treatment effect would emerge with longer follow-up. Neoadjuvant bevacizumab has also been assessed in other tumour types and has been associated with increased clinical and pathological responses;15, 16, 17 however, such an effect was not evident in the ST03 trial. Most (88%) patients in the ST03 trial received 9 weeks of pre-operative chemotherapy, which has previously been shown to enhance tumour down-staging1 and improve overall survival. However, the lack of effect shares similarities with results from studies in other tumour types in which promising results with bevacizumab in the advanced setting have not been replicated in earlier stage disease, although these studies were mainly done in the adjuvant setting, for example in breast18 and colorectal19, 20 cancer. The finding of an increased anastomotic leak rate in patients who had undergone oesophago-gastrectomy was unexpected. The trial was designed such that patients would have at least 8 weeks between their last pre-operative infusion of bevacizumab and surgery. This period extends well beyond the reported half-life of bevacizumab (20 days) and was believed to be sufficient to prevent effects on post-operative outcomes. The primary outcome measures for the phase 2 component were based on tumour perforation rates, cardiac assessments, and post-operative complications.11 In the phase 2 analysis (n=200; 101 patients in the chemotherapy alone group, 99 in the chemotherapy plus bevacizumab group), the anastomotic leak rate was 4% in both groups (five cases in each group) with 107 (54%) patients having gastric tumours, 71 (36%) oesophagogastric junction type III, and 22 (11%) oesophagogastric junction type II. These figures compare to 275 (32%), 141 (16%), and 175 (21%), respectively in the subsequent 863 patients, with the remainder having oesophagogastric junction type I (128 patients) and lower oesophageal (144 patients) tumours (recruited after March, 2011). This change in eligibility criteria increased the proportion of patients undergoing oesophago-gastrectomy, potentially explaining why the increased leak rate was not apparent earlier in the trial. The treatment of anastomotic leaks varies across the UK; however, centres in this study used the same treatment irrespective of which group the patient was in. Surgeons used omentum for anastomosis coverage according to standard practice, although at the time of designing the study, randomised data were not available to support this practice. In this trial, leak was reported by unblinded assessors and clinical, radiological, and surgical definitions were all included. Although there is a possibility that this method overdiagnosed even small (non-clinically significant) leaks, we do not believe that this had a differential effect on rates in each group. Careful review of possible confounding factors including centre and laparoscopic surgical approaches did not provide any clear explanation for the increased leak rate and suggests that there could be a prolonged effect of bevacizumab that impairs wound healing. Findings of one study21 showed that bevacizumab has sustained effects on VEGF inhibition more than 6 weeks after dosing, and findings of several rectal cancer trials in which bevacizumab was used in conjunction with neoadjuvant chemotherapy or chemoradiotherapy showed increased rates of post-operative complications.22, 23, 24, 25 Tumour and blood specimens were collected at baseline in this trial and will be used to investigate whether patients susceptible to long-term effects of bevacizumab such as impaired wound healing can be identified. The potential limitations of our study were the inclusion of both gastric and oesophageal tumours and a generous targeted difference of 10% absolute difference in survival. However, in our subgroup analysis we recorded no indication of a differential effect by tumour site, or any evidence of a differential biological effect based on R0 resection rates, disease-free survival, or progression-free survival. As with the MAGIC trial1 that compared surgery alone with surgery and peri-operative chemotherapy, about half of patients (550 [52%] of 1063) started post-operative chemotherapy and only 119 (22%) of 530 in the chemotherapy plus bevacizumab group completed all chemotherapy cycles plus the six cycles of maintenance bevacizumab. Our data are consistent with findings that R0 resection is an important predictor of long-term survival. In future clinical trials, identification of patients at risk of a positive resection margin might have a role in treatment selection; careful consideration of the clinicopathological features associated with R1 resection will help to inform these decisions. The suggestion from these results that a Mandard grade of 1 or 2 predicts a better survival outcome (as opposed to the usual approach of considering Mandard grades 1–3 as a response and grades 4–5 as no response) requires further evaluation; however, in this trial, patients with a Mandard grade 1 or 2 seem to have improved survival compared with those with grade 3. A similar survival advantage for Mandard grade 1 and 2 responses was seen in the MAGIC trial26 and in the recently reported MRC OE05 trial27 in which two cycles of neoadjuvant cisplatin and docetaxel, oxaliplatin, and fluorouracil were compared against four cycles of neoadjuvant epirubicin, cisplatin, and capecitabine for oesophagogastric junction and oesophageal adenocarcinoma. However, as intensification of chemotherapy in the OE05 trial did not lead to improved overall survival for the group of patients treated with chemotherapy as a whole, it is unclear whether Mandard tumour regression grade 1–2 (or complete pathological response) is a valid surrogate for overall survival as an endpoint in clinical trials. In conclusion, the addition of bevacizumab to peri-operative chemotherapy for patients with resectable oesophagogastric cancer did not lead to an overall survival benefit; therefore these results are not practice changing. Ongoing exploration of novel therapies in order to improve outcomes for oesophagogastric cancer patients is warranted.

TITLE: The effect of motor imagery training for trunk movements on trunk muscle control and proprioception in stroke patients ABSTRACT: [Purpose] The present study was conducted to evaluate the effect of motor imagery training for trunk movements on trunk muscle control and proprioception in stroke patients. [Subjects and Methods] A total of 12 study subjects were randomly assigned to the experimental group (a motor imagery training group) and the control group (a neurodevelopmental treatment, NDT) group. The two groups were treated five times (30 minutes each time) per week for 4 weeks. The experimental group underwent imagery training for 10 minutes and trunk control centered NDT for 20 minutes and the control group underwent only trunk control centered NDT for 30 minutes. The trunk muscle activity and the position sense of the subjects were evaluated before and after the intervention. [Results] The two groups showed significant improvements in muscle activity after the intervention. Only the experimental group showed significant improvements in proprioception. The experimental group showed significant improvements in the variations of muscle activity and proprioception compared to the control group. [Conclusion] Motor imagery training for trunk movements can be effectively used to improve trunk muscle activity and proprioception in stroke patients. BODY.INTRODUCTION: A stroke is a life-threatening neurological disorder. The most common symptom of a stroke is damage to the motor and balance ability of not only the upper and lower limbs, but also the trunk. Loss of balance is an important factor that causes falls, thereby slowing recovery1, 2). A major role of the trunk is to predict the movements of limbs in advance and allow the body to prepare so that normal movements can occur3). However, stroke patients show decreases and delays in the muscle activity of paralyzed trunk muscles4). In addition, the trunk muscles are closely related to the respiratory muscles. The ability to control the trunk plays an important role in promoting respiratory functions5). The trunk stability of stroke patients depends on muscle strength, neural control, and appropriate proprioception6). Stroke patients with impaired proprioception cannot maintain balance and stable postures and have difficulties in performing motor control when their vision is blocked7). Trunk control of stroke patients is an important indicator in predicting their post-stroke activities of daily living, gait, and balance8). Eventually, the lack of postural control can degrade the quality of life9). Many stroke rehabilitation methods have been developed over the last decade. Among them, imagery training is a neural rehabilitation method that has brought about the improvement of motor skills in interventions for stroke patients10,11,12). Imagery training can also be effective if it is used together, with conventional physical therapy, for the functional rehabilitation of the upper and lower limbs, as well as for the recovery of daily activities13). Motor imagery training can be said to be a rehearsal of physical movements14, 15) and is an act of expressing movement internally without externally expressing movement behaviors16, 17). When undergoing imagery training, the same region of the brain that is activated when actual movements are made is greatly activated18) and muscle strength19) and speed20) are improved21). The stability of the trunk is essential for coordination and balance of the upper and lower limbs needed for functional daily living activities. However, studies on trunk control in the rehabilitation of stroke patients have been conducted less often than studies on upper and lower limbs22). Therefore, the present study aimed to investigate the effect of imagery training on stroke patients' trunk control ability. The hypothesis of the study is as follows: The experimental group of stroke patients, given motor imagery training, should have significant improvements in trunk muscle activity and proprioception compared to the control group who received only trunk control centered NDT. BODY.SUBJECTS AND METHODS: The study subjects were 12 stroke patients who were diagnosed with a stroke and were being treated at the welfare center located in S city from July 2016 to August 2016. The criteria for selection were chronic stroke patients with the duration of the disease not shorter than six months, those with a Korean version of the Mini-Mental State Examination (MMSE-K) score not lower than 24 points, those with a score of at least 40 points in a vividness of movement imagery questionnaire, and those with no other neurological or orthopedic disorders. All protocols were approved by the University of Daejeon. Before participation, the procedures, risks, and benefits were explained to the participants, who gave informed consent. Participant rights were protected according to the guidelines of the University of Daejeon. The subjects were randomly assigned to a motor imagery training group of six patients and a control group of six patients for the study. Before the imagery training, a video of normal persons' trunk movements was produced, including lower trunk stabilization exercises using a Swiss ball and balance exercises such as sitting, standing up, and reaching the hand to move a water cup. This video was provided to the patient in a quiet treatment room for 10 minutes using visual and auditory information through a notebook. Thereafter, the patient was instructed to close his/her eyes, while in a comfortable sitting position in a chair, and imagine the movements of the body in the video for 10 minutes with his/her body relaxed. The therapist asked questions in the middle to see if the patient was concentrating on their imagination of the movements of the body. The video was provided only once at the first session and only imagery training was undergone thereafter. The time and method of the imagery training used in the present study was determined by revising and supplementing the study conducted by Dickstein et al. in 201323). Abdominal stabilization exercises and balance training were undergone using a Swiss ball in both lying and sitting positions24). The motor imagery training group underwent imagery training for 10 minutes and trunk control centered NDT for 20 minutes while the control group underwent only trunk control centered NDT for 30 minutes. Wireless electromyography (EMG), conducted with a TELEMYO 2400T G2 (Noraxon, U.S.A. Inc., CA, USA), was used to measure the muscle activity of the trunk muscles. Electromyography electrodes were attached parallel to the muscle bellies of the internal oblique (IO), the external oblique (EO), the rectus abdominis (RA), and the multifidus (MF). The sampling rate was 1,500 Hz, and a 60 Hz notch filter and a 10–350 Hz band pass filter were used. The collected signals were analyzed by full wave rectification, followed by processing with the root mean square (RMS) method. The maximal voluntary isometric contraction (% MVIC) of each muscle was measured to normalize muscle activity values. For the measurement, the patient was instructed to maintain an isometric contraction for five seconds while the shoulder was lifted up to 20° from the floor during a sit-up in a hook lying position. Each measurement was repeated three times and the mean value of the maximum values for the muscle activity for three seconds, not including the first and last second, was calculated. In the present study, to evaluate the position sense of the trunk as a proprioceptive test method, a digital inclinometer (Dualer IQ, J-TECH Medical, Salt Lake City, UT, USA, 2005) was used. To measure the angle, the operating part of the digital inclinometer was placed on the 12th thoracic vertebra and the fixed part was placed on the 1st sacrum vertebra. Each measurement was repeated three times and the mean value was calculated. For the measurements, a lumbar flexion angle of 30° in an upright standing position was set as a target angle, and then the patient was instructed to bend the waist. Thereafter, the patient was instructed to return to the upright position and the difference from 30°, which was set as the target angle, was measured. The data in the present study was analyzed using SPSS 21.0 for Windows. The general characteristics of the study subjects were analyzed using descriptive statistics. A Wilcoxon singlet-rank test, which is a non-parametric test, was performed for comparison between the motor imagery training group and the control group, before and after the intervention. Differences between the two groups were compared using Mann-Whitney U tests. The statistical significance level was set to p=0.05. BODY.RESULTS: The general characteristics of the study subjects are summarized in Table 1Table 1.General characteristics of the subjectsExperimental groupaControl groupbGender (male/female)5/15/1Age (years)60.1 ± 9.2c63.8 ± 7.0Paretic side (right/left)3/32/4Type of stroke (infarction/hemorrhage)3/34/2Stroke duration (months)28.3 ± 7.129.6 ± 4.9amotor imagery training, btrunk control training, cmean ± standard deviation. Tables 2Table 2.Comparison of trunk muscle activation between the two groupsExperimental groupaControl groupbIO (%)pre32.1 ± 12.0c35.0 ± 9.3post61.0 ± 6.0*†50.28 ± 7.5*EO (%)pre41.3 ± 12.339.2 ± 5.2post65.6 ± 7.0*†53.94 ± 8.7*RA (%)pre29.1 ± 6.133.8 ± 7.4post66.4 ± 10.0*†52.6 ± 6.2*MF (%)pre40.3 ± 9.541.6 ± 7.4post69.0 ± 12.1*†54.9 ± 9.1*amotor imagery training, btrunk control training, cmean ± standard deviation. *significant difference within group (p<0.05)†significant difference between group (p<0.05). IO: internal oblique; EO: external oblique; RA: rectus abdominis; MF: multifidus and 3Table 3.Comparison of position sense between the two groupsExperimental groupa(n=6)Control groupb(n=6)RE (°)pre5.8 ± 2.3c6.0 ± 2.7post1.8 ± 0.7*†4.6 ± 2.5amotor imagery training, btrunk control training, cmean ± standard deviation. *significant difference within group (p<0.05). †significant difference between group (p<0.05). RE: reposition errors summarize the differences in muscle activity and proprioception between the experimental group and the control group. There were significant improvements in muscle activity in both the experimental and control groups after the intervention (p<0.05). There was a significant improvement in proprioception in the experimental group after intervention (p<0.05). However, there was no significant difference in proprioception in the control group (p>0.05). The experimental group showed significant improvements in the variations of muscle activity and proprioception compared to the control group (p<0.05). BODY.DISCUSSION: Exercise methods using imagery training have been shown to be effective for the functional rehabilitation of stroke patients through systematic reviews13). The most difficult part in motor imagery training is determining how much the subject can reproduce movements through imagination25). Since the abilities to imagine movements may be different by individual, assessing individuals is important, and tools for assessing healthy people and those for assessing stroke patients may be different10). In past studies, the Movement Imagery Questionnaire (MIQ)26), the Movement Imagery Questionnaire-Revised (MIQ-R)27), and the Vividness of Movement Imagery Questionnaire (VMIQ)28) were used to assess the imaginary abilities of healthy subjects and the Movement Imagery Questionnaire-Revised, Second Edition (MIQ-RS)29) was used to assess the imaginary abilities of stroke patients. Therefore, in the present study, the MIQ-RS, which has been proven to be reliable and valid, was used to assess the imaginary abilities of stroke patients30). The MIQ-RS is composed of a 14-item questionnaire that consists of seven visual and seven kinesthetic items. In this tool, each item is rated on a 7-point Likert scale, ranging from 1=very hard to see/feel to 7=very easy to see/feel. In a study of trunk muscle activity using an EMG, Souza et al.31) reported that the activity of the RA, EO, and IO increased when the arms and legs were moved, suggesting that these muscles play important roles in trunk stabilization. Scott et al.32) reported that the lumbar MF was more active on a Swiss ball than on a rigid chair. This study also examined the trunk muscle activity using the Swiss ball. According to the results, the muscle activation of the RA, EO, IO, and MF significantly increased after intervention in both the motor imagery training group and the control group and the increase in muscle activation was significantly larger in the motor imagery training group than in the control group. The problem of postural control commonly occurs in stroke patients33). To solve problems in balance and postural control, musculoskeletal and neurological elements consisting of the vestibular system, vision, proprioception, muscle strength, and cognitive functions are necessary34). Ryerson et al.6) compared the proprioceptive trunk position sense of stroke patients with that of normal persons based on trunk repositioning error and reported that stroke patients showed larger differences in the proprioceptive trunk position sense. Han and Shin35) reported that, when balance exercise is applied to stroke patients, the patients showed improvement in their trunk position sense. In the present study, when stroke patients underwent trunk control training using Swiss balls, the patients showed improvement in lumbar proprioception. However, whereas the control group did not show any significant difference between before and after the intervention, the motor imagery training group showed significantly larger differences in all results compared to the control group. This suggests that improvement in trunk proprioception and the activation of trunk muscles through motor imagery training can prevent falls that may have occurred and play an important role in activities of daily living and balance. However, there are several problems in interpreting the results of the present study. First, because the number of patients is small, it is difficult to generalize the results to all stroke patients. Second, the present study included only a few muscles not allowing for the evaluation of long-term effects and functions. These limitations should be complemented in future studies.

TITLE: Advice to follow a low-carbohydrate diet has a favourable impact on low-grade inflammation in type 2 diabetes compared with advice to follow a low-fat diet ABSTRACT.BACKGROUND: Inflammation may play an important role in type 2 diabetes. It has been proposed that dietary strategies can modulate inflammatory activity. ABSTRACT.METHODS: We investigated the effects of diet on inflammation in type 2 diabetes by comparing a traditional low-fat diet (LFD) with a low-carbohydrate diet (LCD). Patients with type 2 diabetes were randomized to follow either LFD aiming for 55–60 energy per cent (E%) from carbohydrates (n = 30) or LCD aiming for 20 E% from carbohydrates (n = 29). Plasma was collected at baseline and after 6 months. C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1Ra), IL-6, tumour necrosis factor receptor (TNFR) 1 and TNFR2 were determined. ABSTRACT.RESULTS: Both LFD and LCD led to similar reductions in body weight, while beneficial effects on glycaemic control were observed in the LCD group only. After 6 months, the levels of IL-1Ra and IL-6 were significantly lower in the LCD group than in the LFD group, 978 (664–1385) versus 1216 (974–1822) pg/mL and 2.15 (1.65–4.27) versus 3.39 (2.25–4.79) pg/mL, both P < 0.05. ABSTRACT.CONCLUSIONS: To conclude, advice to follow LCD or LFD had similar effects on weight reduction while effects on inflammation differed. Only LCD was found significantly to improve the subclinical inflammatory state in type 2 diabetes. BODY: Key messagesIn type 2 diabetes, randomization to advice to follow a low-carbohydrate diet or a low-fat diet had similar effects on weight reduction, while effects on inflammation differed.After 6 months, the low-carbohydrate diet, but not the low-fat diet, had a favourable impact on low-grade inflammation in type 2 diabetes. BODY.INTRODUCTION: Inflammation is considered to play an important role in the development of type 2 diabetes as well as in further complications of the disease. At the cellular level, the expression of proinflammatory cytokines like interleukin (IL)-1β and tumour necrosis factor (TNF) is implicated in insulin resistance and beta-cell failure (1). An enhanced inflammatory activity is also detected in peripheral blood. It has been consistently shown in several clinical studies that the levels of inflammatory markers such as C-reactive protein (CRP) and IL-6 are increased in patients with type 2 diabetes compared with healthy individuals, thus indicating a low-grade systemic inflammation (1–5). A few studies have also measured IL-1β and TNF in the circulation but with less consistent results (3,4,6), probably due to the fact that circulating levels of IL-1β and TNF are often marginal. Instead, their major inhibitors, IL-1 receptor antagonist (Ra), TNFR1, and TNFR2, are readily secreted into the blood and considered reliable markers of activity of the cytokines (7–9). Accordingly, elevated levels of IL-1Ra and TNFR2 in plasma have been associated with both insulin resistance and type 2 diabetes (10–12). Interestingly, IL-1Ra levels also predict the incidence of type 2 diabetes independently of CRP levels and other risk factors (5,13). Moreover, circulating levels of TNFR2 predict morbidities such as cardiovascular disease and nephropathy in patients with type 2 diabetes (14,15). Lifestyle interventions play a crucial role in the management of type 2 diabetes and may also lead to reduced inflammation (1,16). The anti-inflammatory effects have been attributed mainly to the concomitant weight loss, whereas the effects of interventional strategies alone, e.g. dietary change, have been more difficult to verify. Traditionally, a low-fat diet has been the recommendation for achieving weight loss and improved glycaemic control in type 2 diabetes. However, although still controversial, evidence is emerging that a low-fat, high-carbohydrate diet has less favourable metabolic effects compared with a low-carbohydrate diet or Mediterranean diet in subjects at high cardiovascular risk (17–22). Moreover, intake of carbohydrates, in particular refined carbohydrates, has been associated with a proinflammatory response (23–25), whereas diets high in unsaturated and polyunsaturated fatty acids may decrease inflammation (16,22). By using a randomized study design, our aim was to investigate the effects of diet on systemic inflammation in type 2 diabetes by comparing a low-fat diet (LFD) with a low-carbohydrate diet (LCD) during weight loss. BODY.MATERIAL AND METHODS: The study was performed in two primary health care centres in the south-eastern part of Sweden. The inclusion criteria were a diagnosis of type 2 diabetes treated with diet, with or without oral glucose-lowering medication or insulin. Exclusion criteria were difficulties in understanding the Swedish language, severe mental disease, malignant disease, or drug abuse. Patients were randomized to advice to follow either a traditional LFD (aiming for 30 energy per cent, E%, from fat) or an LCD (aiming for 20 E% from carbohydrates) over 2 years, as recently described (20). Randomization was performed by drawing blinded ballots. The energy contents of diets were similar, 6694 kJ/day (1600 kcal/day) for women and 7531 kJ/day (1800 kcal/day) for men. Dietary advice was given by physicians on a group basis at baseline, 2, 6, and 12 months, and standardized in regard to energy content and nutrient composition. One dedicated dietician provided all participants with suitable recipes at each group meeting and was also available for questions from participants during the whole study period. Diet records from three consecutive days were performed at baseline, 3, 6, 12, and 24 months. Plasma samples were collected at baseline and 6 months, i.e. the time point when weight loss and compliance was maximal. Plasma samples were also collected from 41 control subjects in the same region, randomly invited from the Swedish Population Register. Subjects who accepted the invitation were included as controls if they were anamnestically healthy and received no medication. Anamnesis was gained by one dedicated nurse co-ordinator who also performed the blood sampling. None of the participants exhibited any clinical signs of acute inflammation at the time of blood collection. The research protocol was approved by the Ethical Review Board of Linköping University. Written informed consent was obtained from all participants. The study was registered with trial number NCT01005498 at ClinicalTrials.gov. Plasma C-reactive protein (CRP) was measured using an immunoassay (Roche Diagnostics GmbH, Vienna, Austria) with a detection limit of 0.03 mg/L. IL-6 and TNFRs were measured in plasma by ELISA assays (RnD Systems Europe, Abingdon, United Kingdom) with detection limits of 0.48 pg/mL (IL-6), 0.8 pg/mL (TNFR1), and 0.6 pg/mL (TNFR2). TNF, IL-1β and IL-1Ra were analysed with Luminex (RnD Systems) with the detection limits 5.4, 2.8, and 7.9 pg/mL, respectively. Inter-assay coefficients of variation were 4.5%, 8.1%, 4.7%, 12%, and 11% for CRP, IL-6, TNFR1, TNFR2, and IL-1Ra, respectively. IBM SPSS Statistics 19 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. Differences within and between groups were analysed with Student's paired and unpaired two-tailed t test except for inflammatory markers that were not normally distributed. For CRP, IL-6, TNFR1, TNFR2, and IL-1Ra, Mann–Whitney U test was used for between-group comparisons differences and Wilcoxon signed ranks test for within-group comparisons. For correlation analyses Spearman's rank correlation was used. A linear multiple regression analysis was performed to assess the independent contribution of different factors to changes in cytokine levels. Effects on weight and HbA1c levels were the main outcome variables. The original statistical power calculation that has been previously described (20) was based on an earlier 6-month pilot study of 28 participants with type 2 diabetes who were randomized to the same diets as in the present study. Twenty patients completed the study, and both diet groups achieved similar reductions in weight, while HbA1c levels tended to decrease in the LCD group only. Based on the results from the pilot study, the number of participants in the present trial was increased to at least 30 in each group. BODY.RESULTS.STUDY PARTICIPANTS: Seventy-two patients were consecutively invited by the study nurses to participate. As shown in the flow diagram (Figure 1), 61 patients entered the study. No patients were lost to follow-up. Demographic and diabetes-related variables of patients in the two groups are presented in Table I. None of the participants were smokers. At baseline, 24 (77%) LFD patients and 22 (73%) LCD patients were treated with cholesterol-lowering drugs (statins). Figure 1.Flow diagram of the study. Table I. Clinical and laboratory characteristics of the two intervention groups at baseline. LFD n = 31 LCD n = 30 P Age, years 63 (11) 61 (9.5) NS Male/female, n 13/18 14/16 NS Duration of diabetes, years 8.8 (6.2) 9.8 (5.5) NS Dietary treatment only, n (%) 2 (6.5) 2 (6.7) NS Oral glucose-lowering treatment only, n (%) 13 (42) 15 (50) NS Oral medication + insulin, n (%) 11 (35) 10 (33) NS Insulin only, n (%) 5 (16) 3 (10) NS Total insulin dose, E 39 (51) 42 (65) NS Metformin dose, mg 1435 (946) 1375 (950) NS Glibenclamide dose, mg 0.4 (1.9) 1.1 (2.6) NS If not stated otherwise, data are given as mean (SD). LCD = low-carbohydrate diet; LFD = low-fat diet. BODY.RESULTS.EFFECTS ON METABOLIC PARAMETERS: The results of dietary records are shown in Table II. Adherence to the proposed diet was similar in both groups. However, the most prominent changes in nutrient intake were seen in the LCD group with a significant reduction in E% intake from carbohydrates and a concomitant increase in E% intake from fat. At 6 months, the E% intake from fat:carbohydrate was 29:49 in the LFD group and 49:25 in the LCD group. The levels of body mass index, HbA1c, lipid levels and insulin doses at baseline and after 6 months are shown in Table III. Both groups showed a significant reduction in body mass index. Also, the reduction in absolute weight was similar between groups and maximal at 6 months: LFD −4.0 (4.1) kg, LCD −4.3 (3.6) kg. Although beneficial changes of HbA1c and HDL cholesterol levels were seen in the LCD group, the levels of HbA1c and HDL cholesterol remained similar between groups. After 6 months, the total insulin dose was significantly reduced in the LCD group but not in the LFD group, while the use of oral glucose-lowering medication did not change. Table II. Dietary outcomes at baseline and 6 months in the two intervention groups ( P values ≥ 0.10 are listed as non-significant, NS). LFD n = 31 LCD n = 30 P between groups Energy intake, kJ  Baseline 1809 (493) 1690 (426) NS  6 months 1553 (427) 1384 (366) NS   P within group < 0.01 < 0.001 Fat, E%  Baseline 32 (5.2) 39 (6.8) < 0.001  6 months 29 (5.4) 49 (7.5) < 0.001   P within group NS < 0.001 Carbohydrate, E%  Baseline 49 (5.6) 41 (11) < 0.01  6 months 49 (5.9) 25 (8.4) < 0.001   P within group NS < 0.001 Protein, E%  Baseline 19 (3.4) 19 (2.8) NS  6 months 20 (3.5) 23 (3.7) < 0.01   P within group < 0.05 < 0.001 Total fat, g  Baseline 66 (23) 74 (23) NS  6 months 53 (24) 79 (25) < 0.001   P within group < 0.05 NS Saturated fat, E%  Baseline 13 (2.7) 16 (4.1) NS  6 months 11 (2.1) 20 (3.7) < 0.001   P within group NS < 0.001 Unsaturated fat, E%  Baseline 12 (2.2) 14 (2.8) NS  6 months 11 (2.5) 18 (3.2) < 0.001   P within group NS < 0.001 Polyunsaturated fat, E%  Baseline 5.2 (1.5) 5.9 (2.6) NS  6 months 5.1 (1.9) 7.7 (2.4) < 0.05   P within group NS < 0.001 Total carbohydrates, g  Baseline 213 (64) 167 (56) < 0.05  6 months 182 (51) 82 (28) < 0.001   P within group < 0.05 < 0.001 Fibres, E%  Baseline 2.6 (0.6) 2.4 (0.7) NS  6 months 2.8 (0.7) 2.2 (0.7) < 0.01   P within group NS < 0.05 Monosaccharides, E%  Baseline 7.2 (3.1) 6.2 (3.0) NS  6 months 8.1 (2.9) 4.0 (2.1) < 0.001   P within group NS < 0.001 Disaccharides, E%  Baseline 8.7 (1.9) 8.3 (2.6) NS  6 months 9.1 (2.8) 5.4 (2.8) < 0.001   P within group NS < 0.001 Saccharose, E%  Baseline 5.5 (1.5) 4.9 (2.5) NS  6 months 5.2 (1.9) 2.5 (1.6) < 0.001   P within group NS < 0.001 Data are given as mean (SD). E% = energy %; LCD = low-carbohydrate diet; LFD = low-fat diet. Table III. Values of body mass index (BMI), HbA1c, and lipids at baseline and 6 months in the two intervention groups. LFD n = 30 LCD n = 29 P between groups BMI, kg/m 2  Baseline 34 (5.7) 32 (5.1) NS  6 months 32 (5.5) 30 (5.1) NS   P within group < 0.001 < 0.001 HbA1c, mmol/mol  Baseline 56 (8.0) 57 (8.6) NS  6 months 55 (9.7) 53 (9.8) NS   P within group NS < 0.01 Total cholesterol, mmol/L  Baseline 4.3 (1.0) 4.5 (1.0) NS  6 months 4.2 (1.1) 4.4 (1.1) NS   P within group NS NS LDL cholesterol, mmol/L  Baseline 2.4 (0.7) 2.7 (0.9) NS  6 months 2.3 (0.8) 2.5 (0.8) NS   P within group NS NS HDL cholesterol, mmol/L  Baseline 1.1 (0.3) 1.1 (0.3) NS  6 months 1.1 (0.3) 1.2 (0.5) NS   P within group NS < 0.05 Triglycerides mmol/L  Baseline 1.8 (0.8) 1.7 (1.4) NS  6 months 1.8 (1.3) 1.5 (1.2) NS   P within group NS NS Data are given as mean (SD). LCD = low-carbohydrate diet; LFD = low-fat diet. BODY.RESULTS.EFFECTS ON INFLAMMATORY PARAMETERS: The levels of IL-1β and TNF were below the limits of detection in all participants. The levels of CRP, IL-1Ra, IL-6, TNFR1, and TNFR2 at baseline and after 6 months are presented in Table IV. At baseline, no differences were seen between the two groups. After 6 months, CRP levels did not show any significant changes within the groups. IL-1Ra levels, on the other hand, decreased significantly in the LCD group, while no change was seen in the LFD group. The levels of IL-6 increased in the LFD group only. After 6 months, both IL-1Ra and IL-6 levels were significantly lower in the LCD group than in the LFD group. TNFR1 levels did not change in any of the groups, while TNFR2 tended to increase in LFD patients (P = 0.064). Table IV. Levels of inflammatory markers at baseline and 6 months in the two intervention groups. LFD n = 30 LCD n = 29 P between groups CRP, mg/L  Baseline 1.41 (0.45–4.01) 1.12 (0.37–3.32) NS  6 months 1.67 (0.80–3.24) 0.87 (0.29–2.74) NS   P within group NS NS IL-1Ra, pg/mL  Baseline 1333 (784–2153) 1298 (818–1873) NS  6 months 1216 (974–1822) 978 (664–1385) < 0.05   P within group NS < 0.01 IL-6, pg/mL  Baseline 2.16 (1.77–3.59) 2.45 (1.39–4.10) NS  6 months 3.39 (2.25–4.79) 2.15 (1.65–4.270) < 0.05   P within group < 0.05 NS TNFR1, ng/mL  Baseline 1371 (1227–1760) 1340 (1149–1637) NS  6 months 1423 (1200–1687) 1320 (1145–1610) NS   P within group NS NS TNFR2, ng/mL  Baseline 2627 (2162–3150) 2530 (2094–2973) NS  6 months 2680 (2322–2968) 2480 (2266–2927) NS   P within group 0.064 NS Data are given as median (interquartile range). P values ≥ 0.10 are listed as non-significant (NS). LCD = low-carbohydrate diet; LFD = low-fat diet. During the study period, statin therapy was initiated in two LCD patients, and, hence, 24 patients in each group were treated with statins at 6 months. At baseline, the levels of CRP, IL-1Ra, IL-6, TNFR1, and TNFR2 did not differ significantly between statin users (n = 46) and non-statin users (n = 15), neither did changes in inflammatory markers differ between statin and non-statin users after 6 months. If the two LCD patients receiving statin during the study period were excluded from the analysis, the reduction of IL-1Ra remained significant in the LCD group (P = 0.002). BODY.RESULTS.CORRELATIONS BETWEEN BASELINE VARIABLES AND BETWEEN CHANGES DURING INTERVENTION: All inflammatory markers were correlated with each other (P values of at least 0.01). Moreover, all inflammatory markers were significantly correlated with BMI (P values of at least 0.01). Levels of CRP, TNFR1, and TNFR2 showed correlations with insulin therapy as well as insulin dose (P < 0.05), while levels of IL-1Ra, TNFR1, and TNFR2 were correlated with HbA1c (P < 0.05). The change in IL-1Ra during intervention was correlated with changes in BMI (r = 0.302, P < 0.05), HbA1c (r = 0.411, P < 0.01), CRP (r = 0.245, P < 0.05), and IL-6 (r = 0.266, P < 0.05) but not with changes in energy intake, HDL cholesterol, or insulin dose. The change in IL-6 correlated with change in CRP (P < 0.05) but not with changes in energy intake, BMI, weight, HbA1c, HDL cholesterol, or insulin dose. In multiple regression analyses using change in IL-1Ra as dependent variable and changes in BMI, HbA1c, CRP, and IL-6 as independent variables, change in IL-1Ra remained associated with change in HbA1c only (standardized regression coefficient β = 0.365, P < 0.05). BODY.RESULTS.COMPARISON WITH A POPULATION WITHOUT KNOWN DIABETES: Forty-one clinically healthy subjects, 24 men and 17 women, all non-smokers (age 64 (7.9) years, BMI 25 (3.2) kg/m2), were recruited as clinically healthy controls in order to provide 'reference' levels of systemic inflammatory markers. The levels in the controls were significantly lower compared with the baseline levels in patients: CRP 0.73 (0.32–1.19) mg/L, IL-1Ra 713 (1996–2502) ng/mL, P values of < 0.05, < 0.001, < 0.001, < 0.001, and < 0.05, respectively. In the LCD group, the levels of IL-1Ra and IL-6 were still significantly higher than in controls after 6 months, P < 0.05 and P < 0.01, respectively, while CRP had reached levels that were similar to controls (Table IV). BODY.DISCUSSION: In the present study, we provide evidence that advice to follow a LCD reduces the subclinical proinflammatory state in type 2 diabetes. Despite similar weight loss and similar total energy intake in patients who were randomized to follow either LFD or LCD, cytokine levels were differently affected in the two groups. After 6 months, IL-1Ra showed a significant decrease in patients who were randomized to follow LCD, while IL-6 increased (and TNFR2 tended to increase) in those who followed LFD. Overall, these changes resulted in significantly lower levels of IL-1Ra and IL-6 in the LCD group compared with the LFD group at 6 months. Clinical studies investigating the effects of carbohydrate restriction on inflammation are sparse, but results that are in line with our findings have been reported. In one study, 40 overweight individuals with atherogenic dyslipidemia were randomized to ad libitum diets very low in carbohydrate (12%) or low in fat (24%) for 12 weeks. Both diets decreased the levels of inflammatory proteins, but overall the anti-inflammatory effect of carbohydrate restriction was larger in the very-low carbohydrate group than in the LFD group (26). A recent study by Davis et al. (27) compared LFD with LCD in 51 patients with type 2 diabetes and, in contrast to our results, did not find any differences in IL-6 after 6 months. On the other hand, these authors observed a decrease in soluble adhesion molecules, like E-selectin, in the LCD arm only. Anti-inflammatory effects of carbohydrate restriction have also been reported in animal models. Guinea pigs assigned to be fed LCD for 12 weeks showed significantly lower levels of inflammatory markers and oxidized cholesterol in the aorta compared with guinea pigs fed LFD (28). A carbohydrate-rich diet itself has also been associated with proinflammatory effects. A regimen of a 4-month eucaloric LFD in 22 healthy women resulted in unfavourable effects on inflammatory markers including CRP and IL-6 (29). In the large PREDIMED Study, subjects at high cardiovascular risk were randomized to either two types of Mediterranean diet or a control diet (21). The control diet group who received advice to follow a low-fat diet reported 44 E% intake from carbohydrates which is rather close to the carbohydrate intake in our LFD group (49 E%). In a minor sub-study of PREDIMED, the levels of IL-6 and soluble adhesion molecules decreased in the Mediterranean diet groups, while a significant increase was observed in the control group after 3 months (30). In a larger sub-study of PREDIMED, the levels of IL-6, TNFR1, and TNFR2 decreased in the Mediterranean diet groups while TNFR1 and TNFR2 increased significantly and IL-6 trended upwards in the control group after 12 months, thus supporting that advice to reduce fat intake, and thereby increase carbohydrate intake, is associated with proinflammatory effects (31). Notably, the PREDIMED Study showed a significantly higher incidence of both type 2 diabetes and major cardiovascular events in the control group compared with the Mediterranean diet group after a median follow-up of 4–5 years (19,21). Several mechanistic studies have reported that acute exposure to carbohydrates induces a proinflammatory response (1,16). This effect is also more pronounced in subjects with obesity or impaired glucose tolerance (32,33). In obese patients presenting for bariatric surgery, a history of high carbohydrate intake was associated with significantly higher odds of inflammation in the liver, while higher fat intake was associated with less inflammation (34). Also, the type of carbohydrate may have a major influence on inflammation. Both population-based and experimental studies have provided evidence that high intake of refined or simple carbohydrates is associated with proinflammatory effects (23–25). In the LCD group of our study, the reduction in consumption of simple carbohydrates (monosaccharides, disaccharides, and saccharose) was more pronounced than the reduction in fibre consumption, thus allowing us to speculate that this might have contributed to the lower cytokine levels. Not only the nature of carbohydrates but also the nature of dietary fats can modulate inflammation. Several studies have reported that a meal enriched in saturated fatty acids is followed by a proinflammatory response. On the other hand, diets high in unsaturated or polyunsaturated fatty acids have been associated with anti-inflammatory effects (22,30,31). Based on dietary records, we found no changes in the proportional intake of saturated, unsaturated, and polyunsaturated fatty acids in the LFD group over 6 months. In the LCD group, the percentage of energy intake from saturated fat showed a significant increase, but, concomitantly, similar increases in intake of unsaturated and polyunsaturated fat were seen. The reduction of IL-1Ra levels in the LCD group constituted the most prominent change in inflammatory biomarkers. The reduction in IL-1Ra was also associated with a reduction in HbA1c after adjustment for weight reduction and changes in CRP and IL-6, indicating a close link between IL-1Ra and glycaemic control. In a previous study, Ruotsalainen et al. (9) used a broad panel of cytokines in order to examine whether levels were abnormal in 129 offspring of patients with type 2 diabetes. Interestingly, only the levels of IL-1Ra were elevated in normoglycaemic offspring and even more so in offspring with impaired glucose tolerance compared with healthy controls, suggesting that IL-1Ra is the most sensitive marker of cytokine response in the prediabetic state. According to current guidelines, the majority of patients were treated with statins, which to some extent may affect the inflammatory response. In vitro, statins exert a wide variety of anti-inflammatory effects, and in randomized trials it is well documented that statins reduce plasma levels of CRP, whereas effects of cytokine levels are less evident (35). We did not find any significant association between changes in inflammatory markers and statin therapy. However, it is still possible that diet-induced effects on CRP levels have been attenuated by the extensive use of statins in our study. Advice to follow LFD or LCD had similar effects on weight reduction in our study. This is not in agreement with previous randomized studies reporting a greater weight loss with LCD than with LFD (36). One explanation may be that our aim was to achieve similar energy restriction in the two groups in order to focus on the effects of macronutrient composition. Accordingly, both groups also reported similar energy intake during the study. One limitation of our study is the limited sample size and possible impact of other lifestyle-related factors, which is a methodological problem in any nutritional intervention. The patients were also informed of randomization results before the diet record at baseline was performed. This may explain why the reported intake of fat and carbohydrates differed between groups at baseline. The participants may already have started to adjust their diets according to allocated diet. No participants were lost to follow-up, which is a strength of the study. The fact that study nurses in the two primary health care centres had been responsible for the care of the participants ahead of the study start may explain this remarkably high participation rate. Efforts to reduce systemic inflammation in patients with type 2 diabetes may be of utmost importance for the prevention of complications, such as cardiovascular disease and nephropathy. Carbohydrate restriction has been associated with anti-inflammatory effects, but, still, evidence is insufficient to support specific amounts of carbohydrate and fat intake in individuals with type 2 diabetes, as stated by American Diabetes Association in their recently published recommendations for dietary therapy (37). Our findings, however, indicate that the use of LCD aiming for 20 E% intake from carbohydrates may be an effective strategy to improve the subclinical inflammatory state in type 2 diabetes.

TITLE: Some Haematological and Biochemical Investigations on Duck Virus Hepatitis following Administration of Glycyrrhizin ABSTRACT: The present study aimed to investigate the protective effect of glycyrrhizin (locally isolated and purified from licorice root) against duck hepatitis virus through the assessment of some hematological and biochemical parameters. One hundred and sixty white Pekin ducklings—one day old—were randomly divided into four equal groups. Group (1) was kept as normal control. Group (2) was inoculated I/P with 10 mg glycyrrhizin/kg BW, three times per week for four weeks. Group (3) was inoculated I/M with 0.5 ml of live attenuated DHV vaccine. Group (4) was inoculated with both glycyrrhizin (10 mg/kg BW I/P, three times per week for four weeks) and live attenuated DHV vaccine (0.5 ml, I/M). Then, all groups of treatment were challenged using virulent DHV except for 20 ducklings from the normal control group which were continued to be kept as negative control. The results revealed that duck hepatitis virus (DHV) caused macrocytic hypochromic anemia, leukopenia, hypoproteinemia, hypoalbuminemia, hyperglycemia, hypercholesterolemia, and marked elevation of liver enzymes and renal parameters. In conclusion, glycyrrhizin injected alone or in combination with DHV vaccine protected or ameliorated the deteriorating effects induced by DHV vaccine and/or duck hepatitis virus infection by improvement of erythrogram and leukogram, as well as liver and kidney functions. BODY.1. INTRODUCTION: Duck virus hepatitis (DVH) is one of the most economic import diseases to all duck-growing farms because of its high potential mortality if the infection is not controlled. It is an acute highly fatal rapidly spreading viral infection of young ducklings. It was first recoded in New York and Taiwan. The morbidity is 100% and the mortality may reach 95–100%, in the first week of age [1]. Survived ducklings after DHV infection have a solid immunity, but it is necessary to protect the duck industry against such fatal disease using the potent-specific vaccine. Live attenuated DVH-1 vaccine which could be administrated through the intramuscular route in breeder ducks 2-3 weeks before lying allowing the transmission of high maternal immunity to the offspring providing them with passive immunity that is able to protect the new hatched birds up to 15 days of age. Also, it could be injected in 2-day-old ducklings followed by a booster dose 2-3weeks later [2]. In Egypt, duck farms are routinely protected against DVH following a vaccination program employing DVH-1 which is the only vaccine recorded in Egypt. Unfortunately, some duck farms stopped vaccination against DVH causing the recurrence of disease outbreaks [1]. Glycyrrhizin (GL) is the major active component extracted from licorice (Glycyrrhiza glabra) roots. GL has anti-inflammatory and antiviral effects that have been used in the treatment of patients with chronic hepatitis B and C [3–7]. GL enhances the production of antibodies through the production of interleukin- 1 (IL-1), IL-2, and IL-12 [8]. Soufy et al. [9] recorded that GL possesses a good immunostimulant and synergistic effect to DVH vaccine through activation of T lymphocyte proliferation. GL with vaccine enhances higher antibody titer against DHV than vaccine alone and high immunity protection. The present study was conducted to investigate the protective effect of glycyrrhizin injected alone or in combination with live attenuated DHV vaccine against experimental infection of ducklings with virulent duck hepatitis virus through the assessment of some haematological and biochemical parameters. BODY.2. MATERIALS AND METHODS: The experiment was done according to guidelines for animal experimentations and approved by the Institutional Animal Care and Use Committee, National Research Center, Animal Care Unite, Dokki, Giza, Egypt. BODY.2. MATERIALS AND METHODS.2.1. EXTRACTION, PURIFICATION, AND IDENTIFICATION OF GLYCYRRHIZIN FROM LICORICE PLANT: Two kg of licorice root (Glycyrrhiza glabra L.) was obtained from Haraz, Abdeen, Cairo, Egypt, for extraction of glycyrrhizin (GL). Purification of glycyrrhizin from licorice was performed according to Bentley and Trimen [10]. The extracted and purified substances were identified using thin layer chromatography (TLC) according to the method of Cui et al. [11]. Glycyrrhizin was administrated as stronger neo-minophagen C (SNMC). It consists of purified glycyrrhizin 2% + cysteine 0.2% + glycine 2% dissolved in physiological saline. It was inoculated 3 times weekly for 4 weeks [12]. Cysteine and glycine were added to avoid side effect of glycyrrhizin by increasing glutathione synthesis and prevent the sodium and water retention effect. BODY.2. MATERIALS AND METHODS.2.2. DUCKLINGS USED: One hundred and sixty white Pekin domestic ducklings one day old, obtained from a private nonvaccinated parent flock without maternal immunity were used in this study. Ducklings were housed under hygienic measures in separate isolators receiving a balanced growing broiler ration, containing protein 21%, fats 3.6%, and fibers 3.4% according to NRC [13]. BODY.2. MATERIALS AND METHODS.2.3. EXPERIMENTAL DESIGN: Ducklings were kept for 4 days for acclimatization and were allocated into 4 equal groups. Group (1) was kept as normal control (without glycyrrhizin treatment or vaccination). Group (2) was inoculated intraperitoneally (I/P) with 10 mg of glycyrrhizin/kg BW as SNMC (3 times weekly for 4 weeks). Group (3) was inoculated intramuscularly (I/M) with 0.5 mL of live attenuated DHV vaccine (obtained kindly from Vet Serum and Vaccine Research Institute, Abbassia, Cairo). Group (4) was inoculated with glycyrrhizin as SNMC (10 mg/kg BW, I/P, 3 times weekly for 4 weeks) and live attenuated DHV vaccine (0.5 mL) I/M. At the end of the 3rd week, all groups were challenged except 20 ducklings from the normal control group were continued to be kept as negative control. BODY.2. MATERIALS AND METHODS.2.4. HEMOGRAM AND BIOCHEMICAL ANALYSIS: Blood samples were collected from each duckling by jugular vein puncture before the challenge test at the end of the 1st, 2nd, and 3rd weeks and after the challenge at the 4th week of the beginning of the experiment. Each blood sample was divided into two portions; the first one was anticoagulated by disodium salt of ethylene diamine tetra acetic acid (EDTA) for determination of hemogram [14]. The second portion was placed in a plain centrifuge tube for serum separation and determination of biochemical constituents; total proteins [15], albumin [16], activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) [17], alkaline phosphatase (ALP) [18], glucose [19], creatinine [20], uric acid [21], and cholesterol [22]. Test kits supplied by bioMérieux-France were used. BODY.2. MATERIALS AND METHODS.2.5. CHALLENGE TEST: The ducklings of each group at the end of the 3rd week of treatment (at the 25th day of age) were challenged I/M with 0.5 mL virulent DHV containing 107 TCID50 per duckling. The strain was kindly supplied from Vet Serum and Vaccine Research Institute, Abassia, Cairo. BODY.2. MATERIALS AND METHODS.2.6. STATISTICAL ANALYSIS: Results are expressed as the mean ± SD. Differences between control and treated groups and differences between control-infected group and other groups after challenge were tested for significance using a one-way analysis of variance followed by least significant difference (LSD). Differences were considered significant at P < 0.05 level [23] using SPSS version 10 computer program. BODY.3. RESULTS.3.1. CLINICAL SIGNS: Before the challenge, ducklings of normal control and glycyrrhizin-treated groups (1 and 2) appeared healthy during the experimental period. However, vaccinated group (3) showed signs of illness in the form of depression, decreased food intake, ruffled feather, and dullness at the 2nd and 3rd days. These signs started to disappear at the 4th and 5th days after vaccination. On the other hand, ducklings of treated and vaccinated group (4) were slightly depressed, mildly anorexic at the 2nd and 3rd days after vaccination, and returned to normal at the 4th and 5th days. After the challenge test (at the 25th day of age), control infected group of ducklings showed severe depression, ruffled feather, and off food. Some ducklings were lying on their sides or breast with leg extended backward and head drown over the back with spasmodic paddling leg movement on the 3rd, 4th, 5th, and 6th days after challenge. The morbidity rate reached 80%, and the mortality rate reached 70%. Glycyrrhizin group (2) showed slight depression and general signs of illness at the 2nd day after challenge; one duckling had spasmodic paddling leg movement but returned healthy at the 3rd day. Neither mortality nor morbidity was recorded, and ducklings appeared healthy with normal size. In addition, ducklings of vaccinated group (3) showed moderate depression and general signs of illness at the 2nd day after challenge; one duckling died at the 3rd day after challenge. The survived duckling was emaciated and maintained stunted growth. In contrast, high protection rate (100%), healthy appearance, normal growth, and size were observed in glycyrrhizin-treated and -vaccinated ducklings (group 4) as shown in Table 1. BODY.3. RESULTS.3.2. ERYTHROGRAM: Compared to normal control group (1) before challenge, red blood cell count (RBC), packed cell volume (PCV) %, and haemoglobin (Hb) concentration showed significant increase in glycyrrhizin-treated group (2) from the 1st till the 3rd week. Glycyrrhizin-treated and vaccinated group (4) showed significant increases in RBCs count and PCV % at the 2nd week. Vaccinated group (3) showed significant decreases in the RBCs count, PCV %, and Hb concentration at the 1st and 2nd weeks. At the 4th week after challenge, RBCs count, PCV %, and Hb concentration showed significant decrease in all groups compared to the normal control group (Table 2). Vaccinated group (3) showed significant an increase in mean corpuscular volume (MCV) and a significant decrease in mean corpuscular haemoglobin concentration (MCHC) at the 1st week before challenge in comparison with normal control group (1). At the 4th week after challenge, significant increase in MCV with marked decrease in MCHC was demonstrated in control-infected and vaccinated groups, while no significant changes were observed in glycyrrhizin-treated group (2) and glycyrrhizin-treated and -vaccinated group (4). This data revealed the presence of macrocytic hypochromic anemia at the 1st week in vaccinated group and at the 4th week (after challenge) in control-infected and vaccinated group (Table 2). BODY.3. RESULTS.3.3. LEUKOGRAM: Before challenge, compared to normal control group (1), glycyrrhizin-treated group (2) showed significant leukocytosis all over the experimental period with lymphocytosis started from the 1st week and monocytosis started from the 2nd week till the end of the experiment. Vaccinated group (3) showed significant leukopenia at the 1st week associated with significant lymphocytosis at the 3rd week and monocytosis from the 1st week till the end of experiment, while significant decreases in heterophil and eosinophil count were detected from the 1st week till the end of the experiment. Glycyrrhizin-treated and -vaccinated group (4) showed leukocytosis, lymphocytosis, and monocytosis from 1st week till the end of the experiment (Table 3). At the 4th week (after challenge), compared to control negative group, significant leukopenia, heteropenia, and eosinopenia were detected in control-infected, vaccinated, treated and vaccinated, and treated groups, respectively, with significant lymphocytosis and monocytosis in glycyrrhizin-treated and -vaccinated, control-infected, and treated groups, respectively (Table 3). BODY.3. RESULTS.3.4. SERUM PROTEINS PROFILE: Before challenge, compared to normal control group (1), glycyrrhizin-treated group (2) showed significant increases in total proteins and globulins at the 2nd week with significant increases in albumin at the 2nd and the 3rd weeks. Vaccinated group (3) showed significant increase in total proteins with significant hyperglobulinemia at the 2nd week, while significant hypoalbuminemia was observed at the 1st and 2nd weeks with significant decrease in A/G ratio from 1st week till the end of the experiment. Glycyrrhizin-treated and -vaccinated group (4) showed significant increase in total proteins with significant hyperglobulinemia at the 2nd and 3rd weeks, while significant decreases were observed in A/G ratio from the 1st week till the end of the experiment except for significant increase at the 3rd week with no significant changes in albumin concentration (Table 4). At the 4th week (after challenge), control-infected group showed significant hypoproteinemia, hypoalbuminemia, and significant decreases in A/G ratio compared to normal control group (1). Glycyrrhizin-treated group (2) showed significant increase in total proteins with hyperglobulinemia and significant decrease in A/G ratio at the 4th week and no significant changes in albumin. Vaccinated group (3) and treated and vaccinated group (4) showed significant hyperglobulinemia, hypoalbuminemia, and significant decrease in A/G ratio at the 4th week (Table 4). BODY.3. RESULTS.3.5. SERUM TOTAL CHOLESTEROL: Before challenge, compared to normal control group (1) serum total cholesterol showed significant increase in vaccinated group (3) at the 1st week and significant decreases in glycyrrhizin-treated group (2) at the 3rd week, while no significant changes were noticed in glycyrrhizin-treated and -vaccinated group (4) (Table 4). At the 4th week (after challenge), compared to normal control group (1), control-infected group and vaccinated group (3) showed significant increase, and treated and vaccinated group (4) showed marked decrease, while treated group (2) showed no significant change in serum total cholesterol (Table 4). BODY.3. RESULTS.3.6. SERUM GLUCOSE: Before challenge, compared to normal control group (1), there is a significant hyperglycemia in vaccinated and treated and vaccinated groups at the 1st week, while significant hypoglycemia was noticed in treated and treated and vaccinated groups at the 3rd week. At the 4th week (after challenge) compared to control group (1), there is a significant hyperglycemia in vaccinated, control-infected, treated and vaccinated, and treated groups, respectively (Table 4). BODY.3. RESULTS.3.7. SERUM LIVER ENZYMES: Before challenge, compared to normal control group (1), activities of AST and ALT showed significant increases in vaccinated groups (3) at the 1st and 2nd weeks. ALP activities showed significant increase in vaccinated group (3) from the 1st week till the 3rd week, and treated and vaccinated group (4) showed significant increase at the 1st week, while no significant change was observed in treated group (2). At the 4th week after challenge, compared to control group (1) there is a significant increase in AST, ALT, and ALP activities in control-infected group, vaccinated group (3), and treated and vaccinated group (4) as shown in Table 5. BODY.3. RESULTS.3.8. SERUM CREATININE AND URIC ACID: Before challenge, compared to normal control group (1), vaccinated group (3) showed significant increase in uric acid and creatinine at the 1st week and at the 1st and 2nd weeks, respectively. On the other hand, no significant changes were noticed in treated group (2) and treated and vaccinated group (4). After challenge, compared to control group (1), control-infected group and vaccinated group (3) showed significant increases in uric acid and creatinine at the 4th week. Treated and vaccinated group (4) showed significant increase in uric acid at the 4th week, while treated group (2) showed no significant changes (Table 5). BODY.4. DISCUSSION: The present work was adopted to investigate the effect of GL as an antiviral and immunostimulant against DHV. This study is based on the assessment of haematological and serum biochemical responses. Glycyrrhizin (GL), the active principle of licorice (Glycyrrhiza glabra) has antiviral, anti-inflammatory, and immunostimulant effects [24]. In the present study, most of vaccinated group and treated & vaccinated group showed symptoms of depression, loss of appetite, ruffled feather, and dullness at the 2nd and 3rd days after vaccination and began to disappear at the 4th and 5th days after vaccination. This period gets along with the incubation period of the DVH which varied from 2 to 7 days and stress effect of vaccination. Similar results were reported by Asplin and Mclauchlan [25] and Mahdy [1]. The symptoms which were observed in treated & vaccinated group were less severe than those of vaccinated group may be referred to the immunostimulant and hepatoprotective effect by GL which might decrease the undesirable effect of live attenuated vaccine [26]. Regarding the results of the challenge test at the 25 day of age, the mortality rate was the highest in control-infected group as 70% of ducklings died at the 3rd, 4th, 5th, and 6th days after inoculation which could be attributed to the immunosuppressive and deteriorated effect of the virus on the liver, kidneys, and thymus gland. These results were parallel to these reported by Saif et al. [2] and Mahdy [1]. Vaccinated group showed 10% mortality which may be attributed to the protective effect of the vaccine, while treated group showed no mortality which may be due to the enterohepatic cycle of GL, antiviral, immunostimulant, anti-inflammatory, and antioxidant effects [24]. Treated and vaccinated group (4) showed no mortality or morbidity due to both efficiency and potancy of GL and vaccine in controlling the virus. Before challenge, results of erythrogram showed significant decrease in vaccinated group (3) at the 1st week and macrocytic hypochromic anemia then normocytic normochromic anemia at the 2nd week. This result could be attributed to loss of RBCs due to extravasation in different organs particularly in the liver and spleen of ducklings as mentioned by Mahdy [1]. Erythrogram of treated and treated and vaccinated groups showed significant increase which could be attributed to the hepatostimulatory and hepatoprotective effect of GL leading to production of more RBCs by the bone marrow under control of erythropoietic factors released by hepatic cells [27]. After challenge, results of erythrogram showed varying types of anemia in all groups which may be attributed to hemorrhage effect of DHV and vaccine on liver and spleen as reported by Campbell and Coles [28] and Mahdy [1]. The presence of different types of anemia may be due to bone marrow response to these hemorrhages [14]. Results of leukogram revealed leukocytosis in treated group (2) and treated and vaccinated group (4) before challenge. These results are similar to those of Al-Okbi et al. [29], who reported that licorice extract increased the total leukocytic count in rat. Leukopenia was demonstrated at the 1st week in vaccinated group (3) before challenge and in all groups after challenge which may be due to the depressing effect of DHV on leukopoietic parameters or the severe leukocytic infiltration in different organs. These results were in agreement with Latimer and Bienzle [30] and Mahdy [1] who reported that leukopenia may be due to some viral infections or live vaccine. Leukopenia was less significant in treated group (2) and treated and vaccinated group (4) which may be due to the immunostimulant effect of GL [9, 31]. The observed lymphocytosis with monocytosis in treated group (2) and treated and vaccinated group (4) may be due to the antigenic stimulation of GL through its stimulation to the mononuclear cells [32]. Moreover, lymphocytosis in ducklings may be secondary to antigenic stimulation by the virus [9], and also monocytosis may be observed in association with antigenic agent (infection) as it has phagocytic function [33]. Results of proteins profile revealed significant hyperproteinemia due to hyperglobulinemia at the 2nd week with significant hypoalbuminemia at the 1st and 2nd weeks in vaccinated group (3) before challenge. After challenge, significant hyperproteinemia due to hyperglobulinemia was observed at the 4th week which may be due to the effect of the virus or vaccine on the proliferation of B lymphocyte [33], while hypoalbuminemia may be due to the adverse effect of vaccine or virus on liver and kidneys. Treated group showed significant hyperproteinemia and hyperglobulinemia at the 2nd week before challenge. The hyperproteinemia may occur due to the effect of GL to preserve the normal functional status of liver [29, 34, 35]. After challenge, it showed significant hyperproteinemia with hyperglobulinemia which may be due to the immunopotentiating action of GL through activation of T cell and the effect of GL as an immune modulating and biological response modifier activities [31]. GL-treated and -vaccinated group showed significant hyperproteinemia at the 2nd week before challenge which may be attributed to the compensatory effect of GL on the deteriorating action of vaccine. After challenge, significant increased hypoalbuminemia was detected at the 4th week due to the effect of DHV infection. The hyperglobulinemia could be attributed to the immunopotentiating effect of GL and vaccine related to the proliferation of B lymphocyte which were converted to plasma cells producing immunoglobolins as a result of vaccine or viral infection [28, 33]. Furthermore, the control-infected group showed significant hypoproteinemia and hypoalbuminemia at the 4th week which may be attributed to the damaging effect of the virus on the liver and kidneys. The observed results in control-infected and vaccinated groups (3) get on the same hand with those obtained by Ahmed et al. [36], Ellakany et al. [37], and Mahdy [1]. Serum total cholesterol showed significant increase in vaccinated group (3) at the 1st and 4th weeks and in the control-infected group at the 4th week which may be attributed to the hepatic injury caused by the DHV or vaccine [38]. GL-treated group (2) showed significant decrease in total cholesterol at the 3rd week which may be due to the antioxidant effect of GL [24] or the effect of GL on increasing the lipid peroxides in liver [39]. Nonsignificant changes after challenge may be due to the hepatoprotective action and antiviral effect of GL [7, 40]. GL-treated and -vaccinated group (4) showed no significant changes in total cholesterol all over the experimental period except significant decrease at the 4th week which may be attributed to the damage of hepatocytes caused by DHV which bounds to lipids and proteins through subcellular fraction [41]. Before challenge, significant hyperglycemia was noticed in vaccinated and treated and vaccinated groups at the 1st week which may be attributed to the stress effect of vaccination which increases gluconeogenesis in the liver [42]. Treated and treated and vaccinated groups showed significant hypoglycemia at the 3rd week which may be due to the inhibitory action of licorice extract on the activity of 11 beta-hydroxysteroid dehydrogenase which converts cortisol to cortisone. This enzyme protects the mineralocorticoid in kidneys [43, 44]. After challenge, significant hyperglycemia was observed at the 4th week in all groups which may be attributed to the stress factor of infection which enhances the gluconeogenes and glycogenolysis in the liver [42]. These results disagree with Mahdy [1] who reported that DHV or vaccine causes hypoglycemia due to damage of liver which resulted in decreased hepatic gluconeogenesis [45]. Regarding liver enzymes, vaccinated group (3) showed significant increase in the activity of ALT and AST at the 1st and 2nd weeks with significant increase in the activity of ALP at the 1st, 2nd, and the 3rd weeks (before challenge) which may be due to the damaging effect of vaccine on liver as hepatic degeneration or necrosis causes leakage of these enzymes, so elevation of the serum levels of AST and ALT were noticed, while the obstruction in bile duct associated with bile duct hyperplasia causes elevation in ALP. After challenge, vaccinated group (3) and control infected groups showed significant increase in the activity of AST, ALT, and ALP at the 4th week which may be attributed to the damaging effect of DHV on liver hepatocyte and biliary canaliculi. This result was rather similar to that of Hochleithner [46], Ellakany et al. [37], and Mahdy [1]. Treated group (2) showed normal AST, ALP, and ALT patterns; which may be attributed to the antiviral, antioxidant, and hepatoprotective effects of GL as it enters the enterohepatic loop, excreted in bile then reabsorbed in the gut to recycle repeatedly through liver [26] or the action of GL in inhibiting the activation of phospholipase A2 [47]. Treated and vaccinated group (4) showed no significant changes in the activity of ALT and AST all over the experiment except a significant increase which was noticed at the 4th week. ALP showed significant increases at the 1st and 4th weeks. The increases in liver enzymes may be attributed to the damage effect of DHV on liver cells and canaliculi [1]. Results were less severe than that recorded in both vaccinated and control-infected groups. These results agreed with Al-Qarawi et al. [34] who reported hepatoprotective effect of GL in rat. Vaccinated group showed significant increase in uric acid at the 1st and 4th weeks and in creatinine at the 1st, 2nd, and 4th weeks. Control-infected group showed significant increase in uric acid and creatinine at the 4th week. Treated and vaccinated group (4) showed no significant changes in uric acid and creatinine at the 1st week but significant increase in uric acid at the 4th week. The increase in uric acid may be due to the damaging effect of DHV or vaccine on kidneys [1, 37]. Finally, DHV infection caused some pathognomonic clinicopathological changes (macrocytic hypochromic anemia, leukopenia, hypoproteinemia, hypoalbuminemia, hyperglobulinemia, hyperglycemia, hypercholesterolemia, and marked elevation of liver enzymes and renal parameters. Live attenuated vaccine gives high protection against DHV but causes some deleterious effects on liver and kidneys. GL has the ability to reduce the adverse changes associated with vaccine or infection through reduction of severity of the clinical signs associated with duck hepatitis vaccine or infection, minimizing the mortalities by the virus, GL alone, or with vaccine can reduce the drastic elevation of liver enzymes with considerable improvement in erythrogram, protein pattern, total cholesterol and glucose, and duckling general health condition. GL gives high protection and prevents the deteriorating action of vaccine and DHV infection. BODY.5. CONCLUSION: Glycyrrhizin injected alone or in combination with DHV vaccine protected or ameliorated the deteriorating effects induced by DHV vaccine and/or duck hepatitis virus infection by improvement of erythrogram and leukogram, as well as liver and kidney functions.

TITLE: Minimally invasive stereotactic puncture and thrombolysis therapy improves long-term outcome after acute intracerebral hemorrhage ABSTRACT: The purpose of this study was to judge the clinical value of minimally invasive stereotactic puncture and thrombolysis therapy (MISPTT) for acute intracerebral hemorrhage (ICH). A randomized control clinical trial was undertaken. According to the enrollment criteria, 122 acute ICH cases were analyzed, of which 64 cases received MISPTT (MISPTT group, MG) and 58 cases received conventional craniotomy (CC group, CG). The Glasgow coma scale (GCS) scores, postoperative complications (PC), and rebleeding incidences were compared. Moreover, 1 year postoperation, the long-term outcomes of patients with regard to hematoma volume (HV) <50 mL and HV ≥50 mL were judged, respectively, by the Glasgow outcome scale (GOS), Barthel index (BI), modified Rankin Scale (mRS), and case fatality (CF). MG patients showed obvious amelioration in GCS score compared with that of CG patients. The total incidence of PC in MG decreased compared with that of CG. The incidences of rebleeding in MG and CG were 9.4 and 17.2%, respectively (P = 0.243). There were no obvious differences between the CFs of MG and CG (17.2 and 25.9%, respectively, P = 0.199). The GOS, BI, and mRS representing long-term outcome for both HV <50 mL and HV ≥50 mL in MG were ameliorated significantly greater than that in CG patients (all P < 0.05). These data suggest that there are advantages with MISPTT not only in trauma and safety, but the MISPTT group had fewer complications and a trend toward improved short-term and long-term outcomes. BODY.INTRODUCTION: Spontaneous intracerebral hemorrhage (ICH) comprises 8–30% of all stroke victims, depending on regional and ethnic differences, and is a devastating form of stroke with the high mortality twofold to sixfold higher than that for ischemic stroke [1] and a 1-year survival rate less than 50% [2]. Hitherto, morbidity and mortality following ICH remain the highest among all forms of cerebrovascular diseases, with a 30-day mortality rate of 35–52%, with 50% of the deaths occurring in the first 2 days [3–5]. After initial irreversible tissue injury is suffered near the hemorrhage nidus, a progressive cascade of elevated local pressure, edema, and excitotoxicity cause additional secondary injury to surrounding areas [6–8]. Secondary brain injury by hematoma often occurs in the days following the initial hemorrhage and is intimately associated with significant neurological deterioration. Despite being considerably frequent, the treatment of hematomas within the basal ganglia continues to be a matter of debate among neurologists and neurosurgeons. The present powerful evidence provided by the results of the International Study of the Treatment of Intracranial Hemorrhage (STICH) corroborates this statement which has been established previously: there was not significant benefit for conventional aggressive surgical treatment over conservative medical treatment for the acute care of ICH [9]. Nevertheless, more than 7,000 patients with ICH in the United States undergo traditional evacuation procedures each year [10]. However, various clinical studies testing the hypothesis that clot burden plays a significant role in several forms of intracranial hemorrhage have been published in recent years, suggesting that clot reduction plays an important role in limiting brain edema and additional neuronal injury, as well as in reducing the severity of neurological deficits following ICH [11–14]. Because of the lack of validated therapeutic options for this form of stroke, the role of minimally invasive surgery (MIS) in the treatment of ICH has gained importance and several different operational methods have emerged over the past decade. In this context, our treatment with a stereotactic technique, which we have termed minimally invasive stereotactic puncture and thrombolysis therapy (MISPTT), is herewith presented. MISPTT is a novel operative technique for ICH, which was developed by Pro Jia in 1997. Although several clinical studies on MISPTT in the acute phase of ICH have been published during the past decade, the impact of MISPTT on the short-term and long-term neurological function of patients who survive the acute phase is less clear. The purpose of the present study was to investigate whether the long-term and short-term benefits of MISPTT are maintained and whether this method improves the ultimate outcome in these ICH patients. Therefore, we compared the long-term outcome 1 year after treatment in a consecutive series of ICH patients treated by MISPTT with the results achieved in a comparable group of patients who were treated by conventional craniotomy (CC). BODY.METHODS: A randomized control clinical trial was undertaken. All ICH patients were inhospital patients from 2005–2008 diagnosed with ICH, according to the ICH criteria drafted by ASA [15]. All cases were monitored in a dedicated stroke unit. The volume of the ICH in milliliters (mL) was estimated on the basis of approximate ellipse volume with the formula A × B × C/2, where A represents the largest diameter of the hematoma on axial CT slices in centimeters, B the diameter of hematoma perpendicular to A on the same slice, and C the number of CT slices in which the hematoma is visible multiplied by the slice thickness in centimeters [16, 17]. BODY.METHODS.INCLUSION AND EXCLUSION CRITERIA FOR PATIENTS: Inclusion criteria were as follows: (1) diagnosed as having spontaneous hemorrhage in the basal ganglion or brain lobe of the brain by CT scan; (2) hemorrhage volume: 30–100 ml; (3) age range: 40–75 years; (4) muscle strength of the paralyzed limbs: grades 0–3 on the muscle strength scale; (5) hemorrhagic duration (from stroke onset to hospital) within 24 h; and (6) informed consent from patients and/or their legal representative. Exclusion criteria were as follows: (1) disturbances of blood coagulation, e.g., thrombocytopenia, hepatitis; (2) traumatic intracranial hemorrhage; (3) intracranial or general infection; (4) complicated with serious heart, liver, renal, or lung disease or functional failure; (5) a previous stroke history with neurological deficits; (6) intracranial aneurysm or arteriovenous malformation complicated with hemorrhage; (7) consent form not obtained from the patient or their legal representative. There were 122 patients analyzed, of which 64 cases were treated with MISPTT and 58 cases with CC. There were no statistically significant differences in sex, age, GCS score, localization of bleeding, hemorrhage volume, or blood pressure level during hospitalization, duration of hypertension and the accompanying diseases in the two groups. Although the accompanying diseases such as diabetes, hyperlipidemia, coronary heart disease and cerebral infarction in the two groups were similar (all P > 0.05), diabetes accounted for 62.5% and 60.3%, respectively, in MISPTT group and CC group, and was perhaps one important risk factor for ICH onset (Table 1).Table 1The baseline characteristics of patientsGroupMISPTT patientsCC patientsP valueNumber of patients6458Gender (m:f)41:2338:200.866Mean age (years)58.7 ± 9.057.6 ± 8.60.483GCS score (n/%)0.998 4–57/10.96/10.3 6–922/34.420/34.5 10–1223/35.919/32.8 13–1512/18.813/22.4Hematoma volume (n/%)0.987 30–49 (mL)27/42.222/37.9 50–79 (mL)23/35.921/36.2 80–100 (mL)14/21.915/25.9Direction of the hematoma (n/%)0.468 Left-sided34/53.327/51.3 Right-sided30/46.731/48.7Location of the hematoma (n/%)0.344 Basal ganglia48/53.339/50.0 Thalamus16/17.819/24.4Mean BP SBP174.3 ± 14.1172.2 ± 11.00.361 DBP96.7 ± 9.798.8 ± 9.40.255Duration of BP (years)7.1 ± 1.97.4 ± 2.00.312Accompanied disease (n/%) Diabetes40/62.535/60.30.807 Hyperlipidemia27/42.222/37.90.632 Coronary heart disease20/31.321/36.20.563 Cerebral infarction14/21.915/25.90.605 BODY.METHODS.TREATMENT METHODS.MINIMALLY INVASIVE STEREOTACTIC PUNCTURE AND THROMBOLYSIS THERAPY: All operations were performed under local anesthesia and intravenous sedation unless the patient was already intubated for medical or neurological indications independent of the procedure. Stereotactic aspiration of the hematomas was performed in the acute phase between the 6th and the 24th hour after onset of stroke as required in the MISPTT protocol. First, the target points were defined according to the CT, and target points were generally chosen in the scan with the largest expansion of the hematoma and special attention was paid to stay away from important cortex function areas (Fig. 1). Puncture situs was measured and marked on the head, again avoiding main blood vessels, then a puncture needle of suitable length (type YL-1) was fixed to the operative electric drill. The puncture needle was entered to the predetermined depth, then the probe core was removed; the hematoma was withdrawn gently using a syringe (diluted with saline if blood thickened) until one-third of hematoma was removed, then a needle-like hematoma disintegrator was inserted. When no more blood could be withdrawn, the hematoma cavity was thoroughly rinsed with saline, until the saline fluid was clear.Fig. 1The slice of the largest hematoma area and puncture point in the CT are illustrated. AB median sagittal line, CD precoronal line, EF lateral sagittal line, P puncture point of hematoma, H center of hematoma, G puncture point of lateral cerebral ventricle, PH puncture depth, L lateral cerebral ventricle To confirm gross hematoma evacuation, an immediate postoperative CT scan was taken to assess the puncture needle placement and residual hematoma volume. If the placement of puncture needle was at the center of hematoma and stable, without rebleeding, a drainage bag connected to the puncture needle was hung 10 cm upon the head and switched for drainage. Upon rebleeding, 1 ku reptilase or 1 mg adrenalin should be injected into the hematoma, drained after 0.5 hour, and rinsed after 6–8 hours. All patients were treated in a dedicated intensive care unit, where subsequent thrombolysis and clot drainage were performed at the bedside using sterile techniques. The hematoma was continuously liquefied by liquefacient (containing 20,000–40,000 U urokinase/2–3 mL saline solution) for 2–4 days (3–5 times/day).On the 1st, 3rd, 5th, and 7th day of postoperation, patients were re-examined using CT. For hematomas that are difficult to liquefy, it should be liquefied as far as possible at first, followed by repeated, gentle aspiration using an agitation maneuver of the liquefacient to achieve vacuity. For hematomas affecting one lateral cerebral ventricle, then only that hematoma cavity should be perforated, but for patients with a hematoma affecting both lateral cerebral ventricles and the casting mould, the hematoma cavity and the contralateral cerebral ventricle should be perforated simultaneously. In addition, for those patients with a hematoma breaking into the lateral cerebral ventricle, lumbar puncture could be performed to repeatedly replace cerebral spinal fluid using NS until the color is pale for 4–5 days (once/day). When the hematoma was cleared or the remaining volume was less than 10 mL, the general condition of the patient was stable and intracranial pressure was normal; the puncture needle was removed, if no intracranial hypertension developed after occluding the drainage tube for 24 hours. The puncture needle was removed at the bedside under sterile conditions, and a single suture was placed at its exit site and covered with an occlusive dressing. In addition, all patients received optimal medical treatment. BODY.METHODS.TREATMENT METHODS.CONVENTIONAL CRANIOTOMY: Between 6 and 24 hours after onset, clearance of the hematoma by traditional craniotomy with large bone flap removal was performed in the Department of Neurosurgery. The medical treatment was routinely performed. BODY.METHODS.TREATMENT METHODS.FOLLOW-UP AND OUTCOME ASSESSMENT: Evaluation of all 122 patients followed identical criteria. Initial assessment included baseline characteristics (age, gender, HV, etc). The pretreatment clinical state of this cohort was assessed according to the GCS. All 122 cases in the two groups could be matched with regard to baseline characteristics. The posttreatment clinical state of the patients was assessed according to the GSC score, incidence of complications, and recurrence of bleeding after surgery. Outcome 1 year after stroke was the major endpoint. Four outcome parameters commonly used to assess outcome were employed to study the full impact of hemorrhagic stroke on long-term follow-up. Total case fatality was defined 1 year after stroke. According to the Glasgow outcome scale (GOS), clinical outcomes were graded, ranging from good recovery (GOS 5) to dead (GOS 1). The performance and functional status of the patients in activities of daily living (ADL) were measured with the Barthel index (BI 0–100). Disability was assessed using the modified Rankin scale (mRS 0–5). The data of all four outcome parameters for the two treatment groups were analyzed and compared. BODY.METHODS.TREATMENT METHODS.STATISTICS: Categorical variables were analyzed using the χ2 test or Fisher's exact test for small samples. Measurement data were analyzed using t tests. All recorded data were input using Epi Info software and statistically analyzed using SPSS 11.5 statistical software. For all analyses, P < 0.05 was considered to be statistically significant. BODY.RESULTS.COMPARISON OF CONSCIOUSNESS LEVEL AFTER SURGERY, GCS SCORE, AND THE INCIDENCE OF COMPLICATIONS BETWEEN THE TWO GROUPS: The GCS scores between the two groups prior to the operation were not significantly different (P = 0.998). The total incidence of complication in the MISPTT group was lower than that of the CC group (P = 0.000). Pulmonary infection, digestive tract hemorrhage, and epilepsy in the MISPTT group were all lower than that observed in the CC group (P = 0.047, P = 0.016, and P = 0.010, respectively). The incidence of bleeding recurrence was not significantly different between the two groups (P = 0.199) (Table 2). Typical examples from the study are shown; for example, CT scans of one coma patient (GCS score 5) with a huge hematoma (>70 mL) are shown in Fig. 2. There were obvious difference in the HV and edema of surrounding brain before MISPTT with a GCS score of 5 and 2 weeks after MISPTT with a GCS score of 14. The patient was self-sufficient 1 year after on-set (Fig. 2).Table 2Consciousness level after surgery, GCS score, and incidence of complicationsGroupMISPTT patientsCC patientsP valueNumber of patients6458GCS score Before operation8.7 ± 1.98.4 ± 2.20.445 After operation11.0 ± 1.28.1 ± 1.00.000Incidence of complication (n/%)20/31.346/79.30.000 Pulmonary infection6/9.413/22.40.047 Digestive tract hemorrhage10/15.620/34.50.016 Epilepsy4/6.313/22.40.010Bleeding recurrence (n/%)6/9.410/17.20.199Fig. 2CT scans of a patient in a coma (GCS score 5) with a large hematoma (>70 mL) before MISPTT (top), and CT scans of the same patient during consciousness (GCS score 14) 2 weeks after MISPTT (bottom). These scans demonstrate the size of the hematoma on serial axial CT images. CT scans 2 weeks after MISPTT showed obvious amelioration in hematoma volume and edema in the surrounding brain compared with that prior to MISPTT. The patient was able to completely return to a normal lie 1 year after on-set BODY.RESULTS.COMPARISON OF LONG-TERM OUTCOME AND TOTAL CASE FATALITY OF THE TWO GROUPS 1 YEAR AFTER STROKE: There were no sharp differences in case fatality between the MISPTT group and the CC group 1 year after stroke (17.2 and 25.9%, respectively, P = 0.243); however, on the whole, the results showed that the survival in the MISPTT group was significantly better than that of the CC group. There were 27 cases with a HV <50 mL in the MISPTT group and there were no deaths 1 year postoperation, and 22 cases with a HV <50 mL in the CC group, of whom 2 had died within 1 year postoperation. Three parameters representing long-term outcome, the GOS (4.4 ± 0.6), BI (82.6 ± 9.5), and mRS (1.8 ± 1.0) in the MISPTT group with a HV <50 mL had significantly ameliorated compared to those in the CG group (GOS 3.4 ± 1.1, BI 73.0 ± 14.6, and mRS 2.7 ± 1.3, respectively) (P = 0.000, P = 0.009, and P = 0.009, respectively). There were 37 cases with a HV ≥50 mL in the MISPTT group, of whom 11 patients had died 1 year postoperation, while there were 36 cases with a HV ≥50 mL in the CC group and 13 patients had died 1 year postoperation. The GOS (3.3 ± 1.6), BI (73.3 ± 15.6), and mRS (2.2 ± 1.2) in the MISPTT group with a HV ≥50 mL had significantly ameliorated compared to those in the CG group (GOS 2.5 ± 1.5, BI 62.2 ± 21.5, and mRS 3.0 ± 1.6, respectively) (P = 0.040, P = 0.042, and P = 0.033, respectively). The results are shown in Table 3.Table 3Outcome 1 year after strokeGroupMISPTT patientsCC patientsP valueNumber of patients6458Case fatality (n/%)11/17.215/25.90.243GOS HV < 50 mL4.4 ± 0.6 (27 cases)3.4 ± 1.1 (22 cases)0.000 HV ≥ 50 mL3.3 ± 1.6 (37 cases)2.5 ± 1.5 (36 cases)0.040BI HV < 50 mL82.6 ± 9.5 (27 cases)73.0 ± 14.6 (20 cases)0.009 HV ≥ 50 mL73.3 ± 15.6 (26 cases)62.2 ± 21.5 (23 cases)0.042mRS HV < 50 mL1.8 ± 1.0 (27 cases)2.7 ± 1.3 (20 cases)0.009 HV ≥ 50 mL2.2 ± 1.2 (26 cases)3.0 ± 1.6 (23 cases)0.033GOS Glasgow outcome scale, mRS modified Rankin Scale, BI Barthel index, HV Hematoma volume BODY.DISCUSSION: The mass effect of a hematoma can lead to the brain damage, including intracranial hypertension or cerebral hernia [18]. There is, however, some evidence that the mass effect caused by a HV (<60 mL) was not the dominant injury mechanism, whereas the toxic substances released from the hematoma were the most important factor in the pathological mechanism of the cerebral hemorrhage [19–21]. It was reported that elevated levels of glutamate were found in the perihematomal region after ICH and these levels decreased after hematoma drainage. Conversely, ischemic LPRs were not found in perihematomal regions and were unchanged after hematoma drainage. These data suggest that excitotoxicity related to glutamate may have an important impact on secondary injury. The data failed to support the role of ischemia in secondary perihematomal damage [6]. Thus, effective removal of the hematoma during the acute phase is a crucial principle in the treatment of ICH in order to save lives and improve long-term quality of life. CC by removing a bone flap is a classical technique for the treatment of ICH, which is characterized by good visualization, complete clearance of the hematoma, easy hemostasia, and complete reduction of pressure, but also has some shortcomings such as length of operation, severe brain damage due to manipulation of the brain during the operation, damage of brain tissue around the hematoma by electrocoagulation, re-bleeding readily, pathophysiological changes postoperation (e.g., disturbance of water and electrolyte balances, fluctuation of blood sugar, instability of life signs), which result in severe impairment of neurological function, multiple complications, higher invalidism rates and fatality rates. With regard to basal ganglia hemorrhages, comparing the outcome of patients treated surgically with that of patients managed conservatively, many earlier and current publications showed no benefit from conventional surgery [9, 10, 22–25]. Only a few reports have demonstrated a trend towards better outcomes with conventional surgery [26–28]. While analyzing the given data, it becomes obvious that the major drawback in all these studies is the heterogeneity of the ICH patient groups with regard to their preoperative neurological status due to the very different degrees of neurological impairment and no uniform consciousness levels, applying different surgical approaches, and different intervals with regard to the onset of the hemorrhagic stroke. Thus, it is an essential issue to select appropriate patients and a homogenous group to determine whether patients truly benefit from the neurosurgical stereotactic evacuation of the hematomas in the acute phase. In many recent studies, the minimally invasive methods have shown to be highly efficient with little risk of re-bleeding and better short-term outcomes [29–31]. Presently, some clinicians are exploring new methods to elevate curative effect of minimally invasive operation techniques. A study by Marquardt and coworkers [32] focused on the use of a novel multiple target aspiration technique in 64 patients to aspirate a "sufficient proportion" of the hematoma with minimal risk for the patient. More than 80% of the hematoma volume was successfully aspirated in 73.4% of the patients with only one episode of re-bleeding. Montes et al. [33] showed that CT-guided thrombolysis and aspiration was safe and effective in the reduction of ICH volume. In the meantime, they proposed further studies were needed to assess optimal thrombolytic dosage, including controlled comparisons of mortality, disability outcome, time until convalescence, and cost of care in treated and untreated patients. MISPTT is a new and novel operative technique that is obviously different from other types of minimally invasive operations in design principle, which was developed by Pro Jia in 1997 with a distinctive thrombolysis installation, and highly safe and efficient function for dissolving and draining coagulated blood. This treatment had been widely applied in China. According to some studies in the past, it was presented that MISPTT in acute ICH efficiently cleared the hematoma, relieved hydrocephalus, reduced intracranial hypertension, and relieved the cytotoxicity of blood thrombin. Furthermore, the washing liquor decreased the levels of cytotoxic substances. In subacute hemorrhage, MISPTT reduced the neurotoxicity of the hemoglobin and its decomposition products. This technique is characterized by its simplicity and is not limited by equipment. Puncturing the brain in MISPTT does little harm to the brain and accelerates recovery of cerebral function, while the liquefaction technique contributes to liquefying the blood coagulum—all helping to shorten the course of disease. In the operations performed, patients were only treated with a 3 mm needle (diameter). Because there is no gap between the needle and skull, the incidence of infection is reduced. Furthermore, it is not necessary to open the skull and use general anesthesia; thus, this procedure is more economical than other operations. The Chinese National Research and Extension Community of the Minimally Invasive Operation suggested that MISPTT is suitable for cases having a hemorrhage volume >30 mL in the basal ganglia and further standardized the operation indication, operation procedure, and the methods to apply the hematoma liquefacient, according to the random sampling of The Ministry of Public Health. Although there are many studies investigating the minimally invasive operation indication for ICH, few specially concerned the indication for the MISPTT. Furthermore, most of these investigations lacked control groups with uniform baseline characteristics prior to operation, did not analyze the correlation factors (e.g., GCS score, hematoma volume, hematoma location, duration of BP and accompanied diseases), and did not observe the long-term outcome for survival. In our study, the above shortcomings were overcome by the inclusion of many factors, e.g., preoperative neurological status, surgical approach, patient selection, GCS score, incidence of complications, rebleeding after surgery, and long-term outcome 1 year after onset. In addition, accompanying diseases in the two groups of ICH patients were observed, such as diabetes, hyperlipidemia, coronary heart disease and cerebral infarction. There were no statistically significant differences between the two groups (all P > 0.05), but it was noticed that diabetes accounted for 62.5% and 60.3%, respectively, in MG and CG, which indicated that diabetes was perhaps one important risk factor for ICH onset (Table 1). The results showed that the level of consciousness and GCS in MISPTT were better than that of the CC group. Of the 13 patients with GCS scores of 4–5 in the two groups, 11 patients died (84.6%); the two survivors were treated with MISPTT (Table 1, one case referring to Fig. 2). The incidence of complications (e.g., pulmonary infection, hemorrhage of the digestive tract, and epilepsy) in MISPTT was obviously reduced compare to the CC group. There were no cases of intracranial infection in either the MISPTT or the craniotomy group. The statistical analysis did not show a significant difference on the rebleeding incidence in the two groups (9.4 and 17.2%, respectively, P = 0.199). There were no obvious differences between the case fatalities in the MISPTT group and the CC group (17.2 and 25.9%, respectively, P = 0.243). The long-term outcome in the MISPTT group surpassed that of the CC group, with respect to GOS, mRS, and BI (all P = 0.000). Currently, the thrombolysis methods have only been partly explored. We used UK as thrombolysis method in our MISPTT study. In another cohort of ICH patients treated using FAST, volumetric analysis of ICH and perihematomal edema seemed to suggest that local use of rtPA for thrombolysis, which differed from the UK used in our MISPTT study, does not exacerbate brain edema formation. Furthermore, there seems to be a strong association between reduction of the ICH volume and reduction of edema volume, as would be expected following the concept of "hemotoxicity" postulated by some investigators [34]. These above results indicate that for the ICH patient meeting MISPTT operation requirements, it is unsuitable to choose CC. However, if the patient has a huge hemorrhage volume, is rapidly deteriorating, or is in the early state of cerebral hernia, then a craniotomy should be selected to reduce cerebral pressure. In addition, another study [33] pointed out that although MIS on patients with cerebral hernia may not result in a good curative effect, the hematoma volume can be partially decrease and the intracranial pressure can be rapidly reduce, thus, gaining time for craniotomy. Of course, CC is occasionally superior in the treatment of ICH with bulk volume. Murthy et al. [35] combined decompressive craniectomy and hematoma clearance to cure 12 patients with hemorrhage volumes >60 mL in the right hemisphere. As a result, 11 survivors were able to leave the hospital (92% survival rate), while in 6 cases, the survivors recovered well. BODY.CONCLUSION: Larger randomized trials proving the increased benefits of MISPTT treatment over traditional craniotomy or purely medical management are currently in process. However, our study and some other treatment trials regarding this treatment modality have reported optimistic results, showing superiority of MMISPTT over CC or PMT. Several methodological issues surrounding this form of treatment remain to be resolved, including comparison of the relative efficacies of various mechanisms of clot thrombolysis and drainage. If successful, MISPTT for ICH may perhaps become an important tool in the growing treatment armamentarium for ICH and the potential for a disease modifying impact in these ICH patients. It is our hope that this ongoing study will bring us closer to other randomized tests regarding more relevant factors with MISPTT as an alternative treatment for ICH. Objectively, MISPTT is currently limited in its ability to achieve hemostasis and completely evacuate the hematoma. Proper selection of the ICH patient group to receive this type of therapy may hold the key to these advances.

TITLE: A randomized pilot phase I study of modified carcinoembryonic antigen (CEA) peptide (CAP1-6D)/montanide/GM-CSF-vaccine in patients with pancreatic adenocarcinoma ABSTRACT.BACKGROUND: CEA is expressed in >90% of pancreatic cancers (PC) and may be an appropriate immunotherapy target. CEA is poorly immunogenic due to immune tolerance; CAP1-6D, an altered peptide ligand can help bypass tolerance. We conducted a pilot randomized phase I trial in PC patients to determine the peptide dose required to induce an optimal CD8+ T cell response. ABSTRACT.METHODS: Patients with a PS 0-1, HLA-A2+ and CEA-expressing, previously-treated PC were randomized to receive 10 μg (arm A), 100 μg (arm B) or 1000 μg (arm C) of CEA peptide emulsified in Montanide and GM-CSF, given every 2 weeks until disease progression. ABSTRACT.RESULTS: Sixty-six patients were screened and 19 enrolled of whom 14 received at least 3 doses of the vaccine and thus evaluated for the primary immunologic endpoint. A median of 4 cycles (range 1-81) was delivered. Median and mean peak IFN-γ T cell response by ELISPOT (spots per 104 CD8+ cells, Arm A/B/C) was 11/52/271 (A vs. C, p = 0.028) for medians and 37/148/248 (A vs. C, p = 0.032) for means. T cell responses developed or increased in 20%/60%/100% of pts in Arms A/B/C. Seven of the 19 patients remain alive at a minimum 32 months from trial initiation, including three with unresectable disease. ABSTRACT.CONCLUSIONS: The T cell response in this randomized phase I trial was dose-dependent with the 1 mg CEA peptide dose eliciting the most robust T cell responses. A signal of clinical benefit was observed and no significant toxicity was noted. Further evaluation of 1 mg CEA peptide with stronger adjuvants, and/or combined with agents to overcome immune inhibitory pathways, may be warranted in PC pts. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT00203892 BODY.BACKGROUND: Pancreatic cancer (PC) is a devastating disease. With an estimated annual incidence of 43,920 in the United States in 2012, it is the fourth leading cause of cancer mortality with 37,390 deaths annually [1]. Currently, the only possibility of cure is through surgical resection, but only approximately 15%–20% of patients are eligible candidates at diagnosis and even with surgery the 5-year overall survival in multiple series is 10%-30% [2]. In those with locally advanced or unresectable disease (~30%–40% of patients) the median overall survival (OS) has been between 8 and 14 months. Patients with metastatic disease (~40%) had a historical median OS of approximately 6 months, although recently substantial progress has been made in chemotherapy options for these patients [3]. Due to the inherent chemotherapy- and radiation-resistance of pancreatic cancer cells, immunotherapy has been explored as a treatment modality in PC patients since the 1990s with a recent review focused on this topic [4]. Multiple vaccination platforms have been developed and tested in advanced solid malignancies including PC, both in metastatic and adjuvant settings. Most of the vaccination techniques have focused on promoting a tumor antigen-specific T cell response using either vaccination or adaptive transfer of tumor-specific T cells in order to increase immune-mediated tumor inhibition [5]. Identification of specific tumor-associated antigens (TAAs) in PC has led to numerous trials of vaccination with these peptides as this approach is safe and relatively simple [6-13]. The goal of this technique is to elicit a CD8+ cytolytic T lymphocyte (CTL) response that is specific for the TAA and subsequent killing of tumor cells harboring that antigen by those CTLs. An example of a TAA, carcinoembryonic antigen (CEA), is a 180-kDa immunoglobulin-like molecule that is expressed on the cell surface, is overexpressed in over 90% of pancreatic adenocarcinoma and functions in cellular adhesion [12,14,15]. CEA as a self-protein has been shown to be poorly immunogenic and thus modifications have been made to enable better binding to the MHC-I complex. CAP-1 (YLSGANLNL), an epitope of CEA, was further modified to CAP1-6D (an Asn to Asp substitution in the CEA sequence; YLSGADLNL), and is an enhanced agonist peptide binding to HLA-A2. Via a change in the interaction with the T cell receptor it produces a more potent CTL response and T cells generated via this approach have been shown to be cross-reactive with wildtype CAP1 and to recognize CEA+ HLA-A2+ tumor cells [16,17]. A number of trials have employed the CEA TAA in various vaccination platforms for patients with CEA-expressing tumors. The early trials showed that the approach was safe in various platforms and with GM-CSF as an adjuvant [18-21]. In the trials which incorporated CEA as an antigen, only one to our knowledge was conducted and reported exclusively in PC patients [22]. In that phase I study, a poxvirus targeting CEA and MUC-1 along with B7.1, ICAM-1 and LFA-3 (TRICOM) (PANVAC-V) was administered followed by booster vaccinations using PANVAC-F (fowlpox virus with same antigens) as well as GM-CSF in ten advanced PC patients. They noted an antibody response in all 10 patients and antigen-specific T cell responses in 5 out of 8 evaluable subjects. A significant increase in OS was observed in those who mounted an anti-CEA and/or MUC-1 response (15.1 m vs. 3.9 m; P = 0.002). Several other studies used CEA vaccination alone or with other epitopes such as MUC-1, with or without radiotherapy and with various platforms, but most patients in those trials did not have PC [23-27]. In general, the vaccines were all safe, elicited an immunologic response in a significant number of patients, and led occasionally to sustained clinical responses. Given the above considerations, we believed it was desirable to explore further CEA-based vaccination in PC patients. To eliminate the necessity of obtaining patient-specific DCs, we utilized tumor antigen peptide emulsification in Montanide adjuvant along with GM-CSF, which has been suggested to stimulate DC differentiation and improve DC recruitment [28,29]. Because the appropriate dose of a CEA-based vaccine to elicit the most robust response was unknown, we designed a randomized phase I study to determine the most appropriate peptide dose. This approach would enable us to ascertain whether an immune response could be elicited, and could establish the most effective dose of the CEA peptide within the vaccine formulation to use in further trials. We report the results of this randomized phase I pilot trial of a CAP1-6D/Montanide/GM-CSF-vaccine in PC patients (ClinicalTrial.gov ID: NCT00203892). BODY.METHODS.TRIAL DESIGN: The study was designed as a randomized pilot trial with the primary endpoint to determine the dose of modified carcinoembryonic antigen (CEA) peptide (CAP1-6D)/Montanide/GM-CSF-vaccine required to induce an optimal CD8+ T cell response and to determine whether this response can be assessed by ELISPOT. Defining the dose limiting toxicities (DLTs), progression free survival and median overall survival were secondary endpoints. The laboratory objective was to determine whether this immunization elicits a specific T cell response as assessed by IFN-γ ELISPOT, against both the CAP1-6D and the native peptide. The study was approved by the University of Chicago Institutional Review Board and conducted at the University of Chicago. All patients were required to give written informed consent in accordance with federal, state, and institutional guidelines. BODY.METHODS.ELIGIBILITY: Patients were required to have an Eastern Cooperative Oncology Group performance status of 0 or 1 and adequate hematologic, hepatic, and renal function. Although originally designed to include only those treated definitively with no evidence of disease (NED) or those with locally advanced PC, the inclusion was later expanded to allow for those with metastatic PC in order to improve accrual. Eligible patients must have expressed HLA-A2 and have histologically or cytologically confirmed adenocarcinoma of the pancreas that expresses CEA either by IHC or serology. Life expectancy of over 3 months, no systemic treatment within 28 days of trial initiation (6 weeks for nitrosoureas or mitomycin C), and age of 18 or older were also required. Exclusion criteria included previous CEA vaccine; history of allergic reaction to compounds of similar chemical or biologic composition to CEA, Montanide ISA-51 or GM-CSF; known autoimmune disorders; conditions of immunosupression such as HIV or treatment with immunosuppressive drugs; pregnancy or breast-feeding; currently active second malignancy; and uncontrolled intercurrent illness. BODY.METHODS.VACCINE FORMULATION AND TREATMENT PLAN: The vaccine contained the modified CEA peptide (CAP1-6D; YLSGADLNL; Multiple Peptide Systems, San Diego, CA) together with Montanide ISA-51 (Seppic, Inc.) as an adjuvant, and sargramostim (GM-CSF; purchased commercially) 250 μg. Vaccine emulsions were prepared in the University of Chicago cGMP facility. Briefly, the appropriate dose of CEA peptide was thawed, combined with 0.9 ml of sterile saline and mixed with GM-CSF and Montanide ISA-51 using the two-syringe method to make an emulsion. To verify creation of an emulsion, a drop was placed in a dish of sterile water and if the drop did not disperse, the emulsion was considered to be a success. Patients were seen for a baseline evaluation which included a history and physical, CT/MRI, blood chemistries, complete blood count with differential, and HLA typing. Confirmation of CEA expression was established either by IHC on the original tumor or by elevated serum CEA levels. Only subjects positive for HLA-A2 and with evidence for CEA expression continued on the study. A cycle was defined as 14 days and vaccine was administered on Day 1 of each cycle until progressive disease or dose-limiting toxicity for a maximum of 24 cycles with delays of greater than 28 days leading to study withdrawal. The vaccine administration site was the proximal thigh with each subsequent administration in the same approximate location. Following the initial administration, patients were seen prior to each vaccine administration with ELISPOT and CA19-9 performed every 4 weeks for the first 8 cycles and a CT/MRI every 8 weeks. After the eighth cycle, CT/MRI and CA 19-9 was repeated every 8 weeks thereafter for one year until disease progression or DLT with ELISPOT performed at the time of disease progression. After one year, CT scans were done every 16 weeks for one year, followed by every 6 months for one year and then annually until progression of disease. BODY.METHODS.RESPONSE CRITERIA AND TOXICITY: Clinical response was evaluated using the Response Evaluation Criteria in Solid Tumors v1.0 [30]. Dose-limiting toxicity was defined as Grade 2 or greater hemorrhage or allergic reaction; any other Grade 3 or greater toxicity or clinical evidence of autoimmune disease. All adverse events were graded according to the CTCAE v2.0. BODY.METHODS.COLLECTION OF PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS) AND PREPARATION OF CD8: Heparinized blood was drawn before treatment, monthly during the vaccination period, and at the end of study. Samples were collected prior to a given treatment administration. PBMCs were isolated using Lymphoprep gradient centrifugation and cryopreserved for immune assays. CEA-specific CD8+ CTL responses were tested by IFN-γ ELISPOT, against both the modified CEA (CAP1-6D) and the wild-type peptide. Poor CEA-specific CD8+ CTL responses were detected by a direct ex vivo assay, so a short in vitro expansion was performed. Briefly, PBMCs were thawed and washed twice with PBS. CD8+ T cells were isolated using anti-CD8 micro beads (Miltenyi Biotech). The non-CD8+ population was pulsed with modified CEA peptide (50 μM) at the presence of β2-microglobulin for 1 hour at 37 ̊C. The cells were then washed twice with AIM-V media and irradiated at 3000 rad. Purified CD8+ T cells were stimulated with irradiated peptide-pulsed CD8- cells along with IL-2 (10 U/ml) for 5 days. On day 5, the CD8+ T cells were collected and re-stimulated with freshly prepared irradiated peptide-pulsed CD8- cells and IL-2 (10 U/ml) for another 5 days. On day 10, the expanded CD8+ T cells were collected, counted, and re-stimulated with peptide-loaded T2 cells for IFN-γ ELISPOT analysis. BODY.METHODS.ELISPOT ASSAY: Briefly, 96 well multiscreen filter plates were prepared by coating overnight with anti-INF-γ mAb (10 μg/ml), washing 3× with PBS, and blocking 1 hour with AIM-V medium containing 10% human AB serum. Expanded CD8+ T cells (10,000/well) were added along with T2 cells (50,000/well) previously loaded with wild type CEA peptide (CAP1), modified CEA peptide (CAP1-6D) and a G250 negative control peptide (50 μM each). Following a 20 hour culture, wells were washed 3 times with ELISPOT wash buffer, incubated 2 hours with a biotinylated anti-IFN-γ secondary Ab, washed 3 times, incubated 1 hour with streptavidin-conjugated AP, washed, and incubated with AP substrate. Excess substrate was removed by rinsing with tap water. Plates were captured and counted using a CTL-ImmunoSpot S6 Core Analyzer from Cellular Technology Ltd (Cleveland, OH). Stimulation with PMA + Ionomycin was used as a positive control for the integrity of the T cell samples. All samples were analyzed in triplicate, and the mean response to the G250 negative control was subtracted from each sample. A positive ELISPOT response for a patient was defined as a minimum increase from baseline in a CTL ELISPOT assay by 50 CEA spots/1×104 CD8+ T cells and a 20% increase over baseline: [(peak ELISPOT-baseline ELISPOT)/baseline ELISPOT] × 100 ≥ 20% and a peak ELISPOT ≥ 50 spots. BODY.METHODS.STATISTICAL CONSIDERATIONS: Patients were randomized into one of three dose levels. A sample size of 15 evaluable patients was planned to obtain sufficient T cell response data in each cohort. The sample size was based on a recombinant avipox vaccine study by von Mehren et al. in patients with recurrent CEA-expressing adenocarcinomas [24]. Acceptable toxicity was defined as a DLT observed in no more than 20% of patients at a given dose. To be included in the analysis a patient must have had a baseline ELISPOT and then at least 3 cycles of the vaccine with an additional ELISPOT done after the 3rd cycle. The maximum T cell frequency achieved was used in the calculation. Descriptive statistics were used to describe the characteristics of the study population. Data was analyzed for overall survival and progression free survival using the Kaplan-Meier estimates from the start of treatment and comparison between the treatment arms using the log-rank test. Elispot responses were compared using Mann–Whitney Rank Sum Test with a P value based on a two-tailed test and P < 0.05 considered statistically significant. Univariate Cox regression models examining CA19-9 levels, ELISPOT and stage (metastatic vs. non-metastatic) were done for PFS and OS. BODY.RESULTS.PATIENT CHARACTERISTICS: Sixty-six patients were screened, twenty-three enrolled, and nineteen received at least a single dose of the vaccine (Figure 1). Patients were randomized to receive 10 μg (arm A), 100 μg (arm B) or 1000 μg (arm C) of peptide in the vaccine preparation. Forty-three subjects were screen failures: 36 due to lack of HLA-A2 expression, four for withdrawing of consent, one for incorrect pathology, one for lack of CEA expression by IHC, and one for an elevated bilirubin. Nineteen patients received at least one dose of the vaccine; three patients experienced a declining performance status and one had a gastrointestinal bleed prior to administration of any vaccine. Fourteen patients received at least three doses of the vaccine and were eligible for the primary immunologic endpoint: 5 in arm A, 5 in arm B and 4 in arm C. The overall and per-group patient characteristics are listed in Table 1. Sixty-eight percent of patients were female. The median age was 60 (range 27–86) and 53% had an ECOG performance status 0. Three-quarters had metastatic disease, with all but one of the remaining patients having no evidence of disease at the time of enrollment. Figure 1Study Enrollment and Treatment. CONSORT Diagram. Table 1 Overall and per-treatment-group patient baseline characteristics Baseline characteristics Overall no. of patients (%) Arm A (%) Arm B (%) Arm C (%) Total 19 5 8 6 Age          Median 60 51 57.5 61.5  Range 27–86 27–71 50–86 47–77 Sex          Male 6 (32) 1 (20) 4 (50) 1 (17)  Female 13 (68) 4 (80) 4 (50) 5 (83) ECOG Performance Status          0 10 2 4 4  1 9 3 4 2 Stage at Trial Entry          No evidence of disease 4 (21) 2 (40) 0 (0) 2 (33)  Locally advanced 1 (5) 0 (0) 0 (0) 1 (17)  Metastatic* 14 (74) 3 (60) 8 (100) 3 (50) Prior Therapy          Chemotherapy alone 5 1 3 1  Chemotherapy/RT alone 2 0 1 1  Surgery total 12 4 4 4   Surgery followed by chemotherapy 7 3 2 2 5 1 2 2   Surgery followed by chemo/RT Prior surgical resection          Yes 12 (63) 4 (80) 4 (50) 4 (67)  No 7 (37) 1 (20) 4 (50) 2 (33) Serum CEA at trial initiation (ng/mL)          Median 4.8 5.3 4.65 7.95  Range 1–246.7 1.1–34.2 2.1–246.7 1–18.1 Serum CA-19-9 at trial initiation (ng/mL)          Median 532 22 1067 27  Range 3–96,900 3–2,710 3–96,900 3–30,300 Abbreviation: Chemo/RT = chemotherapy in combination with radiation; * All received previous chemotherapy. A total of 256 cycles were delivered (median 4, mean, 13.5, range 1–81). The initial protocol did not specify a maximum number of cycles and this was amended after the first patient received 81 cycles, at which time the number of cycles was limited to 24. No dose reductions were allowed nor did any occur. BODY.RESULTS.IMMUNOLOGIC RESPONSE: A positive CD8+ T cell response to CAP1-6D developed in 20% of patients in Arm A, 60% of patients in Arm B, and 100% of patients in Arm C. Five patients were not able to be assessed immunologically due to either less than 3 cycles of the vaccine administered (one patient) or for incomplete sample collection due to early disease progression (4 patients). The mean/median CAP1-6D CD8+ T cell response by ELISPOT in the 14 evaluable patients (spots per 104 CD8+ cells) was 36.5/10.5 in Arm A, 148.45/51.75 in Arm B and 247.69/270.625 in Arm C (Figure 2). A statistically significant difference in median (P = 0.028) and mean (P = 0.032) T cell response against the immunizing mutant peptide was observed between arm A and C. T cell responses to the wild-type CEA peptide followed a similar pattern and also were statistically significantly different between arm A and C (P = 0.028) (Figure 2). The kinetics of the magnitude of the T cell responses were tracked over time for all 14 patients stratified by treatment arm (Figure 3). In general, the peak immune response appeared to occur within the first 100 days and diminished thereafter. In the two of the three patients receiving the vaccine for over 4 months the response was still sustained and remained detectable. Figure 2Baseline and Peak ELISPOT results for 14 evaluable patients and median and mean CAP1-6D (Panel A) and wild-type (Panel B) T Cell response per dose level. Baseline (blue diamond) and peak (green triangle) T cell responses by ELISPOT for each of the 14 evaluable patients. Median and mean T cell response by ELISPOT (spots per 104 CD8+ cells) measured after at least 3 cycles is indicated by the orange bars per each arm. For CAP1-6D (Panel A) median/mean Arm A (0.01 mg) response = 10.5/36.5; median/mean Arm B (0.1 mg) response = 51.75/148.45; median/mean Arm C (1 mg) response = 270.63/247.69. P = 0.028 as measured by Mann–Whitney Rank Sum Test comparing medians. For wild-type (Panel B) median/mean Arm A (0.01 mg) response = 3/22.05; median/mean Arm B (0.1 mg) response = 81/120.5; median/mean Arm C (1 mg) response = 222.5/188.5. P = 0.028 as measured by Mann–Whitney Rank Sum Test comparing medians. Figure 3ELISPOT kinetics of in vitro primed CAP-1 peptide specific-CD8+ T cell responses. T cell responses for all 14 patients at various time points on the trial are presented and stratified by treatment arm. Day 1 is first day of vaccination. BODY.RESULTS.RESPONSE AND SURVIVAL: Individual clinical responses for all 19 patients are listed in Table 2. Seven patients (37%) had stable disease (SD) as their best response and all remain alive today. Among the 14 immunologically evaluable patients, eight had a positive ELISPOT response of which 4 had progressive disease (PD), 3 SD and 1 a complete response (CR) as their best response. Among the 6 patients failing to exhibit positive ELISPOT response, 2 had PD and 4 SD as their best response. There was one CR in a patient with locally advanced, unresectable PC. This patient underwent chemotherapy and radiation and then received 81 doses of the vaccine, randomized to Arm C. She developed a strong ELISPOT response and remains alive and disease free at over 75 months. One patient with metastatic PC in Arm B had SD for 11 months (22 cycles of vaccine administered), a strong CTL response and is alive at over 43 months. Another patient with metastatic PC in Arm C had SD for 6 months, a strong CTL response and is alive at over 48 months. Of the 5 patients that were not able to be evaluated for immunologic response, 3 were in Arm B and 2 in Arm C (Table 2). All 5 had PD within less than 2 months and all died shortly thereafter. Eleven patients had progressive disease (PD) as their best response at or prior to their first scheduled CT evaluation at 8 weeks. Table 2 Response and survival in all 19 patients Arm Stage ELISPOT response (Y/N/NE) Best response No. of cycles completed Time on trial (months) Months alive from trial start A M Y SD 8 3.7 9.4 A M N PD 4 1.8 15.9 A M N SD 24 11.1 47.2* A NED N SD 24 12.4 32.4* A NED N SD 24 12.2 31.9* B M Y PD 4 1.9 3.1 B M Y SD 23 12 43.2* B M Y PD 3 1.4 3.6 B M N SD 22 10 ?* B M N PD 4 1.8 2.2 B M NE PD 1 0.3 0.4 B M NE PD 4 1.2 1.5 B M NE PD 4 1.8 3.0 C M Y PD 4 1.9 11.3 C LA Y CR 81 39.3 75.4* C M Y PD 4 1.8 11.0 C M Y SD 12 6.2 48.5* C M NE PD 3 1.2 2.0 C M NE PD 3 1.4 2.6 Abbreviations: NED = No evidence of disease; M = metastatic; LA = locally advanced; Y = Yes; N = No; NE = not evaluable; * = currently alive; ? = patient is known to be alive but had withdrawn consent; Data as of May 22, 2012. The median OS for the entire cohort of 19 patients was 334 days with a median PFS of 56 days. Overall, 7/19 patients (37%) were alive at a minimum 32 months from trial initiation and of the 17 patients with LA or metastatic disease, five (29%) remain alive. The study was not powered to detect differences in clinical outcome between the three cohorts. BODY.RESULTS.TOXICITY: All 19 patients were evaluable for toxicity. No grade 4 and 5 toxicities were observed; grade 1-3 toxicities are summarized in Table 3. No discontinuation of treatment occurred due to toxicity and no grade 2 or greater hemorrhage or allergic reaction occurred; no autoimmune disorders were noted. The most common toxicities were grade 1-2 skin reactions to the vaccine injection. These did not develop differentially in responders versus non-responders and did not correlate with ELISPOT results. Otherwise common side effects of pain, fatigue, anorexia, nausea/vomiting, diarrhea and constipation were felt to be due to primary disease and not the study vaccine. Table 3 Grade 2 or greater toxicities associated with vaccine   Grade 1 Grade 2 Grade 3 Grade 4 Toxicity No. (%) No. (%) No. (%) No. (%) Injection-site reaction 11 (53) 1 (5) 0 (0) 0 (0)  Erythema 9 (47)        Induration 6 (32) 1 (5)      Pain 3 (16)        Ecchymosis 2 (11)        Discoloration 1 (5)        Edema 1 (5)        Rash 1 (5)        Reaction NOS 1 (5)       Toxicity unrelated to injection site 10 (53) 3 (16) 4 (21) 0 (0)  Pain 10 (53) 3 (16) 2 (11)    Fatigue 10 (53) 1 (5) 4 (21)    Anorexia 4 (21) 0 (0) 2 (11)    Nausea/Vomiting 8 (42) 0 (0) 1 (5)    Constipation 6 (32) 2 (11) 1 (5)    Diarrhea 3 (16) 1 (5) 1 (5)   BODY.DISCUSSION: The diagnosis of pancreatic cancer is associated with approximately a 5-year survival rate of 6% [1]. Innovative therapies are thus desperately needed. Immunotherapy via vaccination has been one approach investigated over the last decade. In pancreatic cancer, several trials have shown that use of a TAA such as MUC-1 in combination with CEA or K-Ras can elicit an immunologic response and possibly improve outcomes in those mounting such a response. To our knowledge, no other trial reported has used CEA as the exclusive TAA vaccination platform in a strictly PC population and the optimal dose of such a vaccine was unknown. The primary objective of this study was to determine the optimal dose of the carcinoembryonic antigen (CEA) peptide (CAP1-6D)/Montanide/GM-CSF-vaccine amongst the three doses administered to induce a maximal CTL response in patients with pancreatic cancer. Based on our data of 14 immunologically assessable patients, the 1 mg dose of peptide in the vaccine emulsion led to the maximal T cell response. A dose response between the vaccine peptide dose and an induced T cell response was observed, with 100% of patients in the 1 mg arm who were eligible for immunologic evaluation demonstrating a CD8+ T cell response by IFN-γ ELISPOT. The median OS and PFS were 334 and 56 days, respectively, and the vaccination was safe and well tolerated. We believe that the randomized phase I trial design proved to be a useful tool, as it allowed determination of the optimal dose of peptide for induction of an antigen-specific CD8+ T cell response in this patient population. Our study does have a number of limitations that highlight some important issues in cancer vaccine development. First, although our sample size was sufficient to answer the primary scientific objective, it was not large enough to have meaningful comparisons of clinical outcomes among the three groups. Also, no obvious correlation between a positive ELISPOT response and clinical outcome was noted, but this is not unexpected due to known resistance mechanisms in the tumor microenvironment and the study's small sample size. Second, due to the inherent need for HLA-A2 restriction that is imposed by using a peptide antigen, many potential subjects were screen failures. In that regard, vaccination platforms that do not restrict based on HLA status, such as those based on whole protein or full length cDNAs incorporated into suitable vectors, do have advantages. Third, CEA is not necessarily utilized by pancreatic cancer cells as part of the malignant process, and it could be argued that gene products contributing to cancer cell growth or survival may be more desirable to target. Finally, in order to enable sufficient accrual, we did not have a homogenous patient population, allowing the inclusion of metastatic, resected, and locally advanced patients, thus not permitting definitive conclusions to be made about any one group. Of note, two patients in group A were enrolled into the study with NED and failed to develop an immune response. We do not have alternate indices of general immune competence for them to try to further explain their anergic state, but the stimulation with PMA + Ionomycin was positive. As noted above and presented in Figure 3, the kinetics of the T cell response suggest that if a response occurred, it happened early with the maximum response occurring within the first 100 days. For those on trial for over 4 months, the response does appear to wane over time, although was sustained at over 300 days for two patients. Also, in regard to vaccine-site delayed-type hypersensitivity as evidenced by skin reactions we did not notice a particular pattern to correlate with ELISPOT results. Out of the 11 patients with a reaction, some developed it without ever mounting a positive ELISPOT results, some at the time of their peak and some at a different point in time. Three phase III trials of PC vaccines have been conducted previously, all of which did not meet their primary objective [31-33]. Over 20 vaccine immunotherapy clinical trials are currently ongoing in various stages of completion (http://clinicaltrials.gov) examining varying iterations of the above approaches for cancers including but not limited to PC, including novel peptides (e.g., mesothelin, VEGFR1/VEGFR2 and survivin), DNA-based vaccines, and combinations of vaccines with chemotherapy. Most results in PC thus far have been underwhelming and the field is still searching for the optimal vaccine approach, clinical context, and predictive biomarkers of clinical benefit. Nonetheless, the favorable clinical outcome observed in a subset of patients treated in our current CEA vaccine trial motivates continued investigations of immunotherapeutic strategies in this disease. Our vaccination approach utilized the adjuvant Montanide ISA-51 along with the cytokine GM-CSF included in the emulsion. It is not clear that this is an optimal vaccine adjuvant for peptide vaccines, and further improvements in the vaccine formulation are conceivable. After this trial was initiated, Slingluff and colleagues reported that the inclusion of GM-CSF with Montanide may result in diminished peptide-induced T cell responses in a melanoma vaccine study [34]. The TLR agonists CpG 7909 and polyIC:LC have recently been explored and should continue to be investigated as a possible adjuvant [35]. The MAGE-3 protein-based vaccine from GSK-Bio utilizes a combination of the TLR9 agonist CpG7909 and the TLR4 agonist MPL [36]. Other cytokines with immune-potentiating activities could be considered, including IL-12 [37-39]. Thus, while our study has identified an optimal dose of CEA peptide, further improvements in vaccine potency might be achievable through optimization of the adjuvant component. In addition to utilizing vaccination to increase the frequency of tumor-reactive T cells, features of the tumor microenvironment can be dominantly suppressive and it may be necessary to inhibit such factors for optimal clinical activity. These include immunosuppressive cytokines such as IL-10 and TGF-β; expression of indoleamine-2,3-dioxygenase; the presence of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs); inhibitory ligands such as B7-H1/PD-L1; and the dense tumor stroma that is characteristic of this disease [40,41]. Recently, using an agonist CD40 mAb in combination with gemcitabine, Beatty et al. demonstrated a partial reversal of immune suppression in PC and highlighted the role of macrophages in this process [42]. Future studies combining an optimized vaccine formulation along with manipulations to favorably alter the PC tumor microenvironment will be attractive to consider. BODY.CONCLUSIONS: We conducted a randomized phase I trial of carcinoembryonic antigen (CEA) peptide (CAP1-6D)/Montanide/GM-CSF-vaccine in 19 pancreatic cancer patients. Our primary objective was to find the optimal dose of the vaccine. The dose that induced a maximal T cell response was 1 mg and no significant toxicity was observed. Several long-term survivors with metastatic or locally advanced disease were noted. Future studies can build on these results by combining the above vaccine peptide dose with stronger adjuvants and/or agents to favorably alter the tumor microenvironment. BODY.COMPETING INTERESTS: Drs. Geynisman, Zha, Kunnavakkam, Aklilu, Catenacci, Polite, Rosenbaum, Namakydoust, Karrison, Gajewski and Kindler declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: DG coordinated and analyzed the data as well as wrote the manuscript; YZ carried out the immunoassays and helped to analyze the data; RK helped with the statistical analysis; MA helped in the original design of the study; DC helped to conduct the study; BP participated in the design of the study and manuscript writing; CR helped to conduct the study; AN helped in the initial data analysis and manuscript preparation; TK helped with the initial statistical design of the trial; TG conceived of the study, participated in the design, immunologic analysis and writing of the final manuscript; HL conceived of the study, oversaw the study's clinical implementation and participated in the design and writing of the final manuscript. All authors read and approved the final manuscript.

TITLE: Comparing the Effects of Hot Pack and Lukewarm-Water-Soaked Gauze on Postoperative Urinary Retention; A Randomized Controlled Clinical Trial ABSTRACT.BACKGROUND:: Urinary retention is a common postoperative complication that mandates urinary catheterization. Urinary catheterization is associated with different physical, mental, and financial problems for both patients and healthcare systems. The patient inconvenience, urinary tract infections, and increase in hospital stay and expenses are common problems of urinary retention and urinary catheterization. Therefore, alternative ways of relieving urinary retention, preferably noninvasive interventions, are of great interest. ABSTRACT.OBJECTIVES:: The aim of this study was to compare the effects of placing hot pack and lukewarm-water-soaked gauze on the suprapubic region on male patients with postoperative urinary retention. ABSTRACT.PATIENTS AND METHODS:: This was a three-group, randomized, controlled trial. A convenience sample of 126 male patients who had undergone general, orthopedic, or urologic surgeries were recruited. The block randomization method was used for allocating patients to either the two experimental groups (the hot pack and the lukewarm-water-soaked gauze groups) or the control one. Patients in the experimental groups were treated by placing either hot pack or lukewarm-water-soaked gauze on the suprapubic region. All patients were monitored for 20 minutes for urinary retention relief. If they did not experience urinary retention relief (starting urine flow and bladder evacuate), urinary catheterization would be performed. The data was collected using information sheet. Elimination of urinary retention was compared among study groups. The one-way analysis of variance and the Chi-square tests were used for analyzing data. ABSTRACT.RESULTS:: Respectively, 59.5%, 71.4%, and 7.1% of patients in the hot pack, the soaked gauze, and the control groups experienced relief from urinary retention and the bladder was emptied. There was a significant difference among study groups in percentage of patients who experienced urinary retention relief. However, the difference between the two experimental groups was not significant. The time to urinary retention relief in hot pack, soaked gauze, and control groups was 15.45 ± 3.15, 13.83 ± 3.80, and 14.59 ± 3.29 minutes, respectively. The difference among the study groups in time to urinary retention relief was not statistically significant. ABSTRACT.CONCLUSIONS:: Both the lukewarm-water-soaked gauze and the hot pack techniques had significant effects on postoperative urinary retention and significantly reduced the need for urinary catheterization. Using these two simple and cost-effective techniques for managing postoperative urinary retention is recommended. BODY.1. BACKGROUND: Urinary retention (UR) refers to the inability of bladder to evacuate urine during voiding (1, 2). Many patients develop UR after surgery. Because of the differences in patient populations, UR definitions, and UR management interventions, the incidence of postoperative UR (POUR) has been reported differently, ranging from 5% to 75% (3-6). All male and female patients from different age groups might develop POUR after different surgeries. POUR is associated with considerable health and financial consequences (1, 2, 4). The causes of UR include decreased bladder sensation, tension on the bladder, anxiety, surgeries, or other factors that affect the bladder (1). The most common surgeries that might cause POUR are the abdominal, anorectal, colorectal, urologic, and gynecologic ones. Surgery-induced alterations in sacral reflex can cause contraction of the bladder sphincter and bring about POUR (5). After surgeries on other parts of the body, POUR may happen due to sedation, anesthesia, decreased mobility, fluid overload, medications, pain, and anxiety (7). Besides the direct effects of surgery on POUR, sedative and anesthetic agents might also suppress the sensation of bladder filling and predispose patients to POUR. Moreover, the increased tone of the bladder internal sphincter, caused by postoperative pain, might significantly contribute to POUR. On the other hand, opioid analgesics, which are administered for managing postoperative pain, might also cause POUR through the same mechanism (4). Developing UR and subsequent urinary stasis is associated with different consequences and complications such as infection, transmural ischemia, decreased bladder sensation and contractility, and temporary or permanent damage to the bladder, detrusor muscle, ureters, and kidneys (1, 2, 4). Moreover, UR can cause pain and anxiety, prolong hospital stay, and increase healthcare costs (4, 5). The key measure for managing UR is urinary catheterization (8). Catheterization is performed when the bladder is palpable, the afflicted patient has great discomfort, and the primary UR treatments are ineffective. Catheterization is an invasive procedure with potential complications, including catheter-associated urinary tract infections (UTIs), urethral trauma, prostatitis, pain, and discomfort. According to Steggall et al. about 80% of all nosocomial UTIs occur due to catheterization. Accordingly, decision on managing UR by catheterization should be made with great caution (4). There are different safe and inexpensive nursing interventions for preventing and managing POUR that can reduce the need for catheterization (1, 2). For instance, placing gauze that is immersed in lukewarm water on the suprapubic region can help alleviate POUR (9). Hosseini et al. found that compared with onion-covered gauze, the water-soaked gauze applied on suprapubic region, was more effective in relieving male patients' UR after cardiac catheterization (9). However, to the best of our knowledge, no study was conducted on comparing the effects of hot pack and water-soaked gauze on POUR. BODY.2. OBJECTIVES: The aim of this study was to compare the effects of placing hot pack and lukewarm-water-soaked gauze on the suprapubic region on male patients' postoperative urinary retention. BODY.3. PATIENTS AND METHODS: This was a three-group, randomized, and controlled trial. The study was conducted in May through July 2014. The study setting was the surgical unit of Milad Hospital, Kashan City, Iran. All male patients who had undergone orthopedic, urologic, or general surgeries in the study setting and according to physician's diagnosis had developed POUR (by lack of urinate after operation, discomfort, and pain in suprapubic region) constituted the study population. Study sample size was calculated after a pilot study and based on α (Type I error) of 0.05, β (Type II error) of 0.2, and effect size of 0.65. Accordingly, 126 patients (42 patients in each group) were recruited through convenience sampling method. The accommodations and care services of the study setting were similar for all patients. The inclusion criteria were being 20 to 75 years old, no history of known urinary tract disorders, no urinary catheterization in their recent surgery, no history of massive bleeding throughout recent surgeries, and a confirmed diagnosis of POUR. Patients who needed emergency urinary catheterization during the study were excluded. Data collection instruments were a demographic questionnaire, a surgery datasheet, and a POUR datasheet. The demographic questionnaire contained items on patients' age, height, weight, and body mass index (BMI). Demographic data were collected by either asking patients or referring to their medical records. The surgery datasheet included items about the kind of anesthesia, the kind of surgery, and the administered opioids and fluids. Finally, the POUR datasheet consisted of questions on the type of the intervention and the interval between starting the intervention and obtaining UR relief. We used a standard (Casio, Tokyo, Japan) chronometer for measuring this time. Study participants were randomly allocated to three groups, namely, the hot pack, the lukewarm-water-soaked gauze, and the control groups, by using the block randomization technique in 21-senary block. Before starting the interventions, we measured the study participants' axillary body temperature by using a mercury thermometer (to eliminate the effect of body temperature on the performance of soaked gauze). Patients in the hot pack group were treated by placing a hot pack (Hospital and Home Care Nanjing, China) containing 200 mL of 50°C water in direct contact on the suprapubic region for 20 minutes. We also treated patients in the second treatment group by putting a standard 10 × 10-cm2 gauze on the suprapubic region for 20 minutes. The gauze had been already soaked in 15 mL of 25°C to 30°C water (The gauzes were not re-wetted). Patients in the control group received no intervention. At the beginning of intervention, the time was measured for 20 minutes. The time of urination was recorded. If UR was not relieved after 20 minutes, the urinary catheterization would be performed immediately. The environmental condition (including room temperature, bed type, room size, and privacy facilities) were same for all patients. BODY.3. PATIENTS AND METHODS.3.1. ETHICAL CONSIDERATION: The University Review Research Ethics Committee at Kashan University of Medical Sciences approved this study. We received formal permissions from the Research Council of Kashan University of Medical Sciences and the administrators of the study setting. The study aims and methods were explained to the participants and written informed consent was obtained. Patients were free to withdraw from the study and ask for receiving urinary catheterization at any time during the intervention. Their privacy and anonymity were guaranteed. BODY.3. PATIENTS AND METHODS.3.2. DATA ANALYSIS: Data analysis was done using the SPSS 11.5 (SPSS Inc, Chicago, Illinois, the United States). Study data were described by using descriptive statistics measures such as mean, standard deviation, and frequency tables. Study groups were compared in variables such as age, BMI, body temperature, administered intravenous opioids and fluids, time to UR relief, percentage of UR relief, type of surgery, and the underlying condition by using the one-way analysis of variance (ANOVA) and the Chi-square tests. BODY.4. RESULTS: The means of patients' age, BMI, body temperature, and administered intravenous fluids during surgery are summarized in Table 1. The results of the one-way ANOVA test revealed that there were no significant differences among the study groups regarding patients' age, BMI, body temperature, and the amount of intravenous fluids administered during surgery (P > 0.05) (Table 1). Table 1. Participants’ Characteristics a Group P Value Hot Pack Soaked Gauze Control Group Age, y 40.97 ± 15.31 45.28 ± 16.52 43.36 ± 15.96 0.45 BMIb, kg/m 2 26.15 ± 2.86 26.40 ± 2.50 26.67 ± 2.35 0.43 Temperature, °C 37.06 ± 0.25 36.13 ± 5.55 36.99 ± 0.23 0.33 Intravenous fluids, mL 370.29 ± 9.53 293.08 ± 8.53 241.25 ± 8.64 0.26 a Data are presented as Mean ± SD. Furthermore, the results of the Chi-square test demonstrated that the three study groups did not differ significantly in terms of variables such as the kind of surgery, administration of opioid agents, and patients' underlying conditions (P > 0.05) (Table 2). Table 2. The Result of Administration of Opioid Agents and Patients’ Underlying Conditions a Groups P Value (Chi-square) Hot Pack Soaked Gauze Control Group Kind of Surgery 0.52 Orthopedic 12 (28.6) 8 (19.0) 7 (16.7) Urologic 12 (28.6) 16 (38.1) 12 (31.7) General surgery 18 (42.9) 18 (42.9) 23 (54.8) Other comorbidities 0.38 Hypertension 36 (85.7) 31 (73.8) 34 (80.2) Other Diseases 6 (14.3) 11 (26.2) 8 (19.0) Administration of opioid s 0.87 Yes 40 (95.2) 39 (92.9) 39 (92.9) No 2 (4.8) 3 (7.1) 3 (7.1) a Data are presented as No. (%). The percentages of patients in the hot pack, soaked gauze, and control groups who experienced UR relief during the 20-minute period of the study intervention without needing urinary catheterization were respectively 71.4%, 59.5%, 7.1%. The results of the Chi-square test revealed that there was a significant difference among the three groups in terms of the percentage of patients who experienced UR relief (P < 0.001). However, the difference between the two experimental groups was not statistically significant (P > 0.05) (Table 3). Table 3. Urinary Retention Relief in the Study Groups After the Intervention a Groups Urinary Retention Relief P Value (Chi-Square) Yes No Hot Pack 25 (59.9) 17 (40.5) < 0.001 Soaked Gauze 30 (71.4) 12 (28.6) < 0.001 Control Group 3 (7.1) 39 (92.9) < 0.001 a Data are presented as No. (%). The means of time to UR relief in hot pack, soaked gauze, and control groups were 15.45 ± 3.15, 13.83 ± 3.80, and 14.59 ± 3.29 minute, respectively. The results of the one-way ANOVA test showed that the three study groups did not differ significantly in terms of the time to UR relief (P > 0.05). BODY.5. DISCUSSION: Study findings revealed that placing both hot pack and lukewarm-water-soaked gauze on the suprapubic region could positively affect POUR. Probably, lukewarm-water-soaked gauze brings UR relief through conveying a sense of exposure with water. Hosseini et al. noted that exposure with water is effective in relieving UR (9). Shafik investigated the effects of warm water bath on 21 patients with POUR after herniorrhaphy surgery and found that 19 patients experienced UR relief (10). In our study, however, only 25 patients in the hot pack group and 30 patients in the soaked-gauze group felt UR relief. This discrepancy between the findings of the two studies can be attributed to patients' direct exposure to warm water by using a warm water basin, which conveyed a greater sense of water exposure, in Shafik's study. Moreover, warm water might have relieved UR through affecting the heat reflex of the bladder sphincter and relaxing it. In contrast, we applied warmth and humidity both separately and locally on the suprapubic region. Shafik's intervention required patients to get out of bed for sitting in a warm water basin while we administered our interventions while patients remained in their beds. Moreover, Shafik only studied the patients who had undergone anorectal surgery while our sample consisted of patients who had had general, orthopedic, or urologic surgeries. in fact, different surgeries might be associated with different levels of POUR (10). Gonullu et al. also reported that applying hot pack to the suprapubic region relieved 77% cases of POUR after general surgeries (11). However, only 59.5% of our participants who had been treated with hot pack experienced UR relief. This discrepancy between the findings of the two studies can be attributed to several factors. First, while Gonullu et al. investigated both male and female patients, we included only male patients. Second, Gonullu et al. recruited the patients who had undergone general surgeries while our participants had had general, orthopedic, or urologic surgeries. Finally, while Gonullu et al. applied 40°C to 45°C hot pack, the temperature of our hot packs was 50°C (11). Hosseini et al also compared the effects of onionskin and soaked gauze on 62 male patients' UR after cardiac catheterization. They found that onionskin and soaked gauze relieved respectively 58.1% and 71% of UR cases. Our soaked gauze intervention also was effective in relieving POUR in 71.4% of patients. However, while Hosseini et al. reported that the mean time to UR relief in the soaked gauze group was 16.63 minutes, this value in the soaked gauze group of our study was 13.70 minutes. The difference in the time of urinary relief can be attributed to the fact that Hosseini et al. recruited a sample of patients who had received cardiac catheterization while we studied the patients who had undergone general, orthopedic, or urologic surgeries. The difference in the type of the surgeries might explain the difference in time to UR relief. In extensive search, we did not find any use hot pack or lukewarm-water-soaked gauze in UR for comparison with our results (9). Study findings indicated that the lukewarm-water-soaked gauze technique is as much effective as the conventional hot pack technique in relieving POUR. Moreover, compared with traditional techniques––such as lying flat and relaxing muscles, getting out of bed and standing in upright position, alleviating anxiety, taking shower, applying ice pack, opening water faucet, and tapping the groin—applying soaked gauze and hot pack is simpler and easier and does not require patient repositioning. Soaked gauze and hot pack techniques can eliminate the need for urinary catheterization and hence, help prevent patients from developing catheterization-related complications such as UTI, trauma, and discomfort. Accordingly, employing the simple and cost-effective techniques of placing hot pack and lukewarm-water-soaked gauze on the suprapubic region is recommended. In this study, some patients avoided of continuing participation that we replaced with new samples. Comparison of soaked gauze with other nursing interventions in relieving UR is recommended.

TITLE: After the diabetes care trial ends, now what? A 1-year follow-up of the RxING study ABSTRACT.INTRODUCTION: There is strong evidence that pharmacist care improves patients' glycaemic control. However, the sustainability and durability of such interventions beyond the research period is not known. RxING was the first trial of pharmacist prescribing in diabetes and it showed an improvement in glycated haemoglobin (HbA1c) of 1.8% over 6 months. ABSTRACT.OBJECTIVE: 1° objective: To evaluate glycaemic control in the RxING study patients 12 months after the end of the formal study follow-up. 2° objective: To assess the patients' risk of cardiovascular events in the next 10 years. ABSTRACT.METHODS: We contacted the participating pharmacists to check if the patients who participated in the RxING study are still taking insulin, the dose of insulin they are taking, and their HbA1c. There were no mandated follow-up visits with the pharmacist after the study completion. ABSTRACT.RESULTS: A total of 100 patients with poorly controlled type 2 diabetes were enrolled in the original RxING study; 93 of them completed the study, while 83 participated in the 12-month follow-up. Seventy-five patients were still taking insulin, with the average dose increasing from 31.1 units (SD 18.4) at study completion to 37.4 units (SD 30.8) (95% CI −13.3 to 0.88, p=0.085). HbA1c was reduced from 9.1% (SD 1) at baseline to 7.3% (SD 0.9) at study completion (95% CI 1.4 to 2, p <0.001), and increased to 8.1% (SD 1.3) 12 months later (95% CI −1.1 to −0.5, p <0.001 vs study completion). ABSTRACT.CONCLUSIONS: Twelve months after completing the intervention, approximately half of the glycaemic control gains were lost. This highlights the importance of structured follow-up with the pharmacist in this patient population. ABSTRACT.TRIAL REGISTRATION NUMBER: clinicaltrials.gov; Identifier: NCT01335763. BODY: Strengths and limitations of this studyThis is the first study to evaluate the sustainability and durability of a pharmacist prescribing intervention for patients with poorly controlled type 2 diabetes beyond the research study period and it demonstrates the importance of ongoing structured pharmacist intervention on glycaemic control in patients with poorly controlled type 2 diabetes.We did not have any data on the patients' cholesterol/high-density lipoprotein ratio and this could affect their risk of cardiovascular events in the next 10 years (which is a surrogate marker): therefore, we used a ratio '4.9' which makes no contribution to the overall risk and assumed that there were no changes to this ratio over time to avoid any effect those changes may have on the risk of cardiovascular events in the next 10 years.We did not construct formal methods to track pharmacist follow-up visits beyond study completion. BODY.INTRODUCTION: The pharmacist's role in diabetes care is well supported in the literature. There is strong evidence, including systematic reviews and randomised controlled trials, that pharmacist interventions improve the patient's glycaemic control alongside other aspects of diabetes care such as medication adherence and knowledge about the disease and its complications.1–5 The RxING study took this evidence one step further, assessing the effect of a community pharmacist independent prescribing intervention on glycaemic control in patients with poorly controlled type 2 diabetes.6 The study showed that such intervention reduced patients' glycated haemoglobin (HbA1c) from 9.1% (SD 1) at baseline to 7.3% (SD 0.9) at 26 weeks (an absolute reduction of 1.8%, 95% CI 1.4 to 2, p<0.001), and fasting plasma glucose was reduced from 11 mmol/L (SD 3.3) at baseline to 6.9 mmol/L (SD 1.8) at 26 weeks (an absolute reduction of 4.1 mmol/L, 95% CI 3.3 to 5, p=0.007). The sustainability and durability of such interventions beyond the research study period is not known, as intervention studies in diabetes usually last between 3 and 24 months.2 There is little information about the long-term effect of pharmacist interventions in diabetes, particularly with prescribing interventions. As such, we conducted a 12-month follow-up of the RxING study patients to evaluate their glycaemic control 12 months after completing the study. The secondary objective was to assess the risk of cardiovascular events in the next 10 years 12 months after completing the study. BODY.METHODS: The methods of the RxING study are reported elsewhere.6 Adult patients with poorly controlled type 2 diabetes (HbA1c between 7.5 and 11%) despite receiving oral hypoglycemic agents, were recruited through 12 community pharmacies across Alberta, Canada.6 Community pharmacists, who had their independent prescribing authority, prescribed 10 units of insulin glargine at bedtime for these patients and asked them to titrate their dose by 1 unit/day until they reached a fasting plasma glucose of 5.5 mmol/L. Patients were followed up at 2, 4, 8, 14, 20 and 26 weeks by their community pharmacists to provide ongoing care and address any issues regarding the treatment. HbA1c was measured at the pharmacy using point of care testing (DCA Vantage, Siemens, Tarrytown, New York, New York, USA) at baseline, 14 and 26 weeks.6 Twelve months after completing the study, we contacted the participating pharmacists to check if the patients who participated in the RxING study are still taking insulin, the dosage of insulin they are taking and their HbA1c level. There were no mandated follow-up visits with the pharmacist after the study completion. Pharmacists have used the Provincial Electronic Health Records (Alberta Netcare) to obtain information about the most recent HbA1c level and their own medication records to check if the patient is still taking insulin glargine and its dosage. We also calculated the patients' risk of cardiovascular events in the next 10 years using the UK Prospective Diabetes Study (UKPDS) calculator, with the assumption that the cholesterol/high-density lipoprotein (HDL) ratio is 4.9. This value was chosen because it makes no contribution to the overall risk,7 as we did not collect any data on the patients' lipid profiles. The level of significance was set at 0.05. The mean HbA1c between the end of the study and 12 months after completion was compared using paired t test. Paired t test and basic frequencies were used to analyse secondary outcomes and demographic and clinical characteristics, respectively. BODY.RESULTS: A total of 100 patients with poorly controlled type 2 diabetes were enrolled in the RxING study. Ninety-three patients completed the study, 86 (93%) of whom participated in the 12-month follow-up (figure 1). Figure 1Patient's enrolment and follow-up flow chart. The demographic and clinical characteristics of the patients are reported elsewhere.6 Table 1 reports some demographic and clinical characteristics of patients at baseline, 6 months and 18 months after baseline. Table 1 Patients’ clinical and demographic characteristics Characteristic Baseline (N=100) 6 months (N=93) 12 months postcompletion (N=86) Age (mean (SD)) 64 (10.4) 64 (10.5) 65 (10.4) Diabetes duration (mean (SD)) 10.2 (7) 10 (6.7) 10.4 (6.8) HbA1c % (mean (SD)) 9.1 (1) 7.3 (0.9) 8.1 (1.3) Insulin dose (mean (SD)) – 31.1 (18.4) 37.4 (30.8) Gender (% male) 58 57 57 Ethnicity (% Caucasian) 89 89 88 Smoking status (% smokers) 22 20 17 HbA1c, glycated haemoglobin. Seventy-five patients (88%) were still taking insulin 12 months after completing the study, with the average dose increasing from 31.1 units (SD 18.4) at study end to 37.4 (SD 30.8), a change of 6.3 units (95% CI −13.3 to 0.88, p=0.085). In the RxING study, HbA1c was reduced from 9.1% (SD 1) at baseline to 7.3% (SD 0.9) at 26 weeks, a change of 1.8% (95% CI 1.4 to 2, p<0.001). Twelve months after completing the study, HbA1c increased from 7.3% (SD 0.9) at study end to 8.1% (SD 1.3), a change of 0.8% (95% CI −1.1 to −0.5, p<0.001; figure 2). Figure 2Glycated haemoglobin (HbA1c) change with the intervention and 12 months postcompletion * Dotted line indicates the completion of the intervention period. Patients' risk of cardiovascular events in the next 10 years was reduced from 31% (SD 17.3) at baseline to 26.7% (SD 14.5) at study end, an absolute reduction of 4.3% (a 14% relative reduction) (95% CI 0.6 to 9.5, p=0.026). Twelve months after completing the study, the risk of cardiovascular events in the next 10 years rose from 26.7% (SD 14.5) at study end to 30.75% (SD 15.5), an absolute increase of 4.05% (15% relative increase) (95% CI −0.09 to 0.005, p=0.083). This increase in risk was mainly caused by the worsening of glycaemic control. BODY.DISCUSSION: Twelve months after completing a pharmacist prescribing intervention for patients with poorly controlled type 2 diabetes, approximately half of the glycaemic control gains and reduction in the risk of cardiovascular events in the next 10 years were lost. Despite impressive early gains in the intervention study, this highlights the need for an ongoing close follow-up of patients with diabetes. The increase in HbA1c and the risk of cardiovascular events in the next 10 years can be explained by the lack of systematic follow-up by the pharmacist. We were informed by the participating pharmacists that patients returned to receiving usual care after the study was completed. Until recently, there was no remuneration system for pharmacists in Alberta, Canada, which was considered as a barrier to follow-up. Disease progression and the lack of optimal insulin dose titration/stopping insulin may have also contributed to this increase. Indeed, it has been reported that patients with type 2 diabetes experience an average loss of 5% of insulin production capacity, per year, after diagnosis.8 The findings of this study highlight the impact of structured pharmacist intervention on glycaemic control in patients with poorly controlled type 2 diabetes. Patients' HbA1c improved by 1.8% (95% CI 1.4 to 2, p<0.001) over 6 months while receiving the pharmacist's intervention; however, their HbA1c increased by 0.8% (95% CI −1.1 to −0.5, p<0.001) 12 months after completing the study. It is also important to note that the gains from the intervention were not completely lost 12 months after completing the study as the baseline HbA1c was 9.1% and 88% of the patients were still taking insulin. If maintained, this change from baseline has the potential to have a significant impact on future diabetes-related complications.7 This study is not without limitations. We did not have any data on the patients' cholesterol/HDL ratio and this could affect their risk of cardiovascular events in the next 10 years, which is a surrogate marker, therefore, we used a ratio '4.9' which makes no contribution to the overall risk7 and assumed that there were no changes to this ratio over time to avoid any effect those changes may have on the risk of cardiovascular events in the next 10 years. Also, no formal methods were constructed to track whether pharmacist follow-up visits had occurred beyond study completion. Pharmacists in Alberta, Canada have access to the Provincial Electronic Health Records (Alberta Netcare), where they can access important health information that was ordered by them or by other healthcare professionals. This system was created to improve the healthcare that is provided to the public. Key health information collected at different interactions with different healthcare professionals at a variety of locations gets recorded in the health records and then will be made available to all the healthcare professionals involved in the patient's care.9 This can affect the generalisability of the study results. The findings of the RxING study took the evidence for the benefits of pharmacist care in diabetes one step further, as it proved that pharmacists can systematically identify patients with poor glycaemic control, educate and support them to achieve better outcomes on top of the known fact that insulin prescribing will improve glycaemic control.6 The 12-month postcompletion data suggest that systematic patient follow-up should be continued in order to maintain glycaemic control improvement. This supports the important role that pharmacists can play in improving glycaemic control in patients with poorly controlled type 2 diabetes.

TITLE: Dermoscopic Changes of Melanocytic Nevi after Psoralen-Ultraviolet A and Narrow-Band Ultraviolet B Phototherapy ABSTRACT.BACKGROUND:: Phototherapy may alter the morphologic features of melanocytic nevi. Dermoscopy is a non-invasive method for evaluation of skin lesions, specifically melanocytic nevi. ABSTRACT.AIMS AND OBJECTIVES:: This study was designed to evaluate the effects of narrowband ultraviolet B (NB-UVB) and psoralen-ultraviolet A (PUVA) therapy on the dermoscopic features of nevi. ABSTRACT.METHODS:: A total of 74 melanocytic nevi were randomly selected from 20 patients. Out of those, 54 nevi received NB-UVB, while 20 received PUVA. 50% of the nevi in each group were exposed to radiation, while the remaining nevi were covered with an opaque tape. All nevi were demoscopically evaluated before and after 30 or 60 sessions of phototherapy. ABSTRACT.RESULTS:: Overall demoscopic changes were observed in 34/37 (91.8%) of the uncovered nevi compared to 16/37 (43.2%) of the covered nevi (P value 0.0001). The most common changes were new dot/globule formation (62.1%), darkening (32.4%), nevus enlargement (27%), and patchy pigmentation (18.8%). Compared to NB-UVB, dermoscopic changes were more frequent in both covered and uncovered nevi of the PUVA group. (P values 0.041 and 0.0172, respectively). New dot/globule formation was observed more frequently in the covered and uncovered nevi of PUVA group. ABSTRACT.CONCLUSION:: PUVA and NB-UVB induce dermoscopic changes in the majority of the irradiated nevi. However, PUVA is associated with higher frequency of dermoscopic changes in both covered and uncovered nevi. BODY.INTRODUCTION: What was known?Melanocytic nevi exposed to ultraviolet light undergo morphologic changes as a result of increase in melanocyte density as well as melanin synthesis. Dermoscopic changes including increase in size and number of nevi and size of globules as well as general darkening may occur after therapeutic doses of NB-UVB or PUVA. Narrowband ultraviolet B (NB-UVB) and psoralen plus UVA (PUVA) have long been used as effective treatment modalities for a broad range of skin diseases. Nevertheless, ultraviolet radiation (UVR) has been recognized as the main environmental cause of skin cancers through different mechanisms including direct DNA damage, suppression of cutaneuos cell-mediated immunity, and the stimulation of melanocyte proliferation.[1] The specific wavelengths responsible for inducing melanoma are unknown. PUVA therapy leads to the formation of PUVA lentigines consisting of atypical melanocytes with increased melanocytic activity.[2] PUVA has also been associated with increased risk of malignant melanoma.[34] However, this finding has not been confirmed in another study.[5] Although animal studies suggest that UVB may be responsible for the induction of cutaneous melanoma,[67] follow-up studies have not found an increased risk of melanoma after therapeutic doses of NB-UVB or broad band UVB.[89] Therefore, studies investigating the effects of UVA and UVB radiation on morphologic and histopathological alterations in melanocytic nevi may help elucidate the mechanisms inovolved in the interaction of UVR and human melanocytes. Dermoscopy is a non-invasive method for evaluation of a variety of colors and structures of the epidermis, the dermoepidermal junction, and the papillary dermis not visible to the naked eye. These specific dermoscopic patterns are closely related to the histopathology of the pigmented lesion and provide a valuable aid in diagnosing the benign or malignant nature of the lesion. During the past two decades, attempts have been made to explore the morphologic alterations in melanocytic nevi induced by UV exposure.[1011121314151617] Since the morphologic appearance of a nevus in individuals undergoing phototherapy could raise suspicion of a melanoma, understanding the different dermoscopic patterns of nevi after therapeutic UVA and UVB is crucial to identify the true high risk nevi that require an excisional biopsy to rule out melanoma and to avoid unnecessary procedures. To the best of our knowledge, the previous studies tended to focus mainly on the effects of sunlight, broad-band phototherapy, and single dose exposure, while there is still limited evidence on the impacts of either NB-UVB or PUVA therapy and their differences on dermoscopic appearance of melanocytic nevi, particularly in the therapeutic doses.[141518] The present study was designed to evaluate the effects of therapeutic doses of NB-UVB and PUVA therapy on the dermoscopic features of acquired melanocytic nevi. The dermoscopic changes induced by NB-UVB or PUVA in the exposed and non-exposed melanocytic nevi have been compared. BODY.MATERIALS AND METHODS: This prospective interventional study was approved by the Tehran University of Medical Sciences Board of Ethics and was conducted at Razi hospital from October 2008 to December 2009. The study population was randomly selected from patients who were referred by the attending dermatologist to receive either NB-UVB or PUVA therapy for dermatologic disorders. The decision for phototherapy was made independently from this study. Individuals with personal or family history of melanoma, atypical nevus syndrome, and those who had previously received any phototherapy treatment were excluded. All patients gave informed consent and were advised to avoid sun exposure during the treatment. The study patients were clinically and dermoscopically examined by an experienced dermatologist. (Dermoscopy device: microDERM D120, Visiomed, Germany). At least two morphologically similar melanocytic nevi per patient (diameter < 5 mm) without clinical or dermoscopic features of atypia were selected. One nevus was randomly selected to remain exposed during the entire phototherapy sessions while the other nevus was covered with an opaque tape. PUVA treatment was given using Waldmann UVA 200 Phototherapy Unit (Germany) two hours after oral ingestion of 0.6 mg/kg of 8-methoxypsoralen. The initial radiation dose was determined based on the patient's skin phototype. The patients received three treatments per week with incrementing dose as tolerated. NB-UVB was given using a Waldmann UV 5040BL UV unit following a three sessions per week protocol. The covered and uncovered nevi were demoscopically evaluated at 30th (n = 5 patients) or 60th (n = 15 patients) sessions of therapy depending on the underlying disease and the scheduled phototherapy protocol. The baseline and post-radiation digital dermoscopy images were evaluated by two independent and blinded investigators and were compared in terms of color, maximal diameter, and structural changes. Statistical analysis was conducted using Statistical Package for the Social Sciences (SPSS, IBM co.) version 17.00. The Chi-square analysis and Fisher's exact test were applied. The statistical significance was set at P < 0.05. BODY.RESULTS.PATIENT DATA: In total, 74 melanocytic nevi of 20 patients, 12 male (60%), with a mean age of 35 years were studied. The patients received phototherapy for various skin conditions including mycosis fugoides (9), Vitiligo (6), psoriasis (3), morphea (1), lichen planus (1). 54 nevi received NB-UVB while 20 received PUVA. 50% of the nevi in each group were exposed to radiation while the remaining nevi were covered. Table 1 shows the anatomical location and type of the studied nevi. Table 1 Anatomical location and type of the studied nevi BODY.RESULTS.EFFECT OF PHOTOTHERAPY ON DERMOSCOPIC FEATURE OF NEVI: Demoscopic changes after phototherapy with either modalities were observed in 34/37 (91.8%) of the exposed nevi compared with 16/37 (43.2%) of the covered nevi (P value 0.0001). The most common dermoscopic changes were new dot/globule formation (62.1%) [Figures 1 and 2], overall darkening (32.4%) [Figure 3], increase in the nevus size (27%) [Figure 4], and patchy pigmentation (18.8%). The enlarging nevi showed significant increase in the maximal diameter form 3.9 mm to 4.6 mm. All these changes were significantly more frequent among the exposed nevi than the covered nevi [Table 2]. Dermoscopic changes were not significantly influenced by patients' age, sex, skin phototype, or anatomical location of the nevi (P > 0.05). Figure 1Dermoscopy of melanocytic nevus before (a) and after (b) phototherapy. New dot and globule formation is seen Figure 2Dermoscopy of melanocytic nevus before (a) and after (b) phototherapy. New dot formation and patchy pigmentatiom is seen Figure 3Dermoscopy of melanocytic nevus before (a) and after (b) phototherapy. Overall increase in pigmentation is seen Figure 4Dermoscopy of melanocytic nevus before (a) and after (b) phototherapy. Increase in nevus size is seen Table 2 Changes in the dermoscopic features of exposed and covered melanocytic nevi after receiving UV radiation BODY.RESULTS.DIFFERENCES BETWEEN NB-UVB AND PUVA-TREATED NEVI: The particular effects of PUVA and NB-UVB phototherapy were compared. The prevalence of overall dermoscopic changes was significantly higher in the PUVA-exposed nevi (100% for PUVA, 88.8% for NB-UVB, P value 0.041). No differences were found between the effects of the two types of UV therapy regarding the frequency of enlarging nevi and the amount of hyperpigmentation. However, new dot-globule formation was noted in all nevi (100%) exposed to PUVA compared to only 48.1% in the NB-UVB group (P value 0.0056) [Table 3]. Table 3 Comparison of the dermoscopic features of exposed and covered nevi in NB-UVB and PUVA-treated patients Among covered nevi, dermoscpoic changes were also more frequently observed in the PUVA-treated patients [7/10 (70%) for PUVA, 2/27 (33.3%) for NB-UVB, P value 0.0172]. In particular, new dot/globule formation was significantly more frequent in the covered nevi of PUVA-treated patients (P value 0.0092) [Table 3]. BODY.DISCUSSION: UV light provokes the secretion of Pro-opiomelanocortin (POMC)-derived peptides including melanocyte-stimulating hormone from exposed skin cells and enhances the proliferative activity by up-regulating the expression of their corresponding receptors.[19] Therefore, the morphologic changes in the irradiated nevi could be best explained by the increase in melanocyte density as well as melanin synthesis.[1320] The induced changes in UV irradiated nevi tend to regress after the cessation of radiation indicating that the increase in size and changes in the dermsocopic feature are less likely to induce or promote the development of melanoma within a melanocytic nevi.[111315] In this study we showed that 91.8% of the nevi openly exposed to either NB-UVB or PUVA undergo dermoscopic alteration. New dot/globule formation (62.1%) was the most common dermoscopic change in our study followed by overall darkening (32.4%), increase in the nevus size (27%), and patchy pigmentation (18.8%). These dermoscopic alterations are in accordance with other studies; Dervis et al. observed a significant increase in size and number and size of globules as well as general darkening in study of 28 melanocytic nevi after PUVA treatment.[18] Study by Manganoni et al. showed that nevus enlargement, increase in number and size of globules/dots, and overall darkening occurs after 4 weeks of UVB or UVA in the melanocytic nevi of healthy individuals.[14] Widening of the pigment network and formation of branched streaks were also noted in these studies, however, we found no changes in the pigment structure or streaks. Pullmann et al. observed an increase in the nevi size and overall pigmentation after photochemotherapy.[21] Pigmented globules histologically correspond to either melanocyte nests at the dermoepidermal junction or melanin storage in melanophages of the papillary dermis while pigmented dots are representative of focal accumulations of free melanin or highly pigmented melanocytes in the epidermis or dermis. Therefore, the new dot/globule formation, the most commonly encountered dermoscopic alteration of melanocytic nevi after phototherapy, corresponds to the increase in melanin synthesis and/or melanocyte proliferation. Importantly, none of the dermoscopic patterns generally associated with malignant melanoma including asymmetry, atypical dot/globule, blue-white veil, pseudopods, radial streaming, atypical network, irregular streaks, or regression structures were noted in the studied nevi. Phototherapists should be familiar with the expected dermoscopic changes of nevi after PUVA and NB-UVB when following upon melanocytic nevi. Emergence of any of the above mentioned high-risk patterns in malonocytic nevi exposed to phototherapy should not be considered a natural consequence of phototherapy and histological evaluation of the suspicious nevi is mandatory. In this study, the effects of NB-UVB and PUVA on the dermoscopic features of the uncovered nevi were compared; we observed that the PUVA-treated nevi were more likely to show morphologic changes (particularly new dot/globule formation) compared to their NB-UVB irradiated counterparts. In a similar study, Karaarslan et al. compared the effect of NB-UVB and PUVA therapy on melanocytic nevi and detected more pronounced and sustained changes in NB-UVB irradiated nevi, suggesting that NB-UVB may have a higher potential to induce melanocytic proliferation. In addition, they observed that increase in the number of dots or globules were exclusively seen in the NB-UVB-treated nevi.[15] However, according to our results, these changes were not only seen in the PUVA exposed nevi, they were in fact more frequent in this group. Induction of dermoscopic alteration in the covered nevi of both groups, especially in the PUVA-treated patients, is another notable finding of this study. Animal studies demonstrated that UVR may result in the proliferation of melanocytes in both shielded and unshielded areas.[22] This concept was further supported in human studies postulating the theory that a certain mitosis stimulating factor originated from the exposed skin cells might spread through a paracrine pathway and consequently alter the histopathologic and morphologic appearance of protected nevi.[23] A wide variety of paracrine factors including mediators and cytokines such as POMC, endothelin-1 (ET-1), stem cell factor (SCF), interleukin-1 (IL-1), interferon-gamma (IFN-g), and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been identified.[202425] The significantly higher frequency of dermoscpoic changes in the covered nevi of the PUVA group as compared with NB-UVB group suggests that physical protection against PUVA may not be as effective as against NB-UVB radiation. This finding may be due to the effect of psoralen photochemistry or specific melanocytic target and distinct photobiological activity induced by different spectrums of UV radiation specifically the paracrine and endocrine signals. However, this concept needs to be validated and further elucidated by future molecular and histopathological studies. The reversibility of the UV-induced dermoscopic changes and follow-up of the patients was not assessed in this study. Small number of nevi in the PUVA-treated group is another limitation. It is plausible that baseline dermoscopic feature of melanocytic nevus (reticular, globular, homogenous) affects the phototherapy-induced changes. Phototherapy parameters including number of treatment sessions and cumulative dose may also influence the result of our study. Due to small number of patients receiving 30 sessions of phototherapy (n = 5), a statistical analysis could not be performed. Further studies are required to elucidate how intrinstic features of each melanocytic nevus, different phototherapy protocols, and doses would possibly affect the response to UV. In conclusion, our study shows that both PUVA and NB-UVB are likely to induce dermoscopic changes in 91.8% of irradiated melanocytic nevi. New dot/globule formation is the most common dermoscopic change in the nevi exposed to phototherapy followed by overall darkening, increase in the nevus size, and patchy pigmentation. We also observed that PUVA is associated with higher frequency of dermoscopic changes in both covered and uncovered nevi. What is new?This study provides a comparision of the dermoscopic changes of melanocytic nevi after exposure to therapeutic doses of NB-UVB or PUVA. Most nevi openly exposed to either NB-UVB or PUVA undergo dermoscopic alteration (91.8%). New dot/globule formation (62.1%) was the most common dermoscopic change followed by overall darkening (32.4%), increase in the nevus size (27%), and patchy pigmentation (18.8%). PUVA induces a higher rate of dermoscopic changes in irradiated nevi as compared to NB-UVB. Among nevi covered form direct irradiation, dermoscpoic changes were more frequently observed in the PUVA-treated patients. This may imply that paracrine factors of melanogenesis are more readily stimulated by PUVA.

TITLE: Impact of Booster Breaks and Computer Prompts on Physical Activity and Sedentary Behavior Among Desk-Based Workers: A Cluster-Randomized Controlled Trial ABSTRACT.INTRODUCTION: The 15-minute work break provides an opportunity to promote health, yet few studies have examined this part of the workday. We studied physical activity and sedentary behavior among office workers and compared the results of the Booster Break program with those of a second intervention and a control group to determine whether the Booster Break program improved physical and behavioral health outcomes. ABSTRACT.METHODS: We conducted a 3-arm, cluster-randomized controlled trial at 4 worksites in Texas from 2010 through 2013 to compare a group-based, structured Booster Break program to an individual-based computer-prompt intervention and a usual-break control group; we analyzed physiologic, behavioral, and employee measures such as work social support, quality of life, and perceived stress. We also identified consistent and inconsistent attendees of the Booster Break sessions. ABSTRACT.RESULTS: We obtained data from 175 participants (mean age, 43 y; 67% racial/ethnic minority). Compared with the other groups, the consistent Booster Break attendees had greater weekly pedometer counts (P < .001), significant decreases in sedentary behavior and self-reported leisure-time physical activity (P < .001), and a significant increase in triglyceride concentrations (P = .02) (levels remained within the normal range). Usual-break participants significantly increased their body mass index, whereas Booster Break participants maintained body mass index status during the 6 months. Overall, Booster Break participants were 6.8 and 4.3 times more likely to have decreases in BMI and weekend sedentary time, respectively, than usual-break participants. ABSTRACT.CONCLUSION: Findings varied among the 3 study groups; however, results indicate the potential for consistent attendees of the Booster Break intervention to achieve significant, positive changes related to physical activity, sedentary behavior, and body mass index. BODY.INTRODUCTION: The workplace presents opportunities for physical activity interventions, because a substantial percentage of US adults work in this setting, most of whom do not get the recommended levels of physical activity (1,2). Furthermore, sedentary workers have increased cardiovascular and metabolic risk and ultimately, premature mortality (3). Besides prolonged sitting during the workday, many employees eat unhealthy foods during the workday and have work-related stress (4). We theorized that promoting physical activity at work, particularly during work breaks, could be beneficial. Despite their potential public health benefits to the workplace and despite workers' desire for physical activity, only a few worksite physical activity interventions have been evaluated (2). A thorough review of the literature on short bouts of physical activity integrated into organizational routines yielded limited results; there is an absence of worksite studies related to physical activity that incorporate a comprehensive set of variables such as self-report and objective measures of physical activity, physiologic indices, sedentary behavior, and psychosocial and organizational factors (2). Because of these limitations, the Booster Break program was proposed, developed, and implemented to test the effects of a comprehensive worksite physical activity intervention (4–8). Booster Breaks are "organized, routine work breaks intended to improve physical and psychological health, enhance job satisfaction, and sustain or increase work productivity" (4). The Booster Break program uses discretionary paid time during the workday to make changes at the individual, environmental, and organizational levels (4–8). We compared behavioral and health outcomes of 2 interventions — a group-based Booster Break intervention and an individual-based computer-prompt intervention — to those of a usual-break control group. We hypothesized that compared with participants in the other 2 study arms, Booster Break participants would have significant improvements in 1) physiologic measures (ie, blood pressure, fasting lipid and triglyceride concentrations, and anthropometrics); 2) physical activity (increase) and sedentary behavior (decrease); and 3) employee measures such as work social support, quality of life, and perceived stress. BODY.METHODS.STUDY DESIGN: We conducted a 3-arm, cluster-randomized controlled trial at 4 worksites in Texas from 2010 through 2013 to evaluate the Booster Break program. Worksite departments were assigned to 1 of 3 groups by using computerized random-number generation with an equal number of departments at each worksite represented in each group. We selected departments and jobs in which employees sat for at least 5 hours per day. Inclusion criteria were English proficiency, full-time employment (35–40 hrs/wk), being aged 18 years or older, and having no physician-specified limits on physical activity. Interventions lasted 6 months. Data were collected from 2010 through 2013; data entry and analyses were conducted from 2014 through 2015. Participants were paid $25 for completing both baseline and follow-up assessments and received their results (weight, height, blood pressure, and cholesterol) from a free worksite health screening. This study was approved by the Committee for the Protection of Human Subjects at The University of Texas Health Science Center and was registered in the ISRCTN Registry (no. ISRCTN2576399). BODY.METHODS.INTERVENTIONS: Both physical activity interventions were consistent with the World Health Organization's Healthy Workplace Framework and Model, which strives to promote effective workplace interventions (9). Booster Break arm. These structured, peer-led group sessions guided employees through a series of stretching, strengthening, and aerobic movements, followed by a 60-second meditation, described elsewhere (5). Daily worksite sessions lasted 13 to 15 minutes during one 15-minute break. Participants signed an attendance sheet. Computer-prompt arm. This individualized intervention interrupted prolonged sedentary time by introducing 3-minute breaks at 5 hourly intervals daily, thus equaling the time used for the Booster Breaks (15 min). Each worksite installed computer software (Workrave version 1.10 [www.workrave.org], Eyes Relax version 0.87 [Centers for Disease Control and Prevention], and Compact Timer version 2.3.2896.29106 [S7, http://compact-timer-free-download.softwares7.com]) and provided training; prompts encouraged workers to get up and walk hallways, stairs, or outdoors. This intervention relies solely on each worker's motivation, attention, and willingness to stop work when prompted. Participants completed a daily log in which they indicated whether they ignored, partially met, or fully met the computer-prompted physical activity breaks. Usual-break arm (control group). This group practiced usual breaks without interventions at any level. Typical patterns were two 15-minute breaks (morning and afternoon) and 30 to 60 minutes for lunch. Previous studies found that usual-break practices rarely include health-promoting behaviors (eg, consuming nutritious foods, practicing meditation, performing any physical activity) (4,7,8). BODY.METHODS.MEASURES: A team from the wellness services department of a local hospital traveled to each participating worksite at baseline and 6 months to complete physiologic assessments. Height, weight, waist circumference, and blood pressure measurements and blood sampling followed standard protocols to ensure validity and reliability (10–15). Fasting blood sampling was performed in the morning before employees started the workday. Lipid assessments (total cholesterol, high-density and low-density lipoprotein cholesterol, and triglycerides) were conducted at a certified laboratory compliant with Clinical Laboratory Improvement Amendments (http://wwwn.cdc.gov/clia/regulatory/default.aspx). To assess objective levels of physical activity, we used step counts from New Lifestyles DigiWalker SW200 pedometers, which indicated movement, a measure of physical activity, following established protocols (16–18). The pedometer's accuracy, reliability, and suitability for applied physical activity research are reported elsewhere (16–18). For one week at baseline and again at 6 months (program completion), each participant wore the pedometer each day — from rising in the morning until retiring at night — except when showering or bathing. Physical activity was self-reported. We used the International Physical Activity Questionnaire long version (0.8 reported reliability and 0.3 criterion validity), which assesses moderate and vigorous physical activity in 5 domains, across which it has strong reliability and validity (19). It assesses time spent sitting — at work, at home, and during leisure time each day — as a measure of total sedentary time. We also used a scale that focuses exclusively on sedentary leisure time; it is a self-reported 7-day checklist, from the Neighborhood Quality of Life Study, that elicits data on the average daily number of minutes of leisure computer and Internet use, video games, telephone use, and television viewing. Its reliability and validity are reported elsewhere (20). Employee and organizational constructs were work social support, quality of life, and perceived stress, which we assessed using the valid and reliable scales of Johnson et al (21), Ware et al (22), and Cohen and Williamson (23). BODY.METHODS.STATISTICAL ANALYSES: Descriptive statistics were computed to characterize the study population. We used χ2 tests of independence to identify any categorical differences in sociodemographic variables between study conditions. Subsequent nonparametric Kruskall–Wallis testing detected any mean or median differences in the continuous outcomes of the study at baseline. Generalized mixed-effects models (SAS Institute, Inc) were used to estimate within- and between-group changes. Because of normality concerns, we specified log normal or Poisson distributions in our mixed-effects models. Fixed effects in these analyses consisted of time, condition, a time-by-condition interaction (ie, between-group changes), and study covariates (age, race/ethnicity, and education). Participants nested within study conditions were treated as a random effect. If the time-by-condition interaction was significant, it was sliced to determine the relevance of within-group mean changes. For simplicity, we report adjusted least-square means and standard errors. All data were analyzed using SAS version 9.3 software (SAS Institute, Inc), and significance was set a P ≤ .05 with a 2-sided test. Posthoc analyses. Our primary analysis led to additional questions that could help enhance the interpretation of our study. The questions related to 1) whether study outcomes differed by race/ethnicity, 2) whether intervention participants had better outcomes than usual-break participants, and 3) whether outcomes differed by program fidelity. We explored whether African American or Hispanic participants in the Booster Break and computer-prompt groups were more or less likely than non-Hispanic white participants to have improvements in quality of life, employee and lifestyle variables, sedentary behavior, or cardiometabolic markers. Adjusted logistic regression models were then used to determine whether the 2 intervention arms were more or less likely to make positive changes in study outcomes than the usual-break arm. Furthermore, changes in outcomes by consistency of participation in the Booster Break intervention were assessed to account for program fidelity, dose, and adherence. Departments considered to have consistent participation for the Booster Break sessions were defined as those with 80% or more of participants attending each session, an acceptable threshold to expect physiologic changes (5). BODY.RESULTS: Participants (N = 185) were randomized by department (N = 35) to 1 of 3 treatment conditions: Booster Breaks (14 departments; 76 participants), computer prompts (9 departments, 61 participants), and usual breaks (12 departments, 48 participants) (Figure). Ten participants did not have any self-reported data at a given assessment and were eliminated from further analyses. Therefore, only 175 participants were used in the analysis. Participants were racially and ethnically diverse (35% African American, 33% non-Hispanic white, and 32% Hispanic) and had a mean age of 43 years; 82% were women, and 55% had a college degree or more (Table 1). The occupations were clerical (25%), nonclerical (55%), and managerial (ie, managers, supervisors, directors, and superintendents) (9%); information was missing for 11%. We found no significant differences in distribution by type of position (nonclerical vs clerical). We found differences in marital status, work social support, and body mass index (BMI) (all P = .02) by intervention condition (Table 2). FigureStages of the Booster Break study for intent-to-treat analyses (all participants);100% of participants were analyzed using intent-to-treat.A total of 196 worksite employees were assessed for eligibility, 185 were randomized to 1 of 3 conditions (ie, Booster Breaks, computer prompts, or usual breaks). Eleven of 196 participants were excluded because they were missing information on department or allocation arm, 10 of 185 participants were lost to follow-up. Of the 69 participants assigned to the Booster Break arm, 41 (59.4%) completed the study. Of the 59 assigned to the computer-prompt arm, 34 (57.6%) completed the study. Of the 47 assigned to the usual-break (control) arm, 26 (55.3%) completed the study. Table 1 Sociodemographic, Lifestyle, and Biological Characteristics of Study Participants (N = 175), by Study Completion Status, Texas, 2010–2013 Characteristic Total (N = 175) Completed (n = 101) Not Completed (n =74) P Value a Study arm, % Usual breaks (control) 27 55 45 .91 Computer prompts 34 58 42 Booster Breaks 39 59 41 Sociodemographics Age, mean (SD), y 43.4 (11.9) 42.5 (12.0) 44.7 (12.1) .21 Married, % 62 63 60 .72 Race/ethnicity, % Non-Hispanic white 33 58 42 .28 African American 35 49 51 Hispanic 32 63 37 Education, % High school diploma or less 9 73 27 .46 Some college 36 56 44 College diploma or higher 55 57 43 Health-related quality of life scores, mean (SD) Physical health b 56.8 (13.7) 57.3 (13.6) 56 (14.0) .61 Mental health c 68.3 (16.9) 68.6 (18.1) 67.8 (15.1) .15 Work social support d 3.9 (0.7) 4.0 (0.7) 3.9 (0.8) .26 Perceived stress e 1.4 (0.6) 1.3 (0.6) 1.4 (0.7) .55 Lifestyle, mean (SD) Body mass index, kg/m 2 30.3 (7.4) 29.4 (8.0) 30.8 (6.7) .17 Television viewing, min/wk 646 (543) 645 (514) 646 (586) .59 LTPA MET, min/wk 612 (1,214) 709 (1,422) 480 (849) .36 Total pedometer steps/wk 45,475 (21,144) 44,869 (23,280) 46,268 (18,122) .17 Selected cardiometabolic markers, mean (SD) Total cholesterol, mg/dL 190 (35.1) 188 (35.4) 193 (34.8) .32 Glucose, mg/dL 96 (27.7) 95.2 (32.0) 97 (20.3) .39 Systolic blood pressure, mm Hg 116 (15.4) 114 (13.5) 120 (17.1) .02 Abbreviations: LTPA, leisure-time physical activity; MET, metabolic equivalents; SD, standard deviation. a P values for continuous variables were estimated with nonparametric testing; P values for categorical variables were estimated by χ 2 testing for independence. b Physical health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). c Mental health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). d Work social support was scored on a scale of 1 to 5, with higher scores indicating greater social support at work ( 21 ). e Perceived stress was scored on a scale of 0 to 4, with higher scores indicating greater stress ( 23 ). Table 2 Baseline Physiologic, Anthropometric, Lifestyle, and Psychosocial Characteristics of Study Participants (N = 175), by Study Arm, Texas, 2010–2013 Characteristic Usual Breaks (n = 47) Computer Prompts (n = 59) Booster Breaks (n = 69) P Value a Sociodemographics Married, % 63 48 72 .02 Race/ethnicity, % Non-Hispanic white 27 42 31 .30 African American 28 24 48 Hispanic 26 33 41 Education, % High school diploma or less 15 3 9 .07 Some college 25 34 45 College diploma or higher 60 63 46 Sociodemographics, median (LCL b , UCL c ) Age, y 42 (32, 49) 44 (35, 56) 43 (36, 52) .26 Weight, lb 186 (162, 211) 163 (152, 173) 197 (176, 218) .08 Body mass index, kg/m 2 29.8 (27.7, 31.8) 28.3 (26.8, 29.9) 32.2 (30.2, 34.3) .02 Waist circumference, in 39. 4 (37.2, 41.6) 36.9 (35.5, 38.3) 39.9 (37.7, 42.1) .11 LTPA MET, min/wk 99 (0, 396) 421 (0, 975) 99 (0, 767) .11 Total pedometer steps/wk 43,558 (37,693, 49,424) 46,860 (41,311, 52,408) 45,633 (39,335, 51,932) .60 Sedentary time Computer use, min/wk 791 (316, 1,267) 293 (138, 448) 1,075 (453, 1,697) .11 Television viewing, min/wk 736 (558, 915) 670 (475, 864) 813 (436, 1,189) .48 Sedentary min/weekday 3,069 (1,801, 4,338) 2,917 (2,193, 3,641) 3,226 (2,336, 4,117) .74 Sedentary min/weekend 950 (426, 1,473) 602 (464, 740) 794 (620, 969) .14 Health-related quality-of-life scores, median (LCL b , UCL c ) Physical health d 82.3 (76.6, 87.9) 83.4 (78.9, 87.7) 81.2 (76.7, 85.6) .72 Mental health e 77.2 (71.4, 82.9) 76.7 (71.8, 81.6) 75.3 (70.9, 79.7) .75 Work social support f 3.9 (3.7, 4.1) 4.1 (4.0, 4.3) 3.8 (3.6, 3.9) .02 Perceived stress g 1.2 (1.0, 1.4) 1.3 (1.1, 1.5) 1.5 (1.3, 1.6) .10 Cardiometabolic markers, median (LCL b , UCL c ) Total cholesterol, mg/dL 191 (184, 199) 182 (175, 190) 196 (185, 207) .15 High-density lipoprotein cholesterol, mg/dL 52 (47, 57) 48 (45, 51) 53 (50, 56) .12 Low-density lipoprotein cholesterol, mg/dL 110 (102, 119) 108 (99, 116) 114 (105, 124) .58 Triglycerides, mg/dL 121 (97, 144) 106 (89, 123) 121 (92, 150) .82 Blood glucose, mg/dL 94 (89, 99) 94 (90, 98) 99 (89, 108) .84 Systolic blood pressure, mm Hg 113 (109, 118) 116 (112, 119) 119 (115, 123) .41 Diastolic blood pressure, mm Hg 70 (67, 73) 70.0 (67, 73) 71 (69, 73) .83 Abbreviations: LCL, lower confidence limit; LTPA leisure-time physical activity; MET, metabolic equivalent; UCL, upper confidence limit. a P values for continuous variables were estimated with nonparametric testing; P values for categorical variables were estimated by χ 2 testing for independence. b Lower confidence limit = 25%. c Upper confidence limit = 75%. d Physical health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). e Mental health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). f Work social support was scored on a scale of 1 to 5, with higher scores indicating greater social support at work ( 21 ). g Perceived stress was scored on a scale of 0 to 4, with higher scores indicating greater stress ( 23 ). Attrition (or drop out) was estimated at 42% overall and was not significantly different among the 3 arms. Attrition did not differ by sociodemographic, quality-of-life, behavioral, or cardiometabolic variables, with one exception: baseline mean systolic blood pressure was greater among those lost to follow-up (P = .02) (Table 1). On average, African Americans had significantly higher BMIs (P = .003), higher systolic blood pressure (P <.001), higher diastolic blood pressure (P < .001), and larger waist circumference (P = .04) than other participants. Also, African Americans reported significantly more sedentary time on weekends (P = .045) and while using the computer (P = .03) and watching television (P = .004). Hispanic participants had higher serum triglyceride concentrations than other participants (P = .007); non-Hispanic whites reported significantly more leisure-time physical activity (P = .02) and work social support (P = .002) than other participants. BODY.RESULTS.CHANGES OVER TIME BY STUDY CONDITION: A PRIORI HYPOTHESES — INTENT-TO-TREAT ANALYSIS: No significant time-by-condition (baseline to change) interactions or main effects were observed among quality-of-life variables in any of the 3 study arms (Table 3). The time-by-condition interaction for waist circumference was significant (P = .05). We found a small increase in waist circumference among computer-prompt participants (P = .047) but no significant change among Booster Break (P = .29) or usual-break (P = .33) participants. Other anthropometric variables did not differ significantly. Significant time-by-condition interactions were observed for pedometer counts (P = .001) and metabolic-equivalent (MET) minutes of physical activity (P = .001). Weekly pedometer counts increased among usual-break participants (P < .001) but decreased among computer-prompt participants (P < .001) and Booster Break participants (P < .001). Similar results were observed for average daily pedometer counts. Leisure-time physical activity per week increased for usual-break (P < .001) and computer-prompt (P < .001) participants but decreased for Booster Break participants (P < .001). Significant time-by-condition interactions were observed for computer use (P = .001), television viewing (P = .001), and both weekday (Monday through Friday, P < .001) and weekend (Saturday through Sunday, P < .001) sedentary time. Computer use decreased for Booster Break participants (P < .001), decreased for usual-break participants (P = .04), and increased for computer-prompt participants (P < .001). Television viewing time decreased among all study participants, with the greatest reductions in usual-break (P < .001), compared with computer-prompt (P < .001) and Booster Break (P < .001) participants. Weekday sedentary behavior increased significantly for usual-break participants (P < .001) and Booster Break participants (P = .04) but not for computer-prompt participants (P = .20). Weekend sedentary behavior decreased for computer-prompt and Booster Break participants (both P < .001) but did not change significantly for usual-break participants (P = .61). A significant time-by-condition interaction was observed for serum triglyceride concentrations, which increased among computer-prompt (P < .001) and Booster Break (P = .001) but not among usual-break (P = .61) participants. Table 3 Comparison of Baseline and Follow-up Characteristics of Study Participants (N = 175), by Study Arm, Texas, 2010–2013 a Variable Usual Breaks (n = 47) Computer Prompts (n = 59) Booster Breaks (n = 69) Time by Group P Value b Baseline Follow-up Baseline Follow-up Baseline Follow-up Health-related quality-of-life scores Physical health c 58.1 (2.2) 61.3 (2.2) 56.7 (2.5) 60.0 (2.5) 59.1 (2.0) 59.7 (2.0) .33 Mental health d 72.4 (3.0) 68.2 (3.0) 70.2 (2.8) 71.0 (2.8) 70.7 (2.6) 70.5 (2.6) .11 Work social support e 3.8 (0.1) 3.9 (0.1) 4.0 (0.1) 3.9 (0.1) 3.7 (0.1) 3.6 (0.1) .38 Perceived stress f 1.1 (0.1) 1.2 (0.1) 1.3 (0.1) 1.2 (0.1) 1.4 (0.1) 1.4 (0.1) .59 Anthropometrics Body mass index, kg/m 2 29.1 (1.1) 29.3 (1.1) 27.9 (1.1) 28.1 (1.1) 31.1 (1.3) 31.0 (1.3) .97 Waist circumference, in 38.6 (1.2) 38.4 (1.2) 36.2 (1.0) 36.5 (1.0) g 38.6 (1.3) 38.4 (1.3) .05 Weight, lb 182.7 (8.5) 183.3 (8.5) 167.0 (8.4) 169.1 (8.4) h 183.2 (7.9) 183.3 (7.9) .22 Physical activity Total pedometer steps/wk 47,591 (4,354) 47,856 (4,345) h 53,444 (4,346) 53,113 (4,229) h 49,514 (4,058) 47,341 (4,040) h .001 Average pedometer steps/d 6,822 (617) 6,904 (615) h 7,836 (603) 7,625 (599) h 7,176 (575) 6,834 (572) h .001 LTPA METs 326 (134) 362 (134) h 647 (156) 683 (156) h 726 (218) 666 (218) h .001 Sedentary behavior Computer usage, min/wk 570 (111) 556 (111) 398 (95) 555 (94) g 644 (99) 406 (100) h .001 Television viewing, min/wk 719 (92) 648 (92) h 674 (95) 632 (94) h 652 (85) 625 (85) h .001 Sedentary min/weekday 3,416 (777) 3,978 (777) h 3,392 (521) 3,405 (521) 3,497 (571) 3,516 (570) g <.001 Sedentary min/weekend 984 (267) 987 (267) 676 (90) 614 (90) h 809 (97) 755 (97) h <.001 Cardiometabolic markers Total cholesterol, mg/dL 197 (4.8) 197 (4.8) 185 (5.3) 191 (5.3) g 199 (6.1) 200 (6.1) h .21 High-density lipoprotein cholesterol, mg/dL 52 (2.6) 53 (2.6) 48 (2.2) 40 (2.2) 53 (2.2) 55 (2.2) h .80 Low-density lipoprotein cholesterol, mg/dL 120 (5.2) 122 (5.2) 114 (5.7) 123 (5.7) h 121 (5.8) 123 (5.8) .09 Triglycerides, mg/dL 126 (12.8) 126 (12.8) 104 (12.2) 112 (12.2) h 121 (16.5) 128 (16.5) h .02 Glucose, mg/dL 96 (3.3) 95 (3.3) 95 (3.0) 94 (3.0) 98 (5.5) 99 (5.5) .60 Diastolic blood pressure, mm Hg 69 (1.7) 70 (1.7) 70 (1.5) 72 (1.5) 70 (1.3) 72 (1.3) .84 Systolic blood pressure, mm Hg 115 (2.5) 118 (2.5) 118 (2.2) 123 (2.2) g 118 (2.4) 120 (2.4) .59 Abbreviations: LTPA, leisure-time physical activity; MET, metabolic equivalent. a Values are mean (standard error) unless otherwise indicated. An intent-to-treat analysis was applied when participants were followed, regardless of adherence to program. b P values calculated by mixed effects models testing the effects of time, condition, time by condition and adjusted for age, education, and race/ethnicity. c Physical health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). d Mental health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). e Work social support was scored on a scale of 1 to 5, with higher scores indicating greater social support at work ( 21 ). f Perceived stress was scored on a scale of 0 to 4, with higher scores indicating greater stress ( 23 ). g P < .05. h P < .01. BODY.RESULTS.POSTHOC ANALYSES: Logistic regression models adjusted for age and education indicated that African American and Hispanic participants were no more or less likely than non-Hispanic white participants to make improvements in study outcomes. Adjusted logistic regression models indicated that Booster Break participants were 6.8 and 4.3 times more likely to have decreases in BMI and weekend sedentary time, respectively, than usual-break participants. Differences in outcomes by consistency of participation in the Booster Break intervention showed that overall, inconsistent attendees were less likely to be married (P = .006) and more likely to be African American (P < .001) or to report lower mean levels of work social support (P = .003) than consistent attendees. Additionally, inconsistent attendees had significantly greater serum concentrations of glucose (P < .001) and triglycerides (P = .009) and higher systolic and diastolic blood pressure (P < .001) than consistent attendees (Table 4). Table 4 Sociodemographic, Lifestyle, and Biological Characteristics of Study Participants (N = 175), by Intervention Attendance Status, Texas, 2010–2013 a Variable Inconsistent Attendance (n = 69) Consistent Attendance (n = 106) P Value b Sociodemographics Married, % 53 67 .006 Race/ethnicity, % Non-Hispanic white 24 39 <.001 African American 52 24 Hispanic 24 38 Education, % High school diploma or less 7 9 .48 Some college 33 38 College diploma or higher 60 53 Age, median (LCL c , UCL d ), y 43 (35, 52) 44 (34, 52) .80 Health-related quality of life scores, median (LCL c , UCL d ) Physical health e 58 (54, 63) 60 (54, 63) .51 Mental health f 75 (64, 80) 72 (60, 80) .55 Work social support g 3.8 (3.5, 4.0) 4.0 (3.8, 4.5) .003 Perceived stress h 1.2 (0.8, 1.9) 1.3 (1.0, 1.8) .48 Lifestyle characteristics, median (LCL c , UCL d ) Body mass index, kg/m 2 29.1 (25.1, 32.9) 28.7 (25.1, 35.2) .95 Waist circumference, in 37.5 (35.4, 40.7) 37.0 (33.3, 43.3) .65 Weight, lb 178.9 (156.0, 213.3) 168.7 (146.4, 214.3) .33 Total pedometer steps/wk 43,325 (32,498, 58,216) 40,709 (30,708, 52,739) .24 Average pedometer steps/d 6,302 (4,991, 8,389) 5,816 (4,387, 7,575) .12 LTPA METs 198 (0, 657) 198 (0, 918) .89 Computer use, min/wk 150 (60, 450) 120 (40, 420) .64 Television viewing, min/wk 630 (270, 840) 420 (240, 840) .40 Sedentary min/weekday 2,400 (1,800, 3,000) 2,250 (1,800, 2,925) .57 Sedentary min/weekend 480 (360, 960) 600 (360, 960) .94 Cardiometabolic markers, median (LCL c , UCL d ) Total cholesterol, mg/dL 186 (168, 213) 185 (163, 211) .54 High-density lipoprotein cholesterol, mg/dL 50 (42, 60) 50 (41, 57) .66 Low-density lipoprotein cholesterol, mg/dL 109 (88, 137) 109 (83, 132) .66 Glucose, mg/dL 98 (89, 109) 97 (82, 94) <.001 Triglycerides, mg/dL 102 (78, 140) 85 (65, 124) .009 Systolic blood pressure, mm Hg 119 (108, 127) 112 (104, 121) <.001 Diastolic blood pressure, mm Hg 73 (68, 127) 68 (62, 75) <.001 Abbreviations: LCL, lower confidence limit; LTPA leisure-time physical activity; MET, metabolic equivalent; UCL, upper confidence limit. a Departments considered to have consistent participation for the Booster Break sessions were those with 80% or more of participants attending each session. b P values for continuous variables were estimated with nonparametric testing; P values for categorical variables were estimated by χ 2 testing for independence. c Lower confidence limit = 25%. d Upper confidence limit = 75%. e Physical health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). f Mental health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). g Work social support was scored on a scale of 1 to 5, with higher scores indicating greater social support at work ( 21 ). h Perceived stress was scored on a scale of 0 to 4, with higher scores indicating greater stress ( 23 ). BODY.RESULTS.CHANGES OVER TIME BY ATTENDANCE STATUS: INTENT-TO-TREAT ANALYSIS: Consistent Booster Break participants had significant time-by-condition interactions for BMI (P = .049), weekly pedometer counts (P < .001), MET-minutes of physical activity (P < .001), computer usage (P < .001), television viewing (P < .001), serum triglycerides (P = .02), and both weekday (P < .001) and weekend sedentary (P < .001) behavior (Table 5). BMI increased significantly among the usual-break participants (P = .02) but did not significantly change among the remaining participants (computer prompts, P = .58; Booster Breaks, P = .49). Weekly pedometer counts increased among Booster Break (P < .001) and decreased among computer-prompt (P < .001) and usual-break (P < .001) participants. Weekly MET-minutes of physical activity increased among usual-break (P < .001) and computer-prompt (P < .001) but decreased among Booster Break (P < .001) participants. Computer use decreased among Booster Break (P < .001) participants, but increased among usual-break (P < .001) and computer-prompt (P < .001) participants. Television viewing time decreased significantly among all groups, with the greatest decrease among Booster Break (P < .001) participants. Weekday sedentary time increased among all participants (all P < .001), with the greatest increase observed among usual-break (P < .001) participants. Significant reductions in weekend sedentary time were observed among Booster Break (P < .001) and computer-prompt (P = .003) participants, whereas no changes were observed among usual-break (P = .07) participants. Lastly, serum triglyceride concentrations increased significantly among Booster Break (P < .001) and computer-prompt (P = .005) participants but did not change significantly for usual-break (P = .06) participants (Table 5). Table 5 Physiologic, Anthropometric, Lifestyle, and Psychosocial Characteristics of Consistent Study Attendees a (N = 106), Texas, 2010–2013 b Variable Usual Breaks (n = 30) Computer Prompts (n = 39) Booster Breaks (n = 37) Time by Group P Value c Baseline Follow-up Baseline Follow-up Baseline Follow-up Health-related quality-of-life scores Physical health d 59.0 (3.2) 60.0 (3.2) 57.4 (3.5) 61.7 (3.5) 58.9 (3.0) 60.2 (3.0) .66 Mental health e 71.1 (4.6) 67.8 (4.6) 72.5 (4.7) 72.0 (4.6) 70.1 (4.3) 69.5 (4.3) .56 Work social support f 3.8 (0.1) 3.9 (0.1) 4.1 (0.2) 4.0 (0.2) 3.8 (0.2) 3.6 (0.2) .14 Perceived stress g 1.2 (0.2) 1.3 (0.2) 1.2 (0.1) 1.1 (0.1) 1.3 (0.1) 1.3 (0.1) .77 Lifestyle characteristics Body mass index, kg/m 2 28.2 (1.9) 29.2 (1.9) h 27.7 (1.9) 27.9 (1.9) 31.4 (1.8) 31.0 (1.8) .049 Waist circumference, in 38.6 (1.9) 38.5 (1.9) 36.4 (1.9) 36.6 (1.9) 39.4 (1.8) 38.7 (1.8) h .08 Weight, lb 187.5 (13.0) 189.3 (13.0) 169.8 (13.7) 171.6 (13.7) 193.6 (12.4) 192.4 (12.4) .16 Total pedometer steps/wk 50,713 (5,461) 48,780 (5,462) i 59,378 (7,563) 57,811 (7,542) i 45,023 (5,139) 46,689 (5,113) i <.001 Average pedometer steps/d 7,250 (781) 6,973 (781) 8,577 (1,081) 8,299 (1,079) 6,465 (731) 6,754 (727) .45 LTPA METs 252 (114) 284 (114) i 891 (198) 934 (198) i 658 (188) 601 (188) i <.001 Sedentary behavior Computer use, min/wk 349 (132) 372 (131) i 258 (132) 437 (132) i 737 (147) 383 (147) i <.001 Television viewing, min/wk 707 (122) 683 (122) i 767 (127) 725 (126) i 804 (114) 743 (114) i <.001 Sedentary min/weekday 2,285 (559) 2,960 (559) i 3,208 (638) 3,307 (638) i 3,415 (621) 3,598 (618) i <.001 Sedentary min/weekend 629 (165) 617 (165) 535 (138) 519 (138) h 819 (141) 681 (141) i <.001 Cardiometabolic markers Total cholesterol, mg/dL 209 (7.1) 211 (7.1) 196 (8.1) 204 (8.1) h 211 (8.8) 214 (8.8) .33 High-density lipoprotein cholesterol, mg/dL 48 (3.8) 50 (3.8) 48 (3.6) 51 (3.6) 50 (3.6) 52 (3.6) .88 Low-density lipoprotein cholesterol, mg/dL 135 (7.7) 139 (7.7) 126 (8.9) 137 (8.9) i 135 (9.2) 140 (9.2) h .12 Triglycerides, mg/dL 131 (19.3) 137 (19.3) 131 (17.0) 137 (19.2) i 119 (18.7) 135 (18.7) i .02 Blood glucose, mg/dL 94 (5.0) 96 (5.0) 97 (5.3) 97 (5.3) 98 (9.1) 100 (9.1) .78 Systolic blood pressure, mm Hg 117 (4.0) 121 (4.0) 117 (3.7) 124 (3.7) h 118 (3.7) 124 (3.7) h .67 Diastolic blood pressure, mm Hg 70 (2.3) 73 (2.3) 70 (2.4) 74 (2.4) i 69 (2.1) 74 (2.1) i .38 Abbreviations: LTPA, leisure time physical activity; MET, metabolic equivalents. a Departments considered to have consistent participation for the Booster Break sessions were those with 80% or more of participants attending each session. b Values are mean (standard error) unless otherwise indicated. c P values calculated by mixed-effects models testing the effects of time, condition, time by condition and adjusted for age, education, and race/ethnicity. d Physical health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). e Mental health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). f Work social support was scored on a scale of 1 to 5, with higher scores indicating greater social support at work ( 21 ). g Perceived stress was scored on a scale of 0 to 4, with higher scores indicating greater stress ( 23 ). h P < .05 i P < .01. Inconsistent attendees had significant time-by-group interactions for physical health (P = .02); weekly pedometer counts (P < .001); MET-minutes of physical activity (P < .001), computer usage (P < .001), television viewing (P < .001), and weekday and weekend sedentary behavior (P < .001); and serum triglyceride concentrations (P = .001) (Table 6). Physical health increased among usual-break (P = .001) but not among computer-prompt (P = .71) or Booster Break (P = .93) participants. Weekly pedometer counts increased among usual-break (P < .001) but decreased among computer-prompt (P < .001) and Booster Break (P < .001) participants. Similarly, weekly MET-minutes of physical activity increased among usual-break (P < .001) and computer-prompt (P = .001) but decreased among Booster Break (P < .001) participants. Computer usage decreased among usual-break (P < .001) and Booster Break (P < .001) but increased among computer-prompt (P < .001) participants. Television viewing decreased among usual-break (P < .001) and computer-prompt (P < .001) participants, but no change was observed among Booster Break (P = .08) participants. Weekday sedentary behavior significantly decreased among Booster Break (P < .001) and computer-prompt (P < .001) but increased significantly among usual-break (P < .001) participants. Weekend sedentary behavior increased significantly among usual-break (P = .02) and Booster Break (P < .001) but decreased among computer-prompt (P < .001) participants. Lastly, serum triglyceride concentrations decreased among usual-break (P = .008) participants, increased significantly among computer-prompt (P = .01) participants, and did not change significantly among Booster Break (P = .16) participants (Table 6). Table 6 Physiologic, Anthropometric, Lifestyle, and Psychosocial Characteristics of Inconsistent Study Attendees a (N = 69), Texas, 2010–2013 b Characteristic Usual Breaks (n = 17) Computer Prompts (n = 20) Booster Breaks (n = 32) Time-by-Group P Value c Baseline Follow-up Baseline Follow-up Baseline Follow-up Health-related quality-of-life scores Physical health d 56.5 (4.5) 63.7 (4.5) e 55.2 (3.9) 56.1 (3.9) 60.6 (3.1) 60.5 (3.1) .02 Mental health f 77.0 (4.7) 71.1 (4.7) g 66.7 (4.1) 70.2 (4.1) 73.7 (3.6) 74.2 (3.6) .06 Work social support h 4.0 (0.1) 3.9 (0.1) 3.7 (0.2) 3.8 (0.2) 3.6 (0.2) 3.5 (0.2) .29 Perceived stress i 1.0 (0.2) 1.0 (0.2) 1.4 (0.2) 1.3 (0.2) 1.4 (0.2) 1.4 (0.2) .60 Lifestyle characteristics Body mass index, kg/m 2 30.2 (1.8) 29.0 (1.8) g 27.2 (1.5) 27.4 (1.5) 30.9 (2.1) 31.0 (2.1) .07 Waist circumference, in 39.0 (2.1) 38.6 (2.1) 35.8 (1.5) 36.5 (1.5) g 38.5 (2.2) 38.8 (2.2) .06 Weight, lb 184.0 (13.9) 182.3 (13.8) 170.3 (13.1) 172.9 (13.1) g 178.6 (11.6) 180.2 (11.6) .16 Total pedometer steps/wk 48,848 (6,202) 52,586 (6,202) e 52,207 (6,135) 51,669 (6,135) e 55,353 (5,038) 50,055 (5,038) e <.001 Average pedometer steps/d 7,037 (878) 7,687 (878) 7,754 (810) 7,344 (810) 8,088 (702) 7,229 (702) .48 LTPA METs 664 (282) 706 (281) e 287 (228) 309 (228) e 808 (408) 744 (408) e <.001 Sedentary behavior Computer use, min/week 803 (235) 721 (235) e 380 (163) 486 (163) e 443 (129) 354 (129) e <.001 Television viewing, min/wk 766 (187) 610 (187) e 570 (189) 539 (189) e 510 (154) 526 (154) <.001 Sedentary min/weekday 4,563 (2,013) 4,913 (2,013) e 3,102 (1,026) 2,952 (1,026) e 3,156 (1,083) 2,993 (1,083) e <.001 Sedentary min/weekend 1,483 (721) 1,515 (721) g 895 (167) 731 (167) e 723 (164) 772 (164) e <.001 Cardiometabolic markers Total cholesterol, mg/dL 187 (7.6) 185 (7.6) 187 (8.6) 189 (8.6) 190 (9.1) 188 (9.1) .71 High-density lipoprotein cholesterol, mg/dL 59 (4.5) 58 (4.5) 45 (3.6) 45 (3.6) 56 (3.3) 58 (3.3) .73 Low-density lipoprotein cholesterol, mg/dL 110 (8.0) 109 (8.0) 115 (8.9) 118 (9.0) 113 (7.6) 112 (7.6) .46 Triglycerides, mg/dL 108 (20.7) 98 (20.7) e 131 (22.1) 140 (22.1) g 105 (31.0) 100 (31.0) .001 Blood glucose, mg/dL 97 (8.9) 89 (6.9) g 93 (5.3) 90 (5.3) 97 (6.5) 97 (6.5) .25 Systolic blood pressure, mm Hg 112 (3.9) 115 (3.9) 121 (3.8) 122 (3.8) e 117 (3.6) 115 (3.6) g .60 Diastolic blood pressure, mm Hg 70 (2.9) 69 (2.9) 72 (2.4) 71 (2.4) 71 (1.9) 70 (1.9) >.99 Abbreviations: LTPA, leisure time physical activity; MET, metabolic equivalent. a Departments considered to have inconsistent participation for the Booster Breaks sessions were those with fewer than 80% or more of participants attending each session. b Values are mean (standard error) unless otherwise indicated. c P Values calculated by mixed-effects models testing the effects of time, condition, time by condition and adjusted for age, education, and race/ethnicity. d Physical health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). e P < .01. f Mental health was scored on a scale of 10 to 100, with higher scores indicating better health ( 22 ). g P < .05. h Work social support was scored on a scale of 1 to 5, with higher scores indicating greater social support at work ( 21 ). i Perceived stress was scored on a scale of 0 to 4, with higher scores indicating greater stress ( 23 ). BODY.DISCUSSION: This trial evaluated the effects of the Booster Break and computer-prompt interventions among a racially and ethnically diverse population by using a complex and novel research design. The results varied and yielded no clear patterns for the a priori hypotheses analyses or for the posthoc analyses of inconsistent attendees of the Booster Break program. As a result of meeting the performance criteria of 80% attendance, the consistent attendees of the Booster Break program received a sufficient dose of the intervention to assess changes. Consistent attendees in the Booster Break study arm increased their weekly pedometer counts and decreased their sedentary behavior as well as maintained BMI status whereas the usual-break group significantly increased their BMI. In both intervention groups, triglyceride concentrations increased although remained within the normal range. The Booster Break intervention is a simple, peer led, 15-minute group-based physical activity performed at work, in work attire, without equipment, and during standard breaks. Despite this convenience and simplicity, having all participants attend the sessions at a high, sustained level was challenging. It is noteworthy that consistent attendees reported having significantly greater work social support than inconsistent attendees reported. This finding may indicate unique and fundamental differences among the worksites related to readiness and receptivity for health promotion initiatives. Because participants were predominantly female, selection bias and the generalizability of our findings to populations of men, other professions, and nonvolunteers are unknown and merit further research. Differences at baseline and between consistent and inconsistent attendees also merit further study because of the unknown effects of these differences and other undetected covariables. Another limitation is that particular results may be beyond covariate adjustment and influenced by the type of participant in the treatment groups or the treatment itself. For example, computer-prompt participants showed increased computer use, which could be an unintended consequence or a byproduct of the intervention itself. Our novel and complex study design extends the literature, because it evaluated a multilevel (ie, individual, physical, social–environmental, and organizational) intervention, an important shift from previous single-focused interventions (2,9). This study has other strengths, including a randomized controlled design, a racially and ethnically diverse study sample, objective measurements of physiologic outcomes, and a 6-month trial period. The racially and ethnically diverse study sample is important, because research predicts that the workforce will become increasingly diverse (24). Previous research has reported cultural and ethnic differences related to cardiovascular risk factors and other health indicators (25); we found racial and ethnic differences in baseline measures but no differences in study outcomes. Previous research has documented that job strain, defined as the ratio of job demands to job control, is a risk factor for elevated blood pressure and is related to lower representation in surveys and research (26,27). Therefore, we recommend that future studies include measures of job strain and comprehensive assessments of psychosocial stressors so that employees experiencing high levels of job strain will be represented in study samples. Additionally, to better understand differences between consistent and inconsistent attendees, thorough analyses are recommended of management's commitment and support for active work breaks versus devaluing such breaks in favor of meeting work demands (7). Workplace Booster Breaks may mitigate major barriers to physical activity, including lack of time, concerns about neighborhood safety, lack of social support, and costs of equipment, workout attire, and gym membership (28). The challenge is to implement workplace interventions with sufficient fidelity and dose. Management support and commitment are essential to provide incentives and to communicate to employees that sustained participation is critical and expected. If practiced routinely during the workday, Booster Breaks can achieve the twin goals of promoting physical activity and reducing sedentary behavior. Given this potential, further research to replicate, refine, and enhance the effects of the Booster Break program is warranted.

TITLE: Comparing McGRATH® MAC, C-MAC®, and Macintosh Laryngoscopes Operated by Medical Students: A Randomized, Crossover, Manikin Study ABSTRACT: We hypothesized that the McGRATH MAC would decrease the time of intubation compared to C-MAC for novices. Thirty-nine medical students who had used the Macintosh blade to intubate a manikin fewer than 3 times were recruited. The participants performed sequential intubations on the manikin in two simulated settings that included a normal airway and a difficult airway (tongue edema). The intubation time, success rate of intubation, Cormack-Lehane grade at laryngoscopy, and difficulty using the device were recorded. Each participant was asked to identify the device that was most useful. The intubation time decreased significantly and by a similar amount to the McGRATH MAC and C-MAC compared to the Macintosh blade (P < 0.001 and P = 0.017, resp.). In the difficult airway, the intubation times were similar among the three devices. The McGRATH MAC and C-MAC significantly increased the success rate of intubation, improved the Cormack-Lehane grade, and decreased the difficulty score compared to the Macintosh blade in both airway settings. The majority of participants selected the McGRATH MAC as the most useful device. The McGRATH MAC and C-MAC may offer similar benefits for intubation compared to the Macintosh blade in normal and difficult airway situations. BODY.1. INTRODUCTION: Tracheal intubation is critical for securing the airway in various situations. The American Society of Anesthesiologists' Closed Claim study showed that difficult intubation or esophageal intubation is the cause of the approximately 35% of life-threatening respiratory events, including death and permanent brain damage [1]. However, the skill needed for a successful direct laryngoscopic tracheal intubation is complex and difficult to acquire because the individual performing the procedure needs to align the oral, pharyngeal, and tracheal axes to obtain a view of the glottis [2, 3]. Moreover, an emergent tracheal intubation in the prehospital setting or an airway of unanticipated difficulty can further complicate the procedure and decrease the success rate [2]. Even when a physician is skilled in the technique, it is difficult to maintain proficiency due to limited clinical opportunities, particularly for nonanesthetists, which results in increased morbidity and mortality [4, 5]. Therefore, an intubating device that can decrease the intubation time and increase the success rate, particularly for medical students and paramedics who have fewer training opportunities, should be an ongoing focus of research and development. The video laryngoscope has recently become popular in operating rooms and emergency departments. Moreover, the importance of the video laryngoscope has been acknowledged, and this instrument has been incorporated into difficult airway management guidelines [6, 7]. The primary advantage of the video laryngoscope is that the camera on the tip of the blade makes overlapping the laryngeal, pharyngeal, and oral axes unnecessary and the intubation time faster [8, 9]. Among many types of video laryngoscopes available, the C-MAC PM video laryngoscope (Karl Storz, Tuttlingen, Germany) has been verified by multiple clinical studies as appropriate for use both in and outside of hospitals [9–11]. C-MAC has been associated with a shorter intubation time than the Macintosh blade [9, 12]. C-MAC's blade angle is regular and similar to that of the Macintosh blade, and its LCD screen rests on the handle, making the device portable. McGRATH MAC (Aircraft Medical Ltd., Edinburgh, UK) video laryngoscope was recently developed [13, 14], and it has a slim, disposable, transparent, regularly shaped blade similar to the Macintosh blade and a wide LCD screen attached to the handle. Although the McGRATH MAC and C-MAC have the same blade angle and portability, the McGRATH MAC differs from C-MAC in that it is lighter and has a more compact screen and handle and a slimmer blade, and its screen has greater proximity to the axis of the blade (Figure 1). These characteristics of the McGRATH MAC may make tracheal intubation easier and faster, particularly for novices such as medical students, than when using C-MAC. To the best of our knowledge, no study has compared the utility of C-MAC and McGRATH MAC for medical students. Therefore, the aim of this study was to compare the efficacy of the McGRATH MAC and C-MAC for use by medical students. We hypothesized that the McGRATH MAC would decrease the time of intubation compared to C-MAC in both normal and difficult airway situations due to its more practical LCD screen position, slimmer blade, and lower weight. To investigate this hypothesis, we compared the time that medical students required for intubation of a manikin using the Macintosh blade, the McGRATH MAC, and C-MAC. BODY.2. METHODS.2.1. STUDY DESIGN AND RECRUITMENT: This randomized, crossover study was approved by the Institutional Review Board of Severance Hospital (ref: 1-2015-0020) in Seoul, Republic of Korea, and the need to obtain written informed consent was waived. The study was registered at ClinicalTrials.gov (ref: NCT02458534, May 26, 2015). Thirty-nine medical students (30 males and 9 females) who had performed fewer than 3 intubations on manikins using the Macintosh blade were recruited. This study was conducted at the Clinical Simulation Center at Yonsei University College of Medicine between May and July 2015. Our hospital offers an elective training course on intubation for the medical students, and this study was conducted during that course. BODY.2. METHODS.2.2. EQUIPMENT: A SimMan manikin (Laerdal Medical Canada Ltd., Toronto, ON, Canada) was used. Three devices, including C-MAC PM (Karl Storz, Tuttlingen, Germany), the McGRATH MAC (Aircraft Medical Ltd., Edinburgh, UK), and the Macintosh laryngoscope, were used for intubation. A blade size of 3 was used for all devices. We used a 7.5 mm cuffed endotracheal tube (MallinckrodtTM TaperGuard Oral/Nasal Tracheal tube, Covidien, MA, USA) for every intubation. A malleable plastic stylet (PortexTM intubation stylet, Smiths Medical ASD, Inc., Norwell, MA, USA) bent with a hockey stick curvature was used for all devices [13, 15]. BODY.2. METHODS.2.3. EXPERIMENT: All participants watched a video demonstration of intubation techniques for the three devices and were provided with an additional 15-minute oral explanation on how to use the intubation devices and how to determine the Cormack-Lehane laryngoscope grade [16]. After the instruction session, the participants were allowed to perform one intubation with each device with a normal airway. The participants then performed sequential intubations on the manikin in two different simulated situations, including a normal airway and a difficult airway. Participants received a 30-minute break between the airway settings. The tongue edema was set to maximum level to simulate a difficult airway of Mallampati grade 3 or 4, which is known as the risk level for a difficult tracheal intubation [7, 17] and is commonly encountered in clinical practice [9]. Each participant was allowed to perform up to three intubation attempts with each device in each simulated airway setting. The order of use for the devices was randomized using six allocation sequences developed before the start of the study. Therefore, each participant was allowed to perform a total of eighteen intubation attempts during the study. Participants were required to grade the laryngeal view according to the Cormack-Lehane classification in each intubation attempt [16]. BODY.2. METHODS.2.4. DATA COLLECTION: The primary outcome was intubation time, which was defined as the time between when the tip of the blade passed the manikin's teeth and when the first chest expansion was observed using the resuscitation bag after removing the intubating stylet. The secondary outcomes were the number of intubation attempts, the success rate of intubation, the Cormack-Lehane grade at laryngoscopy, and the difficulty of using the device. An intubation attempt was halted if the tip of the blade came out of the mouth. A failed attempt was defined as either an intubation that took longer than 120 seconds or the insertion of the tube into the esophagus [18–20]. A failure to intubation was determined if the participant had not succeeded after three attempts [20, 21]. The difficulty of using the device was recorded on a scale of 0 (extremely easy) to 10 (extremely difficult) after the completion of the intubations using three devices in either the normal airway or difficult airway. Each participant was also asked which device was the easiest to use and why they chose that device. BODY.2. METHODS.2.5. STATISTICAL ANALYSIS: This study had a crossover design. Sample size was calculated using α = 0.05 and β = 0.2. A minimum of 4 participants was required in each randomized sequence to detect a 15-second difference [22] with a standard deviation of 18 seconds, which was based on a previous study [13]. Therefore, we estimated that a total of 24 participants would be required. We enrolled 39 participants in the study to minimize data loss. We compared the primary outcome among the three devices using a linear mixed model (LMM). For the secondary outcomes, such as the success rate of intubation, the number of intubation attempts, the Cormack-Lehane grade, and the difficulty score, an LMM was also used. The results were analyzed using SAS version 9.2 (SAS Institute Inc., Cary, NC, USA.). Data are presented as the median (interquartile range) or number (percentage). A P value less than 0.05 was considered statistically significant. BODY.3. RESULTS: Thirty-nine medical students participated in this study. All participants performed nine intubation attempts in each airway setting, and no data were excluded. As a result, a total of 702 intubation attempts were performed in the two airway settings. In the normal airway, one participant failed to intubate the manikin after three attempts using the Macintosh blade, while all intubations were successful after three attempts with the McGRATH MAC and C-MAC (Table 1). The intubation time on the first attempt did not differ significantly among the three devices in the normal airway. However, the intubation times on the second and third attempts decreased significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade in the normal airway (P = 0.005 and P = 0.037 on the second attempt and P < 0.001 and P = 0.001 on the third attempt, resp.). The overall mean intubation time of the three attempts decreased significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade in the normal airway (P < 0.001 and P = 0.017, resp.). The success rate of intubation on the first, second, and third attempts increased significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade in the normal airway (P < 0.001 on the first attempt, P < 0.001 on the second attempt, and P = 0.012 on the third attempt). The success rate of intubation after three attempts did not differ significantly among the three devices. The number of attempts decreased significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade (P = 0.002). The Cormack-Lehane grade improved significantly on the first, second, and third attempts with the McGRATH MAC and C-MAC compared to the Macintosh blade (P < 0.001). The difficulty score of using the device was higher for the Macintosh blade compared to the McGRATH MAC and C-MAC (P = 0.002). No parameters, including intubation time, success rate of intubation, number of attempts, Cormack-Lehane grade, and difficulty score, differed significantly between the McGRATH MAC and C-MAC. Twenty-four participants chose the McGRATH MAC as the most useful device in a normal airway, while fourteen participants chose C-MAC, and one chose the Macintosh blade (Figure 2). In the difficult airway (tongue edema), 12 participants failed to intubate the manikin with the Macintosh blade, while one failed with the McGRATH MAC, and two failed with C-MAC (Table 2). The intubation time on the three attempts did not differ significantly among the three devices. The success rate of intubation on the first, second, and third attempts increased significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade (P < 0.001). The overall success rate increased significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade (P < 0.001). The Cormack-Lehane grade improved significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade (P < 0.001). The difficulty score of using the device was significantly higher for the Macintosh blade compared to the McGRATH MAC and C-MAC (P < 0.001). No parameters, including intubation time, success rate of intubation, number of attempts, Cormack-Lehane grade, and difficulty score, differed significantly between the McGRATH MAC and C-MAC. Twenty-five participants chose the McGRATH MAC as the most useful device in the difficult airway, while thirteen participants chose C-MAC, and one chose the Macintosh blade (Figure 2). Participants chose the McGRATH MAC as the most useful device for intubation because of its lighter weight (53%), comfortable grip (22%), smooth performance during the insertion of the device (12%), close proximity of the camera to the blade (6%), ease of moving the tongue anteriorly with the blade (4%), and simplicity and compact size (2%). Participants selected C-MAC as the most useful device for intubation because of its stability of the blade which eliminates shaking (70%) and its comfortable grip (30%). BODY.4. DISCUSSION: In the manikin with the normal airway, the intubation time decreased significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade, although there was no significant difference between the McGRATH MAC and C-MAC. In the difficult airway caused by tongue edema, the intubation time did not differ significantly among the three devices. The success rate of intubation, the Cormack-Lehane grade, and the difficulty score all improved significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade in both airway scenarios, while there were no significant differences between the McGRATH MAC and C-MAC except in the selection of the McGRATH MAC as the most useful device. In the normal airway, both video laryngoscopes (the McGRATH MAC and C-MAC) significantly reduced the intubation time compared to the Macintosh blade, although there was no significant difference in the intubation time on the first attempt at intubation. This result agrees with a previous manikin study that involved paramedics and demonstrated that the McGRATH MAC significantly decreased the tracheal intubation time compared to the Macintosh blade in a normal airway [23]. The intubation times were similar between the McGRATH MAC and Macintosh blade in another manikin study that involved medical students [13] and were also similar between C-MAC and Macintosh blade in yet another manikin study that involved experienced anesthetists. We think that this is because the video laryngoscope is very easy to learn and requires less than six intubation attempts to achieve a success rate of more than 90%, as shown in a previous study [24], while approximately 50 attempts are needed to achieve proficiency with the Macintosh blade [25]. Moreover, the McGRATH MAC and C-MAC improve the laryngeal view compared to the Macintosh blade. This result is comparable with those of previous studies, which demonstrated that the Cormack-Lehane grade improves with the use of a video laryngoscope because the camera on the blade tip eliminates the need to align the oral, pharyngeal, and laryngeal axes [13, 18, 26]. The success rate of intubation increased, the number of intubation attempts decreased, and the difficulty score decreased significantly with the McGRATH MAC and C-MAC compared to the Macintosh blade. Our result is inconsistent with a previous manikin study showing that C-MAC did not influence the success rate of intubation, the number of the intubation attempts, or the difficulty score compared to the Macintosh blade in a normal airway [26]. We think that this difference depends on the target subjects and that study investigated experienced anesthetists with approximately 17 years of experience with the Macintosh blade and did not necessarily apply the video laryngoscope in normal airways. By contrast, in observational or retrospective studies in the emergency department, C-MAC was associated with an improved laryngeal view along with a higher success rate compared to the Macintosh blade, which is consistent with our results [10, 11]. We found no significant differences in the observed parameters between the two types of video laryngoscopes (the McGRATH MAC and C-MAC) except for the identification of the most useful device. Therefore, the McGRATH MAC and C-MAC seem to be similarly beneficial in improving the performance of intubation in a normal airway compared to the Macintosh blade, particularly for medical students. In the difficult airway, the intubation times were similar among the three devices. Our results agree with a previous manikin study demonstrating that the intubation times were similar between the McGRATH MAC and Macintosh blade in cervical immobilization performed by medical students [13], and this indicates that there may be no benefit to a video laryngoscope on the intubation time in a situation of tongue edema. In another simulated manikin or clinical study of cervical spine immobilization, the intubation time was longer, with a mean of 13 s or similar, when experienced anesthetists used C-MAC compared to the Macintosh blade, and the success rate and laryngeal view were similar between the two devices [27, 28]. By contrast, in our study, the success rate and the laryngeal grade improved significantly with the use of a video laryngoscope, such as C-MAC or the McGRATH MAC, compared to the Macintosh blade. This difference may be due to the lack of familiarity participants had with the Macintosh blade and the more difficult airway situation caused by tongue edema in our study. In another clinical study with patients who were at risk for a difficult intubation, C-MAC provided a higher success rate with an improved laryngeal view [29]. In our study, the difficulty score was significantly lower with C-MAC and McGRATH MAC than with the Macintosh blade. Therefore, based on our study results, it appears that when medical students use the McGRATH MAC or C-MAC, the number of successful tracheal intubations with a difficult airway situation, such as tongue edema, increases, although the intubation times are similar. To the best of our knowledge, this is the first study to compare the intubation conditions between the McGRATH MAC and C-MAC for novices such as medical students. Several previous studies have investigated the McGrath Series 5 (Aircraft Medical Ltd., Edinburgh, UK), which is the previous version of the McGRATH MAC [30]. The McGrath Series 5 has been shown to increase the success rate of tracheal intubation and to avoid complications, such as esophageal intubation and dental trauma, compared to the Macintosh blade in a normal airway when used by medical students with no intubation experience because of its better laryngeal view [18]. However, the McGrath Series 5 was not reported to reduce intubation time or increase the ease of use compared to the Macintosh blade [18]. With difficult airway simulations, such as cervical spine rigidity, the intubation time was even longer, with a mean of 14 to 80 s, when paramedics or experienced anesthetists used the McGrath Series 5 compared to the Macintosh blade, despite the improvement in the laryngeal view [19, 27, 30]. The McGrath Series 5 does not always guarantee an easy and successful intubation, despite the good laryngeal view [14, 31], which may be because of the disadvantages of the acute angle of the blade, including difficulty with the approach of the tube into the vocal cords even though the vocal cords are easily seen, the advancement of the endotracheal tube into the trachea due to contact with the anterior tracheal wall, and the removal of the stylet from the tube [32, 33]. Moreover, the use of the stylet with the acute angle combined with the endotracheal tube can result in an increased risk of pharyngeal or hypopharyngeal perforation [34–37]. These imperfections have appeared in previous clinical and manikin studies comparing the McGrath Series 5 and C-MAC. In a manikin study of cervical spine immobilization, the success rate was lower, at 28%, and the intubation time was longer, with a mean of 67 s, when experienced anesthetists used the McGrath Series 5 compared to C-MAC [27]. In another clinical study of experienced anesthetists who were very familiar with video laryngoscopes, C-MAC resulted in a faster intubation time, fewer intubation attempts, and a lower difficulty score compared to the McGrath Series 5, despite similar success rates in patients with a Mallampati grade of more than 3 [21]. Therefore, in our study, we did not investigate the video laryngoscope with the acute blade angle but rather investigated the two types of video laryngoscopes with regular-shaped blades that were similar to the Macintosh blade. Our results suggest that a video laryngoscope with a regular-shaped blade can reduce the intubation time and the difficulty of device use even in a normal airway setting and for novices such as medical students. The McGRATH MAC was chosen as the most useful device by the most of the participants, while the Macintosh blade was selected as the least useful device. This finding is similar to the results of previous studies showing that a video laryngoscope was rated as more useful than the Macintosh blade by novice users [26]. This result may have occurred because the laryngeal grade improves with the use of the video laryngoscope, and medical students can confirm the passage of the tracheal tube through the vocal cords clearly via the images on the screen. In our study, most participants chose the McGRATH MAC as the preferred device rather than C-MAC. We investigated the reasons why the participants preferred the McGRATH MAC and determined that the main reason was its lighter weight. Most participants were satisfied with the lighter weight, the smaller and slimmer blade size, and the handle of the McGRATH MAC, which made them feel they could manipulate the device easily. The closer position of the LCD screen to the blade was also a reason for this preference because it may allow for better eye-hand coordination, resulting in a better performance of tracheal intubation and less strain of the neck muscles, as shown in previous studies [38, 39]. Although the video laryngoscopes are useful, as shown by our results, there are some limitations of the use of these devices. The optimal glottic view does not guarantee a successful intubation [40] because correct positioning of the endotracheal tube toward the vocal cords is required while monitoring the patient's mouth. During this process, the physicians cannot always see the tip of the endotracheal tube, which results in oropharyngeal complications such as palatopharyngeal arch perforation [37]. In addition, all types of airway situations cannot be resolved with the use of video laryngoscopes. A previous study demonstrated that intubation failure occasionally occurred with the GlideScope video laryngoscope especially in patients with altered neck anatomy and the presence of a surgical scar, radiation changes, or a mass [41]. The authors recommended that methods other than the video laryngoscope should be used for patients with neck pathology. Moreover, oropharyngeal secretions or blood can obscure the view on the monitor and make intubation using the video laryngoscope impossible [42]. The benefits of video laryngoscope use can be maximized by understanding these limitations, and further studies on the role of the video laryngoscope may be required. There are some limitations to our study. First, because the study was performed on manikins, our results may not directly apply to a clinical situation. However, we chose medical students as the target subjects, and, therefore, there was an ethical concern about performing intubations on real patients. In addition, the manikin has been used widely as a validated surrogate in several previous studies that have investigated intubating devices because it allows the intubation environment to be strictly standardized [13, 18, 26, 31]. Second, we did not investigate other difficult intubation conditions, such as cervical immobilization [13, 26, 30], and our results cannot be applied to these situations. We chose the difficult airway setting caused by tongue edema rather than cervical immobilization or pharyngeal obstruction because tongue edema induces the worst laryngeal view, the highest intubation failure rate, and the highest difficulty score of intubation, as shown in a previous study [20], and we think that the utility of the video laryngoscope is most noticeable and important in the most difficult situation. Third, the participants were not blinded to which laryngoscope was being used, and they may have adjusted to the simulated manikin rapidly, making their intubation attempts more successful with the latter devices than with the former [13]. To decrease this bias, the outcomes were clearly defined before the start of the study, and the participants were randomized into 6 allocation sequences. Fourth, a further limitation may be the small number of the participants in this study. However, we calculated the sample size before the start of the study by considering the crossover design. Moreover, the experiences of the participants were consistent, and participants who had experience with any type of video laryngoscope were not recruited to reduce bias in the comparison of the McGRATH MAC and C-MAC. BODY.5. CONCLUSIONS: The McGRATH MAC and C-MAC resulted in a similar decrease in intubation time compared to the Macintosh blade in the normal airway, while the intubation times were similar among all devices in the difficult airway. The McGRATH MAC and C-MAC resulted in similar improvements in the success rate, laryngeal grade, and difficulty of use compared to the Macintosh blade in both the normal and difficult airways. The McGRATH MAC and C-MAC may have similar benefits in improving intubation conditions in normal and difficult airway situations.

TITLE: Does short-term whole-body vibration training affect arterial stiffness in chronic stroke? A preliminary study ABSTRACT: [Purpose] Previous studies have shown that stroke is associated with increased arterial stiffness that can be diminished by a program of physical activity. A novel exercise intervention, whole-body vibration (WBV), is reported to significantly improve arterial stiffness in healthy men and older sedentary adults. However, little is known about its efficacy in reducing arterial stiffness in chronic stroke. [Subjects and Methods] Six participants with chronic stroke were randomly assigned to 4 weeks of WBV training or control followed by cross-over after a 2-week washout period. WBV intervention consisted of 3 sessions of 5 min intermittent WBV per week for 4 weeks. Arterial stiffness (carotid arterial stiffness, pulse wave velocity [PWV], pulse and wave analysis [PWA]) were measured before/after each intervention. [Results] No significant improvements were reported with respect to carotid arterial stiffness, PWV, and PWA between WBV and control. However, carotid arterial stiffness showed a decrease over time following WBV compared to control, but this was not significant. [Conclusion] Three days/week for 4 weeks of WBV seems too short to elicit appropriate changes in arterial stiffness in chronic stroke. However, no adverse effects were reported, indicating that WBV is a safe and acceptable exercise modality for people with chronic stroke. BODY.INTRODUCTION: Post-stroke survivors face the problem of being predisposed to a sedentary lifestyle that restricts their ability to undertake physical activity resulting in further deconditioning, which can significantly increase stroke reoccurrence and cardiovascular disease1). To overcome the potential for cardiovascular disease, physical activity and exercise programs are often implemented. However, for stroke survivors there are numerous challenges to undertaking an exercise regime, such as time, convenience, compliance, ambulation, and postural control. Thus, traditional exercise may not be always feasible. Alternatively, whole-body vibration (WBV) is seen as an attractive and efficient treatment modality to complement other forms of exercise to improve muscle function, force, and power in healthy, compromised, and sporting individuals2). WBV is a rhythmic activity where body-weight static and/or dynamic exercises are performed on an oscillating plate that elicits rapid stretch-reflexes3) involving the tonic vibration reflex4), which causes changes in muscle fiber length5). Consequently, small changes in oxygen uptake occur, suggesting that muscle energy exists6), that may benefit cardiovascular indices. Acute WBV has been shown to significantly augment leg blood flow7), although heart rate (HR) and brachial blood pressure (BP) have been reported to cause only small increases8) or no changes7, 9). Increased brachial-ankle pulse wave velocity (PWV) and arterial stiffness (PWV) are independent predictors of stroke. Other indices of pulse wave analysis of aortic blood pressure (BP) and augmentation index (AIx) are regarded to be more sensitive predictors of cardiovascular events than brachial BP10). AIx is considered an indicator of left ventricular afterload and can increase due to arterial stiffening where wave reflection from peripheral to central arteries rise. It has been reported that following an acute intermittent WBV protocol, brachial-ankle PWV, leg PWV, and AIx were significantly reduced in healthy young men9, 11). Similarly, 10 min of continuous passive vibration, which was defined by lying supine and placing both legs on a vibrating platform, decreased brachial-ankle PWV, leg PWV, and AIx in healthy participants10). In stroke survivors, leg PWV and brachial-ankle PWV were significantly reduced by acute passive vibration (10 min), but it had no effect on HR, brachial BP, and aortic PWV12). Aerobic exercise has the capability to improve arterial stiffness and cardiovascular risk factors13). Thirty min (2×/week, 6 months) of moderate-high intensity aerobic exercise reduces BP and arterial stiffness13). However, as previously stated, traditional exercise such as aerobic conditioning may not be appropriate for certain patients. Thus, WBV may provide an alternative to traditional exercise as it provides an easy and efficient modality, as shown by its improving of health parameters in less time per session compared to resistance training11). Acute WBV has been shown to decrease arterial stiffness in healthy individuals9, 11). Similarly, long-term (3 months) WBV training has been reported to reduce arterial stiffness in middle-aged/older adults14). WBV is similar to mechanical compression, which produces oscillations to elicit muscle contractions15) and causes arterial vasodilation16). The propagation of the vibration stimulus has the ability to transmit mechanical energy to the upper-body and head17) that can enhance upper-body muscular performance18). Thus, WBV may reduce arterial stiffness through the propagation of mechanical stimuli capable of stimulating lower and upper-body arteries. However, to our knowledge, no study has investigated the efficacy of short-term WBV training in post-stroke individuals. Therefore, the aim of this study was to examine the effects of short-term WBV training on carotid arterial stiffness in chronic stroke survivors. We hypothesized that 4 weeks of WBV training would significantly reduce arterial stiffness. BODY.SUBJECTS AND METHODS.SUBJECTS: Four sedentary men (mean and±standard deviation [SD], age 50.5±14.5 years; body mass 106.6±25.9 kg; height 179.3±3.8 cm) and two sedentary women (age 39±2 years; body mass 86.5±15.5 kg; height 168.0±4.0 cm) volunteered to participate in the study. The inclusion criteria required that the participants be aged between 35–65 years and have suffered a clinically diagnosed stroke between 6 months to 5 years prior to the commencement of the study. The type of stroke (ischemic n = 1; hemorrhagic n = 4; transient ischemic attack n = 1, its locality, left hemisphere n = 2; right hemisphere n = 4) and extent of disability were classified by the modified Rankin Scale (range 1–4). The participants had to meet the requirement never having undertaken WBV and were classified as sedentary according to the ACSM guidelines19). Five participants were involved in weekly, organized activities such as boccia, aqua jogging, and physiotherapy, but these activities did not exceed the aforementioned parameters of a sedentary lifestyle. Exclusion criteria included uncontrolled hypertension or hypotension, bone tumors, herniated discs, and recent fractures (<6 months). The study was approved by the University Human Ethics Committee, and written informed consent was obtained from all participants. BODY.SUBJECTS AND METHODS.METHODS: Prior to testing, every participant completed a familiarization session that included WBV, sphygmoCor, and ultrasound. After baseline measurement, participants were randomly assigned to 4 weeks of WBV training or control followed by cross-over after a 2-week washout period. During the control and washout periods, the participants were instructed to continue their normal daily activities. The WBV intervention consisted of 3 sessions of 5 min intermittent WBV per week for 4 weeks, with at least 24 hours of rest between each WBV session. Pre- and post- (4 weeks) WBV and control measurements of arterial stiffness and cardiovascular indices were performed for all participants. Prior to testing, participants refrained from consuming caffeine during the preceding 12 hours, and refrained from taking drugs with known vascular effects. Participants rested in the supine position for 20 min prior to testing and to account for daily biorhythms, all testing was conducted at the same time each day. During the course of the study, participants were informed not to engage in any additional physical activity above what they were currently performing (Table 1Table 1.WBV training protocolSessionVibration frequency (Hz)Peak-to-peak displacement (mm)Peak acceleration (ms−2)Vibration duration (min)1222.129.85 × 12242.130.25 × 13262.134.15 × 14224.348.85 × 15244.352.65 × 16264.360.75 × 17226.567.35 × 18246.574.55 × 19266.586.85 × 110266.586.87 × 111266.586.87 × 112266.586.87 × 1). WBV was performed on a commercial machine (Galileo Sport, Novotec, Pforzheim, Germany), which had a motorized teeterboard that produced side-alternating vertical sinusoidal vibrations of up to 30 Hz, and maximum peak-to-peak displacement (p-p) of 12 mm. With this WBV machine, p-p was dependent on the participant's foot position. The further the feet were from the central oscillating axis the larger the p-p, and vice versa. Thus, a single-axis accelerometer (iMEMS®, ADXL250, Analog Devices, Norwood, MA, USA) was fixed to the edge of the vibrating platform to measure the p-p and determine the three different foot positions (FtP 1–3). To guarantee the location and identification of the different displacements, longitudinal strips of reflective adhesive tape were applied to the WBV plate. This allowed participants to place their second toe and heel midpoint in line with the tape. This helped keep the feet in the correct position during each session. The researcher constantly checked each participant's foot positioning and corrected any deviations. Over the 12 sessions, participants placed their feet in three different positions: FtP 1 = 2.1 mm p-p; FtP 2 = 4.3 mm p-p; and FtP 3 = 6.5 mm p-p. Participants were asked to maintain a static squat stance with 70° knee flexion attained using a manual goniometer. Seventy-degree knee flexion was selected based on evidence that beyond 30° of static squatting maximizes leg extensor activation20) and reduces the negative side effects of vibration being transferred through to the head17). With shoes on, participants were instructed to place their feet in the protocol-defined positions, maintain an upright torso with their eyes and head facing forward, and evenly distribute their body weight through the mid-foot of both feet. They were allowed to use the support bar if required. To date, there has been no scientific test of a short-term WBV training protocol for stroke. A four-week training protocol was implemented, which was based on earlier work that reported improved muscle force output, functional ability, and general well-being in people with multiple sclerosis21). In the current study, the vibration parameters of vibration frequency (Hz), p-p (mm), peak acceleration (ms−2), and session duration (min) were periodized into four blocks (Table 1). Each block consisted of three WBV sessions where the vibration frequency was systematically increased by 2 Hz (22 Hz, 24 Hz, 26 Hz) and p-p was increased at the commencement of a new block of training. The duration remained the same during the first three blocks; five sets of 1-min bouts of WBV with 1-min rests between each. The intermittent nature and volume of WBV exposure was selected on the basis of the Mason, Cochrane, Denny, Firth, Stannard22) aforementioned study, which reported an improvement in mobility parameters in multiple sclerosis. This is a common approach to increase muscular power in healthy individuals23). To maximize the associated benefits of overload, the fourth block of training (session 10–12) was set at 26 Hz, 6.5 mm p-p, and seven sets of 1-min exposures with 1-min rests (Table 1). The vibration frequency was selected from previous research that showed 25–26 Hz to enhance muscular performance24). The vibration duration of 1-min exposure with 1-min rest was prescribed based on previous research demonstrating positive reductions in arterial stiffness9, 11). PWA was performed using a SphygmoCor device (AtCor Medical, Sydney, Australia), which is a non-invasive tool used to assessed arterial stiffness, central blood pressures, and autonomic function. Brachial BP was recorded using an automated oscillometric device (Nissei DS-157; Mentone Educational Centre, Carnegie, Victoria, Australia). Radial artery waveforms were non-invasively recorded with a high-fidelity micromanometer (SPC-301; Millar Instruments, TX, USA) from the wrist of the non-affected stroke arm using applanation tonometry. These waveforms were calibrated against brachial systolic blood pressure (SBP) and diastolic blood pressure (DBP) from comparable hemodynamic properties of the upper limb arteries. A corresponding aortic pressure waveform was generated using a validated transfer function25), from which central SBP (cSBP), central DBP (cDBP), central pulse pressure (cPP), and AIx was calculated using the integrated software (SCOR Px 7.1, AtCor Medical, Sydney, Australia). AIx was normalized to a heart rate (HR) of 75 beats/min (AIx@75). HR was measured from the time between pulse waveforms, and participants remained in the supine position for all PWA measurements. If the first two consecutive AIx@75 values differed by more than 4% or blood pressures of greater than 5 mmHg, a third measurement was taken and the mean of the closest two values was recorded. Data were collected directly via a personal computer (Toshiba, Intel® CoreTM i5, Windows 7 operating system). PWV was measured using the SphygmoCor device. The pulse pressure wave of the common carotid and radial arteries were recorded non-invasively using applanation tonometry as described above. The recordings were gated using the integrated electrocardiogram, with the velocity of the pulse wave being calculated using the integrated software (SCOR Px 7.1, AtCor Medical, Sydney, Australia). Participants remained in the supine position for BP measurements that were obtained prior to PWV measurements. Prior to electrode placement, any excess hair was removed with a razor and the area cleaned with alcohol wipes. Surface pre-gelled Ag-AgCl electrodes (Ambu, Ballerup, Denmark) were placed on the participant's chest: 1 cm below the suprasternal notch on the sternum, 1 cm above the xiphoid process on the sternum, and 3 cm above the left iliac crest. Distal and proximal measurements were then measured and recorded for each participant. The measurements of PWV and carotid-to-radial pulse transit time standard deviation (PTT SD) were used for subsequent analysis. The common carotid artery was imaged non-invasively using commercial B-mode ultrasound (Sonoite Micromaxx) equipped with a 6–13 MHz linear array transducer. Ultrasound was applied to find local arterial stiffness (β), where β = Ln(SBP/DBP)/(∆D/Dd) and is defined by the arterial diameter over the cardiac cycle relative to change in blood pressure26). Blood pressure of the common carotid artery was recorded using applanation tonometry as described above. Blood pressure at the brachial artery, systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse pressure (PP) were sampled from four 6-second video recordings. For each participant, the probe was placed on the common carotid artery opposite to that of the stroke-affected side at a perpendicular (90°) angle to the vessel, 1 to 2 cm below bifurcation. Ultrasound global and probe-dependent settings were all standardized. Participants held their breath during each of the four 6-second video recordings with the common carotid artery diameters extending across the entire imaging plane. The four 6-second diameter measurements of systolic diameter (Dsys), diastolic diameter (Ddia), and the difference between systolic diameter and diastolic diameter (Dist) were collected and averaged. Additionally, distensibility coefficient (DC) and compliance coefficient (CC) measurements were obtained via ultrasound; where DC is the relative rise of arterial cross-sectional area for a given increase in pressure, and CC is the absolute rise in cross-sectional area for a given increase in arterial pressure, and assumes that the length of the vessel is unaltered by the pulse wave. A repeated measures Analysis of Variance (ANOVA) was performed to determine differences in dependent variables versus time (time [pre-WBV, post-WBV, pre-control, post-control] x intervention). Data gathered was analyzed using SPSS software (version 21, SPSS Inc., Chicago, IL, USA). The level of statistical significance was set at p < 0.05 and all values are expressed as mean±standard deviation (SD). BODY.RESULTS: All six participants completed the required 12 sessions of WBV and four weeks of control, on average it took 4.5±0.5 weeks (mean±SD) to complete 12 WBV sessions. Following a one-month follow-up, no participant suffered any further strokes, symptoms of stroke, or any other complications associated with WBV training. There was no significant interaction effect of intervention and time, and no main effect of intervention or time for any of the PWA measurements (Table 2Table 2.PWV, PWA, blood pressure, and carotid artery diameter values for pre and post WBV and controlWBVControlPrePostPrePostPWV (m/s)7.0±2.16.6±1.86.7±1.36.5±0.7Carotid to radial PTT SD (%)6.5±3.26.9±1.96.8±2.65.5±2.8Arterial stiffness (Beta)12.1±2.510.2±2.710.0±3.111.6±2.7DC (10-3 /kPa)19.6±3.2 22.0 ±6.523.1±6.420.0±6.2CC (mm2 /kPa)0.8±0.21.0±0.31.0±0.30.9±0.3AIx@7518.1±5.017.5±5.914.2±6.818.3±5.8cSBP (mmHg)110.8±9.0112.5±12.7111.9±13.3110.3±11.7cDBP (mmHg)76.4±10.077.9±8.677.9±10.076.6±10.1cPP (mmHg)34.3±3.734.6±7.534.0±6.733.8±6.4HR (beats/min)60.3±9.863.3±11.662.0±12.564.3±13.7SBP (mmHg)120.2±10.1123.5±14.5122.8±15.4120.8±14.1DBP (mmHg)75.7±9.777.0±9.777.3±9.476.0±9.8PP (mmHg)44.5±6.446.5±9.845.5±8.744.8±8.2Dsys (mm) *7.6±0.97.8±0.87.7±0.67.8±0.7Ddia (mm) *7.3±0.97.5±0.87.3±0.77.5±0.6Dist (mm)0.3±0.10.4±0.10.4±0.10.3±0.1PWV: pulse wave velocity; Carotid to radial pulse transit time standard deviation (PTT SD); DC: distensibility coefficient; CC: compliance coefficient; AIx@75: augmentation index normalized HR 75 bpm; cSBP: central systolic blood pressure; cDBP: central diastolic blood pressure; cPP: central pulse pressure; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; PP: pulse pressure (SBP-DBP); Dsys: systolic diameter; Ddia: diastolic diameter; Dist: difference between Dsys and Ddia* Significant time effect post intervention for Dsys (p = 0.039) and Ddia (p = 0.040)). However, a decrease in AIx@75 was displayed for WBV and the control illustrated an increase over time but this time effect was not significant (p = 0.225). There was a significant time effect post intervention for systolic (p = 0.039) and diastolic diameter (p = 0.040) (Table 2) but there was no interaction or intervention effect. Table 2 illustrates the interaction effect of PWV over time, demonstrating a decrease over time for both WBV and control but these measurements were not significant (p = 0.474). Furthermore, there was no significant difference (p = 0.299) in carotid to radial pulse transit time standard deviation between WBV and control (Table 2). Arterial stiffness (Table 2) decreased over time for WBV. In contrast, arterial stiffness increased over time in the control group, but the interaction effect was not significant (p = 0.166). For the distensibility coefficient (DC) (Table 2) there was no significant interaction effect (p = 0.124) or main effect between the WBV and control groups (p = 0.431). Similarly, there was no significant interaction effect (p = 0.237) or main effect for compliance coefficient (CC) (Table 2) between the WBV and control groups (p = 0.496). BODY.DISCUSSION: The primary aim of this study was to assess the efficacy of short-term WBV training on indices of arterial stiffness (carotid arterial stiffness, augmentation index, pulse wave velocity) in individuals who have experienced chronic stroke. The results of the current WBV training protocol did not produce any significant positive or negative effects on central BPs, AIx@75, HR, PWV, carotid arterial stiffness (β), DC, and CC. These results are supported by recent research, which found no significant changes in blood pressure (SBP and DBP) following 3 months of WBV in middle-aged and older adults14). However, some studies of acute WBV have shown significant changes in BP. For example, Figueroa, et al.11) and Rittweger, Beller, Felsenberg27) reported significant increases in SBP after WBV with body-weight squats, where values returned to baseline 15 min post intervention, with Rittweger et al.27) showing a significant decrease in DBP after WBV compared to control (bicycle ergometry). Additionally, an acute WBV study by Otsuki et al.9) found no significant changes in blood pressure (SBP, DBP, and PP) after 60 min of WBV or control (no WBV). In contrast, short-term WBV (6 weeks) showed a significant decrease in SBP 3 min after WBV when compared to control (no exercise)28). However, the discrepancies between the results of Gil28) and the present study may be attributed to exercise protocols and the participants' health conditions (overweight and obese women vs. chronic stroke). In the present study, AIx@75 appeared to decrease post-WBV (0.7±3.1%) and increase post-control (4.3±4.8%). A non-significant decrease in AIx post-WBV has been reported by Figueroa, et al11). The authors reported that AIx decreased significantly during 15 and 30 min of recovery following an acute WBV bout involving a static squat compared to a significant increase in AIx during post-recovery from no-WBV (static squat only). According to the authors, the decrease in AIx wave magnitude following WBV was unlikely to be influenced by HR, but rather a result of vasodilation in the peripheral arteries11), which may explain the decrease in central AIx seen in the present study. Previously, Otsuki, et al.9) reported that following acute intermittent WBV (10 × 60 s, interspersed with 60 s rest) performed by healthy men, brachial-ankle PWV decreased during recovery at 20 and 40 min and returned to baseline 60 min after compared to no WBV (control), indicating that WBV acutely decreased arterial stiffness. In a similar study, the vibration protocol was modified slightly to include a higher frequency (40 Hz) that acquired PWV from carotid-femoral, brachial-ankle, and femoral-ankle sites11). Femoral-ankle PWV decreased significantly at 5 min in both the WBV and without WBV (control) groups, where it continued to remain low during the 30-min recovery post-WBV period and the control returned to baseline. However, there were no significant changes in carotid-femoral PWV or brachial-ankle PWV after either trial. It should be noted that brachial-ankle PWV involves both central and peripheral arterial stiffness, where aortic and leg PWV are the main independent correlates, explaining 58% and 23% of the total variance in brachial-ankle PWV respectively29), and as a result any changes in this measure of arterial stiffness should be interpreted carefully9). The theory of a local effect may help to explain the current findings. If WBV generates a local effect on PWV, it would elicit changes in leg PWV rather than aortic or carotid-radial PWV. This has been demonstrated by changes in femoral-ankle PWV11) and brachial-ankle PWV9), where brachial-ankle PWV was independently correlated with leg PWV29). The changes seen by Otsuki et al.9) may represent changes in both aortic and leg arterial stiffness. However, Otsuki et al.9) did not measure aortic and leg PWV separately, and it cannot be determined whether leg PWV alone contributed to the improvement. Likewise, Figueroa et al.11) did not display any significant changes in brachial-ankle PWV, and it is therefore possible that a local effect could explain the insignificant findings of the present study. The findings of the current study revealed no significant change in carotid arterial stiffness (β), DC, or CC. Although, a significant time effect was evident for both diastolic diameter (p = 0.040) and systolic diameter (0.039), these significant time effects did not show any significance for the relative stroke change in diameter (Dist). A previous study reported that low-intensity resistance exercise in healthy individuals30) acutely increases carotid arterial compliance, systolic and diastolic diameters, and decreased β stiffness index 30 and 60 min post-exercise. However, HR and carotid and brachial BPs showed no significant changes. The differences between the previous and the present study may be attributed to the duration of intervention and consequently when assessments took place after intervention (acute vs. short-term), mode of exercise (bench press vs. WBV), and participant characteristics (healthy individuals compared to chronic stroke). The aim of this study was to recruit eight participants to undergo a trial based on previous work that studied six weeks of WBV in overweight/obese women that improved brachial-ankle PWV by 8%31) and achieved a power of 0.89 with α = 0.05. However, the research was conducted in an area with a small stroke community, where only six participants volunteered for the study. Thus, the non-significant findings of the current study were possibly due to the small sample size. Furthermore, a longer duration of WBV may have elicited the desired changes. This assumption is supported by Gil28), who suggested that it may take longer than 6 weeks to detect changes in PWV. In conclusion, while there were no significant findings, the present study did identify a non-significant decrease in carotid arterial stiffness post-WBV compared to pre-WBV, while control decreased between pre and post. However, no adverse effects were reported, indicating that WBV is a safe exercise modality for chronic stroke patients.

TITLE: Effect of dietary advanced glycation end products on inflammation and cardiovascular risks in healthy overweight adults: a randomised crossover trial ABSTRACT: Diets high in advanced glycation end products (AGEs) are thought to be detrimental to cardiovascular health. However, there remains uncertainty about the beneficial effect of a low AGE diet on cardiovascular risk factors and inflammatory markers in overweight individuals. We thus performed a randomised, double blind, crossover trial to determine whether consumption of low AGE diets reduce inflammation and cardiovascular risks in overweight and obese otherwise healthy adults. All participants (n = 20) consumed low and high AGE diets alternately for two weeks and separated by a four week washout period. Low AGE diets did not change systolic (p = 0.2) and diastolic blood pressure (p = 0.3), mean arterial pressure (p = 0.8) and pulse pressure (p = 0.2) compared to high AGE diets. Change in total cholesterol (p = 0.3), low-density lipoprotein (p = 0.7), high-density lipoprotein (p = 0.2), and triglycerides (p = 0.4) also did not differ and there was no difference in inflammatory markers: interleukin-6 (p = 0.6), monocyte chemoattractant protein-1 (p = 0.9), tumour necrosis factor α (p = 0.2), C-reactive protein (p = 0.6) and nuclear factor kappa beta (p = 0.2). These findings indicate that consumption of low AGE diets for two weeks did not improve the inflammatory and cardiovascular profiles of overweight and obese adults. BODY.INTRODUCTION: Advanced glycation end products (AGEs) are formed endogenously from non-enzymatic reactions of amino acids with sugars1. Consumption of foods high in sugar and/or foods exposed to high temperature cooking methods such as deep-frying, broiling, roasting, baking and grilling can increase the total daily AGE intake by 25% compared to the average adult daily intake2, 3. High AGE intake from food accelerates the production of endogenous AGEs and increases the level of circulating AGEs in the blood stream4, 5. This intake of high AGE contributes to the progression of type 2 diabetes (T2DM) and cardiovascular diseases (CVD) although the exact mechanisms are not clearly understood6–11. AGE formation can be reduced by shortening the cooking time and lowering temperature to reduce food browning, or by addition of acidic (low pH) ingredients and by high humidity or food moisture content7, 12. Given the ease of such changes, reduction of AGE intake may be a promising intervention to lower cardiovascular risk3, 13–15. In humans, the impact of low AGE diets on inflammation and cardiovascular risk factors is not clearly understood. In some studies involving healthy obese individuals, low AGE diets reduced inflammatory markers11, 16, 17 but did not change plasma lipid levels17, 18 or blood pressure17, 19. Whereas, other studies showed a reduction in plasma lipid levels after low AGE diets19–21 but not of markers of inflammation18, 22. In patients with T2DM, low AGE diets improved inflammatory markers6, 8, 9, but not blood pressure9, and lipid levels6, 9. Cai and colleagues however reported a reduction in oxidised low-density lipoprotein after 6 weeks of consumption of low AGE diets (5 fold lower in AGE content) in patients with T2DM23. In all these studies, test and control diets were either not matched for energy and macronutrient content or it was not stated whether similar macronutrient content was achieved between the diets, which could potentially influence the results24. We have therefore investigated the impact of low and high AGE diets carefully matched for both energy and macronutrient profile. These matched diets were given for two weeks each to healthy obese adults in a randomised cross-over design trial to determine effects on cardiometabolic parameters. We have previously reported the main outcomes of the trial which showed that dietary AGEs improved insulin sensitivity but not insulin secretion in overweight and obese non-diabetic individuals24. In this study, we conducted analyses of secondary outcomes of the trial and investigated if dietary AGEs improve blood pressure, plasma lipid profiles and inflammatory markers. BODY.RESULTS.BASELINE CHARACTERISTICS: As previously described24, the mean age, body mass index, waist-to-hip ratio and % body fat of the participants were 34 (10) years, 31.3 (3.8) kg/m2, 0.9 (0.1) and 31.1 (6.7), respectively. There was no difference in levels of circulating AGEs and metabolic profiles such as insulin sensitivity and secretion between group 1 and 2 before each test diet24. Anthropometric and blood pressure measurements, plasma lipid levels and inflammatory markers were also not different between the two interventions at the beginning of each dietary period (Table 1). The cumulative dietary AGE intakes were significantly lower in the low AGE dietary period compared to the high AGE dietary period (all p < 0.002), as reported previously24. The mean consumption of carboxymethyllysine, carboxyethyllysine and methylglyoxal-derived hydroimadazolidine were decreased by 27%, 38%, and 21%, respectively, in the low AGE group compared to the high AGE group.Table 1Participants' baseline characteristics at the beginning of each study period (n = 20).ParametersLow AGE groupHigh AGE groupP-valueWeight (kg)94.2 ± 15.294.1 ± 14.30.94Body mass index (kg/m2)31.4 ± 3.831.3 ± 3.70.85*% body fat30.7 ± 6.530.6 ± 6.60.83Waist-to-hip ratio0.9 ± 0.070.9 ± 0.070.35*Systolic BP (mm Hg)123.1 ± 10.8122.3 ± 12.30.66Diastolic BP (mm Hg)77.9 ± 8.976.7 ± 90.38Mean arterial pressure (mm Hg)92.9 ± 9.191.9 ± 90.37Pulse pressure (mm Hg)45.1 ± 6.445.6 ± 10.10.90*Total cholesterol (mmol/l)4.8 ± 14.6 ± 10.06LDL (mmol/l)3.1 ± 0.83.1 + 0.70.61HDL (mmol/l)1 ± 0.21 ± 0.20.35Triglycerides (mmol/l)1.5 ± 11.3 ± 0.70.11*CRP (mg/l)2.1 ± 1.92.4 ± 2.70.69*TNFα (pg/ml)2.8 ± 3.71.6 ± 0.90.18MCP-1 (pg/ml)126 ± 99121 ± 660.92*IL-6 (ng/ml)1.5 ± 2.21.6 ± 2.40.64*NFκβ p65 activity (ng/μg protein)14 ± 1012 ± 100.53All values are presented as means ±SDs. Paired t-tests were conducted to determine the p-values. *Data transformation was done. BP, blood pressure; CRP, C-reactive protein; HDL, high-density lipoprotein; IL-6, interleukin -6; LDL, low-density lipoprotein; MAP, mean arterial pressure; MCP-1, monocyte chemoattractant protein-1; NFκβ; Nuclear factor kappa beta; PP, pulse pressure. BODY.RESULTS.EFFECT OF LOW AGE DIET ON INFLAMMATORY MARKERS: Inflammatory markers such as tumour necrosis factor α (TNF α), C-reactive protein (CRP), monocyte chemoattractant protein 1 (MCP-1) and interleukin 6 (IL-6) levels and nuclear factor kappa beta (NFκβ) activity in peripheral blood mononuclear cells (PBMCs) did not change after consumption of either low or high AGE diets. Between groups comparison also showed that the changes in these markers were not significantly different between low and high AGE diets (Table 2).Table 2Effects of low and high AGE diets on markers of inflammation and cardiovascular risk factors (n = 20).ParametersChange from baseline in low AGE groupChange from baseline in high AGE groupDifference between the groupsMean ± SDP-value†Mean ± SDP-value†Mean ± SDP-value††Weight (kg)−0.69 ± 1.340.03−0.16 ± 1.170.54−0.52 ± 1.520.17BMI (kg/m2)−0.22 ± 0.450.03−0.06 ± 0.380.49−0.16 ± 0.50.20% body fat−0.06 ± 1.20.82−0.08 ± 1.390.80+0.02 ± 1.560.37WHR+0.004 ± 0.030.55−0.004 ± 0.0320.54+0.008 ± 0.0310.49Systolic BP (mm Hg)−1.2 ± 7.70.49−1.3 ± 8.30.47+0.1 ± 11.40.24Diastolic BP (mm Hg)−0.8 ± 8.40.67−1.9 ± 6.20.18+1.1 ± 11.60.33Pulse Pressure (mm Hg)−0.4 ± 7.70.81+0.5 ± 9.70.80−0.9 ± 9.20.16MAP (mm Hg)−0.9 ± 7.30.57−1.7 ± 5.20.16+0.8 ± 10.70.80Total cholesterol (mmol/l)−0.4 ± 0.50.0006−0.15 ± 0.30.04−0.3 ± 0.60.26LDL (mmol/l)−0.3 ± 0.30.002−0.01 ± 0.30.86−0.3 ± 0.50.73HDL (mmol/l)−0.06 ± 0.10.03−0.008 ± 0.080.66−0.05 ± 0.10.21Triglycerides (mmol/l)−0.1 ± 0.30.08−0.2 ± 0.30.01+0.04 ± 0.50.44IL-6 (ng/ml)−0.4 ± 1.90.71*−0.2 ± 1.80.94*−0.2 ± 2.90.62*MCP-1 (pg/ml)−5.4 ± 37.40.52+1.9 ± 44.60.84+7.41 ± 65.80.94TNFα (pg/ml)−0.4 ± 4.40.84*+1.8 ± 5.50.16*−2.2 ± 7.10.15*CRP (mg/l)+0.08 ± 0.660.57+0.19 ± 1.550.57+0.1 ± 1.60.60NFκB p65 activity (ng/μg protein)−3.4 ± 9.90.14−1.3 ± 11.40.61−2.1 ± 16.70.18†Paired t-tests were conducted to determine the p-values within each groups. ††A 2 × 2 ANOVA for cross over study was used to determine the overall difference between the diets. *Data transformation was done. BP, blood pressure; CRP, C-reactive protein; HDL, high-density lipoprotein cholesterol; IL-6, interleukin -6; LDL, low-density lipoprotein; MAP, mean arterial pressure; MCP-1, monocyte chemoattractant protein-1; NFκβ; nuclear factor kappa B; SD, standard deviation. BODY.RESULTS.EFFECT OF LOW AGE DIETS ON CARDIOVASCULAR PARAMETERS: Anthropometric measures such as weight and body mass index were significantly reduced only in the low AGE group compared to high AGE group. Percentage body fat and waist-to-hip ratio did not change after consumption of either diet. Between groups comparison have shown that none of these anthropometric measures were different between the diets (Table 2). Systolic and diastolic blood pressure and mean arterial pressure decreased after consumption of both diets whereas pulse pressure was reduced only after intake of low AGE diets. Between groups analyses (after controlling for period and sequence effect) did not show any significant change in these measurements between the diets (Table 2). The total plasma cholesterol levels decreased in both groups, however, the change between the two diets was not significant. Both plasma low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels decreased with low AGE diet but not changed with high AGE diet whereas the overall difference between the diets was not significant (Table 2). Plasma triglyceride levels decreased with high AGE diet but there was no difference between low and high AGE diets (Table 2). BODY.RESULTS.CORRELATION ANALYSES BETWEEN INFLAMMATION MARKERS AND ANTHROPOMETRIC MEASURES AND CARDIOVASCULAR RISK FACTORS: The correlation analyses indicated that the change in diastolic blood pressure and MCP-1 is positively correlated with % body fat (r = 0.43, p = 0.05) and waist-to-hip ratio (r = 0.46, p = 0.03), respectively. Change in systolic blood pressure is also positively correlated with NFκβ (r = 0.47, p = 0.04) whereas plasma lipid levels negatively correlate with CRP (total cholesterol: r = −0.53, p = 0.01; HDL: r = −0.59, p = 0.006; LDL: r = −0.56, p = 0.008) and IL-6 (total cholesterol: r = −0.51, p = 0.03; HDL: r = −0.58, p = 0.01; LDL: r = −0.54, p = 0.01). After adjusting either for change in weight, body mass index, % body fat or waist-to-hip ratio, neither systolic blood pressure nor lipid parameters correlated with inflammatory markers. Other inflammation parameters did not show any correlation with cardiovascular parameters (all p > 0.1). Circulating and urinary AGE levels also did not correlate with any cardiovascular parameters (all p > 0.09). BODY.DISCUSSION: The present study aimed to measure the effect of consuming low AGE diets for 2 weeks on markers of inflammation and cardiovascular risk factors in healthy obese and overweight individuals. No significant difference was found in changes in blood pressure, plasma lipid levels (total cholesterol, LDL, HDL, triglycerides) or inflammatory markers (CRP, TNFα, MCP-1, IL-6, NFκβ activity) between low and high AGE diets. Our findings on blood pressure measurements were consistent with other studies conducted in healthy overweight individuals17, 19, prediabetes21, obese people with metabolic syndrome25, 26, and patients with T2DM9. Regarding plasma lipid profile, we did not find a significant difference between low and high AGE diets. Some studies conducted in healthy individuals17, 18, obese individuals with metabolic syndrome25, 26, and patients with T2DM6, 9 have also reported that consumption of low AGE diets did not change plasma lipid levels compared to high AGE diets. In contrast, two other studies that involved young healthy obese volunteers20 and prediabetic individuals21 noted a reduction in plasma lipid levels after a low AGE diet. In the first study, the difference between the 2 diets could have been due to the higher intake of dietary fat and carbohydrate on the high AGE diets20. The patients in the second trial in the low AGE arm of the study had relatively lower intima-media thickness compared to those who were in the high AGE diet21. This may exaggerated the effect of high AGE diets on lipid profile. In addition, consumption of low AGE diets combined with aerobic exercise program for 12 weeks showed a decrease in plasma lipid levels in overweight and obese men19. This effect however could have been due to the synergistic effect of physical activity with low AGE diets as it has been widely known for its beneficial effect on metabolic changes27. Therefore, these studies19–21 were not able to differentiate between the true effect of low and high AGE diet as they were confounded by other differences in diet, exercise or disease state of the patients. In this study, intake of low AGE diets for 2 weeks did not change inflammatory markers. This finding was consistent with some studies conducted in healthy overweight and obese individuals18, 22. Other studies conducted in healthy individuals showed an improvement in inflammatory markers after intake of low AGE diets11, 16, 17. However, in these studies, the diets were not matched for macronutrient content, which might have influenced the findings. Studies in obese people with metabolic syndrome26, prediabetes21 and patients with T2DM8, 9 showed an improvement in inflammatory markers after intake of low AGE diets compared to high AGE diets. Therefore, it could be suggested that intake of low AGE diets was beneficial for those who had higher baseline values in inflammatory markers such as patients with metabolic syndrome or T2DM. Compared to the previous studies, our study has several strengths that include standardised diets in energy and macronutrient content were provided, study personnel including the investigators were unaware of the dietary allocation, and baseline characteristics such as anthropometric, circulating AGEs and metabolic parameters were not different at baseline during each dietary period. We have also used a crossover design to control for possible confounding factors in addition to the rigorous randomisation process. The small sample size and relatively short duration could be considered as main limitations of this study as one can argue that the change in physiologic processes after intake of low AGE diets may require longer duration. In conclusion, we have shown that consumption of low AGE diets for 2 weeks did not have beneficial effects in reducing markers of inflammation and cardiovascular risk factors in healthy overweight and obese adults. Long-term well designed trials with larger sample sizes are needed to confirm our findings. BODY.METHODS.STUDY PARTICIPANTS: Twenty overweight and obese but otherwise healthy and normoglycemic adults, aged 18–50 years completed the study24, according to a protocol as previously published elsewhere28. Participants did not have diabetes as indicated by a 75 g oral glucose tolerance test (OGTT) (WHO 1999 criteria). All were non-smokers and had no clinical and laboratory signs of infection and none took supplements/medications during the study period. The participants were recruited from the general community through advertisements between January 2006 and December 2010. Ethical approval was obtained from the Alfred Hospital Ethics Committee, Melbourne, Australia and complied with the Declaration of Helsinki. All participants provided written informed consent prior to participation. BODY.METHODS.STUDY DESIGN: This study employed a two-period randomised cross-over double-blind design. All participants underwent both diets; one low in AGE content and one high in AGE content (typical of a modern Western diet) and diets were matched in energy and macronutrient content. Participants commenced the study after a two-week run-in on their habitual diet but with limitation of their alcohol, fast food and coffee intake. Test diets were consumed for two weeks separated by a four-week washout period (habitual diet). The primary outcomes of this study that focused on insulin resistance/sensitivity have been published24. BODY.METHODS.RANDOMISATION AND MASKING: Randomisation occurred for 7 blocks of 4 participants stratified by gender and diet order. Participants were masked to the allocation of diet type and to how the diet might affect glucose metabolism. Clinical and laboratory investigators were also masked to diet allocation. BODY.METHODS.STUDY PROCEDURES: All participants underwent medical and laboratory screening including a 75 g oral glucose tolerance test (OGTT). Prior to metabolic testing, participants were asked to abstain from strenuous exercise and caffeine for 3 days. BODY.METHODS.DIETARY INTERVENTION: Prior to starting the 'run-in' to the first diet, participants kept a 3-day diet record of their habitual diet (2 weekdays, 1 weekend day) based on household measures. Nutrient content was analysed with SERVE (SERVE Nutrition Systems, St Ives, NSW), based on Australian food composition tables. With the use of Australian food composition data from SERVE as well as data from the United States on the N (ε)-carboxymethyllysine (CML – the most common AGEs used in clinical studies as an indicator of dietary AGE intake) content of common foods, a menu of carefully matched alternative food choices (of low AGE vs high AGE content) individualised to suit the preferences and habitual diet of each participant was designed. These alternative food choices were matched for macronutrient content and total energy but greatly differed in calculated AGE content, and were provided for each meal of the day, including snacks and beverages. Food choices on the high AGE diet had higher dietary AGE content, while the isoenergetic low AGE diet, matched for macronutrient content, had reduced AGE content through altered cooking techniques4 and use of differently processed foods. All food for the two test diets was provided weekly to the participants as ready-to-eat items or as packed food portions, to assist with dietary compliance. For food that required cooking, detailed instructions for storage and heating (method, temperature, and duration) were provided. Participants were asked to eat to appetite throughout both dietary periods to maintain constant body weight. They were required to keep a detailed dietary record indicating cooking method and number of portions eaten for each food item supplied, unconsumed foods or additional foods eaten. Later foods incorporated in these diets were also chemically analysed for their AGE content. The dietician made telephone contact twice a week to provide support and resolve difficulties as well as to ensure dietary compliance. To analyse the dietary AGE content, food items were obtained from local supermarkets and prepared according to the instructions provided to the participants in the study. BODY.METHODS.CARDIOVASCULAR MEASURES: Systolic and diastolic blood pressure were measured using an automated oscillometric measurement system (Omron) after a 30 minute rest. Pulse pressure was calculated by subtracting the diastolic blood pressure from the systolic blood pressure. Mean arterial pressure was computed as diastolic blood pressure + pulse pressure/329. Plasma lipid levels such as total cholesterol, LDL, HDL and triglycerides were measured using a standard commercial enzymatic assay (Beckman Coulter LX20PRO Analyser and Synchron) and Systems Lipid and Multi Calibrators (Beckman Coulter Diagnostics). BODY.METHODS.MEASUREMENT OF INFLAMMATORY MARKERS AND AGES: Markers of inflammation (TNFα, MCP-1 and IL-6 levels) were analysed using a commercial automated chemiluminescent enzyme immunoassay and immulite analyser (Diagnostic Products Corporation). Whereas, highly sensitive near infrared particle immunoassay rate methodology was used to measure plasma CRP levels. NFκβ activity in PBMCs was detected and quantified using TransAM NF- κβ DNA-binding activity assay (Active Motif, Carlsbad, CA, USA). Measurement of urinary and serum AGEs were quantified with the use of ultraperformance liquid chromatography–tandem mass spectrometry as previously described30. BODY.METHODS.STATISTICAL ANALYSIS: Change from baseline values was calculated for each parameter in both dietary periods. Paired t-tests were used to compare the participants' characteristics at the beginning of each dietary period and to determine the change in parameters after each study diet. Repeated measures ANOVA was employed to determine the change difference between the two diets after adjusting for carryover, sequence and period effects. The observed carryover effect in this study was very minimal (29.8%) and thus the data in both study periods were combined. Results are presented using means and standard deviations unless and otherwise stated. Appropriate data transformation was undertaken in the event of skewed data to approximate a normal distribution. Statistical analyses were performed using Stata 14 software. P-value of ≤ 0.05 is used to describe statistical significant difference. BODY.METHODS.DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

TITLE: Innovative psycho-educational program to prevent common postpartum mental disorders in primiparous women: a before and after controlled study ABSTRACT.BACKGROUND: Universal interventions to prevent postnatal mental disorders in women have had limited success, perhaps because they were insufficiently theorised, not gender-informed and overlooked relevant risk factors. This study aimed to determine whether an innovative brief psycho-educational program for mothers, fathers and first newborns, which addressed salient learning needs about infant behaviour management and adjustment tasks in the intimate partner relationship, prevented postpartum mental health problems in primiparous women. ABSTRACT.METHODS: A before and after controlled study was conducted in primary care in seven local government areas in Victoria, Australia. English-speaking couples with one-week old infants were invited consecutively to participate by the maternal and child health nurse at the universal first home visit. Two groups were recruited and followed sequentially: both completed telephone interviews at four weeks and six months postpartum and received standard health care. Intervention group participants were also invited to attend a half-day program with up to five couples and one month old infants, facilitated by trained, supervised nurses. The main outcome was any Composite International Diagnostic Interview (CIDI) diagnosis of Depression or Anxiety or Adjustment Disorder with Depressed Mood, Anxiety, or Mixed Anxiety and Depressed Mood in the first six months postpartum. Factors associated with the outcome were established by logistic regression controlling for potential confounders and analysis was by intention to treat. ABSTRACT.RESULTS: In total 399/646 (62%) women were recruited; 210 received only standard care and 189 were also offered the intervention; 364 (91%) were retained at follow up six months postpartum. In women without a psychiatric history (232/364; 64%), 36/125 (29%) were diagnosed with Depression or Anxiety or Adjustment Disorder with Depressed Mood, Anxiety, or Mixed Anxiety and Depressed Mood in the control group, compared with 16/107 (15%) in the intervention group. In those without a psychiatric history, the adjusted odds ratio for diagnosis of a common postpartum mental disorder was 0.43 (95% CI 0.21, 0.89) in the intervention group compared to the control group. ABSTRACT.CONCLUSIONS: A universal, brief psycho-educational group program for English-speaking first time parents and babies in primary care reduces de novo postpartum mental disorders in women. A universal approach supplemented by an additional program may improve effectiveness for women with a psychiatric history. ABSTRACT.TRIAL REGISTRATION: ACTRN 12605000567628. BODY.BACKGROUND: Postnatal mental health problems in women are associated with disability, reduced social participation and diminished caretaking capacity [1] and constitute a significant public health problem [2], which has proved difficult to prevent [3]. BODY.BACKGROUND.NATURE AND PREVALENCE OF POSTPARTUM MENTAL HEALTH PROBLEMS: The predominant focus of research, policy initiatives, clinical practice recommendations and health education has been postnatal depression, but there is increasing evidence that postnatal anxiety disorders are at least as common, but less well recognised than depression [4,5]. Brockington [1] in a review of postnatal psychiatric disorders concludes that women identified through screening as depressed actually have heterogeneous conditions including posttraumatic stress disorder, panic, phobic, obsessional and generalised anxiety disorders, adjustment disorders and depression. These are situation-focused, disabling and often reflect adversity [6]. Even among those who meet diagnostic criteria for major depression, severity ranges from mild to severe and most depression after childbirth is minor and not major [7]. Brockington [1] argues that 'postnatal depression' therefore has value as a lay term, but is imprecise as a clinical or a research construct. This lack of clarity is reflected in widely divergent estimates of prevalence for probable depression as assessed by the Edinburgh Postnatal Depression Scale [8] in women in high income countries from 3.7% to 36% [9] and for diagnoses of postnatal generalised anxiety disorder from 4.4 to 8.5% [10]. There is less evidence about the prevalence of panic disorders in women after childbirth, but a much higher de novo onset of panic attacks in the first twelve weeks postpartum (10.9%) than expected by chance (0.92%) is reported [10]. In women with a history of panic attacks, symptoms tend to increase after childbirth, but not during pregnancy [10]. The prevalence of adjustment disorders arising in response to the birth of a baby has not been established. However, there is recognition that a proportion of mothers-of-infants who seek help for early parenting difficulties do not meet diagnostic criteria for depression or an anxiety disorder, but do have higher than population average scores on the Edinburgh Postnatal Depression Scale (EPDS) [8]. An expanded conceptualisation, including adjustment disorders, is required to recognise their needs [11]. BODY.BACKGROUND.UNIVERSAL INTERVENTIONS TO PREVENT POSTNATAL DEPRESSION: In addition to treatment for people with current mental health problems, a comprehensive approach to mental health service delivery must include mental health promotion and the prevention of mental disorders [12]. A range of interventions to prevent postnatal mental health problems, principally 'postnatal depression', have been tested in randomised controlled trials. These have included secondary prevention via indicated interventions for women with current clinically significant depressive symptoms, selective interventions for women identified by screening as at risk of developing depression and universal interventions offered to all women to reduce population prevalence [13-17]. Universal strategies are preferred; because even small reductions in population prevalence have a greater public health benefit than treating individuals who are already symptomatic [18]. They are also less stigmatizing and more likely to be used [19]. Systematic reviews have concluded that screening measures administered in pregnancy have low positive predictive values, probably because events after childbirth are more salient determinants of postnatal depression [15]. There have been seven trials of universal postnatal interventions offered to unselected populations of women who have recently given birth. Five tested strategies for use with individual women: debriefing with a psychologist about childbirth experiences [20] or a midwife listening visit during the postnatal hospital stay [21]; earlier-than-usual postnatal visit to a primary care physician [22]; ten three-hour home visits involving practical assistance and emotional care from a specifically trained support worker in the first postpartum month [23]; and an information pack containing specific, salient written information about maternal health, sleep and support needs and management of infant crying, with or without an invitation to attend a facilitated new mothers' group [24]. The other two trials assessed comprehensive community-based interventions which involved increasing the skills of primary health care nurses to identify women's physical and mental health conditions and initiate referral to appropriate health services [25,26]. Lumley et al [26] also provided specific training for general practitioners and community development aimed at enhancing local facilities and services for parents of newborns. Of these varied strategies, only Lavender et al's midwife-listening visit [21] and MacArthur et al's primary health care intervention [25] were associated with reduced mental health problems in the treated compared to the control group. In Lavender et al's study, there was apparent selection bias in that 60% of participants were single women and the very high rates of self-reported depression and anxiety in the control arm (50% classified as having clinically significant symptoms) led reviewers to conclude that it is a 'true outlier' [27]. McArthur et al's intervention [25] was embedded in the UK National Health Scheme in which women are more likely to have an established relationship with a primary health care practice than in other health systems. All studies were adequately powered, analyzed by intention to treat, had properly concealed random allocation to trial arms and blinded assessment of outcomes. There were some methodological limitations: attrition greater than 20% at final assessment [22-25] and in one trial there was poor compliance with the intervention [24]. Dennis [3] concludes that these studies are generally of good methodological quality. The findings raise questions about why most of the interventions were not effective. A number of explanations emerge. First, most conceptualised postnatal mental health problems as depression and/or general mental health morbidity and role functioning assessed by the SF-36 [28]. Anxiety was an outcome in only two trials. Lavender [21] reported reduced anxiety symptoms, but Priest [20] found no differences between groups in acute stress disorders. It is possible therefore that the interventions might have had undetected benefits for mental health problems other than depression or acute stress disorders. Second, none of the trials analysed outcomes by prior psychiatric history, so it is also possible that the interventions had differential effects in women with and without a history of mental health problems, which were not detected [27,29]. Third, the mechanisms by which the interventions were proposed to reduce mental health morbidity did not target modifiable risk factors directly. Of the four well-established risk factors for depression after childbirth: personal psychiatric history, coincidental adverse life events, quality of relationship with the intimate partner and insufficient social support [30], the latter two are the most readily modified in the postpartum period. Most of the universal interventions addressed low social support, but through the provision of enhanced professional care outside the domestic sphere particularly in primary health care consultations, and not by seeking to improve the quality of a woman's intimate relationships [20,21,23-26,29]. BODY.BACKGROUND.NEW AVENUES FOR PREVENTION.INTIMATE PARTNER RELATIONSHIP: There is consistent evidence that the quality of relationship with the intimate partner is associated with postnatal mental health in women. It has been found to act both protectively and to increase risk. Women, who experience their partners as welcoming the pregnancy, and providing empathic support and encouragement, have better mood [31-33]. In contrast, women, who feel unable to confide in their partners or are experiencing conflict, poor communication or dissatisfaction in the relationship have worse mood [31,34-42]. Although the evidence is consistent, few investigators have operationalised how difficulties in the intimate partner relationship are enacted in day-to-day behaviours. Only two randomised controlled trials of universal interventions for the prevention of postnatal mental health problems, both offered during pregnancy in North America, included partners. Fifty years ago Gordon et al [43] included men in two additional childbirth education classes, not only modelling that the work of parenting is a shared obligation, but also providing guidance to assist men to be sensitised to the demands of this life change for women. There were significantly fewer 'emotional upsets' in women in the intervention than the standard care group six months postpartum. Midmer [44] tested the effects of two additional 3-hour classes which focused on increasing: couples' appreciation of potential feelings of isolation, ambivalence, conflict, resentment and guilt in new mothers; and skills for managing relationships with extended family, a fretful baby, and the redistribution of household chores, using role-play and practice in problem solving and communication techniques in a standard childbirth education program for women and men. There was lower anxiety in women and men in the intervention than in the standard care group six weeks and six months postpartum. Gordon et al used non-standardised outcome assessments and neither study controlled for cluster effects, but Gordon et al's study is cited as evidence of the importance of including women's partners in strategies to promote postpartum mental health [27]. BODY.BACKGROUND.NEW AVENUES FOR PREVENTION.UNSETTLED INFANT BEHAVIOUR: To date, most investigations in this field have presumed that infants' behaviour reflects parenting factors [45], in particular that prolonged infant crying is a consequence of maternal depression [46]. Few have acknowledged that the relationship might be reciprocal and that infant behaviour might exert an adverse effect on a mother's confidence and affect. Infant behaviour, especially prolonged inconsolable crying, frequent night-time waking, short daytime sleeps and feeding difficulties are very common reasons for mothers of infants to seek help [47,48]. Mothers of infants who cry excessively report significantly more parenting stress and less sense of competence and efficacy than other mothers, and do not experience their infants as a source of positive reinforcement [49]. BODY.BACKGROUND.NEW AVENUES FOR PREVENTION.OCCUPATIONAL FATIGUE: Profound fatigue is widespread among mothers of newborns but is often normalised or trivialised, despite the adverse impact it exerts on normal daily functioning [50]. It has been regarded as symptomatic of depression, but an alternative view is that it arises because the unpaid workload of mothering a newborn is severely underestimated [51]. Poor infant sleep and maternal fatigue have been shown in a prospective investigation to precede the onset of depressive symptoms in women [52] BODY.BACKGROUND.NEW AVENUES FOR PREVENTION.SOCIAL THEORY OF DEPRESSION: Brown and Harris's [53] social theory proposes that depression in women arises from experiences of entrapment and humiliation, which we argue are salient to the circumstance of mothering a newborn. The work of infant care is intrinsically confining. If the baby is responsive and rewards the mother by quieting to her soothing, smiling, interacting, suckling easily, and developing along at least an average trajectory, the baby provides gratification. In contrast, an infant who resists soothing, cries inconsolably, or is difficult to breastfeed can be experienced as critical and unappreciative. The work of mothering an infant and managing a household in which an infant lives is repetitive, isolated, never complete, and can be ungratifying. A mother of a newborn depends on her partner for recognition and affirmation of her endeavours and is especially vulnerable to his criticism, which can be humiliating. At this life phase women have increased dependence on intimate relationships, and reduced interactions with workplaces and the broader community. None of the universal postnatal interventions included partners or attempted to modify day-to-day interactions in this relationship, included infants and addressed infant behaviour, or attempted to prevent occupational fatigue. We postulate that depression and anxiety in mothers of newborns can be conceptualised as reflecting poorly functioning intimate relationships, which are potentially modifiable mediated by fatigue. The aim of this study was to assess whether What Were We Thinking! (WWWT) a brief, novel, highly structured, universal psycho-educational intervention for mothers, fathers and a first newborn, which addresses the intimate partner relationship, infant behaviour management, and thereby the mediating effects of occupational fatigue (see [29] for a detailed description) is effective in reducing the common maternal mental health problems of Depression or Anxiety or Adjustment Disorder with Depressed Mood, Anxiety, or Mixed Anxiety and Depressed Mood. BODY.METHODS.STUDY DESIGN: The What Were We Thinking! intervention was designed to be highly diffusible amongst families and social networks and includes attractive take home materials for ongoing reference. To prevent contamination of the standard care group with the intervention, we used a before and after controlled study design [54]. We first recruited and followed a control group who received standard primary postnatal care. Immediately after this, a second group was recruited and followed in the same way, but in addition to standard postnatal care, this group was invited to attend the intervention. Outcomes in the two groups were compared, controlling for baseline differences. BODY.METHODS.SETTING: The study was conducted in seven local government areas (LGAs) in the Australian state of Victoria (population 5.2 million [55]). Diverse LGAs were selected by Socio-Economic Indices for Areas (SEIFA) to represent a range of areas across the spectrum of socioeconomic advantage and disadvantage [56]; three were from rural Victoria and four were in metropolitan Melbourne. Recruitment to the control group took place from February to December 2006, and to the intervention group from February to December 2007. The intervention was conducted when babies were approximately four weeks old in easily accessed Maternal and Child Health Centres in the participating local government areas. Six-month follow-up of control group participants was completed in June 2007 and of the intervention group in June 2008. BODY.METHODS.PARTICIPANTS: All couples with healthy firstborn infants, sufficient English language proficiency to complete telephone interviews, both partners of which were willing to participate in the study and aged over 18 years, were eligible. Maternal and child health nurses provided verbal and written information about the study at the universal home visit and in the postnatal ward of a private maternity hospital. Interested couples provided contact details and were telephoned by research staff within one week. Women and men who agreed to participate returned individual signed consent forms by mail. BODY.METHODS.THE INTERVENTION PROGRAM: What Were We Thinking! (WWWT) is a highly structured, interactive, gender-informed, couples-based, psycho-educational program for parents and a first newborn to promote confident parental caretaking, optimise functioning in the intimate partner relationship, improve infant manageability and reduce common postnatal mental disorders in women [29]. BODY.METHODS.THE INTERVENTION PROGRAM.THEORETICAL PRINCIPLES OF THE INTERVENTION: The theoretical principles of the WWWT program are, first, that postpartum anxiety is as important as depression and requires explicit attention; however, as depression and anxiety are not easily distinguished, they are addressed most effectively together. Second, that partner and infant behaviours can be modified to decrease those that contribute to maternal depression and anxiety and increase those that promote maternal confidence and sense of competence. Third, that women desire care from and gratification within these relationships and not increased care from health professionals. Fourth, that improvements in on-going day-to-day interactions are of fundamental importance to mental health promotion. Fifth, that this knowledge needs to be made available at a critical developmental stage and in a readily comprehensible form. Finally, that the language of the intervention is crucial and needs to challenge gender stereotypes and honour the work of mothering newborns. BODY.METHODS.THE INTERVENTION PROGRAM.PSYCHO-EDUCATIONAL APPROACH: WWWT is psycho-educational in addressing theoretically plausible psychological mechanisms using an educational approach to meet parents' learning needs. The program aims to: minimise experiences of humiliation through increasing fathers' understanding and empathy; counter experiences of entrapment by promoting infant care as a shared endeavour in which parents with comparable competence can permit each other independent or shared leisure [53]; and promote cognitive- rather than emotion-focused responses to infant crying by building skills to respond in non-avoidant ways. Together these strategies are expected to promote gratifying and rewarding intimate interactions rather than frustrating and diminishing ones, minimise maternal fatigue and thereby lead to increased parental confidence; more settled infant behaviour; and reduced depression, anxiety and adjustment disorders [29]. The educational approach addresses the provision of salient knowledge and opportunities to learn new skills. These are difficult to acquire through self-learning at this life stage because of fatigue, and because it is difficult for most people to distinguish between resources that are evidence-based, and those that constitute personal experience or opinion. Principles of adult learning are used and include group discussion, focused tasks to be undertaken individually and then discussed as a couple; practice in problem solving and negotiation, hands-on supported practice in infant settling, short talks and practical demonstrations. Anxiety is contained by a supportive, non-judgemental and knowledgeable facilitator. Very careful language-use is prescribed so that gender stereotypes are challenged, fathering and mothering are positioned as different but of equal importance and emotions are named and normalised without the use of psychiatric labelling. BODY.METHODS.THE INTERVENTION PROGRAM.SPECIFIC CONTENT: WWWT has 13 sections, grouped into two components: "About Babies" and "About Mothers and Fathers". About Babies includes sections about infant temperament, crying and fussing, recognition of tired cues, sleep needs, establishing feed-play-sleep routines of daily care and sustainable settling strategies. Opportunities to practise wrapping their babies and settling them to sleep are provided in the session. About Mothers and Fathers includes sections about differences between how parenthood had been imagined and reality; recalling the difficult and pleasing aspects of the baby's birth; recognising, naming and renegotiating the unpaid workload; acknowledging the losses and gains of parenthood; thinking about experiences within their families of origin that they wish to duplicate or to leave behind; and identifying gaps in support. This component provides language and strategies to assist couples to understand and respond effectively to changed needs for support, re-negotiate the paid and unpaid household workload fairly and manage the losses and gains associated with becoming parents. A folder containing a short book covering program content in accessible plain language and illustrations, and worksheets for each section is used during the program and taken home by couples for later reference. BODY.METHODS.THE INTERVENTION PROGRAM.PROGRAM DELIVERY: Antenatal education for women and their partners is well established and there are high participation rates in Australia, but it does not continue postpartum when parents have high learning needs. Programs were held on Saturday mornings to maximise fathers' participation with groups of up to five couples and their babies. Materials were sent by mail to those who did not attend the face-to-face intervention. BODY.METHODS.THE INTERVENTION PROGRAM.PROGRAM FIDELITY: Program facilitators were three maternal and child health nurses, experienced in leading groups, who had attended a half day training session conducted by JF (clinical psychologist) and HR (adult educator). Training included didactic sessions to address relevant theory, role play to promote use of appropriate language to describe household work and challenge gendered stereotypes, and practice in supporting parents in infant settling techniques. The program was pilot tested with eleven couples and their infants and feedback was incorporated prior to implementation. Fidelity to the program was upheld by adherence to the Facilitators' Handbook containing program theory, learning outcomes, group strategies and interactive worksheets for each section. Telephone and email communication with lead investigators was available to facilitators for an immediate response to unanticipated problems. The research coordinator provided informal weekly supervision and support, and formal supervision took place in face-to-face settings with JF and HR at bi-monthly intervals throughout the implementation phase. BODY.METHODS.STANDARD CARE: Participants in the control group received usual primary health care. BODY.METHODS.DATA SOURCES: Data were collected by computer-assisted-telephone-interviews (CATIs) conducted at approximately two weeks (baseline interview) and six months (follow up interview) postpartum. The primary outcome was any diagnosis of a disorder meeting DSM IV criteria [57] for a Specific or Social Phobia, Panic with or without Agoraphobia, Generalized Anxiety Disorder, Dysthymia, Major or Minor Depressive Episode in the first six months postpartum as assessed by the relevant module of the Composite International Diagnostic Interview (CIDI) [58]. Adjustment disorders were diagnosed as the DSM IV criteria of feeling low and sad most of the day, nearly every day for at least two weeks since the birth of the baby and that it had not followed bereavement (Adjustment Disorder with Depressed Mood); a period of at least a month since the baby's birth of feeling worried, tense or anxious about everyday problems such as work, family or life with the baby (Adjustment Disorder with Anxiety), or both (Adjustment Disorder with Mixed Anxiety and Depressed Mood). These diagnoses were not made if the participant's other symptoms met criteria for Major or Minor Depressive Episode or Generalised Anxiety Disorder. Psychiatric history was assessed by both study-specific questions and the CIDI as self reported lifetime history of treatment for alcohol or drug dependence, depression, eating disorder, or symptoms meeting criteria for panic attack in non life-threatening situations. Potential confounders were assessed using study-specific questions at the baseline interview. Maternal factors included: age, country of birth, language spoken at home, marital, educational and occupational status, self-rated health, gravidity, appraisal of partner support and self-rated confidence on discharge from maternity hospital. Infant factors included: multiple birth, sex, birthweight, gestation at birth, age, health status and method of feeding. Standardised psychometric instruments were used to assess personality; depressive symptoms, infant behaviour and quality of relationship with the intimate partner (see Table 1). Table 1 Standardised instruments and their psychometric properties Variable Instrument Scale description Psychometric properties Primary outcome Common mental disorders Composite International Diagnostic Interview (CIDI) [ 58 ] Widely used, completely structured lay-administered clinical interview that yields DSM-IV and ICD-10 diagnoses through algorithms. Concordance between CIDI diagnoses and Structured Clinical Interview for DSM-IV (SCID) diagnoses of depression (κ = 0.54) and anxiety disorders (κ = 0.48) [ 70 ]. Baseline factors Personality factors which might increase vulnerability to mental health problems Vulnerable Personality Style Questionnaire (VPSQ) Vulnerability Subscale measures over-sensitivity to the opinions of others and lack of assertiveness Range of scores 6 to 30 Cronbach's α for internal consistency 0.77; test - retest reliability 0.82 p < 0.01, in a model predicting postnatal depression sensitivity 0.14 and specificity 0.94 [ 71 ]; correlation with self-esteem 0.58 [ 72 ]. Depressive symptoms Edinburgh Postnatal Depression Scale (EPDS) [ 8 ] 10-item self- report scale to screen for probable depression during the postnatal year in research and health care settings. Range of scores 0 to 30 Standardised α 0.87; sensitivity 0.85 and specificity 0.77; positive predictive value 0.83[ 8 ]. Quality of relationship with intimate partner Intimate Bonds Measure (IBM) Subscales: Care, Control Care subscale assesses sensitivity, warmth, emotional responsiveness, trust, physical gentleness and kindness. Control subscale assesses coercion, dominance, exertion of power and extent of criticism. Range of scores 0 to 36 for each subscale Care: Cronbach's α 0.94; correlation with clinical interview ratings of quality of relationship 0.68. Control: Cronbach's α 0.89 and correlation with clinical interview ratings of quality of relationship 0.74 [ 73 ]. Duration and frequency of infant crying and fussing in a 24 hour period Barr Chart [ 74 ] Possible range 0 to 24 hours Parental diary of duration of episodes of crying, fussing, sleeping, and content infant behaviours. Reliably completed by parents, high correlation with tape recordings: for frequency (r = 0.85, p = 0.002) and duration (r = 0.90, p = 0.001) of episodes [ 74 ]. Fidelity of intervention delivery was assessed by standard program evaluation checklists, completed by facilitators after each implementation. Facilitators rated how well the objectives of each of the 13 individual components of the program had been achieved, using a scale of 1 to 5 (1 = not at all; 5 = completely), and recorded details of unforeseen events that influenced delivery of the intervention. Potential effect modifiers occurring between interviews were assessed at the follow up interview including: self-reported adverse life events, which were rated according to number and severity of events [59], and use of mental health or early parenting services. BODY.METHODS.PROCEDURE: Interviews were conducted uniformly by trained telephone interviewers who had no other involvement in the study. Repeat contact attempts were made for up to one month at the preferred time that had been nominated at recruitment to participants who were unavailable. Attrition was minimised by the use of participant-provided additional contact telephone numbers. An AUD 25 shopping voucher to compensate for inconvenience was posted to participants who completed all interviews. CATIs were identical for intervention and control participants with the exception of specific questions in the follow up interview about the intervention program for participants in the intervention group. BODY.METHODS.SAMPLE SIZE: The intervention program was designed for groups of five couples. The sample size calculation included a correction to adjust for any correlation between responses within the same group. Assuming an intraclass correlation of 0.1 and an average cluster size of 5, the inflation factor for the intervention group was 40%. Thus the required ratio of control group participants to intervention group participants was 1:1.4. For a change of 10% in the prevalence of common mental disorders in women in the first six months postpartum, a two group continuity corrected chi-sq test with a 0.05 two-sided significance level will have 80% power to detect the difference between a control group proportion of 0.2 (20%) and a WWWT program group proportion of 0.1 (10%) (OR 0.444), with groups of 193 and 246, respectively, a total sample size of 439. BODY.METHODS.ETHICS: Approval to conduct the study was provided by the Department of Human Services Victoria Human Research Ethics Committee and the University of Melbourne's Human Research Ethics Committee. BODY.METHODS.DATA MANAGEMENT AND ANALYSIS: Maternal age and psychometric measures of maternal mood, personality and infant crying and fussing were continuous variables. Binary variables were constructed for: occupation, which was coded by the Australian Standard Classification of Occupations (ASCO 1 to 4 and ASCO 5 - 8) [60]; whether English or another language was spoken at home; any or no psychiatric history; primi- or multigravid; unexpected or intended pregnancy; self rating as confident or anxious on discharge from maternity hospital; any breastfeeding or formula feeding and sufficient or insufficient help and support from partner at baseline. Study group was intervention or control and primary outcome was presence or absence of depression, or anxiety or adjustment disorders in the first six months postpartum. Baseline characteristics of those retained in the study were compared to those lost to follow-up. Univariate analyses were used to establish all significant differences in baseline variables between control and intervention groups, and to test associations between baseline variables and the primary outcome. Chi-square tests were used for binary variables; Mann-Whitney tests for ranked and ordered categorical variables, and t-tests for continuous variables. Statistical significance was set as p < 0.05. Between group differences are presented as means (95% confidence intervals) for continuous variables, and proportions (95% confidence intervals) for categorical variables The relationship between participation in the intervention and the outcome was tested by logistic regression in STATA [61], adjusting for potential confounders selected a priori from the univariate analyses. Variance Components Estimation was specified in the model and robust standard errors were used to adjust for clustering of individuals attending the same intervention program. Analysis was by intention to treat. Univariate analysis confirmed that the relationship between study group and the primary outcome was mediated by psychiatric history. An interaction term for psychiatric history and study group was therefore included in the model. The model was adjusted for variables that differed between groups at baseline and for those associated independently with the primary outcome. All baseline data from a small set of cases were lost owing to server failure. Under an assumption of Missing Completely at Random, we used complete case analysis, thus excluding these cases and an additional ten cases with a small amount of missing data. Results of the model are presented as adjusted odds ratios and 95% confidence intervals and according to the TREND guidelines [62]. As a sensitivity analysis, we re-ran the model on the same number of cases, but excluding from the analysis the variable with the most missing cases. The odds ratios in this analysis were compared with the results of the original analysis. BODY.RESULTS.PARTICIPANTS: Of the 646 eligible couples invited to participate, 399 women completed the first interview giving an overall recruitment fraction of 61.8%. Of these, 364 (91.2%) women completed the follow up interview. Women who could not be contacted by telephone for the follow up interview (n = 35) had significantly lower educational attainment, higher self-rated confidence on discharge from maternity hospital and reported fewer breastfeeding problems at the baseline interview than those who had completed both interviews (n = 364). Server failure led to loss of baseline data from eight participants and data for at least one baseline variable in the final model was missing for eighteen participants (5%). These were excluded, leaving 346 cases with complete data in the final model (Figure 1). Figure 1Flow chart of recruitment and participation. The baseline interview was conducted at mean (SD) 4.1 (2.3) weeks and the follow up interview at 27.6 (5.5) weeks postpartum. There were no significant between-group differences in infant age at either interview. Women in the intervention group were significantly older, more likely to speak English at home, to have completed post-secondary education, to be in professional or managerial employment and to be multigravid, and less likely to report that the index pregnancy had been unintended than women in the control group. Babies in the intervention group were more likely to be breastfed and cried and fussed for longer periods than those in the control group (see Table 2). Table 2 Baseline characteristics of participants by study group 95% CI for the difference Control (n = 210) Intervention (n = 189) p Lower Upper Sociodemographic characteristics Age Mean (s.d.) 30.2 (5.31) 31.62 (4.79) 0.004 -2.47 -0.47 Aboriginal/Torres Strait Islander n (%) 2 (1) 1 (0.5) 0.625 English spoken at home n (%) 198 (94.3) 186 (98.4) 0.03 Married n (%) 148 (70.5) 135 (71.4) 0.834 Completed post-secondary education n (%) 135 (64.3) 157 (83.1) <.001 Employment ASCO classification *:  1 and 2 n (%) 78 (37.3) 111 (59.0) <. 001  3 and 4 n (%) 35 (16.7) 31 (16.5)  5 and 6 n (%) 73 (34.8) 34 (18.1)  7, 8 and 9 n (%) 24 (11.5) 12 (6.4) Health and reproductive history Self-rated health good or excellent n (%) 184 (87.7) 177 (93.6) 0.321 Psychiatric history (panic episodes only) n (%) 58 (27.6) 42 (22.2) 0.401 Psychiatric history (other disorders) n (%) 16 (7.6) 25 (13.2) 0.065 More than one previous pregnancy n (%) 40 (19.1) 53 (28) 0.036 Unexpected pregnancy n (%) 56 (26.8) 29 (15.3) 0.005 Conceived by ART + n (%) 18 (8.6) 17 (9) 0.893 Caesarean birth n (%) 69 (32.9) 59 (31.2) 0.726 Psychological factors Confident on leaving hospital n (%) 123 (58.9) 91 (50.0) 0.079 Can confide in partner n (%) 185 (88.5) 163 (89.6) 0.742 Feels supported by partner n (%) 200 (96.2) 173 (95.1) 0.596 Vulnerable personality style (VPS vulnerability subscale score) Mean (s.d.) 12.75 (4.16) 12.7 (3.96) 0.904 -0.75 0.85 Current mood (EPDS) Mean (s.d.) 5.81 (4.01) 6 (3.75) 0.646 -0.96 0.59 Partner relationship (IBM Care subscale score) Mean (s.d.) 33.34 (3.76) 32.76 (4.12) 0.149 -0.21 1.36 Partner relationship (IBM Control subscale score) Mean (s.d.) 4.58 (4.26) 4.76 (4.75) 0.694 -1.08 0.72 Infants Twins n (%) 3 (1.4) 1 (0.5) 0.368 Female n (%) 109 (52.7) 92 (48.9) 0.46 Birth weight (g) Mean (s.d.) 3503.6 (437.5) 3406.9 (510.4) 0.054 -1.76 195.30 Gestation at birth (weeks) Mean (s.d.) 39.86 (1.45) 39.62 (1.56) 0.116 -0.06 0.54 Infant age at interview (weeks) Mean (sd) 4.3 (2.5) 3.9 (2.2) 0.19 -0.17 0.81 Rating of baby's health Good or Excellent n (%) 201 (96.2) 174 (95.6) 0.401 Currently breastfeeding n (%) 163 (78) 160 (87.9) 0.01 Had breastfeeding problems n (%) 50 (23.8) 46 (24.3) 0.902 Length of time baby cried or fussed in previous 24 hours (hours) Mean (s.d.) 2.86 (2.04) 3.52 (2.04) 0.005 -1.11 -0.2 * ASCO Australian Standard Classification of Employment classification [ 60 ] 1 and 2: Managers, Administrators, Professionals 3 and 4: Associate Professionals, Tradespersons, Associated Workers 5 and 6: Advanced/Intermediate Clerical, Sales, Service Workers 7, 8 and 9: Intermediate Production and Transport Workers, Elementary Clerical, Sales and Service Workers, Labourers & Related Workers, students & unemployed + ART assisted reproductive technology BODY.RESULTS.INTERVENTION PROGRAM FIDELITY, PARTICIPATION AND SATISFACTION: A total of 37 intervention programs were implemented with women, their partners and babies, with a mean (SD) group size of 2.7 (1.6) families, at mean (SD) 6.6 (2.5) weeks postpartum. Facilitator evaluation checklists, which were available for 36/37 (97%) programs, showed that in each program all the individual sections had been delivered, and that the objectives of the individual sections were achieved (rated at least 4/5) in almost all of these (491/504; 97%). A total of 120/189 (63.5%) of women in the intervention group attended the program and received a folder of written materials for take-home reference. Most of those who booked and confirmed, but did not attend a program, did not provide a reason, but those who did, cited unexpected illness or partner work or sporting commitments. The folder of written materials was posted to all those who did not attend in person. Most of the women (54/61; 89%) who were sent materials by mail reported at the follow up interview that they had received them. Anonymous participant evaluation questionnaires completed by 98/120 (82%) women at the end of the intervention program revealed that 92 (94%) reported increased understanding of infant sleep needs, 81 (83%) increased understanding of infant temperament, 91 (93%) increased understanding of infant sleep and settling strategies, 71 (72%) could now talk more effectively about parenting with their partners and 64 (66%) already reported increased confidence in infant care. BODY.RESULTS.MENTAL HEALTH OUTCOMES: The primary outcome was a CIDI diagnosis of Depression or Anxiety or Adjustment Disorder with Depressed Mood, Anxiety, or Mixed Anxiety and Depressed Mood in the first six months postpartum (Table 3). Of the 117 women diagnosed as having experienced a disorder, 52 (44.4%) had no psychiatric history and were classified as having a de novo condition. The remainder (65/117; 55.5%) had histories of depression, panic, eating or substance abuse disorders and were classified as having a recurrent mental health problem. Table 3 Women meeting diagnostic criteria for common mental disorders in the first six months postpartum by psychiatric history and study group (n = 364) Whole sample (n = 364) Without psychiatric history n = 232 (63.7%) With psychiatric history n = 132 (36.3%) Disorder Control n = 196 Intervention n = 168 Total n = 364 Control n = 125 Intervention n = 107 Control n = 71 Intervention n = 61 None 129 118 247 89 91 40 27 Adjustment disorder with anxious mood 36 28 64 22 12 14 16 Adjustment disorder with depressed mood 4 2 6 2 1 2 1 Adjustment disorder with mixed mood 6 6 12 1 3 5 3 Dysthymia 0 2 2 0 0 0 2 Anxiety disorder 20 12 32 10 0 10 12 Depression 1 0 1 1 0 0 0 Total disorders n (%) 67 (34.2) 50 (29.8) 117 (32.1) 36 (28.8) 16 (15.0) 31 (43.7%) 34 (55.7%) In the group without a psychiatric history, the absolute risk reduction associated with the intervention was 0.14 (14%), and the relative risk reduction was 0.48 (48%). The original effect size on which our power calculation was based was conservative (10% difference); we showed a larger-than-postulated effect size, which reached significance in our smaller-than-necessary sample size. BODY.RESULTS.FACTORS ASSOCIATED WITH MENTAL HEALTH OUTCOMES AT 6 MONTHS: Use of mental health and residential early parenting services since the birth were potential effect modifiers as they had been used by significantly more participants in the intervention than in the control group. Univariate analysis revealed that use of both these services was associated with psychiatric history. Specifically, 69% of those who consulted a mental health practitioner (p < 0.001) and 83% (p < 0.01) of those who attended a residential early parenting service had a psychiatric history. There were no significant differences between study groups in the use of these services by women without a psychiatric history and therefore these were not included in the model. There were no significant between-group differences in number or severity of coincidental adverse events experienced between baseline and follow up. In the final model, adjusting for all other factors, three variables remained significant predictors of the primary outcome: EPDS score at baseline interview, study group, and the interaction term for psychiatric history and study group, indicating that the effect of the intervention varied by whether or not respondents reported a psychiatric history (see Table 4). Table 4 Factors associated with diagnosis of common mental disorders at outcome (n = 346) Variable Reference Category Odds Ratio Robust Standard. Error p 95% CI . Respondent age 0.97 0.03 0.31 0.90 1.03 Language at home English 1 Other than English 0.88 0.65 0.87 0.21 3.78 Occupation Skilled 1 Unskilled 0.81 0.24 0.47 0.44 1.44 Number of pregnancies First 1 More than one 1.23 0.38 0.50 0.67 2.26 Unexpected pregnancy No 1 Yes 1.28 0.42 0.45 0.67 2.45 EPDS total score 1.14 0.05 0.00 1.05 1.24 VPS vulnerability score 1.07 0.04 0.06 1.00 1.16 Support from partner Yes 1 No 2.57 1.63 0.14 0.75 8.88 Breastfeeding Yes 1 No 1.19 0.40 0.61 0.61 2.31 Confidence at discharge from maternity hospital Yes 1 No 1.10 0.30 0.74 0.64 1.88 Time baby cried or fussed in 24 hours 0.96 0.07 0.62 0.83 1.11 Psychiatric history No 1 Yes 1.59 0.54 0.17 0.82 3.09 Study group Control 1 Intervention 0.43 0.16 0.02 0.21 0.89 Study group × psychiatric history Interaction term 4.27 2.21 0.01 1.53 11.78 For participants with no psychiatric history, being in the intervention group was associated with a significantly reduced odds (OR 0.43; 95% CI 0.21, 0.89; p = 0.022) of a diagnosis of a mental disorder. A linear combination of estimates was used to calculate the odds ratio associated with diagnosis of a mental disorder for participants with a psychiatric history in the intervention group (OR = 1.8; 95% CI 0.92, 3.71; p = 0.082) and demonstrated that being in the intervention group was not associated with significantly increased odds, compared to the control group, and thus the intervention did not cause harm. BODY.RESULTS.SENSITIVITY ANALYSIS: The model was re-run on data from these same 346 respondents, excluding as predictor the number of hours the infant had cried or fussed in the past 24 hours, which had the highest number of missing values and was not a significant predictor. None of the odds ratios changed by more than 5%. The model was therefore re-run on all cases for which all the remaining predictors were available (n = 353) and the results were not different from those already reported. BODY.DISCUSSION: This before and after controlled study provides the first evidence that a brief, structured, universal, salient, gender-informed psycho-educational intervention offered in local settings within the first month postpartum appears to be effective in reducing the onset of the common postpartum mental health problems of Depression or Anxiety or Adjustment Disorder with Depressed Mood, Anxiety, or Mixed Anxiety and Depressed Mood in partnered mothers of a first infant who have no history of psychiatric illness. There are insufficient comprehensive data to establish current and lifetime prevalence estimates of DSM IV Axis I disorders, including, anxiety and adjustment disorders in women in the first six months after childbirth. However, the lifetime prevalence (36%) and 6-month prevalence (32%) reported in this study are consistent with available evidence. A systematic investigation of 1066 women attending for routine antenatal care in Pisa, Italy reported that lifetime prevalence was 50.4% and that 26.3% met criteria for current disorders [63]. In Australia Hiscock and Wake [64] found in a systematic community-based survey of 738 mothers of seven- month-old infants that 15% scored more than 12 and another 18% scored 10 - 12 on the EPDS. It is well established that evaluation of complex health promotion interventions in real world settings is challenging [65]. In accordance with the criteria for evaluation of before and after controlled studies [66], we argue that the findings of this study are important, of notable magnitude and that relevant determinants were not ignored; participants in the first and second group met identical eligibility criteria and were recruited systematically from the general population of primiparous mothers and there was no co-occurring service change that might have contributed to a general trend in improvement of common maternal mental health problems over the time these data were collected. It is reasonable therefore to attribute the outcome to the intervention. We acknowledge nevertheless that this study has limitations. Although attrition was low and participation fractions were adequate, the strength of this evidence is limited by potential selection bias because couples were not randomised to intervention or control conditions. There were differences in baseline characteristics which might have influenced the outcomes. Some of these might have been protective of mental health: women in the intervention group were older, were more likely to be in higher status occupations and were more likely to speak English at home and therefore to have easier social participation. Fewer had unintended pregnancies and more had established breastfeeding than women in the control group. However, the babies of women in the intervention group cried and fussed for longer in twenty-four hours than those in the control group, which might have increased the risk of depression in these women [51]. Although differences in a comprehensive set of relevant baseline characteristics were controlled for in analyses, the possibility that people with an unknown, but better adaptive capacity and lower likelihood of developing a common postpartum mental disorder, were recruited to the intervention group remains. However, the well-established determinants of postpartum depression: past psychiatric history, quality of intimate relationship (IBM Care and Control scores and ability to confide in and perceived support from the partner), current mood (EPDS scores and self-rated confidence in infant care) and vulnerable personality characteristics (VPSQ Vulnerability score) did not differ between groups at baseline. In addition, all participants received the "benevolent attention" of participation in detailed structured interviews about matters of direct relevance to their current experiences. Overall, we believe that the potential for bias is unlikely to account for the magnitude of the effect that was found. However, the findings should be interpreted with caution. It is perhaps unsurprising that a brief (half day) intervention was insufficient to reduce the elevated risk of postpartum mental disorders in women with a history of mood, panic or eating disorders. However, there was very high satisfaction with the program in those who attended the face-to-face session: almost all participants found the knowledge and learning opportunities it provided relevant, timely and valuable. It did not cause harm. The limited effectiveness for prevention of postnatal mental health problems in women with a past psychiatric history, suggests that a stepped approach in which a universal program is one element in a comprehensive mental health care system might be beneficial. The group with additional needs can be readily identified by primary care professionals by simple questioning, and referred for additional assistance, perhaps including specifically tailored supplementary programs. These could include other psychosocial interventions, for example, structured peer support which has been shown by Dennis et al [67] to be effective in preventing postnatal depression in women identified by screening as being at high risk; and individual consultations with maternal and child health nurses about how to manage infant sleep problems, found by Hiscock et al [68] to lead to significantly lower levels of depressive symptoms. However, this novel intervention has merit. It appears that the approach is sound, and that offering a salient, acceptable, well-theorised, gender-informed, timely, non-stigmatising, psycho-educational program to couples and their first babies promotes optimal interactions with the benefit of reduced postpartum anxiety, depression and adjustment disorders in the majority of women. It suggests too that our hypothesised mechanisms of seeking to optimise interactions between mother, father and newborn so that empathy and affirmation are increased and criticism, irritability and insensitivity decreased; and to encourage both partners to participate more equally in the increased unpaid workload and infant care were effective. This intervention is innovative in several ways. First, it includes partners and babies, and focuses on the modification of social risk factors, specifically the quality of day-to-day interactions in a woman's intimate relationships with her partner and her first infant. Second, it recognises that postpartum anxiety and adjustment disorders are common, but less well recognised than depression and require direct attention. Third, it is informed by plausible causal mechanisms that have not been delineated in previous trials. These include: that infant crying and resistance to soothing can arouse anxiety, helplessness and a sense of incompetence; that women experience many unrecognised losses in having a baby; and that disabling occupational fatigue is widespread. Together these can be conceptualised as experiences of entrapment, humiliation and grief which increase potential for depression and anxiety [53]. Rather than positioning men and infants as victims of a woman's mental state, it conceptualises intimate relationships as reciprocal and modifiable. Fourth, the intervention is gender-informed in naming infant care and household tasks as work and making it explicit that failure to recognise the unpaid workload or to share it fairly contributes to occupational fatigue and interpersonal conflict. Finally, rather than just offering support, the intervention was psycho-educational in providing salient knowledge, active learning opportunities and skills training at a critical life-stage. It is framed as a health promotion activity of universal relevance in response to heightened learning needs common to all new parents and is intended not to be stigmatising. This intervention sought to address possible limitations in the existing prevention trials and distinguished between de novo and recurrent mental health problems. It addressed diverse mental health problems including anxiety and adjustment disorders and not just depression. It aimed to modify salient aspects of a woman's intimate social environment, rather than aspects of her individual functioning. It is integrated into primary health care in a local setting and capitalises on an optimal shared learning environment [69]. The study was conducted in seven study sites, which were chosen to achieve diversity in socioeconomic status and in rural and metropolitan locations, and involved systematic recruitment of all couples meeting inclusion criteria. Participation in the study and the intervention appears to have been more appealing to people who were better educated and occupied higher socioeconomic positions. Although the program is intended to be universal, it is unlikely that the face-to-face professionally-facilitated model will reach everyone. This suggests that other modalities might be required to make this knowledge and these skills more widely available to people with lower language skills and emotional literacy. The effectiveness of the intervention was tested when implemented by trained, highly skilled practitioners and it is unknown whether this intervention will be effective when integrated into existing standard primary health care. This novel approach now requires testing in a pragmatic cluster randomised controlled trial. BODY.CONCLUSIONS: A universal, brief psycho-educational group program for English-speaking, first-time parents and babies in primary care appears to reduce de novo common postpartum mental health problems in women. A universal approach supplemented by an additional program may improve effectiveness for women with a psychiatric history. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: JF and HR conceptualised and developed the intervention, obtained funding and conducted the study. JF, HR and KW were involved with the acquisition and management of study data. KW undertook the analysis and all authors were involved in interpretation of the data, with additional expert statistical advice from the Statistical Consulting Centre, University of Melbourne. JF and HR drafted the manuscript with critical revision from KW. JF is the guarantor. All authors have 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/1471-2458/10/432/prepub

TITLE: Polaprezinc combined with clarithromycin-based triple therapy for ABSTRACT: The efficacy and safety of polaprezinc combined with triple therapy was compared with triple therapy alone in the eradication of Helicobacter pylori. A randomized, parallel-group, open-label, controlled, prospective multicenter study was conducted in 11 cities in China. Treatment-naive patients with H. pylori–associated gastritis were randomly assigned to one of three arms for a 14-day treatment: Arm A triple therapy (omeprazole 20 mg, amoxicillin 1 g, and clarithromycin 500 mg, each twice daily) plus polaprezinc 75 mg twice daily; Arm B triple therapy plus polaprezinc 150 mg twice daily, or Arm C triple therapy alone. The rate of H. pylori eradication was the primary endpoint. Secondary endpoints were symptom improvement and lower incidence of adverse events. 303 patients completed the study– 106, 96, and 101 patients in Arms A, B, and C, respectively. Intention-to-treat (ITT) analysis showed that the rate of H. pylori eradication was significantly higher for Arms A (77.0%) and B (75.9%) compared to Arm C (58.6%) (P < 0.01), whereas there was no difference between Arms A and B (P = 0.90). Per-protocol (PP) analysis showed that the rate of H. pylori eradication was significantly higher for Arms A (81.1%) and B (83.3%) compared to Arm C (61.4%) (P < 0.01), whereas there was no significant difference between Arms A and B (P = 0.62). All three groups reported significant symptom improvement at 7, 14, and 28 days after treatment, compared to baseline (P < 0.0001). The adverse event rate for Arm B (5.1%) was higher than for Arms A (2.8%) (P = 0.04) and C (1.9%) (P = 0.02). There were no serious adverse events in any group. It appears that standard dose polaprezinc combined with triple therapy can significantly improve the H. pylori eradication rate, without an increase in toxicity. BODY.INTRODUCTION: Helicobacter pylori (H. pylori), one of the most globally prevalent pathogens, colonizes about 50% of the world's population [1]. It is transmitted from human to human and causes chronic gastritis in all colonized subjects. It can lead to peptic ulcer, atrophic gastritis, gastric adenocarcinoma, and MALT lymphoma. H. pylori eradication cures gastritis and can alter the development of long-term complications [2]. The Kyoto Global Consensus recommends that individuals infected with H. pylori should be offered eradication therapy unless there are competing considerations [3]. In China, the rate of H. pylori infection remains elevated at 40%–60%, in part due to increasing rates of antibiotic resistance and decreasing rates of efficacy with proton pump inhibitor (PPI)–based triple therapy regimens [4]. Among the antibiotics recommended for H. pylori eradication therapy, rates of resistance to metronidazole are as high as 60%–70%, to clarithromycin 20%–38%, and to levofloxacin 30%–38%. Resistance to amoxicillin, furazolidone, and tetracycline remains low at 1%–5%. As rates of antibiotic resistance have increased, the rates of H. pylori eradication using standard triple therapy (PPI, clarithromycin, and amoxicillin) have declined well below 80%. Extending therapy from 7 to 10 days increases the eradication rate by only about 5%. Common therapies, such as sequential therapy, concomitant therapy, and levofloxacin-based triple therapy have demonstrated no advantage for native Chinese patients [4]. Currently, bismuth-containing quadruple therapy is the recommended front-line treatment in China. However, some patients have an intolerance to bismuth (e.g., allergy) and experience gastrointestinal symptoms such as nausea, vomiting, and darkened stools. Also, the ideal regimen for bismuth (i.e., compound, formulation, dose, dosing interval, and relation to meals) remains unclear [5]. The Fourth Chinese National Consensus Report on the management of H. pylori infection suggested that combining a mucosal protective agent with triple therapy may have the same efficacy as bismuth-containing quadruple therapy [6]. However, few clinical studies in China have evaluated the efficacy of adding a mucosal protective agent to enhance eradication of H. pylori. Polaprezinc is a chelate compound of zinc and l-carnosine, with a history of clinical use in Japan spanning more than 20 years. In addition to its recognized role as a gastric mucosal protective agent that promotes the healing of peptic ulcers, polaprezinc also improves H. pylori eradication rates [7, 8]. In 1999, Kashimura and colleagues [7] reported that polaprezinc combined with triple therapy comprised of lansoprazole, amoxicillin, and clarithromycin can increase the H. pylori eradication rate from 24/31 (77.4%, triple therapy alone) to 33/35 (94.3%), with no increase in the incidence of adverse events [7]. Based on this single-center, small sample size study in Japan, we designed and conducted a randomized, parallel-group, controlled, open-label prospective multicenter study to evaluate the clinical efficacy and safety of adding polaprezinc to triple therapy to improve the rate of H. pylori eradication in Chinese patients with gastritis. We also compared the clinical efficacy of two different doses of polaprezinc combined with triple therapy in the eradication of H. pylori and improvement of clinical symptoms. BODY.MATERIALS AND METHODS.STUDY DESIGN: This was a randomized, parallel-controlled, open-label, prospective multicenter clinical study. The study protocol and the CONSORT checklist are available as supporting documents; see S1–S3 Files. The study adhered to the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all participants prior to enrollment. This study protocol was approved by the Peking Union Medical College Hospital Ethics Committee (HS2013002) (S4 and S5 Files). The study has been retrospectively registered with the Chinese Clinical Trial Registry (ChiCTR-IIR-16009688). Reason for not registering prior to enrollment of patients was due to the fact that it was not a mandatory requirement to have this study registered by our approving Ethics Committee. The authors confirm that all ongoing and related clinical trials for this intervention will be registered prospectively. BODY.MATERIALS AND METHODS.PARTICIPANTS: We prospectively enrolled patients infected with H. pylori who visited one of the 11 recruited geographically diverse hospitals between January 2014 and June 2015: Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union Medical College; First Clinical Hospital of Jilin University; First Affiliated Hospital of Nanchang University; Jiangsu People's Hospital; People's Hospital of Wuhan University; First Affiliated Hospital of Zhengzhou University; Zhongshan Hospital of Xiamen University; Xiangya Hospital of Central South University; Qilu Hospital of Shandong University; First Affiliated Hospital of Zhejiang University; Renji Hospital Shanghai Jiaotong University School of Medicine. Patients with upper gastrointestinal symptoms who had been referred for a 13C or 14C urea breath test and esophagogastroduodenoscopy (EGD) were recruited into the study. Patients aged 18–70 years with documented H. pylori infection and without prior eradication therapy were eligible for enrollment. Patients who met one or more of the following criteria were excluded: prior standard eradication therapy for H. pylori; EGD-confirmed gastric or duodenal ulcer, gastric cancer, lymphoma, and obvious erosive or hemorrhagic gastritis within 6 months; prior antibiotic or bismuth therapy within 4 weeks of enrollment; history of gastrostomy; prior PPI therapy within 2 weeks of enrollment; penicillin skin test positive; contraindications or allergy to any of the study drugs; severe liver, heart, or kidney disease, alcoholism, malignancy, or other serious diseases; psychosis, severe neurosis; pregnancy or lactation; participation in other clinical trials within 3 months of enrollment. BODY.MATERIALS AND METHODS.INTERVENTION: All enrolled participants underwent a physical examination and laboratory studies as specified by the protocol, including a complete blood count (CBC), comprehensive metabolic panel, electrocardiogram (ECG), and for women of childbearing age, a urine pregnancy test. Participants were then randomly assigned to Arm A, B, or C. Arm A: polaprezinc 75 mg + omeprazole 20 mg + amoxicillin 1 g + clarithromycin 0.5 g orally twice daily for 14 days; Arm B: polaprezinc 150 mg + omeprazole 20 mg + amoxicillin 1 g + clarithromycin 0.5 g orally twice daily for 14 days; Arm C: omeprazole 20 mg + amoxicillin 1 g + clarithromycin 0.5 g orally twice daily for 14 days. All participants repeated the 13C or 14C urea breath test, physical examination, and laboratory studies 4 weeks after therapy. Study drugs were polaprezinc (Ruilaisheng, Broadwell Pharmaceutical, Jilin, China), omeprazole (Losec®, AstraZeneca Pharmaceutical, Shanghai, China), amoxicillin (Federal Amoxil, Zhuhai Federal Pharmaceutical, Zhuhai, China), and clarithromycin (Limaixian, Xi'an Lijun Pharmaceutical, Xi'an, China). BODY.MATERIALS AND METHODS.OUTCOMES: The primary endpoint of this study was H. pylori eradication 4 weeks after the end of intervention, as measured by a 13C or 14C urea breath test. The secondary endpoints were the improvement of gastrointestinal symptoms by day 7, 14, and 28 after the completion of treatment and lower incidence of adverse events. Each symptom, including upper abdominal pain, acid reflux, belching, heartburn, bloating, nausea and vomiting, was graded according to its severity: 0 none, 1 mild, 2 moderate, or 3 severe. Participants were asked to assign and record the severity score of each symptom before treatment and on day 7, 14, and 28 after treatment. The participants were also informed of the common adverse events before treatment and were asked to record these during treatment. There were follow-up interviews on day 7, 14, and 28 after treatment. The physical examination and laboratory studies were repeated on day 28 after therapy, including CBC, urine test, comprehensive metabolic panel, and ECG. BODY.MATERIALS AND METHODS.SAMPLE SIZE: The calculation of the sample size was based on the primary endpoint. A previous single-center, small sample size study conducted in Japan determined that polaprezinc combined with triple therapy increased the H. pylori eradication rate from 77.4% to 94.3% [7]. Using the χ2 test, with a two-sided α value of 0.05 and β value of 0.20, we estimated that a sample size of 112 participants in each group would detect a 15% difference in eradication rates. BODY.MATERIALS AND METHODS.RANDOMIZATION AND BLINDING: When an eligible participant was ready to be randomized, the investigator at each recruited site provided the participant's name and study ID to an independent researcher. Participants were randomly assigned 1:1:1 to Arm A, B, or C according to the unique randomization schedule that was generated by Department of Epidemiology and Health Statistics, Peking Union Medical College and supplied to each center. The independent researcher was in charge of drug distribution and recovery. This trial was designed as open-labled, for the primary endpoint eradication rate of H.pylori is objective data. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSES: All statistical analyses were performed by the Department of Epidemiology and Health Statistics, Peking Union Medical College using SAS 9.3 software (SAS Institute, Irvine, CA). The primary endpoint H. pylori eradication rate and one of the secondary endpoints improvement of gastrointestinal symptoms were analyzed in an intention-to-treat (ITT) population and a per-protocol (PP) population. The secondary endpoint the incidence of adverse events was analyzed with a safety analysis (SAS) population. The ITT population included all participants who were enrolled, randomized, and who took at least one dose of drug. Missing observations were accounted for using the last observation carried forward (LOCF) method. The PP population included all participants who completed the entire protocol with good compliance. The SAS population included all participants who were randomized, took drugs, and were assessed for safety at least once. Categorical variables were described as percentages, while continuous variables were described as means and standard deviations. For the continuous variables that meet and do not meet the parameter test, analysis of variance (ANOVA) and the Kruskal-Wallis test were used, respectively. For the categorical variables that meet and do not meet the χ2 test, the χ2 test and Fisher exact test were used respectively. The Cochran–Mantel–Haenszel (CMH)–χ2 test was used for group comparisons, and the Mantel–Heanszel method was used to calculate the 95% confidence interval (CI) of the H. pylori eradication rate in each group, and the difference in eradication rates between two groups. All P values were two-tailed and P < 0.05 was considered statistically significant. BODY.RESULTS.DEMOGRAPHICS AND CHARACTERISTICS: Participant flow for this study is shown in Fig 1. From January 2014 to June 2015, 456 patients from 11 hospitals were screened for eligibility. Of these, 124 were excluded: 86 did not meet inclusion criteria, 26 declined to participate, and 12 were excluded for other reasons. 332 patients were enrolled and randomized into three groups for ITT analysis: 113 in Arm A (polaprezinc 150 mg/d combined with triple therapy), 108 in Arm B (polaprezinc 300 mg/d combined with triple therapy) and 111 in Arm C (triple therapy). 316 patients accepted the intervention and first-time safety assessment for the safety analysis set. 303 patients completed the study for PP analysis: 106 in Arm A, 96 in Arm B, and 101 in Arm C. The total dropout rate was 8.8%. No statistically significant differences were found between the three groups regarding baseline demographic data including age, gender, body mass index (BMI), and gastrointestinal symptoms in the ITT and PP populations (P > 0.05) (Table 1). 10.1371/journal.pone.0175625.g001Fig 1Participant flow. 10.1371/journal.pone.0175625.t001 Table 1 Demographic data and baseline characteristics of participants. ITT population P value Arm A Arm B Arm C Number 113 108 111 Age, mean ± SD (years) 41.0 ± 12.2 40.5 ± 13.6 41.0 ± 11.8 0.95 Gender (female/male) 65/48 50/58 53/58 0.19 BMI, mean ± SD (kg/m 2 ) 22.56 ± 3.06 21.92 ± 4.16 23.02 ± 3.49 0.11 Gastrointestinal symptoms at baseline Upper abdominal pain[median (min, max)] 1.0 (0.0–3.0) 1.0 (0.0–2.0) 1.0 (0.0–3.0) 0.61 Acid reflux [median (min, max)] 0.0 (0.0–2.0) 0.0 (0.0–2.0) 0.0 (0.0–3.0) 0.87 Belching[median (min, max)] 0.0 (0.0–3.0) 0.0 (0.0–2.0) 0.0 (0.0–3.0) 0.72 Heartburn[median (min, max)] 0.0 (0.0–2.0) 0.0 (0.0–3.0) 0.0 (0.0–3.0) 0.34 Bloating[median (min, max)] 1.0 (0.0–2.0) 1.0 (0.0–2.0) 0.0 (0.0–3.0) 0.98 Nausea[median (min, max)] 0.0 (0.0–2.0) 0.0 (0.0–2.0) 0.0 (0.0–2.0) 0.23 Vomiting[median (min, max)] 0.0 (0.0–1.0) 0.0 (0.0–2.0) 0.0 (0.0–2.0) 0.25 PP population P value Arm A Arm B Arm C Number 106 96 101 Age, mean ± SD (years) 40.5 ± 12.2 40.1 ± 13.6 40.5 ± 11.9 0.97 Gender (female/male) 61/45 44/52 49/52 0.22 BMI, mean ± SD (kg/m 2 ) 22.56 ± 3.11 22.00 ± 4.36 22.93 ± 3.48 0.23 Gastrointestinal symptoms at baseline Upper abdominal pain[median (min, max)] 1.0 (0.0–3.0) 1.0 (0.0–2.0) 1.0 (0.0–3.0) 0.72 Acid reflux[median (min, max)] 0.0 (0.0–2.0) 0.0 (0.0–2.0) 0.0 (0.0–3.0) 0.61 Belching[median (min, max)] 0.0 (0.0–3.0) 0.0 (0.0–2.0) 0.0 (0.0–3.0) 0.73 Heartburn[median (min, max)] 0.0 (0.0–2.0) 0.0 (0.0–2.0) 0.0 (0.0–3.0) 0.58 Bloating[median (min, max)] 1.0 (0.0–2.0) 1.0 (0.0–2.0) 0.0 (0.0–3.0) 0.94 Nausea[median (min, max)] 0.0 (0.0–2.0) 0.0 (0.0–2.0) 0.0 (0.0–2.0) 0.26 Vomiting[median (min, max)] 0.0 (0.0–1.0) 0.0 (0.0–1.0) 0.0 (0.0–2.0) 0.07 ITT, intention-to-treat; PP, per-protocol; BMI, body mass index; min, minimums; max, maximums. Arm A, polaprezinc 150 mg/d combined with omeprazole, amoxicillin, clarithromycin for 14 days. Arm B, polaprezinc 300 mg/d combined with omeprazole, amoxicillin, clarithromycin for 14 days. Arm C, omeprazole, amoxicillin, and clarithromycin for 14 days. BODY.RESULTS: The ITT analysis revealed H. pylori eradication rates of 77.0%, 75.9%, and 58.6% in Arms A, B, and C, respectively. The H. pylori eradication rate of polaprezinc combined with triple therapy was much higher than triple therapy alone, with 18.4% (P < 0.01, 95% CI [6.4–30.4]) improvement in Arm A and 17.4% (P < 0.01, 95% CI [5.2–29.6]) improvement in Arm B. There was no significant difference in H. pylori eradication rates between the two doses of polaprezinc– 150 mg/d (Arm A) and 300 mg/d (Arm B)–combined with triple therapy (P = 0.90) (Table 2, Fig 2). 10.1371/journal.pone.0175625.g002Fig 2H. pylori eradication rates in the ITT and PP populations.ITT, intention-to-treat; PP, per-protocol Arm A, polaprezinc 150 mg/d combined omeprazole, amoxicillin, clarithromycin for 14 days Arm B, polaprezinc 300 mg/d combined omeprazole, amoxicillin, clarithromycin for 14 days Arm C, omeprazole, amoxicillin, and clarithromycin for 14 days *P < 0.01 10.1371/journal.pone.0175625.t002 Table 2 H . pylori eradication rates in the ITT and PP populations. ITT population PP population H . pylori eradication rate: n/N (%) Arm A 87/113 (77.0%) 86/106 (81.1%) Arm B 82/108 (75.9%) 80/96 (83.3%) Arm C 65/111 (58.6%) 62/101 (61.4%) H . pylori eradication rate difference (%) Arm A vs Arm C 18.4% P < 0.01,95% CI(6.4, 30.4) 19.7% P < 0.01, 95% CI (7.7, 31.8) Arm B vs Arm C 17.4% P < 0.01,95% CI (5.2, 29.6) 21.9% P < 0.01, 95% CI (9.9, 34.0) Arm A vs Arm B 1.1% P = 0.90,95% CI (-10.1, 12.3) 2.2% P = 0.62, 95% CI (-12.7, 8.3) ITT, intention-to-treat; PP, per-protocol. Arm A, polaprezinc 150 mg/d combined with omeprazole, amoxicillin, clarithromycin for 14 days; Arm B, polaprezinc 300 mg/d combined with omeprazole, amoxicillin, clarithromycin for 14 days. Arm C, omeprazole, amoxicillin, and clarithromycin for 14 days. The PP analysis revealed H. pylori eradication rates of 81.1%, 83.3%, and 61.4% in Arms A, B, and C, respectively. The difference of H. pylori eradication rates between polaprezinc combined with triple therapy and triple therapy alone can further increase to reach 19.7% (Arm A vs. Arm C, P < 0.01, 95% CI [7.7–31.8]) and 21.9% (Arm B vs Arm C, P < 0.01, 95%CI [9.9–34.0]), respectively. There was no significant difference in the H. pylori eradication rates between the two polaprezinc doses combined with triple therapy (Arm A vs. Arm B) (P = 0.62) (Table 2, Fig 2). BODY.RESULTS.SYMPTOM IMPROVEMENT: ITT and PP analyses evaluated the severity of gastrointestinal symptoms at day 7, 14, and 28 after treatment. Symptoms included abdominal pain, acid reflux, belching, heartburn, bloating, nausea, and vomiting. All three groups exhibited significant symptom improvement at day 7, 14, and 28 after treatment compared to baseline (P < 0.0001). However, there was no significant difference in symptom improvement between the three groups at day 7, 14, and 28 (P > 0.05) (Table 3). 10.1371/journal.pone.0175625.t003 Table 3 Symptom improvement in the ITT and PP populations. Decrease in severity rate ITT population PP population Arm A Arm B Arm C P value Arm A Arm B Arm C P value N 113 108 111 106 96 101 Abdominal pain Day 7 52 (46.0%) 44 (40.7%) 40 (36.0%) 0.37 52 (49.1%) 43 (44.8%) 39 (38.6%) 0.35 Day 14 63 (55.8%) 54 (50.0%) 47 (42.3%) 0.29 61 (57.5%) 52 (54.2%) 47 (46.5%) 0.37 Day 28 63 (55.8%) 54 (50.0%) 54 (48.6%) 0.49 61 (57.5%) 52 (54.2%) 54(53.5%) 0.39 Acid reflux Day 7 34 (30.1%) 31 (28.7%) 28 (25.2%) 0.84 34 (32.1%) 27 (28.1%) 27 (26.7%) 0.74 Day 14 46 (40.7%) 36 (33.3%) 36 (32.4%) 0.83 46 (43.4%) 33 (34.4%) 35 (34.7%) 0.58 Day 28 47 (41.6%) 36 (33.3%) 33 (29.7%) 0.58 47 (44.3%) 34 (35.4%) 34 (33.7%) 0.51 Belching Day 7 25 (22.1%) 29 (26.9%) 28 (25.2%) 0.58 25 (23.6%) 27 (28.1%) 28 (27.7%) 0.77 Day 14 31 (27.4%) 35 (32.4%) 32 (28.8%) 0.60 31 (29.2%) 34 (35.4%) 32 (31.7%) 0.67 Day 28 32 (28.3%) 35 (32.4%) 36 (32.4%) 0.59 32 (30.2%) 34 (35.4%) 35 (34.7%) 0.64 Heartburn Day 7 28 (24.8%) 26 (24.1%) 24 (21.6%) 0.41 28 (26.4%) 23 (24.0%) 23 (22.8%) 0.96 Day 14 31 (27.4%) 31 (28.7%) 28 (25.2%) 0.50 31 (29.2%) 28 (29.2%) 26 (25.7%) 0.76 Day 28 31 (27.4%) 29 (26.9%) 29 (26.1%) 0.70 31 (29.2%) 27 (28.1%) 27 (26.7%) 0.82 Bloating Day 7 46 (40.7%) 38 (35.2%) 34 (30.6%) 0.82 46 (43.4%) 34 (35.4%) 33 (32.7%) 0.63 Day 14 54 (47.8%) 45 (41.7%) 41 (36.9%) 0.84 53 (50.0%) 42 (43.8%) 40 (39.6%) 0.71 Day 28 57 (50.4%) 47 (43.5%) 44 (39.6%) 0.88 56 (52.8%) 45 (46.9%) 42 (41.6%) 0.84 Nausea Day 7 22 (19.5%) 13 (12.0%) 17 (15.3%) 0.42 21 (19.8%) 12 (12.5%) 17 (16.8%) 0.40 Day 14 23 (20.4%) 16 (14.8%) 22 (19.8%) 0.55 22 (20.8%) 15 (15.6%) 21 (20.8%) 0.61 Day 28 24 (21.2%) 16 (14.8%) 23 (20.7%) 0.44 23 (21.7%) 15 (15.6%) 23 (22.8%) 0.44 Vomiting Day 7 10 (8.8%) 7 (6.5%) 14 (12.6%) 0.70 10 (9.4%) 5 (5.2%) 14 (13.9%) 0.16 Day 14 10 (8.8%) 7 (6.5%) 14 (12.6%) 0.70 10 (9.4%) 5 (5.2%) 14 (13.9%) 0.16 Day 28 10 (8.8%) 7 (6.5%) 14 (12.6%) 0.70 10 (9.4%) 5 (5.2%) 14 (13.9%) 0.16 ITT, intention-to-treat; PP, per-protocol. Arm A, polaprezinc 150 mg/d combined with omeprazole, amoxicillin, clarithromycin for 14 days. Arm B, polaprezinc 300 mg/d combined with omeprazole, amoxicillin, clarithromycin for 14 days. Arm C, omeprazole, amoxicillin, and clarithromycin for 14 days. BODY.RESULTS.ADVERSE EVENTS: The incidence of adverse events was 3/107 (2.8%) in Arm A, 5/99 (5.1%) in Arm B, and 2/103 (1.9%) in Arm C. The incidence of adverse events in Arm B was much higher than in Arms A (P = 0.04) and C (P = 0.02). No participant experienced serious adverse events. One female participant in Arm B experienced increased salivation and involuntary shaking of the lower limbs on the second day of treatment. She discontinued treatment. Four weeks later, her alanine aminotransferase (ALT) level rose from 27 to 250 U/L and her aspartate aminotransferase (AST) level rose from 22 to 146 U/L. She was given oral polyene phosphatidylcholine and compound glycyrrhizin for 3 weeks, at which time her ALT and AST levels returned to normal. Another female patient complained of nonspecific symptoms including dizziness, palpitations, flushing, thirst, abdominal pain, numbness, and a gurgling sound after the first dose. This patient withdrew spontaneously and the symptoms promptly disappeared. Other adverse events included mild leukopenia, elevated liver enzymes or bilirubin, mildly elevated serum uric acid, and high blood pressure (Table 4). 10.1371/journal.pone.0175625.t004 Table 4 Adverse events in SAS population. SAS population Arm A Arm B Arm C SAS patients (n) 107 99 103 Adverse events 3 (2.8%) * 5 (5.1%) 2 (1.9%) * Mild leukopenia 1 (0.9%) 0 (0.0%) 0 (0.0%) Liver enzymes slightly elevated 2 (1.9%) 1 (1.0%) 1 (1.0%) Bilirubin slightly elevated 0 (0.0%) 1 (1.0%) 0 (0.0%) Serum uric acid mildly elevated 0 (0.0%) 1 (1.0%) 0 (0.0%) Blood pressure slightly increased 0 (0.0%) 0 (0.0%) 1 (1.0%) Nervous system symptoms 0 (0.0%) 1 (1.0%) # 0 (0.0%) Nonspecific symptoms 0 (0.0%) 1 (1.0%) † 0 (0.0%) SAS, safety analysis set. Arm A, polaprezinc 150 mg/d combined with omeprazole, amoxicillin, clarithromycin for 14 days. Arm B, polaprezinc 300 mg/d combined with omeprazole, amoxicillin, clarithromycin for 14 days. Arm C, omeprazole, amoxicillin, and clarithromycin for 14 days. *P<0.05 compared to Arm B. # Participant in Arm B experienced increased salivation and involuntary shaking of the lower limbs on the second day of therapy. The participant discontinued treatment because of these adverse events. †Participant in Arm B complained of nonspecific symptoms, including dizziness, palpitations, flushing, thirst, abdominal pain, numbness, and gurgling sound after the first dose. The participant withdrew spontaneously. BODY.DISCUSSION: Our study shows that the mucosal protective agent polaprezinc when combined with clarithromycin-based triple therapy can significantly improve the rate of H. pylori eradication compared to standard clarithromycin-based triple therapy, without significant side effects. We showed that a higher dose of polaprezinc when combined with clarithromycin-based therapy did not further significantly increase efficacy. These results support the use of an adjuvant to improve the therapeutic efficacy of current anti–H. pylori treatment regimens, even in areas of high antibiotic resistance. Polaprezinc is a chelate compound consisting of 23% zinc and 77% l-carnosine. The typical clinical oral dose is 150 mg/d, which contains 34 mg zinc and 116 mg l-carnosine [8]. Its pharmacological activity is attributable mainly to the zinc ion [8]. The exact mechanism by which polaprezinc enhances anti–H. pylori therapy is unclear. Previous studies have shown that zinc chloride inhibits H. pylori growth and reduces the expression of interleukin 1 beta (IL-1β) by gastric epithelial cells [9]. Spectrophotometric examination has revealed that polaprezinc scavenges monochloramine in H. pylori–infected Mongolian gerbils [10]. Some studies have shown that the zinc complex with famotidine can inhibit both the activity of the human urease enzyme and the growth of H. pylori; the anti-H. pylori activity observed was comparable in antibiotic-resistant and antibiotic-sensitive strains [11]. Another potential mechanism is that zinc replaces the nickel ions at the urease active site, which interferes with the complex-forming ability of the two metal ions and results in inhibition of urease activity and growth retardation of H. pylori [12]. Polaprezinc has been used as an effective anti-ulcer drug in Japan for more than 20 years. It is also used to reduce small intestine mucosal injury from low-dose aspirin [13], accelerate healing of pressure ulcers [14], and prevent oral mucositis associated with radiotherapy [15]. Polaprezinc had previously shown promise as a gastric mucosal protective agent in combination with triple therapy to improve the rate of H. pylori eradication. Yakoob and colleagues first demonstrated the susceptibility of H. pylori to zinc chloride in 116 clinical isolates of H. pylori; and further showed that the susceptibility of these strains to zinc chloride 40 μg/mL or bismuth subsalicylate 20 μg/mL was similar– 95% vs 98% in vitro [16]. Subsequently, Kuwayama and colleagues reported that polaprezinc in combination with low-dose metronidazole (750 mg/d) and amoxicillin (750 mg/d) completely eradicated H pylori [17]. Moreover, Kashimura and colleagues found that polaprezinc combined with triple therapy can significantly improve H. pylori eradication rates from 77.4% to 94.3% [7]. Our study confirmed that 75 mg twice daily polaprezinc combined with triple therapy significantly increases the H. pylori eradication rate to 77% (ITT)– 81.1% (PP), much higher than traditional triple therapy at 58.6% (ITT)– 61.4% (PP). Increasing the twice daily dose of polaprezinc from 75 mg to 150 mg does not further improve the clinical outcome. The present high resistance rates to clarithromycin (37.5%), metronidazole (67.2%), and levofloxacin (33.5%) have made empiric treatment difficult in Chinese patient populations [18]. Although the resistance rates of amoxicillin (6.8%), tetracycline (3.5%), rifampicin (14.2%), and furazolidone are low, their side effects and limited availability have restricted their use [18]. Tailored therapy is generally unavailable in China, and H. pylori culture and antibiotic susceptibility tests are invasive and costly, as a biopsy specimen must be obtained during gastroscopy. Our study showed that in regions with high rates of antibiotic resistance, H. pylori eradication rates can be greatly improved by combining a safe mucosal protective agent with standard triple therapy. This study also showed significant improvement in the severity of gastrointestinal symptoms at 7, 14, and 28 days after eradication therapy in all groups, compared to baseline symptoms. However, polaprezinc combined with triple therapy did not show an advantage compared to triple therapy alone. This is likely due to the short duration of follow-up. We would propose extending the follow-up period to 6 to 12 months to compare differences in symptoms. Lastly, we observed a higher incidence of adverse events in the high-dose polaprezinc group compared to the standard dose polaprezinc or triple therapy alone groups. Among the high-dose polaprezinc combined with triple therapy group, one participant experienced excessive salivary secretion and involuntary shaking of the lower limbs on the second day of therapy, along with a transient elevation of liver enzymes. These adverse events were likely due to the toxic effect of the high zinc concentration when polaprezinc is used at a high dose, but may also be related to individual drug sensitivity. Other rare side effects, including mild leukopenia, elevated liver enzymes, elevated serum uric acid, and high blood pressure were inconsequential and normalized after completion of the study. Overall, the standard dose of polaprezinc when combined with clarithromycin-based triple therapy was shown to be safe and well tolerated. Our study has several limitations. First, patients with a positive 13C or 14C urea breath test and without prior eradication therapy were enrolled in this study. We did not use a second non-invasive diagnostic method to confirm the H.pylori infection, because, at the time of our study, H.pylori serology kits with high local validation and H.pylori stool antigen tests were not widely available in China. Second, bismuth-containing quadruple therapy for 14 days is currently the first-line treatment in China due to the high dual resistance of clarithromycin and metronidazole [4]. Noninferiority comparison trials to assess the relative merits of polaprezinc-containing versus bismuth-containing quadruple therapy are needed. A previous randomized, open-label, multicenter prospective study reported a bismuth-containing quadruple therapy H. pylori eradication rate of 68% (ITT)– 77% (PP) in China [6], and sequential and concomitant therapy eradication rates of 72.1% (ITT)– 76.5% (PP) [19] and 78.3% (ITT)– 87.4% (PP) [20], respectively. Also, in vitro susceptibility of H. pylori clinical isolates to zinc chloride 40 μg/mL was similar to bismuth subsalicylate 20 μg/mL (95% vs 98%) [16]. Thus, we believe that polaprezinc-containing quadruple therapy with a 77.0% (ITT)– 81.1% (PP) eradication rate would obtain similar or better results in future comparisons to bismuth-containing quadruple therapy, sequential therapy, or concomitant therapy. Further, polaprezinc-containing quadruple therapy may achieve higher compliance than sequential therapy, and it requires fewer antibiotics than concomitant therapy. Third, we compared the triple therapy of omeprazole, amoxicillin and clarithromycin, with and without the addition of polaprezinc, for the treatment of H. pylori infections. Resistance to amoxicillin is rare; however, resistance to clarithromycin is a critical factor. We regret that we did not determine the clarithromycin resistance in order to predict the eradication rate in other populations with known clarithromycin resistance, using the Hp-normogram [21], which is based on the eradication rates in both clarithromycin-sensitive and clarithromycin-resistant patients. Overall, we confirmed the effectiveness of the zinc compound, polaprezinc, in improving the H. pylori eradication rate, and we verified the results of the prior small study in Japan [7]. Acknowledging that the improvement in the eradication rate was less than ideal in the current study, we intend to conduct the head-to-head comparison of polaprezinc-containing quadruple therapy and bismuth-containing quadruple therapy, with the testing of clarithromycin resistance. We will replace omeprazole with rabeprazole to increase acid inhibition, which may further improve the eradication rate. We will also investigate the mechanisms by which polaprezinc enhances the rate of H. pylori eradication when combined with triple therapy. BODY.SUPPORTING INFORMATION: S1 FileThe CONSORT Checklist.(DOC)Click here for additional data file. S2 FileThe protocol in Chinese.(DOC)Click here for additional data file. S3 FileThe protocol in English.(DOCX)Click here for additional data file. S4 FileThe primary ethical approval in Chinese.(PDF)Click here for additional data file. S5 FileThe translation of primary ethical approval in English.(PDF)Click here for additional data file. S1 TableThe data set of clinical trial result.(XLSX)Click here for additional data file.

TITLE: Influence of Inter-Set Stretching on Strength, Flexibility and Hormonal Adaptations ABSTRACT: Adequate levels of strength and flexibility are important for the promotion and maintenance of health and functional autonomy as well as safe and effective sports participation. The aim of the present study was to analyze the effects of 8 weeks of strength training with or without inter-set static stretching on strength, flexibility and hormonal adaptations of trained men. Sixteen trained men were randomly divided into 2 groups: the static stretching group (SSG) and passive interval group (PIG). All participants performed 24 training sessions 3 times a week. The test and retest of 8RM, strength, flexibility, cortisol and growth hormone concentration in pre and post test conditions were also evaluated. To compare the differences between and within groups in pre- and post-training tests, ANOVA with repeated measures was performed (SSGpre x SSGpost; PIGpre x PIGpost; SSGpost x PIGpost). An alpha level of p<0.05 was considered statistically significant for all comparisons. Both groups showed significant increases in strength (SSGpre vs. SSGpost; PIGpre vs. PIGpost) in the same exercises for leg extension (LE) and Low Row (LR). Specifically, in the SSG group, the parameters for LE were (p = 0.0015 and ES = 2.28 - Large), and the parameters for LR were (p = 0.002 and ES = 1.95 - Large). Moreover, in the PIG group, the parameters for LE were (p = 0.009 and ES = 1.95 - Large), and the parameters for LR were (p = 0.0001 and ES = 2.88 - Large). No differences were found between the groups (SSGpost vs. PIGpost). Both groups showed significant increases in flexibility but in different joints (SSGpre vs. SSGpost; PIGpre vs. PIGpost). In the SSG group, only three joints showed significant increases in flexibility: shoulder extension (p = 0.004 and ES = 1.76 - Large), torso flexion (p = 0.002 and ES = 2.36 - Large), and hip flexion (p = 0.001 and ES = 1.79 -Large). In the PIG group, only three joints showed increases in flexibility: horizontal shoulder abduction (p = 0.003 and ES = 2.07 - Large), hip flexion (p = 0.001 and ES = 2.39 – Large), and hip extension (p = 0.02 and ES = 1.79 - Large). In-between group analyses (SSGpost x PIGpost) revealed differences in two joints: shoulder extension (p = 0.001) and horizontal shoulder abduction (p = 0.001). Hormonal profiles showed no significant differences in cortisol secretion or growth hormone concentration. In conclusion, both studied strength protocols (with and without inter-set static stretching) resulted in flexibility and strength gains without an effect on the anabolic and catabolic hormonal profile. BODY.INTRODUCTION: Adequate levels of strength and flexibility are important for the promotion and maintenance of health and functional autonomy, as well as safe and effective sports participation (ACSM, 1998; Simão et al., 2011). In this context, strength training (ST) is considered an integral component of a well-rounded exercise program, contributes to the treatment and prevention of injuries, and improves sports performance (ACSM, 2002; ACSM, 2009). The combinations of different types of stretching modes on athletic performance have been previously studied (Mikolajec et al., 2012; Shrier, 2004; Bacurau et al., 2009; Beckett et al., 2009; Little and Williams, 2006; Yamaguchi and Ishii, 2005; Behm et al., 2001; Dalrymple et al., 2010). All of these studies, with the exception of the study by Dalrymple et al. (2010), observed a decrease in explosive sport skills, such as sprinting and vertical jumps. However, Dalrymple et al. (2010) did not explain the influence of the two different stretching models (passive and dynamic stretching) on the countermovement jump. Gomes et al. (2010) observed a decrease in the capacity to maintain force on strength training exercises before proprioceptive neuromuscular facilitation (PNF). In this study, static stretching did not affect endurance or strength performance. Research has also demonstrated that a different inter-set rest interval length can produce different acute responses and chronic adaptations in neuromuscular and endocrine systems (Salles et al., 2009). However, little research has focused on the activity performed during these recovery periods (Caruso and Coday, 2008; Garcia-Lopez et al., 2010). It is common to see lifters performing ST inter-set stretching to improve the muscular recovery in sports or recreational-related exercises (Garcia-Lopez et al., 2010). Additionally, it has been suggested that inter-set stretching influences the time under tension and associated neuromuscular, metabolic, and/or hormonal systems. Recent data have shown that ST inter-set static stretching negatively affected the bench press acute kinematic profile compared with inter-set ballistic stretching and non-stretching conditions (Garcia-Lopez et al., 2010). In a chronic manner, static stretching performed before ST sessions resulted in similar strength gains to ST alone, suggesting that strength and stretching can be prescribed together to achieve optimal improvements in flexibility (Simão et al., 2011). Based on these results, the performance of inter-set static stretching may lead to additional improvements in flexibility levels and muscular recovery without additional time expended in the gym. However, to date, only Simão et al. (2011) have observed the chronic effects of ST inter-set stretching on flexibility. Therefore, the aim of the present study was to analyze the effects of eight weeks of strength training with and without inter-set static stretching on strength, flexibility and hormonal adaptations. BODY.MATERIAL AND METHODS.PARTICIPANTS: The initial sample was composed of 16 trained men. All participants underwent a routine clinical evaluation. To be included in the experiment, volunteers had to meet the following criteria: (a) be trained for at least 24 months with a weekly frequency of three days; (b) agree to not perform any type of regular physical activity other than the prescribed strength training and flexibility training during the experiment; (c) be free from any condition that would influence the collection or interpretation of data; and (d) be free from the intake of ergogenic aids that could influence the collection or interpretation of the data. The 16 men were randomly assigned to 2 groups: the static stretching group (SSG; n = 8) or passive interval group (PIG; n = 8) (Table 1). Study details were explained verbally and in writing, and all participants signed an informed consent form before participation in the study in accordance with the declaration of Helsinki. The study protocol was approved by the Research Ethics Committee of the State University of Pará (Brazil). BODY.MATERIAL AND METHODS.EIGHT REPETITION MAXIMUM TEST (8RM): After a strength training familiarization period (4 sessions), all participants performed 2 familiarization sessions with the 8RM test protocol, with 72 hours between sessions. The 8RM tests were performed for the following exercises: machine bench press (BP), leg extension (LE), low row (LR), leg curl (LC), shoulder press (SP), and leg press (LP) (Techno Gym®-Gambettola, Italy) using a counterbalanced order. On day 1, the first 8RM test was performed, and then, after 72 hours, the 8RM test was repeated to determine test-retest reliability. The heaviest load achieved on the two test-retest days was considered the 8RM load. No exercise was allowed in the 72 hours between the 8RM tests. To minimize error during the 8RM tests, the following strategies were adopted (Simão et al., 2005; ACSM, 2010): (a) standardized instructions concerning the testing procedures were given to participants before the test; (b) participants received standardized instructions on exercise technique; (c) standard verbal encouragement was provided during the testing procedure; d) verbal stimuli were used to maintain a high exercise intensity; e) the additional weights used in the study were previously calibrated on a precision scale (Filizola, Brazil); f) for a repetition to be validated, a complete range of motion had to be performed. The 8RM was determined in fewer than 5 attempts, with a rest interval of 5 minutes between them; g) no pause was allowed between the eccentric and concentric phase of a repetition or between repetitions, and the velocity was controlled with a metronome (Qwik Time Quartz Metronome, Evets Corp., Laguna Beach, CA) calibrated to 60 beats × min−1. After the 8 weeks of training, the 8RM test was performed with the same procedures of the pre-training test to observe the possible strength gains within and between groups. The 8RM tests were consistently conducted during the morning for each participant. BODY.MATERIAL AND METHODS.FLEXIBILITY MEASUREMENT (GONIOMETRY PROTOCOL): Flexibility was measured before and after 8 weeks of the experiment in 8 maximum stretching articular movements (ACSM, 2010). The flexibility measurements were taken 72 hours after the last 8RM test. The maximum flexibility measurement registered in 3 attempts with an interval of 10 seconds between attempts was considered for further evaluation (ACSM, 2011). The same procedure was executed post-training. All flexibility tests were conducted at the same time of the day. The data collected during the first evaluation were not made available to the evaluator to prevent information bias during measurements taken after training. Before the flexibility test, a warm-up was performed for the muscle groups involved in the evaluation. Two sets were used for the static stretching warm-up protocol, holding the position for 10 seconds in each set, until a point of slight discomfort was reached. A 10-second interval was provided between the warm-up stretching sets. The 8 maximum stretching articular movements were: a) shoulder flexion; b) shoulder extension; c) horizontal shoulder abduction; d) horizontal shoulder adduction; e) torso flexion; f) torso extension; g) hip flexion; and h) hip extension. BODY.MATERIAL AND METHODS.BLOOD EVALUATIONS (CORTISOL AND GROWTH HORMONE): Both the SSG (n = 8) and PIG (n = 8) group participants underwent two blood collections: one at baseline and the second at the end of the eighth week of the exercise program. Blood was collected by a trained professional (approximately 5-ml blood samples from the antecubital vein) at 8 am to avoid the different concentrations of the hormonal circadian rhythm, with 12 h of rest. Blood samples were shipped in conditions suitable for laboratory analysis. Growth hormone was analyzed using the chemiluminescent enzyme immunometric method, while cortisol was analyzed using a chemiluminescent enzyme immunoassay. BODY.MATERIAL AND METHODS.PROCEDURES: Before the 8-week training program (24 total sessions), 16 trained men were randomly assigned to 2 groups: the static stretching group (SSG; n = 8) and passive interval group (PIG; n = 8). The SSG and PIG groups performed 4 familiarization sessions with the exercises included in the training program. After familiarization with the exercises and before 8RM tests and retests, the subjects performed 2 sessions covering the 8RM procedures. Individuals performed the test and retest of 8RM, test and retest of flexibility and had their cortisol and growth hormone (GH) evaluated under the pre-test and post-test conditions. The baseline measurements of the hormonal responses, strength and flexibility tests were taken 72 hours apart. After the flexibility measurement, both groups underwent 8 weeks of training under the supervision of experienced fitness professionals. After the 8-week training program, flexibility, strength, and hormone concentrations were evaluated again. BODY.MATERIAL AND METHODS.TRAINING PROTOCOLS: The training protocol for all groups included 3 weekly sessions on alternate days, for a total of 24 sessions. All 16 subjects completed the study. All sessions were supervised by experienced fitness professionals. Strength training was composed of 6 exercises executed in 4 sets with 8RM. The order established for the strength training was as follows: BP, LE, LR, LC, SP, and LP. Before each training session, the subjects executed a specific warm-up involving 15 repetitions with 50% of the load used in the first and second exercises of the sequence. The rest interval between sets was 2 min and included passive rest or static stretching exercises for the muscle involved in the ST exercises for the PIG and SSG group, respectively. Static stretching was performed at the point of mild discomfort, with stretches held for 30 seconds (ACSM, 2011). The rest interval between an exercise was 5 min. The initial training loads were adjusted for all participants and increased when the volunteers were able to perform more than 8RM by readjustment of 5% of the initial loads. All testing and training sessions were performed in the morning hours and were consistent throughout the study. To protect against bias, the investigators that conducted the training program did not conduct the testing measurements, and the staff involved in the strength and flexibility tests were blinded to the group assignment. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSES: The statistical analysis was initially performed using the Shapiro–Wilk normality test and homoscedasticity test (Bartlett criterion). As mentioned, the 8RM tests were found to be similar when tested on two occasions prior to performing the different sequences. To compare the differences between and within groups in pre-training and post-training tests, ANOVA with repeated measures was performed (SSGpre × SSGpost; PIGpre × PIGpost; SSGpost × PIGpost). An alpha level of p<0.05 was considered statistically significant for all comparisons. All statistical analyses were conducted using SPSS statistical software package, version 20.0 (SPSS Inc., Chicago, IL). The calculation of effect size (the difference between pretest and posttest scores divided by the pretest standard deviation) and scale proposed by Rhea (2004) were used to examine the magnitude of any treatment effect. BODY.RESULTS.STRENGTH RESULTS: The results obtained show an intraclass coefficient of SSG group: BP = 0.97; LE = 0.97; LR = 0.93; LC = 0.98; SP = 0.99; LP = 0.98 and in PIG group: BP = 0.96; LE = 0.98; LR = 0.80; LC = 0.94; SP = 0.97; LP = 0.98. A paired-samples t-test was performed and did not demonstrate any significant difference (p < 0.05) between 8RM tests on separate testing occasions. Both groups showed significant increases in strength (SSGpre vs. SSGpost ; PIGpre vs. PIGpost), in same exercises; LE and LR. In SSG group in LE (p =0.0015 and ES = 2.28 - Large) and LR (p = 0.002 and ES = 1.95 -Large) and in PIG group in LE (p = 0.0090 and ES = 1.95 – Large) and in LR (p = 0.0001 and ES = 2.88 - Large). No differences were showed between groups (SSGpost vs. PIGpost). All results are presented in Table 2. BODY.RESULTS.FLEXIBILITY MEASUREMENTS: Both Groups showed significant increases in flexibility, but in different joints (SSGpre vs. SSGpost; PIGpre vs. PIGpost). In SSG Group, only three joints showed significant increases in flexibility: shoulder extension (p = 0.004 and ES = 1.76 - Large); torso Flexion (p = 0.002 and ES = 2.36 – Large) and hip flexion (p = 0.001 and ES = 1.79 – Large). In PIG group, only three joints showed increases in flexibility: horizontal shoulder abduction (p = 0.003 and ES = 2.07 – Large); hip flexion (p = 0.001 and ES = 2.39 – Large) and hip extension (p = 0.02 and ES = 1.79 – Large). In between groups analyses (SSGpost x PIGpost) differences were found only in two joints: shoulder extension (p = 0.001) and horizontal shoulder abduction (p = 0.001). All results are presented in Table 3. BODY.RESULTS.HORMONE PROFILE: The results showed no significant differences in the concentration of cortisol and growth hormone (Table 4 – p > 0.05). Effect size data demonstrated trivial results in both hormones in SSG and PIG group (pretest vs. posttest). BODY.DISCUSSION: The purpose of the present study was to analyze the effects of eight weeks of ST in 2 experimental groups (SSG and PIG) with and without inter-set static stretching on strength, flexibility and hormone profile of trained men. It was hypothesized that ST performed with inter-set static stretching would not result in additional strength and flexibility and also not change the anabolic-catabolic hormone profile after 24 weeks of training. The key finding of the present study was that both training groups presented significant strength and flexibility gains after 24 weeks and showed no differences in the anaboliccatabolic hormone profile, which confirmed the initial hypothesis. Additionally, the inter-set static stretching ST group demonstrated larger strength gains in two exercises and larger flexibility gains in three joints compared with ST alone. However, the results revealed a significant increase in muscle strength for only a few exercises in the SSG (LP, LR) and PIG (LR) experimental conditions. This indicates that stretching between sets does not compromise increases in strength achieved by resistance training. Inter-set static stretching significantly changed the strength of LE in the SSG group and LR in both groups. These findings are consistent with previous studies on this issue (Nelson et al., 2005; Bacurau et al., 2009; Gomes et al., 2011) and indicate that stretching does not modify the strength gains promoted by resistance training. These findings have potentially important implications for strength and conditioning professionals who may commonly use stretching exercises as an integral part of a warm-up routine or during the training session itself. Perhaps, these results could be applied to the resistance training protocol with a long-term rest interval, 2 minutes between sets and 5 minutes between exercises and high loads with 8RM. To our knowledge, this is the first study that analyzed the chronic effects of inter-set stretching. However, previous studies have analyzed the chronic effects of pre- (Simão et al., 2011) or post-ST (Nóbrega et al., 2005) stretching on strength and flexibility gains. Nevertheless, the effect size in the current study showed that strength training with stretching inter-set intervals increases flexibility in previously recreationally trained men, and strength and flexibility can be prescribed together to achieve better flexibility results. Indeed, Simão et al. (2011) analyzed strength and flexibility gains achieved through isolated or simultaneous strength and flexibility training in adult sedentary women. The sedentary women were randomly assigned to ST, flexibility training, the combination of both, or a control group. All of the groups performed preand post-training sit and reach tests to verify the flexibility level and 10RM tests for leg press and bench press exercises. The training protocol for all of the groups, except for the control group, was composed of three sets of eight exercises for upper and lower limbs three times per week. The flexibility training was composed of static stretching exercises that involved the upper and lower limbs performed before ST sessions. The results showed that the ST, ST + flexibility, and flexibility groups had significantly increased flexibility compared with baseline and the control group. The strength tests demonstrated that ST and ST + flexibility significantly increased 10RM when compared with baseline flexibility and the control group. The authors suggested that strength and flexibility can be prescribed in the same session to increase flexibility to a greater extent. Similar to our findings, the strength and flexibility training group presented larger flexibility gains than the ST alone group; however, there were no additional strength gains when compared with the ST alone group. Kokkonen et al. (2007) conducted research to verify the differences in lower limb strength gains in physically active individuals by comparing strength training in isolation versus strength training combined with static stretching exercises for the hip, thigh muscles and plantar flexors. They found significant strength increases in the lower limbs for both groups. However, the greatest differences were observed in the group that performed strength training in combination with stretching exercises (16%, 27% and 31% in the 1RM test for knee flexion, knee extension, and leg press exercises, respectively). The data from our study showed an increase of 27.79% in the knee extension exercise in the SSG group, which was in agreement with the study by Kokkonen et al. (2007). Moreover, the present study showed that increases in strength could be observed in the SSG group in the LR with an increase of 23.19% in the 8RM test; the PIG group demonstrated increases in strength in the LR (18.72%) in the 8RM test. Mohamad et al. (2011) reported that stretching between sets of ST could increase muscle hypertrophy and suggested the possibility of additional increases in muscle strength in relation to strength training alone. However, it is important to emphasize that our study did not assess muscle hypertrophy, but only one anabolic growth hormone and one catabolic hormone (cortisol). These hormones showed no significant differences between pre- and post-ST evaluations between the groups. It is also important to note that the inclusion of stretching during intervals between sets did not reduce the gains at the end of the experiment or the intensity at which our participants were able to train. There are multiple studies that have suggested acute strength impairments following the performance of static stretching. This phenomenon could easily have affected chronic adaptations and daily session loading. Any decrease in the acute training load could have contributed to the differing amounts of strength gains between the groups. Nóbrega et al. (2005) investigated the interaction of ST and flexibility training in young sedentary men and women. The subjects followed an ST protocol with intensities initially set at 60% of 1RM which was continuously adjusted so that fatigue was achieved after 8–12 repetitions. Static stretching exercises were performed after the ST sessions. At the end of 12 weeks, the authors verified that resistance training improved muscle strength either alone or in combination with flexibility training; however, ST alone did not change flexibility. Flexibility increased with specific training alone or in combination with ST. Similar to Simão et al. (2011) and our results, Nóbrega et al. (2005) found strength gains with ST alone and in combined strength and flexibility training groups. In our study, the finding that larger strength increases in two exercises were observed in the inter-set stretching group is new. Previous literature has not presented larger strength gains with the inclusion of stretching exercises in a ST routine, which may be related to the fact that the static stretching exercises were performed between sets. Inter-set stretching will influence the time under tension and the associated neuromuscular, metabolic, and/or hormonal responses (Mohamad et al., 2011), which are related to the larger strength improvements presented by the inter-set stretching group. The increased time under tension increases the effect of various neuromechanical and metabolic stimuli that are thought to be important for hypertrophic adaptation (Mohamad et al., 2011). It has been suggested that during ischemic conditions, such as during inter-set stretching, metabolites and ions accumulate rather than dissipate, which in turn leads to GH secretion and increased levels of IGF-1 (Mohamad et al., 2011). The limitations of our study include no inclusion of a control group, the short experimental period, and reduced adaptation to ST in the trained sample. BODY.CONCLUSIONS AND PRACTICAL IMPLICATIONS: In conclusion, both studied ST protocols (with and without inter-set static stretching) resulted in flexibility and strength gains without influencing the anabolic-catabolic hormone profile. However, the results suggest that inter-set static stretching can be adopted to achieve additional strength and flexibility gains. Future studies analyzing the flexibility and strength gains in response to different inter-set stretching strategies, longer intervention periods, and different samples are necessary to confirm the results. Further research is also necessary to verify the effects of these strategies on hypertrophic adaptation, as previously suggested. The results of the present study indicate that inter-set static stretching leads to additional improvements in strength and flexibility without additional time expended in the gym. The time saved by omitting separate stretching routines may help increase adherence to training by recreational fitness practitioners who have limited exercise time.

TITLE: The impact of Cognitive Processing Therapy on stigma among survivors of sexual violence in eastern Democratic Republic of Congo: results from a cluster randomized controlled trial ABSTRACT.BACKGROUND: Sexual violence is associated with a multitude of poor physical, emotional, and social outcomes. Despite reports of stigma by sexual violence survivors, limited evidence exists on effective strategies to reduce stigma, particularly in conflict-affected settings. We sought to assess the effect of group Cognitive Processing Therapy (CPT) on stigma and the extent to which stigma might moderate the effectiveness of CPT in treating mental health problems among survivors of sexual violence in the Democratic Republic of Congo. ABSTRACT.METHODS: Data were drawn from 405 adult female survivors of sexual violence reporting mental distress and poor functioning in North and South Kivu. Women were recruited through organizations providing psychosocial support and then cluster randomized to group CPT or individual support. Women were assessed at baseline, the end of treatment, and again six months later. Assessors were masked to women's treatment assignment. Linear mixed-effect regression models were used to estimate (1) the effect of CPT on feelings of perceived and internalized (felt) stigma, and (2) whether felt stigma and discrimination (enacted stigma) moderated the effects of CPT on combined depression and anxiety symptoms, posttraumatic stress, and functional impairment. ABSTRACT.RESULTS: Participants receiving CPT experienced moderate reductions in felt stigma relative to those in individual support (Cohen's D = 0.44, p = value = 0.02) following the end of treatment, though this difference was no longer significant six-months later (Cohen's D = 0.45, p = value = 0.12). Neither felt nor enacted stigma significantly moderated the effect of CPT on mental health symptoms or functional impairment. ABSTRACT.CONCLUSIONS: Group cognitive-behavioral based therapies may be an effective stigma reduction tool for survivors of sexual violence. Experiences and perceptions of stigma did not hinder therapeutic effects of group psychotherapy on survivors' mental health. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT01385163. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13031-018-0142-4) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: In conflict-affected areas of Eastern Democratic Republic of Congo (DRC), nearly 40% of women participating in a community survey reported an experience of sexual violence (SV) in the 16 years following the 1994 civil war [1]. SV is associated with serious physical health consequences, including gynecological injuries, unintended pregnancy, and sexually transmitted diseases [2]. SV also has extensive psychological effects; elevated rates of depression, post-traumatic stress disorder (PTSD), and suicidality are well documented among survivors in DRC and elsewhere [1, 3, 4]. Due to the stigma associated with rape and its effects, SV can also profoundly affect survivors' relationships with their partners, family, and community [5, 6]. Link and Phelan define stigma as a social labeling and distancing process that occurs in the context of a power differential [7]. Stigma can be experienced as perceived negative attitudes (i.e. perceived stigma) or changes in the way individuals are treated in their communities (i.e. discrimination or enacted stigma). Enacted stigma has been reported by SV survivors in DRC as taking various forms [5, 6, 8]. For example, approximately a third of SV survivors reported abandonment by their husbands or communities due to their experience of SV [9]. Survivors may also internalize the stigma they experience, for instance, by developing feelings of shame [5, 11]. Further exploration of the stigma associated with SV in DRC demonstrates a complex interrelationship with mental health. SV survivors who are rejected by their families have been found to experience higher levels of posttraumatic stress and depression symptoms on average relative to those whom remain accepted within their families [8]. Findings among adolescents in DRC support that stigma may be a mechanism by which SV produces poor mental health outcomes [10]. Mental distress may also exacerbate the effects of stigma on survivors either by leading to additional stigma or by increasing social isolation [11]. For instance, posttraumatic stress symptoms among women living in conflict-affected areas of DRC were found to be associated with fewer visits from family and community members to women's homes, but also fewer visits by affected women to others' homes [12]. This interrelationship raises the possibility that interventions to improve mental health among SV survivors may also be effective in reducing stigma. Peer support and counseling for individuals who are the target of stigma have been identified as promising anti-stigma interventions [13, 14]. A recent systematic review of HIV-related stigma reduction interventions identified only seven evaluations of this type of program in high or low and middle-income settings; information and contact-based programs (where individuals have an opportunity to interact with someone living with HIV/AIDS) for the general community were far more common [15]. Overall, the majority of these studies found that stigma decreased over the course of the program, but not all used a validated measure of stigma and several studies included fewer than 100 individuals. Evaluation of the impact of mental health programs on stigma is also warranted to ensure that interventions for marginalized populations do not unintentionally cause harm. Cognitive Processing Therapy (CPT) is an evidence-based psychotherapy specifically developed for survivors of rape that includes psychoeducation and cognitive restructuring [16]. We previously found that group-based CPT had large effect sizes (ranging from 1.1–1.8) on trauma, depression and anxiety, and functional impairment for survivors of sexual violence in DRC [17]. Group CPT was also observed to have small effects on emotional support seeking and membership and participation in community groups in this same sample of women [18]. We hypothesized that CPT might be effective in addressing felt (i.e. perceived and internalized) stigma among SV survivors due to its focus on cognitive reframing, reducing avoidance of trauma-related cues and social isolation, and specific emphasis on identifying and challenging maladaptive beliefs (such as self-blame and negative beliefs about self and others) that stem from SV experiences [16]. An example of a maladaptive belief is a woman feeling that "I don't have a voice in my house" on account of being raped. In a prior study of rape survivors in the United States, maladaptive beliefs were found to significantly change as a function of CPT [19]. Further, by helping survivors identify sources of support or to recognize evidence that disconfirms their beliefs, CPT might address women's overgeneralized perceptions or negative beliefs about how others view them. Stigma may also affect survivors' willingness to join or participate fully in group therapies. Stigma related to intimate partner violence and SV in general has been described by women as interfering with daily functioning and care seeking, leading to self-isolation [5, 20]. For example, shame over being raped has been described as a reason for delaying seeking medical care in DRC [21]. In the United States, mental health-related stigma has shown a negative association with ability or willingness to continue pharmacological treatment once initiated [22]. Disruption in care seeking for a variety of problems associated with sexual violence may be a mechanism by which stigma negatively impacts overall health outcomes [23, 24]. We therefore hypothesized that enacted and felt stigma could moderate the effect of CPT on SV survivors' mental health. Given the dearth of research on the anti-stigma potential of counseling programs, the multifaceted negative consequences of stigma for SV survivors, and the promise of CPT as an effective intervention, we sought to answer the following research questions: what is the impact of group-CPT on felt stigma among survivors of sexual violence in Eastern DRC; and, does enacted or felt stigma moderate the effect of CPT on mental health? BODY.METHODS: This secondary analysis uses data from a prior randomized controlled trial of lay-worker administered group Cognitive Processing Therapy for female SV survivors in Eastern DRC. Detailed information on trial design is available in a report of primary trial outcomes: posttraumatic stress and combined depression and anxiety [17]. In brief, sixteen villages (14 in South Kivu and 2 in contiguous areas of North Kivu) were purposefully selected out of 23 in which a psychosocial assistant (PSA) was providing supportive counseling to female SV survivors through one of three non-governmental organizations (NGOs) supported by the International Rescue Committee. PSA availability and whether a village could be consistently and safely accessed guided selection. These villages were grouped by location and language and cluster randomized to cognitive processing therapy (CPT; n = 7) or a continuation of individual support (IS; n = 8). One village was excluded post-randomization due to concerns about its PSA's ability to provide CPT. Women were recruited in December 2010 by PSA review of current and former client lists. To be eligible, women needed to (a) have witnessed or experienced SV ("rape" in local translations); (b) be experiencing poor mental health (defined as a score of at least 55 on the total symptom scale, equivalent to an average response of 1 to scale items); and (c) be experiencing some impairment in daily functioning (defined as a score of 10 on the functioning scale, equivalent to indicating at least some dysfunction on half the scale items). Women were excluded if they expressed suicidal ideation that was deemed to need immediate intervention. In areas randomized to CPT, recruitment ceased when 28–30 eligible women were identified in each village. Women closest to the NGO offices were then selected to fill three groups of up to 8 women per village. In IS villages, all eligible women were invited to join the study [17]. Follow-up assessments administered by research assistants masked to treatment assignment took place within one month of the end of CPT and again six months later. Oral informed consent was obtained from all individual participants included in the study. The Johns Hopkins Bloomberg School of Public Health and the Kinshasa School of Public Health Institutional Review Boards provided ethical approval. BODY.METHODS.INTERVENTION CONDITIONS: Cognitive processing therapy (CPT) is a manualized evidence-based psychotherapy developed specifically for treating mental health problems in rape survivors. To improve the reach of the intervention in DRC where the gap between available services and need is large, CPT was delivered in a group format. In addition, based on findings of similar efficacy [25], a version of CPT without a trauma narrative component was used. Treatment occurred over 12 sessions (of approximately 2 h in length), the first of which was delivered individually. PSAs in villages randomized to CPT received two weeks of training from US-based practitioners. A multi-tiered apprenticeship structure (described elsewhere [26]) was used to support and monitor CPT implementation with oversight by local and US-based supervisors. For the individual support (IS) arm, PSAs continued to be available to women with the same psychosocial support services that they provided before study initiation, including referrals for medical, economic, and legal problems. BODY.METHODS.INSTRUMENTS: The study instrument was translated into five local languages (Kibembe, Kifuliro, Kihavu, Mashi, and Swahili) and included the following sections: demographics, trauma exposure, daily functioning, and psychosocial and mental health problems. BODY.METHODS.INSTRUMENTS.DEMOGRAPHICS: Participants self reported their age, language, marital status, the total number of people living in their household, and whether they had lived in the same village for 10 years or more. For analysis, marital status was treated as a binary variable of currently married or not. BODY.METHODS.INSTRUMENTS.TRAUMA EXPOSURE: Women self-reported whether they had either (a) witnessed or (b) experienced the following events: sexual violence (locally translated as rape), a severe violent attack such as a beating or attack with a weapon, looting or burning of home or property, abandonment or having been thrown out, and abduction. Women also reported whether they had witnessed murder. Two composite variables were created by separately summing the types of traumas experienced (maximum possible score of 5) and witnessed (maximum possible score of 6). BODY.METHODS.INSTRUMENTS.FUNCTIONING: Following a process described elsewhere [27], a 20-item local functionality inventory was created based on participants' descriptions of what activities women needed to be able to complete daily to support themselves, their families, and their communities (see Additional file 1 for a full list of activities). Participants indicted how much difficulty they experienced in doing each activity on a Likert scale of 0 (none) to 4 (often cannot do). Item responses were averaged so a woman's score could range from 0 to 4, with higher scores reflecting greater impairment. Cronbach's alpha for the functioning scale was 0.93 [17]. BODY.METHODS.INSTRUMENTS.PSYCHOSOCIAL AND MENTAL HEALTH PROBLEMS: Women in three Eastern DRC communities initially described mental health and psychosocial problems experienced by SV survivors during a qualitative study. Participants in this qualitative study constituted a separate sample from the women enrolled in the RCT. Based on these findings, a 55-item mental health symptom and psychosocial problem questionnaire was created that included 15 depression and 10 anxiety items from the Hopkins Symptom Checklist-25 (HSCL), 16 posttraumatic stress items from the Harvard Trauma Questionnaire (HTQ), and additional items described in the qualitative study not covered by these measures (see [17] for a list of all items). The HSCL-25 and HTQ scales were used to measure the primary outcomes of combined depression and anxiety and posttraumatic stress in the original RCT analysis. For the HSCL and HTQ, women were asked to indicate on a Likert scale how often in the last four weeks they experienced each problem ranging from 0 (not at all) to 3 (a lot). Responses to scale items were averaged so mental health symptom scores could range from 0 to 3 with a higher score indicating greater severity. For this secondary analysis, the HTQ item "feeling detached of withdrawn from others" and the HSCL item "feelings of worthlessness, no value" were excluded from the average posttraumatic stress and combined depression and anxiety average scores respectively due to the inclusion of these items on the felt stigma scale (see below). Cronbach's alpha for the revised HTQ and HSCL scale were 0.87 and 0.89, respectively. BODY.METHODS.INSTRUMENTS.FELT AND ENACTED STIGMA: The stigma scale development and testing process, conducted with baseline data from all women who indicated having directly experienced sexual violence in this sample (n = 383), is described in detail elsewhere [28]. Briefly, an initial exploratory factor analysis was conducted using 16 psychosocial problems mentioned by participants that were not captured on the combined depression and anxiety or PTSD scale. Six of these 16 items loaded together on one factor, distinct from items representing more general mental distress such as anger, being cold, or thinking too much. To support the content validity of the measure, three items from HSCL or HTQ (two of which were ultimately retained) and six on traumatic experiences (four of which were ultimately retained) were combined with these 6 initial items and included in further factor analyses. Ultimately, these analyses supported the existence of two related but distinct constructs: one, composed of experiences of rejection and discrimination, we termed enacted stigma (4-items); and a second, composed of indicators of perceived and internalized stigma, we termed felt stigma (8-items) (see Additional file 1 for a list of items) [28]. For each felt stigma item, women were asked to indicate on a Likert scale how often in the last four weeks they experienced each problem ranging from 0 (not at all) to 3 (a lot). Item responses were averaged so felt stigma scores could range from 0 to 3 with a higher score indicating greater severity. Cronbach's alpha for the felt stigma scale was 0.86, indicating strong internal consistency [28]. Data on enacted stigma was only collected at baseline. The enacted stigma scale asked women to indicate whether they had ever experienced particular traumatic events. Participant responses were averaged and rescaled to range from 0 to 10 for ease of interpretability. The Kuder-Richardson coefficient for the enacted stigma scale was 0.68, indicating borderline acceptable internal consistency [28]. BODY.METHODS.ANALYSIS: Our sample included women who reported witnessing but not directly experiencing SV (n = 22, < 6%) and women who reported a SV experience. We chose to include both sets of women because, as women were recruited from the rosters of organizations providing services to survivors of sexual violence, it is likely that women who only indicated witnessing violence had (a) in actuality also experienced SV or (b) still experienced the effects of SV (including stigma) to a certain extent as witnesses. Longitudinal maximum likelihood estimated mixed-effect linear regression models with a robust variance estimator were used for all analyses with an intent-to-treat approach. Random effects included the participant (to account for multiple measures over time), CPT group, and village. Differences in baseline demographics by treatment group were assessed using Student's t-, Wilcoxon rank-sum, and Pearson's chi-squared tests. Linear regression was used to assess predictors of change in felt stigma over time. Results from these tests and consistency with prior analyses of data from this trial [17, 18] guided covariate selection. Multiple imputation with chained equations (MICE) [29] was used to account for item-level missing data and loss to follow up. Out of 405 participants, 135 (33%) and 92 (23%) missed assessments at the first and second follow up respectively (participant flow diagram is available; [17]). One item on the enacted stigma sub-scale was only relevant to married women and another only to women with children (see Additional file 1). If a woman's response was missing for more than two items, her enacted stigma score was considered missing (n = 22, 5.43%). For assessing the impact of CPT on felt stigma, time and treatment group were included as fixed effects. Treatment effect was the difference in change in felt stigma score over time by treatment group (time by treatment group interaction). For moderation analyses, we examined the difference between intervention and control participants' mean changes on study outcome scales (HSCL, HTQ, and functioning) over time by level of enacted and felt stigma reported at baseline (three-way interaction between intervention arm, time, and stigma variable). Cohen's d effect sizes were calculated by standardizing regression coefficients by the pooled standard deviation of the outcome at baseline [30]. All analyses were implemented with Stata 13 [31]. BODY.RESULTS.DEMOGRAPHICS AND BASELINE CHARACTERISTICS OF STUDY WOMEN: Women randomized to group-CPT were older on average (37 vs. 34 years of age), more likely to speak Mashi or Kihavu, and a greater proportion were currently married (59% vs. 43%) (Table 1). Women randomized to CPT were also, on average, living in larger households at baseline (7.4 vs. 6.8 people) and more likely to have lived in their village for ten years or more (75% vs. 60%). Overall, women had approximately 2 years of formal education and were responsible for an average of four children. Women assigned to continue IS reported greater functional impairment, combined depression and anxiety, and posttraumatic stress symptoms than women assigned to CPT at baseline; however, they reported experiencing and witnessing fewer types of traumas than women in the CPT arm (3.4 vs. 3.9 and 4.1 vs. 5.2, respectively). Across the trial arms, 40 women (10%) reported knowing the person(s) who perpetrated the act of sexual violence. There was no significant difference in enacted stigma by group, though women in IS did report a slightly greater number of discrimination experiences. As reported previously, 141 women (90%) in CPT completed treatment (i.e. attended at least 9 sessions). In the IS arm, 182 women (73%) met with the APS at least once; on average, these 182 women attended 5 sessions with the APS [17].Table 1Demographics, traumatic experiences, and mental health of women in Democratic Republic of Congo at study baseline, April 2011VariableCPT(n = 157)IS(n = 248)Demographic characteristics Age in years, Mean (SD)*36.89 (13.44)33.77 (12.43) Years of education completed, Mean (SD)1.76 (2.76)2.25 (3.14) Number of people living in home, Mean (SD)*7.41 (3.15)6.81 (3.32) Number of children responsible for, Mean (SD)3.96 (2.67)4.06 (2.76)Marital Status, No. (%)* Single20 (12.74)35 (14.11) Married93 (59.24)107 (43.15) Divorced1 (0.65)11 (4.44) Separated19 (12.10)43 (17.34) Widowed24 (15.29)52 (20.97)Living in territory of origin, No. (%)130 (82.80)194 (78.23)Lived at current home for 10 years or more, No. (%)*118 (75.16)148 (59.68)Language, No. (%)* Kibembe0 (0.0)46 (18.55) Kifuliro30 (19.11)64 (25.81) Kihavu58 (36.94)81 (32.66) Mashi45 (28.66)0 (0.0) Swahili24 (15.29)57 (22.98)Perpetrator of sexual violence known to womana12 (8.11)28 (11.91)Trauma and Discrimination Experiences Average different traumas experienced, Mean (SD)*3.91 (1.08)3.36 (1.36) Average different traumas witnessed, Mean (SD)*5.20 (1.28)4.06 (1.96) Enacted stigma, Mean (SD)4.67 (3.43)5.13 (3.50)Mental health Functioning score, Mean (SD)*1.65 (0.69)2.48 (0.82) Depression and anxiety score, Mean (SD)*1.95 (0.51)2.18 (0.46) Posttraumatic stress score, Mean (SD)*1.86 (0.58)2.21 (0.49)Note: CPT Cognitive Processing Therapy, IS individual support, SD standard deviation; *Between arm difference significant at p < 0.05. aDenominator is comprised of the 383 women who indicated personally experiencing sexual violence BODY.RESULTS.THE EFFECT OF COGNITIVE PROCESSING THERAPY (CPT) ON FELT STIGMA: Overall, women at baseline had a mean felt stigma score of 1.98, equivalent to experiencing a moderate amount of felt stigma on average. Women randomized to IS reported more severe felt stigma than women in CPT, a difference that reached statistical significance (Table 2). While women in both arms experienced a reduction in felt stigma over time, participants in group-CPT experienced a significantly greater decline in felt stigma relative to IS participants at the end of treatment (B = − 0.44, p value = 0.02). This magnitude but not the statistical significance of this effect was maintained six months post-treatment (B = − 0.45, p value = 0.12).Table 2Effect of CPT on felt stigma among women in Democratic Republic of Congo at end of treatment and after six-month maintenance period, April 201 l–February 2012 (n = 405)Time pointCPTaMean (SD)ISaMean (SD)b (SE)bBcP-valueBaseline1.72 (0.67)2.15 (0.62)< 0.001dPost intervention0.57 (0.68)1.60 (0.82)−0.30 (0.13)− 0.440.0246 mo. post-intervention0.60 (0.65)1.43 (0.74)−0.31 (0.20)−0.450.119Note: CPT Cognitive Processing Therapy, IS individual support, SD standard deviation, SE standard error. aUnadjusted mean scores. bβ is the coefficient for the interaction between treatment group (CPT or IS) and assessment time point and represents the difference in the change in felt stigma over time between women in the CPT and IS arms. Estimated using longitudinal mixed-effect linear regression; random effects included participant, CPT group, and village. Model covariates were baseline age, marital status (currently married yes or no), language, having lived in the current village for at least 10 years or less, total number of people living in the household, number of types of traumas experienced and witnessed, average baseline functioning score, and average baseline score on all mental health symptom items not included on the felt stigma scale. cCohen's D effect size standardized using the pooled baseline standard deviation of the felt stigma outcome. dP-value is for the Wilcoxon rank-sum test of difference in felt stigma mean score at baseline by treatment group BODY.RESULTS.MODERATION OF CPT EFFECTIVENESS FOR MENTAL HEALTH PROBLEMS BY STIGMA: Reported felt stigma at baseline did not significantly moderate the effect of CPT on combined depression and anxiety (b = − 0.07; 95% confidence interval (CI): − 0.40, 0.27) at the end of treatment (see Table 3). Higher felt stigma at baseline was associated with a greater, but non-significant, treatment effect of CPT relative to IS for posttraumatic stress (b = − 0.18; 95% CI: -0.49, 0.14) and functional impairment (b = − 0.17; 95% CI: -0.57, 0.24) at the end of treatment. Enacted stigma at baseline did not significantly moderate the effect of CPT on functional impairment (b = − 0.02; 95% CI: -0.07, 0.04) or posttraumatic stress (b = 0.02; 95% CI: -0.04, 0.07). Higher reported experiences of enacted stigma at baseline were most strongly associated with a reduction in the effect of CPT relative to IS for combined depression and anxiety (b = 0.04; 95% CI: 0.005, 0.08), though this difference was also non-significant.Table 3Moderation of the effect of CPT on the mental health of women in Democratic Republic of Congo by stigma reported at treatment initiation, April 2011–February 2012 (n = 405)OutcomeFelt stigmaeb (SE) aFelt stigmap-valueEnacted stigmae (SE) a,b,cEnacted stigma p-valueDepression and Anxiety d Post intervention−0.07 (0.17)0.710.04 (0.02)0.08 6 mo. post-intervention0.01 (0.18)0.940.03 (0.03)0.36Posttraumatic stress d Post intervention−0.18 (0.16)0.270.02 (0.03)0.56 6 mo. post-intervention0.01 (0.20)0.95−0.004 (0.03)0.88Functioninge Post intervention−0.17 (0.21)0.42−0.02 (0.03)0.55 6 mo. post-intervention0.18 (0.25)0.48−0.01 (0.04)0.73Note: CPT Cognitive Processing Therapy, SE standard error. ab is the beta-coefficient for the three-way interaction between treatment group (CPT or IS), assessment time point and moderating variable (felt-stigma or enacted stigma scale score) and represents the difference associated with a one unit increase in stigma score of the change in outcome over the time period between women in the CPT and IS arms. Estimated using longitudinal mixed-effect linear regression; random effects included participant, CPT group, and village. Model covariates were baseline age, marital status (currently married yes or no), currently pregnant, language, having lived in the current village for at least 10 years or less, total number of people living in the household, number of children for which the participant is responsible, and number of types of traumas experienced and witnessed. bSample size for the analysis of enacted stigma as a moderator is reduced to n = 383 due to some women missing more than 50% of the scale as one item was only relevant to married women ("rejected by husband") and another to those with children ("forced to live away from your children"). cFor estimation of beta coefficients for the outcome of posttraumatic stress, 10 imputations were used instead of 11 due to a convergence failure in one imputed data set. dThe HTQ item "feeling detached of withdrawn from others" and the HSCL item "feelings of worthlessness, no value" were excluded from the average posttraumatic stress and combined depression and anxiety average scores respectively due to the inclusion of these items on the felt stigma scale. eLocally developed scale, based primarily on qualitative research with women in DRC BODY.DISCUSSION: We sought to explore the potential of a group-delivered evidence-based psychotherapy, Cognitive Processing Therapy, to reduce felt (i.e. internalized and perceived) stigma among female survivors of sexual violence in eastern DRC. Experiences and feelings of stigma were common among survivors at baseline. Group CPT participants experienced moderately greater reductions in felt stigma relative to control participants who only had access to basic individual psychosocial support. The magnitude, though not the statistical significance, of this effect was maintained six months-post treatment. Neither felt nor enacted stigma significantly moderated the effect of CPT on mental health outcomes or functionality. However, women reporting more discrimination at baseline did experience slightly lower treatment effects. The group format of the intervention is one possible mechanism behind the reduction observed in felt stigma; findings on effects of group psychosocial interventions on stigma are mixed. Some limited evidence exists that group-delivered Acceptance and Commitment Therapy, a psychotherapy based in principles of mindfulness, can reduce substance abuse-related internalized stigma [32]. Conversely, a study of group Cognitive Behavioral Therapy found improvements in self-esteem among people with schizophrenia, but not perceived stigma [33]. The mixed nature of these findings from the broader literature suggests that while social contact may be an important factor to consider in efforts to reduce stigma, the nature of that contact, the characteristics of the participants, and the content of group activities must be given critical consideration. An emphasis on cognitive restructuring, a key component in CPT, is another potential mechanism of the observed impact on felt stigma. This focus may explain differences in stigma reduction effects observed between CPT and other trauma-focused treatments that place greater emphasis on gradual desensitization or exposure [25, 34]. CPT was found to be more effective than Prolonged Exposure Therapy in reducing guilt related to hindsight bias (feeling like the person could have done something to stop what happened) and lack of justification (for what they did surrounding the trauma) among SV survivors in the US [25]. A second exposure based therapy for PTSD, Narrative Exposure Therapy, delivered individually to child soldiers in northern Uganda was found to have no impact on perceived stigma [34]. This supports the idea that cognitive restructuring may be a skill that helps survivors' not only deal with their own and others' beliefs regarding trauma experiences and mental disorders, but other interrelated domains important to overall wellness. A group-based microfinance program (with no psychotherapeutic components) in the same area of DRC also resulted in a reduction in felt stigma for female survivors of sexual violence, but this reduction was smaller in magnitude [35]. Although one potential pathway of this effect could have been social interaction and acceptance by women in the group, other pathways specific to economic activity (e.g. empowerment) could also explain this change. Differences in size of the effect between CPT and this microfinance program could be due to the programs impacting different aspects of the stigma experience. For instance, a microfinance program might be more effective in reducing stigma specifically associated with perceptions of exclusion from economic activities or non-specific feelings of low worth. For individuals who primarily experience stigma as shame or blame, this type of program may not be effective. Alternatively, as a general skill with potential broad utility, cognitive restructuring may be effective in helping women address multiple components of the stigma experience. The lower degree of felt stigma reported by CPT participants was largely maintained six months post-treatment, though the effect lost statistical significance. One potential area for future research is whether booster sessions may be needed to promote the long-term well-being of women in a context of continued adversity. As women may continue to be exposed to acts of SV and enacted stigma, the feasibility of combining CPT with a broader community-based campaign for social change, and whether this type of multilevel intervention could bolster the moderately sized reduction in stigma observed in this study, is worthy of investigation. As the enacted stigma scale items asked about having ever experienced these acts of discrimination, we were not able to assess the effect of CPT on enacted stigma since we could not accurately detect change in this outcome. Future study should assess if CPT has the potential to impact other forms of stigma. Our lack of significant findings regarding moderation of CPT treatment effects by enacted or felt stigma may be due to several different factors. Our primary hypothesis was that both forms of stigma might impact care seeking, as in DRC women who experienced stigma on average took longer to seek services for sexual violence than those who did not [21]. Given that our sample was drawn from women who had already chosen to seek support services specifically designed for sexual violence survivors, these women may have been less likely to delay or decide not to seek care when invited by trusted providers a second time, inhibiting our ability to see an effect. We also hypothesized that ultimately stigma might impact treatment success because it could lead to lower engagement in care. This hypothesis was based on a prior study in the US that found lower perceived stigma predicted greater adherence to antidepressant medication [22]. Overall, women in CPT were highly engaged in therapy. Our hypothesis therefore could simply be incorrect in this context where the need is great and opportunities for mental health services are otherwise practically non-existent. BODY.DISCUSSION.LIMITATIONS: As this represents a secondary analysis of an evaluation of the effect of CPT on mental distress, the original trial was not designed to assess stigma as an outcome. Accordingly, there may be important parts of the stigma experience in this context that are not included in the measure used. As an example, survivors in DRC have described rejection as a complex concept that extends beyond physical separation from family and partners to include emotional and financial changes in relationships [5]. As most of the items on our stigma scales arose from qualitative research in DRC, our measure may also be specific to the context. Further, as the enacted stigma scale included some items only relevant to women who have children or are married, findings regarding moderation of mental health treatment effects of CPT by enacted stigma may have limited generalizability. Women were recruited for this trial through services providing psychosocial assistance to sexual violence survivors, and thus our results may not be generalizable to non-careseeking women. As felt stigma includes an individual's own assessment of their worth and how others view them, it is likely that factors related to mental health problems (e.g. negative affect or attribution bias) can shape felt stigma. This makes it challenging to distinguish between general emotional distress and internalized or perceived stigma in the measurement of the construct. We did find in earlier sensitivity analyses that depression and felt stigma items largely loaded onto distinct latent constructs in an exploratory factor analysis [28]. In addition, after removing the three items from the felt stigma scale that we hypothesized would most strongly overlap with aspects of depression ("feelings of worthlessness", "wanting to avoid others or hide", and "feeling detached from others"), our findings were not meaningfully changed (results available upon request). It is also notable that the effect of CPT on felt stigma was attenuated relative to the effects on depression, anxiety, and trauma, which we expected given the intervention was designed to explicitly address mental health problems rather than stigma specifically. A further limitation is our inability to assess the long-term impact of the effects of CPT on felt stigma. BODY.CONCLUSIONS: Despite these limitations, our findings suggest that psychotherapies may be an effective tool for reducing feelings of stigma in women affected by sexual violence. Important for practitioners, group mental health therapies delivered to sexual violence survivors may be effective regardless of the level of stigma women perceive or internalize or past experiences of discrimination. Future research should compare individual and group delivered therapies to tease out the impact of group participation from that of the actual psychotherapy elements (e.g., cognitive restructuring). In addition to exploring the mechanisms by which CPT may impact stigma, another important direction for future research is assessing whether reductions in stigma may facilitate long-term improvements in women's mental health. BODY.ADDITIONAL FILE: Additional file 1:List of items included in locally-developed scales. (DOCX 68 kb)

TITLE: The impact of a disease management program (COACH) on the attainment of better cardiovascular risk control in dyslipidaemic patients at primary care centres (The DISSEMINATE Study): a randomised controlled trial ABSTRACT.BACKGROUND: To evaluate the efficacy of Counselling and Advisory Care for Health (COACH) programme in managing dyslipidaemia among primary care practices in Malaysia. This open-label, parallel, randomised controlled trial compared the COACH programme delivered by primary care physicians alone (PCP arm) and primary care physicians assisted by nurse educators (PCP-NE arm). ABSTRACT.METHODS: This was a multi-centre, open label, randomised trial of a disease management programme (COACH) among dyslipidaemic patients in 21 Malaysia primary care practices. The participating centres enrolled 297 treatment naïve subjects who had the primary diagnosis of dyslipidaemia; 149 were randomised to the COACH programme delivered by primary care physicians assisted by nurse educators (PCP-NE) and 148 to care provided by primary care physicians (PCP) alone. The primary efficacy endpoint was the mean percentage change from baseline LDL-C at week 24 between the 2 study arms. Secondary endpoints included mean percentage change from baseline of lipid profile (TC, LDL-C, HDL-C, TG, TC: HDL ratio), Framingham Cardiovascular Health Risk Score and absolute risk change from baseline in blood pressure parameters at week 24. The study also assessed the sustainability of programme efficacy at week 36. ABSTRACT.RESULTS: Both study arms demonstrated improvement in LDL-C from baseline. The least squares (LS) mean change from baseline LDL-C were −30.09% and −27.54% for PCP-NE and PCP respectively. The difference in mean change between groups was 2.55% (p=0.288), with a greater change seen in the PCP-NE arm. Similar observations were made between the study groups in relation to total cholesterol change at week 24. Significant difference in percentage change from baseline of HDL-C were observed between the PCP-NE and PCP groups, 3.01%, 95% CI 0.12-5.90, p=0.041, at week 24. There was no significant difference in lipid outcomes between 2 study groups at week 36 (12 weeks after the programme had ended). ABSTRACT.CONCLUSION: Patients who received coaching and advice from primary care physicians (with or without the assistance by nurse educators) showed improvement in LDL-cholesterol. Disease management services delivered by PCP-NE demonstrated a trend towards add-on improvements in cholesterol control compared to care delivered by physicians alone; however, the improvements were not maintained when the services were withdrawn. ABSTRACT.TRIAL REGISTRATION: National Medical Research Registration (NMRR) Number: NMRR-08-287-1442 Trial Registration Number (ClinicalTrials.gov Identifier): NCT00708370 BODY.BACKGROUND: Chronic diseases pose a significant disease burden causing 60% of all deaths worldwide [1]. Of these, 50% are attributed to cardiovascular diseases [1]. Low-and middle-income countries are the biggest contributors to the increase in cardiovascular disease burden [2]. Although it varies among countries, the factors contributing to the escalating prevalence of chronic diseases are an ageing population, tobacco use, unhealthy diet practices and physical inactivity, urbanisation and global marketing [3], where half of these risk factors are modifiable through behaviour modification. Primary healthcare plays a pivotal role in gearing patients towards positive behaviour management [4]. This can be achieved through the use of a chronic disease management (CDM) model, which emphasises the integration of several elements including multidisciplinary care delivery, patient education and provider decision support, self-management and patient empowerment support, clinical information technology, social support and quality incentives within the primary health care system [5]. Although studies have shown that chronic disease management are associated with marked improvements in many clinical outcomes associated with cardiovascular diseases [6-9]; many developing countries have yet to integrate CDM into their primary healthcare systems due to limited resources and systems orientated towards acute symptomatic care [4]. In addition, there is paucity of literature that addresses the sustainability of chronic disease management programmes in developing countries, in terms of its efficacy and cost. Malaysia is not immune to the rising tide of chronic diseases, where cardiovascular diseases account for more than 25% of all-cause mortality [10] and prevalence of cardiovascular risk factors such as hypertension, diabetes mellitus and dyslipidaemia has reached epidemic proportions [11]. As a developing country, Malaysia is faced with the expected challenges of implementing chronic disease management in a resource-limited environment [12]. Although there have been several disease management programmes implemented in Malaysia, evidence on their cost-effectiveness, applicability and sustainability is lacking [13]. Despite the often-repeated recommendations to incorporate multidisciplinary healthcare teams in chronic disease care, access to allied health services is usually limited in a developing country like Malaysia. In addition, evidence to support the use of nurse-assisted dyslipidaemia management has been conflicting [14,15]. In view of the current lack of evidence on CDM efficacy in a developing country such as Malaysia, we designed a randomised controlled trial to assess the impact of a chronic disease management programme, COACH (Counselling and Advisory Care for Health) in managing dyslipidaemia. The COACH programme used in this study was intended to be modelled after the original COACH study [16,17]; however it was adjusted to the local situation and limitations. The primary objective of the DISSEMINATE study was to evaluate the efficacy of the COACH programme led by primary care physicians with assistance from nurse educators (PCP-NE) compared to that led by the primary care physicians alone (PCP) in improving serum low density level-cholesterol (LDL-C) in dyslipidaemic subjects over a period of 24 weeks. Secondary objectives included the impact of the COACH programme in the two arms in improving (i) patients' lipid profiles i.e. High Density Lipoprotein-Cholesterol (HDL-C), LDL-C, Total Cholesterol (TC), Triglycerides (TG) and TC: HDL ratio, (ii) blood pressure (systolic and diastolic), (iii) Framingham cardiovascular risk, (iv) lifestyle modification (smoking behaviour, diet, alcohol consumption, physical activity), (v) programme satisfaction using a visual analogue scale (VAS) and (vi) statin compliance. All outcomes were assessed at 24 and 36 weeks of study duration. The outcomes assessment at 36 weeks was designed to evaluate the sustainability of programme effectiveness after the programme ended at week 24. BODY.METHODS.TRIAL DESIGN: This was a multi-centre, open-label, parallel, randomised trial of a disease management programme (COACH) led by primary care physicians for dyslipidaemic patients. Comparison was made between primary care physicians who managed patients alone (PCP Group-Control arm) versus those who were assisted by nurse educators (PCP-NE Group-Intervention arm). Patients were allocated on a 1:1 ratio. The study was approved by Malaysian Medical Research Ethics Committee (MREC) (National Medical Research Registration (NMRR) Number: NMRR-08-287-1442) and was conducted in compliance with the Declaration of Helsinki and International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines, as well as local regulatory requirements. No major changes were made to the trial methodology after study initiation. BODY.METHODS.SITE SELECTION: Sites were selected based on their ability to recruit patients and to commit to the project. In order to achieve the recruitment rate and to provide a fair representation of the patient population in Peninsula Malaysia, licensed primary healthcare centres around the Klang Valley and other suburban towns were invited. Other recruitment factors were availability of infrastructure such as internet line or fax machine, the investigator's understanding of GCP, the availability of a site coordinator and previous clinical trial experience. Thirty five sites were approached and 21 sites actively participated in the study. BODY.METHODS.SUBJECT RECRUITMENT: Patients who enrolled were newly diagnosed with dyslipidaemia. Subjects could be either male or female age 18 years or older. Besides having given informed consent, subjects must have been contactable via telephone, live or through short messaging services (SMS). Communication with patients through these methods was a key element of the COACH programme. Exclusion criteria applied included participation in any other clinical research studies in the preceding 6 months, history of mental illness, hypothyroidism, or presence of any other condition, which the investigators judged could increase risk to the subject and could interfere with the conduct of the study or interpretation of the data. Eligibility was defined by dyslipidaemia divided according to 3 cardiovascular risk groups. Group I subjects were dyslipidaemic with an LDL-C level within 160 to 250mg/dL without concomitant cardiovascular risk factors. Group II subjects had at least one additional cardiovascular risk factor, excluding coronary heart disease (CHD) and diabetes mellitus (DM) with serum LDL-C level between 130 to 250mg/dL; Group III subjects consisted of those with serum LDL-C level between 100 to 250mg/dL with pre-existing CHD or CHD risk equivalent, such as DM or other atherosclerotic diseases. The study subjects were required to be lipid drug naïve and eligible for statin therapy prior to study enrolment. Random numbers were generated by computer and printed in sealed envelopes. The envelopes were subsequently allocated to each new patient enrolled into the programme. A person independent of the study was tasked to assign a randomisation number to a subject with known study identification number. The allocation of the individual study group was then made known when the envelopes were unveiled. Since COACH was administered by the nurse educators independent of the primary care doctor, knowledge of treatment allocation only became evident to the doctor when the subject returned for his/her third scheduled visit (week 12). The study duration was 36 weeks. BODY.METHODS.STUDY PROCEDURE: All subjects received standard care and advice from their primary care physicians as per the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel [ATP] III) guidelines [18]. As part of the standard care, all subjects received a COACH health booklet which was completed by the investigator during clinic visits. Subjects were expected to attend clinic visits at week 12, 24 and 36 after baseline randomisation. Statin dose was titrated according to cardiovascular risk group as recommended by NCEP ATP III Guidelines. Demographic data, primary diagnosis, medical history, family history, smoking status, alcohol, diet, physical activity, drug allergies and all other concomitant medications were captured in the study case report form (CRF). Blood pressure reading, pulse rate, general physical examination, 12-lead ECG, and blood sampling were also conducted during study visits. All blood samples were sent to a central laboratory for analysis. Telephone follow-up were made by site coordinators at week 6 and 18 to ensure all patients completed the health booklet correctly and complied with statin treatment. Subject compliance to prescribed statin was assessed by the primary care doctor and percentage compliance was noted in the CRF. BODY.METHODS.STUDY INTERVENTION: In addition to the standard care, subjects randomised to the PCP-NE COACH Programme received bi-weekly telephone follow-up by trained nurse educators for 24 weeks. The main purpose of the telephone call was to provide patient's self-management support and patient empowerment and the discussion was guided by the health education booklet. During the telephone follow-up, the nurse educators provided reinforcement of the health education information and reminded patients to adhere to counselling advice and prescribed medications, as well as to discuss any problems of adherence encountered with their physicians. Each telephone call lasted an average of 15 min. Subjects also received phone calls and SMS to remind them about forthcoming follow-up visits. The study intervention was designed to support the doctor-patient relationship and reinforce the prescribed care plan. BODY.METHODS.STUDY CONTROL: All subjects randomised to PCP COACH Programme only received care from the site investigators as per normal practice. There was no additional telephone follow-up and reinforcement by nurse educators. At the end of 24 weeks, the study period ended. However, all subjects were required to attend follow-up visit at week 36, an extension phase, during which both arms received standard care only. This was to evaluate whether there would be sustained lipid control without reinforcement. BODY.METHODS.OUTCOME MEASURES: The primary efficacy endpoint was the mean percentage change from baseline LDL-C at week 24 between the two treatment arms. Other efficacy endpoints included mean percentage change from baseline of lipid profile (TC, LDL-C, HDL-C, TG, TC: HDL ratio), Framingham Cardiovascular Heart Risk Score, and absolute risk change from baseline in blood pressure parameters (systolic and diastolic) at week 24 and week 36. Lifestyle change assessment of smoking behaviour, food consumption, physical activity, alcohol consumption, and medication compliance were also evaluated at the end of the study. BODY.METHODS.STATISTICAL ANALYSIS: The sample size was calculated to detect a clinically meaningful difference of 10% in percentage change from baseline in LDL-C levels with a standard deviation of 22.5%, a 2-sided, 5% significance level and 85% power. During the implementation of the study, it was found that there was potential treatment contamination of the standard care arm where, behaviour modification occurred due to subjects from both arms interacting with each other. To overcome this, the sample size was adjusted to account 20% of treatment contamination (changing the effect size to 0.08%) and 10% attrition rate, giving the study a total of 320 subjects. The analysis was conducted using intention-to-treat (ITT) analysis, which was defined as all lipid drug naïve subjects who were newly diagnosed with dyslipidaemia, eligible for statin therapy and were randomised with at least 1 post-baseline response (efficacy or outcomes research endpoints). Last observation carried forward was used for missing data in the ITT analysis. Statistical analysis was based on the mean percentage change from baseline using a restricted maximum likelihood (REML) model with repeated measures approach (mixed model repeated measures [MMRM]). Significance tests were based on the least squares (LS) mean difference between treatment group to compare treatment contrast for week 24 and week 36. The analyses conducted at 24 weeks evaluated the effectiveness of the programme while analyses at 36 weeks assessed the medium-term effectiveness of care by physicians assisted by nurse educator (PCP-NE) compared with primary care physician care alone (PCP). BODY.RESULTS: In order to achieve a sample size of 320 subjects, 364 potential subjects were screened however, only 297 subjects met the eligible criteria and were randomised. The period of patient recruitment was from September 2008 to July 2009. All patients were followed up for 36 weeks and the study was concluded in May 2010. One hundred and forty nine subjects were randomised to the PCP-NE COACH programme (Intervention) and 148 subjects to PCP COACH group (control). Of these, 122 (81.9%) from the intervention group and 123 (83.1%) from the standard care group completed the 36 weeks study. Figure 1 illustrates the trial profile for the DISSEMINATE study. Figure 1Study trial profile. There were no significant differences in subjects' baseline demographic characteristics and cardiovascular risk factors (Table 1). All subjects had a primary diagnosis of dyslipidaemia with a mean duration of 0.6 years from disease onset in both groups. All subjects were prescribed with HMG-CoA reductase inhibitors (statins). The most common statins prescribed in each group were lovastatin, simvastatin and atorvastatin. The median duration of statin treatment was 246 days. Approximately 99% and 95% of the patients in the intervention and control arm respectively reported compliance to statin treatment. Table 1 Baseline subjects demographics & characteristics   Intervention Arm (n=149) Control Arm (n=145) Number of subjects; n (%)    Male 86 (57.7%) 83 (57.2%)    Female 63 (42.3%) 62 (42.8%) Age, years; Mean (SD) 49.4 (11.1) 48.8 (9.9) Race; Asian 149 (100.0) 145 (100.0) Weight, kg; Mean(SD) 72.7 (15.2) 70.2 (15.1) Height, cm; Mean (SD) 160.2 (8.2) 159.6 (9.3) Body mass index, kg/m 2 ; Mean (SD) 28.2 (4.9) 27.4 (4.9) Mean duration of dyslipidaemia since onset (years) 0.6 0.6 Cardiovascular risk group a ; n (%)    Group I 14 (9.4) 24 (16.6)    Group II 87 (58.4) 74 (51.0)    Group III 48 (32.2) 47 (32.4) Age risk; n (%)    Male aged >45; Female aged >55 72 (48) 60 (41)    Male aged ≤45; Female aged ≤55 77 (52) 85 (59) Family history b ; n (%)    Yes 29 (19) 32 (22)    No 120 (81) 113 (78) Blood pressure ≥ 140/90 mm Hg or antihypertensive medication at baseline; n (%)    Yes 89 (60) 88 (61)    No 60 (40) 57 (39) Diabetes Mellitus; n (%)    Yes 47 (32) 47 (32)    No 102 (68) 98 (68) CHD or CHD risk equivalent a ; n (%)    Yes 1 (1) 1 (1)    No 148 (99) 144 (99) Smoking Status    Never smoked 101 (68) 97 (67)    Ex-smoker 13 (9) 19 (13)    Current smoker 35 (23) 29 (20) COACH = Counselling and Advisory Care for Health; SD = standard deviation; CHD = coronary heart disease; PCP = Primary Care Physicians; PCP-NE = Primary Care Physicians assisted by Nurse Educators. a Group I: subjects with dyslipidaemia with no other cardiovascular risk factors; Group II: subjects with dyslipidaemia with at least 1 additional cardiovascular risk factor, excluding CHD and DM; Group III: subjects with dyslipidaemia with CHD or CHD risk equivalent. b A history of premature coronary heart disease in a first-degree relative (parent or sibling). If the afflicted family member was male, premature coronary heart disease would have occurred before age 55, and if female, before age 65. At the end of the study, the ITT set consisted of data from 132 and 131 subjects, from the intervention and control arms respectively. At week 24, there was a trend towards greater improvement in the intervention group. The least squares (LS) mean changes from baseline LDL-C were −30.09% and −27.54% for the intervention group and control groups, respectively. The difference of mean change in the intervention group was 2.55% lower than the control group, however this was not significant (p=0.288). At week 36, the LS mean of LDL-C between the 2 study arms was comparable at −25.88 and −26.86, for the intervention and control groups respectively. On the other hand, there was a statistically significant difference when comparing the LS mean in the intervention arm at week 24 and week 36 (p=0.016). This was due to withdrawal of the COACH programme which resulted in a diminution of the earlier LDL-C improvements seen in the intervention arm. Similar outcomes were observed with total cholesterol (TC) level. The trend towards improved TC seen in the intervention group compared with the control arm at 24 weeks did not sustain at 36 weeks. No significant difference was detected in triglyceride (TG) levels at week 24 and 36 between the 2 study arms. Table 2 and 3 illustrates the percentage change from baseline in LDL-C and TC between the intervention and control group. Table 2 Percent change from baseline in LDL-C, TC and HDL-C between intervention and control groups Time point Outcomes LS Mean (SE) Difference in LS Means (%) PCP-NE (n=132) PCP (n=131) PCP-NE versus PCP 95% Cl (%) p-value Week 24 LDL-C −30.09 (2.03) −27.54 (2.03) −2.55 −7.26, 2.16 0.288 TC −22.97 (1.55) −20.80 (1.54) −2.17 −5.72, 1.38 0.229 HDL-C −2.60 (1.30) −5.61 (1.30) 3.01 0.12, 5.90 0.041 Week 36 LDL-C −25.88 (2.03) −26.86 (2.03) 0.98 −3.68, 5.65 0.679 TC −19.94 (1.53) −19.97 (1.52) 0.04 −3.43, 3.51 0.984 HDL-C −2.97 (1.42) −5.44 (1.41) 2.48 −0.79, 5.74 0.136 LS = Least Squares; SE = Standard Error; 95%; CI = 95% Confidence Interval; LDL-C = Low Density Lipoprotein Cholesterol; TC = Total Cholesterol; HDL-C = High Density Lipoprotein Cholesterol; PCP = Primary Care Physicians; PCP-NE = Primary Care Physicians assisted by Nurse Educators. Table 3 Percent change from baseline in LDL-C, TC and HDL-C between week 24 and 36 Study Arm Outcomes LS Mean (SE) p-value Week 24 Week 36 PCP-NE (n=132) LDL-C −30.09 (2.03) −25.88 (2.03) 0.016 TC −22.97 (1.55) −19.94 (1.53) 0.018 HDL-C −2.60 (1.30) −2.97 (1.42) 0.768 PCP (n=131) LDL-C −27.54 (2.03) −26.86 (2.03) 0.695 TC −20.80 (1.54) −19.97 (1.52) 0.515 HDL-C −5.61 (1.30) −5.44 (1.41) 0.896 LS = Least Squares; SE = Standard Error; 95% CI = 95% Confidence Interval; LDL-C = Low Density Lipoprotein Cholesterol; TC = Total Cholesterol; HDL-C = High Density Lipoprotein Cholesterol; PCP = Primary Care Physicians; PCP-NE = Primary Care Physicians assisted by Nurse Educators. In contrast, a significant difference was detected in high density lipoprotein-cholesterol (HDL-C) at week 24 between the intervention and control group with a greater decrease in HDL. As illustrated in Table 2, the difference in LS means of HDL-C between the intervention and control group was 3.01 (95% CI 0.12 – 5.90), p=0.041 at week 24. However, this effect did not persist beyond the duration of the intervention (p=0.136) after week 24 (Table 3). These results at and after week 24 were also reflected similarly in the total cholesterol (TC): high-density lipoprotein cholesterol (HDL-C) ratio. There were no significant differences between both study arms in relation to statin treatment compliance, systolic and diastolic blood pressure and Framingham Coronary Heart Disease risk scores at week 24. In terms of lifestyle modification (i.e. cigarettes smoking, dietary changes, and physical activity), no difference was observed between the intervention and control group. In general, more than 80% of the subjects were satisfied with the health booklet provided. Approximately 90% of subjects from the intervention arm also expressed satisfaction with the programme in helping them achieve health care goals through telephone follow-up by nurse educators. BODY.DISCUSSION: The COACH programme in this study utilised patient education and empowerment as well as decision support as interventional strategies to improve cholesterol control. Both study arms had shown improved LDL-C and TC level throughout the study period. The results were a trend towards add-on improvements in both LDL-C and TC levels when patients were co-managed by nurse educators, even though this was not statistically significant. The lack of statistical significance was most likely attributed to dilution of treatment effects with the use of similar patient education methods in the control group as well as in the intervention group, specifically, using the COACH health booklet. The COACH programme applied in our study delivered more comprehensive care than usual compared to the typical Malaysian primary care settings, which usually have little time or resource to provide comprehensive disease management care as part of daily practices [19]. As a result, in this study, patients from both study arms would have benefited from increased knowledge of their health conditions. Although the COACH programme used in this study was similar to other studies published by Vale et al. [16] and Allen et al. [17], there are significant differences in terms of study methodology. In the study conducted Vale et al. [16], patients were continuously coached based on previous assessment visit and progress monitored via negotiated action plan. In addition, the Vale's study focused on hospital based disease management programme. On the other hand, the COACH programme by Allen et al. [17] utilised the model of community-based participatory research methodology to design the disease management programme, which consisted of enhanced usual care with or without intensive disease management by nurse practitioner/community health workers. Both studies had adopted many essential elements from the Chronic Care Model [3], which had been proven to improve chronic disease outcomes. In contrast to this study, the authors had only adapted the previous COACH studies as a patient support programme rather than a disease management programme due to severe resource limitation i.e. lack of trained nursing support resulting in resistance from local doctors to adopt a shared-care model on patient disease management counselling. As a result, the nurse educators in this study had no access to patient's medical records and health education was reinforced using the health education booklet only. Evidence for the success of disease management programmes involving additional support from nurses or other disciplines is mixed. Our findings are comparable with some studies [14,20] where reduction in blood cholesterol was similarly equivocal due to the Hawthorne effect among patients and healthcare providers, a change in people's behaviour when being observed. In addition, the study may have heightened the awareness of disease management practices among family physicians involved. However, studies published by Vale et al. [16] and Allen et al. [17] that utilised a similar programme, demonstrated significant improvement in LDL-C and TC change from baseline when patients with coronary heart diseases underwent the programme. As opposed to our study, the positive outcomes demonstrated in both the studies may be due to: (1) the population enrolled i.e. the study recruited CHD patients experiencing acute coronary syndrome who were more motivated to change; (2) difference in setting i.e. developed versus developing country; (3) difference in prescription behaviour i.e. higher proportion of patients prescribed with statin and with higher doses of atorvastatin in the Vale et al. study (not commonly prescribed among the doctors in the DISSEMINATE study). Besides the 2 studies discussed above, there are also other studies that have had positive results with multi-disciplinary support [21-24]. The DISSEMINATE study had also revealed interesting findings with regards to HDL-C decrease over the 6 months duration. This finding was inconsistent with many clinical studies that reported mild improvement of HDL-C with both drug treatment and disease management care [25,26]. Several theories are hypothesised for this outcome. First of all, there is some evidence that a low fat diet reduces not just LDL-C but also HDL-C [27-29]. Secondly, the COACH programme did not provide detailed nutritional education with information on how to reduce high dietary fat intake while substituting dietary fat with polyunsaturated fatty acids (PUFA) such as olive oil. It is possible that both of these factors could have resulted in the unexpected reduction in HDL-C levels detected in our study. Several limitations to our study are identified. In an effort to standardise patient care between different sites, a health booklet was distributed to all subjects. As discussed above, this had diluted the difference in interventional effects between control group and intervention group. Also, the findings in the control arm would not be generalise to the current primary care settings as it had deviated from the normal local practice; however, the outcomes from this study has demonstrated that current chronic disease care in Malaysia primary care practices are suboptimal and some improvements in patient's disease outcome can be achieved by simply spending extra time to educate the patients on chronic disease self-management and treatment compliance. And furthermore, our study has also shown that the task of personalised patient education does not have to be purely the domain of the physicians but can also be provided by trained nurses. The study might have elicited a more significant finding with addition of a third study arm assessing the lipid outcomes among patients who received "actual" standard care delivery by local physicians. Though the primary outcome of the study was not achieved, an additional post-hoc analysis may be able to determine the proportion of uncontrolled dyslipidaemic patients who achieved cholesterol target at the end of the study follow-up. In the current analysis, the proportion of patients who achieved target cholesterol level is not known. A higher than expected attrition rate (initial sample size calculation only accounted for 10% loss to follow-up) could have also affected the final results of the study. Although our study showed that complementary nurse support services in a disease management programme had a positive trending impact on LDL-C and TC level compared to physician management alone, the effect was not sustainable after the intervention was withdrawn at week 24. Continuous therapeutic behavioural change seems to be mandated to ensure long-term sustainable lipid control. BODY.CONCLUSION: This study was designed as a trial in a "real-world" setting and thus faced with its share of expected challenges. The limitations of our study are acknowledged, and some of the lack of effect may be attributable to similarities between the management of patients in the intervention and control group. Further study would be warranted to build upon the results of this study while addressing some of the limitations. Despite the lack of statistical significance, the trend towards improved dyslipidaemia can be considered a success at some level. As there is a paucity of published evidence on disease management programmes in this region, the data provided by this study may be considered an indicator about the potential role of disease management programmes in developing Asian countries. BODY.ABBREVIATIONS: AE: Adverse events; ATP: Adult Treatment Panel; BMI: Body mass index; BP: Blood pressure; CDM: Chronic Disease Management; CHD: Coronary heart disease; CI: Confidence interval; Cm: Centimeter; COACH: Counselling and Advisory Care for Health; CORFIS: Community based cardiovascular risk factor intervention strategy; CRF: Case report form; DISSEMINATE: Disease management programme on the attainment of better cardiovascular risk control; DM: Diabetes mellitus; ECG: Electrocardiogram; ESRD: End stage renal disease; FAS: Full analysis set; GCP: Good Clinical Practice; GP: General practitioner; HDL-C: High density lipoprotein cholesterol; HMG-CoA: 3-Hydroxy-3-Methyl-Glutaryl-CoA; ICH: International Conference on Harmonization; Kg: Kilogram; LDL-C: Low density lipoprotein cholesterol; LS: Least squares; mg/dL: Milligram per deciliter; mmol/L: Millimol per liter; mmHg: Millimeter mercury; MMRM: Mixed model repeated measures; MREC: Medical Research Ethical Committee; NCEP: National Cholesterol Education Program; NIH: National Institute of Health; PUFA: Polyunsaturated fatty-acid; REML: Restricted maximum likelihood model; SD: Standard deviation; SE: Standard error; SMS: Short messaging service; TC: Total cholesterol; TG: Triglycerides; VAS: Visual analogue scale; WHO: World Health Organization. BODY.COMPETING INTERESTS: The preparation of this manuscript was funded by a grant from Pfizer (Malaysia) Sdn Bhd. However, Pfizer has no influence over the drafting of the manuscript, nor the decision to publish. The responsibility for the contents of this manuscript rests entirely with the authors. The authors and investigators in this study have no association with the original COACH programme. All authors were independent investigators of the study with the exception of JFS who was previously an employee of Pfizer (M) Sdn Bhd. BODY.AUTHORS’ CONTRIBUTIONS: All authors have contributed significantly to the success of this study. JFS was responsible for the conception and the design of the study as well as reviewed the manuscript. MM, KO, SN, ZK, SYS, NA, KK and FA demonstrated significant input and dedication during patient recruitment and data collection. WL was responsible for conducting the literature review and manuscript write up. 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/1471-2296/13/97/prepub

TITLE: Neural Rhythms of Change: Long-Term Improvement after Successful Treatment in Children with Disruptive Behavior Problems ABSTRACT: Neural changes were investigated for children with disruptive behavior problems one year after a treatment program ended. Thirty-nine children and their parents visited the research lab before, after, and a year after treatment ended. During those lab visits, electroencephalography (EEG) was recorded during a challenging Go/No-go task. Treatment consisted of intensive 14-week combined cognitive behavioral therapy and parent management training sessions. For the analysis, participants were divided into long-term improvers (IMPs) and long-term nonimprovers (NIMPs) based on changes in their externalizing problem scores. The results showed early no-go theta power (4–8 Hz, 100–250 ms) decreased for long-term IMPs compared to NIMPs. When participants were divided based on changes in their comorbid internalizing symptoms, effects were stronger and reductions in theta power were found for early as well as later phases (250–650 ms). We provided preliminary evidence that theta power is a useful neural measure to trace behavioral change linked to improved self-regulation even up to a year after treatment ended. Results may have implications for the characterization of children with disruptive behavior problems and may lead to the development of novel markers of treatment success. BODY.1. INTRODUCTION: The ultimate goal of treatment is to show benefits long after treatment ends. However, follow-up studies examining treatment outcomes for children with disruptive behavior problems (DBP) are rare and typically show small effect sizes [1, 2]. Furthermore, treatment studies often focus on testing the efficacy of a particular intervention by comparing an experimental treatment group with a control group (e.g., treatment as usual). However, this approach does not reveal the variability of outcomes within the treatment group itself: some children improve while others do not. Examining how improvers and nonimprovers differ in terms of the processes of change might be key in finding cost-saving predictors for effective treatment, the refinement of current treatment models, and the development of more reliable indices of long-term treatment success. To explain what causes variability in long-term treatment outcomes, researchers have proposed political and socioeconomical as well as family and peer-relationship factors [3–5]. For example, Reyno and McGrath [6] showed that low family income was an important factor in predicting treatment outcomes for children with externalizing behavior problems. But the ability to change these factors is often small, especially for a child. What individuals can change, however, is how they cope with and manage to control the tonic stressors that surround them. This ability, to flexibly control one's own emotions and cognition in the service of (long-term) internal goals, is broadly defined as self-regulation [7]. Effective self-regulation skills are believed to function as buffers against stressors and build resilience in multiple domains. Studies have shown, for example, that effective self-regulation is associated with better academic performance and social functioning [8, 9]. Conversely, problems with self-regulation can manifest in severe internalizing or externalizing problem behaviors such as those related to DBP, anxiety, and attention disorders [7, 10, 11]. Increasing our measurement precision of self-regulation is important and the field has begun to look at neuroscientific techniques to provide the tools to measure and enrich our understanding of neural processes underlying self-regulation. The electroencephalography (EEG) technique is particularly sensitive in measuring the rapid cognitive processes that allow us to monitor and inhibit responses. For those reasons, event-related potentials (ERPs, averaged electrophysiological waveforms locked to an event), derived from the EEG, have traditionally been associated with self-regulatory processes during inhibition tasks. Differences in amplitudes of ERPs, for example, have been able to distinguish individuals with externalizing behavior problems from their typically developing peers [12–14]. Recently, researchers focus on event-related spectral perturbations (ERSPs; oscillatory patterns in the EEG waveform locked to events at the level of individual trials) to better understand human cognition. Such techniques capture rhythmic patterns derived from assemblies of neural populations which are widely considered intrinsic to brain function in general and crucial for the neural integration and processing of information in particular [15–17]. For example, alpha power (8–12 Hz) has been related to an active inhibition of task irrelevant brain areas during working memory tasks [18]. In the present paper our main objective was to focus on theta power. Theta power (4–8 Hz), derived from long-range, intercortical, or corticolimbic pathways, is understood as a binding rhythm that synchronizes multiple limbic and (neo)cortical brain regions [16, 19, 20]. In a review, Knyazev [16] concluded that theta power was mostly involved in memory and emotion regulation. In the field of working memory, for example, changes in theta power have been associated with the integration and maintenance of information [21, 22]. In the area of cognitive control, one recent study from our lab found that variations in later frontomidline theta power (after 250 ms) during the no-go trials of a Go/No-go task related to developmental changes in self-regulation [23]. Early frontomidline theta power, though prominent in the data, was not examined in this study. In another study, Lenartowicz et al. [24] compared children with ADHD and their typically developing peers using a working memory task and found differences in early frontomidline theta power during the high memory load. We propose that, related to self-regulation, at an early stage of visual processing, theta power could act in recruiting attentional vigilance towards the encoding or monitoring of goal-relevant stimuli; and, at a later stage, theta power may be increasingly executive in nature and directly relate to dedicating attentional resources towards the inhibition of a response and subsequent evaluative processing (see also [25], for a recent review). Such an interpretation of early and later processing in the temporal dynamics of emotion regulation processing is in line with the recent ERP literature [14, 26] as well as a study exploring the chronometry of source activations localized to the medial prefrontal cortex [27]. In the latter study, an emotional Go/No-go task was used to track the chronometry of activation. Early window activity (around 200 ms) was interpreted as a bottom-up vigilance to perceived stimuli (i.e., manifested as a threat bias) whereas later activation was seen as more regulatory in nature. Very little is known about the plasticity of the neural systems underlying self-regulation after successful treatment of DBP. To date, only two studies have been conducted that investigated neural changes using EEG with successful treatment of self-regulation. Lewis et al. [28] and Woltering et al. [13] used a Go/No-go task to examine children with DBP using ERP and source localization methods and found changes in neural activation after successful treatment. The treatment consisted of a combined cognitive behavioral therapy (CBT) and parent management training (PMT) program and used a broad range of evidence-based clinical techniques to train self-regulation skills (see [29], for overview). To the best of our knowledge, no treatment study has yet investigated long-term brain plasticity effects in children with DBP nor has any study examined changes in theta power after an intervention. The present study built on two former studies from our lab that found neural changes in children who showed improvement on externalizing problem behaviors directly after treatment ended [13, 28]. In addition to examining improvements in externalizing behavior, we also aimed to investigate long-term changes in internalizing problems because emerging research indicates that anxiety may underlie the manifestation of DBP behaviors (see [30], for overview). Anxiety is also frequently comorbid with DBP, with up to 75% of children clinically referred for aggression exhibiting clinically elevated anxiety symptoms [30]. Although treatment was geared towards externalizing problems, therapeutic techniques that foster emotional control could also benefit underlying emotions of anxiety. A number of families indicated that they were interested in being contacted a year after treatment ended, allowing us to investigate whether theta power would change for children who showed long-lasting improvement from their baseline session. Based on our previous studies [13, 28], which consistently found reductions in neural activation with successful treatment, directional hypotheses could be formulated. We hypothesized that long-term improvers would show a continuous reduction in frontomidline theta power from baseline to after treatment to follow-up compared to long-term nonimprovers. Consistent with a chronometry study using the same task, which found heightened neural activation for DBP children in early and later processing, we expected to find reductions in early and as well as later theta power [27]. Furthermore, we also hypothesized that long-term improvements in internalizing symptoms may be equally, or even more, sensitive to concomitant reductions in frontomidline theta activation. BODY.2. METHODS.2.1. PARTICIPANTS: Participants were recruited from two agencies that provided treatment for DBP. A total of 150 children between 8–12 years old and their families agreed to participate in the study. Sixty participants completed the follow-up assessment a year after treatment ended. Of these 60 participants, 39 (31 boys, 80%) had usable neural and behavioral data at pretreatment and follow-up sessions. These 39 participants constituted the sample for the present study. This select group did not differ from the initial sample at baseline on measures of age, sex, medication use, demographics, or on severity of problem behavior as determined by t-tests. See Table 1 for a more detailed description of the sample, including socioeconomic and ethnic demographics. The vast majority of medication use consisted of stimulant medication (either Concerta or Ritalin). Participants were referred to treatment agencies by mental health professionals, teachers, police, and/or parents. Inclusion criteria consisted of scores on or above the borderline-clinical range (84th percentile) on the externalizing scale of the child behavior checklist (CBCL; [31]). Exclusion criteria consisted of significant cognitive impairment, such as a persistent developmental delay (no participants were excluded on these grounds). The study was approved by the research Ethics Board of the University of Toronto. BODY.2. METHODS.2.2. INTERVENTION: An evidence-based treatment program called SNAP (Stop Now and Plan; Augimeri, Walsh, Levene, Sewell, and Rajca, 2014) was delivered to children (CBT) and parents (PMT). The SNAP program has undergone evaluations demonstrating positive treatment outcomes evidenced at least to 6- and 12-month follow-up periods [32, 33]. Three-hour-long weekly group therapy sessions were held separately for the children and their parents at the community agencies for 14 weeks. CBT targeted effective regulation of emotional and other behaviors through well-documented strategies such as cognitive restructuring, problem solving, role-playing, and social and token reinforcements, as well as generalization activities [34, 35]. PMT promoted positive parenting practices such as skill encouragement, problem solving, and monitoring, as well as the replacement of coercive or lax discipline strategies with mild sanctions targeting misbehavior [36–38]. Therapists were either social workers, child-care workers, and M.A.- or Ph.D.-level clinical psychology students. Therapists were trained in the PMT and CBT protocols and regularly supervised to ensure compliance with the treatment model. BODY.2. METHODS.2.3. PROCEDURE: Behavioral and EEG data were collected 2 weeks both before and after the 14-week treatment sessions. Follow-up sessions occurred 12 months after treatment had ended. During lab sessions, children were accompanied to the lab by a parent. After a brief introduction to the testing environment, parental consent and child assent were obtained (first session only) in accordance with the guidelines of the 1964 Declaration of Helsinki (World Medical Organization). Parents were seated in an adjacent room and asked to complete the CBCL. For the first two lab sessions, a procedure followed which involved a discussion with the parent and a battery of executive function tasks. The results of these data are not used for this report (see [39, 40], for more details). Next, children were informed that they could win a big prize for playing the EEG "computer game" and were shown two toy bins. One of the bins contained small, undesirable toys. A second, "big prize" bin contained a wide selection of more desirable, age-appropriate toys such as action figures, stuffed animals, games, and $10 gift certificates from a local music/computer game store. As part of a mood induction, the children were informed that successful performance (accumulation of points) in the game would allow them to pick a prize from the big prize bin but poor performance would limit their choice to the less desirable bin. In the EEG recording room, children were seated in front of a computer screen. The height of the seat and the angle of the chin rest were adjusted to align the children's eyes to the center of the computer screen after which children were instructed on the Go/No-go task. At the end of the session, all participants received the big prize, regardless of performance. BODY.2. METHODS.2.4. MEASURES: Questionnaire Measure. The CBCL [31] is a highly reliable and commonly used parent report of child problem behavior yielding standardized T-scores for scales such as internalizing and externalizing behavior problems. Borderline-clinical scores for these subscales constitute T-scores ranging from 60 to 63 (starting from 84th percentile) and anything above 63 is considered clinical (98th percentile). Go/No-go Task. An adapted version of a previously developed Go/No-go task was used for the present study [41]. The task was presented using E-Prime software (Psychological Software Tools, Pittsburgh, PA). Participants were required to press a button as fast as possible whenever a letter appeared on the screen (the go condition) and withhold responding whenever a letter was repeated a second time in succession (the no-go condition). Children used the index finger of their dominant hand. In order to provide the same level of challenge for all participants at all ages and to obtain a sufficient number of correct no-go trials for our analyses, a dynamic adjustment of the stimulus time was used in the task. The no-go error-rate for the task was maintained at 50% ± 10% by dynamically adjusting the stimulus duration. For example, stimulus duration was increased with each erroneous response made on no-go trials and decreased following correct no-go trials, but only when the no-go trial followed a correct go trial. For each block, accumulated points were displayed approximately every 20 trials in the center of the computer screen. Points were added for correct no-go responses and deducted for response errors on both go and no-go trials. Error feedback was provided by a red bar in the middle of the screen for 200 ms following incorrect responses, omitted responses, and late responses. To minimize intertrial interference clear time was introduced at the end of each trial during which no stimulus was presented (500 ms after response; 400 ms after no response). Children were presented with a practice block followed by three blocks of trials in a fixed order (blocks A, B, and C). In blocks A and C children gained points quite steadily. These blocks were structurally identical, each consisting of 200 trials, including 66 no-go trials (2 : 1 ratio of go to no-go), in pseudorandom sequence. In block B, children immediately began losing all (or almost all) points due to a change in the point-adjustment algorithm as well as a reduction in overall stimulus duration (we point out that this has not always been clear in previous publications from our lab; this prevents direct comparisons between block B and other blocks on performance). The loss of points was intended to induce negative emotion, such as anxiety and/or frustration. To limit the intensity and duration of children's distress, block B consisted of only 150 trials, including 40 no-go trials. Children were reminded at the beginning of the task and at the onset of each block that a high number of points were required to win the "big prize." At the end of the task, all children were told that they would receive the big prize. At the end of the last and third session, a debriefing procedure explained to the child that the second block was rigged and that it was impossible to accumulate points. Block B was not analyzed for the present study due to low trial counts (the mean no-go trial count was 9) and because it was structurally different from the other blocks. BODY.2. METHODS.2.5. EEG ANALYSIS: EEG data were collected using a 129-channel sensor net (GSN 200; Electrical Geodesics Inc., Eugene, OR). The sampling rate of the data digitization was 250 Hz and impedance values had to be below 50 KΩ before recording could begin [42]. All channels were referenced to Cz during recording. Then, data were filtered to the frequency range of 1–30 Hz off-line, using an FIR band-pass filter. The filtered data were then segmented into trial segments from 400 ms before to 1000 ms after the stimulus onset. Data cleaning followed a standard procedure where an automated algorithm was run using an EGI Netstation artifacting tool (Electrical Geodesics, Inc., Eugene, OR). Channels were automatically marked bad when they exceeded a transition threshold of 200 μv over the entire segment (max–min). After running a 20 ms moving-average smoothing algorithm, remaining eye blinks were detected when the vertical eye channels exceeded a threshold of 150 μv (max–min) within a 160 ms (moving) time window within each trial. Eye movements were detected when horizontal eye channels exceeded a threshold of 100 μv (max–min) over a 200 ms time window (HEOG and VEOG channels were recorded simultaneously with the EEG). Furthermore, each segment of the EEG was excluded from averaging if 15 or more channels were rejected. If a channel was marked bad in 25% of the trials, it was considered suspicious and marked bad for all trials. Trained research assistants, blind to the hypotheses, checked each file for accuracy. Next, bad channels were interpolated using spherical splines on a trial by trial basis. Data were subsequently average-referenced (this method most closely approaches a reference-free montage using a dense array system; [43]) and exported to MATLAB (The Mathworks, Inc.). ERP averaging and baseline correction was also conducted for the P3 component (mean of 500–800 ms, for the midline electrodes: 6, 11, and Cz). To calculate theta power, time-frequency decomposition was performed on individual no-go trial data using short-time fast Fourier transformation (FFT) with a moving Hanning window (EEGLAB, [44]). Through a logarithmic transformation, power values were in decibel (dB) units. The output frequency ranged from 1.95 Hz to 29.3 Hz divided into 15 linear-spaced frequency bands with 1.95 Hz steps. Theta power was centered around 3.9, 5.8, and 7.8 Hz. In the time domain, the FFT output was calculated for 280 time points which covered a range of 272 ms before to 872 ms after the stimulus. Baseline correction was done by subtracting prestimulus theta power (defined at −150–0 ms) from the poststimulus period (see also [23]). To prevent unbalanced trial counts across sessions from influencing the results for within-subjects analyses, all trial counts were set to the lowest value across all sessions (a random selection was made when trials had to be reduced for certain time points in order to make them equal across all time points). There was no difference in trial counts between IMPs (m = 22.5, sd = 8.2) and NIMPs (m = 22.3, sd = 7.2). No subjects had fewer than 10 trials. A grand average (GA) waveform of theta power in the 4–8 Hz range was produced to aid in determining the region and time windows of interest before any of the improver status analyses were explored. Based on previous research (see [13]) and the GA-plot, frontomidline electrodes Cz, FCz, and Fz were chosen as the sites of interest. For the time windows of interest, two peaks were distinguished from the GA-plot: an early one, ranging from 100 to 250 ms, and a later one, ranging from 250 to 650 ms. As shown in Figure 1, the early peak (100–250 ms) of frontomidline activation also features theta activation in posterior sites suggestive of occipitoparietal activation. We will interpret variation in theta power during this early period as differences in attentional resources being dedicated to binding percepts (e.g., the stimuli) with motivational states. This may be reflected in behavior by a vigilance of attention in the monitoring of the task. Although not initially planned, based on Figure 1, we will also conduct additional analyses investigating theta power at posterior sites during the earlier time window (theta power was most consistently centered around O1, Oz, and O2). The correlation between theta power in frontal and posterior sites was r(39) = .82, p < .001, suggesting communication between these regions. The later theta power peak (250–650 ms) will be interpreted as more executive in nature and can reflect attentional resources being dedicated to inhibiting the response as well as an evaluation of outcomes (see also rationale in introduction). BODY.2. METHODS.2.6. GROUP CLASSIFICATION: To classify our sample into long-term improvers (IMPs) and nonimprovers (NIMPs), we performed a median split on the change-scores from baseline to follow-up on the externalizing subscale of the CBCL. Considering our low sample size, this method ensured that we had a sizable amount of IMPs and NIMPs to conduct our planned analyses. A similar method was used to classify IMPs and NIMPs based on long-term changes in internalizing symptoms. Seventy-two percent of all participants were consistently classified as IMPs or NIMPs regardless of whether the grouping was performed by externalizing or internalizing symptom improvement. No statistically significant differences were found between the IMPs and the NIMPs in age, sex, medication, and demographic variables related to ethnicity, parental education, and family income (see also Table 1). BODY.2. METHODS.2.7. STATISTICAL ANALYSIS: Outlier analyses were performed on each of the variables whereby data points were removed from further analysis if they were more than three standard deviations from the mean. No outliers were present for the variables in the present study. Block was explored as a factor in all main neural analyses but did not appear to be significant in any of our analyses. Therefore, to reduce degrees of freedom, we have combined the data for blocks A and C. Greenhouse-Geisser corrected statistics were reported when assumptions of sphericity were violated for the Repeated Measures ANOVA. Partial eta-squared values (η 2) were computed to ascertain effect size. According to [45], partial η 2 = .01 corresponds to a small effect, partial η 2 = .10 corresponds to a medium effect, and partial η 2 = .25 represents a large effect. BODY.3. RESULTS.3.1. QUESTIONNAIRE AND BEHAVIORAL PERFORMANCE: Externalizing symptoms generally decreased for the whole sample across the three sessions as shown by a one-way within-subjects ANOVA, F(2,34) = 21.60, p < .001, partial η 2 = .56. A similar pattern could be seen for internalizing symptomatology, F(2,34) = 7.82, p = .002, partial η 2. We note that although our sample was selected for externalizing symptomatology, the mean T-score for internalizing symptomatology was well in the borderline-clinical range and almost reached standard clinical levels of impairment (m = 63.9, sd = 6.9). Table 2 shows the CBCL T-scores broken down for long-term improver status group for each of the sessions. Differences between long-term improver status were tested at each session using t-tests. To investigate changes in the behavioral performance measures during the Go/No-go task, mixed model Repeated Measures ANOVAs were run with session (3 levels: pretreatment, posttreatment, and follow-up) and group (2 levels: IMPs and NIMPs) as within- and between-subject factors, respectively. Whether groups were divided by changes in their externalizing or internalizing scores, no statistically significant interaction effects were found between IMPs and NIMPs. In these analyses, main effects of session were found for go and no-go accuracy as well as for reaction time, showing that accuracy increased and reaction times became faster (all p's < .01). Table 2 shows the behavioral performance data at each time point with differences of long-term improver status tested using t-tests. BODY.3. RESULTS.3.2. THETA POWER.3.2.1. GROUPING BY EXTERNALIZING PROBLEMS: To investigate whether IMPs, grouped by their changes in externalizing problems, were showing differences in frontomidline theta power across time compared to NIMPs, mixed model Repeated Measures ANOVAs were run with session (3 levels: pretreatment, posttreatment, and follow-up) and group (2 levels: IMPs and NIMPs) as factors. For the first theta power peak, a main effect of session was found, F(2,33) = 3.72, p = .035, partial η 2 = .18, showing a general decrease in theta power. There was no statistically significant group-by-session interaction effect (p = .17), but because we hypothesized a decrease in IMPs compared to NIMPs, we were justified in investigating the planned contrasts. As predicted, IMPs showed a significant decrease in theta power from pretreatment to follow-up (p = .002) whereas this was not the case for NIMPs (p = .56). Moreover, a marginally significant effect was also found for IMPs from pretreatment to posttreatment (p = .095). Figure 2 shows the theta power across sessions for IMPs and NIMPs for the first peak. As an additional analysis, posterior theta power was also investigated. Though patterns in the data were similar, there was no statistically significant effect group-by-session interaction effect. No statistically significant main or interaction effects were found for the second theta peak. Figure 3 shows the time-frequency plots across all time points for session and group. BODY.3. RESULTS.3.2. THETA POWER.3.2.2. GROUPING BY INTERNALIZING PROBLEMS: When children were grouped by their level of change in theta power based on internalizing problems a main effect of session was found for the first peak, F(2,33) = 3.87, p = .031, partial η 2 = .19, showing a decrease in theta power across sessions. A session-by-group interaction effect was found at the level of a trend, F(2,33) = 3.00, p = .064, partial η 2 = .19, whereby planned contrasts revealed a statistically significant decrease from pretreatment to follow-up (p = .001) for IMPs only (NIMPs, p = .81). No statistically significant effects were found for posterior theta. For the second peak, a statistically significant session-by-group interaction effect was found, F(2,33) = 4.57, p = .018, partial η 2 = .19, showing decreases in theta power for IMPs from pretreatment to follow-up (p = .001) and from posttreatment to follow-up (p = .01). Figure 4 plots the theta power across sessions for IMPs and NIMPs for the second peak. Figure 5 shows the time-frequency plots across all time points for each session and group when participants were grouped by changes in internalizing scores. Correlations were also run to test whether the changes in externalizing or internalizing scores were directly associated with changes in early and later theta power. As expected, marginally significant decreases in later theta power were associated with reductions in internalizing symptoms, r(39) = .28, p = .08. Remaining correlations did not reach standard levels of statistical significance. To show that results of theta were not solely due to relatively slow ERP components such as the P3, the main analyses were repeated with the P3 ERP component. No significant interaction effects were found for the externalizing or internalizing grouping analyses, suggesting that theta power adds unique variance over and above the ERP. BODY.4. DISCUSSION: We set out to test whether long-term behavioral improvement would be reflected in changes in neural theta power in children with DBP who participated in a treatment aimed at improving self-regulation. Our findings show reductions in neural activation only for those children who showed long-term improvement in their externalizing symptoms. These findings are in line with previous studies from our lab investigating neural changes directly after treatment [13, 28] and suggest increased neural efficiency when children improve. That these effects occurred during early theta power may suggest that the brains of long-term improvers have a more efficient communication between occipitoparietal regions involved in perception and the frontolimbic systems mediating motivation and monitoring. And that these effects occurred in the absence of behavioral performance differences suggests increased neural efficiency as a similar performance is achieved with an equal amount of neural resources. These findings add to the extant literature as previous studies, using ERP, have typically found changes at a later stage of processing [13]. When participants were grouped on the basis of long-term improvement in their internalizing symptomatology, a similar but stronger pattern was found for long-term improvers. Next to improvers showing decreased theta power activation during the early phase of processing, we also found decreases during later theta, suggesting increased neural efficiency in more executive processing related to inhibition and evaluation. The finding that theta power was more sensitive to changes in internalizing than externalizing symptoms suggests that theta power may be more strongly connected to underlying effects of anxiety. We think that this latter finding has consequences for the characterization of our sample as well as the explanation of our effects. We interpret our neural effects in long-term IMPs to mean an increased efficiency in neural systems mediating a hypervigilant and overcontrolling style of self-regulation. There is a growing consensus that comorbid anxiety in children with DBP is not just an auxiliary phenomenon but that it may drive and maintain externalizing problems (see [30], for overview). Anxiety problems are characterized by a continuous hypervigilance to threat, excessive worry, and an overcontrolled style of self-regulation [11, 46]. The externalizing problem behaviors in our sample could be explained through a depleted self-regulatory capacity due to prolonged anxiety which could result in aggressive outbursts (e.g., consistent with Baumeister's ego-depletion model, [47]). At a neural level, the heightened theta power may reflect overactive frontolimbic circuits interacting at an early stage with perceptual processes which could underlie the threat-focused attentional biases [48, 49] and at a later stage with executive processes underlying the inefficient inhibitory control and evaluative processes common in anxiety [11, 50]. The reduction of theta power after successful treatment could reflect normalization in the overactive frontolimbic systems that underlie these hypervigilant states and the overcontrolled style of self-regulation. We suggest, at a behavioral level, that this would relieve some of the tension and rigidity these children bring to social situations and, according to the anxiety hypothesis of aggression [30, 51], lead to fewer bouts of aggression and other externalizing problem behaviors. BODY.5. IMPLICATIONS: These results, particularly if they are replicated by independent groups, have implications for the characterization and treatment of children with DBP. The finding that internalizing symptoms, as opposed to externalizing symptoms, seemed more sensitive to long-lasting neural changes reinforces the notion that anxiety may lie at the root of DBP. This may suggest that DBP treatment should focus on targeting anxiety as that may more effectively relieve children of the underlying problems they experience. We are also a step closer to finding reliable neural markers of treatment efficacy. Such measures directly tapping into the neural systems underlying the cognitive processes of self-regulation could, in the future, complement traditional indices of treatment success. We suggest a multimethod approach, where a set of indices based on ERP [13, 14], source localization [28, 52], psychophysiology [39, 40], and power [23] may together form a powerful toolkit for predicting and understanding treatment success. BODY.6. LIMITATIONS AND CONSIDERATIONS: A number of considerations apply to this study. First, the current study was not intended nor conducted as a randomized controlled trial (RCT). Our primary interest was to look at individual differences in responses to treatment rather than in assessing the impact of the model of treatment delivery on outcomes. The clinical population thus functioned as their own "control." This meant, however, that caution must be taken when drawing conclusions about treatment efficacy and statements about what caused these children to show improvement in the present study. We would also like to point out that it was not possible, due to the low sample size, to get reliable data on maintenance effects, that is, taking only the improvers at post and investigating only their progress a year later. A future study, using a much larger sample size and an RCT design, would be able to more definitively attribute changes to treatment and provide finer delineation, at neural and behavioral level, of the treatment effects across time. Second, a limitation of the study is the lack of formal clinical diagnoses in accordance with DSM criteria for conduct disorder, oppositional defiant disorder, ADHD, and/or mood disorders. This was a limitation that was hard to remedy for practical reasons. To ensure that our results were relevant to "real world" practice, the study was conducted in partnership with community-based child and family agencies that implement evidence-based interventions. As a result, the study was constrained to the protocols that these agencies already had in place. Children referred for treatment to these agencies did not regularly undergo full psychiatric assessments. Instead, a number of standardized measures were used to assess children's and parents' clinically relevant symptoms and functioning. Third, we did not have complete verifiable information on treatment programs that participants may have signed up for in the year between our second and third lab sessions. This information could have helped interpret what made treatment successful. Regardless, the strength of the current design is geared towards investigating what changes with successful compared to unsuccessful outcomes and is less suitable for determining the causal reasons for that change. Last, although we have focused on theta power, we acknowledge that other oscillations could also play a role in the neural plasticity of self-regulatory processes. In fact, researchers have also suggested these different oscillations are not independent but interact with each other in reciprocal ways [53, 54]. We believe such approaches, incorporating multiple bands and their interactions, are a fruitful approach for future studies. BODY.7. CONCLUSION: To the best of our knowledge, this is the only intervention study to date investigating neural correlates of self-regulation in children with DBP a year after treatment ended. The study provides preliminary support for the idea that long-term changes can be found in frontomidline theta power activation patterns mediating behavior underlying self-regulation. The results may help provide more comprehensive understanding of children with DBP and work towards neural indices that aid with the diagnosis and efficacy of treatment.

TITLE: Effectiveness of Golimumab in Clinical Management of Patients with Rheumatoid Arthritis ABSTRACT.BACKGROUND AND OBJECTIVES: Limited data are available regarding the use of golimumab (100 mg) every 4 weeks, with or without methotrexate (MTX). The aim of this retrospective analysis was to evaluate the effectiveness and safety of golimumab following usual clinical practice in Japanese patients with rheumatoid arthritis (RA) according to the recommendations given in the Japanese package insert. ABSTRACT.PATIENTS AND METHODS: Japanese RA patients with moderate-to-high disease activity, according to the 28-joint disease activity score based on C-reactive protein (DAS28-CRP) criteria, despite treatment with MTX or another biological agent, were enrolled. Patients were assigned to 50 mg golimumab plus MTX or 100 mg golimumab monotherapy every 4 weeks for 24 weeks. All patients were given MTX if it was not contraindicated. The primary endpoint was the proportion of patients achieving clinical remission (defined as a DAS28-CRP <2.3 or a simplified disease activity index [SDAI] score <3.3) at 24 weeks. ABSTRACT.RESULTS: Most patients received combined 50 mg golimumab plus MTX (41/43). In these patients, the primary endpoint, clinical remission, was attained in 83 % of patients according to DAS28-CRP criteria (p < 0.001) and 69 % according to SDAI criteria (p < 0.001) by week 24. Adverse events were reported in 11.6 % of patients receiving golimumab. ABSTRACT.CONCLUSIONS: Golimumab (50 mg) plus MTX effectively reduced the signs and symptoms of RA and was generally well tolerated in patients with an inadequate response to MTX and other biological agents. BODY.INTRODUCTION: In recent years, methotrexate (MTX) therapy at high dose levels and tumor necrosis factor (TNF) inhibitor therapy have been applied to treatment of rheumatoid arthritis (RA). Anti-TNF therapy, either alone or in combination with MTX (apart from infliximab, which should only be used in combination with MTX), is recommended in patients with active RA with inadequate response to MTX or another disease-modifying antirheumatic drug (DMARD) or combination of DMARDs or another anti-TNF agent [1–3]. These new methods of treatment are expected to yield not only the alleviation of disease activity, but also structural improvement of the affected joints and improvement in daily life for patients. The three most widely used anti-TNF agents in Japan are infliximab, etanercept, and adalimumab, and numerous reports have been published on these agents [4–6]. Golimumab (GLM), a new human anti-TNF antibody agent created using transgenic mice, has been shown to exert effectiveness comparable to that of existing anti-TNF antibody agents when injected subcutaneously at 4-week intervals [7–13]. This drug was introduced in Japan in September 2011, thus providing a new treatment option for Japanese patients with RA. GLM can be administered either as monotherapy at a dosage of 100 mg or in combination with MTX at dosages of 50 or 100 mg every 4 weeks [14]. It is indicated not only in patients who have not previously received treatment with biological agents but also in patients who have experienced difficulties with infliximab or adalimumab therapy; for example, problems with neutralizing antibodies. In Japan, there have been no published reports on the use of GLM in clinical practice to date. When patients are enrolled into clinical studies, age and disease activity are often taken into account to ensure safety and continued use of the investigational agent, so the populations studied differ from the population managed in real life. Therefore, this analysis evaluates the use of GLM in patients with RA receiving real-life clinical care at our clinic. BODY.METHODS.SUBJECTS: This retrospective analysis included patients with baseline moderate-to-high disease activity according to a 28-joint disease activity score based on C-reactive protein (DAS28-CRP) >3.2 despite treatment with MTX or another biological agent. Patients had RA according to American College of Rheumatology (ACR) criteria [15] and had received GLM treatment as outpatients at our hospital, the Shono Rheumatology Clinic, Fukuoka, Japan, between September 2011 and April 2012. Consent in writing was obtained from each patient in advance. BODY.METHODS.TREATMENT: Patients received combination therapy with GLM plus MTX, with GLM administered at a dose of 50 mg or 100 mg every 4 weeks plus MTX administered at a dose of up to 8 mg/week; or GLM monotherapy, with GLM administered at 100 mg every 4 weeks, for a total of 24 weeks. All patients were prescribed MTX if it was not contraindicated. GLM was administered subcutaneously in accordance with the Japanese package insert [14]. BODY.METHODS.OUTCOME MEASURES: The primary endpoint of this retrospective analysis of effectiveness was to evaluate the proportion of patients achieving remission defined as a DAS28-CRP <2.3 or a simplified disease activity index (SDAI) score <3.3. Mean changes in the DAS28-CRP from baseline to 4 weeks were also evaluated. Safety was evaluated on the basis of adverse events and laboratory test data. For each parameter, additional stratified analyses were conducted, dividing the patients into two groups; that is, bio-naïve patients who had not received biological agents prior to receiving GLM, and patients who had received prior biological agents (i.e., those switching from other biological agents to GLM). BODY.METHODS.STATISTICAL ANALYSIS: All data were included for efficacy and safety analyses. The last observation carried forward (LOCF) method was used to allow for missing data. Comparison of groups was performed using the Student's t test with statistical significance set at p < 0.05. BODY.RESULTS.PATIENT BASELINE DEMOGRAPHICS AND CLINICAL CHARACTERISTICS: Of all patients studied, 18 were bio-naïve cases and 25 had received prior biological agents, including infliximab (n = 4), etanercept (n = 10), adalimumab (n = 6), and tocilizumab (n = 5). Of the 25 patients previously treated with biological agents, 19 had received one prior biological agent and 6 had received two or more agents. Table 1 shows the baseline demographics and disease characteristics of the patients enrolled into the study. Patient characteristics were generally well balanced between bio-naïve patients and those who had received a prior biological agent, except the proportion of women was slightly greater (96.0 vs 83.3 %) and disease duration was slightly longer (122.6 vs 105.3 months) in the bio-switching group.Table 1Baseline demographics and disease characteristics in bio-naïve patients and patients who had received prior biological agentsTotal (n = 43)Bio-naïve (n = 18)Prior biologicals (n = 25)Sex [n (%)] Female39 (90.7)15 (83.3)24 (96.0) Male4 (9.3)3 (16.7)1 (4.0)Age [years]59.1 (32–79)55.8 (37–79)61.4 (32–76)Disease duration [months]115.3 (7–708)105.3 (7–708)122.6 (12–252)DAS28-CRP4.14 (1.28–7.04)4.16 (2.61–6.39)4.12 (1.28–7.04)SDAI22.2 (2.81–62.30)22.30 (6.70–56.29)22.20 (2.81–62.30)CDAI20.92 (2.50–60.90)20.94 (6.50–56.00)20.90 (2.50–60.90)Tender joint count [0–68]6.3 (0–24)6.7 (1–22)6.0 (0–24)Swollen joint count [0–68]5.9 (0–22)5.4 (0–18)4.8 (0–22)mHAQ [0–24]0.65 (0–2)0.44 (0–2)0.72 (0–2)CRP [mg/dL]1.5 (0.1–13.5)1.6 (0.1–13.5)1.4 (0.1–8.4)RF positive [n (%)]34 (79.0)13 (72.2)21 (84.0)ACPA positive [n (%)]22 (51.1)11 (61.1)11 (44.0)All values are presented as means with ranges given in parentheses unless specified otherwiseACPA anti-citrullinated protein autoantibody, CDAI clinical disease activity index, CRP C-reactive protein, DAS28-CRP disease activity score 28 based on C-reactive protein, mHAQ modified health assessment questionnaire; RF rheumatoid factor, SDAI simplified disease activity index BODY.RESULTS.INTERVENTIONS: In total, 41 patients received GLM at a dose of 50 mg every 4 weeks in combination with MTX (mean dose 6.23 mg/week) and 2 patients received GLM monotherapy at a dose of 100 mg every 4 weeks. Four patients were unsatisfied with the inconvenience of self-injection and injection pain of prior biological treatment, despite sufficient clinical response; therefore, those patients in clinical remission at baseline were switched to GLM treatment as a result of patients' wishes. Of the 43 patients, 35 completed the 24-week treatment period. BODY.RESULTS.EFFECTIVENESS: Remission rates, defined as the proportion of patients achieving a DAS28-CRP <2.3 and an SDAI score <3.3, steadily improved over the course of treatment with GLM (Fig. 1). After 8 weeks of treatment, 71.4 % of patients achieved a DAS28-CRP <2.3 and 50.0 % achieved an SDAI score <3.3. After 8 weeks of treatment, the DAS28-CRP and SDAI remission rates were higher in patients who had not received prior biological agents versus those who had (55.6 vs 50.0 % and 61.1 vs 41.7 %, respectively).Fig. 1Remission rate in 43 patients with rheumatoid arthritis treated with golimumab alone or in combination with methotrexate. Remission was defined as a 28-joint disease activity score based on C-reactive protein (DAS28-CRP) of <2.3 or a simplified disease activity index (SDAI) score of <3.3. DAS28-CRP remission and DAS28-CRP plus SDAI remission (ALL) are shown. BL baseline, W weeks The mean DAS28-CRP 4 weeks after the start of treatment was significantly improved compared with the pretreatment score [mean DAS28-CRP at week 4 = 1.80 vs 4.14 (range 1.28–7.04) at baseline; p < 0.001]. Improvements in DAS28-CRP and SDAI scores throughout the treatment period were similar in bio-naïve patients and those who had received prior biological agents (Figs. 2, 3). Changes in DAS28-CRP and SDAI scores at 4 and 8 weeks were statistically significant compared with baseline in both bio-naïve patients and those who had received prior biological agents (all p < 0.001).Fig. 2Changes in mean 28-joint disease activity score based on C-reactive protein (DAS28-CRP) in bio-naïve or previously treated patients with rheumatoid arthritis receiving golimumab alone or in combination with methotrexate. The dashed line represents the defined remission cutoff value of 2.3. BL baseline, W weeksFig. 3Changes in mean simplified disease activity index (SDAI) score in bio-naïve or previously treated patients with rheumatoid arthritis receiving golimumab alone or in combination with methotrexate. The dashed line represents the defined remission cutoff value of 3.3. BL baseline, W weeks BODY.RESULTS.TOLERABILITY: GLM was generally well tolerated with no unexpected safety issues observed. Adverse events (shown in Table 2) were reported in five patients, most of whom were receiving GLM (50 mg) in combination with MTX (6 or 8 mg). Two patients reported fractures (one ankle and one femur); one patient was hospitalized due to renal impairment, chest pain, dyspnea, bronchial asthma, acute upper respiratory tract inflammation, and bronchitis; one patient (treated with GLM monotherapy at 100 mg) experienced venous thromboembolism and lower limb edema; and one patient reported renal impairment, hepatic function, and nephrogenic anemia. Consistent with other GLM safety data reported in Japanese clinical trials, no unknown adverse event was reported in this clinical analysis. All adverse events were resolved with treatment.Table 2Adverse events and course reported in five patients with rheumatoid arthritis treated with golimumab every 4 weeks for 24 weeksCaseAdverse eventsCourse1Ankle fractureTreated by another clinic2Femur fractureTreated by another clinic3Renal impairment, chest pain, dyspnea, asthma bronchial, acute upper respiratory tract inflammation, bronchitisRecovered as inpatient4Embolism venous, edema lower limbResolved, in remission5Renal impairment, hepatic function disorder, nephrogenic anemiaRecovered BODY.DISCUSSION: The present analysis in Japanese patients with RA in real-life clinical care revealed high effectiveness and safety of GLM alone or in combination with MTX, with significant improvements in mean DAS28-CRP and SDAI scores observed in bio-naïve patients 16 weeks after the start of treatment (p < 0.001). The reason for the high remission rate was considered to be the difference in average patient body weight between western countries and Japan (75 vs 50 kg, respectively). These effectiveness data are consistent with efficacy data from clinical studies [7–10, 12, 13, 16]. Most GLM studies are designed to permit rescue of patients at 16 weeks with alternative pharmacological therapy for those meeting the nonresponse criteria for early escape [8–10, 12, 13]. Similar to the GO-FORTH study [13], our clinical analysis involved patients treated with MTX at 8 mg/week, which is the maximum dose approved in Japan at the time that the patients were receiving treatment [17]. This is lower than the current recommended MTX dose in RA [3, 14, 18] and lower than the MTX dose used in combination with GLM in other published studies [7, 9, 10]. Despite the low doses of MTX used, overall remission rates with GLM were high. Evidence suggests that, for patients receiving MTX who fail to achieve clinical remission based on SDAI or disease activity score scales, increasing the MTX dose needs to be considered at this time [17]. In the GO-FORWARD study, GLM was shown to be effective in patients who showed lower responses or who were refractory to prior MTX therapy [9, 10]. In the present retrospective analysis, manifestation of effectiveness appeared to be delayed in the bio-switching group compared with the bio-naïve group, suggesting the necessity for longer follow-up when evaluating effectiveness in patients who switch between biological therapies. In a post-hoc analysis of the effectiveness in relation to the reasons for switching, the effectiveness did not differ significantly according to the reason (data not shown). This suggests that patients undergoing switching will respond to this therapy, regardless of the reasons for switching. This supports findings by Smolen et al. [12] that switching from other anti-TNF agents to GLM was effective regardless of the reasons for switching, indicating that GLM can serve as the second anti-TNF agent when patients are switched from another TNF agent. Of the five anti-TNF agents available, including certolizumab pegol, all have different affinities to TNF-α; therefore, switching from one anti-TNF agent to another is likely to be effective. Expression of antibodies to anti-TNF antibody agents such as infliximab, adalimumab, and certolizumab pegol monotherapy is not uncommon; however, incidences of anti-GLM antibodies in the GO-FORWARD [9] and GO-FORTH [13] studies were remarkably low. Because GLM is prepared by the transgenic mouse technique, it is an antibody with high affinity for the antigen [19], which means that formation of unstable proteins or aggregations, which can serve as immunogens, is unlikely. Studies of GLM (100 mg) monotherapy were conducted in Caucasian and South American countries in GO-FORWARD [9, 10] and in Japan in GO-FORTH [13] and GO-MONO [16], and showed that GLM is an appropriate biological agent for preventing the loss of effectiveness in Caucasian, South American, and Japanese populations receiving long-term RA treatment [9, 10, 13, 16]. As a result of findings from the GO-FORTH [13] and GO-MONO [16] studies, in addition to the 50-mg dose, GLM (100 mg) every 4 weeks—as monotherapy and in combination with MTX—has been approved in Japan. Further studies at this dose level in larger numbers of patients are necessary. Apart from the usual limitations relating to observational data and retrospective analyses, particularly with regard to selection and enrolment bias, a major limitation of our analysis is the small patient numbers, especially for patients receiving GLM (100 mg) monotherapy. In addition, evaluation of levels of anti-GLM antibodies and the effects of GLM on structural joint damage in this real-life setting would have been useful; however, this was not evaluated in the original study. Despite suppression of joint destruction being an important factor in improving the quality of life of patients with RA, the number of published reports evaluating this effect in patients treated with GLM is small, because of its relatively recent development. Emery et al. [7] reported that treatment with GLM suppressed joint destruction significantly 52 weeks after the start of treatment, and further long-term observation is needed. However, due to the short follow-up period in our analysis, such observation was not possible. In the present analysis, there were no serious adverse events arising from the use of GLM, although deterioration in renal function was reported in two patients. An association with the development of malignant tumors has been suggested with GLM, and further clinical confirmation is warranted [20]. However, long-term observation of the patients in our study is needed before any definite conclusions can be made. It is important to select a type of biological agent taking into account the lifestyle of individual patients. Despite reported problems with pain and administration site reactions, subcutaneous injection of drugs offers greater convenience than intravenous infusion, which requires physical immobilization for many hours at a hospital, and a longer dosing interval is also advantageous. Because GLM contains only small amounts of stimulating acidic additives and requires only a small volume of dosing solution, reported incidences of pain and administration site reactions are low [14]. BODY.CONCLUSION: In the present analysis, GLM plus MTX or GLM monotherapy used in clinical practice in Japanese patients with RA was confirmed to have high effectiveness and safety, comparable with existing biological agents. Thus, we conclude that GLM is a promising new alternative for the treatment of RA in Japanese patients showing poor response, those in whom the use of other biological agents is contraindicated, and cases where the use of MTX in combination with biological agents is difficult.

TITLE: Stroke Outcomes in Thai Elderly Patients Treated with and without Intravenous Thrombolysis ABSTRACT: Higher mortality was found in very old patients with acute ischemic stroke treated with intravenous recombinant tissue-plasminogen activator (rtPA) as compared to younger patients. The benefit of thrombolytic treatment in this particular subgroup is still a subject of debate. The purpose of this study was to compare stroke outcomes in Thai patients aged over 70 years treated with and without intravenous rtPA. This was a retrospective review of sequential cases and was not a randomized controlled study. One-hundred and five patients with acute ischemic stroke aged over 70 years who were treated with intravenous rtPA and 105 patients without rtPA treatment (control group) were included in the study. Patients' base-line characteristics and study outcomes of interest were compared. There were significant differences in the base-line characteristics of the two groups. However, for the subgroup of patients aged over 80 years, these characteristics were similar. Those who were treated with intravenous rtPA had a higher rate of favorable outcomes (40% vs 16%; P=0.137) and a lower rate of mortality (22% vs 44%; P=0.128) than patients who did not receive rtPA treatment. In well-matched subgroups of patients aged over 80 years, our retrospective review revealed there was a trend of a higher rate of favorable outcome and lower mortality in patients receiving rtPA treatment. More study is needed to further confirm the suggested benefit of thrombolysis in Asian octogenarian acute stroke patients. BODY.INTRODUCTION: Although intravenous thrombolysis has become a standard treatment for acute ischemic stroke in eligible patients, the recommendations for treating patients aged over 80 years are still a subject of debate. Many studies have revealed a higher mortality rate and lower favorable outcome rate in patients aged over 80 years. In some studies, the rate of symptomatic intracerebral hemorrhage (ICH) was higher in patients aged over 80 years while in others it was similar to that observed in younger patients.1-6 However, a large study and a meta-analysis have both recently shown that older patients benefit from treatment at least as much as younger patients.7,8 Most of the data are from Western countries. Menon et al. assessed risk score for intracranial hemorrhage after intravenous thrombolytic treatment in 10,242 patients with acute ischemic stroke and found that Asian race was one of the independent risk predictors.9 Data in Asian patients may be different from that in Western populations. Thrombolytic studies from some Asian countries, such as Taiwan and Vietnam, have not included patients aged over 80 years.10,11 Previous studies in Thai patients showed that older age (≥70 years old) was inversely associated with early improvement. Significantly higher rates of mortality and symptomatic ICH were also found in patients aged 70 years or over as compared with younger patients after treatment with recombinant tissue-plasminogen activator (rtPA).12,13 However, whether treatment with thrombolysis will have a greater beneficial effect on outcomes than treatment without rtPA in Thai elderly patients is unknown. The purpose of this study was to compare stroke outcomes in patients aged over 70 years treated with and without intravenous thrombolysis. BODY.MATERIALS AND METHODS: This was a case-control study. The number of patients on each arm was calculated by the following formula (equal to 82): where: P1=0.40, P0=0.20, power of the test 80%, confidence level 95%. One-hundred and five patients aged over 70 years with acute ischemic stroke treated with intravenous rtPA at Thammasat University Hospital, Thailand, between June 2007 and January 2011 were identified from the Thammasat Stroke Registry and were included in the study. One-hundred and five patients aged over 70 years who were seen from May 2010 to January 2011, and who were not treated with intravenous thrombolysis, were included as control. Intravenous rtPA was prescribed for acute ischemic stroke patients within 3 h of onset if there were no contraindications. However, after the publication of the European Cooperative Acute Stroke Study III (ECASS III), and the recommendations with regard to widening the time window for the treatment of acute ischemic stroke with intravenous rtPA issued by the American Heart Association/American Stroke Association,14,15 we extended the time window for treatment with rtPA to 4.5 h. Most contraindications were the same as in the guidelines for treatment of acute ischemic stroke from the American Heart Association/American Stroke Association.14 However, older age (>80 years old) was not an exclusion criterion. Patients with high blood pressure (systolic blood pressure >185 mmHg or diastolic blood pressure >110 mmHg) were not excluded as long as blood pressure could be controlled by intravenous nicardipine (as defined by achieving target systolic blood pressure <185 mmHg and diastolic blood pressure <110 mmHg) before administration of rtPA. Abnormal findings from brain computed tomography (CT) including: i) hyperdense lesions suggestive of bleeding; and ii) hypodense lesions accounting for more than one-third of the middle cerebral artery (MCA) distribution were also contraindications for thrombolysis. Patients who were treated with intravenous rtPA (0.9 mg/kg) were admitted to an intensive care unit or stroke unit to monitor and control blood pressure with a clinical follow up for the first 24 h. The severity of the stroke was evaluated using the National Institutes of Health Stroke Scale (NIHSS) by the same doctors before and after receiving rtPA. Another CT brain scan was performed within 36 h in all patients who received rtPA. However, follow-up brain CT was not routinely performed in all of those patients who were not treated with intravenous thrombolysis. The modified Rankin scale (mRS) was used to assess outcomes of the patients three months after the stroke onset. Symptomatic ICH was also classified by the National Institute of Neurological Disorders (NINDS) stroke rtPA criterion, i.e. hemorrhage associated with worsening of 1 point or more in the NIHSS score.16 Early improvement was defined as having complete recovery or marked clinical improvement (decrease in NIHSS from baseline >8 points) 24 h after admission. The base-line characteristics of patients, including age, gender, cardiovascular risk factors, blood glucose at presentation, platelet count, prothrombin time, severity of stroke, and stroke subtypes were studied. The measured outcome variables of this study were early improvement at 24 h, symptomatic intracerebral hemorrhage, favorable outcome (mRS 0.1) and death at three months. Patients' demographics, vascular risk factors and measured outcome variables were compared between those who were treated with and those without intravenous thrombolysis; an independent-samples t-test for continuous variables and the χ2 test for dichotomous variables were used. The data are presented as means for continuous variables and percentage (number) for dichotomous variables. This study was approved by the human ethics committee of the Faculty of Medicine, Thammasat University, Thailand. BODY.RESULTS: One-hundred and five patients aged over 70 years were not treated with intravenous rtPA during the study period; of these, 49 patients presented within 4.5 h after stroke onset. The reasons for not prescribing rtPA were: delays in the intervention process, i.e. late presentation, delay in laboratory or CT process (n=14, 28.6%); excessively mild stroke severity, i.e. NIHSS under 4 (n=12, 24.5%); rapid improvement (n=5, 10.2%); abnormal CT findings i.e. hypodense lesion over one-third of the MCA distribution (n=16, 32.7%); recent major surgery (n=1, 2%); and history of intracerebral hemorrhage, ICH (n=1, 2%). Follow-up CT or magnetic resonance imaging (MRI) was performed in 48 patients who did not receive rtPA. The base-line characteristics of the patients with and those without rtPA treatment are presented in Table 1. There were significant differences in base-line characteristics associated with older age (78 vs 76; P=0.03), milder stroke (10 vs 13; P=0.001), lower blood sugar at presentation (116 vs 135; P=0.026) and reduced frequency of cardioembolic stroke (25% vs 42%; P=0.009) in patients without the rtPA treatment as compared with those with the rtPA treatment. However, there was no significant difference in the rates of favorable outcomes (41% vs 37%; P=0.56) or mortality (25% vs 22%; P=0.69) in association with a marginally higher rate of symptomatic intracerebral hemorrhage (4% vs 14%; P=0.09) in those receiving rtPA treatment. A significantly higher rate of early improvement at 24 h was found in patients treated with rtPA (4% vs 23%; P=0.001). Univariate analysis and multiple logistical regression analysis were carried out to look for the factors associated with early improvement. Only rtPA treatment was related to early improvement (OR 14.1, 95% CI: 1.6-124.7) after adjustment for gender, stroke subtypes, hyperlipidemia, coronary artery disease, atrial fibrillation, antithrombotic treatment and symptomatic intracerebral hemorrhage. Stroke outcomes at three months as measured by the modified Rankin Scale (mRS) are presented in Figure 1. There were significant differences in some base-line characteristics in the subgroup of patients aged over 80 years, with a greater history of hypertension and higher mean age, milder stroke, and a reduced risk of cardioembolic stroke in patients not receiving rtPA treatment (Table 1). Patients (>80 years old) treated with intravenous rtPA had a higher rate of favorable outcomes (40% vs 16%; P=0.137) and a lower rate of mortality (22% vs 44%; P=0.128) than patients not receiving rtPA treatment. BODY.DISCUSSION: The number of elderly patients is increasing in most countries and consequently strokes in the very old (>80 years) subgroup occur more often; approximately one-third of all stroke patients are in this subgroup.17,18 Mortality rates and non-dependency were higher in patients aged over 80 years as compared with the younger patients. Higher 1-month mortality rates were 31-34.6% in the very old patients (>80 years) as compared with the younger patients (13.4-16.7%); one study reported a 1-year mortality rate of 51.6%.19,20 Disability after stroke (mRS ≥3) was reported in 78% of the very old patients who survived.20 Our study showed that the 3-month mortality rate was 44% in patients aged over 80 years who did not receive rtPA treatment. Causes of high mortality may result from the reduced utilization of health care resources or the comorbidity and high proportion of atrial fibrillation reported in association with stroke.18,19,21 Comorbidity, cognitive impairment, marital status, absence of caregivers and a lack of motivation that reduced the effectiveness of rehabilitation programs were potential contributors to the high level of disability observed in stroke survivors.22-25 Thrombolytic treatment in very old patients with acute ischemic stroke is still a subject of debate. The National Institute of Neurological Disorders and Stroke (NINDS) included all age ranges of the patients whereas the European Cooperative Acute Stroke Study (ECASS) excluded patients aged over 80 years.14,16 Thus, the European Medicine Evaluation Agency has not approved thrombolysis with alteplase for use in patients aged over 80 years.26 A systemic review and meta-analysis included 13 studies comparing outcomes after intravenous thrombolysis among 764 elderly patients (age ≥80 years) and 2792 patients under 80 years of age. Elderly patients achieved less favorable outcomes (OR 0.49, 95% CI: 0.40-0.61) and showed higher mortality rates (OR 2.77, 95% CI: 2.25-3.40) but not significantly higher symptomatic ICH rates (OR 1.31, 95% CI: 0.93-1.84) as compared with patients under 80 years old.27 Ford et al. studied outcomes and symptomatic ICH rates in 19,411 patients aged 80 years or under and 1831 patients aged over 80 years in the Safe Implementation of Treatment in Stroke - International Stroke Thrombolysis Register (SITS-ISTR).3 Here patients over 80 years of age had a higher mortality rate (30% vs 12%, OR 1.53, 95% CI: 1.43-1.65) and were less independent (35% vs 57%, OR 0.73, 95% CI: 0.68-0.78). There was a nonsignificant increase in the symptomatic ICH rate according to NINDS criteria in the older subgroup (9.5% vs 7.8%, OR 0.96, 95% CI: 0.87-1.06). Mishra et al. assessed the effect of age in response to alteplase by comparing 23,334 patients who received thrombolysis from the SITS-ISTR (during 2002-2009) with a control group made up of 6166 patients who participated in VISTA neuroprotection trials (during 1998-2007) and were not treated with rtPA.28 Among the subgroup of patients aged over 80 years, 2235 and 1237 patients were treated with and without rtPA, respectively. As compared with a control subgroup, very old patients (>80 years) treated with rtPA had a higher rate of favorable outcomes (mRS 0-2) (OR 2.1, 95% CI: 1.7-2.5) and a marginally lower mortality rate (OR 0.89, 95% CI: 0.76-1.0). The third international stroke trial (IST-3) compared the efficacy and safety of rtPA vs placebo, with a sample size of 3035 patients and including 1617 patients aged over 80 years.7 In a subgroup of patients aged over 80 years, the rate of favorable outcome was higher in those receiving rtPA treatment (27.3% vs 23.5%, OR 1.35, 95% CI: 0.97-1.88) and this did not differ significantly from the younger patients. Our study showed non-significant differences in the rates of favorable outcomes, symptomatic intracerebral hemorrhages and mortality in patients aged over 70 years with or without thrombolytic treatment. However, there were significant differences in several base-line characteristics between the groups. Older age, severe stroke, high blood glucose, cardioembolic stroke had been reported to be the independent factors of poor outcomes and death. Thus, it is not possible to conclude that thrombolytic treatment is ineffective in aging patients. Differences in favorable outcomes and death between those with and without rtPA treatment were obvious in subgroups of patients aged over 80 years. Patients with the rtPA treatment had a higher rate of favorable outcomes (40% vs 16%; P=0.137) and a lower rate of mortality (22% vs 44%; P=0.128) compared with those without the rtPA treatment. However, the difference did not reach statistical significance, which may largely be due to the small number of patients in this subgroup. The mechanism of early recovery or improvement is not clearly understood. A transcranial Doppler (TCD) study in patients treated with rtPA found that the timing of arterial recanalization, as determined by TCD, correlated with early clinical recovery from stroke.29 Several studies reported a similar proportion of successful recanalization in patients aged over 80 years to that achieved in younger patients.3 Our study showed that early improvement within 24 h after stroke onset was found more frequently in patients who received the rtPA treatment, which may represent a higher rate of recanalization. BODY.CONCLUSIONS: Our study has some limitations. Firstly, this was a retrospective review of sequential cases and was not a randomized controlled study. Non-thrombolytic patients (>70 years of age) were classified by the duration of time required to reach similar numbers to those achieved in patients treated with rtPA. Significant differences in base-line characteristics of the two groups were noted. The differences in stroke severity between the case and control groups may represent more rapid stroke awareness in patients with more severe stroke, as this subgroup of patients was more likely to receive thrombolytic treatment. However, for the subgroup of the patients aged over 80 years, characteristics were fairly similar. Second, a follow-up brain CT or MRI was not performed in some of the patients who did not receive rtPA treatment; therefore, the rate of asymptomatic intracerebral hemorrhage may be underestimated. This is the first study in Thailand comparing stroke outcomes in aging patients with and without rtPA treatment. The study showed that the very old patients receiving rtPA did not have poorer outcomes. Further randomized controlled studies are still needed to confirm the suggested benefit of thrombolysis in aging Asian patients. In conclusion, in subgroups of patients aged over 80 years, this retrospective review revealed that there was a trend of higher rate of favorable outcome and lower mortality in patients receiving rtPA treatment.

TITLE: Repeated testing improves achievement in a blended learning approach for risk competence training of medical students: results of a randomized controlled trial ABSTRACT.BACKGROUND: Adequate estimation and communication of risks is a critical competence of physicians. Due to an evident lack of these competences, effective training addressing risk competence during medical education is needed. Test-enhanced learning has been shown to produce marked effects on achievements. This study aimed to investigate the effect of repeated tests implemented on top of a blended learning program for risk competence. ABSTRACT.METHODS: We introduced a blended-learning curriculum for risk estimation and risk communication based on a set of operationalized learning objectives, which was integrated into a mandatory course "Evidence-based Medicine" for third-year students. A randomized controlled trial addressed the effect of repeated testing on achievement as measured by the students' pre- and post-training score (nine multiple-choice items). Basic numeracy and statistical literacy were assessed at baseline. Analysis relied on descriptive statistics (histograms, box plots, scatter plots, and summary of descriptive measures), bootstrapped confidence intervals, analysis of covariance (ANCOVA), and effect sizes (Cohen's d, r) based on adjusted means and standard deviations. ABSTRACT.RESULTS: All of the 114 students enrolled in the course consented to take part in the study and were assigned to either the intervention or control group (both: n = 57) by balanced randomization. Five participants dropped out due to non-compliance (control: 4, intervention: 1). Both groups profited considerably from the program in general (Cohen's d for overall pre vs. post scores: 2.61). Repeated testing yielded an additional positive effect: while the covariate (baseline score) exhibits no relation to the post-intervention score, F(1, 106) = 2.88, p > .05, there was a significant effect of the intervention (repeated tests scenario) on learning achievement, F(1106) = 12.72, p < .05, d = .94, r = .42 (95% CI: [.26, .57]). However, in the subgroup of participants with a high initial numeracy score no similar effect could be observed. ABSTRACT.CONCLUSION: Dedicated training can improve relevant components of risk competence of medical students. An already promising overall effect of the blended learning approach can be improved significantly by implementing a test-enhanced learning design, namely repeated testing. As students with a high initial numeracy score did not profit equally from repeated testing, target-group specific opt-out may be offered. BODY.BACKGROUND: Tests can force learners to activate knowledge or skills. This activation impacts learning: Test-enhanced learning has been shown to have marked effects on achievement and retention [1]. Test-enhanced leaning could be used in medical education to effectively improve the training of competences, which are still underrepresented, despite their relevance. Diagnostic advice and therapeutic decisions rely on good risk estimation and risk communication (RE&C) skills. Nonetheless, there is striking evidence of a marked lack of RE&C skills in many physicians [2]. RE&C learning objectives are underrepresented in medical curricula. Therefore, effective and sustainable training approaches are required. Consequently, it is promising to adopt test-enhanced learning to curricular training of RE&C skills. Test-enhanced learning was defined as "increased retention of knowledge or skills that is produced by the act of retrieval during testing" [3]. Justifications of test-enhanced learning explain its effect by two main factors: first, the enhanced exposure to learning content ("total-time hypothesis") [4, 5] and, second, the stimulation of cognitive effort due to recalling knowledge in order to solve the test ("retrieval hypothesis") [6]. Dedicated instructional designs implement test-enhanced learning by repeated testing and by feedback leading to self-regulation [7]. Repeated testing can be further improved by spaced learning. Spaced learning fosters retention effects by a careful adjustment (increasing) of time-intervals between test repetitions [6, 8]. Studies in a laboratory setting suggested that repeated testing has a strong effect on long-term retention and improves the ability to allocate cognitive effort effectively (meta-level learning) [6, 9]. Test-enhanced learning was successfully established in medical education on graduate and postgraduate level, in dental education, and in nursing education [10–13]. Test-enhanced learning was investigated in the context of clinical reasoning and human error prevention [14, 15], but so far no study addressed repeated testing in the context of RE&C training. Test-enhanced learning may profit largely from blended learning (BL). BL combines "different modes of delivery, models of teaching and styles of learning" [16]. Driven by technology, many BL approaches focus on complementing face-to-face teaching with computer/web based learning in order to combine their respective strengths. Many learning management systems support online tests with learner feedback. Therefore, blended test-enhanced learning can be implemented efficiently by combining face-to-face teaching, online delivery of learning content, and online self-tests in a BL scenario. Statistical literacy is a necessary condition for avoiding critical misinterpretations of statistical data, clinical studies and quantitative tests. The term was coined by Gigerenzer et al. [17], who urged dedicated effort to improve the risk estimation and communication skills (RE&C) of physicians. Different studies showed consistently that many physicians fail in interpreting screening statistics and cannot estimate even roughly the predictive values given the sensitivity and specificity of a diagnostic test [2, 18, 19]. A lack of statistical literacy jeopardizes adequate consultation, informed consent, and shared decision making [18, 20, 21]. As an example, an obvious lack of RE&C competence is held responsible for suicides induced by misinterpreted HIV-test results: As doctors assumed that high test sensitivity always implies equally high predictive values they (incorrectly) told test-positive patient they were almost certainly infected [17]. A recent study yielded that many counselors still communicate utterly incorrect interpretations of HIV test performance to their clients [22]. RE&C skills, therefore, need to be addressed systematically and effectively by medical curricula. Training of RE&C skills can profit from given evidence: Studies yielded, that instructing medical students to use natural frequencies as a representational means for probabilities led to marked and sustainable achievements in RE&C skills [23]. Recently, Caverly et al. [24] developed an instrument for assessing skills in critical risks interpretation. In addition, the choice of adequate means for communicating risks/chances to patients has been thoroughly investigated in the recent years yielding evidence of 1) marked effects and 2) reasons for preferring specific formats of communication [25–27]. Medical education can, therefore, use these results in order to specify adequate learning objectives for RE&C training. Despite the need for RE&C training, no dedicated RE&C program regularly integrated in a medical curriculum has been reported. Furthermore, no study has yet addressed the effect of test-enhanced learning in curricular RE&C training for medical students. Blended, test-enhanced learning seems an effective approach to curricular RE&C training. As online tests can be easily delivered in a BL scenario, repeated testing can be considered a particularly suitable form of blended, test-enhanced learning. In view of the opportunities of applying repeated testing to blended RE&C training of medical students, a thorough investigation of its impact on learning achievement was needed. Our study aimed to investigate this impact by a randomized controlled trial. BODY.METHODS.COHORT RECRUITMENT AND FLOW OF THE STUDY: Participants were recruited from students of the Aachen medical school enrolled in the mandatory course "Evidence-based Medicine" (EBM). The learning objectives of the EBM course include a subset of RE&C related topics. For organizational reasons the complete year of medical students is regularly split in two sub-cohorts, which attend the course subsequently due to course rotation. The study included the complete course cohort of the summer term 2015 and, therefore, represented a typical cross-section of medical students in the third year of our medical curriculum. The repeated testing scenario was piloted in the term preceding the study (winter term 2014/15) in order to test the reliability of the technical platform, student compliance, and the comprehensibility of the self-test items. No control group was established; all students enrolled in the EBM course were included in repeated testing. In the following term the complete course cohort of the EBM course was invited to take part in the study and was asked for consent. The participants filled out a profiling questionnaire (see below) and answered the baseline test. Subsequently, the participants were randomly assigned to the intervention group and the control group. After the EBM course, i.e. 9 weeks after the start of the study, post-testing took place. Figure 1 shows the flow of the study.Fig. 1Intervention integrated in the previously existing course "Evidence-based Medicine" (EBM) BODY.METHODS.SAMPLE SIZE CALCULATION: Sample size estimation relied on Borm's approach [28, 29]: The calculation found that n = 46 was needed for a power of .8 at significance level α = .05 if a middle-sized effect (d = .4) and correlation (R-squared = .7) were assumed. BODY.METHODS.RANDOMIZATION: Balanced randomization was carried out by ordering the list of participants by random numbers (generated by the R environment for Statistical Computing) and dividing the list equally. BODY.METHODS.INTERVENTION: This study introduced repeated testing of risk estimation/communication (RE&C) skills in the context of an existing BL module on EBM. The EBM course combined attendance teaching (lectures), training in the computer lab, online tutorials, and online tasks and tests. The control group as well as the intervention group followed this BL approach. The primary intervention of the study was to let students work on a sequence of six short online-tests (10–15 min, one test per week), which provided RE&C related items. The sequence implemented a repeated testing approach, based on multiple choice items and items asking for numerical input. The students received electronic feedback on their test-performance immediately after submitting the completed test. All students were informed that the results of the tests would not influence their grades. They were also informed that completion of all tests was required for admittance to the final course exam. The online-tests (and additional learning material) were delivered by the Learning Management System (LMS) Moodle (https://moodle.org/). The LMS logged the students' activities, which enabled assessing of their compliance with the online-tests afterwards. The tests were accessible only after individual login. Each test was only available for a predefined interval of 6 days after each lecture, which enabled spaced learning as specified in the introduction. The LMS allowed to check for compliance and to send a reminder to students, who failed to complete the test in time. In these cases the deadline was extended by 5 days. Both, the intervention group and the control group, had to work equally on short online tasks each week. The only difference between intervention and control was the presence vs. absence of repeated online tests concerning RE&C competencies. In the case of the control group these tests were substituted by other online tasks. Figure 2 gives an overview of the EBM module, which includes nine lectures (90 min) and four units of computer practice (90 min per student) of contact time and additional online training. Parts of the online training were always available to the students. Other parts were strictly scheduled and, therefore, enabled spaced and repeated testing. Differences between the test and control group in the course of the existing EBM module are highlighted in Fig. 2.Fig. 2Flow of participants BODY.METHODS.OUTCOME AND MEASUREMENT: The primary endpoint (dependent variable) of this study was the participants' learning achievement concerning a set of operationalized learning objectives RE&C. Learning achievement was measured by the difference in the scores of a pre- and post-training test. Details of the test instrument are given below. The primary independent variable was the participants' assignment to either the control or intervention group. The study used two different instruments for data acquisition: a short profiling test (five items) and a test of RE&C skills (nine items). The profiling test was used only at baseline, whereas the nine RC&E items formed the pre- and post-training test. The first three items of the profiling test represent the Basic Numeracy Test introduced by Schwartz et al. [30] and used previously in studies concerning statistical literacy. The fourth profiling item addressed the concept of statistical independence using roulette as an example. The last profiling item asked the students to decide, which of five given methods yield 100% correct results, thereby investigating the students' awareness of the general lack of absolute certainty. The total number of correct answers to the five profiling items served as a score (profiling score) with values ranging between zero and five. The nine items on RE&C skills were inspired by the aspects of basic statistical literacy introduced by Gigerenzer et al. [17]. Table 1 gives an overview of the items and the learning objectives addressed.Table 1Overview of the items used for profiling basic numeracy and awareness of probability (profiling items) and the items addressing risk estimation and communication (RE&C items) a ItemTopicObjectiveTypeP1Transform a percentage into an integer value(Profiling participants' basic numeracy)NumP2Transform integer values into percentages(Profiling participants' basic numeracy)NumP3Give expectation for the result of the next coin flip in a sequence of coin flips(Profiling participants' concept of probability)MCP4Estimate the chance of the next color being black in roulette(Profiling participants' concept of probability)BAP5Rate certainty of detection methods (e.g. certainty of a DNA-test)(Profiling participants' awareness of uncertainty)BARE&C itemsObjectiveTypeI1Define five-year mortality ("In the context of a study on the mortality of a disease you are going to investigate the five-year mortality. When does the five-years interval start?")Interpret and explain five-year mortalityMCI2Substantiate dual formulation for risk communication ("For communicating risks to patients it is recommended to adopt "dual wording". Which effect should be avoided by this measure?")Explain framing effect and its consequencesMCI3Interpret specificity of diagnostic test ("In court proceedings concerning medical malpractice it is discussed, which diagnostic tests should be accepted. Which feature would make a test unfavorable to a defendant hospital, because the test is more likely to indicate a non-existing complication than other tests?")Interpret and explain test characteristicsMCI4Communicate the risk of being ill, given a positive test result ("You intend to tell a patient, how likely it is to actually have a certain disease, in case that the test yields a positive result. Which of the following five formulations is NOT suitable here?")Communicate risks adequatelyMCI5Compare absolute vs. relative risk ("You have to make a therapeutic decision. By literature research you find two relevant RCTs. The first study found out that therapy A leads to a relative risk reduction of 0.1%. The second yielded an absolute risk reduction of 1%. Which of the following statements is correct?")Interpret risks adequatelyMCI6Interpret five-year mortality (screening context) ("The implementation of a screening program increased five-year mortality from 30% to 100%. Clinical Studies found out, that, nonetheless, there is no difference in mean life expectancy between patients included in the screening program and patients not included. What is the reason behind this discrepancy?")Explain benefits and harms of interventionsMCI7Calculate true positives from prevalence and sensitivity ("A given disease has 1% prevalence in standard population. The sensitivity of a diagnostic test is 85%. In how many cases out of 10,000 the test correctly indicates the disease?")Interpret and explain test characteristicsMCI8Calculate absolute risk given mortality of test vs. control group ("Experts discuss the implementation of a screening program, which examines the participants every three years over an interval of 10 years. Participating in the program reduces the relative risk to die from the disease by 50%. Without screening, four out of 1000 patients die from the disease. What is the correct absolute risk reduction here?")Interpret and communicate risks adequatelyMCI9Calculate predictive values given prevalence and test characteristics ("Trisomy 21 is present in 1% of pregnancies of 40 year old women. Non-invasive tests have a sensitivity of about 90% and a specifity of about 95%. What is the probability of a trisomy 21 in case of a positive test result?")Understand and communicate test resultsNum aItem types: BA binary alternative (Y/N), MC multiple choice, Num numerical input Except for one item expecting a numerical answer, all items were multiple choice questions (Type A: single positive choice out of five possible answers). Four items required the students to calculate numbers from given values before answering the question (e.g. values for prevalence, mortality, sensitivity, specifity). In these cases the values used in the final exam differed from the values used in the baseline test to prevent the students from reusing numerical results learned by heart. Apart from that, the wording of the items was kept exactly the same. As mentioned above: all nine items of the baseline RE&C test (pre-training test) were repeated in the final exam (post-training test). The total number of correct answers to the nine RE&C items served as pre-training score (baseline) and post-training score, respectively, with values between zero and nine. BODY.METHODS.DATA ANALYSIS: To be able to associate the individual baseline score to the corresponding score of the final exam the baseline data were pseudonymized by applying the secure hash algorithm (SHA-1) to the students' matriculation numbers. After combining the baseline data and the final assessment, the joined dataset was completely anonymized before being further processed by statistical analysis. Histograms and a summary of descriptive statistics were used to compare the characteristics of the intervention group and the control group. In order to examine the primary endpoint of the study 95% confidence intervals for the mean scores of the pre- and post-test were calculated by non-parametric bootstrapping and plotted as a line chart. Non-parametric bootstrapping constructs confidence intervals by resampling (randomly drawing sub-samples with replacement) from the observed data [31]. Internal consistency reliability of the test was calculated using Cronbach's alpha. Subsequently, an analysis of covariance (ANCOVA, Type III sums of squares) served as a means for testing the initial hypothesis. The ANCOVA used the group as the independent variable and the final assessment score as the dependent variable while treating the baseline score as a covariate. Effect sizes (Cohen's d, r) were calculated using adjusted means and standard deviations, respectively. Statistical analysis used the R environment (The R Project for Statistical Computing, https://www.r-project.org/). For nonparametric bootstrapping the R-package Hmisc [32] was adopted using the default of 1000 bootstrap resamples. The R-package Psych [33] was used for calculating Cronbach's alpha. The profiling score (ranging between zero and five) was used to compare participants with a low profiling score to those with a high score. Originally, we planned to define the high performing subgroup by the third quartile of the distribution of profiling scores. After data acquisition it turned out, that this approach was not feasible (see the results section): Most of the participants in both groups achieved a profiling score of four. Therefore, this value represented median, first, and third quartile of the distribution. As a consequence, we had to define high performers in a different way and decided to compare the participants with a score ranging between zero and four (i.e. a profiling score ≤ 80%) to those, who achieved a score of five (100%). This was the least arbitrary choice and roughly approximates the top- 25% of performers. We compared the difference between pre- and post-training RE&C scores of the high profiling scores subgroup (profiling score > 80%) to that observed in the other subgroup (profiling score ≤ 80%). Again we used confidence intervals calculated by non-parametric bootstrapping and line charts. BODY.RESULTS: The repeated testing scenario was piloted in the winter term preceding the actual study (2014/15). All students enrolled in the EBM course (N = 150) participated in the pilot intervention; no control group was established. The pilot confirmed the technical reliability of the LMS. There was no critical failure during the course. Five students failed to submit two out of six self-tests, and 16 students failed to submit one self-test in time (yielding a total rate of 14% non-compliants). Compliance decreased over time: in the case of test 1 there was only one non-compliant, while there were 3, 2, 4, 4, and 13 for tests 2–6, respectively. As a result of distractor analysis one test-item (concerning the interpretation of predictive values) was reworded due to a random (i.e. approximately equal) distribution of answers over all answer options including the correct one. The study took place during the summer term 2015 (April 7th to July 17th, 2015). Figure 1 shows the resulting flow of participants: All 114 students enrolled in the EBM course consented to take part in the study and were assigned to either the intervention or control group (both: n = 57) by balanced randomization. The intervention group included 47 female and 10 male students; the average age was 21.7 (SD: 2.1). The control group included 44 female and 13 male students; here the average age was 22.2 (SD: 2.45). Thus, there were no marked differences between the groups concerning these features. Five participants dropped out due to non-compliance with the online activities of the blended learning course.Profiling scores (with possible values between zero and five) indicated very good performance: Only 18.87% and 14.28% of the control group and intervention group, respectively, had a score of less than 4 (i.e. fewer than 80% of the items), 58.49% and 64.29% had a score of exactly 4 (i.e. 80% of the items), while 22.64% and 21.42% earned the best possible score of 5. The distribution and cumulative frequencies of the profiling scores for intervention vs. control yielded no marked differences. The score of 4 (i.e. 80% of the items) represented the median, first, and third quartiles as well. As a consequence, comparison of low and top performers based on quartiles was not feasible. As argued in the methods section, choosing the participants with a profiling score above 80% was the least arbitrary choice and roughly approximated the top- 25% of performers. Thus, we used this criterion for subgroup definition. With respect to pre-training test performance, i.e. the RE&C score, there were differences between the intervention group and control group. Table 2 gives an overview on the pre-training and post-training scores achieved by the intervention group and the control group, respectively. As already considered by choosing ANCOVA, these differences require control of the pretest performance when testing the study hypothesis.Table 2Overview of the RE&D Scores (before training, after training, and difference)GroupPre-training RE&D ScorePost-training RE&D ScoreDifferenceIntervention3.61 (SD: 2.86)8.18 (SD: 1.46)4.57 (SD: 3.48)Control3.04 (SD: 1.88)6.98 (SD: 3.71)3.94 (SD: 4.82) Based on the post-training test results, the calculation of Cronbach's alpha was .71 indicating acceptable (but rather moderate) reliability based on internal consistency. Independent from the intervention, all participants profited considerably from the program (Cohen's d for overall pre vs. post scores: 2.61). ANCOVA indicated that the covariate (pre-training score at baseline) was not related to the post-training score, F(1, 106) = 2.88, p > .05, while there was a significant impact of the intervention (repeated testing) on learning achievement, F(1106) = 12.72, p < .05, d = .94, r = .42 (95% CI: [.26, .57]). These results show that, even when controlling for the effect of the baseline (treated as a covariate), the post-training test score depends significantly on the group (intervention vs. control group). Thus, including repeated testing into an existing blended learning design led to significantly higher learning achievement, which cannot be explained otherwise i.e. by differences between prior knowledge/skills in the intervention group and the control group, respectively. Calculation of the adjusted means yielded 7.03 (95% CI: [6.60, 7.47]) and 8.13 (95% CI: [7.71, 8.56]) for the control and intervention group, respectively. Figure 3 shows means for pre- and post-intervention scores with bootstrapped 95% confidence intervals for all participants (combined) and for subgroups scoring high (score > 80%,) and low (≤80%) in the profile items concerning basic numeracy and statistical literacy, respectively.Fig. 3Mean scores of the pre- vs. post-training test for the intervention (blue) vs. control (grey). The three diagrams compare subgroups with a different score in the profiling test, which addressed basic numeracy plus the awareness of statistical independence and general uncertainty. The vertical lines show the 95% confidence intervals calculated by bootstrapping. The star symbol indicates significant differences between intervention and control group BODY.DISCUSSION: This study showed that some relevant RE&C skills of medical students can be trained efficiently by a dedicated blended learning program integrated into the medical curriculum. With respect to the aim of the study our data show that the marked overall effect of the blended learning approach (as measured by RE&C related MC- and numerical items) can be improved significantly by implementing repeated testing on top of the blended learning approach. Students with high profiling score did not profit equally from repeated testing: In this group the study did not find a positive effect of repeated testing on top of the effect of the standard training. For the complete cohort the results confirm our initial hypothesis: The ANCOVA results and the separate 95% confidence intervals of the mean scores at post-training time clearly show the effect of the intervention. The non-parametric bootstrapping procedure used for calculating the confidence intervals does not rely on specific assumptions concerning the shape of the underlying distribution, which strengthens the result. Repeated testing, therefore, can add significantly to the achievement of risk estimation and communication related learning goals in our BL scenario. Obviously, the marked learning effect of the training module in general (pre-training vs. post-training) exceeds the additional effect of the repeated testing intervention. Our instrument used to measure the learning outcome contained only multiple choice items and numerical items. Assessing risk communication in a realistic context (e.g. assessment based on standardized patients) was far beyond the scope of the study. Nonetheless, some of the RE&C items required the participants to calculate and estimate risks starting from information given in a typical clinical decision context. Consequently, these items covered the skills of risk estimations relevant to clinical situations. Validity was also fostered by deriving the items systematically from RE&C related learning objectives defined by the catalogue of operationalized learning objectives of our faculty. The standard training in both groups yielded a high effect size. Compared to this effect, the additional effect of repeated testing was relatively small. Therefore, one could argue that the benefit of the intervention was too small to justify the additional effort required to implement the intervention or even the additional workload for the students. In contrast, both the implementing effort and the additional workload would be overrated, if only considered in a short-term perspective. Since the electronic self-assessment could be reused in future courses, the implementation costs per term decrease drastically. And, as suggested by informal feedback from the students, the additional workload induced by the repeated tests reduced the workload necessary for preparing for the final exam. As an additional effect, obligatory repeated tests included in the BL design and offered during short intervals forced the students to access the online training regularly. Repeated testing may, thus, foster adherence to blended learning in general. The study showed that there was a significant effect of repeated testing on the outcome in general. Nonetheless, the additional investigation of subgroups of the initial profiling items raises doubts as to whether high performers of the profiling items profit from the intervention at all. Given the considerable workload of repeated testing, one could well argue, that repeated testing is unjustified in the case of these high performers. The matter might be solved by establishing the profiling test as a routine and then allowing high performers to opt out of repeated testing. Cantillon [34] pointed out possible problems of test-enhanced learning in medical education and stressed that it "needs to be evaluated as a curriculum-wide strategy to avoid skewing learning study behaviour". He questioned the compatibility of test-enhanced learning with multiple assessment formats used in the context of competency-based programs. In addition, he stressed the need to investigate the effect of improved retention on actual problem solving. These questions are still relevant and should be addressed by further research on the subject. BODY.DISCUSSION.LIMITATIONS: The study context was defined not only by the learning objectives and content of the RE&C training, but also by the BL design and environment. BL had specific implications for the experimental manipulation: The introduction of repeated tests might have been hampered or more costly, if no BL infrastructure had been available. Without the electronic BL environment it would have been much more difficult to track students' adherence to the repeated test protocol. Timely individual feedback concerning adherence might also not have been feasible. In contrast, the BL environment allowed implementing repeated tests, instant feedback to students about test results, and tracking adherence easily and without unreasonable effort. Blinding was not feasible due to the students' different learning activities induced by the intervention. In order to keep the bias as low as possible, the repetitive self-tests were delivered automatically to the participants by the LMS, while nearly everything in the virtual course room looked the same for the intervention and control group. Most items of our baseline-/post-training test address factual knowledge and, thus, learning objectives at a low level of competence. The items requiring calculation go beyond the (cognitive) level of factual knowledge. Nonetheless, one could still argue that risk estimation and communication skills on higher competence levels are not adequately addressed by this study. For instance, the training did not address the need of assessing the quality of information given by a patient (Did he/she lie?) or the ability to judge whether a patient actually understands the risk figures. Furthermore, the study design did not include long-term follow-up. An investigation into long-term retention was beyond the scope of the study. Obviously, these important aspects need to be addressed by future investigations. BODY.CONCLUSIONS: Including repeated online tests in a BL scenario can improve learning achievement in medical curricula. Our study showed that the training of RE&C competences profited considerably from this repeated testing. Nonetheless, this study did not address complex RE&C skills on higher competence level such as detecting false information deliberately given by patients. Thus, it is not clear so far, whether suitably designed repeated testing can support training in higher level RE&C skills as well. Students with high initial numeracy score did not profit equally from repeated testing in RE&C. As a consequence, opting out of repeated testing could be offered to this subgroup in the future with the provision of initial student profiling. Our results suggest promoting the use of repeated testing in order to foster fields still underrepresented in medical curricula despite their relevance.

TITLE: The effect of budesonide/formoterol maintenance and reliever therapy on the risk of severe asthma exacerbations following episodes of high reliever use: an exploratory analysis of two randomised, controlled studies with comparisons to standard therapy ABSTRACT.BACKGROUND: Divergent strategies have emerged for the management of severe asthma. One strategy utilises high and fixed doses of maintenance treatment, usually inhaled corticosteroid/long-acting β2-agonist (ICS/LABA), supplemented by a short-acting β2-agonist (SABA) as needed. Alternatively, budesonide/formoterol is used as both maintenance and reliever therapy. The latter is superior to fixed-dose treatment in reducing severe exacerbations while achieving similar or better asthma control in other regards. Exacerbations may be reduced by the use of budesonide/formoterol as reliever medication during periods of unstable asthma. We examined the risk of a severe exacerbation in the period after a single day with high reliever use. ABSTRACT.METHODS: Episodes of high reliever use were quantified and exacerbations occurring post-index day with these episodes were examined post hoc in two double-blind studies comparing the efficacy and safety of budesonide/formoterol maintenance and reliever therapy (Symbicort SMARTTM, Turbuhaler®) 160/4.5 μg twice daily plus as needed with similar or higher maintenance doses of ICS/LABA plus SABA or formoterol. ABSTRACT.RESULTS: Budesonide/formoterol maintenance and reliever therapy significantly reduced the risk of episodes of high reliever use (>6 inhalations/day) vs. all alternative ICS/LABA regimens. With conventional fixed-dose treatment the need for exacerbation treatment within 21 days ranged from 6.0–10.1% of days post-index for all regimens compared with 2.5–3.4% of days with budesonide/formoterol maintenance and reliever therapy. ABSTRACT.CONCLUSIONS: Budesonide/formoterol maintenance and reliever therapy reduces the incidence of high reliever episodes and the exacerbation burden immediately following these episodes vs. alternative ICS/LABA plus SABA regimens at up to double the maintenance dose of ICS. ABSTRACT.TRIAL REGISTRATION: These studies do not have registration numbers as they were conducted before clinical trial registration was required BODY.BACKGROUND: Short-acting β2-agonists (SABAs) are the mainstay of asthma reliever medication, but patients who over-rely on SABAs have an increased risk of fatal or near-fatal asthma [1-3]. In contrast, inhaled corticosteroids (ICS) used as maintenance therapy reduce this risk [1,2,4]. Regular use of long-acting β2-agonists (LABA) is established as the preferred second-line maintenance therapy in combination with ICS. The beneficial effects of ICS/LABA on exacerbation rates, lung function, SABA use and daily symptoms compared with ICS alone are well documented [5]. However, even in patients using ICS/LABA, escalating SABA use remains an indicator of the potential for a severe exacerbation [6,7]. Budesonide/formoterol as maintenance and reliever therapy is well established and incorporated in guidelines [8]. Without compromising any measure of asthma control, compared with other higher fixed-dose steroid-containing regimens, its great attractiveness is in reducing severe exacerbations [9-15].The mechanism for this effect is currently unknown [9,10,15-17]. One explanation is that events initiating unstable asthma, and potentially an exacerbation, occur less frequently or are promptly aborted and thus not recognisably different from the usual asthma state. Alternatively, the exacerbation sequence may commence, but, in some instances, the additional as-needed budesonide/formoterol modifies the course of the event to prevent an exacerbation. The 'window of opportunity' represents the period during which symptom-driven increases in reliever and ICS use may act [18]. Study patients were symptomatic, using reliever medication on most days in the run-in period. If an exacerbation were to develop in this population, further increases in reliever use should be seen. The utility of high reliever use events as a marker for an impending exacerbation could be tested by examining the short-term increase in the conversion to a severe exacerbation. A preferred management strategy would reduce the frequency of periods with high reliever use or lower the conversion rate into a severe exacerbation. We examined episodes of high reliever use and the clinical course in the subsequent 21 days in two large studies. These studies compared the efficacy and safety of budesonide/formoterol maintenance and reliever therapy (160/4.5 μg twice daily [bid] plus as needed) with: the same maintenance dose of ICS/LABA, budesonide/formoterol, with terbutaline or formoterol as reliever medication and with budesonide/formoterol or salmeterol/fluticasone at a higher maintenance dose with terbutaline as needed [10, 12]. BODY.METHODS: Patients came from two double-blind, parallel-group exacerbation prevention studies (SD-039-0734; Study A [12]; SD-039-0735; Study B [10]). Severe exacerbations were defined as deteriorations in asthma control resulting in hospitalisation, emergency room (ER) treatment or the need for oral corticosteroids for ≥3 days. Patients were aged ≥12 years with an asthma diagnosis for ≥6 months and ≥1 exacerbation in the last year. All patients used ICS for ≥3 months prior to enrolment and had a forced expiratory volume in 1 second (FEV1) ≥50% of predicted normal. In Study A, [12] patients received budesonide/formoterol 160/4.5 μg (Symbicort® Turbuhaler®, AstraZeneca, Lund, Sweden) bid during a 2-week run-in and throughout the 12-month randomised treatment period. Terbutaline (Bricanyl®, AstraZeneca, Lund, Sweden; 0.4 mg/inhalation) was used as needed during run-in and either formoterol (Oxis®, AstraZeneca, Lund, Sweden; 4.5 μg/inhalation), terbutaline (0.4 mg/inhalation) or budesonide/formoterol (160/4.5 μg/inhalation) as needed during the treatment period ( 1). Figure 1Patient flow. *Study A, treatment period = 12 months; Study B, treatment period = 6 months; bid = twice daily; BUD/FORM = budesonide/formoterol; inh = inhalation; SAL/FLU = salmeterol/fluticasone. In Study B, [10] following a 2-week run-in on ICS plus terbutaline without LABA, patients were randomised to one of three 6-month regimens: budesonide/formoterol (160/4.5 μg bid) for maintenance and reliever as needed, fixed-dose budesonide/formoterol 320/9 μg one inhalation bid plus terbutaline (0.4 mg/inhalation) as needed or fixed-dose salmeterol/fluticasone 25/125 μg (SeretideTM EvohalerTM, GlaxoSmithKline, Uxbridge, UK) two inhalations bid plus terbutaline (0.4 mg/inhalation) as needed (Figure 1). BODY.METHODS.INCREASING SABA USE AND EXACERBATIONS: Analyses focused on confirming the relationship between the first (index) day with a defined threshold of SABA exposure (>2, >4, >6 and >8 inhalations of terbutaline/day for at least one day) and the increased exacerbation risk in subsequent days [10,12]. BODY.METHODS.EPISODES OF >6 INHALATIONS/DAY OF RELIEVER: A pre-specified threshold for high reliever use was examined for each regimen. This threshold was selected because maintenance therapy with budesonide/formoterol is approved up to a dose of 1,280/36 μg/day [10,12]. On days when >6 inhalations/day of as-needed therapy were used this limit would be exceeded. We determined the use of >6 as-needed inhalations/day in the intention-to-treat (ITT) populations and the time to the first (index) day with such episodes. BODY.METHODS.EXACERBATIONS WITH >6 INHALATIONS/DAY OF RELIEVER: Exacerbation rates post-index day and the number of subsequent exacerbation days were assessed after the index to study end and post-index for ≤21 days. The 21-day window would allow severe exacerbations coinciding with high reliever use to be captured in reasonable proximity to the index day. The average time in an exacerbation state was defined as the number of days with oral steroid use initiated by the physician or days of hospital treatment, without double-counting if this overlapped. Differences in the annualised exacerbation rate and hospitalisations/ER treatments in these periods were compared between regimens and with reference to analyses in the ITT populations [10,12]. BODY.METHODS.TOLERABILITY: Descriptive statistics examined patients with an index day of >6 inhalations/day of reliever, to assess the incidence of serious adverse events (SAEs) and discontinuations due to adverse events (DAEs). BODY.METHODS.STATISTICAL ANALYSIS: The risk profile for patients requiring >6 inhalations/day of reliever in the ITT population was described by Kaplan–Meier plots and evaluated using a Cox proportional hazards model with treatment as factor. All analyses comparing exacerbation rates in the ITT population and post-index rate ratios used a Poisson regression model with treatment as factor and study or post-index time as offset, with confidence intervals adjusted for overdispersion. As data on the percentage of exacerbation days were skewed, post-hoc analyses to compare this outcome between treatment regimens were conducted using a bootstrap procedure. This procedure was selected because it does not make assumptions about different data distributions and provides a more controlled estimate of variance. BODY.RESULTS.SABA USE AND EXACERBATIONS: Sensitivity analyses showed that regardless of the ICS/LABA regimen, the higher the number of as-needed inhalations (>2, >4, >6 and >8 inhalations/day) of terbutaline on the index day, the greater the incidence of exacerbations during the following 21 days. Across both studies ~15% of patients using fixed-dose maintenance therapy with budesonide/formoterol (160/4.5 or 320/9 μg bid) and ~24% using salmeterol/fluticasone (50/250 μg bid) experienced severe exacerbations in the 21 days following an index day with >6 inhalations/day of terbutaline (Figure 2). Figure 2Incidence of asthma exacerbations occurring 21 days after the first episode with high terbutaline use. Kaplan–Meier plots of time from first use of >2, >4, >6 and >8 as-needed inhalations/day of terbutaline to first exacerbation on alternative fixed-dose ICS/LABA regimens: A) budesonide/formoterol (BUD/FORM) 160/4.5 μg twice-daily over 12-months (Study A) (N=1,138); B) budesonide/formoterol 320/9 μg twice-daily over 6-months (Study B) (N=1,099); C) salmeterol/fluticasone (SAL/FLU) 50/250 μg twice-daily over 6-months (Study B) (N=1,119). Day 0 is the first day with the specified level of as-needed inhalations of terbutaline. The number of patients with exposure to each level of terbutaline and the percentage of the total randomised population this represents is shown in parenthesis above each Kaplan–Meier curve. BODY.RESULTS.>6 INHALATIONS/DAY OF RELIEVER: Patients who used >6 inhalations/day on at least one occasion had a similar baseline demography across treatment regimens (Table 1) and were similar to the ITT populations, although baseline daily reliever use was higher in this subgroup (Additional file 1: Table S1, online supplement). Table 1 Demographics and asthma control during run-in among the patient subgroups with episodes of >6 inhalations/day of as-needed medication use on at least 1 day in Study A and Study B   Study A (12-month assessment)* Study B (6-month assessment) † All patients used BUD/FORM maintenance SAL/FLU FD BUD/FORM FD BUD/FORM maintenance Terbutaline as needed (n = 278) Formoterol as needed (n = 228) BUD/FORM as needed (n = 148) Terbutaline as needed (n = 156) Terbutaline as needed (n = 167) BUD/FORM as needed (n = 124) Male, n (%) 108 (39) 95 (42) 58 (39) 67 (43) 66 (40) 57 (46) Age, years 43 (12–78) 42 (12–81) 42 (12–77) 38 (12–73) 41 (12–81) 39 (12–74) Smokers, n (%) 20 (7) 18 (8) 17 (12) 13 (8) 23 (13) 11 (8) ICS at entry, μg·day -1 777 (250–1,600) 789 (400–1,600) 755 (400–1,600) 811 (500–1,600) 796 (300–2,000) 791 (500–1,600) FEV 1 , % predicted 70 (39–100) 70 (50–99) 71 (50–99) 71 (51–132) 70 (50–117) 72 (50–114) Reversibility, % (range) 23 (11–90) 24 (12–81) 24 (12–132) 23 (12–79) 23 (11–84) 22 (12–74)   Control assessed in run-in   Morning PEF, l/min 325 (111–619) 334 (106–689) 328 (123–621) 324 (127–885) 326 (113–607) 337 (155–558) Reliever use, inh./day 2.8 (0.5–9.7) 2.9 (0.5–9.1) 3.1 (0.6–8.9) 3.6 (0.7–10.8) 3.7 (0.8–9.5) 3.8 (0.8–8.8) Symptom-free days, % 7 (0–78) 7 (0–60) 6 (0–100) 6 (0–90) 4 (0–80) 5 (0–60) *Patients used BUD/FORM maintenance plus terbutaline as needed during run-in. † Patients used pre-study ICS + terbutaline during run-in (no LABA). Data are presented as mean (range) unless otherwise stated. BUD/FORM = budesonide/formoterol; FD = fixed-dose maintenance; FEV 1  = forced expiratory volume in 1 second; inh. = inhalation; ICS = inhaled corticosteroids; LABA = long-acting β 2 -agonist; PEF = peak expiratory flow; SAL/FLU = salmeterol/fluticasone. In the 12-month Study A, an index day of >6 inhalations/day was observed in 278 (24%) of patients on terbutaline, 228 (20%) of patients on formoterol and 148 (13%) of patients on budesonide/formoterol reliever therapy respectively (Figure 1). In the 6-month Study B, an index day of >6 inhalations/day of reliever was observed in 156 (14%) patients on salmeterol/fluticasone, 167 (15%) on budesonide/formoterol and 124 (11%) patients on budesonide/formoterol maintenance and reliever treatment, respectively (Figure 1). In both studies the risk of any episode with >6 inhalations/day of reliever was reduced with budesonide/formoterol maintenance and reliever therapy vs. comparators (P ≤ 0.014; Table 2 and 3). Table 2 Annualised exacerbation rates and hospitalisations/ER treatments in the full ITT population and the subgroups with episodes of high as-needed reliever use (>6 inhalations/day on ≥1 study day) (Study A)   Reliever treatment groups Risk or rate ratios (95% CI), comparing reliever groups Terbutaline BUD/FORM BUD/FORM vs. terbutaline BUD/FORM vs. formoterol Formoterol vs. terbutaline   Number of patients with episodes with   >   6 inh./day as needed All patients, n (%)* † 278 (24.4) 228 (20.1) 148 (13.4) 0.51 (0.42–0.62) 0.66 (0.53–0.81) 0.77 (0.65–0.92)         P  < 0.001 P  < 0.001 P  = 0.004 Exacerbation rate. patient-year -1 All patients, annualised rate (events)* ‡ 0.37 (377) 0.29 (296) 0.19 (194) 0.52 (0.44–0.62) 0.67 (0.56–0.80) 0.78 (0.67–0.91)         P  < 0.0001 P  < 0.0001 P  = 0.0012 High as-needed group, annualised rate (events) § Post-index day to study end 0.97 (180) 0.76 (120) 0.41 (42) 0.42 (0.29–0.62) 0.54 (0.36–0.80) 0.79 (0.61–1.03) ≤21 days post-index 3.04 (48) 2.72 (35) 1.67 (14) 0.55 (0.30–1.00) 0.61 (0.33–1.14) 0.90 (0.58–1.38) Severe exacerbation days All patients, % rate (total days)* ║ 0.8 (3030) 0.6 (2214) 0.4 (1353) 0.45 (0.35–0.58) 0.63 (0.48–0.82) 0.72 (0.57–0.91)         P  < 0.0001 P  < 0.001 P  < 0.001 Post-index day to study end 2.4 (1616) 1.7 (998) 0.8 (300) 0.34 (0.21–0.50) 0.46 (0.29–0.71) 0.73 (0.52–1.01) ≤21 days post-index 7.9 (458) 6.1 (287) 2.5 (77) 0.32 (0.14–0.57) 0.41 (0.18–0.81) 0.77 (0.44–1.28) Hospitalisation/ER visit rate, patient-year –1 All patients, rate (events) *‡ 0.07 (115) 0.05 (98) 0.04 (70) 0.61 (0.45–0.82) 0.73 (0.54–0.99) 0.83 (0.63–1.08)         P  < 0.001 P  = 0.046 P  = 0.17 High as-needed group, rate (events) § Post-index day to study end 0.27 (50) 0.24 (37) 0.18 (18) 0.65 (0.38–1.12) 0.75 (0.42–1.31) 0.88 (0.57–1.34) ≤21 days post-index 1.14 (18) 1.09 (14) 0.96 (8) 0.84 (0.36–1.93) 0.88 (0.37–2.09) 0.95 (0.47–1.92) * All patients (ITT population); † Cox proportional hazard model (post hoc analysis); ‡ Poisson regression (a priori analysis); § Poisson regression ( post hoc analysis); ║ Bootstrap procedure ( post-hoc analysis). BUD/FORM = budesonide/formoterol; CI = confidence interval; ER = emergency room; inh. = inhalations. Table 3 Annualised exacerbation rates and hospitalisations/ER treatments in the full ITT population and the subgroups with episodes of high as-needed reliever use (>6 inhalations/day on ≥1 study day) (Study B)   Treatment groups Risk or rate ratios (95% CI) SAL/FLU FD + terbutaline BUD/FORM FD + terbutaline BUD/FORM maintenance and reliever therapy BUD/FORM maintenance and reliever therapy vs. SAL/FLU FD + terbutaline BUD/FORM maintenance and reliever therapy vs. BUD/FORM FD + terbutaline BUD/FORM FD + terbutaline vs. SAL/FLU FD + terbutaline Number of patients with episodes with   >   6 inh./day as needed All patients, n (%) *† 156 (13.9) 167 (15.1) 124 (11.2) 0.74 (0.59–0.94) 0.65 (0.52–0.83) 1.13 (0.91–1.41)         P  = 0.014 P  < 0.001 P  = 0.26 Exacerbation rate. patient-year -1 All patients, annualised rate (events)* ‡ 0.38 (208) 0.32 (173) 0.24 (125) 0.61 (0.49–0.76) 0.72 (0.57–0.90) 0.85 (0.69–1.04)         P  < 0.001 P  = 0.0048 P  = 0.10 High as-needed group, annualised rate (events) § Post-index day to study end 1.92 (94) 1.50 (80) 0.92 (37) 0.48 (0.31–0.75) 0.61 (0.39–0.97) 0.78 (0.55–1.11) ≤21 days post-index 4.78 (41) 3.02 (28) 2.00 (14) 0.42 (0.23–0.77) 0.66 (0.35–1.25) 0.63 (0.39–1.02) Severe exacerbation days All patients, % rate (total days)* ║ 0.7 (1327) 0.6 (1143) 0.4 (692) 0.53 (0.38–0.73) 0.60 (0.42–0.86) 0.88 (0.64–1.21)         P  < 0.001 P  < 0.01 NS High as-needed group, % rate (total days) ║ Post-index day to study end 3.7 (667) 2.8 (539) 1.8 (259) 0.47 (0.27–0.78) 0.64 (0.34–1.15) 0.74 (0.44–1.21) ≤21 days post-index 10.1 (315) 6.0 (203) 3.4 (88) 0.34 (0.14–0.66) 0.57 (0.23–1.16) 0.60 (0.35–1.00) Hospitalisation/ER visit rate, patient-year –1 All patients, rate (events) *‡ 0.16 (106) 0.10 (72) 0.10 (64) 0.61 (0.44–0.83) 0.88 (0.63–1.24) 0.68 (0.51–0.92)         P  = 0.0015 P  = 0.47 P  = 0.013 High as-needed group, rate (events) § Post-index day to study end 0.96 (47) 0.74 (40) 0.40 (16) 0.41 (0.23–0.73) 0.53 (0.30–0.95) 0.78 (0.51–1.19) ≤21 days post-index 2.44 (21) 1.08 (10) 0.86 (6) 0.35 (0.14–0.86) 0.79 (0.29–2.18) 0.44 (0.21–0.94) * All patients (ITT population); † Cox proportional hazard model (post hoc analysis); ‡ Poisson regression (a priori analysis); § Poisson regression ( post hoc analysis); ║ Bootstrap procedure ( post hoc analysis). BUD/FORM = budesonide/formoterol; CI = confidence interval; ER = emergency room; FD = fixed-dose maintenance; inh. = inhalations; SAL/FLU = salmeterol/fluticasone. BODY.RESULTS.EXACERBATION CONVERSION RATE FROM EPISODES OF >6 INHALATIONS/DAY OF RELIEVER: In the Study A ITT population, 180/377 (48%) exacerbations in the as-needed terbutaline group and 120/296 (41%) in the as-needed formoterol group occurred post-index day (Table 2). The corresponding figure for as-needed budesonide/formoterol was 42/194 (22%). Similar results were seen in Study B, where 94/208 (45%) and 80/173 (46%) exacerbations occurred post-index day in the fixed-dose salmeterol/fluticasone and budesonide/formoterol groups, respectively, with terbutaline used as reliever therapy. In contrast, 37/125 (30%) exacerbations in the budesonide/formoterol maintenance and reliever therapy group occurred post-index day (Table 3). Figure 3 shows the time to first exacerbation during the 21 days post-index with each reliever regimen. The overall exacerbation rates from the index day to study end are presented in Tables 2 and 3. Figure 3Asthma exacerbations associated with episodes of high reliever use (>6 inhalations/day) A) in Study A, B) in Study B. i) Proportion of all patients (ITT population) with an index day of >6 inhalations/day in the three treatment arms over the study period; ii) Kaplan–Meier plot of time from first use of >6 as-needed inhalations/day to first exacerbation during the following 21 days; iii) percentage of patients with ongoing treatment for an exacerbation for each day following the index day. Note in panels ii) and iii) only a minority of the at risk subgroup of patients having an episode of high reliever use identified in panel i) developed a severe exacerbation requiring additional treatment. The majority of high reliever episodes resolved spontaneously on all regimens.4 BODY.RESULTS.EXACERBATIONS RATES IN THE WINDOW OF OPPORTUNITY: The rate of severe exacerbations per patient during the 21-day post-index period was increased at least eightfold in all groups from the mean ITT rates in the full dataset. Rates increased from 0.37 to 3.04 per patient-year with terbutaline, 0.29 to 2.72 with formoterol and 0.19 to 1.67 with budesonide/formoterol in Study A (Table 2), and from 0.38 to 4.78 with fixed-dose salmeterol/fluticasone, 0.32 to 3.02 with fixed-dose budesonide/formoterol and 0.24 to 2.00 per patient-year with budesonide/formoterol maintenance and reliever therapy, in Study B (Table 3). In both studies exacerbation rates favoured budesonide/formoterol maintenance and reliever therapy vs. each fixed-dose regimen using SABA. Nevertheless, reliever use remained a robust marker for deteriorating asthma control irrespective of reliever regimen. BODY.RESULTS.EXACERBATION DAYS IN THE WINDOW OF OPPORTUNITY: In the terbutaline as-needed group (Study A) post-index, the daily profile of the percentage of patients with actively treated exacerbations peaked at ~7 days, waning to a more stable level at ~16 days (Figure 3A). In Study B, the peak incidence of exacerbation treatment in patients using budesonide/formoterol with terbutaline as needed was of similar magnitude and duration as in Study A. In both studies, patients using budesonide/formoterol maintenance and reliever therapy had an attenuated peak (Figures 3A and B). The number and duration of individual events contributing to the mean profiles are available online (Additional file 2: Figure S1 and Additional file 3: Figure S2). The average time in an exacerbation state during the 21-day post-index period was 7.9% of days with as-needed terbutaline, 6.1% with as-needed formoterol and 2.5% of days with as-needed budesonide/formoterol, respectively in Study A (Table 2). In Study B, the corresponding values were 10.1% of days with fixed-dose salmeterol/fluticasone plus as-needed terbutaline, 6.0% with fixed-dose budesonide/formoterol plus as-needed terbutaline and 3.4% of days with budesonide/formoterol maintenance and reliever therapy, respectively (Table 3). BODY.RESULTS.ICS DOSE IN THE WINDOW OF OPPORTUNITY: Only a minority of patients reported high reliever use; however, because of the regimen nature, patients receiving budesonide/formoterol maintenance and reliever therapy were exposed to increased ICS doses during the window of opportunity. In Study A, total ICS exposure based on median average beclomethasone dipropionate (BDP) equivalents in the 21-day period was 1,363 μg/day with budesonide/formoterol maintenance and reliever therapy vs. 500 μg/day for both comparator groups. In Study B, the corresponding values were 1,357 μg/day with budesonide/formoterol maintenance and reliever therapy vs. 1,000 μg/day for both fixed-dose control groups. Overall, ICS exposure was not excessive: the median ICS doses for budesonide/formoterol maintenance and reliever therapy for the ITT population were 657 and 642 μg/day BDP equivalents in Study A and B, respectively. BODY.RESULTS.HOSPITALISATIONS/ER VISITS: In Study A, the need for hospitalisation/ER visits was not significantly different between regimens during the 21-day period. This was in contrast to the ITT population. Nevertheless, hospitalisation/ER visits were less frequent with budesonide/formoterol vs. terbutaline (8 vs. 18 events; Table 2). The risk of hospitalisation/ER visits in Study B during the 21-day period was significantly (P < 0.05) lower for both budesonide/formoterol regimens compared with fixed-dose salmeterol/fluticasone (Table 3). This mirrored previous findings in the ITT population. BODY.RESULTS.TOLERABILITY: In both studies, the number of patients with SAEs and DAEs was lower in the budesonide/formoterol maintenance and reliever therapy arms vs. comparator arms in patients using >6 inhalations/day of reliever (Table 4). Asthma was the only common cause of SAEs and DAEs by event type; all other events occurred with an incidence ≤1%. In Study A, patients were also discontinued due to pre-specified exacerbation/safety criteria (i.e. ≥3 severe exacerbations during any 3 months, five during the whole study or any severe exacerbation lasting ≥20 days). This resulted in an additional 10, 2 and 1 patient(s) in the terbutaline, formoterol and budesonide/formoterol as needed groups, respectively, being withdrawn from the study (Figure 1). In Study A, two deaths were reported – one in the as-needed terbutaline arm (cardiac arrest) and one in the as-needed formoterol arm (brain neoplasm). No deaths were reported in Study B. Table 4 Safety among the subgroups of patients with episodes of >6 inhalations/day of as-needed therapy in Study A and Study B   Study A (12-month assessment) Study B (6-month assessment) BUD/FORM + terbutaline (n = 278) BUD/FORM + formoterol (n = 228) BUD/FORM maintenance and reliever therapy (n = 148) SAL/FLU FD + terbutaline (n = 156) BUD/FORM FD + terbutaline (n = 167) BUD/FORM maintenance and reliever therapy (n = 124) Deaths, n (%) 1 (<0.5) 1 (<0.5) 0 (0) 0 (0) 0 (0) 0 (0) Patients with an SAE, n (%) 26 (9) 25 (11) 21 (14) 13 (8) 18 (11) 6 (5) Asthma-related SAE, n (%) 15 (5) 15 (7) 8 (5) 7 (4) 5 (3) 2 (2) Patients with a DAE, n (%) 12 (4) 12 (5) 2 (1) 3 (2) 6 (4) 0 (0) *Asthma-related DAEs, n (%) 6 (2) 9 (4) 1 (1) 3 (2) 4 (2) 0 (0) *An additional 10, 2 and 1 patients receiving: terbutaline, formoterol and budesonide/formoterol reliever regimen, respectively in Study A were withdrawn due to predefined asthma-related treatment failure/safety criteria. DAE = adverse events leading to discontinuation from the study; BUD/FORM = budesonide/formoterol; FD = fixed-dose maintenance; SAE = serious adverse event; SAL/FLU = salmeterol/fluticasone. BODY.DISCUSSION: We have shown that episodes of high reliever use (>6 inhalations/day on at least 1 day) predict an increased risk of near-term and later exacerbations, and also that most of these episodes do not develop into severe exacerbations. Our findings suggest that budesonide/formoterol maintenance and reliever therapy can reduce the incidence of episodes of high reliever use and the severe exacerbations that coincide with many of these events compared with ICS/LABA and reliever regimens at similar or higher maintenance ICS doses. We examined the exacerbation risk immediately following an episode with high reliever use. Unlike previous studies retrospectively examining the association between SABA use from prescription records and the incidence of severe exacerbations, [7,18,19] this analysis focused on the temporal association between the first day with high reliever use and the onset of exacerbation. All patients used standard maintenance ICS/LABAs. When as-needed reliever use exceeded 6 inhalations on at least 1 day, exacerbation rates in the 21 days post-index increased at least eightfold compared with background rates in the ITT populations for all regimens. This suggests that, irrespective of the level of maintenance ICS/LABA treatment and reliever type, a day with high as-needed use is a robust measure of disease instability. On a background of budesonide/formoterol maintenance therapy, it has been reported that both formoterol and budesonide contribute to decreased severe exacerbations [12] .We have shown that formoterol is superior to terbutaline and that budesonide/formoterol is superior to both in reducing the risk of a high reliever use episode [12]. Budesonide/formoterol is also superior to both alternative relievers in reducing the conversion rate of these episodes to severe exacerbations in the 21 days post-index day. This suggests that both budesonide and formoterol have airway stabilising effects that explain the preventative effect observed in this study and in the ITT analysis. Budesonide/formoterol as maintenance and reliever therapy was also compared with budesonide/formoterol at twice the maintenance dose and to salmeterol/fluticasone at a similarly higher ICS dose [10]. Notwithstanding the more intense maintenance treatment, patients using budesonide/formoterol maintenance and reliever therapy were less likely than other groups to have an index high reliever use episode. They were also less likely than those on salmeterol/fluticasone fixed maintenance dose to develop a severe exacerbation. There was no significant difference between budesonide/formoterol maintenance and reliever therapy and fixed-dose budesonide/formoterol in this comparison but there was a significant reduction in the number of severe exacerbation days in the 21-day window. Therefore, compared with budesonide/formoterol maintenance and reliever therapy, all tested comparators were associated with an increased risk of events of concern and a higher risk of conversion to a severe exacerbation. A criticism of the budesonide/formoterol maintenance and reliever studies was that the maintenance dose of the higher dose comparator arms was not adjusted to allow even higher doses, which would be possible in ordinary clinical care. However, even on the higher fixed-dose regimes, the majority of events resolve without a change in treatment. The institution of an even higher maintenance dose would thus have been unnecessary in the majority of occasions. Studies have not shown any advantage of doubling the dose of maintenance ICS during an asthma worsening [20,21]. However, quadrupling or quintupling ICS maintenance doses is reported as effective, [22,23] but these are based on intensive multi-modality asthma monitoring that may be possible in trials but not in ordinary care. In any case, the dose increases with budesonide/formoterol in the 21 days after the onset of the event are less than a trebling of the regular maintenance dose. The precise mechanism behind this acute protection is uncertain but may rely on several factors, including reductions in the late asthmatic response after allergen inhalation, eosinophilic inflammation, bronchial hyperreactivity and/or reducing pulmonary blood flow, all of which occur within hours of administering higher ICS doses [24-27]. In the current analysis, patients receiving budesonide/formoterol maintenance treatment plus formoterol as reliever had a lower risk of severe exacerbations compared with a control subgroup using equally high levels of terbutaline. Furthermore, in a smaller group of patients with episodes of high as-needed budesonide/formoterol use, there was a further reduced incidence of exacerbations including asthma-related SAEs and DAEs compared with the larger groups using LABA or SABA as reliever. A meta-analysis of seven studies[9-15] has also confirmed that the incidence of asthma-related SAEs and DAEs with budesonide/formoterol maintenance and reliever therapy is significantly lower than with fixed-dose regimens using ICS or ICS/LABA combination therapy plus terbutaline or salbutamol as needed [28]. Thus, budesonide/formoterol maintenance and reliever therapy appears to be well tolerated and reduces the risk of severe exacerbations following exposure to high levels of SABA that could mask worsening inflammation. The present analysis also provided an insight to the a priori significant increase in ER visits/hospitalisations with fixed-dose salmeterol/fluticasone compared with both budesonide/formoterol regimens independent of reliever therapy (Table 4) [10]. Patients receiving salmeterol/fluticasone had a longer peak period with increased exacerbation treatment and a significant doubling in ER visits/hospitalisations during the 21 days post-index vs. both budesonide/formoterol regimens (Figure 3C; Table 3). This difference occurred without any increase in the number or duration of episodes of high terbutaline use with fixed-dose salmeterol/fluticasone vs. fixed-dose budesonide/formoterol. Furthermore, there was no difference in asthma control between the fixed-dose subgroups at baseline or on treatment outside of the 21-day window. Thus, the current analyses highlight a difference between fixed-dose ICS/LABAs, not during stable periods, but impacting outcomes during periods of instability. The varying pharmacological properties of the different LABAs, primarily the lower efficacy of salmeterol vs. formoterol leading to less broncho protection, [29,30] antagonism of SABA-induced smooth muscle relaxation [31] and broncho protection [30] with salmeterol but not formoterol, may provide a rationale for the differences in exacerbation rates seen between LABAs when added to ICS in two meta-analyses [32,33] .Our observations may warrant additional prospective investigation. This analysis does have limitations. The number of high reliever use events can be compared between treatment arms. However, subsequent comparisons may be influenced by the efficacy of randomised treatment and patients included in further analysis may be dissimilar between groups if groups are diminished in size due to differences in the prior efficacy of the regimens. Despite these difficulties it is an important observation that high reliever use episodes are more likely to resolve without severe exacerbation if they occur in a person who is receiving budesonide/formoterol maintenance and reliever therapy than in someone who remains on standard reliever therapy. In summary, this study confirms that a window of opportunity exists for preventing exacerbations associated with high reliever use. When compared with alternative fixed-dose ICS/LABA plus SABA regimens, at similar or higher maintenance ICS doses, budesonide/formoterol maintenance and reliever therapy reduces the incidence of episodes of high reliever use and exacerbations that frequently coincide with acute periods of asthma instability. Budesonide/formoterol maintenance and reliever therapy is a logical evolution from the fixed-dose maintenance treatment strategy that is more efficient and effective in patients with moderate or severe asthma. BODY.ABBREVIATIONS: FEV, Forced Expiratory Volume; ICS, Inhaled CorticoSteroid; LABA, Long-Acting β2-Agonist; SABA, Short-Acting β2-Agonist. BODY.COMPETING INTERESTS: RB has received reimbursement for attending scientific conferences, and/or fees for speaking and/or consulting from AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Novartis, Nycomed and Pfizer. The Pulmonary Department at Mainz University Hospital received financial compensation for services performed during participation in clinical trials organised by various pharmaceutical companies. PK has received a fee for speaking at a company-sponsored symposium from AstraZeneca and travel to an international congress was funded by AstraZeneca. MJP has received honoraria from AstraZeneca for CME lectures and has acted on advisory boards for AstraZeneca, GlaxoSmithKline and Nycomed. TLGA, IPN and SP are employees of AstraZeneca and hold shares in AstraZeneca. KR has participated in advisory boards and received lecture fees from AstraZeneca, Boehringer, Chiesi Pharmaceuticals, Pfizer, Nycomed, MSD and GlaxoSmithKline. The Department of Pulmonology at Leiden University Medical Centre has received grants from various pharmaceutical companies. BODY.AUTHOR CONTRIBUTIONS: RB was involved in the study design and the drafting of the manuscript. SP performed the statistical analysis. All authors read and approved the final manuscript. BODY.SUPPLEMENTARY MATERIAL: Additional file 3Figure S2: P Incidence, type and duration of asthma exacerbations associated with episodes of high reliever use of >6 inhalations/day in Study B.Click here for file Additional file 1Table S1: Patient baseline demography in full ITT populations).Click here for file Additional file 2Figure S1: Incidence, type and duration of asthma exacerbations associated with episodes of high reliever use of >6 inhalations/day in Study A.Click here for file

TITLE: Comparison of hemodynamic and metabolic stress responses caused by endotracheal tube and Proseal laryngeal mask airway in laparoscopic cholecystectomy ABSTRACT.BACKGROUND:: We aimed to compare hemodynamic and endocrine alterations caused by stress response due to Proseal laryngeal mask airway and endotracheal tube usage in laparoscopic cholecystectomy. ABSTRACT.MATERIALS AND METHODS:: Sixty-three ASA I-II patients scheduled for elective laparoscopic cholecystectomy were included in the study. Patients were randomly allocated into two groups of endotracheal tube and Proseal laryngeal mask airway. Standard general anaesthesia was performed in both groups with the same drugs in induction and maintenance of anaesthesia. After anaesthesia induction and 20 minutes after CO2 insufflations, venous blood samples were obtained for measuring adrenalin, noradrenalin, dopamine and cortisol levels. Hemodynamic and respiratory parameters were recorded at the 1st, 5th, 15th, 30th and 45th minutes after the insertion of airway devices. ABSTRACT.RESULTS:: No statistically significant differences in age, body mass index, gender, ASA physical status, and operation time were found between the groups (p > 0.05). Changes in hemodynamic and respiratory parameters were not statistically significant when compared between and within groups (p > 0.05). Although no statistically significant differences were observed between and within groups when adrenalin, noradrenalin and dopamine values were compared, serum cortisol levels after CO2 insufflation in PLMA group were significantly lower than the ETT group (p = 0.024). When serum cortisol levels were compared within groups, cortisol levels 20 minutes after CO2 insufflation were significantly higher (46.1 (9.5-175.7) and 27.0 (8.3-119.4) in the ETT and PLMA groups, respectively) than cortisol levels after anaesthesia induction (11.3 (2.8-92.5) and 16.6 (4.4-45.4) in the ETT and PLMA groups, respectively) in both groups (p = 0.001). ABSTRACT.CONCLUSION:: PLMA usage is a suitable, effective and safe alternative to ETT in laparoscopic cholecystectomy patients with lower metabolic stress. BODY.INTRODUCTION: Surgical trauma causes reaction and damage in the organism in proportion to the extent of the operation. Triggering the physiologic mechanism off causes local inflammation and hypermetabolism process due to the general substrate mobilization and accelerated biochemical reaction.[1] It is also associated with complex stress response characterized by neuro-humoral, immunological and metabolic alterations.[2] Activation of sympathetic nervous system and increased release of catabolic and immuno-suppressive pituitary hormones can be attributable to surgical stress response.[3] In clinical practice, these activations cause changes in heart rate and blood pressure and alterations in biochemical measurements like noradrenalin, adrenalin, dopamine and cortisol levels. The type of surgical procedure, either open or laparoscopic, reflects the severity of traumatic stress imposed to the organism. Open surgery may develop much more stress than laparoscopic surgical procedures due to long surgical time, big surgical incision and local and systemic effects.[245] Endotracheal intubation has been demonstrated to impose the most intense stress to organism under general anaesthesia.[67] Proseal laryngeal mask airway (PLMA), a supraglottic airway device, is a modification of classical laryngeal mask airway (LMA) which was introduced to clinical practice in 2000. Having a gastric drainage system to provide a channel for regurgitated fluid, named gastric tube placement, and thus preventing gastric insufflation is the major difference between PLMA and classical LMA. PLMA is considered to cause minimal stress response as it is a supraglottic device, less invasive, used without muscle relaxants and used in short procedures.[89] In this study, we aimed to compare hemodynamic and metabolic alterations caused by stress response due to PLMA and endotracheal tube (ETT) usage in laparoscopic cholecystectomy. BODY.MATERIALS AND METHODS: After approval of the Ethics Committee and obtaining patients' informed consents, 63 ASA I-II patients, aged between 20-70 years scheduled for elective laparoscopic cholecystectomy, were included in the study. Exclusion criteria were a known or predicted difficult airway, hypertension, diabetes mellitus, chronic obstructive pulmonary disease, cardiac disease, history of allergic reaction, history of corticosteroid drug usage, and body mass index greater than 35 kg/m2. Forty-five minutes before operation, 0.07 mg/kg intramuscular midazolam was given to the patients. Routine monitoring including electrocardiogram, noninvasive blood pressure and pulse oximetry were applied to each patient in the operating room. Standard general anaesthesia was performed in both groups by using 4-7 mg/kg sodium thiopental, 1-2 μg/kg fentanyl citrate and 0.1 mg/kg vecuronium bromide during induction. Patients were randomly allocated into two groups. Randomization was performed before anaesthesia by using a sealed opaque envelope with a computer-generated block random allocation. In the ETT group (n = 31), endotracheal intubation was performed. Endotracheal tubes numbers 7 and 7.5 were used in females and numbers 8 and 8.5 in males. ETT placement was defined as difficult if a guide was used or if cricoid pressure was applied. The cuff pressure of ETT was maintained in a range of 25-30 cm H2O by using a cuff manometer. In the PLMA group (n = 32), PLMA (Laryngeal Mask Company, Henley-on-Thames, UK) was inserted by using index finger insertion method. PLMA 3 and 4 were used in females and PLMA 5 in males. PLMA insertion was defined as easy if insertion within the pharynx was completed without resistance through a single manoeuvre. However, in case of any resistance to insertion or more than one necessary manoeuvre, the procedure was considered as difficult. The position of PLMA was confirmed by bilateral chest movements, a square waveform on capnography, chest auscultation, expired tidal volume > 8 ml/kg and insertion of nasogastric tube and aspiration of gastric contents. The cuff pressure of PLMA was maintained at 60 cm H2O. ETT and PLMA insertions were performed by the same anaesthetist who had an anaesthetic experience more than 3 years and had successfully used classical LMA more than 500 times and PLMA for at least 100 times. Sevoflurane 1-2% and 50% NO2 + 50% O2 were used in the maintenance of anaesthesia. Patients were mechanically ventilated with a tidal volume of 8 ml/kg and a respiratory rate of 12 beat/min. Oxygen saturation (SpO2) and end-tidal carbon dioxide (EtCO2) were adjusted to > 95% and < 45 mmHg, respectively. If SpO2 < 95%, the fraction of inspired oxygen (FiO2) and tidal volume were increased respectively. If EtCO2 > 45 mmHg, respiratory rate was increased. Intra-abdominal pressure was maintained < 15 mmHg during the operation. Mean arterial blood pressure (MAP), heart rate (HR), SpO2, airway pressures especially peak airway pressure, EtCO2 and tidal volume were recorded at the 1st, 5th, 15th, 30th and 45th minutes after the insertion of airway devices. Side effects like bronchospasm, laryngospasm, coughing, gagging, hoarseness, and aspiration were also evaluated. Hemodynamic alterations like hypotension (> 25% decrease in MAP from the baseline), hypertension (> 25 % increase in MAP from the baseline), tachycardia (heart rate > 120 beat/min), and bradycardia (heart rate < 50 beat/min) were recorded during the operation and manipulated by titration of inhalation anaesthetics at 0.5% concentrations, volume infusion and administration of intravenous 0.5 mg atropine sulphate. After induction of anaesthesia and 20 minutes after CO2 insufflation, venous blood samples were obtained for measuring adrenalin, noradrenalin, dopamine and cortisol levels. Blood samples were centrifuged immediately and plasmas were stored at -80°C until hormonal analysis. For analyzing catecholamine levels, high pressure liquid chromatography (HPLC) method was applied using Eureka kits (Enrico Fermi 25 60033 Chiaravalle (An) Italy). Serum cortisol levels were measured by Modular Analytics E170 Module (Roche Diagnostics, Indianapolis, USA). Cortisol levels were measured in 10 patients of each group before the procedure and the values were found to be 17.74 ± 13.8 in the ETT and 16.03 ± 12.6 in the PLMA groups. Therefore, with a 100% increase in the ETT and a 50% increase in the PLMA groups, a preliminarily sample size of 25 patients per group with a type 1 error of 0.05 and a type 2 error of 0.20 was calculated. Statistical analyses was performed by SPSS11.5 (Statistics Package for Social Sciences) for Windows. Data was presented as mean ± standard deviation, median and frequency (%). Comparisons between groups were performed by unpaired student t-test and Mann-Whitney U-test for continuous variables and chi-square test for intermittent variables. Paired sample t-test and Wilcoxon test were used for comparisons within each group. A p value less than 0.05 was considered as statistically significant. BODY.RESULTS: No statistically significant differences were found between the groups when demographic data was taken into account (p > 0.05) (Table 1). Duration of operation was 46 ± 11.26 minutes and 45.43 ± 13.78 minutes in the ETT and PLMA groups, respectively (p = 0.875). ETT number 7 was placed for 7, number 7.5 for 20 and number 8.5 for 8 patients. PLMA size 3 was inserted for 19, size 4 for 11 and size 5 for 2 patients. First attempt success rate was 90.32% and 93.75% in the ETT and PLMA groups, respectively. Second attempt success rate was 9.645 in the ETT group and 6.25% in the PLMA group. Respiratory parameters like SpO2, peak airway pressure, EtCO2, and tidal volume were not statistically significantly different when compared between and within groups at all evaluation times (p > 0.05) (Table 2). Peak airway pressures were slightly increased in both groups after carboperitoneum, but it did not disrupt ventilation. Ventilation was optimal (EtCO2 < 45 mmHg) in all patients in both groups before and after CO2 insufflation. Changes in hemodynamic parameters were not statistically significant when compared between and within groups at all evaluation times (p > 0.05) (Table 3). Table 1 Demographic parameters Table 2 Respiratory parameters Table 3 Hemodynamic parameters Although no statistically significant difference was observed between and within groups when adrenalin, radrenalin and dopamine values were compared, serum cortisol level after CO2 insufflation in the PLMA group was significantly lower than the ETT group (p = 0.024) (Table 4). When serum cortisol levels were compared within groups, cortisol levels 20 minutes after CO2 insufflation were significantly higher than cortisol levels after anaesthesia induction in both groups (p = 0.001) (Table 5). Table 4 Comparison of stress hormones between groups Table 5 Comparison of stress hormones within groups BODY.DISCUSSION: Achieving a safe and effective airway is the principal aim of the anaesthesiologists. During laparoscopic cholecystectomy, safe airway management is necessary because intrathorasic pressure increases due to increased intra-abdominal pressure, gastroesophageal and biliary reflux which may in turn be a result of obesity and chronic systemic illnesses of the patients. ETT is considered as the safest anaesthetic procedure for laparoscopic cholecystectomy. However, it has also some intraoperative and postoperative disadvantages like laryngospasm, hoarseness, and sore throat. In addition, supraglottic compression during laryngoscopy procedure increases systolic and diastolic blood pressure.[9] Maltby et al. postulated that laparoscopic surgery was an important test for evaluating the effectiveness of supraglottic airway devices which were used in positive pressure ventilation.[10] It is already known that LMA is not safe in some procedures like laparoscopic cholecystectomy, as the patients have high intra-abdominal pressure which may cause gastroesophageal reflux and aspiration. Lu et al. compared PLMA and LMA in laparoscopic cholecystectomy patients and found PLMA to be a more effective ventilatory device for laparoscopic cholecystectomy than LMA. They did not recommend LMA for laparoscopic cholecystectomy.[11] Evans et al.[12] and Keller et al.[13] researched whether PLMA could prevent the aspiration of regurgitated fluid or not. They found that a correctly placed PLMA allowed fluid in the esophagus to bypass the pharynx and mouth when the drainage tube was open and thus provided a safe airway management. According to the recommendations of the abovementioned studies, we preferred to use PLMA in the current study to gain a safe and intact airway. We inserted nasogastric drainage tube in our PLMA and ETT groups and did not observe gastric distension and aspiration of gastric content due to gastric regurgitation in any patient of our study. Lalwani et al. evaluated PLMA as an alternative to ETT in pediatric patients for short duration surgical procedures. They found hemodynamic responses to insertion of PLMA to be lower than ETT.[14] Piper et al. compared PLMA and ETT in gynaecologic laparoscopies and concluded that PLMA was a safe and effective ventilation device with low stress imposed to patient and low occurrence of pharyngeal and laryngeal complications.[15] Lim et al. compared PLMA and ET intubation on laparoscopic gynaecologic procedures and concluded that PLMA as effective as ETT with an attenuated hemodynamic response.[16] In a multicenter study, Evans et al. found that PLMA caused no detectable responses to insertion and heart rate and mean arterial pressure decreased after insertion.[17] In the current study, no adverse changes in hemodynamic variables were seen in either group. Although mean arterial blood pressure slightly increased in some of the patients in the ETT group after laryngoscopy, these increases were not statistically significant. Maltby et al. assessed if LMA and PLMA were good alternatives to ETT in laparoscopic gynaecology procedures with respect to pulmonary ventilation. There were no statistically significant differences among the groups for SpO2, EtCO2, and airway pressure before or during peritoneal insufflation. They recommended that ETT could safely be substituted with correctly placed LMA and PLMA in gynecologic laparoscopy.[10] Lalwani et al. found no significant differences in SpO2 and EtCO2 levels between their PLMA and ETT groups in pediatric patients.[14] Sinha et al. compared ETT and PLMA in pediatric laparoscopy patients by means of EtCO2, peak inspiratory pressure and SpO2. They did not report any statistically significant differences in SpO2, EtCO2 and positive inspiratory pressure between the groups. They concluded that the two devices had comparable ventilator efficacy in laparoscopic pediatric surgeries.[18] Similar to the above research, synchronous evaluations of SpO2 and EtCO2 levels affecting hemodynamic and stress responses were not significantly different between the groups in our study. Although increases in EtCO2 levels after CO2 insufflation were seen in both groups, they did not cause considerable changes in hemodynamic parameters. EtCO2 increased in the PLMA group similar to the ETT group which indirectly implied PLMA to be as efficient airway device as ETT. In our study, we administered the same drugs in both groups. Since comparison of the effects of anaesthetic agents on stress hormones was not our aim, we did not assess the effects of drugs. Although general anaesthesia was performed to all of our patients, increases in cortisol levels were significantly lower in the PLMA group than in the ETT group. Therefore, the important factor was not the general anaesthesia but the way it was applied. Walder and Aitkenhead evaluated if pneumoperitoneum affected hemodynamic and stress responses in laparoscopic cholecystectomy patients. They observed that although plasma vasopressin concentration significantly increased during pneumoperitoneum, noradrenalin and adrenalin concentrations, and renin activity did not change with pneumoperitoneum.[19] CO2 insufflation for pneumoperitoneum in long periods may cause stress hormone increase due to CO2 diffusion. In our study, mean duration of operation was nearly 45 minutes in both groups. Therefore, it is considered that the reason of increase in cortisol level only in the ETT group was not CO2 insufflation. Lentschener et al. found that both humoral and hemodynamic responses initiated in the pneumoperitoneum by contact with CO2 have been prevented by continuous adequate depth of anaesthesia and normovolaemia.[20] In the current study, the patients were operated in elective circumstances and depth of anaesthesia and volume were controlled easily and strictly so humoral and hemodynamic responses caused by pneumoperitoneum were lower than the estimations. We observed an increase in serum cortisol levels in the PLMA group after CO2 insufflation. In addition, cortisol levels 20 minutes after CO2 insufflation were significantly higher than the first measurements in both groups. As dopamine, adrenalin and noradrenalin have short duration of activity with the plasma half time shorter than 2 minutes, they were not efficient enough for reflecting the metabolic stress response in our study. Thus, more sensitive new parameters and challenges need to be considered in addition to present laboratory tests. BODY.CONCLUSION: Correctly placed PLMA is an efficient and safe airway option to ETT in laparoscopic cholecystectomy operations with lower incidence of metabolic stress responses and postoperative complications.

TITLE: Randomized Phase Ib/II Study of Gemcitabine Plus Placebo or Vismodegib, a Hedgehog Pathway Inhibitor, in Patients With Metastatic Pancreatic Cancer ABSTRACT.PURPOSE: Sonic hedgehog (SHH), an activating ligand of smoothened (SMO), is overexpressed in > 70% of pancreatic cancers (PCs). We investigated the impact of vismodegib, an SHH antagonist, plus gemcitabine (GV) or gemcitabine plus placebo (GP) in a multicenter phase Ib/randomized phase II trial and preclinical PC models. ABSTRACT.PATIENTS AND METHODS: Patients with PC not amenable to curative therapy who had received no prior therapy for metastatic disease and had Karnofsky performance score ≥ 80 were enrolled. Patients were randomly assigned in a one-to-one ratio to GV or GP. The primary end point was progression-free-survival (PFS). Exploratory correlative studies included serial SHH serum levels and contrast perfusion computed tomography imaging. To further investigate putative biologic mechanisms of SMO inhibition, two autochthonous pancreatic cancer models (KrasG12D; p16/p19fl/fl; Pdx1-Cre and KrasG12D; p53R270H/wt; Pdx1-Cre) were studied. ABSTRACT.RESULTS: No safety issues were identified in the phase Ib portion (n = 7), and the phase II study enrolled 106 evaluable patients (n = 53 in each arm). Median PFS was 4.0 and 2.5 months for GV and GP arms, respectively (95% CI, 2.5 to 5.3 and 1.9 to 3.8, respectively; adjusted hazard ratio, 0.81; 95% CI, 0.54 to 1.21; P = .30). Median overall survival (OS) was 6.9 and 6.1 months for GV and GP arms, respectively (95% CI, 5.8 to 8.0 and 5.0 to 8.0, respectively; adjusted hazard ratio, 1.04; 95% CI, 0.69 to 1.58; P = .84). Response rates were not significantly different. There were no significant associations between correlative markers and overall response rate, PFS, or OS. Preclinical trials revealed no significant differences with vismodegib in drug delivery, tumor growth rate, or OS in either model. ABSTRACT.CONCLUSION: The addition of vismodegib to gemcitabine in an unselected cohort did not improve overall response rate, PFS, or OS in patients with metastatic PC. Our preclinical and clinical results revealed no statistically significant differences with respect to drug delivery or treatment efficacy using vismodegib. BODY.INTRODUCTION: Pancreatic cancer (PC) is the fourth leading cause of cancer mortality in the United States, with 38,460 deaths annually.1 Five-year survival for all stages combined is only 6%. Gemcitabine had beenthe backbone treatment for years in advanced disease,2 until the introduction of FOLFIRINOX (fluorouracil, leucovorin, irinotecan, and oxaliplatin)3 and gemcitabine plus albumin-bound nab-paclitaxel4 regimens, both reported after initiation of our trial. Despite numerous attempts, most gemcitabine combinations with molecularly targeted therapies have failed to demonstrate a significant improvement in overall survival (OS),5–8 with the exception of gemcitabine plus erlotinib, which has demonstrated a statistically significant but clinically modest benefit.9 Vismodegib (Erivedge; Genentech, South San Francisco, CA), a synthetic small-molecule inhibitor of smoothened (SMO) in the hedgehog (Hh) pathway,10,11 has demonstrated clinical benefit in basal cell carcinoma and medulloblastoma—both harboring recurrent Hh pathway mutations in SMO or protein patched homolog 1 (PTCH1).12,13 Vismodegib is US Food and Drug Administration approved for the treatment of patients with advanced basal cell carcinoma. Trials applying various SMO inhibitors to other tumors harboring genomic activation of the Hh signaling pathway are under way, some within novel clinical trial designs.14 However, no single-agent activity was observed in early phase I studies within molecularly unselected patients with advanced and pretreated PC.15,16 Nevertheless, the Hh pathway has been reported to be critical for tumor progression in preclinical PC models17,18 and has been considered a potential therapeutic target.19–21 Sonic Hh (SHH) is overexpressed in approximately 70% of PCs and has been shown to be an early and late mediator of PC tumorigenesis.22–24 Tumor-derived SHH has influenced and promoted tumor growth in preclinical PC systems by activating Hh signaling in stroma.25–28 This paracrine Hh signaling may establish and maintain the desmoplastic stroma observed in PC, creating a barrier for proper drug penetration.29 Hh pathway inhibition with IPI-269609, an SMO inhibitior,30–32 reportedly led to increased tumor perfusion, enhanced tumor delivery of gemcitabine when coadministered, and improvement in survival in a genetically engineered murine PC model,29 forming the rationale for human clinical trials in PC. Given this background, we hypothesized that inhibition of the Hh pathway would be synergistic with gemcitabine and would lead to improved progression-free survival (PFS) compared with gemcitabine alone for metastatic PC.33 This article reports the final results of a phase Ib (n = 7) and multicenter randomized phase II trial (n = 106) comparing gemcitabine plus vismodegib (GV) with gemcitabine plus placebo (GP) in patients with advanced PC; there were no significant differences in overall response rate (ORR), PFS, or OS between these two groups. To further investigate the putative biologic mechanisms of SMO inhibition, we used two autochthonous PC models (KrasG12D; p16/p19fl/fl; Pdx1-Cre [KPP] and KrasG12D; p53R270H/wt; Pdx1-Cre [KR]) to recapitulate the phase II clinical trial. In contrast to recent reports with IPI-926 (saridegib),29,34–36 our preclinical and clinical results with vismodegib are concordant and demonstrate no statistically significant differences with respect to tumor growth, drug delivery, or treatment efficacy. BODY.PATIENTS AND METHODS: The Data Supplement provides detailed information on methods. BODY.RESULTS.CLINICAL TRIAL.PATIENT CHARACTERISTICS.: Seven patients were enrolled onto the phase Ib open-label GV portion, and no safety issues were identified. For the randomized phase II part of the trial, 111 patients were enrolled at 13 sites between February 2010 and June 2012, stratified by Karnofsky performance score (80 v 90 or 100) and disease status (newly diagnosed v recurrent; Fig 1). Of these, four patients withdrew consent before starting treatment (two from each arm), and one patient (randomly assigned to GV) was subsequently found to have been ineligible and never started therapy. Analyses were based on the remaining 106 patients. Patient characteristics were similar between treatment arms, except for the incidence of peritoneal metastases, which were higher in the GP arm (9% v 23%; Table 1). Fig 1.CONSORT diagram of clinical trial enrollment and treatment in phase II trial. gem, gemcitabine. Table 1. Baseline Patient Demographic and Clinical Characteristics Characteristic No. (%) GV (n = 53) GP (n = 53) Total (N = 106) Age, years     Median 64 64 64     Range 49-82 39-84 39-84 Sex     Male 31 (58) 27 (51) 58 (55)     Female 22 (42) 26 (49) 48 (45) Race     White 40 (77) 45 (88) 85 (83)     African American 10 (19) 6 (12) 16 (16)     Asian 1 (2) 0 (0) 1 (1)     Other 1 (2) 0 (0) 1 (1)     Missing 1 2 3 Karnofsky performance score     100 19 (36) 17 (32) 36 (34)     90 14 (26) 20 (38) 34 (32)     80 20 (38) 16 (30) 36 (34) Disease status at enrollment     Newly diagnosed 48 (91) 48 (91) 96 (91)     Recurrent metastatic 5 (9) 5 (9) 10 (9) Primary tumor location     Head 23 (43) 24 (46) 47 (45)     Neck/uncinate 1 (2) 2 (4) 3 (3)     Body 16 (30) 11 (21) 26 (25)     Tail 13 (25) 13 (25) 26 (25) Site of metastasis *     Liver 41 (77) 44 (83) 85 (80)     Lung 12 (23) 14 (26) 26 (25)     Peritoneum 5 (9) 12 (23) 17 (16)     Other 8 (15) 10 (19) 18 (17) Crossover to GV 22 (42) Abbreviations: GP, gemcitabine plus placebo; GV, gemcitabine plus vismodegib. * Patients may have > one primary or metastatic site. BODY.RESULTS.CLINICAL TRIAL.SAFETY.: Median number of cycles was four (range, one to 12 cycles) in the GV arm and three (range, zero to 14 cycles) in the GP arm. Combination therapy with GV was generally well tolerated and did not result in unexpected toxicities (Table 2). There were no statistically significant differences in the rate of adverse events (AEs) between the arms. Four patients in the GV arm and two in the GP arm withdrew from treatment as a result of AEs. Sixteen patients (GV, n = 10; GP, n = 6) died while receiving treatment. Table 2. Grade 3 to 5 Toxicities at Least Possibly Related to Treatment Toxicity No. (%) P GV (n = 53) GP (n = 53) Neutropenia 15 (28) 12 (23) .66 Fatigue 7 (13) 4 (8) .53 Anorexia 5 (9) 2 (4) .44 Vomiting 5 (9) 2 (4) .44 WBC decreased 5 (9) 7 (13) .76 Platelet count decreased 6 (11) 5 (9) 1.0 Anemia 4 (8) 7 (13) .53 Nausea 4 (8) 3 (6) 1.0 Elevated AST 5 (9) 3 (6) .72 Elevated ALT 3 (6) 2 (4) 1.0 Blood bilirubin increased 3 (6) 1 (2) .62 Hyperglycemia 3 (6) 3 (6) 1.0 Hypokalemia 4 (8) 2 (4) .68 Alkaline phosphatase increased 2 (4) 2 (4) 1.0 Lymphocyte count decreased 2 (4) 5 (9) .44 Hyponatremia 1 (2) 5 (9) .20 Abbreviations: GP, gemcitabine plus placebo; GV, gemcitabine plus vismodegib. BODY.RESULTS.CLINICAL TRIAL.EFFICACY.: ORRs were similar in the two arms (GV: complete response [CR], 0 [0%]; partial response [PR], 4 [8%]; stable disease [SD], 27 [51%]; disease control [ie, CR + PR + SD], 31 [58%]; GP: CR, 1 [2%]; PR, 6 [11%]; SD, 20 [38%]; disease control, 27 [51%]). The difference in response rates (8% v 13%) was not significant (P = .53; Data Supplement). The primary end point of the study was PFS. At the final analysis, events (progression or death) occurred in 48 patients (91%) receiving GV and 51 (95%) receiving GP. Median PFS was 4.0 months for GV and 2.5 months for GP (adjusted hazard ratio [HR], 0.83; 95% CI, 0.55 to 1.23; Fig 2A; Data Supplement). Fig 2.(A) Progression-free and (B) overall survival by treatment arm. Blue, gemcitabine plus vismodegib; gold, gemcitabine plus placebo. Hazard ratio (HR) after adjusting (adj) for Karnofsky performance score and disease status (newly diagnosed v recurrent). Median OS was 6.9 months for GV and 6.1 month for GP (adjusted HR, 0.96; 95% CI, 0.64 to 1.44; Fig 2B; Data Supplement). No survival differences were noted in a preplanned secondary analysis of OS that censored patients receiving GP at first progression, before crossover to GV (P = .69). Note that patient crossover did not affect the primary end point (ie, PFS), because crossover happened after the event occurred. For patients receiving GP who crossed over at progression (n = 22 [42%]), median PFS was 1.8 months, and median OS was 2.9 months (Data Supplement). One-year survival rates in the GV and GP arms were 15% and 25%, respectively (P = .3). OS and PFS did not differ significantly by Karnofsky performance score (P = .66 and .42, respectively; Data Supplement). Mortality and disease progression rates were consistent and uniformly high across all centers. BODY.RESULTS.CLINICAL TRIAL CORRELATIVE RESULTS.SHH SERUM LEVELS.: Median pretreatment plasma SHH level pooled for both treatment arms was 1.01 ng/mL (GV arm, 1.01 ng/mL; GP arm, 1.06 ng/mL). SHH levels did not change significantly with subsequent cycles (P = .087), nor was there a difference between treatment groups (P = .85) or patients with cancer (n = 89) and normal controls (n = 40; P = .4) (Figs 3A and 3B). SHH serum levels did not correlate with age in either patients with cancer (r = 0.17; P = .13) or controls (r = 0.03; P = .87; Figs 3C and 3D). Fig 3.Clinical trial translational correlatives. Serum SHH levels (A) comparing controls (n = 40) with patients with pancreatic cancer enrolled onto trial (n = 89), (B) by treatment group (gemcitabine plus vismodegib [GV] or gemcitabine plus placebo [GP]) with increasing treatment (TX) cycle, and association with age in (C) patients with cancer and (D) controls. (E) Radiologic correlatives evaluating association of baseline tumor perfusion with tumor response to therapy. BODY.RESULTS.CLINICAL TRIAL CORRELATIVE RESULTS.RADIOLOGIC TUMOR PERFUSION.: By univariable analysis, baseline computed tomography (CT) perfusion of the primary tumor was not associated with response to therapy, expressed as percent change in tumor size (r = −0.09; P = .77; Fig 3E; Data Supplement). BODY.RESULTS.MURINE TRANSLATIONAL CORRELATIVES.SMO INHIBITION DOES NOT REVEAL QUANTIFIABLE CHANGES TO TUMOR STROMA IN VIVO.: Previous preclinical studies have suggested that SHH is abnormally expressed in genetically engineered models of PC.29,37 SHH expression and pathway activation were confirmed in the KPP genetically engineered mouse model of PC by transcriptional gene analysis and immunohistochemistry. SHH immunoreactivity exhibited focal staining throughout tumors, predominantly in mucinous and well-defined ductal epithelial cells (data not shown), consistent with previous reports.26 Comparative transcriptional analysis of normal pancreas and tumor revealed significantly elevated expression of Hh ligands SHH and Indian Hh (IHH), as well as SMO, glioma-associated oncogene family zinc finger 1 (GLI1), GLI2, Hh Interacting Protein (HHIP), and PTCH1 (Data Supplement). To determine whether inhibition of SMO leads to changes in tumor stroma, we used a small molecule, HhAntag,38 a potent orally available preclinical surrogate of the US Food and Drug Administration–approved vismodegib (GDC-0449).10 Previous reports have shown that antagonism of Hh signaling resulted in increased mean microvessel density (MVD).29 HhAntag treatment on tumor-bearing KPP animals revealed no significant change in MVD (Fig 4A; P = .54; Data Supplement), despite observing pathway inhibition (Data Supplement). Moreover, HhAntag treatment did not affect intratumoral extracellular matrix deposition assessed through trichrome staining (Fig 4B; P = .29). Fig 4.Effects of hedgehog pathway antagonism (HhAntag) on vasculature, stromal content, and intratumoral gemcitabine metabolites in KrasG12D; p16/p19fl/fl; Pdx1-Cre (KPP) tumors. (A) Quantitation of immunohistochemical staining for meca-32 expression in pancreatic tumors of KPP mice treated with vehicle (circles; n = 13) or smoothened (SMO) inhibitor (squares; n = 13) for 10 days. Data presented as percentages of meca-32–positive areas over analyzed tumor areas for each tumor (Mann-Whitney P = .54; scale bar, 200 um). (B) Quantitative analysis of stromal content by trichromatic stain in pancreatic tumors of KPP mice treated with vehicle or SMO inhibitor for 10 days. Data presented as percentages of positive stain areas over analyzed tissue areas (Mann-Whitney P = .29). (C) Mass spectromic quantitation of intratumoral concentration of 2′,2′-difluorodeoxycytidine triphosphate (dFdCTP; active form of gemcitabine) from each tumor after treatment for 10 consecutive days with vehicle or SMO inhibitor and gemcitabine 50 mg/kg 30 minutes before tumor collection (Mann-Whitney P = 1.000). (D) Ratios of 2′,2′-difluoro 2′-deoxycytidine (dFdC) to diflurodeoxyuridine (dFdU) in pancreatic tumors from each tumor (Mann-Whitney P = .48). NS, not significant. BODY.RESULTS.MURINE TRANSLATIONAL CORRELATIVES.SMO INHIBITION DOES NOT AFFECT GEMCITABINE DRUG DELIVERY IN VIVO.: The modest response rates observed in patients with PC treated with gemcitabine has, in part, been attributed to poor drug delivery associated with a prohibitive tumor microenvironment. Gemcitabine undergoes intracellular conversion, from 2′,2′-difluoro 2′-deoxycytidine (dFdC) to the active triphosphate form 2′,2′-difluorodeoxycytidine triphosphate (dFdCTP), responsible for inhibition of DNA synthesis and repair. To directly measure drug delivery, dFdCTP levels were analyzed in PC tumors of KPP mice after continuous 10-day HhAntag treatment and a single dose of gemcitabine. Liquid chromatography–tandem mass spectrometry analysis revealed that dFdCTP concentrations were similar in both vehicle- and HhAntag-treated KPP mice (Fig 4C; Mann-Whitney P = 1.00). Intracellular gemcitabine is also converted to an inactivated form, diflurodeoxyuridine (dFdU). The ratio of the unprocessed form (dFdC) to inactive form (dFdU) estimates intratumoral gemcitabine exposure. The average ratio of dFdC to dFdU in KPP tumors of HhAntag-treated mice was not significantly different between vehicle and SMO inhibitor groups (Fig 4D; P = .48). Together, these results indicated that HhAntag did not increase gemcitabine delivery in this genetically engineered PC mouse model. BODY.RESULTS.MURINE TRANSLATIONAL CORRELATIVES.PANCREATIC TUMOR PROGRESSION AND OS REMAIN UNALTERED BY HH PATHWAY INHIBITION.: Next, we investigated the therapeutic impact of HhAntag in KPP mice. Growth rates of tumors, as determined by serial ultrasound imaging, were not significantly different when comparing HhAntag and vehicle arms, indicating no single-agent effect of Hh pathway inhibition (Figs 5A and 5B). Although the combination of gemcitabine plus HhAntag significantly decreased tumor growth relative to control treatment (log-rank P = .0052), the growth rate was not different from that of gemcitabine alone, known to affect tumor growth in this model.39 Consistent with the lack of tumor growth effect, the HhAntag plus gemcitabine combination did not provide significant improvement in OS when compared with gemcitabine alone (Fig 5C). In summary, the addition of HhAntag did not affect tumor growth or animal longevity in the KPP model. Fig 5.Smoothened (SMO) inhibitor does not affect tumor progression or overall survival in Kras LSL-G12D; p16/p19 fl/fl; Pdx1-Cre (KPP) mice. (A) Individual tumor growth rates plotted by from serial ultrasound images as volumes depicted longitudinally by animal within each regimen. (B) Antilogged values of slopes in each longitudinal plot are graphed, and average tumor burden fold changes per day in each study group of KPP mice are shown, with approximate 95% CIs (vehicle v SMO inhibitor, P = .86; vehicle v combination, P = .0156; gemcitabine v combination, P = .18) (C) Kaplan-Meier plots of KPP mice treated with vehicle (blue, n = 15; median, 1.9 weeks), SMO inhibitor (gold, n = 16; median, 1.2 weeks), gemcitabine (gray, n = 14; median, 3.8 weeks), and gemcitabine plus SMO inhibitor combination (red, n = 12; median, 3.4 weeks; gemcitabine v vehicle, P = .0059; combination v vehicle, P = .0179; gemcitabine v combination, P = .10 [all P values from log-rank test]). HhAntag, hedgehog pathway antagonism. BODY.RESULTS.MURINE TRANSLATIONAL CORRELATIVES.ANALYSIS OF SMO INHIBITION IN KR MICE.: Although the KPP model represents the genetics of a subset of the PC population, previous findings were generated in a model composed of mutant KRAS and mutant p53 expression in the pancreas.40 Therefore, we reproduced all in vivo studies in the KR model. HhAntag treatment on tumor-bearing KR animals revealed a significant decrease in MVD using meca-32 (Data Supplement; P = .0418) and trend toward decrease using CD31 (Data Supplement; P = .29). Trichrome staining revealed no significant changes to the stroma of HhAntag-treated tumors (Data Supplement; P = .83). Together these findings indicated that as in the previous KPP model, the stroma of the developed KR tumors was not significantly affected by HhAntag treatment. Finally, we observed no significant change in concentration of the gemcitabine metabolite dFdCTP in tumors after HhAntag (Data Supplement; P = .25) and no significant change in the ratio of dFdC to dFdU (Data Supplement). Furthermore, neither HhAntag nor the gemcitabine plus HhAntag combination affected tumor growth rates in KR animals compared with gemcitabine alone (Data Supplement). Consistent with the tumor growth study, HhAntag did not lead to survival benefit over vehicle or gemcitabine treatment (Data Supplement). Taken together, neither of the murine PC model genotypes (KPP or KR) demonstrated any measurable differences between gemcitabine delivery to tumors, tumor growth rate, or OS with HhAntag treatment. A significant decrease in microvessel density, consistent with previous work in transplantable models using vismodegib, was observed.41 BODY.DISCUSSION: Effective molecularly targeted treatment for advanced PC remains an unmet need. When we began this trial, extensive preclinical evidence provided rationale for the clinical evaluation of Hh pathway inhibition along with concurrent gemcitabine treatment.19,22,24,25,29,32 The addition of vismodegib to gemcitabine in this clinical study was well tolerated, no unexpected toxicities were observed, and there were no significant toxicity differences compared with the GP arm (Table 2). Unfortunately, this trial failed to meet its primary objective of a statistically significant improvement in median PFS, achieving a median PFS of 4.0 and 2.5 months in the GV and GP arms, respectively (HR, 0.83; 95% CI, 0.55 to 1.23). It should be noted, however, that the study was powered (85%) to detect an HR of 0.6, and therefore, a smaller effect, if present, may not have been detectable. Similarly, there were no differences between arms in median OS (6.9 v 6.1 months) or disease control rate (58% v 51%). Despite a recent report suggesting that serum SHH levels were decreased in patients with PC compared with age-matched controls, we did not observe this association.42 Moreover, we hypothesized that serum SHH might change over time, potentially differentially with or without exposure to vismodegib, but as noted, this was not observed either. Consistent with these findings, Kim et al43 recently reported no change in tumor SHH protein expression in matched biopsies from patients treated for 3 weeks with vismodegib. To investigate the discrepancy between our clinical data and previous reports,24,25,29 we sought to assess baseline primary pancreatic tumor perfusion and its association with response to therapy. Perfusion CT is a clinical technique used to provide regional maps and obtain quantitative measurements of hemodynamic parameters on the basis of the linear relationship between CT enhancement and iodinated contrast material concentration.44,45 Whole-organ perfusion of the pancreas using dynamic contrast-enhancement imaging revealed significantly lower perfusion in the tumor compared with adjacent normal pancreatic tissue.46 In addition, enhancement patterns of PCs on conventional dynamic multidetector row CT correlated with degree of angiogenesis, and these patterns were reportedly modified by degree of fibrosis.47 However, we did not observe a correlation between higher baseline tumor perfusion and improved treatment response to gemcitabine (pooled analysis, GV + GP) in this univariable analysis of a small exploratory cohort. Because there were no significant differences in clinical outcomes between the GV and GP arms, evaluation of serial changes in perfusion over time was not performed. Much of the preclinical work supporting Hh inhibition for PC was performed with IPI-296 (saridegib).29,31 It is noteworthy that a randomized phase II trial of saridegib in PC, conducted simultaneously with our trial, was halted at interim analysis in January 2012 because of worse median PFS and median OS compared with the placebo arm.36 A recent report attempting to discern the preclinical29 to clinical36 discrepancy suggested that prolonged exposure to IPI-296 before frank tumor development (PanIN stage) ultimately led to tumors with undifferentiated histology, increased vascularity, and heightened proliferation.34 Importantly, clinical correlates to confirm the original29 or newly reported34 preclinical findings, including increased vascularity, improved drug delivery, and worsened disease resulting from IPI-296 treatment, are still lacking or unreported. Subsequently, a similar preclinical study evaluating vismodegib treatment, also at the tumor precursor stage (PanIN), reported accelerated tumor progression.35 Here, further support for the critical role of stroma early in pancreatic tumor formation was provided with elegant studies, where SHH was genetically deleted coincident with tumor suppressor loss and oncogene activation. In the absence of epithelial-derived SHH secretion, the resulting pancreatic tumors were phenotypically distinct, devoid of desmoplastic stroma, more vascularized, and more proliferative than controls.34,35 The early treatment design of these experiments is consistent with a large body of work describing the preventive nature of stroma in early tumor formation,48–50 but it does little to reconcile the preclinical and clinical discrepancy with Hh inhibition in established pancreatic tumors. The discordance between the preclinical29 (benefit) and clinical36 (detriment) results with IPI-926 in established tumor scenarios may be a result of overinterpretation of the preclinical gemcitabine model data,29 because small yet statistically significant effects do not ensure a biologically meaningful effect in patients. This lack of predictive correlation contrasts the strong predictive value of preclinical work in models harboring mutationally driven Hh pathway signaling.51 Patient-derived xenograft models may also promote better understanding in the future. Patients with PC frequently present with advanced disease, so to model the clinical treatment scenario, we treated two independent, genetically engineered PC models when defined, measurable tumors were readily detectable. Not only did SHH pathway inhibition not improve survival, no measurable changes in gemcitabine delivery or tumor growth rate were observed in either murine model. An increase in vascular density was not observed with vismodegib treatment, in contrast to IPI-926.29 In our study, HhAntag treatment showed a significant or trending decrease in microvessel density, depending on the marker used, which is consistent with previous work in preclinical tumor models where vismodegib treatment reduced tumor growth and mean vessel density.41 It is also possible that the preclinical efficacy differences may be attributed to unique molecular properties of the agents, given that IPI-926 is a cyclopamine derivative, whereas GDC-0449 (vismodegib) is a synthetic inhibitor of SMO. For example, although both molecules occupy a similar pocket within the transmembrane domain of SMO, they may mechanistically diverge, because cyclopamine treatment leads to accumulation of SMO in the primary cilium, whereas vismodegib prevents it.52,53 Moreover, cyclopamine has been shown to have off-target effects that may lead to enhanced toxicity and/or potentially cause the stromal effects observed. Notably, a clinical trial with IPI-926 in PC was stopped because of worse survival outcome in the investigational arm.36 In our study, we did not observe increased toxicity or a detriment in survival with GV. Nevertheless, neither molecule demonstrated statistically significant improvement when combined with gemcitabine in PC. In conclusion, we found no benefit in adding vismodegib to gemcitabine in this randomized phase II trial of molecularly unselected patients with metastatic PC; we corroborated these findings in two independent genetic murine PC models (KPP and KR). Given the discrepant results between the two tested SMO inhibitors, their selectivity may uniquely diverge, where off-target effects might explain altered outcomes. Importantly, our preclinical findings were ultimately concordant with the outcome of our clinical trial. Given that newer chemotherapy regimens have demonstrated benefit over gemcitabine, a number of ongoing trials are evaluating the role vismodegib in PC using these more active cytotoxic backbones.54

TITLE: P02.23. The efficacy of prolotherapy using dextrose-morrhuate for lateral epicondylosis: a pilot randomized controlled trial ABSTRACT: BODY.PURPOSE: Chronic lateral epicondylosis (CLE) is common, expensive and debilitating. A substantial number of patients are refractory to existing therapy and "watchful waiting." Prolotherapy is a CAM injection therapy for chronic musculoskeletal pain including tendinopathy. We assessed dextrose prolotherapy for CLE in a pilot-level study. BODY.METHODS: The study design was a 2-arm non-blinded randomized controlled trial. Group 1 received dextrose prolotherapy and Group 2 was a waitlist control. Nineteen adults seen with at least 3 months of symptomatic CLE in 22 elbows refractory to prior care received ultrasound-guided injections of 20% dextrose-morrhuate sodium (Group 1) solution at baseline, 4, and 8 weeks. Waitlist subjects (Group 2) were followed and discouraged from starting new care. Primary outcome measure was Patient-rated Tennis Elbow Evaluation [PRTEE, (100 points) assessed at baseline, 4, 8 and 16 weeks]. Prolotherapy participants were additionally assessed at 32 weeks. Secondary measures included dynamometer-assessed pain free grip strength and participant satisfaction. BODY.RESULTS: No baseline differences existed between the groups in gender, duration of elbow pain, prior therapy or baseline PRTEE scores. Prolotherapy participants (n=10) reported improved PRTEE composite scores compared to Waitlist (n=12) at 4 and 16 weeks (p<0.05), and improved pain and function PRTEE subscale scores (p<0.05) at 4 and 16 weeks, respectively. Prolotherapy participants reported improvement in composite PRTEE scores from baseline at 16 and 32 weeks of 17.9±11.64 and 24.8±10.58 points, a difference of 49.7% and 70.2% respectively, far in excess of the 11-point PRTEE-based minimal clinical important difference. Grip strength improved in all groups without between-group difference. Satisfaction with prolotherapy was high; there were no adverse events. BODY.CONCLUSION: Prolotherapy using dextrose and morrhuate sodium resulted in safe, significant, sustained improvement of PRTEE-based elbow composite, pain and function scores compared to baseline status and waitlist control subjects. The results of this pilot study suggest the need for a definitive clinical trial.

TITLE: Prevention of Type 2 Diabetes in Subjects With Prediabetes and Metabolic Syndrome Treated With Phentermine and Topiramate Extended Release ABSTRACT.OBJECTIVE: To evaluate over 108 weeks the effect of phentermine and topiramate extended release (PHEN/TPM ER) treatment on progression to type 2 diabetes and/or cardiometabolic disease in subjects with prediabetes and/or metabolic syndrome (MetS) at baseline. ABSTRACT.RESEARCH DESIGN AND METHODS: Subanalysis of a phase 3, randomized, placebo-controlled, double-blind study of overweight/obese subjects (BMI ≥27 to ≤45 kg/m2) with two or more comorbidities. Subjects were randomized to placebo, PHEN 7.5 mg/TPM ER 46 mg (7.5/46), or PHEN 15 mg/TPM ER 92 mg (15/92) plus lifestyle modifications for 108 weeks. Percent weight loss in the intent-to-treat population using multiple imputation (ITT-MI), annualized incidence rate of progression to type 2 diabetes, and changes in glycemia, lipid parameters, blood pressure, and waist circumference were evaluated. ABSTRACT.RESULTS: At baseline, 475 subjects met the criteria for prediabetes and/or MetS. After 108 weeks, subjects with prediabetes and/or MetS in the placebo, 7.5/46, and 15/92 groups experienced mean percent weight loss of 2.5, 10.9, and 12.1%, respectively (ITT-MI; P < 0.0001 vs. placebo), associated with reductions of 70.5 and 78.7% in the annualized incidence rate of type 2 diabetes for those receiving 7.5/46 and 15/92, respectively (ITT, P < 0.05), versus placebo. The ability of PHEN/TPM ER to prevent diabetes was related to degree of weight lost and was accompanied by significant improvements in cardiometabolic parameters. PHEN/TPM ER was well tolerated by this subgroup over 2 years. ABSTRACT.CONCLUSIONS: PHEN/TPM ER plus lifestyle modification produced significant weight loss and markedly reduced progression to type 2 diabetes in overweight/obese patients with prediabetes and/or MetS, accompanied by improvements in multiple cardiometabolic disease risk factors. BODY.INTRODUCTION: The increased prevalence of type 2 diabetes, together with its burden of patient suffering and societal costs, underscores the importance of finding effective strategies for both treatment and prevention of this disease (1,2). Two clinical constructs for identifying individuals at high risk of developing type 2 diabetes are prediabetes and metabolic syndrome (MetS). Prediabetes is a state of dysglycemia defined by impaired fasting glucose (IFG) and/or impaired glucose tolerance (1,3). It is estimated that 79 million Americans aged 20 years or older have prediabetes (2), with 25% of them progressing to type 2 diabetes within 3–5 years (3,4). Type 2 diabetes is associated with abdominal obesity and insulin resistance (diagnostic criteria were established by the Advanced Treatment Panel III of the National Cholesterol Education Program); MetS is a cluster of risk factors for cardiovascular disease (5–8). Individuals with MetS are at a fivefold increased risk of developing type 2 diabetes (5). Because IFG is one of the constituent traits used to identify MetS, overlap with criteria for prediabetes exists, and the risk of progression to type 2 diabetes is further increased in individuals who satisfy both sets of criteria (9). Thus, effective treatment of these at-risk individuals is imperative for the prevention of type 2 diabetes. Sustained loss of 5–10% of body weight in obese and overweight patients has proven to be effective in preventing progression from prediabetes (3,10–13) and MetS (10,14) to type 2 diabetes. It also ameliorates the cardiometabolic disease process, as shown by an increase in insulin sensitivity and a reduction in cardiovascular disease risk factors (12,13,15). However, achieving sustained weight loss at a clinically meaningful level sufficient to reduce risk remains a challenge for many patients (16,17). The primary approach to treating obesity and its related complications involves lifestyle modifications, including reductions in caloric intake (by 500–1,000 calories/day) combined with increases in physical activity (18). Bariatric surgery can also be an effective weight loss option for patients meeting specific criteria (19) and may reduce the incidence of type 2 diabetes (20–22), but the approach entails risks associated with surgery, nutritional deficiencies, and weight regain in some patients (23). In patients for whom lifestyle changes alone are insufficient and bariatric surgery is not an option, pharmacotherapies may be considered. Phentermine and topiramate extended release (PHEN/TPM ER; Qsymia; VIVUS, Inc., Mountain View, CA) have been shown to induce significant weight loss when combined with lifestyle modification in overweight/obese adults (24–26). The CONQUER study assessed effectiveness of PHEN/TPM ER for weight loss in overweight/obese adults with two or more weight-related comorbidities over 56 weeks (clinicaltrials.gov, NCT00553787) (25) and was followed by SEQUEL, a 52-week blinded extension study (NCT00796367) (26). In order to assess the ability of PHEN/TPM ER to reduce progression to type 2 diabetes and improve cardiometabolic parameters in patients at high risk of developing type 2 diabetes, we analyzed the subpopulation of patients meeting the criteria at baseline for prediabetes and/or MetS who elected to enroll in SEQUEL. BODY.RESEARCH DESIGN AND METHODS: SEQUEL was a 52-week extension of the 56-week, phase 3, randomized, double-blind, parallel-group, placebo-controlled CONQUER trial (25,26). The selection process for the 36 SEQUEL sites was based on high initial CONQUER enrollment and subject retention. Subject outcomes and randomization remained blinded during this process. All subjects who completed CONQUER on treatment at this subset of 36 sites were eligible to enroll in the SEQUEL extension study (26). All subjects entering SEQUEL maintained their original randomized treatment assignment from CONQUER (in a 2:1:2 ratio, stratified by sex and diabetes status) of once-daily oral placebo, PHEN 7.5 mg/TPM ER 46 mg, or PHEN 15 mg/TPM ER 92 mg (placebo, 7.5/46, and 15/92, respectively), plus lifestyle modification counseling based on the LEARN (lifestyle, exercise, attitudes, relationships, and nutrition) program (27), for an additional 52 weeks, resulting in 108 weeks of treatment. A computer-generated algorithm had been used to randomize subjects to study treatment at the beginning of the CONQUER study. Investigators and subjects remained blinded to treatment assignment. Study drug compliance (assessed by count of capsules returned by subject) and lifestyle counseling were addressed at each study visit, conducted every 4 weeks. At baseline (CONQUER week 0), subjects were overweight or obese adults (aged 18–70 years), with BMIs of 27–45 kg/m2, and two or more of the following weight-related comorbidities: central adiposity, dyslipidemia, hypertension, or type 2 diabetes. Subjects were actively managed to standard of care for their comorbidities, including the option to add, discontinue, or dose-adjust medications. The trials were approved by each center's institutional review board and overseen by an independent data safety review board. All subjects provided written informed consent. The first subject was enrolled into this study on 6 December 2008, and the last subject completed the study on 8 June 2010. The subgroup analyses presented in this article were performed on the subset of subjects with prediabetes and/or MetS at baseline who elected to enroll in the SEQUEL study. Subjects with a medical history of type 2 diabetes at baseline were excluded from this analysis. The criteria for prediabetes were as defined by the American Diabetes Association: IFG (fasting glucose levels 100–125 mg/dL [5.6–6.9 mmol/L]) or impaired glucose tolerance (blood glucose 140–199 mg/dL [7.8–11.0 mmol/L] 2 h after 75-g glucose load during an oral glucose tolerance test [OGTT]) (3). The diagnosis of MetS was made when three or more of the following five criteria were met: waist circumference ≥102 cm in men or ≥88 cm in women; triglycerides ≥150 mg/dL (1.7 mmol/L) or taking one or more lipid-lowering medications; HDL cholesterol (HDL-C) <40 mg/dL (1.0 mmol/L) in men or <50 mg/dL (1.3 mmol/L) in women or taking one or more lipid-lowering medications; systolic blood pressure ≥130 mmHg or diastolic blood pressure ≥85 mmHg or taking one or more antihypertensive medications; and fasting glucose ≥100 mg/dL (5.6 mmol/L) or taking drug treatment for elevated glucose (5). The primary end point was percent weight loss from baseline, which was assessed after 108 weeks (or early termination) in the SEQUEL study. Prespecified secondary end points were assessed at baseline, week 56, and week 108 (or early termination) and included annualized incidence rate of progression to type 2 diabetes and changes in glycemia, lipid parameters, blood pressure, and waist circumference (25,26). Remission of MetS (i.e., no longer meeting the diagnostic criteria as evidenced by satisfying only two or less of these criteria) at week 108 was also assessed. Finally, at week 56, high-sensitivity C-reactive protein (hs-CRP) and fibrinogen, both of which are inflammatory markers associated with MetS, were measured, as was adiponectin, which is decreased in subjects with obesity and cardiometabolic disease (28). For analyses of glucose and insulin as measured by OGTT (75-g loading dose), the change in each parameter from the preglucose loading dose sample to the sample obtained 2 h after the glucose loading dose at each applicable visit was calculated. OGTT was measured at baseline, week 4, week 56, and week 108. Fasting blood glucose was measured at baseline and weeks 4, 16, 28, 40, 56, 48, 96, and 108. Subjects were considered to have progressed to type 2 diabetes if their blood glucose was ≥126 mg/dL under fasting conditions during two or more consecutive measurements and/or ≥200 mg/dL at 2 h after an OGTT. BODY.RESEARCH DESIGN AND METHODS.STATISTICAL ANALYSIS: In this subanalysis, primary and secondary end points were assessed in the intent-to-treat (ITT) population using ANCOVA with terms for treatment group and baseline value. To accommodate missing data, multiple imputation (MI) was applied to all end points where missing data were apparent using, specifically, a two-step imputation process with m = 5 imputations per step (29). In the first step, data were imputed to create a monotone missing data pattern by using a Markov chain Monte Carlo algorithm. In the second step, remaining missing data were imputed using Rubin regression method (30). The complete imputed data sets were then analyzed by ANCOVA as described above, and the results from analysis of the separate imputed data sets were pooled into single estimates and tested as described by Schafer (31). The annualized incidence rate of type 2 diabetes was calculated as the number of newly diagnosed subjects divided by the number of subject-years of follow-up for each treatment group. The number of subject-years of follow-up was calculated as the sum of the number of days across all subjects from the randomization date in CONQUER to the onset date of type 2 diabetes or to the date of study completion or discontinuation (for subjects who did not develop type 2 diabetes) divided by 365.25. Absolute risk was calculated as the number of subjects progressing to type 2 diabetes divided by the number of subjects in each treatment group. The rates of progression to type 2 diabetes among the treatment groups were compared using a χ2 test. Analyses of the primary and secondary end points were also performed on the ITT sample with last observation carried forward (ITT-LOCF), consisting of all subjects who were randomized, took one or more doses of the study drug or placebo, and had one or more postbaseline body weight measurements; protocol-prespecified statistical assessments have been described elsewhere (25,26). BODY.RESULTS: Of the 866 subjects who completed CONQUER at eligible SEQUEL sites, 675 (77.9%) elected to enroll in the SEQUEL extension study (Supplementary Fig. 1) (26). The SEQUEL cohort included 145 (21.5%) subjects with type 2 diabetes at baseline and 55 (8.1%) subjects who did not meet criteria for either prediabetes or MetS; these individuals were excluded from the current analysis, leaving 475 (70.4%) at-risk subjects as defined by either prediabetes or MetS criteria, including 316 with prediabetes, 451 with MetS, and 292 meeting criteria for both prediabetes and MetS. Baseline demographics and clinical characteristics for subjects with prediabetes and/or MetS were similar among the treatment arms (Table 1). Table 1 Baseline demographics and clinical characteristics of the cohort with prediabetes and/or MetS at baseline (ITT)* BODY.RESULTS.WEIGHT LOSS: Treatment with PHEN/TPM ER induced significantly greater weight loss versus placebo in subjects in the prediabetes and/or MetS cohort. After 108 weeks of treatment, this cohort lost 10.9 and 12.1% of their body weight in the 7.5/46 and 15/92 treatment arms, respectively, vs. 2.5% in those subjects receiving placebo (ITT-MI; P < 0.0001), with similar results in the ITT-LOCF analysis (Fig. 1). The degree of weight loss in the placebo and PHEN/TPM ER treatment arms was similar in subjects with prediabetes or MetS at baseline and in the overall SEQUEL population at week 108 (26). No subjects experienced a BMI <18.5 kg/m2 at study end. Figure 1Percent weight loss from baseline to week 108 in the cohort with prediabetes and/or MetS at baseline. Least squares mean percent weight loss in the ITT population of subjects with prediabetes and/or MetS. P < 0.0001 vs. placebo for all time points assessed. LS, least squares. BODY.RESULTS.PROGRESSION TO TYPE 2 DIABETES: Although subjects in all treatment arms with prediabetes and/or MetS were administered a moderate lifestyle intervention program, the cumulative incidence rates of type 2 diabetes (Fig. 2A) were markedly reduced in subjects randomized to PHEN/TPM ER when compared with placebo over 108 weeks. The annualized incidence rate of type 2 diabetes in this population was 6.1, 1.8, and 1.3 for placebo, 7.5/46, and 15/92 (reductions of 70.5% with 7.5/46 and 78.7% with 15/92; P < 0.05 vs. placebo; ITT). The absolute risk reduction of progression to type 2 diabetes was 11.4, 3.5, and 2.5% for placebo, 7.5/46 (95% CI 1.8–13.9% vs. placebo), and 15/92 (3.5–14.3% vs. placebo). In subjects meeting criteria for prediabetes, subjects receiving 7.5/46 had a 48.6% reduction in the annualized incidence rate of type 2 diabetes and those receiving 15/92 had an 88.6% reduction versus placebo (Fig. 2B). Furthermore, subjects with MetS receiving 7.5/46 had a 76.6% reduction and those receiving 15/92 had a 79.7% reduction (Fig. 2B). Figure 2Incidence rates of type 2 diabetes from baseline to week 108 in SEQUEL study. A: Cumulative incidence rates of type 2 diabetes at study end (Kaplan-Meier) in the prediabetes and/or MetS cohort (ITT). B: Annualized incidence rates of type 2 diabetes at study end in the prediabetes cohort and the MetS cohort (ITT). C: Relationship between weight loss and type 2 diabetes incidence at study end in the prediabetes and/or MetS cohort (ITT-MI). Error bars represent 95% CI. Annualized incidence rate of type 2 diabetes was based on first occurrence of two consecutive fasting glucose ≥7.0 mmol/L, two consecutive OGTT ≥11.1 mmol/L, or taking antidiabetes medications at end point. *P = 0.0125 vs. placebo; †P = 0.0093 vs. placebo; ‡P = 0.0007 vs. placebo; §P < 0.05 vs. <5% weight loss for all comparisons. The magnitude of effect for type 2 diabetes prevention was related to the degree of weight loss achieved at 108 weeks in the ITT-MI population (Fig. 2C). Greater weight loss was associated with a greater reduction in incidence of type 2 diabetes regardless of randomization group. Subjects achieving <5% weight loss had the highest annualized type 2 diabetes incidence rate: 6.3. The lowest incidence rate, 0.9, was observed with weight loss of ≥15%; an intermediate type 2 diabetes incidence rate of 1.3 was seen among those with ≥5 to <10% or ≥10 to <15% weight loss (ITT-MI; P < 0.05 vs. <5% weight loss for all comparisons). In the ITT-LOCF analysis, annualized incidence rate of type 2 diabetes was 6.1 (SD 1.3), 1.8 (0.9), 0.6 (0.6), and 1.3 (0.8) for the <5, ≥5 to <10, ≥10 to <15, and ≥15% groups, respectively. BODY.RESULTS.EFFECTS ON CARDIOMETABOLIC DISEASE PARAMETERS: PHEN/TPM ER also significantly improved cardiometabolic disease risk factors versus placebo in subjects with prediabetes and/or MetS. When compared with placebo, fasting glucose, fasting insulin, 2-h post-OGTT glucose, fasting triglycerides, and HDL-C were all improved in the PHEN/TPM ER groups over 108 weeks (ITT-MI) (Fig. 3). Reductions in systolic blood pressure (mmHg) of –3.9 (SE 0.98), –5.0 (1.14), and –5.1 (0.91) and reductions in diastolic blood pressure of –3.7 (0.73), –3.6 (0.82), and –3.8 (0.61) were observed with placebo, 7.5/46, and 15/92, respectively (not significant vs. placebo; ITT-MI) (Supplementary Table 1). Subjects treated with PHEN/TPM ER also had reduced waist circumference, HbA1c, and homeostasis model assessment of insulin resistance and increased whole body insulin sensitivity index versus placebo at week 108 (ITT-MI) (Supplementary Table 1). Similar results were seen in the ITT-LOCF analysis (Supplementary Table 2). Figure 3Glycemic and lipid parameters at week 108 in the cohort with prediabetes and/or MetS at baseline (ITT-MI). A: Least squares mean percent change from baseline in glucose in subjects in the prediabetes and/or MetS cohort. B: Least squares mean percent change from baseline in insulin in the prediabetes and/or MetS cohort. C: Least squares mean percent change from baseline in lipid parameters in the prediabetes and/or MetS cohort. Error bars represent 95% CI. *P = 0.0474; †P < 0.0001; ‡P = 0.0028; §P = 0.0126; ¶P = 0.0012, #P = 0.0419; ‖P = 0.0004; **P = 0.0262; ††P = 0.0009 vs. placebo for all comparisons. LS, least squares. Among those with MetS at baseline, by week 108, a significantly greater percentage of subjects treated with 7.5/46 (22.4%) and 15/92 (27.6%) achieved remission of MetS compared with placebo (9.2%; P = 0.0001 vs. placebo). Also, at week 56 in subjects with prediabetes and/or MetS, PHEN/TPM ER was associated with lower hs-CRP values (–1.7, –2.7, and –2.2 mg/dL in placebo, 7.5/46, and 15/92, respectively; P = not significant vs. placebo; ITT-MI), lower fibrinogen levels (–10.1, –11.3, and –15.2 mg/dL in placebo, 7.5/46, and 15/92; P = not significant vs. placebo; ITT-MI), and increased adiponectin concentrations (0.4, 2.2, and 2.9 μg/mL in placebo, 7.5/46, and 15/92; P < 0.0001 vs. placebo; ITT-MI). BODY.RESULTS.ADVERSE EVENTS: Reported adverse events (AEs) in the prediabetes and/or MetS groups indicated that PHEN/TPM ER was generally well tolerated; more subjects receiving PHEN/TPM ER experienced paraesthesia, sinusitis, dry mouth, constipation, headache, and dysgeusia than those receiving placebo (Supplementary Table 3). The types and severity of AEs seen in this subgroup analysis were similar to those seen in the overall SEQUEL populations and in other clinical trials investigating PHEN/TPM ER for the treatment of obesity (24–26). Among subjects with prediabetes and/or MetS, two (1.3%), five (4.3%), and three (1.5%) subjects in the placebo, 7.5/46, and 15/92 groups, respectively, experienced palpitations, and zero, one (0.9%), and two (1.0%) subjects, respectively, experienced tachycardia. In the placebo, 7.5/46, and 15/92 groups, respectively, discontinuation of study medication due to treatment-emergent AEs occurred in 3.1, 6.1, and 5.5%, and serious treatment-emergent AEs occurred in 5.0, 7.0, and 8.5% at week 108; only appendicitis occurred in ≥1% of subjects receiving any treatment dose (two subjects in the 15/92 group) (Supplementary Table 4). No deaths occurred during the SEQUEL study. BODY.CONCLUSIONS: This subgroup analysis of patients participating in the CONQUER and SEQUEL studies allowed for assessment of the ability of PHEN/TPM ER to prevent progression to type 2 diabetes in at-risk patients during a 2-year period. In patients with prediabetes and/or MetS, PHEN/TPM ER was highly effective in inducing and sustaining weight loss and had a profound effect on prevention of type 2 diabetes, as measured by cumulative and annualized incidence rates. There was a 71 and 79% reduction in progression to type 2 diabetes among patients treated with 7.5/46 and 15/92 compared with placebo over 108 weeks. Additional studies are needed to determine whether weight loss associated with PHEN/TPM ER treatment will be maintained beyond 2 years or lead to sustained lower rates of progression to type 2 diabetes as compared with patients treated with placebo. However, most cases of type 2 diabetes in PHEN/TPM ER–treated patients occurred in the first year of the study, whereas cases continued to accumulate into the second year in the placebo group (Fig. 2A); thus, the difference in cumulative incidence between the PHEN/TPM ER and placebo groups, and the relative degree of type 2 diabetes prevention, may continue to increase over time. The ability to prevent type 2 diabetes was greatly dependent on the magnitude of weight loss, independent of randomization group. The annualized incidence rate for type 2 diabetes was progressively reduced as weight loss increased, with the lowest value realized at ≥15% weight loss, suggesting that greater weight loss is associated with greater benefits. Previous studies of lifestyle intervention, such as the Diabetes Prevention Program (DPP) (13), have also indicated that the degree of weight loss was a predominant determinant of type 2 diabetes prevention (32), although the Finnish Diabetes (12,33) and Da Qing (11) studies demonstrated that both weight loss and exercise exerted independent effects. The DPP study, wherein patients achieved ∼6% mean weight loss at 2 years and ∼4% weight loss at 4 years in the lifestyle intervention arm, reported a progressive 16% reduction in type 2 diabetes risk with every kilogram of weight loss but without an indication that there was a threshold of weight loss for maximal type 2 diabetes prevention (13,32). The current study is in agreement with the DPP, demonstrating that greater weight loss leads to greater reductions in the rate of type 2 diabetes. All categories with ≥5% weight loss experienced greater reductions in cumulative type 2 diabetes incidence when compared with the weight loss category of <5%. Thus, although modest weight loss of ∼5%, as recommended by the ADA (3), is beneficial, greater degrees of weight loss appear to lead to greater prevention of type 2 diabetes. Although the current study was limited to 2 years, the DPP, Finnish Diabetes, and Da Qing studies all demonstrated that after changes in or discontinuation of active treatment, the incidence of new type 2 diabetes diagnoses remained reduced compared with placebo or usual care over longer periods of follow-up (11,34–36). Based on these data, we theorize that reduced rates of type 2 diabetes may continue to be observed in the PHEN/TPM ER treatment arms compared with placebo, even after discontinuation of study drug. Of course, this is just speculation, but it does constitute a compelling consideration for future studies. Importantly, weight loss and prevention of type 2 diabetes as a consequence of PHEN/TPM ER therapy were accompanied by an increase in insulin sensitivity, as manifested by reduced glucose and insulin values, and improvements in cardiometabolic risk factors (blood pressure, waist circumference, triglycerides, and HDL-C). Furthermore, systemic inflammation, as measured by hs-CRP and fibrinogen at week 56, was reduced, and levels of the insulin-sensitizing adipocytokine adiponectin, at week 56, were increased. Since insulin resistance, dyslipidemia, inflammation, and dysregulated secretion of adipocytokines are all hallmarks of cardiometabolic disease, these findings are indicative of the potential reversal of this pathophysiologic process (37,38). It should be noted that in clinical trials assessing PHEN/TPM ER, all patients received advice on lifestyle modification, and the current benefits reflect the combination of PHEN/TPM ER and the lifestyle program (25,26). The LEARN program is similar to the DPP lifestyle intervention in that it strongly emphasizes behavior modification; however, the LEARN program has a less stringent calorie reduction requirement (decrease of 500 vs. 750–1,000 kcal in DPP) and encourages a progressive increase in exercise, rather than specifying a minimum amount of physical activity, as in DPP (27,39). Although the differences between lifestyle intervention alone (placebo group) and PHEN/TPM ER with lifestyle intervention to promote weight loss and prevent type 2 diabetes were relatively small in the SEQUEL trial, treatment with PHEN/TPM ER should nevertheless be combined with lifestyle modification to realize the full clinical benefits demonstrated in this study. These findings have particular relevance to real-world treatment decisions, since maintaining clinically meaningful weight loss through lifestyle changes alone is challenging (16,17). The robust clinical benefits observed with an effective pharmacologic agent combined with lifestyle modification thus may confer a significant advantage to improve outcomes in patients at high risk of developing type 2 diabetes. In general, PHEN/TPM ER was well tolerated, with no meaningful differences in safety in the prediabetes and/or MetS cohort during 108 weeks when compared with the overall SEQUEL population, and no differences between years 1 and 2 (26). Given the high risk of type 2 diabetes, which confers extensive patient suffering and high societal costs, the potential benefit-to-risk ratio of weight-loss treatment could be particularly favorable in patients with prediabetes and/or MetS. This study had certain limitations. SEQUEL was limited to high-enrolling centers with high patient retention from CONQUER, so not all patients were eligible for the extension (26). Patients enrolled at sites eligible to participate in SEQUEL had slightly greater weight loss (∼1% across treatment arms) at CONQUER end point than patients at non-SEQUEL sites. In addition, a higher percentage of PHEN/TPM ER–treated patients elected to continue in the study, so the original 2:1:2 randomization ratio was not maintained in the SEQUEL trial. The overall enrolled population for the SEQUEL clinical trial was larger than the subset of patients evaluated in this subanalysis; even so, baseline demography, efficacy, and safety were similar to the overall population, suggesting continuity across populations (25,26). Because patients with type 2 diabetes were excluded, there were some significant differences, mostly in glycemic parameters, between the cohort included in this analysis and those who were excluded (Supplementary Table 5). Also, because the study involved active management to standards of care, changes in concomitant medications for treatment of hypertension, dyslipidemia, and hyperglycemia are likely to have affected related study variables, often narrowing the gap between PHEN/TPM ER–treated patients and those taking placebo. However, active management was applied by treatment-blinded clinicians across placebo and PHEN/TPM ER treatment groups. Although these medication adjustments may affect some parameters, this also means that the study is largely representative of the type of care given in routine clinical practice, indicating that clinical benefits observed here may also be achieved in a real-world setting (3). In a separate analysis of the overall SEQUEL population, including those with type 2 diabetes, the weight loss associated with PHEN/TPM ER treatment induced improvement in cardiometabolic parameters even as use of medications to treat dysglycemia, hypertension, and dyslipidemia was reduced as compared with placebo (40). This suggests that weight loss associated with PHEN/TPM ER may lead to reduced medication burden for the treatment of weight-related comorbidities. Lastly, although 2 years is longer than any registration studies, it would be beneficial to have longer-term data to add to our understanding of the benefits and risks of prolonged PHEN/TPM ER use. This study demonstrates that PHEN/TPM ER plus lifestyle modification was generally well tolerated and produced significant weight loss through 108 weeks in patients with prediabetes and/or MetS at baseline. The ability of PHEN/TPM ER to prevent progression to type 2 diabetes was profound, with both PHEN/TPM ER treatment groups exhibiting statistically significant reductions in incidence rate in these high-risk individuals with prediabetes and/or MetS, with greater weight loss leading to greater reductions in progression to type 2 diabetes. Concomitant improvements in glucose homeostasis, insulin sensitivity, and cardiometabolic disease biomarkers were also observed. These data indicate that adding PHEN/TPM ER to lifestyle modification may constitute a new and effective therapeutic approach in patients with obesity and cardiometabolic disease, even as an alternative to bariatric surgery, by virtue of the ability of PHEN/TPM ER to produce substantial weight loss and to reduce risk of progression to type 2 diabetes in patients at high risk. BODY.SUPPLEMENTARY MATERIAL: Supplementary Data

TITLE: Phase I randomized double-blind placebo-controlled single-dose safety studies of Bowman-Birk inhibitor concentrate ABSTRACT: In previously performed animal studies and Phase I–II human trials, Bowman-Birk inhibitor concentrate (BBIC) appeared to be a promising cancer chemopreventive agent. The present study describes the results of two phase I randomized double-blind placebo-controlled trials performed in male subjects to assess the safety and toxicity of the original and new formulations of BBIC administered in a single dose as a suspension in orange juice. The dose of BBIC varied from 800–2,000 chymotrypsin inhibitor (CI) units. The BBI concentration in the serum samples collected from the subjects was analyzed by a dot-blot analysis procedure using the 5G2 monoclonal antibody, which is specific for reduced BBI. A total of 41 subjects were enrolled, 20 in the initial BBIC study and 21 in the second BBIC study. In these human trials, no clinically relevant changes in hematological or biochemical parameters were observed. Overall, BBIC was found to be well-tolerated. For these BBIC single-dose phase I trials, there was no dose-limiting toxicity for BBIC, even at the highest dose evaluated, and there were no apparent differences between the clinical trial results for the two formulations of BBIC. The bioavailability of BBI in the second clinical trial, which used the new BBIC formulation, was approximately 40 to 43% of the BBI bioavailability reached in the first clinical trial, which used the original BBIC formulation. The observed bioavailability difference was attributed to the different BBIC formulations used in these two clinical trials. These trials demonstrated that BBIC is safe when administered in a single dose of up to 2,000 CI units. Therefore, the results from the two trials indicate that a multi-dose trial of BBIC may be safely performed with doses of up to 2,000 CI units per day. BODY.INTRODUCTION: Increasing evidence indicates that individuals with a high dietary intake of soybean-derived products have low incidence and mortality rates from common cancers in the Western hemisphere, including cancers of the colon, breast and prostate (1). A number of different agents in soybeans may act as cancer chemopreventive agents in human populations (2). These agents include the soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), inositol hexaphosphate (phytic acid), the sterol, β-sitosterol, and the isoflavone, genistein, which have been demonstrated to suppress the development of cancer in animal carcinogenesis assay systems. BBI has been shown to have the strongest anticarcinogenic activity in animal carcinogenesis model systems in comparison to other potential cancer chemopreventive agents in soybeans (2). BBI, as a purified compound and an extract of soybeans in which BBI has been concentrated, has been shown to suppress carcinogenesis in a wide variety of in vivo and in vitro carcinogenesis assay systems (3). BBI is an 8-kDa soybean-derived protein containing 71 amino acids with two functional domains. One domain inhibits trypsin, the other inhibits chymotrypsin and several other serine proteases with chymotrypsin-like specificity, including elastase (4,5), cathepsin G (5,6) and chymase (7). BBI has been shown to have several therapeutic activities (reviewed in 8–10). BBI concentrate (BBIC) is a soybean extract enriched in BBI (11). It is believed that the chymotrypsin inhibitory activity of BBI conveys these therapeutic activities, therefore, the potency of BBIC is measured in chymotrypsin inhibitor (CI) units. One CI unit is defined as the amount of a substance required to inhibit 1 mg of bovine pancreatic chymotrypsin (11). Like BBI, BBIC inhibits trypsin and chymotrypsin and is anticarcinogenic, as measured by its ability to prevent malignant transformation in vitro and suppress carcinogenesis in vivo (reviewed in 3,9,11,12). In phase I clinical trials performed previously, no toxicity was observed when BBIC was orally administered in a single dose of up to 800 CI units in patients with premalignant lesions known as oral leukoplakia (13) or in daily doses of up to 800 CI units for 6 months in patients with benign prostatic hyperplasia (14). A subsequent phase IIa clinical trial in patients with oral leukoplakia demonstrated a dose-dependent reduction in oral lesion size after a one-month treatment with BBIC at doses of up to 1,066 CI units (15). In the clinical trial with benign prostatic hyperplasia patients, statistically significant decreases were observed in the serum prostate-specific antigen (PSA) level, serum triglyceride level and prostate volume following a 6-month treatment period with BBIC at doses of up to 800 CI units (14). BBIC tablets have also been administered to patients with active ulcerative colitis at a dose of 800 CI units per day for 12 weeks (16). In this study, the Sutherland Disease Activity Index (SDAI) was used to assess disease activity, response (index decrease >3) and remission (index <1 with no rectal bleeding). Favorable trends were observed in the rates of remission and clinical response, and no severe adverse events were observed. The results of the trial indicated a potential advantage over the placebo for achieving a clinical response and the induction of remission in patients with active ulcerative colitis, without apparent toxicity. Based on the non-toxicity and positive clinical responses observed in the previous clinical trials, two additional clinical trials were performed for the present study, using single BBIC doses of up to 2,000 CI units to determine the pharmacokinetics and safety of BBIC administered orally as a suspension in orange juice (OJ). Males were chosen for these trials as it was predicted that this would be the beginning of a prostate cancer prevention program utilizing BBI as the prostate cancer chemopreventive agent. One of these trials used the original formulation of BBIC and the other trial used a new formulation of BBIC. The primary objectives were to determine i) the dose-limiting toxicities for single doses of BBIC and expansion of the range of doses tested in humans, ii) the recommended doses of BBIC for a subsequent phase I multiple-dose study, and iii) pharmacokinetic characterization of the original and new BBIC formulations. BODY.MATERIALS AND METHODS: Two sequential randomized, double-blind, placebo-controlled trials were performed in healthy male subjects (NCT00287833 and NCT00679094). The trials were approved by the Institutional Review Board at the University of Pennsylvania (Philadelphia, PA, USA) and all subjects provided informed consent. The subjects were male, aged 18–65 years and assessed to be in good health by physical exam, electrocardiography and standard hematological tests. The inclusion criteria consisted of an Eastern Cooperative Oncology Group performance status (17) of 0–2, the lack of chronic medical conditions, no evidence of psychiatric problems and a weight within 15% of the ideal body weight. The subjects were excluded if they had a history of heart disease, chemotherapy in the last 12 months, tobacco smoking, allergies or prior adverse reactions to soybeans or if they had a prior diagnosis of pancreatic or other gastrointestinal diseases. Those who reported taking more than two vitamin supplements or non-steroidal anti-inflammatory drug on a regular basis, and vegetarians and others with a large soy component to their diet were also excluded. BODY.MATERIALS AND METHODS.BBIC: The original BBIC formulation was manufactured by Central Soya (Fort Wayne, IN, USA) according the procedure described previously (11). The new BBIC formulation was manufactured originally by Central Soya, but purified by Dynamic Extractions Ltd., (DE; Slough, Berkshire, UK) under direction from the National Cancer Institute (NCI) Division of Cancer Prevention (DCP) and supplied to the University of Pennsylvania School of Medicine Investigational Drug Service by the NCI Repository (McKesson Bioservices, Rockville, MD, USA). The contents of the original BBIC formulation have been described in detail previously (11). While the original BBIC formulation did contain low levels of normal food bacteria that are deemed non-pathogenic and are not expected to lead to adverse health effects, the NCI DCP produced a more purified formulation of BBIC that would not contain the normal food bacteria and represented a more concentrated version of BBIC. A more concentrated form of BBIC was expected to be considerably more useful in future human trials in which the total BBIC doses could be increased substantially. The company chosen to purify BBIC was DE, which is a UK specialist chromatography company. The DE product was called freeze-dried BBIC (BBIC-700). The final CI activity of the DE product was 562 CI units/g, while the original BBIC product was approximately 100 CI units/g (11). The microbiological content of the DE product was as follows: Aerobes <1 cfu/g (upper limit, 100 cfu/g); yeasts and molds <1 cfu/gram (upper limit, 100 cfu/g); E. coli and Salmonella were absent. The methods used in the preparation of freeze-dried BBIC were consistent with current good manufacturing principles. The new BBIC formulation from DE was stored in the refrigerator at 2 to 8°C. The original BBIC formulation was stored at room temperature. BODY.MATERIALS AND METHODS.STUDY DESIGN AND ENDPOINTS: Each study aimed to enroll a total of 20 healthy male volunteers, who were sequentially assigned to four different cohorts (drug levels), with five subjects per cohort. Subjects were recruited from the city of Philadelphia using print advertisements in local newspapers and posters around the campus of the University of Pennsylvania. One subject per cohort was assigned to receive a placebo instead of the active medication. Assignment to placebo or active medication was performed in a random and double-blind manner, the study pharmacist used a random number generator to assign treatments. The assigned treatment group was revealed at the completion of the trial. The following drug levels were investigated: 800, 1,200, 1,600 and 2,000 CI units. The first and second BBIC trials used the original formulation and the new formulation of BBIC, respectively. The subjects assigned to receive the placebo received 11.5 fl oz of OJ (Minute Maid Original 100% Orange Juice from Concentrate; Minute Maid, Sugar Land, TX, USA) with no additives. For subjects receiving BBIC, the measured dose of study medication was suspended in 11.5 fl oz of OJ from a single container. Both of the above ingredients were added to a subsequently sealed container and agitated until the suspension mixed uniformly. The total volume of the preparation was approximately 12 fl oz (350 ml). BBIC was administered orally to subjects in the form of a suspension at a concentration of 6% (w/v) in OJ. The CI activity is preserved in this formulation at this medication concentration for at least 3 h after suspension in the OJ, so the study medication was administered immediately after suspension in OJ. BODY.MATERIALS AND METHODS.SUBJECTS AND SAMPLING SCHEDULE: The subjects arrived at the Clincial and Translational Research Center (Philadelphia, USA) after fasting overnight. The subjects swallowed a single dose of BBIC suspension or placebo and immediately ate a defined low-fat breakfast. The subjects remained in the clinical research facility for the first 48 h of the study to facilitate the required frequent blood draws and to ensure that all subjects consumed the same low-fat diet. Subsequent to the first 48 h, the subjects returned to their homes, ate their normal diets without restrictions and then returned to the clinic for the required blood draws. Blood and urine samples were obtained for BBI pharmacokinetic evaluation at the following times following completion of drug ingestion: 0 (immediately prior to BBIC administration), 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24 and 48 h. Additional blood samples were obtained at 12, 24 and 48 h, and on or around days 7, 14, 21 and 28 for clinical blood chemistry and toxicity evaluation. Serum for pharmacokinetic analysis was separated from blood cells and frozen at −80°C in 1-ml aliquots. Urine samples were obtained at the same times as blood samples. At each collection time, the subjects were directed to void their bladder. All urine collected during that time interval was pooled, total volume recorded and a sample was frozen at −20°C for pharmacokinetic analysis. Amylase, lipase and low density lipoprotein/high density lipoprotein (LDL/HDL) were also assessed at times 0h, 12 h, 1 week and 4 weeks. Safety and toxicity were scored using the NCI Common Toxicity Criteria scoring system version 2.0. Statistical analysis consisted of tabulation of graded toxicities by dose level. BODY.MATERIALS AND METHODS.REAGENTS USED FOR BBI MEASUREMENT: A mouse monoclonal antibody, designated 5G2, was used as a primary antibody for BBI measurement by a dot-blot analysis in this study. The 5G2 antibody was produced and characterized as previously described (18). A horseradish peroxidase-conjugated goat anti-mouse IgG2b antibody was purchased from Southern Biotechnology Associates (Birmingham, AL, USA) and used as a secondary antibody for the BBI measurement. BBI was purchased from Sigma Chemicals (St. Louis, MO, USA) and radio-chemically reduced by exposure to 137Cs γ-rays under anoxic conditions in a buffer containing 100 mM formate and 10 mM phosphate buffer (PB; pH 5.5), as previously described (18). The reduced BBI was diluted to a concentration of 50 μg/ml and used as a stock solution of BBI standard antigen for BBI quantitation. Enhanced chemiluminescence (ECL) reagent was purchased from GE Healthcare (Piscataway, NJ, USA) for dot visualization in the dot-blot analysis. BODY.MATERIALS AND METHODS.BBI MEASUREMENT BY DOT-BLOT ANALYSIS: The BBI in serum samples was measured using a dot-blot analysis procedure. The serum samples were diluted in a buffer containing 70% 10 mM sodium PB (pH 7.5) and 30% absolute ethyl alcohol. For each dot-blot analysis, the BBI standard was serially diluted in a buffer containing 70% 10 mM PB (pH 7.5) and 30% absolute ethyl alcohol to BBI concentrations of 0 (baseline), 10, 30, 50, 100, 150 and 200 ng/ml and analyzed along with the serum samples to generate a standard curve for BBI quantitation. To perform the dot-blot analysis, three 10-μl aliquots of each sample or BBI standard were spotted onto an immobilion-PSQ membrane (Millipore, Billerica, MA, USA), with each membrane containing the entire set of serum samples from two or three subjects in addition to the BBI standards. The membranes were allowed to dry completely at room temperature. The membranes were rinsed with 10 mM PB (pH 7.5) for 2 min, blocked with 5% milk for 30 min, rinsed with PB three times for 5 min each and incubated with the primary antibody for 1 h. Following incubation, the membranes were rinsed again with PB three times for 5 min each and incubated with the secondary antibody for 1 h. The membranes were washed with PB and incubated with ECL reagent for 1 min, and then exposed to X-ray film. The integrated density of each spot on the membrane was obtained by scanning the X-ray films using ImageJ software (Sigmaplot version 12.0) from the National Institutes of Health (http://rsbweb.nih.gov/ij/index.html) following background subtraction. For each dot-blot analysis, triplicate values of each BBI standard were averaged and a standard curve was established by a linear regression analysis using the BBI concentration as the independent variable and the averaged integrated density as the dependent variable. The BBI concentration in each serum sample was determined using the standard curve generated on the same dot-blot membrane. BODY.MATERIALS AND METHODS.PHARMACOKINETIC AND STATISTICAL ANALYSIS: The serum BBI levels were analyzed using pharmacokinetic function macros developed for Microsoft Excel (http://www.boomer.org/pkin/soft.html). The data for the area under the curve (AUC) in each clinical trial were analyzed by linear regression analyses and compared among different treatment groups by one-way ANOVA followed by Tukey's test. The AUC data from the two clinical trials were further analyzed by two-way ANOVA using the BBIC dose and BBIC formulation (original vs. new) as the independent variables. P<0.05 was considered to indicate a statistically significant result. BODY.RESULTS.FIRST BBIC STUDY: A total of 37 patients were screened and a total of 20 subjects were enrolled in the initial BBIC study between December 2005 and March 2007. The characteristics of the patients enrolled are listed in Table I. No subject was lost to follow-up. Adverse events were observed in placebo- and BBIC-treated subject groups at approximately equal frequency. A total of 50 adverse events were observed in the 16 BBIC-treated subjects (mean, 3.125 per subject) vs. 13 in the 4 subjects receiving the placebo (3.25 per subject). The rates of adverse events did not increase with dose, and in the majority of cases were lower in the highest dose group than in the other groups. The exceptions to this were incidents of grade 1 hyperglycemia [6 reported incidents in the 16 treated subjects (3 in subjects receiving 2,000 CI units) vs. 1 incident in a subject receiving placebo] and a single report of hyperkalemia in a subject receiving 2,000 CI units (Tables II and III). The only grade 3 adverse event was a high alanine aminotransferase (ALT) level in a subject who received 800 CI units. The subject's ALT levels were observed to be normal during the inpatient portion of the study (48 h post-ingestion time period). During the subject's one week follow-up visit, the grade 3 high ALT was observed, but by the second week, the ALT had resolved to a normal level. BODY.RESULTS.SECOND BBIC STUDY: A total of 34 subjects were screened and a total of 21 subjects were enrolled in the second BBIC trial between June 2007 and March 2009. No subject was lost to follow-up. A dosing error occurred for one subject, who received 1,500 CI units BBIC instead of 1,200 CI units BBIC. This subject's data were reported separately in this analysis and the subject was replaced by an additional patient in the 1,200 CI units BBIC dose cohort. Hence, there were a total of 21 subjects in the second study instead of the initially intended 20 subjects. No other dosing anomalies occurred. No serious adverse events were reported during the study. The only grade 2 toxicities reported were abnormal triglyceride and aspartate aminotransferase (AST) levels and hypoglycemia. The single occurrence of a grade 2 abnormal triglyceride level occurred in a patient receiving the placebo (Table IV). The single occurrence of a grade 2 abnormal AST level occurred in a patient in the 1,600 CI units BBIC dose level (Table V). Ten occurrences of grade 2 hypoglycemia were experienced at 800 CI units BBIC (3 subjects), 1,200 CI units BBIC (3 subjects), 1,600 CI units BBIC (2 subjects) and 2,000 CI units BBIC (2 subjects), and therefore did not appear to be correlated with dose. BODY.RESULTS.PHARMACOKINETIC CHARACTERIZATION OF THE ORIGINAL AND NEW BBIC FORMULATIONS: The BBI concentration in the serum samples collected from subjects orally administered with BBIC at doses of up to 2,000 CI units was analyzed by a dot-blot analysis procedure using the 5G2 monoclonal antibody, which is specific for reduced BBI. Based on the signal responses of the BBI standards included in the assay, the dot-blot analysis was linear in a BBI concentration range of 0–200 ng/ml (Fig. 1). Preliminary analyses of 1:200 diluted serum samples produced lower BBI results in the dot-blot analyses for the subjects who received the highest dose of BBIC than for the subjects in the other treatment groups (data not shown), which indicated the presence of a hook effect of falsely low values in immunoassays when an excess of antigen affects the binding capacity of the detection antibody (18–20). To confirm the presence of the hook effect, a serum sample from a subject treated with the highest dose of BBIC (2,000 CI units) was mixed with a serum sample from a subject treated with placebo in various proportions and subjected to the dot-blot analysis. The results indicated that the measured BBI values increased with the proportion of serum from the BBIC treated subject up to 60% and then declined (Fig. 2). The results confirmed the presence of a hook effect in the dot-blot analysis for the 1:200 diluted serum samples from the subject in the highest BBIC dose group. To avoid a possible hook effect on the serum BBI measurement, the serum samples from subjects in the two highest BBIC dose groups were diluted 1:500 for the dot-blot analysis. The serum samples from subjects treated with placebo or BBIC at the two low doses (800 and 1,200 CI units) were diluted at 1:200 for the dot-blot analysis, since the BBI concentration in these samples was not predicted to be high enough to cause a hook effect. For the first phase I trial, the normalized AUC for the serum BBI level was moderately correlated with the BBIC dose, with a correlation coefficient of 0.65 and a slope value of 0.0007 (Fig. 3). The mean normalized AUC values for subjects treated with placebo or BBIC at doses of 800, 1,200, 1,600 or 2,000 CI units were 1.00±0.13, 1.08±0.31, 1.60±0.27, 2.53±0.27 and 2.03±0.34, respectively (Fig. 4). The difference among the treatment groups was statistically significant (P=0.006 by one-way ANOVA). For the second phase I trial, the normalized AUC for the serum BBI level was only weakly correlated with the BBIC dose, with a correlation coefficient of 0.36 and a slope value of 0.0003 (Fig. 3). The mean normalized AUC values for the subjects treated with placebo or BBIC at doses of 800, 1,200, 1,600 or 2,000 CI units were 1.00±0.24, 0.91±0.39, 1.51±0.29, 1.39±0.12 and 1.49±0.33, respectively (Fig. 4), and the difference was not statistically significant (P=0.446 by one-way ANOVA). To determine whether the dose-responses of the serum BBI levels were different between the first clinical trial, which used the original BBIC formulation, and the second clinical trial, which used the new BBIC formulation, the AUC data obtained in the two clinical trials were analyzed by a two-way ANOVA. The results indicate that the serum BBI level expressed as the AUC was significantly affected by the BBIC dose (P=0.022) and the BBIC formulation (P=0.031) received by the subjects. The mean normalized AUC value for BBIC-treated subjects (all doses combined) was 1.808 and 1.325 for the first and second clinical trials, respectively, and the difference was statistically significant (P=0.031). After subtracting the baseline normalized AUC value of 1 for the placebo treatment group, the net increases in the mean normalized AUC value for BBIC treated subjects in the first and second clinical trials were 0.808 and 0.325, respectively. BODY.DISCUSSION: Two phase I randomized double-blind pharmacokinetic and safety trials were conducted using two different formulations of BBIC, a candidate chemopreventive agent, administered orally as a suspension in OJ. These clinical trials were initiated in preparation for a prostate chemoprevention program in prostate cancer at the University of Pennsylvania. The main objectives of the pharmacokinetic analyses were to measure and characterize the levels of BBI in serum and urine following a single dose of BBIC or placebo in healthy male subjects; these analyses will be reported separately. No serious adverse events were observed. No clinically significant laboratory abnormalities were reported for any dose level. Four human trials utilizing BBIC have been completed previously (13–15,22–24). Phase I and phase IIa studies of BBIC in patients with oral leukoplakia were also performed (13,15,22,23). The results from these trials indicated no toxicity from BBIC at any dose level studied. Over the dose range of 200–1,000 CI units per day, BBIC caused a reduction of total oral leukoplakia lesion size that was linearly correlated with increase in dose. The compound was well-tolerated with no evidence of laboratory, symptomatic or clinical side-effects (23). A randomized, double-blind trial of BBIC in patients with benign prostatic hyperplasia (BPH) was also performed (14,24). This trial involved 6 months of BBIC treatment involving dosage levels of 100–800 CI units per day in oral tablet form. There was no dose-limiting toxicity of BBIC observed in this study. For the BBIC single-dose phase I trials reported in the present study, the dose range chosen was higher than any that had been used previously in BBIC human trials (800–2,000 CI units). No dose-limiting toxicity was observed for BBIC, even at the highest dose evaluated (2,000 CI units), and the results from the two trials were comparable for the studies involving two different formulations of BBIC. Thus, the results from the two trials indicate that a multidose trial of BBIC may be performed with doses of up to 2,000 CI units per day. The pharmacokinetic studies using urine samples from previously completed clinical trials have shown that BBI is excreted rapidly in the urine between 3 and 12 h after BBIC administration (13). Linear regression analyses of the BBI results demonstrated dose-dependent increases in mean BBI concentration, peak BBI concentration, peak minus mean BBI concentration and the peak to mean ratio of BBI concentration in the urine samples of subjects following BBIC treatment (14). These results indicate that BBI is absorbed systemically in human subjects following oral administration of BBIC. In the present study, serum BBI levels were determined for subjects enrolled in two new clinical trials using the original and new BBIC formulations. The results demonstrate that the AUC value for the serum BBI level was significantly correlated with the dose of BBIC received by the subjects in the two trials, however, the slope of the linear regression line for the first trial (slope=0.0007) was more than twice as steep as the slope of the linear regression line for the second clinical trial (slope=0.0003). In addition, the mean normalized AUC value for the serum BBI level in the BBIC-treated subjects was significantly higher in the first clinical trial than in the second clinical trial. Based on the ratio of regression line slopes between the two clinical trials (0.0003/0.0007=0.4286) and the ratio of the net increase in the mean normalized AUC values (0.325/0.808=0.4022), the bioavailability of BBI in the second clinical trial was approximately 40 to 43% of the BBI bioavailability reached in the first clinical trial. Since the two clinical trials were performed using the same experimental design, with the exception of the BBIC formulation, the differences observed are attributable to the different BBIC formulations used in these two clinical trials. While the CI activity in the original and new formulations of BBIC appeared to be comparable, there are numerous variables that may affect the biological activities of BBI/BBIC, as previously discussed (11). Two of these factors are the refrigeration and freezing of BBIC samples, which result in a reduced ability of the samples to affect radiation-induced transformation in vitro (11). The new formulation of BBIC was exposed and maintained under refrigerated and frozen conditions during the purification procedure used by DE or the subsequent storage of the new product, while it is recommended that the original formulation of BBIC should not be exposed to either refrigerated or frozen conditions. It is likely that the temperatures, or the new solvents (ethyl acetate and methanol) used by DE in the production of the new formulation of BBIC, may have altered the bioavailability of BBI compared to the BBI in the original formulation of BBIC. Prior to the BBIC clinical trials in humans, information about the absorption, distribution and excretion of BBI was primarily based on animal studies utilizing radiolabeled BBI. These studies indicated that approximately half of the BBI administered orally is excreted in the feces in an unaltered form, whereas the remainder enters intestinal epithelial cells (25) or crosses the intestinal lumen via a paracellular mechanism (26). In animal studies, BBI is able to survive the digestive process, reach the colon in an active form and is capable of interacting with proteases in the same manner as expected for BBI (26,27). The measurement of BBI in biological samples by immunoassay has proven to be technically challenging. BBI is readily detectable using monoclonal antibodies that have been generated by Brandon et al (28) in food samples (29) or in human serum and urine samples spiked with purified BBI in its native form (30). Oral administration of BBI results in a form of BBI in the bloodstream and urine that cannot be detected with the antibodies against BBI in its native form (18), despite the fact that the BBI appearing in blood and urine following oral intake has the same molecular weight and the same ability to inhibit trypsin and chymotrypsin as BBI (26). Since it is necessary to use antibodies reactive with reduced BBI to detect BBI in blood and urine samples from subjects following BBI oral intake (18,31), it is assumed that BBI is present in a reduced form in body fluids. Pharmacokinetic studies of BBI have previously been performed in rodents, dogs and humans with antibodies that react with reduced BBI (reviewed in 32). As part of subchronic toxicity studies of BBIC in rats and dogs, serum concentrations of BBI were measured using one of the antibodies that reacts specifically with reduced BBI, known as 5G2 (18), by a dot-blot method. As summarized previously (32), the serum BBI level was 32–48% higher in rats treated with oral daily doses of 500 or 1,000 mg/kg BBIC for 3 months and 35–50% higher in dogs treated with oral daily doses of 500 or 1,000 mg/kg BBIC for 45 or 89 days compared to their respective control groups. The highest dose of BBIC administered to human subjects in the present study was at least one order of magnitude below the dose used in the previous rat and dog toxicity studies on a kg-body weight basis. However, the magnitude of increase in the mean normalized AUC value for the serum BBI level of the highest BBIC dose groups in the present study was of the same order of magnitude as previously observed in animal toxicity studies, indicating that there may be an upper limit for BBI absorption following oral administration. Based on the shape of the dose-response curves of the serum BBI level (Fig. 4), the serum BBI level appeared to reach a plateau at the dose of 1,600 CI units, and further increases in the BBIC dose did not result in an additional increase in the BBI level in the circulation. This may have practical implications for future clinical trials using BBIC or other soybean-based dietary supplements with BBI as the main active ingredient. The results from the first and second trials of BBIC utilizing the original and new BBIC formulations indicate that a dose-limiting toxicity for BBIC was not observed up to a dose of 2,000 CI units. Therefore, it is proposed that a multidose BBIC study may be extended up to a high dose of 2,000 CI units per day.

TITLE: Laparoscopic closure of perforated gastro-duodenal ulcer: 15 years’ experience in our centre ABSTRACT.INTRODUCTION: The objective of the study is to share the results and development findings on the laparoscopic closure technique applied in our centre during a 15-year period (1998–2012). ABSTRACT.AIM: To compare statistically the standard parameters (hospitalization, duration of operation) versus conventional surgery, and at the same time we compared mainly morbidity and mortality. ABSTRACT.MATERIAL AND METHODS: During the period under review we operated on a total of 259 patients, 115 (44.4%) of them laparoscopically, and 144 (55.6%) of them conventionally. The sample was divided into two groups: patients with ASA physical status classification system 1–3, and patients with ASA 4–5. ABSTRACT.RESULTS: The results favour laparoscopy within the group with ASA 1–3 in terms of several parameters, namely: duration of hospitalization – 7.7 days in the case of laparoscopic intervention, vs. 10.6 days for conventional surgery (p < 0.05); and duration of operation – 61 min vs. 85.1 min respectively (p < 0.05). Total morbidity was 27.5% in the case of patients with conventional surgery, vs. 10.9% with laparoscopic intervention (p < 0.05). The sample of patients with ASA 4–5 suffered a high mortality of 82.7%. ABSTRACT.CONCLUSIONS: Laparoscopic closure of perforated ulcer is a safe therapeutic method, as confirmed by the results of many other studies around the world, which in many aspects favour the laparoscopic technique. BODY.INTRODUCTION: Johan Mikulicz-Radecki (1850–1905), often referred to as the first surgeon who closed a perforated peptic ulcer by simple closure, said: 'Every doctor, faced with a perforated duodenal ulcer of the stomach or intestine, must consider opening the abdomen, sewing up the hole, and averting a possible inflammation by careful cleansing of the abdominal cavity' [1]. Perforation occurs in approximately 2–10% of patients with ulcer disease [2]. It comprises approximately 5% of all abdominal emergencies, and perforation incidence is 7–10 per 100 000 capita [3, 4]. Patients with perforated ulcer are predominantly men aged 40 to 60 years [4]. They could have ulcer disease anamnesis (29%), or consumption of NSAID (20%). Around 5–10% of patients arrive at the hospital in a condition of shock [5]. On the other hand, one of the rarest causes of stomach perforation is incarceration of hiatus hernia (< 0.01%) [6]. X-ray examination performed in a standing position will in 80–85% of cases prove the presence of free air under the diaphragm, and the subsequent radiological techniques confirm the diagnosis in 80–90% of cases [7]. It clinically manifests as a sudden, from the very beginning unusually intense, shocking pain, with abdominal guarding clinical symptoms. Clinically we distinguish three stages [8]:Chemical peritonitis – acidic content sterilises the content of stomach and duodenum, which freely flows to the peritoneum, and causes the chemical peritonitis.Transitory stage – after 6–12 h there occurs an arbitrary improvement – pain relief, caused by dilution of gastro-duodenal liquid with peritoneal exudate.Intra-abdominal infection emerges after 12 to 24 h. Post-surgery mortality in the case of perforated ulcer still remains high, around 6–10% [9]. Boey or Irvin score systems (0–3 scale, Table I) relate mortality risk to such factors as:Condition of shock before surgery – 1 point +,Dominant associated disease – 1 point +,Postponement of surgical intervention by more than 24 h from the beginning of the disease – 1 point +. Table I Boey score system with respect to morbidity and mortality Boey Morbidity [%] Mortality [%] 0 17.4 1.5 1 30.1 14.4 2 42.1 32.1 3 100 BODY.INTRODUCTION.LAPAROSCOPIC TREATMENT OF PERFORATED ULCER: The first laparoscopic suture closure was carried out by Nathanson et al. in 1990 [10]. At the same time Mouret carried out the first laparoscopic omentoplastic surgery using a fibrin sealant [11]. From available references on the data collected through questionnaires, in 2004 in Slovak Republic only 24.2% of relevant surgery departments applied laparoscopic suture closure of perforated gastro-duodenal ulcer [12], whereas in the Czech Republic the same figure in 2002 was already 56% [13]. In the beginning the laparoscopic approach is indicated in each suspected case of perforated ulcer, but it also has counter indications, such as a high-risk patient with ASA 5, and ileus. BODY.INTRODUCTION.POSITION OF A PATIENT AND POSITION OF THE OPERATING TEAM: In this intervention the patient lies on his/her back, with the left upper extremity adducted. The operating surgeon is on the left side of the patient, and the surgeon's assistant is on the left side of the surgeon. The operating surgeon may eventually be between the patient's legs, and the surgeon's assistant in this case remains on the left side of the patient [14]. The laparoscopic tower is on the right side of the patient, next to his/her chest, or head. This set-up is good for preparation in the epigastric region. The position of the patient, and/or his/her rotation during the intervention should be as follows: During the preparation carried in the epigastric region, the patient should be in an anti-Trendelenburg position at 20–30° [15], which provides a better view of the operating field, since the viscera fall down. In case of cleansing the patient can be leaned in different directions, depending on the necessity to visually inspect individual abdominal quadrants. BODY.INTRODUCTION.POSITION OF TROCARS: We usually insert the optical 10 mm port into the umbilicus. The working 5 mm port for the operating surgeon's left hand is located in the anterior axillary line at the level of the umbilicus, for the atraumatic grasper. Another 5 mm working port for the operating surgeon's right hand is located in the medio-clavicular line above the level of the umbilicus for the suture holder, and the suction and irrigation device (Figure 1). Figure 1Position of ports during suturing In the case of obese patients, the position of ports can be adjusted and moved closer to the operating field. In the case of a bad view of the local situation, a fourth port can be located in the epigastrium, for a retractor for the liver and viscera. BODY.INTRODUCTION.SURGICAL PROCEDURE DURING SUTURE CLOSURE OF A PERFORATED ULCER: The next step after the introduction of an optical port and confirmation of the diagnosis is the introduction of working ports as described above. First we take a sample of exudate for bacteriological tests, and then we carry out the inspection of the abdominal cavity, in order to localise precisely the perforation spot, and the extent of peritonitis. Quite often the gall bladder and the liver adhere with fibrin accretions in the vicinity of the ulcer, which is most frequently located on the frontal side of the first part of the duodenum. The follow-up step is the cleansing of the abdominal cavity, where irrigation with a warm physiological solution is followed by evacuation of the exudate and removal of fibrin accretions to the maximum possible extent. In terms of methodology we start at the right upper quadrant, proceeding to the left upper one, then we continue to the left lower quadrant, and we end in the right lower one. We need to be especially diligent in the area of the Douglas cavity, and in the space between intestinal loops. BODY.INTRODUCTION.PERFORATION SUTURE CLOSURE TECHNIQUE: After the suture closure of the ulcer, there is no need for biopsy of the duodenal ulcer, but on the other hand, in the case of gastric ulcer, it is recommended to take a biopsy of the ulcer margin. Suture is carried out by a slowly absorbable or non-absorbable material applied with atraumatic needle 2/0 or 3/0. Usually two or three transverse sutures are applied (Photos 1 and 2). After the perforation is suture-closed, it is possible (using a part of the large omentum) to cover the suture closure, and fix it to the upper suture. Some surgeons apply for omentoplastic intervention a fibrin sealant. In the case of a chronic callous ulcer, it is problematic to sew together ulcer margins, and we can therefore apply a thicker thread, 1/0, in order to avoid it cutting through the tissue in a fibrous environment. Photo 1Perforation suture A Photo 2Perforation suture B In the case of larger ulcers it is possible to sew-in within the defect, with several stitches, the free end of the omentum, in order to close the defect. It is the very size of the defect that causes the conversion [16]. We can check the tightness of the suture closure with a patient in the Trendelenburg position, following the application of physiological solution and blowing air into the stomach, which should not cause air leakage into the free abdominal cavity. Flushing of the abdominal cavity is performed until the clear liquid comes out, and then we end the operation by applying drain tubes [15], which we place in the following order. One goes into the subhepatic space, which monitors the area of suture, which goes into the incision used as a port in the right mesogastrium. The remaining two drain tubes are inserted from the left, through incisions used as ports into the left subphrenic space and to the Douglas cavity (in the case of 3-port intervention we introduce into the Douglas cavity a drain tube through an incision on the right side). After the operation we keep in place the nasogastric tube until the peristaltic restart onset. Provided the clamping test was successful, the tube can be extracted. In complicated ulcer-suturing cases, or protracted onset of passage, we can carry out examination using a water contrast agent (UroDiagramin). The gastro-fibroscopic examination is planned following 4 to 6 weeks after the operation. BODY.MATERIAL AND METHODS: During the 15-year period from 1998 until 2012, we carried out in our centre altogether 259 operations of perforated gastro-duodenal ulcer. We did operations laparoscopically in the case of 115 patients (44.4%), and conventionally in the case of 144 patients (55.6%). The best method for comparing the two groups is the comparison of morbidity and mortality. For group homogeneity reasons we compared separately patients with ASA 4–5, whom we put aside from the compared groups. These patients suffer high mortality and mortality, with a high number of cases with high entry cardio-pulmonary premorbidity as shock-affected patients, who were in most cases classified as patients with contraindicated laparoscopic revision, which would substantially change the outcome for the group of patients operated conventionally (Table II). There were altogether 29 patients with ASA 4–5 (11.1%). Table II Distribution of the group of patients with respect to ASA Group ASA 1–3 ASA 4–5 Together Conventional 121 24 145 Laparoscopic 110 5 115 The number of conventionally operated patients with ASA 1 during the period from 1998 until 2012 was 120, and for laparoscopically operated patients it was 110. The homogeneity of the group ensures a smooth transition to laparoscopy (Figure 2), when the surgeons in our centre (following sufficient training) started to choose laparoscopic inspection as the preferred choice. Figure 2Ratio of laparoscopic intervention versus conventional surgical therapy of the perforated ulcer in our department BODY.MATERIAL AND METHODS.GROUP OF CONVENTIONALLY OPERATED PATIENTS WITH ASA 1–3: The majority of patients from the group of 120 conventionally operated patients were operated on between 1998 and 2004–2005. In those years in the course of conventional surgical therapy of perforated ulcer the most frequent intervention was excision of the ulcer followed by pyloroplasty (93 patients: 77.5%), as well as associated truncal subdiaphragmatic vagotomy (60 patients, 50%). Not in all cases of pyloroplasty did we also do vagotomy, and vice versa (Table III). In a smaller number of cases we did partial resection of the stomach and gastroenterostomy (altogether 6 patients, 5%). Vagotomy had a complicated course in 2 patients with spleen lesion, i.e. in 3.3% of all vagotomies, with the subsequent need to carry out splenectomy. The average duration of hospitalisation was 10.6 days, and the average duration of surgical intervention was 85 min. Table III Percentage of individual types of interventions in conventionally operated patients with ASA 1–3 ( n = 120) Intervention Patients Percentage Pyloroplasty 93 77.5 Vagotomy 60 50 BII + GEA 6 5 The total number of patients with complications within the group of conventionally operated patients, with ASA 1–3, was 33 (27.5%). Among them 4 patients died (3.33%). Within this group (in several cases there were multiple complications per single patient) the most frequent complication was wound infection, and more specifically in the case of 14 (11.7%) patients. Extra-abdominal complications (bronchopneumonia or cardiac decompensation) occurred in 11 (9.2%) patients, of whom 2 patients died. We re-operated 11 (9.2%) patients, of whom 2 died. The reason for re-operation was 2 × ileus, 3 × intra-abdominal abscess, 2 × disruptions of wound, and 3 × leakages. Other complications occurred in 2 patients (bleeding). We identified leakage in 3 patients (of whom 1 died), whereas in all cases re-operation was required, which comprises 2.5% of the total number. The average duration of hospitalisation of a patient with complications in conventionally operated cases with ASA 1–3 within this group was 16.8 days vs. 8.2 days on average for patients without complications. BODY.MATERIAL AND METHODS.GROUP OF LAPAROSCOPICALLY OPERATED PATIENTS WITH ASA 1–3: The majority of patients from the group of 110 laparoscopically operated patients were operated on between 2004–2005 and 2012 (Figure 2). In the case of laparoscopically operated patients with perforated ulcer, 81 patients were suture closed (73.6%), sutured ulcer with subsequent omentoplasty was carried out in 17 patients (15.5%), and the remaining 12 patients (10.9%) were treated only with flushing of the abdominal cavity, and a targeted drain tube without suture due to failure to find the perforation (with already healing ulcer – application of methylene blue via nasogastric tube) (Table IV). Table IV Percentage of individual types of interventions in laparoscopically operated patients with ASA 1–3 ( n = 110) Intervention Patients Percentage Simple closure 81 73.6 Suture + omentoplasty 17 15.5 Targeted drainage 12 10.9 The average duration of hospitalisation was 7.7 days, and the average duration of intervention was 61 min. The total number of patients with complications within the group of laparoscopically operated patients with ASA 1–3 was 12 (10.9%). Among them 1 patient died (0.9%) (heart failure, pulmonary embolism). We did not record any wound infection within the group of 12 patients with complications. Bronchopneumonia or cardiac decompensation occurred in 4 patients (3.6%). Operative re-intervention was required in 4 (3.6%) patients. The reason for surgical revision was in 2 cases an intra-abdominal abscess, and in 2 others leakage. Of the remaining 4 patients (33%), conservative therapy was applied in 2 patients with leakage, and in 2 patients with an intra-abdominal abscess. Altogether, leakage occurred in 4 patients, which comprises 3.6% of the total group of patients, but only in 2 cases, i.e. 1.8%, did it require re-operation. Conversion occurred in 2 (1.8%) patients. The reasons for conversions were polyps in 1 case, and the size of the ulcer in another single case. The average duration of hospitalisation in patients with complications among laparoscopically operated patients with ASA 1–3 within this group was 15.7 days vs. an average of 6.7 days in patients without complications. BODY.MATERIAL AND METHODS.GROUP OF OPERATED PATIENTS WITH ASA 4–5: From 1998 until 2012, 29 (11.1% of all) patients with ASA 4–5 underwent operations. They suffered high morbidity and mortality. This was the case due to cardiac reasons, or the associated status of shock during the protracted course of peritonitis (within our group the postponement of hospitalisation by patients themselves) (Table V). The mortality within this group was 82.7%, i.e. death occurred in 24 patients. In the case of the majority of patients we could not carry out laparoscopy due to the anaesthesiology risk of capnoperitoneum (the main reason behind the exclusion of this group). The laparoscopy in the case of the remaining 5 of these patients did not influence the haemodynamics during the course of the operation. Table V Mortality comparison for patients with ASA 4–5 Group ASA 4–5 Exitus Exitus [%] Conventional 24 20 83.30 Laparoscopic 5 4 80 BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: For the statistical assessment of parametric quantitative values we used Student's t test (bilateral distribution with uneven dispersions), and for the assessment of qualitative values we used the χ2 test with a 2 × 2 contingency table, with the statistical significance limit level being p < 0.05. The minimal number of patients within a single contingency table should be at least 5, in order to maintain the test accuracy. Provided the number of patients was below 5, we did not assess the said qualitative parameter from the viewpoint of statistical significance. BODY.RESULTS: In addition to the basic parameters such as comparison of hospitalisation duration, and operating time (Table VI), we focused mainly on the comparison of morbidity and mortality, which are the main distinctions in comparing the two methods, i.e. laparoscopic and conventional operation approaches (Table VII). As mentioned above, for group homogeneity reasons, we only compared patients with ASA 1–3 (88.9% of all patients). Table VI Statistical comparison of individual parameters in laparoscopic and conventional groups (operating time, hospitalisation duration, average age) Parameter Conventional Laparoscopic Value of p Statistical significance Operating time [min] 85.1 61 0.0000000004 p < 0.05 Hospitalisation [days] 10.6 7.7 0.00013 p < 0.05 Average age [years] 48.6 47.2 0.52 Table VII Statistical comparison of individual parameters in laparoscopic and conventional groups (morbidity and mortality) Parameter Conventional ( n = 120) Laparoscopic ( n = 110) Value of p Statistical significance Complications: 33 (27.5%) 12 (10.9%) 0.0015 p < 0.05  Exitus 4 (3.3%) 1 (0.9%) –  Wound infection 14 (11.7%) 0 0 p < 0.05  Bronchopneumonia 11 (9.2%) 4 (3.6%) 0.09  Re-intervention 11 (9.2%) 4 (3.6%) 0.09  LEAK 3 (2.5%) 4 (3.6%) – These statistical comparison results show a significant difference in the hospitalisation duration in favour of laparoscopy, on average 7.7 days vs. 10.6 days for conventionally operated patients. Similarly the operation intensity in terms of operation duration confirmed a statistically significant difference in favour of laparoscopy, namely 61 min vs. 85 min for conventionally operated patients. The average age was comparable. We found a statistically significant difference in the number of complications in favour of laparoscopy, 10.9% vs. 27.5% for conventional operations. In the case of laparotomy we did not record even a single wound infection complication compared to the conventional group with the occurrence of 11.7%. The differences in favour of laparoscopy, 3.6% vs. 9.2% in the case of postoperative occurrence of extra-abdominal complications, such as bronchopneumonia and cardiac complications, were statistically insignificant. The differences are apparent, but for statistical confirmation they would normally require a larger group of patients. Similarly to the complications with the follow-up necessity of postoperative re-intervention, laparoscopy seems to be the preferred choice, with 3.6% vs. 9.2% in the case of conventionally operated patients. Even in this case, for the confirmation of a significant difference we would require a larger group. With very low numbers of patients in leakage and exitus groups, we were unable to compare the results, although the results were similar. BODY.DISCUSSION: The best parameters for comparison of the two operating techniques are mortality and morbidity. Perforated ulcer is still associated with a high rate of morbidity and mortality. Several studies have been published, although only 3 of them are prospectively randomized [17–19], with a high (degree of evidence). The comparison of results in the aforementioned studies shows significant differences in morbidity (22% in laparoscopy group vs. 36% in conventional group), and mortality (2.5% vs. 5.8%). In these studies postoperative leakage occurred more frequently in the laparoscopic group (3%), versus the conventional group, with only 1.1% of operated patients with leakage [17–19]. However, it is necessary to realise that laparoscopic suture closure of the perforated ulcer is a relatively complicated operation, and it requires manual skills in mini-invasive surgery. The reason for conversion, 12.4% on average, is in most cases the size of the perforation [16]. Additional reasons were inability to visualise the perforation, location of perforation, and bleeding. In one of the most recent studies, there were not found any significant differences in terms of post-operative leakage from the suture closure, when the surgeons applied a single suture without omentoplasty, or with it [20]. Alternative options are the application of fibrin glue, which is limited by the size of the perforation, or the application of biodegradable lactide-glycolide-caprolactone patches, which are still at the stage of development. During the recent period, we can observe a gradual cessation of the final surgical intervention (resection, pyloroplasty, vagotomy) with low re-occurrence of the ulcer disease following the eradication of Helicobacter pylori (double combination of antibiotics and proton-pump inhibitor), and elimination of NSAID usage [21]. BODY.CONCLUSIONS: Laparoscopic suture closure of the perforated gastro-duodenal ulcer performed in a centre with sufficient history in laparoscopic surgery is a safe method, which offers better results compared to conventional therapy. The potential advantages of the mini-invasive approach are as follows: better view of the trans-operative situation, limited surgical trauma, smaller operating wound, limited intestinal manipulation and earlier recovery to baseline activities [22]. These advantages contribute to the lower occurrence of complications, especially such as wound complications, and the method is associated with reduced postoperative pain, and the possibility of faster mobilisation. In economic terms, laparoscopy provides a shorter hospitalisation time, and last but not least, a better cosmetic outcome. Even the conclusion of the EAES consensus as of 2006 reads that in the case of suspected perforated ulcer it is recommended to apply a mini-invasive approach as a safe therapeutic method [23]. This has also been confirmed by available prospective randomised studies, and in our centre the laparoscopic suture closure became the option of choice.

TITLE: Flexible, dual-form nicotine replacement therapy or varenicline in comparison with nicotine patch for smoking cessation: a randomized controlled trial ABSTRACT.BACKGROUND: Extended use of combined pharmacotherapies to treat tobacco dependence may increase smoking abstinence; few studies have examined their effectiveness. The objective of this study was to evaluate smoking abstinence with standard nicotine patch (NRT), extended use of combined formulations of nicotine replacement therapy (NRT+), or varenicline (VR). ABSTRACT.METHODS: A total of 737 smokers, including those with medical and psychiatric comorbidities, were randomly assigned to one of the above three treatment conditions. The NRT group received 10 weeks of patches (21 mg daily maximum); the NRT+ group received patches (35 mg daily maximum) and gum or inhaler for up to 22 weeks; and the VR group received 1 mg twice daily for up to 24 weeks (22 weeks post target quit date). All participants also received six standardized 15-minute smoking cessation counseling sessions by nurses experienced in tobacco dependence treatment. The primary outcome was carbon monoxide-confirmed continuous abstinence rates (CAR) from weeks 5–52. Secondary outcomes were: CAR from weeks 5–10 and 5–22, and carbon monoxide-confirmed 7-day point prevalence (7PP) at weeks 10, 22, and 52. Adjusted and unadjusted logistic regression analyses were conducted using intention-to-treat procedures. ABSTRACT.RESULTS: The CARs for weeks 5–52 were 10.0 %, 12.4 %, and 15.3 % in the NRT, NRT+, and VR groups, respectively; no group differences were observed. Results with 7PP showed that VR was superior to NRT at week 52 (odds ratio (OR), 1.84; 97.5 % Confidence Interval (CI), 1.04–3.26) in the adjusted intention-to-treat analysis. Those in the VR group had higher CAR at weeks 5–22 (OR, 2.01; CI, 1.20–3.36) than those in the NRT group. Results with 7PP revealed that both NRT+ (OR, 1.72; CI, 1.04–2.85) and VR (OR, 1.96; CI, 1.20–3.23) were more effective than NRT at 22 weeks. As compared to NRT monotherapy, NRT+ and VR produced significant increases in CAR for weeks 5–10 (OR, 1.52; CI, 1.00–2.30 and OR, 1.58; CI, 1.04–2.39, respectively); results were similar, but somewhat stronger, when 7PP was used at 10 weeks (OR, 1.57; CI, 1.03–2.41 and OR, 1.79; CI, 1.17–2.73, respectively). All medications were well tolerated, but participants in the VR group experienced more fatigue, digestive symptoms (e.g., nausea, diarrhea), and sleep-related concerns (e.g., abnormal dreams, insomnia), but less dermatologic symptoms than those in the NRT or NRT+ groups. The frequency of serious adverse events did not differ between groups. ABSTRACT.CONCLUSIONS: Flexible and combination NRT and varenicline enhance success in the early phases of quitting. Varenicline improves abstinence in the medium term; however, there is no clear evidence that either varenicline or flexible, dual-form NRT increase quit rates in the long-term when compared to NRT monotherapy. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01623505; Retrospectively registered on July 13, 2011 BODY.BACKGROUND: Despite the well-established consequences [1], smoking remains prevalent worldwide [1–3]. Cessation attempts are common; 40 % of smokers try to quit at least twice annually [4], but only 3–5 % will be successful [5]. Pharmacotherapy and behavioral interventions improve quit rates [6, 7]. Evidence based on indirect comparisons derived from meta-analyses favors combinations of nicotine replacement therapy (NRT) or varenicline [6–8] over NRT monotherapy. To date, most studies have compared standard-dose NRT or varenicline to placebo; only a handful of studies have compared NRT monotherapy to combinations of NRT products or varenicline [6–11], and only one study [12] has included NRT monotherapy, combination NRT, and varenicline in a single trial, with the results proving inconsistent with previous meta-analyses and clinical practice guidelines [7]. Additional comparisons of these treatments are required to provide direct evidence of their relative effectiveness. Little is known about the efficacy of smoking cessation treatments in populations with significant medical or psychiatric comorbidities [13–15]; these groups are typically excluded from trials. Cardiac patients, for example, who may gain immediate benefits from quitting are often deemed ineligible for participation in smoking cessation research due to concerns of potential adverse events. In fact, a recent review reported that only two randomized controlled trials (RCTs) investigated varenicline prescribed to patients with active cardiovascular disease and 11 studies included patients with a history of cardiovascular disease [16]. Studies examining cessation among those with psychiatric diagnoses are limited by small sample size, have omitted formal diagnostic procedures, or have examined only select psychiatric populations. This is particularly true of trials assessing varenicline – only six RCTs examining psychiatric patients have been published and three included fewer than 50 smokers [17–22]. As rates of smoking are disproportionately higher in those with psychiatric disorders, RCTs including such patients should be a priority. The present trial includes patients with physical and psychiatric comorbidities and it is the first to directly compare NRT and varenicline in smokers with and without psychiatric illness. The primary objective of this randomized controlled trial was to compare the effectiveness of three cessation treatment strategies: standard-dose monotherapy NRT (NRT); extended duration of combinations of NRT products (NRT+); and extended varenicline (VR). These interventions were chosen as they are first-line treatments for smoking cessation, and the use of flexible and dual-form NRT reflects contemporary clinical practice and the choices of smokers in the real world; the efficacy of this treatment and varenicline compared to standard-dose nicotine replacement therapy requires further testing. We hypothesized that NRT+ and/or VR would each be superior to standard-dose NRT in achieving smoking cessation. BODY.METHODS.STUDY DESIGN: Written consent forms and study procedures were approved by the Ottawa Health Sciences Network Research Ethics Board, and the trial was registered on ClinicalTrials.gov (identifier# NCT01623505). All participants provided voluntary written informed consent. This parallel, three-group randomized controlled trial was conducted at a single center (Ottawa Heart Institute) between June 2010 and July 2014. Participants were randomized to one of the three treatment conditions: NRT, NRT+, or VR. Six standardized 15-minute smoking cessation counseling sessions by nurses experienced in tobacco dependence treatment were provided to all participants; sessions were offered in-person on an individual basis. BODY.METHODS.ELIGIBILITY AND SCREENING: Participants were recruited by advertising (i.e., radio, local newspaper, and posters), from those presenting to the Quit Smoking Program at our institution, and from referrals by local physicians. Interested smokers contacted the study coordinator by phone or in-person and were screened for eligibility. A baseline visit was scheduled at which eligibility was reconfirmed. Eligible participants were 18 years or older, smoked ≥ 10 cigarettes per day, and were willing to make a quit attempt in the next 2–4 weeks. Participants were initially excluded if they had used any of the study medications in the previous 6 months; this proved to be overly restrictive and unnecessary due to the short medication wash out period and, subsequently, was revised. Exclusion criteria included the use of NRT or varenicline for more than 72 consecutive hours in the past month; the presence of contra-indications to the use of study medications; serious cardiac arrhythmias or a myocardial infarction or cerebral vascular accident within the previous 10 days; severe or unstable angina pectoris; end-stage renal disease or use of cimetidine; alcohol or substance abuse in the previous 3 months; unstable psychiatric symptoms precluding informed consent (i.e., active, untreated psychosis or suicidality); refusal to be randomized; unable to attend follow-up appointments; and an inability to understand English or French. Women were excluded if pregnant, lactating, or likely to become pregnant in the next year. No more than one person from the same household was permitted to participate. BODY.METHODS.STUDY PROCEDURES: Participants attended a baseline assessment, which included a medical and psychiatric assessment, and completed questionnaires assessing demographics, smoking history, and nicotine dependence. Women provided a urine sample to test for pregnancy. After eligibility was confirmed by one of the principal investigators (HT, AP), participants were randomized to receive NRT, NRT+, or VR using a computer-generated block randomization schedule by a statistical consultant not involved in the trial; block sizes varied from 6–12. Randomization was stratified by psychiatric status (yes/no). Participants were seen on eight occasions including a baseline assessment and first counseling session at week 0, and follow-ups at 1, 3, 5, 8, 10, 22, and 52 weeks post-target quit date. Counseling and medication distribution occurred at the first six visits; a 5-week supply was provided at baseline and week 5 to prevent missed doses if a participant was absent at a follow-up appointment. Participants selected their target quit date; those using nicotine replacement therapies set this date 1–14 days from the baseline assessment, and participants using varenicline set a date on day 8–14 after baseline. At each appointment, withdrawal symptoms, smoking status, medication usage, and adverse events were assessed. Exhaled carbon monoxide (CO) levels were determined at weeks 5, 10, 22, and 52. Study nurses collected outcome data during the treatment phase, and the research coordinator, who was blind to treatment condition, collected follow-up data (week 22 and 52). Further details regarding study design, randomization procedures, counseling content, resources and staff training have been described previously [23]. BODY.METHODS.PHARMACOLOGICAL INTERVENTIONS: All medications were purchased with study funds from the Ottawa Heart Institute Pharmacy, and were provided to participants on an open label basis. The research coordinator collecting follow-up data at weeks 22 and 52 was blind to treatment condition. BODY.METHODS.PHARMACOLOGICAL INTERVENTIONS.NRT GROUP: A 10-week supply of Nicoderm® patches was provided to participants. The first patch was applied on the target quit date. Initial dosing was determined by the daily average of cigarettes smoked: ≥ 20 cigarettes/day received 21 mg/day for 6 weeks, 14 mg/day for 2 weeks, and 7 mg/day for 2 weeks, while those smoking less were prescribed 14 mg/day for 6 weeks and 7 mg/day for 4 weeks. BODY.METHODS.PHARMACOLOGICAL INTERVENTIONS.NRT+ GROUP: Treatment for the NRT+ group was similar to that in the NRT group; however, the fixed-dose strategy and 10-week duration of therapy was not applied. Instead, participants were encouraged to address withdrawal symptoms by titrating their NRT use up to a daily maximum of 35 mg via patches and to use Nicorette® gum or inhalers ad libitum. The Minnesota Nicotine Withdrawal Scale [24] was used to assist participants and staff to titrate dosing at each visit; scores ≥ 2 on any item signaled a need to increase the dosage. If interested and it was recommended by the study nurse or physician, participants could continue to receive treatment for up to 22 weeks. BODY.METHODS.PHARMACOLOGICAL INTERVENTIONS.VR GROUP: Participants assigned to the VR group began the medication (i.e., Champix) at the baseline assessment. The dosage was 0.5 mg once daily for 3 days, increasing to 0.5 mg twice daily for days 4–7, followed by a maintenance dose of 1 mg twice daily for 11 weeks. If interested and it was recommended by the study nurse or physician, participants could receive a second 12-week supply of varenicline at the week 10 counseling session. Thus, treatment could be provided for up to 24 weeks (i.e., 22 weeks post-target quit date). BODY.METHODS.MEASURES: At baseline, age, sex, ethnicity, marital and employment status, education level, income (Canadian dollars), number of smokers in the household, the age of onset of smoking, number of years as a daily smoker, number of previous quit attempts, number of cigarettes smoked per day, and motivation and confidence to quit (1–10 scale) were collected from each smoker. A self-reported medical history was obtained by the study nurse or physician and medication use documented. The presence of a psychiatric diagnosis was determined using the Mini International Neuropsychiatric Interview 6.0.0. (MINI) [25]. Nicotine dependence was assessed with the 6-item Fagerstrom Test for Nicotine Dependence [26]; scores ≥ 6 indicate high dependence. During the treatment phase, the Minnesota Nicotine Withdrawal Scale was used to assess withdrawal symptoms during the previous 24 hours [24]. Adherence to the study medication was assessed by dividing the amount used by the amount dispensed. Adverse events were noted at each visit, and the study nurse (treatment phase) or study coordinator (follow-up phase) inquired about changes in the participants' health, medications, and any recent hospitalizations. The primary efficacy end point was the CO-confirmed continuous abstinence rate (CAR) during weeks 5–52. As per the Russell Standard [27], a participant was considered abstinent if he or she smoked five cigarettes or less during that period and had an exhaled CO level of ≤ 9 ppm at the 52-week visit. Participants were considered smokers if a visit was missed or dropped out from the study. Secondary outcomes included CO-confirmed CAR from 5–10 and 5–22 weeks post-target quit date and CO-confirmed 7-day point prevalence abstinence (7PP) at weeks 5, 10, 22, and 52 (i.e., smoked no cigarettes, even a puff, in the last 7 days, confirmed with CO exhalation). BODY.METHODS.STATISTICAL ANALYSES: The primary endpoint used to determine sample size was the CAR measured from weeks 5–52. The sample size was calculated based on the assumption that the proportion to quit in the NRT group would be 0.20 compared to 0.30 in the VR group and 0.35 in the NRT+ group [7]. A priori analyses indicated that 854 participants were required to detect differences in abstinence rates between the groups; we planned to increase this sample size to 1068 to account for attrition (20 %). The primary analysis was based on an intent-to-treat approach but, as per the Russell Standard [27], was extended such that participants who had missing outcome data were considered smokers [27]. Participants that died or were no longer reachable (i.e., moved, phone not in service) were removed from the analysis [27]. An unadjusted logistic regression model that included treatment group as the independent variable (NRT as reference group) was fit to the smoking status (abstinent or not) from weeks 5–52 for the primary analysis. These analyses were repeated with the secondary outcomes (CAR from weeks 5–10 and 5–22, as well as 7PP collected at weeks 10, 22, and 52). An adjusted logistic regression model was conducted including baseline variables that were potential univariate predictors of cessation outcome (conservative univariate P value set at 0.15). As the assumption that missing data is equivalent to smoking may bias the treatment effect analysis, we also conducted a sensitivity analysis by assuming an imperfect relationship between missing and smoking status. As per Hedeker et al. [28], we assumed different odds ratios for this relationship and compared these to the intention-to-treat analysis. In addition, we conducted the logistic regression analyses with responders only. Finally, exploratory logistic regression analyses were conducted to investigate the effect of medication extension on smoking status. For all analyses of smoking-cessation outcomes, an alpha level of 0.025 was used to adjust for multiple comparisons. As such, odds ratios (OR) with 97.5 % confidence intervals (CI) are reported throughout. χ2 analyses with Bonferroni-adjusted post hoc tests (alpha level 0.05) were performed to examine the relationships between adverse events and treatment condition. BODY.RESULTS.ENROLLMENT AND FOLLOW-UP: From 1700 potentially eligible participants, 737 were randomly assigned to treatment: NRT (n = 245), NRT+ (n = 245), and VR (n = 247). With 245 participants per group and an alpha of 0.025 to account for multiple comparisons, we had 63 % power to detect a 10 % improvement in quit rates between the NRT and VR groups and 93 % power to detect a 15 % improvement in quit rates between NRT and NRT+. Follow-up rates at the end of treatment (week 22) were 62.4 % (n = 153) in the NRT group, 73.4 % (n = 180) in the NRT+ group, and 70.4 % (n = 174) in the VR group. At study completion, follow-up rates were 62.0 % (n = 152), 70.2 % (n = 172), and 65.2 % (n = 161), respectively; these rates were 66.0 % (n = 152), 73.4 % (n = 171), and 68.2 % (n = 161), respectively, using the Russell Standard excluding 15, 12, and 11 participants, respectively, due to death or moving/phone out of service (Fig. 1).Fig. 1Recruitment and retention for the FLEX trial BODY.RESULTS.DEMOGRAPHIC, MEDICAL AND SMOKING PROFILE: Sample characteristics were similar between the treatment groups at baseline (see Table 1 and previous publication [23]). Participants had a mean (M) age of 48.6 (standard deviation (SD), 10.8; Median (Mdn), 50; interquartile range (IQR), 42–56) years, 53.6 % (n = 395) were male, 91.8 % (n = 669) were white, had 14 (SD, 3.0; Mdn, 14; IQR, 12–16) years of education and 52.0 % (n = 381) were employed full-time. Most participants (82.9 %; n = 611) had at least one medical condition; the most prevalent conditions were chronic pain (39.1 %; n = 286), respiratory illness (34.7 %; n = 255), and arthritis (31.2 %; n = 229). Many participants (59.0 %; n = 435) met criteria for a lifetime psychiatric diagnosis; major depressive disorder (64.6 %; n = 281) and anxiety disorders (21.4 %; n = 93) were most prevalent. Smoking had begun during adolescence (M age, 14.5; SD, 4.0 years; Mdn, 20; IQR, 15–25), and participants smoked for 31 (SD, 11.7) years on average consuming 23.2 (SD, 10.8; Mdn, 32; IQR, 23–32) cigarettes per day. Participants tended to live with one smoker (M, 1.04; SD, 1.02); 66.2 % (n = 475) lived with one or more smokers. High nicotine dependence (M, 6.1; SD, 2.2; Mdn, 6; IQR, 5–8) and confidence (M, 7.38; SD, 2.16; Mdn, 8; IQR, 6–8) and motivation (M, 8.69; SD, 1.47; Mdn, 9; IQR, 8–9) to quit were evident at the baseline assessment.Table 1Baseline demographic information n (%)OverallNRTNRT+VRAge (M, SD)48.61 (10.8)48.13 (11.1)48.57 (10.5)49.11 (10.8)Sex Male395 (53.6)137 (55.9)131 (53.5)127 (51.4) Female342 (46.6)108 (44.1)114 (46.5)120 (48.6)Marital status Married/common law340 (46.3)120 (49.0)107 (43.9)113 (46.3) Divorced/separated/widowed232 (31.7)69 (28.1)79 (32.3)84 (34.4) Single/never married161 (22.0)56 (22.9)58 (23.8)47 (29.2)Years of education (M, SD)14.13 (3.0)13.97 (2.7)14.19 (3.0)14.21 (3.2)Employment status Full/part time449 (61.3)157 (64.4)145 (59.5)147 (60.1) Homemaker/retired/unemployed158 (21.6)49 (20.0)51 (20.8)58 (23.6) Disability leave126 (17.1)38 (15.6)48 (19.7)40 (16.3)Annual household income (Canadian dollars) 19,999 or less162 (22.7)50 (20.7)61 (25.8)51 (21.6) 20,000–39,999147 (20.6)51 (34.7)47 (19.9)49 (20.8) 40,000–69,999216 (30.3)76 (31.5)67 (28.4)73 (30.9) 70,000 or more188 (26.4)64 (26.6)61 (26.8)63 (26.7)Lifetime psychiatric diagnosis (%)435 (59.0)144 (58.5)143 (58.4)148 (58.9)Smoking characteristics (M, SD) Cigarettes smoked per day23.2 (10.8)22.4 (11.3)24.0 (10.9)23.3 (10.1) Cumulative years smoked31.0 (11.7)30.5 (12.2)30.9 (11.3)31.7 (11.5) Fagerstrom test of nicotine dependence6.1 (2.2)6.0 (2.2)6.3 (2.2)6.1 (2.3) Number of previous quit attempts4.6 (5.4)4.3 (4.5)4.2 (4.5)5.2 (6.7) Motivation to quita 8.7 (1.5)8.7 (1.4)8.7 (1.6)8.7 (1.4) Confidence to quita 7.4 (2.2)7.3 (2.2)7.4 (2.1)7.4 (2.2) Number of other smokers in the household1.0 (1.0)1.1 (0.9)1.0 (0.9)1.1 (1.2)M, mean; SD, standard deviation; NRT, standard dose, nicotine replacement therapy; NRT+, flexible, dual-form nicotine replacement therapy; VR, varenicline aMotivation and confidence to quit were reported on 10-point scales where 0 = not at all confident/motivated to and 10 = completely confident. Please note that this table is an adapted version of a previously published table [23] BODY.RESULTS.INTERVENTION: The median medication dose at week 5 was 21 mg, 21 mg, and 1 mg for NRT, NRT+, and VR, respectively. At week 10, reductions were noted for the NRT group (Mdn, 7 mg), but remained the same for the NRT+ and VR groups. The maximum average daily dosage of nicotine patches used by NRT+ participants was 24.4 mg daily (SD, 6.7 mg; range, 14–35 mg). Most participants (80.4 %) in the NRT+ group chose to use inhalers over gum. Adherence rates were similar across groups (P = 0.08): 84.3 % (n = 194), 79.7 % (n = 198), and 78.9 % (n = 200), respectively. BODY.RESULTS.SMOKING ABSTINENCE: Table 2 displays the abstinence rates by treatment condition as well as results from the unadjusted and adjusted logistic regression models. The CARs for weeks 5–52 were 10.0 % (n = 24), 12.4 % (n = 30), and 15.3 % (n = 37) in the NRT, NRT+, and VR groups, respectively; they were not significantly different between groups (P > 0.025). Results with 7PP showed that VR was superior to NRT at week 52 (OR, 1.84; CI, 1.04–3.26) in the adjusted intention-to-treat analysis. Those in the VR group had higher CAR at weeks 5–22 (OR, 2.01; CI, 1.20–3.36) than those in the NRT group. Results with 7PP revealed that both NRT+ (OR, 1.72; CI, 1.04–2.85) and VR (OR, 1.96; CI, 1.20–3.23) were more effective than NRT at 22 weeks. As compared to NRT monotherapy, NRT+ and VR produced significant increases in CAR for weeks 5–10 (OR, 1.52; CI, 1.00–2.30 and OR, 1.58; CI, 1.04–2.39, respectively); results were similar, but somewhat stronger, when 7PP was used at 10 weeks (OR, 1.57; CI, 1.03–2.41 and OR, 1.79; CI, 1.17–2.73, respectively). Overall, the logistic regression model adjusting for predictors variables (i.e., marital status, income, nicotine dependence, and motivation) strengthened the observed results (Table 2). Results from the sensitivity analysis were not different from the primary analysis (i.e., intention-to-treat approach with missing data coded as smokers). In comparisons between NRT and NRT+, analysis including responders only produced weaker odds ratios and below significant levels for the NRT+ group at weeks 10 and 22. Responder-only analyses including VR were consistent with the intention-to-treat analyses.Table 2Abstinence rates and odds ratios by treatment conditionNRTNRT+VRPrimary outcome: continuous abstinence at week 5–52 Abstinence, n (%)23 (10)29 (12.4)36 (15.3) OR (97.5 % CI)REF1.28 (0.67–2.43)1.62 (0.87–3.01) Unadjusted AdjustedREF1.34 (0.67–2.70)1.84 (0.94–3.58)Secondary outcome: 7-day point prevalence abstinence at week 52 n (%)34 (14.8)43 (18.5)51 (21.7) UnadjustedREF1.31 (0.76–2.25)1.60 (0.94–2.70) AdjustedREF1.31 (0.79–2.37)1.84 (1.04–3.26)*Secondary outcome: continuous abstinence at week 5–22 n (%)38 (15.8)59 (24.5)65 (27.1) UnadjustedREF1.51 (0.88–2.57)2.01 (1.20–3.36)** AdjustedREF1.67 (0.95–2.94)2.18 (1.25–3.80)**Secondary outcome: 7-day point prevalence at week 22 n (%)38 (15.8)59 (24.5)65 (27.1) UnadjustedREF1.72 (1.04–2.85)*1.96 (1.20–3.23)** AdjustedREF1.87 (1.09–3.20)*2.09 (1.22–3.57)**Secondary outcome: continuous abstinence week 5–10 n (%)72 (29.4)94 (38.7)98 (39.8) UnadjustedREF1.52 (1.00–2.30)1.58 (1.04–2.39)* AdjustedREF1.62 (1.03–2.56)*1.66 (1.04–2.63)*Secondary outcome: 7-day point prevalence at week 10 n (%)65 (26.5)88 (36.2)97 (39.4) UnadjustedREF1.57 (1.03–2.41)*1.79 (1.17–2.73)** AdjustedREF1.65 (1.04–2.61)*1.89 (1.19–3.01)**REF, reference group; OR, odds ration; 97.5 % CI, 97.5 % confidence interval; NRT, standard dose, nicotine replacement therapy; NRT+, flexible, dual-form nicotine replacement therapy; VR, vareniclineAdjusted for marital status, income, nicotine dependence, psychiatric status and motivation to quit. *P < 0.025; **P < 0.01 Over half of the participants in the NRT+ (64.5 %; n = 158) and VR (53.4 %; n = 132) groups extended treatment beyond the initial intervention period (i.e., 10 weeks). Those who chose to extend were more likely to be older (P = 0.007) and smoke-free at 10 weeks (P < 0.001); no group differences were found for any of the remaining baseline demographic and smoking-related variables. In order to evaluate the effects of the extended treatment (i.e., the use of study medications past 10 weeks) above and beyond the type or combination of medication, we conducted an exploratory logistic regression analysis comparing five conditions: NRT alone (reference group, n = 244), NRT+ without extension (n = 85), NRT+ with extension (n = 158), VR without extension (n = 91), and VR with extension (n = 132). Results showed that CAR from 5 to 22 weeks in the non-extension arms were not significantly different from the 10-week NRT monotherapy; quit rates were 14.3 % (n = 34) for NRT, 9.4 % (n = 8) for non-extended NRT+, 25.3 % (n = 40) for extended NRT+, 21.9 % (n = 20) for non-extended VR, and 30.3 % (n = 40) for extended VR. Participants in the extended conditions, however, had more success in their quit attempts from weeks 5–22 as compared to NRT monotherapy (OR, 2.05; CI, 1.17–3.61 for extended NRT+ and OR, 2.69; CI, 1.51–4.79 for extended VR). Participants who extended their varenicline use were more likely to be continuously abstinent from weeks 5–52 (9.4 %, n = 23, NRT; 12.1 %, n = 11, non-extended VR; 18.9 %, n = 25, extended VR; OR, 2.14; CI, 0.92–4.97) as compared to NRT. Results were similar, but slightly stronger, in the adjusted analyses controlling for marital status, income, nicotine dependence, psychiatric status and motivation to quit. BODY.RESULTS.ADVERSE EVENTS: Table 3 presents the adverse events reported by participants by treatment condition. Participants in the VR group experienced more fatigue, digestive symptoms (e.g., nausea, diarrhea), and sleep-related concerns (e.g., abnormal dreams, insomnia) than those in the NRT or NRT+ groups. Those in the VR group were less likely to have dermatologic symptoms (e.g., skin rash or irritation). Adverse events resulting in treatment discontinuation by the qualified investigator were not significantly different between the groups (1.6 % (n = 4) NRT group; 2 % (n = 5) NRT+ group; and 2 % (n = 5) VR group; P = 0.93). The frequency of serious adverse events did not differ between groups (3.7 % (n = 9) in the NRT group; 2.4 % (n = 6) in the NRT+ group; and 3.2 % (n = 8) in the VR group; P = 0.073); only three were deemed to be possibly related to the study medication (VR, 2; NRT+, 1).Table 3Adverse events by treatment conditionAdverse eventTreatment group n (%)NRTNRT+VR P Cardiovascular (e.g., palpitations, tachycardia, chest pain)5 (2.0)3 (1.2)3 (1.2)0.687Digestive (e.g., indigestion, nausea, diarrhea, constipation, flatulence)48 (19.6)64 (26.1)139 (56.3)<0.001a Muscular (e.g., hypertonia, join, neck or jaw pain)7 (2.9)5 (2.0)4 (1.6)0.632Nervous system (e.g., dizziness, light headedness, tingling fingers)24 (9.8)27 (11.0)31 (12.6)0.622Psychiatric (e.g., anxious, disturbed concentration, suicidal ideation)12 (4.9)9 (3.7)18 (7.3)0.267Sleep (e.g., abnormal dreams, insomnia, sleep disturbance)92 (37.6)115 (46.9)149 (60.3)<0.001a Fatigue (e.g., drowsy, lethargic)9 (3.7)19 (7.8)43 (17.4)<0.001a Metabolic (e.g., increased appetite, taste perversion)13 (5.3)14 (5.7)23 (9.3)0.151Respiratory (e.g., coughing, congestion, shortness of breath)6 (2.4)9 (3.7)10 (4.0)0.592Skin (e.g., rash, itchiness, dry skin, redness)94 (38.4)81 (33.1)12 (4.9)<0.001a Other (e.g., eyes difficult to focus, hot flashes, low sex drive)21 (8.6)19 (7.8)48 (19.4)<0.001a NRT, standard dose, nicotine replacement therapy; NRT+, flexible, dual-form nicotine replacement therapy; VR, varenicline aStatistically significant BODY.DISCUSSION: Among smokers with and without medical and psychiatric diagnoses, no differences in CAR from weeks 5–52 were observed between the treatment groups; VR, however, significantly improved the odds of quitting when 7DPP was the outcome variable in the adjusted model at one year (P = 0.019). Both NRT+ and VR produced statistically significant increases in CAR from weeks 5–10; and VR resulted in greater abstinence rates from weeks 5–22 when compared to NRT monotherapy. Using 7PP, NRT+ was also more effective than NRT at 22. Of note, the analysis with responders only found insignificant results for all time points in the comparisons between NRT and NRT+, likely indicating a possible overestimation of the effectiveness of the NRT+ intervention. Nonetheless, the NRT+ quit rates were 10 % higher at 10 weeks and 8.7 % higher at 22 weeks, which are clinically significant. Our results suggest that both varenicline and extended and combination NRT enhance success in the early phases of quitting; varenicline improves abstinence in the medium-term and that there is no clear evidence that varenicline or combined NRT increase quit rates in the long-term in comparison to NRT monotherapy. Similar results were reported in a recent study with these treatment groups, albeit with reduced treatment duration (i.e., 12 weeks) [12]. Using the secondary outcome of 7DPP, only VR produced improved quit rates in comparison to NRT at 1 year. Previous investigations have demonstrated the safety of this medication [17, 29–32]. In our sample, differences in the frequency of adverse events were observed: VR use was related to higher levels of fatigue and sleep and digestive problems, and fewer reports of skin problems as compared to the groups using nicotine replacement therapies. The experience of these symptoms, however, did not translate into an unbalanced distribution of treatment discontinuation by condition. Further, no differences in psychiatric adverse events were detected, including between the psychiatric and non-psychiatric groups in our sample. We argue that smokers with psychiatric illness may be offered treatment options similar to those in the general population; their choice should not be restricted by the presence of a psychiatric diagnosis. In addition, the cost of varenicline is at least half that of combination NRT, a factor that is important to many smokers attempting to quit. Our quit rates are similar but slightly lower than those reported in previous studies [9, 10, 17, 33] and a recent meta-analysis reporting abstinence at 6 months or more [6]. Lower rates of cessation may reflect the current population of smokers: those with higher nicotine dependence and significant comorbidities who have greater difficulty quitting [34, 35]. The sociodemographic and medical profile of our participants provides evidence of higher nicotine dependence and significant comorbidities among current smokers. Our sample had multiple medical conditions (82.9 % (n = 611) reported at least one; 12.6 % (n = 93) reported > 5; 59.0 % (n = 435) had a lifetime psychiatric diagnosis; 64.3 % (n = 455) fell in the high nicotine dependence category (i.e., > 6 in the Fagerstrom Test for Nicotine Dependence test); and 22.7 % (n = 162) live in poverty (i.e., annual income < $20,000). As we attempted to provide an intervention that might be amenable to real-world settings, we provided fewer counseling sessions than typically provided in efficacy trials [33], which may be another explanation for our lower quit rates. Adverse events reported in our study appear higher than those reported in previous investigations [17, 33]. There are two potential explanations: first, adverse events were categorized and summed in our study, while other studies often count each symptom occurrence independently. For example, sleep disturbances (e.g., abnormal dreams, insomnia, and hypersomnia) were tallied in our study, but presented individually in previous studies. Second, participants in our trial were not blind to treatment condition nor was a placebo control group established; this may have contributed to participants seeking, detecting, perceiving, and/or interpreting symptoms more frequently and assigning causality to the study medication leading to increased reporting of adverse events. Nonetheless, these symptoms were well-tolerated as treatment discontinuation was limited and did not differ by condition. The major strengths of this trial were its randomized design, validated outcomes, and the particular inclusion of smokers typically excluded from cessation studies. It is one of few studies to compare monotherapy NRT to extended, combined NRT or extended varenicline [6, 8, 11, 12, 36, 37], and the second to include these treatments in a single trial. Our results add to the literature regarding more effective use of smoking cessation pharmacotherapy; the long-term results of flexible, dual-form NRT and varenicline are less clear and put into question the early indicators derived from meta-analyses and clinical experience. Our study is not without limitations. Although we employed conservative estimates for sample size calculations, it remains possible that clinically important differences in quit rates between the treatment groups were not detected due to an inadequate sample size which fell below our planned levels (i.e., 737 participants enrolled versus 854 required to detect differences and 1068 planned to recruit to account for attrition); this is particularly true for the comparison between VR and NRT+ (63 % power). The generalizability of these three treatment methods to a broader population of smokers with comorbid conditions is unknown. For example, we are limited by our sample being comprised mainly of white participants. Unequal sample sizes were evident in the exploratory analyses investigating length of treatment. Further, this analysis no longer included the balancing of potential confounders because patients self-selected whether to extend treatment or not; however, no differences were detected between groups on any baseline demographic or smoking-related variables, and we controlled for variables which significantly predicted outcomes in the analysis. A randomized controlled trial including these five treatment conditions would solidify the findings reported herein. Such a trial would clarify if the treatment extension or therapy dose produced the enhanced success in quitting. At present, however, our study design was limited by the use of both increased medication dosage and duration of treatment in the NRT+ group (≤35 mg, maximum 22 weeks) than in the NRT group (≤21 mg, maximum 10 weeks); increases in one or the other would have allowed for more definitive conclusions regarding treatment efficacy. Nonetheless, similar results to those reported herein have been reported by recent trials which kept duration constant [12] or restricted treatment to single-form only [37]. We were also limited by the lack of a placebo condition. The study staff was aware of treatment allocation during the treatment phase; there may have been reporting bias in favor of one treatment versus another or in the expectation of adverse events. This effect was reduced, however, by the fact that multiple nurses were involved in the assessment and staff collecting follow-up data at weeks 22 and 52 were blind to treatment condition. There may have been a reporting bias of adverse events by participants. Future research might record participants' treatment preference prior to randomization to better understand the effects of preference on efficacy outcomes and adverse advent reporting. Our study may have been strengthened by identifying potential confounders for the adjusted analysis a priori. Although we made concerted efforts to retain and follow all randomly assigned participants throughout the 52-week trial, high attrition rates were noted across all treatment groups, and missing data could have affected outcomes. The assumption that all drop-outs have resumed smoking could underestimate abstinence rates; however, empirical evidence from smoking cessation studies suggests that drop-outs tend to relapse to smoking [27] and our sensitivity analysis did not indicate differences from the intention-to-treat analysis. The analyses with responders only, however, did reveal that results may be overestimated with regards to the extended and combined NRT group. BODY.CONCLUSION: Our trial suggests that, in comparison to a standard nicotine patch, flexible and dual-form NRT and varenicline enhance cessation in the short-term but, unfortunately, the effect appears to wane over time. Although both NRT+ and VR led to increased odds of quitting from 5 to 10 weeks and VR produced improved cessation rates from 5 to 22 weeks, only clinically significant differences between the NRT and VR groups (5.3 %) were observed at 1 year. The results presented herein, therefore, do not fully support the use of flexible, dual-form nicotine replacement therapies or varenicline rather than standard nicotine patch for smoking cessation maintenance. Future research is needed to clarify the effectiveness of these treatments; trials that investigate both the dose and duration of each treatment condition are needed. BODY.ABBREVIATIONS: NRT, nicotine replacement therapy; NRT+, flexible and dual-form nicotine replacement therapy; VR, varenicline; CAR, carbon monoxide confirmed, continuous abstinence rate; 7PP, carbon monoxide confirmed, 7 day point prevalence abstinence; OR, odds ratio; CI, confidence interval; RCT, randomized controlled trial; MINI, Mini International Neuropsychiatric Interview; Mdn, median; IQR, interquartile range; FTND, Fagerstrom test of nicotine dependence; CO, carbon monoxide

TITLE: Honey Mitigates Radiation-Induced Oral Mucositis in Head and Neck Cancer Patients without Affecting the Tumor Response ABSTRACT: Radiation-induced mucositis is a dose-limiting factor in the effective treatment of head and neck (H & N) cancers. The objective of this study was to understand the efficacy of honey in mitigating radiation-induced mucositis and whether it would interfere with tumor control. This was a single-blinded, randomized, controlled study and was carried out in patients with H & N cancer requiring curative radiotherapy (>62 Gy (Gray)). The patients meeting the inclusion criteria were randomly assigned to receive either honey (n = 25) or povidone-iodine (active comparator) (n = 25) during radiotherapy. Oral mucositis was assessed using the RTOG (Radiation Therapy Oncology Group) grading system before the start, during, and at the end of the treatment by an investigator unaware of the treatment. The results indicate that when compared with the active comparator, honey reduced the radiation-induced oral mucositis, decreased the incidence of intolerable mucositis, treatment breaks, loss of treatment days (p < 0.0001 and < 0.0003) and did not affect the radiation-induced tumor response. The clinical observations indicate that honey mitigates the radiation-induced mucositis and does not interfere with tumor cell killing. BODY.1. INTRODUCTION: Mucositis is an unavoidable side effect observed in most cancer patients undergoing radiotherapy for head and neck cancers (H & N) [1]. Depending on the severity, oral mucositis is classified as tolerable (grade 1 and 2 mucositis) and intolerable mucositis (grade 3 or 4) [1]. In severe conditions, mucositis also contributes to local and systemic infections [1]. This will affect the treatment schedule, dose, and therapeutic outcome [1]. From a therapeutic perspective, there are no drugs for the avoidance/mitigation of radiation-induced mucositis and preventive procedures consist of symptom management and adherence to basic oral care to prevent infections and alleviate the mucosal symptoms [1]. In most hospitals colloidal silver solutions, salt and soda rinses, or hydrogen peroxide rinses are used [1]. In recent years, the recombinant human keratinocyte growth factor-1 (KGF-1) (palifermin) has also been reported to be useful in mitigating mucositis [1]. However, its exorbitant cost is a major deterrent from widespread use in developing countries [1]. In the recent past, studies from around the world have shown that honey was effective at mitigating radiation- and chemotherapy-induced mucositis [2,3,4,5,6,7,8,9,10,11,12,13,14,15]. Met analyses have substantiated the observed beneficial effects [13,14,15]. Honey is arguably one of the world's oldest dietary agents with medicinal use, and scientific studies have shown it to possess wound healing, antimicrobial, analgesic, and anti-inflammatory effects in various experimental studies [2,3,4,5,6,7,8,9,10,11,12,13,14,15]. The present study was principally carried out to observe whether honey interfered with the radiation-induced tumor response while having a positive effect against oral mucositis. BODY.2. PATIENTS AND METHODS.2.1. PATIENT POPULATION: The study was carried out with histopathologically confirmed H & N cancer patients requiring radiotherapy. Patients who had radical surgery prior to six weeks at the start of radiation treatment were also included. The exclusion criteria included patients younger than 18 years of age; women who were pregnant or lactating; patients on high doses of non-steroidal anti-inflammatory drugs; poor oral hygiene and xerostomia; and significant comorbidity such as poorly controlled diabetes mellitus, hypertension, schizophrenia, bipolar disorder, and severe depression. Patients who had had oral surgery less than six weeks previously, those who had had chemotherapy within the past six weeks, and those who had previously been treated with radiotherapy for H & N cancers were excluded from the study. The study was approved by the Hospital Ethics Committee (MIO/IEC/2010/12/05). Before the start of the study, patients and their caregivers were informed about the treatment schedule, the benefits, and the possible adverse effects of the study by a trained professional. They were also informed of their right to withdraw during the course of the study. The queries from both patients and caregivers were answered and written informed consent was obtained from the patients. BODY.2. PATIENTS AND METHODS.2.2. STUDY DESIGN: This was a randomized, investigator-blinded clinical study conducted at Mangalore Institute of Oncology from January 2012 to June 2012. A sample size of 50 subjects was considered from previous studies [7,11]. Before initiation of the study, the volunteers were examined for decayed teeth, ulcers, or lesions in the oral mucosa by an orodental physician. Of the eligible 56 patients, six did not meet the inclusion criteria on account of metabolic disease (four) or very poor oral hygiene (two). The remaining 50 volunteered to participate in the study. The randomization was performed using opaque envelopes by a staff member unaware of whether they were distributing the active comparator (povidone-iodine Group A) or the test group (honey Group B). BODY.2. PATIENTS AND METHODS.2.3. RADIATION TREATMENT: The curative radiation treatment dose (62–70 Gy (Gray)) was planned as per the international guidelines [16] using the linear accelerator (Varian Medical Systems, Palo Alto, CA, USA). All patients received a fraction of 2 Gy, five consecutive days a week, for seven consecutive weeks). Some patients also received carboplatin infusion (70 mg/m2/day intravenously (IV)) prior to the scheduled radiation. The patients were provided with the standard oral, dental, medical, and supportive care [16]. A feeding tube was placed only when necessary. Routine screening was performed every other day for oral infections and antimicrobial agents were prescribed based on the culture sensitivity reports. The participants were informed to clean their teeth thrice a day (early morning, after lunch, and at night) using a soft toothbrush. Patients with spontaneous gum bleeds were provided with cleaning solutions. As all patients were in the hospital during the treatment period it was easy to monitor their adherence to diet, medications, practice of oral hygiene, and the interventional agents. BODY.2. PATIENTS AND METHODS.2.4. STUDY MOUTHWASHES: In this study, group A were assigned to povidone-iodine (Betadine 1 mL and 100 mL water), while those in group B were allocated to honey (Dabur India, New Delhi, India). The honey used in the study was of polyfloral origin. To avoid between-batch variation, both honey and povidone-iodine were purchased in a single lot and used for the study. The patients in the povidone-iodine group were asked to follow the schedule as per the protocol of Madan and co-workers [17]. The patients in the honey cohort were taught to apply honey three times a day (1 h prior to radiation, and 2 and 6 h after radiation). The patients were taught to swish the oral cavity and to eat at least 30 minutes after the honey treatment. BODY.2. PATIENTS AND METHODS.2.5. OUTCOME MEASURES: The assessment for the degree of mucositis was done first at the time of mold preparation and then on a weekly basis during the course of radiation treatment. The objective scoring was done by a senior orodental pathologist unaware of the intervention received by each patient. A regular torchlight was used to grade the degree of mucositis in the oral cavity, while a laryngoscope was used to assess the oropharyngeal areas. The grading was scaled from 0 to 4, depending on the severity of oral mucositis based on the The radiation therapy oncology group (RTOG) guidelines. Grades 0, 1, and 2 were "tolerable" and grades 3 and 4 were "intolerable" forms of mucositis, as described earlier [5]. The calibration of assessment was not required because the blinded researcher evaluated the patients throughout the study period [5]. BODY.2. PATIENTS AND METHODS.2.6. TREATMENT RESPONSE EVALUATION AFTER TREATMENT COMPLETION: The response to radiotherapy was assessed during the first follow-up (i.e., four weeks after completion of treatment). The clinical assessment was done by a senior radiation oncologist in accordance to the guidelines prescribed by World Health Organization [18]. Degree of tumor volume shrinkage was considered an index of radio responsiveness. Patients with 100% regression of tumor at the primary site were considered complete responders (CR), whereas partial responders (PR) had a higher than 50% regression and non-responders (NR) had a lower than 50% regression [18]. BODY.2. PATIENTS AND METHODS.2.7. STATISTICAL ANALYSIS: Analysis of variance (ANOVA) was used to compare the extent of severe mucositis score on a weekly basis, testing the equality of proportion for the delay in incidence and the number of tolerable and intolerable mucositis, while the χ2 test was used to compare the total incidence of worst-ever grades of ulceration, the number of treatment days lost due to intolerable mucositis, weight loss, and tumor response. A p value < 0.05 was considered significant. BODY.3. RESULTS.CLINICAL STUDY: The details on age, sex, location of cancer, stage of cancer, type of treatment used, dose, incidence of breaks, and loss of days due to mucositis for the two groups are listed in Table 1 and Table 2. The study population consisted of 13 women and 37 men, with ages ranging from 34 to 73 years (56.29 ± 10.3). The mean age for women was 49.92 ± 9.34 (range 36–67), while that for men were 58.31 ± 10.69 (range 34–73). One patient in the control group succumbed to the disease during the third week of the treatment (Figure 1). The remaining 24 patients in the control group and 25 in the honey group continued with the planned treatment (Figure 1). Radiation exposure caused mucositis in both the cohorts, and the mean mucositis scores are represented on a weekly basis in Figure 2. The onset of tolerable and intolerable mucositis was delayed in the patients using honey and was significant for week 2 (p < 0.0001) and 3 (p < 0.003). The early signs of radiation-mucositis were first seen at the end of first week after exposure to 10 Gy (5 fractions) in both the groups (Figure 2). At the end of the first week, 84% of the patients in the control and 24% in the honey group showed signs of grade 1 mucositis (p < 0.05). With continuation of the treatment, the incidence of grade 2 mucositis appeared only in the controls (32% (8/25) vs. 0% (0/25)) and was significant (p = 0.002) at week 2. The incidence of intolerable mucositis (only grade 3) were seen earlier and at the end of week 3 in controls (16% (4/24), p = 0.03) and reached a peak in both the groups at the end of week 7 (12/24 in controls vs. 8/25 in honey (p = 0.2)). There was no incidence of grade 4 mucositis in either group. With respect to the number of treatment days lost, of the 25 patients in the honey group, three (15%) experienced treatment interruption between the 4th and 5th week of radiation. The corresponding number for patients in the povidone-iodine group was five (20.83%), of whom two needed a treatment break at an early stage before completion of 40 Gy (four weeks) and three after completing 40 Gy between the 5th and 7th weeks. The number of treatment days lost were 7.4 ± 0.08 and 6.33 ± 0.94 days, respectively, for the povidone-iodine and honey groups, and was not significant (Table 2). Additionally, it was observed that when compared to the povidone-iodine (4.86 ± 1.73) group, the weight loss was less in the honey cohorts (2.77 ± 0.85) and was statistically significant (p < 0.001). With respect to assessing the effect of honey on radiation-induced cell killing and tumor regression, the clinical analysis performed four to five weeks after the last fraction of radiation showed that there was no significant difference in the observed cases of the complete response (CR), partial response (PR), and no response (NR). Cumulative results indicated that in the control group 45.8% (11/24) of CR, 25% (6/24) of PR, and 29.2% (7/24) NR were observed, while in the honey-treated group it was 52% (13/25) of CR, 20% (5/25) of PR, and 28% (7/25) of NR and was not significant. BODY.4. DISCUSSION: From a clinical perspective, the resulting inflammation and ulceration seen in mucositis are very painful [1]. The denuded epithelium can also provide access to the oral microbial flora and the resulting infection aggravates the condition [1]. Additionally, exposure to fractionated doses of radiation compromises the wound healing process and this aggravates and complicates the situation [1]. In the present study honey was observed to be effective at mitigating radiation mucositis, which is in agreement to earlier reports [2,3,4,5,6,7,8,9,10,11,12,13,14,15]. The observed effects could be attributed to the honey's analgesic [9,12], antimicrobial [12], and wound-healing effects [19,20]. At a cellular level, exposure to ionizing radiation causes the generation of free radicals (reactive oxygen species and reactive nitrogen species), DNA strand breaks, and activation of transcription factors (NF-κB) [1]. Additionally, the immune cells also produce pro-inflammatory cytokines (Tumor necrosis factor-α (TNF-α), IL-1, and IL-6), which aggravate tissue injury and cell death [1]. Honey has been shown to possess free radical scavenging effects in various experimental systems of study; this could have contributed to the observed protective effects, at least in part [19,20]. The most important observation of our study was that, while honey was effective in mitigating radiation-induced mucositis, it did not interfere with the treatment response in H & N cancer patients. BODY.5. CONCLUSIONS: The results of this study indicate that honey mitigates radiation-induced mucositis and that the protective effect does not interfere with tumor cell killing. The cell culture details indicate that treatment with honey enhanced the radiation-induced cell killing. Further studies are planned to validate these observations with appropriate study models, controls, and end point assays.

TITLE: ABSTRACT.CONTEXT:: Denosumab 60 mg sc injection every 6 months for 36 months was well tolerated and effective in reducing the incidence of vertebral, nonvertebral, and hip fracture in predominantly Caucasian postmenopausal women with osteoporosis. ABSTRACT.OBJECTIVE:: The objective of this phase 3 fracture study was to examine the antifracture efficacy and safety of denosumab 60 mg in Japanese women and men with osteoporosis compared with placebo. ABSTRACT.DESIGN AND SETTING:: A randomized, double-blind, placebo-controlled trial with an open-label active comparator as a referential arm was conducted. ABSTRACT.PATIENTS:: Subjects were 1262 Japanese patients with osteoporosis aged 50 years or older, who had one to four prevalent vertebral fractures. ABSTRACT.INTERVENTION:: Subjects were randomly assigned to receive denosumab 60 mg sc every 6 months (n = 500), placebo for denosumab (n = 511), or oral alendronate 35 mg weekly (n = 251). All subjects received daily supplements of calcium and vitamin D. ABSTRACT.MAIN OUTCOME MEASURE:: The primary endpoint was the 24-month incidence of new or worsening vertebral fracture for denosumab vs placebo. ABSTRACT.RESULTS:: Denosumab significantly reduced the risk of new or worsening vertebral fracture by 65.7%, with incidences of 3.6% in denosumab and 10.3% in placebo at 24 months (hazard ratio 0.343; 95% confidence interval 0.194–0.606, P = .0001). No apparent difference in adverse events was found between denosumab and placebo during the first 24 months of the study. ABSTRACT.CONCLUSION:: These results provide evidence of the efficacy and safety of denosumab 60 mg sc every 6 months in Japanese subjects with osteoporosis. BODY: Denosumab, a fully human monoclonal IgG2 antibody against the receptor activator of nuclear factor-κB ligand, given as a sc at a dose of 60 mg every 6 months increased bone mineral density (BMD) at the lumbar spine and total hip and reduced the incidence of new vertebral, nonvertebral, and hip fractures in predominantly Caucasian postmenopausal women with osteoporosis in the Fracture REduction Evaluation of Denosumab in Osteoporosis every 6 Months (FREEDOM) study (1). However, the subgroup analyses did not show significant reduction in the incidence of new vertebral fracture in nonwhites and subjects living in North and Latin America, presumably due to the small number of subjects and/or the low incidence of fracture in these subgroups (2). Biannual sc of denosumab 60 mg was selected as an optimum dose in Japanese postmenopausal women with osteoporosis in the dose-response study (3). The objective of this study was to assess the efficacy of denosumab on fracture risk reduction and safety in Japanese subjects with osteoporosis compared with placebo for 24 months. The exploratory objective was set to consider the clinical positioning of denosumab for the osteoporosis treatment in Japanese patients comparing the data of BMD and bone turnover markers (BTMs) with alendronate 35 mg weekly as the recommended dosage in the prescribing information in Japan. Fracture and safety data were also collected in the alendronate group. BODY.SUBJECTS AND METHODS.STUDY DESIGN: Denosumab fracture Intervention RandomizEd placebo Controlled Trial (DIRECT) was a randomized, double-blind, placebo-controlled, multicenter trial with an open-label active comparator as a referential arm. Subjects were randomly assigned in a 2:2:1 ratio to receive one of the following three treatments for 24 months: denosumab 60 mg sc every 6 months, placebo for denosumab, or open-label oral alendronate 35 mg weekly. Randomization was stratified by gender. All subjects who received the investigational product (IP) were administered daily supplements containing at least 600 mg calcium and 400 IU vitamin D throughout the study period. The study was designed by the Steering Committee (T.Nakam., T.Matsu., T.Su., and T.H.) and sponsor (H.T., K.W., and T.O). The sponsor had responsibility for data collection and quality control. The Safety Monitoring Board (T.M., I.G., H.Y., Y.T., S.T., S.M., and T.Y.) met semiannually to monitor subject safety, based on blinded data. Radiographs were assessed by the Central Committee (T.Nakan., M.I., T.So., and M.F.). The oral events reported from the local investigators were reviewed by the dental expert (T.Y.). M.S. oversaw the study conduct. Analyses for publication were the responsibility of the sponsor. The manuscript was contributed to and approved by all authors. BODY.SUBJECTS AND METHODS.SUBJECTS: Japanese subjects with osteoporosis including postmenopausal women and men aged 50 years or older were eligible for the study if they had one to four prevalent vertebral fractures with a BMD T-score of less than −1.7 (Young Adult Mean in Japan 80%) at the lumbar spine or −1.6 (Young Adult Mean in Japan 80%) at the total hip by dual-energy X-ray absorptiometry based on the diagnostic criteria of primary osteoporosis in Japan (4). Subjects were excluded if they had more than two moderate and/or any severe vertebral fractures on lateral spine radiographs by semiquantitative (SQ) grading (5) or if they had evidence of the conditions such as hyperparathyroidism, hypoparathyroidism, hypercalcemia, hypocalcemia, rheumatoid arthritis, or Paget's disease of the bone. The subjects were ineligible if they had taken oral bisphosphonates for more than 3 years. Those who had taken bisphosphonates for less than 3 years were eligible if they had received bisphosphonates for less than 2 weeks or had no dosing before 6 months prior to the study enrollment. Subjects were excluded if they had taken selective estrogen receptor modulators, calcitonin, hormone replacement therapy, or teriparatide within 6 weeks before the study enrollment. Additional exclusion criteria included evidence of 2.0 mg/dL or greater serum creatinine, 80 IU/L or greater aspartate aminotransferase, 90 IU/L or greater alanine aminotransferase, or other conditions judged to be inadequate for participation in the study. The institutional review boards at all study sites approved the protocol and consent process for this study, and all subjects provided written informed consent before participation. This study was conducted in compliance with the ethical principles of the Declaration of Helsinki and in accordance with good clinical practice. BODY.SUBJECTS AND METHODS.EFFICACY ENDPOINTS: The primary endpoint was the 24-month incidence of radiographically determined morphometrical new or worsening vertebral fracture for denosumab vs placebo. The secondary end points included the incidence of new vertebral fracture and nonvertebral fracture; the percentage change from baseline in BMD at the lumbar spine (L1-L4), total hip, femoral neck, and distal one third radius; and the percentage change from baseline in serum concentrations of the BTMs, C-telopeptide of type 1 collagen (CTX-1), and bone-specific alkaline phosphatase (BSAP). The exploratory endpoint included the incidence of major nonvertebral fracture known to be significantly associated with decreased BMD (6), consisting of the proximal humerus, forearm, ribs/clavicle, pelvis, hip, distal femur, and proximal tibia. The incidence of nonmajor nonvertebral fracture, which included all nonvertebral fracture except for major nonvertebral fracture, was assessed as a post hoc analysis. Fractures of the skull, face, metacarpus, finger, and toe phalanges as well as pathological fractures and those that were associated with severe trauma defined as a fall from a height higher than a stool, chair, or first rung of a ladder or severe trauma other than a fall were excluded (7). BODY.SUBJECTS AND METHODS.EFFICACY MEASURES: Spine radiographs, antero-posterior and lateral, were taken at baseline and every 6 months over 24 months. To identify morphometrical vertebral fracture, the vertebral bodies of the lateral projection from Th4 to L4 were assessed using both the SQ and quantitative morphometry (QM) methods by the experts of the Central Committee who were blinded to treatment as previously reported (4, 5). Both SQ and QM requirements had to be met for prevalent vertebral fractures at baseline. A new vertebral fracture was defined as an increase of at least 1 SQ grading scale in a vertebral body that was normal at baseline and showing loss of height at the anterior, posterior, or central vertebra by at least 20% from baseline. A worsening fracture was defined in the same manner as a new vertebral fracture, with the above criteria applied to a vertebral body with a prevalent fracture. In cases of disagreement between SQ and QM methodologies, a binary SQ assessment was made to adjudicate the discordant results by the committee. For assessment of nonvertebral fracture, investigators took radiographs at any time during the study to identify the fracture in a subject reporting clinical symptoms. The committee reviewed the radiographs in a blinded manner. BMD as evaluated by dual-energy X-ray absorptiometry was measured at the above-mentioned four sites at baseline and 3, 6, 12, 18, and 24 months except for the distal one third radius at 3 months. The QDR instrument (Hologic) was used in this study. Quality control and BMD scan analysis were performed centrally (Synarc). Concentrations of the above two BTMs were measured from fasting serum samples collected in the morning around the same time at baseline and 1, 3, 6, 12, and 24 months before the administration of the IP. Serum CTX-1 and BSAP were evaluated by the central laboratory using an ELISA and chemiluminescent enzyme immunoassay, respectively (Mitsubishi Chemical Medience Corp). BODY.SUBJECTS AND METHODS.ADVERSE EVENTS (AES): All subjects were questioned concerning AEs at each visit, and all AEs were assessed, regardless of the determinations of causality by the investigators. The Medical Dictionary for Regulatory Activities (MedDRA, version 14.0) was used to categorize reported AEs. Safety laboratory tests including serum chemistry, hematology, and urinalysis were assessed at baseline and 1, 6, 12, 18, and 24 months. Safety was assessed by recording all AEs, serious AEs, fatal AEs, AEs leading to study discontinuation, and AEs leading to discontinuation of IP. AEs of interests, such as hypocalcemia, bacterial cellulitis, infection, eczema, events potentially related to hypersensitivity, cardiovascular disorder, malignant or unspecified tumors, fracture healing complication, atypical fracture of femur, and osteonecrosis of the jaw (ONJ), were prespecified. A dental expert in this study (T.Y.) reviewed each potential case of ONJ in a blinded manner. Study investigators clinically assessed the healing of nonvertebral fractures within 6 months after their occurrence. Antidenosumab binding antibodies were assessed in those samples of subjects randomized in the denosumab or placebo group. BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSES: This study had 90% power to detect a 50% reduction in new or worsening vertebral fracture in the denosumab group as compared with the placebo group, assuming a 16% incidence in the placebo group at 24 months and 20% subject discontinuation rate. The study was planned to enroll 440 subjects per double-blind treatment group (denosumab or placebo). The number of subjects for the alendronate group was set to be 220. Comparisons for alendronate vs. denosumab or placebo were post hoc analyses and not prespecified in the protocol. Efficacy analyses were performed using the full analysis set, which includes all randomized subjects except for those who did not have osteoporosis at screening, did not receive the IP or had no available efficacy data after the first dose of the IP. All tests were performed at a two-sided 5% significant level, and all confidence intervals (CIs) are given as two-sided 95% CIs (α = .05). The 24-month incidence of subjects with new or worsening vertebral, new vertebral, nonvertebral, major nonvertebral, and nonmajor nonvertebral fracture was estimated by the Kaplan-Meier method with 95% CIs. A log-rank test was used to compare the incidence between the denosumab and placebo groups. A proportional hazard model was used to estimate the hazard ratios (HRs). The grouped survival data approach (8) was applied to the log-rank test and the estimation of the HR for the statistical analysis of vertebral fracture because most vertebral fractures were observed at the scheduled visits. The subgroup analyses in the women and men were conducted for new or worsening vertebral fracture and new vertebral fracture. A Student's t test was used to compare the percentage changes from baseline in BMD between the denosumab and placebo groups. The Wilcoxon rank-sum test was used to compare the percentage changes from baseline in the BTMs between the denosumab and placebo groups. The safety analysis set included all subjects who received at least one dose of IP, and AEs were summarized by the randomized treatment groups. For the exploratory objective, the referential comparisons for the alendronate vs the denosumab or placebo group were conducted for fractures, BMD and BTMs using the same method as mentioned above. BODY.RESULTS.SUBJECTS: A total of 1262 subjects were randomized at 119 study sites, 500, 511, and 251 in the denosumab, placebo, and alendronate groups, respectively (Figure 1). The numbers of subjects who completed the study at 24 months were 414 (82.8%), 416 (81.4%), and 204 (81.3%), respectively. Figure 1.Disposition of study subjects. *, Three subjects were excluded due to lack of efficacy data after IP administration; **, one subject was excluded due to lack of efficacy data after IP administration. FAS, full analysis set. Baseline characteristics of the subjects were similar across the three groups (Table 1). The proportion of subjects with at least one vertebral fracture at baseline was 98.3%. The mean BMD T-score at the lumbar spine, total hip, and femoral neck was −2.74, −1.98 and −2.32, respectively. Table 1. Baseline Characteristics of the Subjects Double Blind Open Label Denosumab (n = 472) Placebo (n = 480) Alendronate (n = 242) Gender, n, %     Female 449 (95.1) 456 (95.0) 230 (95.0)     Male 23 (4.9) 24 (5.0) 12 (5.0) Age, y     Mean-year a 69.9 ± 7.36 69.0 ± 7.67 70.2 ± 7.31     Group, n, %         <65 99 (21.0) 126 (26.3) 56 (23.1)         65–74 246 (52.1) 231 (48.1) 112 (46.3)         ≥75 127 (26.9) 123 (25.6) 74 (30.6) Body mass index, kg/m 2a 22.6 ± 2.9 22.4 ± 3.1 22.3 ± 3.0 Prevalent vertebral fractures, n, %     0 6 (1.3) 9 (1.9) 5 (2.1)     1 315 (66.7) 319 (66.5) 157 (64.9)     2 113 (23.9) 105 (21.9) 62 (25.6)     ≥3 38 (8.1) 47 (9.8) 18 (7.4) T-score a     Lumbar spine (L1-L4) −2.78 ± 0.89 −2.73 ± 0.88 −2.69 ± 0.94     Total hip −2.01 ± 0.79 −1.95 ± 0.73 −1.96 ± 0.79     Femoral neck −2.38 ± 0.70 −2.29 ± 0.71 −2.29 ± 0.69 Serum CTX-1, ng/mL b 0.64 (0.44, 0.78) 0.63 (0.43, 0.78) 0.61 (0.42, 0.77) Serum 25-hydroxyvitamin D, ng/mL a 20.97 ± 6.08 20.63 ± 5.91 21.10 ± 6.30 Twenty subjects were originally assessed as having one or more vertebral fractures during an initial single reviewer assessment, and subsequent blinded adjudication by the committee determined that no prevalent vertebral fracture was present at baseline. Subjects were excluded if they had less than 12 ng/mL serum 25-hydroxyvitamin D concentration at screening. a Data are mean ± SD. b Data are mean (quartiles 1 and 3). Consequently, five subjects (two, one, and two in the denosumab, placebo, and alendronate groups, respectively) were not classified as osteoporosis based on the diagnostic criteria in Japan. BODY.RESULTS.FRACTURES: The 24-month incidence of new or worsening vertebral fracture was 3.6% in the denosumab group and 10.3% in the placebo group, with the reduction in risk by 65.7% (P = .0001) (Figure 2A and Supplemental Table 1). In the subgroup analysis in women, the risk of new or worsening vertebral fracture at 24 months was reduced by 63.2% in the denosumab group compared with the placebo group (HR 0.368, 95% CI 0.207–0.653, P = .0004). The 24-month incidence of new vertebral fracture was 2.2% in the denosumab group and 8.6% in the placebo group, with the reduction in risk by 74.0% (P < .0001) (Figure 2B). In the subgroup analysis in women, the risk of new vertebral fracture at 24 months was reduced by 72.6% in the denosumab group compared with the placebo group (HR 0.274, 95% CI 0.136–0.553, P = .0001). Figure 2.Cumulative incidence of vertebral fracture. The cumulative incidence of new or worsening vertebral fracture (A) and new vertebral fracture (B) in all subjects in the denosumab and placebo groups. For panels A and B, the percentages given were incidence by the Kaplan-Meier estimate over a 24-month treatment period. The HR (95% CI) and P value of new or worsening vertebral fracture (A) for the denosumab vs the placebo group was 0.343 (0.194, 0.606) and P = .0001. The HR (95% CI) and P value of a new vertebral fracture (B) for the denosumab vs placebo group was 0.260 (0.129, 0.521) and P < .0001. In the subgroup analysis in men at 24 months, the incidence of new or worsening vertebral fracture in the denosumab and placebo groups was 0% and 12.5%, respectively, and that of new vertebral fracture in the denosumab and placebo groups was 0% and 8.3%, respectively. The P value of a new or worsening vertebral fracture and new vertebral fracture for the denosumab vs placebo group was P = .0748 and P = .1478, respectively. The HRs (95% CI) were not estimated because there were no subjects with a vertebral fracture in the denosumab group. The 24-month incidence of nonvertebral fracture was 4.1% in both the denosumab and placebo groups (P = .9951). The 24-month incidence of major nonvertebral fracture was 1.6% and 3.7% in the denosumab and placebo groups, respectively (P = .0577). The 24-month incidence of nonmajor nonvertebral fracture was 2.5% and 0.4% in the denosumab and placebo groups, respectively (P = .0120). In the alendronate group, the 24-month incidence of new or worsening vertebral, new vertebral, nonvertebral, major nonvertebral, and nonmajor nonvertebral fracture was 7.2%, 5.1%, 2.7%, 2.3%, and 0.4%, respectively. As the referential comparison, the HR (95% CI) and the P value of new vertebral fracture for the alendronate vs placebo group was 0.641 (0.335, 1.226) and P = .1749, and for the denosumab vs alendronate group was 0.416 (0.180, 0.962) and P = .0344. BODY.RESULTS.BONE MINERAL DENSITY: Mean BMD percentage change from baseline at 24 months was 9.1% and 0.1% at the lumbar spine in the denosumab and placebo groups, 4.6% and −1.1% at the total hip, 4.0% and −1.1% at the femoral neck, and 0.5% and −1.8% at distal one third radius, respectively (Figure 3, A–D). The difference between the two groups was significant as early as 3 months at the lumbar spine, total hip, and femoral neck (P < .0001) and 6 months at the distal one third radius (P < .0001). Figure 3.Mean BMD percentage changes from baseline. Mean BMD percentage changes from baseline over a 24-month treatment period at the lumbar spine (L1–L4) (A), total hip (B), femoral neck (C), and distal one third radius (D) are shown. a, P < .0001 based on the Student's t test at each time point for the denosumab vs placebo group; b, a referential comparison, P < .01 based on the Student's t test at each time point for the alendronate vs placebo group; c, P < .05 based on the Student's t test at each time point for the denosumab vs alendronate group. The bars show 95% CIs of the mean values at each time point. A central vender performed all BMD analyses. Abnormal vertebrae, such as those with an abnormality, fracture, or artifact, were excluded from analyses. In the alendronate group, the mean BMD percentage change from baseline at 24 months was 7.5%, 3.6%, 2.9%, and −0.2% at the lumbar spine, total hip, femoral neck, and distal one third radius, respectively. As the referential comparison, the difference between the alendronate and placebo groups was significant as early as 3 months at the lumbar spine, total hip, and femoral neck (P < .01) and 6 months at the distal one third radius (P < .01). The difference between the denosumab and alendronate groups was significant as early as 3 months at the lumbar spine and total hip (P < .05), at 12 and 24 months at the femoral neck (P < .05), and from 18 months at the distal one third radius (P < .05). BODY.RESULTS.BONE TURNOVER MARKERS: The median percentage change from baseline in serum CTX-1 and BSAP in the denosumab group was reduced by 70.9% at 1 month and 50.2% at 3 months, respectively, and maintained significant reduction levels thereafter (Figure 4, A and B). The difference in serum CTX-1 and BSAP between the denosumab and placebo groups was significant as early as 1 month (P < .0001). Figure 4.Median BTM percentage changes from baseline. Median BTM changes from baseline over a 24-month treatment period for serum CTX-1 (A) and serum BSAP (B) are shown. a, P < .0001 based on the Wilcoxon rank-sum test for the denosumab vs placebo group; b, as a referential comparison, P < .01 based on the Wilcoxon rank-sum test at each time point for the alendronate vs placebo group; c, P < .05 based on the Wilcoxon rank-sum test at each time point for the denosumab vs alendronate group. The bars show the interquartile range of the percentage changes from baseline at each time point. In the alendronate group, the median percentage change from baseline in serum CTX-1 and BSAP was reduced by 66.3% and 46.4%, respectively, at 6 months and maintained these reduction levels after 6 months. As the referential comparison, the difference between the alendronate and placebo groups was significant as early as 1 month in serum CTX-1 (P < .01) and as early as 3 months in serum BSAP (P < .05), respectively. The difference in serum CTX-1 and BSAP between the denosumab and alendronate groups was significant as early as 1 month (P < .05), except at 6 months in serum CTX-1. BODY.RESULTS.SAFETY: No differences were found in all AEs, serious AEs, fatal AEs, or AEs leading to discontinuation among the denosumab, placebo and alendronate groups during the first 24 months of the study (Table 2). The incidences of AEs of interest and serious AEs including hypocalcemia, bacterial cellulitis, infection, eczema, malignant or unspecified tumors, hypersensitivity, or cardiovascular disorder were not different among the groups. The most frequent infections were nasopharyngitis (44.4%, 42.2%, and 38.4% in the denosumab, placebo, and alendronate groups, respectively) and cystitis (5.9%, 6.0%, and 3.7%, respectively). These infections were all mild in severity. Two subjects in both the denosumab and alendronate groups experienced biochemical hypocalcemia without clinical symptoms. No cases of delayed fracture healing, ONJ, or atypical fracture of the femur were reported in any of the treatment groups. No subjects developed neutralizing antibodies to denosumab in the denosumab or placebo groups. Table 2. Summary of Adverse Events Adverse event Double blind Open label Denosumab (n = 475), n, % Placebo (n = 481), n, % Alendronate (n = 242), n, % All 448 (94.3) 446 (92.7) 229 (94.6) Serious 66 (13.9) 68 (14.1) 30 (12.4) Fatal 5 (1.1) 5 (1.0) 0 (0.0) Leading to study discontinuation 5 (1.1) 2 (0.4) 2 (0.8) Leading to discontinuation of IP 23 (4.8) 31 (6.4) 18 (7.4) AEs of interest     Hypocalcemia 2 (0.4) 0 (0.0) 2 (0.8)     Bacterial cellulitis 6 (1.3) 3 (0.6) 0 (0.0)     Infection 286 (60.2) 269 (55.9) 131 (54.1)     Eczema 70 (14.7) 81 (16.8) 31 (12.8)     Potentially related to hypersensitivity 90 (18.9) 105 (21.8) 45 (18.6)     Cardiovascular disorder 68 (14.3) 63 (13.1) 21 (8.7)     Malignant or unspecified tumors 9 (1.9) 11 (2.3) 2 (0.8)     Fracture healing complication 0 (0.0) 0 (0.0) 0 (0.0)     ONJ 0 (0.0) 0 (0.0) 0 (0.0)     Atypical femoral fracture 0 (0.0) 0 (0.0) 0 (0.0) Serious AEs of interest     Hypocalcemia 0 (0.0) 0 (0.0) 0 (0.0)     Bacterial cellulitis 0 (0.0) 0 (0.0) 0 (0.0)     Infection 5 (1.1) 7 (1.5) 3 (1.2)     Eczema 0 (0.0) 0 (0.0) 0 (0.0)     Potentially related to hypersensitivity 0 (0.0) 0 (0.0) 1 (0.4)     Cardiovascular disorder 6 (1.3) 7 (1.5) 2 (0.8)     Malignant or unspecified tumors 7 (1.5) 10 (2.1) 2 (0.8) The analysis of AEs included those of all subjects who received at least one dose of IP. Reported AEs were coded by using Medical Dictionary for Regulatory Activities, version 14.0. BODY.DISCUSSION: Denosumab 60 mg sc every 6 months for 24 months significantly decreased the risk of new or worsening vertebral fractures by 65.7% compared with placebo in Japanese subjects with osteoporosis. Subsequent post hoc sensitivity analyses showed that these results were not affected by the presence of a small number of subjects who were not classified as osteoporosis (data not shown). The reduction in the risk of vertebral fracture in postmenopausal women was significant and similar to that observed in the overall population. The incidence of new or worsening vertebral and new vertebral fracture in older men in the denosumab group was lower than that in the placebo group, although the difference in these incidences did not reach statistical significance, possibly due to the small sample size (Figure 2). The risk reduction of denosumab on vertebral fracture to placebo in Japanese women with postmenopausal osteoporosis is similar to that observed in FREEDOM, including predominantly Caucasian postmenopausal women (1, 2). The HR (95% CI) for new or worsening vertebral fracture (denosumab vs placebo) in postmenopausal women in DIRECT was 0.37 (0.21–0.65) at 24 months. The risk ratio (95% CI) for the new or worsening vertebral fracture (denosumab vs placebo) in postmenopausal women in FREEDOM was 0.29 (0.21–0.39) at 24 months (9). The 95% CI was narrower in FREEDOM due to the larger sample size than DIRECT, but the range of CIs of the two studies overlap considerably. Thus, the efficacy of denosumab on the relative risk reduction of vertebral fracture in postmenopausal osteoporosis is consistent between Japanese and predominantly Caucasian women. Results of the analysis on the incidence of nonvertebral fractures were complex: the incidence of total nonvertebral fracture was similar between the denosumab and placebo groups; there was a trend for a lower incidence of major nonvertebral fracture in the denosumab group compared with the placebo group; and the incidence of nonmajor nonvertebral fracture was higher in the denosumab group compared with the placebo group. The risk of nonvertebral fracture was consistently lower in the denosumab group compared with the placebo group at 2 and 3 years in FREEDOM (1). The incidence of nonvertebral fracture in the denosumab group in DIRECT (4.1%) was apparently similar to that in FREEDOM. Therefore, the difference in the effect of denosumab on the risk of nonvertebral fracture between DIRECT and FREEDOM is unlikely due to the low incidence of nonvertebral fracture in Japanese subjects, but it may be related to the smaller sample size of DIRECT, as compared with FREEDOM. The gains of BMD from baseline at the lumbar spine, total hip, and femoral neck in this study were comparable with those in FREEDOM and other studies (1, 10). In DIRECT, however, the BMD gain from baseline at the distal one third radius with denosumab was 0.5% on average at 24 months, smaller than the gains of 1.0%–2.0% on average observed in other studies. At the distal one third radius, the BMD loss in the placebo group was −1.8% on average at 24 months in DIRECT. Thus, the difference in the mean values at the distal one third radius between the denosumab and placebo groups was 2.3%, which is comparable with the gains obtained by denosumab compared with placebo in the trials of predominantly Caucasian women during the period of 2 years (10). Changes in BTMs in this study were similar to the effects of denosumab reported in previous studies in Caucasians postmenopausal women with low bone mass (10) and Japanese postmenopausal women with osteoporosis (3). The 24-month treatment with denosumab was well tolerated. No apparent differences in the AEs of the interest were observed between the denosumab and placebo groups. The results of the BMD and BTMs as a referential comparison for the alendronate vs denosumab or placebo group were consistent with previous studies (11–13). The HR for new vertebral fracture between the alendronate and placebo groups in DIRECT (0.641) was within the range of 95% CI (0.41–0.68) observed in the Fracture Intervention Trial (14). The apparent lower incidence of vertebral fracture in the denosumab group compared with the alendronate group may be related to the earlier spine BMD increase and stronger inhibition of bone resorption by denosumab but also to use of the alendronate dosage approved in Japan, which is half the dosage used in the United States. The incidence of nonmajor nonvertebral fracture was numerically higher in the denosumab group than the alendronate group. This difference may be caused by the relatively small sample size, and is unlikely to be clinically significant due to inconsistency of the results of the vertebral fracture, BMD, and BTMs. No apparent difference in the safety profile was observed between the denosumab and alendronate groups. Therefore, denosumab could be used safely in the treatment of osteoporosis for Japanese patients, as is alendronate. This study included a relatively small number of subjects, and the study duration was only 24 months, which may have limited the ability to detect rare AEs such as hypocalcemia, bacterial cellulitis, and ONJ. The study was also underpowered to test the efficacy of denosumab on the prevention of nonvertebral and hip fracture. The interpretation of the results of the referential comparison for alendronate vs denosumab or placebo is limited by the fact that this study was not designed to assess the difference in fracture incidence between the alendronate and denosumab or placebo groups. In addition, the alendronate dosage that was used in this study, ie, 35 mg weekly, which is approved in Japan, is different from the globally approved dose of 70 mg weekly. In conclusion, denosumab 60 mg every 6 months significantly reduced the risk of vertebral fracture in Japanese subjects with osteoporosis who had existing mild or moderate vertebral fractures. These results provide evidence for the efficacy and safety of denosumab 60 mg sc every 6 months in the treatment of Japanese subjects with osteoporosis.

TITLE: Bioelectrical impedance vector analysis for evaluating zinc supplementation in prepubertal and healthy children ABSTRACT.BACKGROUND: The prevalence of abnormal nutritional status has increased in children and adolescents. Nutritional assessment is important for monitoring the health and nutritional status. Bioelectrical impedance vector analysis (BIVA) combines changes in tissue hydration and structure and body composition that can be assessed. ABSTRACT.OBJECTIVES: The objective of this study was to use BIVA to evaluate nutritional status in 60 prepubertal children, aged between 8 and 9 years, supplemented with zinc, to detect possible changes in body composition. ABSTRACT.DESIGN: We performed a randomized, controlled, triple-blind study. The children were divided into the control group (CG; sorbitol 10%, n=29) or the experimental group (EG; 10 mg Zn/day, n=31), and the duration of the experiment was 3 months. Anthropometric assessments were performed for all of the children. ABSTRACT.RESULTS: The body mass index-for-age increased after oral zinc supplementation in the EG (p=0.005). BIVA indicated that the CG demonstrated a tendency for dehydration and decreased soft tissue and the EG demonstrated a tendency for increased soft tissue, primarily the fat-free mass. After analyses of BIVA ellipses, we observed that this method could detect improvements in body composition in healthy children supplemented with zinc. ABSTRACT.CONCLUSIONS: These results suggest that BIVA could be an auxiliary method for studying a small population undergoing zinc intervention. BODY: The prevalence of nutritional disorders has increased in children and adolescents in developed and developing countries, indicating deficiencies of essential vitamins and minerals (1, 2). This prevalence has resulted in a significant impairment of growth and development in this population (3). Nutritional assessments are important for monitoring the health and nutritional status of children. Among the many nutritional assessment methods, anthropometry and body composition provide acceptable accuracy with similar discriminative ability when measured by dual-energy X-ray absorptiometry (4, 5). Methods that accurately assess body composition in children are scarce. International body mass index (BMI)-for-age cutoffs have been proposed to classify overweight and underweight children (6, 7). However, BMI levels among children should be interpreted with caution. Although a high BMI-for-age is a good indicator of excess fat mass (FM), BMI cannot differentiate whether the weight change is due to variations of FM, fat-free mass (FFM), or water (8, 9). Bioelectrical impedance (BIA) is a user-friendly, non-invasive, low-cost, portable method that can be used to calculate the total body fat in children and adults, and it is considered to be a useful tool for assessing body composition (10–13). However, when using conventional BIA, it is difficult to establish the effect of body weight in prediction equations, and no single equation has been developed to calculate the total body fat in children of different ages. Therefore, an accurate evaluation using the bioelectrical impedance vector analysis (BIVA) is necessary, and the patterns are based only on the electrical properties of the tissues. Combined changes in tissue hydration (resistance component) and structure (reactance component) can be monitored with BIVA. Both components of the impedance vector are considered simultaneously, and body composition can be interpreted (14). The graphical method of resistance and reactance corrected by body length (RXc) is based on analysis of the bivariate impedance vector distribution in a healthy population with specific features (10). BIVA is a qualitative method and does not provide quantitative measurements of corporal volumes (15–18). BIVA is useful for clinical purposes because of its ability to detect changes in hydration or body composition in children (9). Therefore, BIVA may be helpful in identifying those children who are at risk of pathological changes in body composition, specifically during chronic conditions (i.e. chronic obstructive pulmonary disease, anorexia nervosa, cancer, and chronic renal failure) (14, 19–24). Supplementary zinc exerts a positive effect on nutritional status through positive weight gain (25). Moreover, zinc is an essential nutrient required for numerous metabolic functions, and its deficiency results in growth retardation, cell-mediated immune dysfunction and cognitive impairment, and decreased protein and nucleic acid synthesis (26). This study used a BIA vector to evaluate nutritional status in prepubertal healthy and eutrophic children supplemented with zinc to detect changes in body composition status. BODY.METHODS.SUBJECTS: The participants included 60 healthy and eutrophic prepubertal children aged between 8 and 9 years from three municipal schools in the city of Natal, Brazil. Informed consent was obtained from all of the children and their parents or guardians before data collection. This study was approved by the Onofre Lopes University Hospital Research Ethics Committee of Federal University of Rio Grande do Norte (UFRN) (protocol number 542/11). The subjects were recruited through an advertisement on school noticeboards and meetings with parents. The children were divided into control and experimental groups, and the pairing was performed randomly. The parents and children did not know the status of their experimental group (EG; or oral solution). Only one member of the team controlled the experiments and revealed children's experimental status (control or experimental group) at the time of data collection. BODY.METHODS.INCLUSION AND EXCLUSION CRITERIA: The children were healthy, eutrophic, and at Tanner stage 1 for genital, breast, and pubic hair growth, which was evaluated by a medical doctor. The exclusion criteria were missing or incomplete dietary data; early pubarche, thelarche, or menarche; nutritional disorders; history of disease (neoplasia; diabetes mellitus; liver, kidney, and thyroid disorders; and acute infectious or inflammatory diseases); undergoing surgery; using vitamin or mineral supplements; and children outside the interval −2 to 2+Z-score for sex-specific BMI-for-age, weight-for-age, and height-for-age indices, according to the 2006 World Health Organization curves (27). BODY.METHODS.EXPERIMENTAL DESIGN: This study was a randomized, controlled, triple-blind study and was based on non-probability sampling (convenience sample). The control group (CG) (n=29) was supplemented with placebo (10% sorbitol, the same vehicle used to prepare zinc solution) and the EG (n=31) was supplemented daily with 10 mg of elemental zinc for 3 months. The CG consisted of 16 males and 13 females, and the EG consisted of 16 males and 15 females. All children underwent anthropometry, BIA measurements, and blood collection to analyze serum zinc at the beginning and end of this study. BODY.METHODS.ANTHROPOMETRY: Anthropometric measurements were performed after an overnight fast. Weight (kg) and height (m) were measured using an electronic balance (Balmak, BK50F, São Paulo, SP, Brazil) and a stadiometer (Stadiometer Professional Sanny, American Medical do Brasil, São Paulo, SP, Brazil), respectively. To measure weight, the child stood on the scale wearing light clothing without shoes. To measure height, the child remained standing without shoes with free head props, heels together, arms extended along the body, and an upright body posture. The heels, buttocks, shoulders, and head touched the wall or vertical surface of the measuring equipment. All measurements were performed by the same examiner to avoid bias (28). New growth curves provided by the World Health Organization for children aged 5 to 19 years were used to classify malnutrition, eutrophic state, and obesity (29). BMI was calculated as the ratio between the body weight (kg) and the square of height in meters (kg/m2). BMI was evaluated by the AnthroPlus v1.0.4 program (available at www.who.int/growthref/en/). BODY.METHODS.BIOELECTRICAL MEASUREMENTS: Bioimpedance (BIA) is a nutritional assessment method that estimates body composition and therefore nutritional status. Resistance (R) and Reactance (Xc) are the components of this method that will provide this information. After the child had emptied his/her bladder, BIA was performed with a BIA analyzer (Quantum II, RJL Systems, Comp Corp., Clinton Township, MI, USA). The Houtkooper equation (30) (Table 1), which was validated and currently recommended for use in children, was utilized (31). This technique requires the precise placement of four electrodes (standard tetrapolar placement on the right hand and foot) strictly following the method reported by Lukaski et al. (32). The two components of the whole-body impedance vector were recorded from single representative stable measurements conducted by the same operator. Table 1 Results of body composition obtained in the control (CG, n =29) and experimental (EG, n =31) groups before and after placebo or oral zinc supplementation in prepubertal and healthy children CG-before vs. CG-after CG-before vs. EG-before CG-before vs. EG-after CG-after vs. EG-before CG-after vs. EG-after EG-before vs. EG-after BMI-for-age (kg/m 2 ) Mean difference −0.1966 −0.08854 −0.4982 0.1080 −0.3017 −0.4097 95% CI of difference −1.444 to 1.051 −1.316 to 1.139 −1.726 to 0.729 −1.119 to 1.335 −1.529 to 0.925 −1.617 to 0.797 Significance NS NS NS NS NS p =0.005 R/H (Ohm/m) Mean difference −1.098 1.905 10.30 3.002 11.40 8.398 95% CI of difference −53.10 to 50.90 −49.25 to 53.06 −40.85 to 61.45 −48.15 to 54.15 −39.75 to 62.55 −41.89 to 58.69 Significance NS NS NS NS NS NS Xc/H (Ohm/m) Mean difference −0.5828 1.163 0.7157 1.746 1.298 −0.4474 95% CI of difference −5.614 to 4.448 −3.786 to 6.112 −4.234 to 5.665 −3.203 to 6.695 −3.651 to 6.248 −5.313 to 4.419 Significance NS NS NS NS NS NS BMI, body mass index; R , resistance; H , height; Xc , reactance. NS, not significant ( p >0.05) and significant ( p =0.005), using Tukey's multiple comparisons test. BODY.METHODS.RESISTANCE (R): The R is the opposition to flow of an alternating current through intra- and extracellular ionic solutions representing the real portion of the impedance (Z). Lean tissues are excellent conductors of electric current due to the large amount of water and electrolytes, that is, they have low R to the passage of electric current. On the contrary, fat, bone, and skin constitute a means of low conductivity, and therefore have high R. BODY.METHODS.REACTANCE (XC): The Xc is the capacitance produced by tissue interfaces and cell membranes representing the imaginary portion of the Z across tissues (33). Xc means the opposition to an electric current caused by the capacitance (property of storing energy in the form of an electrostatic field). The combination of these two values provides information about total body water, FFM, and FM. BODY.METHODS.BIVA: The BIVA plots direct measurements of the vectors R and Xc from the impedance analyzer (RXc graph). According to the RXc graph, impedance measurements standardized by the height of the subject are represented as bivariate vectors with their confidence and tolerance intervals, which are ellipses in the RXc plane. These vectors do not depend on equations (34). The BIVA according to the RXc method is a good indicator of clinical outcomes and for clinical research studies aiming to identify disorders in body composition (35, 36). To investigate the differences between groups, we calculated and plotted the 95% confidence intervals for the average bivariate vector impedance for each group. Furthermore, we calculated and plotted the tolerance ranges of 95, 75, and 50% for the children, which were divided according to the group and time of the study. Healthy Italian children were used as a reference population to compare bioelectrical data (36). BODY.METHODS.ORAL ZINC SUPPLEMENTATION: The EG was supplemented with 10 mg Zn per day for 3 months in the form of zinc sulfate heptahydrate (ZnSO4·7H2O; Merck, Darmstadt, Germany). Oral zinc solution (152.97 μmol Zn/day) was prepared at the Pharmacotechnical Laboratory of the Department of Pharmacy, UFRN. Each drop contained 1 mg of elemental zinc. The CG received an oral placebo as sorbitol 10%. These solutions were added to milk or juice every morning at breakfast. Zinc supplementation was monitored every 2 weeks during home visits by the same observer. BODY.METHODS.MATERIALS: Vacuette Z serum clot activator tubes (Greiner Bio-One, Monroe, NC, USA) were used for biochemical analyses. Becton Dickinson tubes (Trace Element, Serum, Franklin Lakes, NJ, USA) were used for zinc analyses. Polypropylene plastic syringes were purchased from BD (Hercules, CA, USA), and plastic tips and tubes (metal-free) were purchased from BioRad Laboratories (Hercules, CA, USA). Zinc sulfate heptahydrate (ZnSO4·7H2O) and Titrisol zinc standard were purchased from Merck (Darmstadt, Germany). BODY.METHODS.LABORATORY PROCEDURES: All blood samples were collected into the appropriate tubes, and the procedures related to the handling of zinc samples were performed in accordance with international standards (37). After sample collection, the laboratory procedures were performed at the Multidisciplinary Laboratory of Chronic Degenerative Diseases. The blood samples were placed in trace metal-free tubes without anticoagulants and remained in a stainless steel incubator (FANEM 502, São Paulo, SP, Brazil) for 120 min until clot formation occurred. A 500 mL volume of serum was collected with plastic, trace-metal-free pipettes and was transferred to plastic tubes containing 2,000 μL ultra-pure water (Milli-Q Plus, Millipore, Billerica, MA, USA) to dilute the serum (1:4) for zinc analyses. The samples were stored at −80°C for subsequent analyses (Ultralow Freezer, Nuaire, MN, USA). The serum zinc samples were analyzed in triplicates within the same assay by atomic absorption spectrophotometry (SpectrAA-240FS, Varian, Mulgrave, Victoria, Australia) according to the manufacturer's instructions. Zinc sensitivity was 0.01 μg/mL. The intra-assay coefficient of variation was 2.37%. The normal reference range was 0.7–1.2 μg/mL. A standard zinc solution (1,000 mg/mL) was obtained by diluting a Titrisol zinc standard in ultra-pure water. The wavelength was 213.9 nm. The lamp current was 10 mA, and all other procedures, such as calibrations and measurements, were performed according to the manufacturer's instructions. BODY.METHODS.STATISTICAL ANALYSES: The D'Agostino–Pearson omnibus normality test was used to analyze the normality of all study data. Paired Student's t-test was used to compare the data obtained in the control and experimental groups. The Wilcoxon matched-pairs signed rank test was used to complement the paired, non-parametric test. All comparisons were considered to be significant at the 5% significance level. All analyses and figures were performed and created with the GraphPad Prism 6.0 software (GraphPad Software, Inc., San Diego, CA, USA). The BIVA statistical analyses were performed using BIVA software 2003. To determine the group differences, the bivariate 95% confidence interval for the mean impedance vector was calculated and plotted using the bivariate normal distribution of R divided by height (R/H) and Xc divided by height (Xc/H). The unpaired, two-sample Hotelling's T 2 test for vector analysis was performed. Separate 95% confidence ellipses indicated a significant difference between the mean vector positions on the RXc plane (equivalent to a significant two-sample Hotelling's T 2 test, p<0.05). The paired, one-sample Hotelling's T 2 test was performed to determine if the changes in the mean group vectors (measured at the first and second time points) were significantly different from zero (null vector). A 95% confidence ellipse excluding the null vector indicated a significant vector displacement (31). These tests are a multivariate extension of Student's t-test. To determine whether the results were true for the population studied, we used a sample size calculation for the comparison of two means (paired samples) as follows: n=(Zα+Zβ)2.σ2D/δ2, where α=0.05, P (test power)=0.90, β=2(1−P)=0.20, σ2D (standard deviation of the difference)=0.105043, and δ>−0.09 (maximum permissible difference). BODY.RESULTS: This study was conducted with 29 children in the CG and 31 children in the EG who underwent anthropometric and bioelectrical assessments. The sample size (60 children) was adequate for the conclusions in this study. For any value, δ≥−0.09 (i.e. the minimum sample size required was 15 patients). According to the BMI-for-age classification, all children were healthy during the 3-month study (Table 1). Assuming a bivariate normal distribution of R/H and Xc/H, we calculated the bivariate 95% confidence limits for the mean impedance vectors of the different classification groups (i.e. the ellipse within the two-dimensional mean vector falls with a 95% probability). The 'RXc mean graph' was the average of R/H and Xc/H recorded the groups (38). The mean vectors for the CG and EG were plotted before and after placebo and supplementation. The 95% confidence ellipses of the three mean vectors of each group overlapped, which indicates that the position between the vectors at the CG, EG, and reference population (healthy Italian children) were not significantly different in the RXc plane. The paired one-sample Hotelling's T 2 test indicated a difference in the mean vectors between the first measurement (before placebo or supplementation) and the second measurement (after placebo or supplementation). The EG had increased in the soft tissue (primarily FFM) body composition after oral zinc supplementation compared with before supplementation (p<0.0001). However, the p value of the paired one-sample Hotelling's T 2 test for the CG was 0.9 (Fig. 1). Fig. 1The 95% confidence ellipses of impedance vectors measured by the difference between before and after placebo (control group) and before and after zinc supplementation (experimental group). We drew bivariate 50, 75, and 95% tolerance intervals of the impedance vector in the reference population. We then plotted the distribution of individual vectors on the reference ellipses, which allowed for the comparison of the bivariate, intersubject impedance variability. These ellipses were separated by groups before and after placebo or zinc supplementation versus the reference population (healthy Italian children) (Fig. 2). Fig. 2Distribution of impedance vectors with the 50, 75, and 95% tolerance ellipses for (A) the control group before placebo, (B) the control group after placebo, (C) the experimental group before zinc supplementation, and (D) the experimental group after zinc supplementation. R/H, resistance/length; Xc/H, reactance/length. The distribution of individual vectors reflected the heterogeneity of hydration status in eutrophic children ranging from severe dehydration (vectors beyond the upper pole of the 95% tolerance ellipse) to pre-edema fluid overload (vectors close to the lower pole of the 75% tolerance ellipse) in both groups. The CG demonstrated a tendency for dehydration and decreased soft tissue. The EG demonstrated a tendency for increased soft tissue (primarily the FFM). The serum zinc levels were not significantly different between the groups (Fig. 3). However, zinc intake plus zinc supplementation was significantly different in the EG when compared with a CG (p<0.0001). Moreover, the tolerable upper zinc intake level did not exceed the expected values of 23 mg Zn per day for children in the EG. There were no side effects associated with the 10 mg Zn per day. Fig. 3Serum zinc levels in the control group before placebo (CG-B) and after placebo (CG-A) and in the experimental group before zinc supplementation (EG-B) and after zinc supplementation (EG-A). BODY.DISCUSSION: In this study on 60 healthy and eutrophic children, we compared the effect of zinc supplementation on nutritional status as evaluated by BIVA for assessing body composition. We obtained the tolerance intervals of ellipses in children aged between 8 and 9 years. BIVA is a clinically useful method and can be used for routine monitoring of variations in body fluids and nutritional status in children. The length of the vector indicates hydration status, and a left/right shift of the vector indicates soft tissue mass (35). In clinical validation studies in adults, vectors falling out of the 75% tolerance ellipse indicate abnormal tissue impedance. An upper pole or lower pole displacement of the vectors parallel to the major axis of tolerance ellipses indicates tissue dehydration or hyper-hydration, respectively. Extremes of soft tissue mass, such as in obese people and athletes, or lean and wasting conditions are associated with a vector displacement to the left or to the right, respectively, along the minor axis of tolerance ellipses (36, 37, 39). To date, longitudinal data regarding the use of BIA vector for evaluating children after zinc supplementation have not been reported in the literature. We found positive changes in the body composition with zinc supplementation. BIVA in healthy neonates provides good results for obtaining nutritional status and body fluids, and their clinical state could be predicted depending on their location within the quadrants of the graphic ellipses (38). BIVA has been used for detection, monitoring, and controlling hydration and nutritional status using vector displacement for feedback among patients with Alzheimer's disease (40), cachexia (41), stable and non-stable heart failure patients (42), critically ill and cardiorenal patients (43), hemodialysis patients (44), and cancer patients (28, 45). However, there are no references in the literature regarding zinc intervention and BIVA to compare with our study. Therefore, we did not have a reference method or gold standard for the estimation of body composition, and the results should be interpreted with caution. Concerning anthropometric assessment, the BMI-for-age increased after oral zinc supplementation in the EG. This physiological effect is expected, even in healthy children (46). All children were classified as eutrophic throughout the study using the BMI-for-age. Some studies have demonstrated that BIVA has an advantage compared with other methods because BIVA is a good identifier of individual vectors indicating changes in tissue hydration and body structure in subjects from any BMI class. Guida et al. (9) reported a BMI-specific difference in vector position in an 8-year-old age group with progressive vector shortening in groups with increasing BMI class. We used BIVA to observe whether there were changes in body composition with oral zinc supplementation. We suggest that this supplementation changed the body composition of children because the p value in the EG was<0.0001 (calculated by the BIVA software). BODY.DISCUSSION.LIMITATIONS: The main limitation of this study was its relatively small sample size. Nevertheless, the rate of compliance was satisfactory. As our sample was not probabilistic, other studies with a more representative population are required. Moreover, this was an innovative study that used a novel methodological approach, and there were no previous references in the scientific literature using BIVA to assess a nutritional intervention. BODY.CONCLUSIONS: Following the analyses of ellipses by BIVA, we found that this method may detect improvements in body composition (primarily the FFM) in healthy children supplemented with zinc. This finding suggests that BIVA can be used to study a small population undergoing zinc (or other micronutrients) intervention. However, longitudinal data are required to investigate vector migration during zinc supplementation. Monitoring the vector displacement trajectory toward the reference target vector position may represent useful feedback during nutritional therapy. Other studies are required to confirm our results. Currently, BIVA is still not considered to be a gold standard.

TITLE: Insulin Secretion and Its Determinants in the Progression of Impaired Glucose Tolerance to Type 2 Diabetes in Impaired Glucose-Tolerant Individuals ABSTRACT.OBJECTIVE: We investigated the effect of early-phase insulin secretion on the incidence of type 2 diabetes in individuals with impaired glucose tolerance (IGT) participating in the Finnish Diabetes Prevention Study (DPS). We examined how a lifestyle intervention affected early-phase insulin secretion (ratio of total insulin area under the curve [AUC] and total glucose AUC [AIGR] from 0 to 30 min) during a 4-year follow-up intervention trial and whether AIGR0–30 response was modified by insulin sensitivity (IS) and obesity. ABSTRACT.RESEARCH DESIGN AND METHODS: A total of 443 participants with IGT originally randomized to a lifestyle intervention or control group were studied. IS and AIGR0–30 were estimated from an oral tolerance glucose test administered annually during the 4-year follow-up trial and were related to the risk of diabetes onset over a 6-year follow-up. ABSTRACT.RESULTS: Lifestyle intervention resulted in higher IS (P = 0.02) and lower unadjusted AIGR0–30 (P = 0.08) during the 4-year follow-up. A higher IS and a lower BMI during the follow-up were associated with a lower unadjusted AIGR0–30 during the follow-up, independently of study group (P < 0.001). A greater increase in IS on the median cutoff point of a 0.69 increase was associated with higher IS-adjusted AIGR0–30 during the follow-up (P = 0.002). In multivariate models, IS and IS-adjusted AIGR0–30 were both inversely associated with diabetes incidence (P < 0.001). Participants who progressed to type 2 diabetes were more obese and had lower IS and Matsuda IS index-AIGR0–30 than nonprogressors. ABSTRACT.CONCLUSIONS: Our results indicate that the reduction in the risk of developing type 2 diabetes after lifestyle intervention is related to the improvement of IS along with weight loss. Improved IS may also have beneficial effects on preservation of β-cell function. BODY: Genetic and environmental factors both contribute to the development of type 2 diabetes (1). Current evidence indicates that an underlying defect in insulin secretion in the presence of insulin resistance leads to the development of diabetes. Impaired glucose tolerance (IGT) is already characterized by impaired first-phase insulin secretion, a determinant for further progression to diabetes (2–5). Lifestyle changes involving healthy diet, moderate weight loss, and increased physical activity reduce the risk of diabetes (6–8). The extent to which this is due to reduced insulin resistance or improved insulin secretion is not known. Improvements in insulin secretion and insulin sensitivity after 1 year of lifestyle intervention were associated with lower diabetes risk in the Diabetes Prevention Program (DPP) study during a follow-up of 3.2 years (6). In a substudy of the Finnish Diabetes Prevention Study (DPS) in persons with IGT who did not progress to diabetes, insulin secretion measured during a frequently sampled intravenous glucose tolerance test (IVGTT) remained stable for years (9). However, data from long-term intervention trials on the mechanisms that may result in improvement of glucose metabolism and prevention of diabetes associated with healthy lifestyle changes are scarce. Therefore, we investigated the effect of surrogate indices of early-phase insulin secretion and insulin sensitivity from an oral glucose tolerance test (OGTT) on diabetes incidence in individuals participating in the Finnish DPS. We also evaluated whether insulin secretion response in the OGTT was modified by insulin sensitivity and obesity, and how lifestyle intervention may affect the β-cell function. BODY.RESEARCH DESIGN AND METHODS.DESIGN OF THE DPS: The DPS was a randomized, controlled, multicenter study in Finland between the years 1993 and 2000 (ClinicalTrials.govNCT00518167) in which 522 individuals with IGT were randomized into an intervention or control group in five centers. The study design and methods of the DPS have been reported in detail elsewhere (8,10). The study protocol was approved by the ethics committee of the National Public Health Institute of Helsinki, Finland, and all of the study participants gave written informed consent. The main inclusion criteria were BMI >25 kg/m2, age 40–64 years, and IGT based on the mean values of two OGTTs according to the World Health Organization 1985 criteria. Random allocation to one of the two study groups was stratified according to the center, sex, and the 2-h glucose at the screening OGTT. At baseline and at annual visits, individuals completed a medical history questionnaire and underwent a physical examination that included anthropometric measurements and an OGTT. For this study, analyses were limited to those 443 participants who had at least one measurement of glucose and insulin at 30 min during the 4-year follow-up trial because samples for 30-min insulin and glucose were not collected at baseline (Supplementary Fig. 1). The median length of the study was 4 years (range 1–6). During this 4-year follow-up period, all participants were undergoing the randomized intervention. Participants who developed diabetes discontinued the study (n = 12, n = 14, and n = 19 at years 2, 3, and 4, respectively), and the measurements from their previous annual visits before diagnosis were used for the analyses. BODY.RESEARCH DESIGN AND METHODS.PROGRAM FOR THE INTERVENTION GROUP: The intervention program has been described previously (8,10). Briefly, the individuals in the intervention group received individually tailored dietary advice aiming at reducing weight and the intake of total and saturated fat and increasing the intake of dietary fiber. Individuals in the intervention group also received individual guidance to increase their level of physical activity. The most intensive period of intervention was during the first year of the study, when the participants showed improvement in main lifestyle indicators (e.g., body weight and glucose and lipid concentrations). The control group received general advice on the benefits of weight reduction, physical activity, and a healthy diet. BODY.RESEARCH DESIGN AND METHODS.GLUCOSE AND INSULIN HOMEOSTASIS: During 1993 to 1996, a baseline 2-h OGTT was performed (75 g glucose load). In the OGTT performed during follow-up visits starting from the middle of 1996, samples were also taken for 30-min insulin and glucose and for 60-min glucose measurements (Supplementary Fig. 1). BODY.RESEARCH DESIGN AND METHODS.LABORATORY DETERMINATIONS: Glucose levels were measured locally by standard methods, and the measurements were standardized by the central laboratory in Helsinki (8). Serum insulin was determined with a radioimmunoassay (Pharmacia, Uppsala, Sweden) that shows 41% cross-reactivity with proinsulin. BODY.RESEARCH DESIGN AND METHODS.CALCULATIONS: Glucose area under the curve (AUC) during the OGTT was calculated using the trapezoidal method. As surrogate indices of the first/early-phase insulin secretion and of peripheral insulin sensitivity, the ratio of total insulin AUC and total glucose AUC during the 0–30 min OGTT (AIGR0–30) and the Matsuda index of insulin sensitivity (Matsuda ISI: 10,000/square root of [fasting glucose × fasting insulin × (arithmetic mean of glucose × arithmetic mean insulin both during an OGTT at 0, 30, and 120 min)]) were calculated according to published equations (11,12). These indices were chosen based on a previous large population study conducted by our local collaborators in which Matsuda ISI and AIGR0–30 were considered the best indices of insulin sensitivity and secretion (11). In addition, a frequently sampled IVGTT was performed in a subsample of the DPS, and the insulin sensitivity index (SI) and acute-phase insulin response (AIR) were calculated by the MINMOD Millennium software (9). AIR and SI measured at year 4 were used for validation of the AIGR0–30 (n = 53) and Matsuda ISI (n = 47). The Pearson correlation coefficient (r) of AIGR0–30 with AIR was 0.67 (P < 0.001). Matsuda ISI had a correlation of r = 0.73 (P < 0.001) with SI. We also calculated Matsuda ISI according to the latest publication (13) to assess insulin sensitivity at baseline without using the 30-min glucose and insulin values. Its correlation coefficient was also significant (r = 0.74, P < 0.001) versus SI in IVGTT. Both Matsuda ISIs were strongly correlated at all 4-year follow-up visits (r between 0.94 and 0.95, P < 0.001 for all). Because of the known nonlinear relationship between insulin secretion and insulin sensitivity, we also adjusted, by regression analysis, the log of AIGR0–30 by the log of the Matsuda ISI at each year of the study to obtain insulin secretion independently of insulin sensitivity (3,4,14,15). We then transformed the adjusted AIGR0–30 back into untransformed values by taking the antilog (14) to obtain Matsuda ISI-adjusted AIGR0–30 (ISI-adjusted AIGR0–30). ISI-adjusted AIGR0–30 was strongly correlated with SI-adjusted AIR (rs = 0.63, P < 0.001). The outcome variables were averaged from the available yearly measurements of each participant during the 4-year follow-up (16). For those individuals developing diabetes during the first 4 years, the measurements at the time of conversion to diabetes and thereafter were excluded. BODY.RESEARCH DESIGN AND METHODS.STATISTICAL ANALYSES: Variables with a non-normal right-skewed distribution were log transformed for statistical analyses and given as geometric mean with a 95% CI. To test the association of the BMI and Matsuda ISI with insulin secretion at the 4-year follow-up and to test whether there was a group effect, univariate general linear models adjusted for age and sex were constructed. The association of insulin secretion and insulin sensitivity during the first 4 years of follow-up with the risk of incident diabetes during a mean of 6 years of follow-up was assessed by Cox proportional hazards regression models adjusted for age, sex, and study group (intervention or control). For those participants who developed diabetes during the first 4 years, the measurements taken at the year of diagnosis were excluded. Univariate general linear modeling was used for comparisons between progressor and nonprogressors to type 2 diabetes during a mean of 6 years of follow-up for the main variables measured during the 4-year follow-up. A value of P < 0.05 was considered statistically significant. Analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL). BODY.RESULTS.PARTICIPANTS’ CHARACTERISTICS: Individuals in the intervention and control groups included in this analysis had similar body weight, BMI, age, sex distribution, and glucose and insulin levels at baseline (Supplementary Table 1). The decrease in fasting and 2-h glucose, body weight, and BMI were already larger in the intervention group than in the control group after the first year of intervention (Supplementary Table 2) and were in line with the results reported for the entire DPS population (n = 522) (8,10). Insulin sensitivity tended to increase more in the intervention than in the control group (P = 0.07). When divided by the median cutoff point for the change in Matsuda ISI (0.69), the proportion of participants in the higher (mean change, 2.13) and in the lower (−0.41) ranges was significantly different between the intervention (58% and 42%, respectively) and the control (41% and 59%, respectively) groups (P = 0.002). BODY.RESULTS.FASTING AND POSTLOAD GLUCOSE AND INSULIN VALUES AND BMI DURING THE 4-YEAR FOLLOW-UP: Overall, belonging to the intervention group was associated with a better glucose and insulin profile during the follow-up (Supplementary Table 3). Participants in the intervention group had lower body weight and BMI during the follow-up than those in the control group, but the difference between the groups was not significant (P = 0.18 and P = 0.25, respectively). BODY.RESULTS.EFFECT OF LIFESTYLE INTERVENTION ON INSULIN SECRETION AND INSULIN SENSITIVITY DURING THE 4-YEAR FOLLOW-UP: During the follow-up, although the average value (geometric mean [95% CI]) for the unadjusted AIGR0–30 tended to be lower in the intervention than in the control group (30.9 [28.7–33.0] vs. 33.0 [30.8–35.3], P = 0.08), the average Matsuda ISI was significantly higher in the intervention group than in the control group (4.24 [3.99–4.49] vs. 3.85 [3.60–4.11], P = 0.02). However, ISI-adjusted AIGR0–30 was not different between the study groups (29.2 [27.9–30.5] vs. 29.5 [28.1–30.9], P = 0.82). BODY.RESULTS.ASSOCIATIONS OF BMI AND INSULIN SENSITIVITY WITH EARLY-PHASE INSULIN SECRETION DURING THE 4-YEAR FOLLOW-UP IN THE COMBINED INTERVENTION AND CONTROL GROUPS: In different models, BMI was directly associated (β = 0.27; P < 0.001) and Matsuda ISI was strongly (β = −0.71; P < 0.001) inversely associated with AIGR0–30 during the follow-up, and both attenuated the group effect on AIGR0–30 (Table 1). BMI was inversely associated with ISI-adjusted AIGR0–30 during this period (β = −0.12; P = 0.03). In models where Matsuda ISI was placed as the dependent variable, higher BMI was inversely associated with Matsuda ISI (β = −0.49; P < 0.001) independently of study group, and BMI also attenuated the effect of lifestyle intervention on Matsuda ISI (P = 0.07 for the group effect). Table 1 Associations of BMI and Matsuda ISI with AIGR 0–30 and with ISI-adjusted AIGR 0–30 during the 4-year follow-up in participants from the Finnish DPS BODY.RESULTS.IMPACT OF CHANGES IN BMI AND INSULIN SENSITIVITY AFTER THE FIRST YEAR OF INTERVENTION ON THE INSULIN SECRETION DURING THE SUBSEQUENT 4-YEAR FOLLOW-UP: A greater decrease in BMI or a greater increase in Matsuda ISI after the first year of intervention (the most intensive period of the study) was associated with lower AIGR0–30 at the subsequent follow-up (P < 0.001) and again attenuated the group effect on AIGR0–30 during the follow-up (Table 1). A greater decrease in BMI during this period had a weaker effect on ISI-adjusted AIGR0–30 (P = 0.03) than in AIGR0–30. In similar models, being in the group of participants with a greater increase in Matsuda ISI based on the median cutoff (0.69) was associated with higher ISI-adjusted AIGR0–30 during the follow-up than those with less improvement in Matsuda ISI (β = 0.29, P = 0.002). BODY.RESULTS.EARLY-PHASE INSULIN SECRETION AND GLUCOSE RESPONSE IN THE COMBINED INTERVENTION AND CONTROL GROUPS DURING THE 4-YEAR FOLLOW-UP: During the follow-up, Matsuda ISI and ISI-adjusted AIGR0–30 were independently associated with lower concentrations of fasting glucose, 2-h glucose, and glucose AUC0–120 in models adjusted for age, sex, and study group (P < 0.001, Supplementary Table 4). BODY.RESULTS.EARLY-PHASE INSULIN SECRETION AND INSULIN SENSITIVITY AND DEVELOPMENT OF DIABETES: During a mean follow-up of 6 years (range 1–10), the number of diabetes cases was 71 in the control and 60 in the intervention groups. After taking into account the effects of the study group, age, and sex, participants who progressed from IGT to diabetes compared with those who did not progress to diabetes during the mean 6-year follow-up had lower Matsuda ISI and ISI-adjusted AIGR0–30 and higher BMI on average during the 4-year follow-up study, but AIGR0–30 was identical in both groups (Table 2). Participants who developed diabetes also reduced less their body weight and BMI during the first year of the trial (Table 2). Table 2 BMI, AIGR 0–30 , Matsuda ISI, and ISI-adjusted AIGR 0–30 during the 4-year follow-up and body weight and BMI change during the first year of the intervention study in participants of the Finnish DPS who did or did not progress from IGT to type 2 diabetes after a mean follow-up of 6 years Cox regression analyses showed that average Matsuda ISI and ISI-adjusted AIGR0–30 during the follow-up were inversely associated with the incidence of diabetes during the mean 6-year follow-up (Table 3). Although average AIGR 0–30 during the 4-year follow-up alone did not predict diabetes, when Matsuda ISI was included in the model, lower insulin secretion and insulin sensitivity both predicted progression to diabetes. Higher BMI during the follow-up or lower BMI change during the first year of the lifestyle intervention study alone or independently of AIGR0–30 predicted diabetes. This association was no longer significant when Matsuda ISI or ISI-adjusted AIGR0–30 was included in the model. Table 3 Hazard ratios for the risk of developing type 2 diabetes during a mean of 6-year follow-up according to a 1-SD change in AIGR 0–30 , Matsuda ISI, and ISI-adjusted AIGR 0–30 at the 4-year follow-up in participants from the Finnish DPS Of note, in similar models assessing the regression from IGT to normal glucose tolerance (NGT) during a mean 6-year follow-up (Supplementary Table 5), a 1-SD increase in ISI-adjusted AIGR0–30 was associated with a 1.3 increase in the regression from IGT to NGT (P = 0.002). AIGR0–30 and Matsuda ISI alone were not associated with the regression from IGT to NGT (P = 0.29 and P = 0.14, respectively), but when they were entered in the same model, a 1-SD increase in AIGR0–30 and Matsuda ISI was associated with a 1.5 increase in the regression from IGT to NGT (P = 0.001), independently also of BMI. BODY.CONCLUSIONS: Nonpharmacologic lifestyle intervention in high-risk individuals prevented or at least postponed the onset of type 2 diabetes in the Finnish DPS. Participants in the intervention group showed greater reductions in fasting and postchallenge glucose levels and in body weight compared with those in the control group (8,10). In the current study, we demonstrated that higher insulin sensitivity estimated as Matsuda ISI and higher insulin sensitivity-adjusted insulin secretion (ISI-adjusted AIGR0–30) during the 4-year follow-up study were both associated with lower diabetes incidence during a mean follow-up of 6 years. Regression to NGT was more strongly associated with higher ISI-adjusted AIGR0–30 than insulin sensitivity. Individuals who developed type 2 diabetes reduced less BMI during the first year of the intervention, were more obese, and had lower Matsuda ISI and ISI-adjusted AIGR0–30 values during the study than the individuals who did not develop type 2 diabetes. We found that AIGR0–30 per se did not predict diabetes during the mean 6-year follow-up, unless Matsuda ISI contribution was taken into consideration in the models. In contrast, low Matsuda ISI and ISI-adjusted AIGR0–30 were both associated with a higher risk of developing diabetes, independently of BMI. Previous prospective studies have shown that insulin resistance and impaired early-phase insulin secretion predicted the conversion from IGT to diabetes (2–5,17,18). Moreover, in insulin-resistant states, improvement of insulin resistance protected from diabetes and was associated with lower endogenous insulin requirement and preservation of β-cell function (19). Therefore, some improvement in insulin secretion adjusted for insulin sensitivity seems to be possible in the IGT phase, resulting in better β-cell function and possibly lowering the risk of developing diabetes. Altogether, these findings clearly demonstrate the importance of insulin secretion in the development of type 2 diabetes and that the effect of insulin sensitivity on insulin secretion needs to be taken into account due to their complex interaction. In line with published observations, we observed that insulin sensitivity, which was higher in the intervention than in the control group during the 4-year follow-up, was inversely associated with the risk of developing type 2 diabetes (3,5,17) and also inversely associated with BMI during this same interval. In previous findings from a subsample of individuals participating in the DPS, the improvement in insulin sensitivity between baseline and the fourth year of the study was strongly correlated with the magnitude of weight loss (9). These findings were independent of randomization group, and the associations found could be partly explained, for example, by a possible decrease in nonesterified fatty acid release that accompanies loss of body mass (20,21). Although at first glance paradoxical, the lower AIGR0–30 observed in the lifestyle intervention group can be explained by the improvement in Matsuda ISI, which remained higher during the follow-up in this group, and reductions in body weight and BMI in the intervention arm during the first year of the study compared with the control group. Our study, along with other studies but with shorter follow-up duration, shows that a lower BMI and better insulin sensitivity are associated with a decrease in the demand of insulin in obese, insulin-resistant, and glucose-intolerant individuals (22–24). We could not find any difference between the intervention and control groups concerning ISI-adjusted AIGR0–30 that could indicate a direct beneficial effect of lifestyle intervention on β-cell function. Excluding data from the year diabetes was diagnosed may have underestimated the effect of lifestyle intervention. Nevertheless, higher ISI-adjusted AIGR0–30 during the follow-up was associated with a lower risk of developing diabetes and a higher chance of regressing from IGT to NGT. Moreover, participants who had higher increase in Matsuda ISI during the most intensive period of the intervention trial, and who mostly belonged to the intervention group by study design, had higher ISI-adjusted AIGR0–30 during the follow-up. Therefore, weight loss achieved with the lifestyle intervention may be a mediating factor on preserving β-cell function by improving insulin sensitivity and perhaps by avoiding lipotoxicity resulting for example, from ectopic fat accumulation, higher release of nonesterified fatty acid, and activation of inflammatory cascades, all factors related to obesity (20,21). Overall, our findings emphasize the importance of targeting reduction in BMI to improve insulin sensitivity and preserve insulin secretion capacity to prevent or postpone the conversion from IGT to diabetes. A major limitation of our study was that we could not estimate early-phase insulin secretion and sensitivity from OGTT at baseline before the intervention began. Nonetheless, the main clinical and metabolic features related to insulin and glucose metabolism, such as BMI, age, sex proportion, glucose, and insulin parameters did not differ between the intervention and control groups. Therefore, the differences between groups reported in this study are likely to be a reflection of the intervention itself. Of note, diabetes incidence was similarly lower in the original intervention group as in the population included in this study. Insulin sensitivity and insulin secretion were not measured by the hyperinsulinemic-euglycemic clamp or the IVGTT. We did not use the oral glucose minimal model indices for estimating insulin secretion (25), which also includes the incretin response, because we did not measure C-peptide. However, we used an IVGTT for validation of the indices used in the current study in a subsample of the DPS. In DPS, early-phase insulin secretion (AIR) has high repeatability, which, as the insulin sensitivity-adjusted AIR, was also associated with diabetes risk (16). In a similar population, AIGR0–30 and Matsuda ISI were considered the best indices of insulin sensitivity and secretion (11). The averaged values of measures of obesity, insulin sensitivity, and insulin secretion during the 4-year follow-up were used for the analyses. The main changes in lifestyle and body weight occurred during the first year of the study and were largely maintained thereafter. To decrease the variability of the crude measurements and increase the statistical power, we therefore averaged values (16). Strengths of the current study include the well-characterized and homogenous study population (obese individuals with IGT) and yearly measurements during a relative long period of follow-up of a large and carefully conducted lifestyle intervention study population. Our results indicate that the reduction in the risk of developing type 2 diabetes after lifestyle intervention is related to the improvement of insulin sensitivity. The weight loss achieved with the lifestyle intervention, which also improved insulin sensitivity, might have beneficial effects on better preservation of β-cell function. Because weight loss results from joint effects of changes in diet and physical activity and is possibly modified by genetic factors, the interplay between their effects on insulin secretion and risk of developing type 2 diabetes requires further and more detailed investigation. BODY.SUPPLEMENTARY MATERIAL: Supplementary Data

TITLE: THE EFFECT OF BODY AWARENESS THERAPY AND AEROBIC EXERCISES ON PAIN AND QUALITY OF LIFE IN THE PATIENTS WITH TENSION TYPE HEADACHE ABSTRACT.BACKGROUND:: This study is to investigate the effect of Body Awareness Therapy (BAT) and Aerobic Exercises on pain and quality of life in patients with Tension-Type Headache (TTH). ABSTRACT.MATERIALS AND METHOD:: Sixty individuals with TTH diagnosis who referred Neurologist were incorporated into study. The individuals were randomly grouped into 3 as BAT (n=20), aerobic exercise (n=20) and control group (n=20). Pain severity of the individuals was evaluated by Visual Analog Scale (VAS) and pain diary, disability with ache; by Pain Disability Index (PDI) and Headache Impact Tests (HIT) and quality of life was evaluated by SF-36. Subsequent to first assessments, 3 sessions of 60 minutes per week throughout 6 weeks totally. ABSTRACT.RESULTS:: When the groups were compared at the end of the study, a significant decrease was observed in VAS, PDI and HIT values in the individuals in the BAT and aerobic exercise groups. With the individuals in group BAT and aerobic exercise all parameters of quality of life were observed to be increased significantly. ABSTRACT.CONCLUSION:: BAT and aerobic exercise programs to be applied on TTH patients were concluded to be important in decreasing the pain, in increasing the quality of life and in reducing pain-related daily constraints of the individuals. BODY.INTRODUCTION: Headache disorders are the most frequent group of diseases seen in the community (Niere et al., 2009; Biondi et al., 2004). Among primary headache disorders, the most frequent one is the tension-type headache (TTH) (Groot et al., 2011; Ropper et al., 2009; Olesen, 2006). The lifelong prevalence of TTH was stated to be between 46-86% in different studies (Stovner et al., 2007; Torelli et al., 2004). While TTH is seen in female gender more often (Bendsten et al., 2009; Crystal et al., 2010), the age of onset varies from childhood to adulthood (Dismond, 1999; Blaschek et al., 2012). Disorders of general state of health (Söderberg et al., 2011) inadequate rest, sleep disorders (Söderberg et al., 2011; Sierpina et al., 2007), irregular meal times, anxiety, depression (McGneeney, 2009; Jensen et al., 2003), fatigue (Millea et al., 2002; Center, 2000), postural defect (Giacomini et al., 2004), stress and menstrual period for women (Sierpina et al., 2007), are risk factors for headache (Bendsten et al., 2009; Boes et al., 2004). Although the main mechanism to induce TTH is still unknown at the present time (Jensen et al., 2000; Toro-Velasco et al., 2009), TTH has been claimed to be stress-related constitutively in the first performed studies (Bendsten et al., 2009; Jensen, 2001). In the recently performed studies, TTH was shown to be not only due to alterations in the peripheral mechanisms but also to be due to alterations in central mechanisms (Bendsten et al., 2009; Jensen, 2001; Bendsten et al.,2011). In the treatment of TTH patients, basically; the aims are; to relieve the pain, to relieve the patient, to bring the patient's daily-life into minimally affected position and to increase the quality of life. In these patients, it is necessary that the treatment methods comprise all the pathologies and there is a multidisciplinary approach (Davis et al., 2008). In the treatment approaches, pharmacological and non-pharmacological methods are applied (Vernon et al., 1999; Lenssinck et al., 2004). Nowadays, in patients with TTH, as frequently applied, non-pharmacological approaches such as local cold and hot application, classical or connective tissue massage, Transcutaneous electrical nerve stimulation (TENS) (Melzack et al., 2001), ultrasound (US), electromyographic (EMG) biofeedback (Paiva et al., 1982), exercises (such as aerobic exercises, stretching exercises, posture exercises) and relaxation techniques, cognitive therapy, manual techniques as physical therapy such as soft tissue mobilization and manipulation (Fernandez-de-las, 2008; Biondi, 2005; Kanji et al., 2006) and alternative methods such as acupuncture, yoga, meditation, T'ai-Chi, and Body awareness therapy (BAT) are used (Söderberg et al., 2011; Center, 2000; Davis et al., 2008). BAT, body awareness state, means one's awareness of self-entity. It contributes to the physical, mental and spiritual development of the individual and increases the individual's awareness of these three dimensions. Also, it provides regain and enhancement of posture, balance and natural reflexes of motion to the body. By providing body (sensory) and mind (motor) integrity; a healthy and quality life, the tension occurring by stress and chronic pain are managed effectively (Dittrich et al., 2008; Gard, 2005). BAT is widely used in the treatment of chronic pain rehabilitation (such as musculoskeletal diseases, fibromyalgia, headache, general chronic pain) and is still being improved (Dittrich et al., 2008; Roxendal 1985). When the frequency of TTH is considered, it is seen to cause a significant disability, workday loss and economic cost for the community (Davis et al., 2008; Lenssinck et al., 2004). Besides, TTH, can lead to a decline in quality of life and work capacity, significant inefficacy in one's daily life activities and functions (Niere et al., 2009; Stovner et al., 2007; Jensen, 2003). In this study, investigating the effect of Body Awareness Therapy (BAT) and aerobic exercises upon pain and quality of life in patients with TTH is aimed. BODY.METHODS.DATA SOURCE AND DESIGN: Eighty individuals with chronic TTH diagnosis who referred to Bolu Izzet Baysal Public Hospital Koroglu Unit Neurology outpatient clinics for headache complaint, were incorporated into the study. This study followed a thesis project conducted by one of the authors. The criteria to be included in the study were determined to be as: being between 18-55 years of age, being diagnosed with chronic TTH, being only headache, being able to visit the hospital independently, not having a communication difficulty or problem, being volunteer to participate in the study and the pain severity value being between 4-7 according to VAS. During treatment sessions, the patients whose pain increased positively, who had cardiac disease, cardiac arrhythmia, cardiovascular disease, who had malignancy and who received chemotherapy, radiotherapy that caused malignancy, who had any unbalancing neurologic or orthopedic disturbance, who were pregnant, who received antidepressive and antipsychotic treatment, who were alcohol and drug addict and who had mental disturbances that caused them to not to understand the exercise to be done were excluded from the study. Power analysis was conducted to determine the number of people to participate in the study. Examination was done by a medical doctor specialist for neurosurgery. After applying the inclusion and exclusion criteria, those included were referred to the physiotherapy department. Of the 80 patients with chronic TTH, 20 patient did not meet the inclusion criteria (3 patients were cardiac disease, 3 patients had antidepressive treatment, 4 patients were pregnant, 5 patients had transport difficulty and 5 patients had due to family problems not incorporated in the study). The study included 60 chronic TTH patients. Block randomization was done by a computer-generated random number list prepared by an investigator with no clinical involvement in the trial. The volunteers were randomly assigned to three groups: BAT, aerobic exercise and control group. Sixty individuals quit the study due to being unsuitable for the inclusion criteria (Figure 1). The first and the second groups were determined to be the study group; as to the third group, it was determined to be the control group. Within the study, BAT was applied to the first group; as for the second group, the aerobic exercise program was applied. Throughout the treatment period, 2 people from aerobic exercise group had to quit due to transport difficulty, health and family problems. The study was completed with a total of 58 people (Figure 1). BODY.METHODS.INSTRUMENTS: The people who were included in the study, their sociodemographic data, and their TTH knowledge were interrogated with an assessment form prior to the study and 6 weeks after the beginning of the study. The pain severity of the patients was evaluated by using VAS. VAS is a pain severity measurement scale and its reliability was demonstrated (Strong et al., 2002). Besides, a pain diary was kept to determine the headaches of the patients during the treatment period. The patients were asked to mark the days of their headache on the given chart. Pain-related disability was assessed by Pain Disability Index (PDI) and by Headache Impact Tests (HIT). PDI is a simple and rapid means to measure the constraint in normal daily functions due to pain in patients who have chronic pain. It is used to determine the patient ratings in the beginning, and the effectiveness of the interventions. This index was developed by St. Louis University Medical Center. PDI is comprised of a total of 7 parameters as follows; family and household responsibilities, entertainment, social activity, occupation, sex life, self-care and daily life activities. These parameters fall within 0-10. Zero means "I have no problems with pain", 10 means "I have severe constraint due to pain". The total score is calculated between 0-70 by the sum of 7 parameters (Osün et al., 2003; Biçer et al., 2004). HIT on the other hand, is implemented for questioning pain severity, work and spare time activities, fatigue, and cognitive features. It is a test composed of 6 questions applied on paper. Each item is answered in 5-item Likert scale and by summing up the points, and thus, the total score is obtained. Score and respond level are directly proportionate to each other. HIT scale is a favorable test to use because it measures both short-time and also widespread effect (Kosinski et al., 2003; Yang et al., 2010). When carrying out the evaluation, digits at the bottom of the columns are summed up and the total score is found. The total score is between 36-78. Scoring is done as follows: 60 points and over: Headaches affect the life extremely. It restricts daily activities more severely compared to others who suffer from a headache. 56-59 points: Headaches affect daily activities significantly. 50-55 points: Headaches affect daily activities mildly. 49 points and less: Headaches do not affect daily activities yet (HIT-6T Turkey version, 2000). SF-36 (The MOS 36-item short form health survey) form is used as a general quality of life scale. This form was developed by Ware and his co-workers and its Turkish validity and reliability adaptation was made by Kocyigit and his co-workers (Kocyiğit et al., 1999). The form is composed of a total of 36 items that can be filled by the patient. These items include 8 different dimensions concerning health. Physical function (10 items), social function (2 items), physical problems-related role constraints (3 items), emotional problems-related role constraints (3 items), mental health (5 items), liveliness (4 items), pain (2 items) general health [general perspective (5 items) and alteration in health (1 item)]. Items are scored (0 = poorest health state, 100 = best health state) and are evaluated one by one. By subscales, it evaluates the health between 0-100; and 0 indicates poor health state, 100 indicates good health state. BODY.METHODS.TREATMENT PROGRAM: Individuals in the treatment group were taken under two different training programs. To the first group of the individuals in the treatment group (20 individuals), BAT program was applied, to the second group (20 individuals), the aerobic exercise program was applied. We were divided further into 2 more separate groups of 10 each, because 20 individuals are hard to control and difficult to get into the training program at the same time. The treatment procedure for BAT-applied group was implemented as follows: the three sections of BAT; relaxation, motion and massage were applied by a physiotherapist educated on BAT to the individuals as they were divided into 2 groups, 10 people each on 3 sessions of 60 minutes each per week for total 6 weeks. The second group of individuals were also divided into 2 separate groups; 10 people each in the same way and were taken under aerobic exercise program. The exercise protocol was consisted of 3 phases as; 5 min warm-up phase, 5 min cooling phase and 30 min aerobic exercises. Aerobic exercises, accompanied by music, were performed via step-dance board by beginning with 30 min and progressively increasing the time. Aerobic exercise intensity was tasked at the submaximal level. To keep the aerobic exercise intensity at the submaximal level, original 6-20 points Rating of Perceived Exertion (BORG scale) was used.94 Each session intensity was designated to correspond to the 13-14 points of 6-20 points BORG scale. This point is adapted to exercise intensity which corresponds to 65-70% of maximum heart rate by the American College of Sports Medicine. By following the BORG scale, the exercise intensity was regulated with the exercise repeat times and increasing its duration progressively (Borg, 1998). As for the control group, there was no application practiced. However, as the study ended, the individuals of the control group were asked whether they wanted to participate in BAT or aerobic exercise programs. Those who wanted to participate were included in the treatment program. BODY.METHODS.ETHICAL APPROVAL OF THE STUDY PROTOCOL: The study was assessed by the Abant Izzet Baysal University Ethical Committee and was approved ethically (2011/56). Each patient was informed of the method and the goal of the study and the patient consent form was signed by them with regard to participating in the study willingly. BODY.METHODS.STATISTICAL ANALYSES: The descriptive statistics belonging to obtained measurements were given as mean ±Standard Deviation, number and % frequencies. In the comparison of the groups in terms of numerical/quantitative type demographic measurements and clinical features, single direction variance analysis was used and for determination of the different groups, post hoc Turkey test was used. In categorical measurements done prior to and after the treatment program, chi square analysis was utilized to determine the difference between the groups. The relationship between the Numerical/Quantitative measurements was examined via correlation analysis. Last value carried forward" method was used. Kolmogorov-Smirnov test was used for checking of normality. The statistical significance level in the analysis of the data was determined as p<0,05. For statistical operations, PASW (SPSS, 18) package was used. BODY.RESULTS: Sixty chronic TTH diagnosed individuals whose average of age was 39.26±9.23 years, were incorporated in the study. The physical features of the individuals who were incorporated into the study were given in Table 1. Table 1 Physical Features of the Participants BAT Group X±SS AE Group X±SS Control Group X±SS F p Age (year) 42.6±9.5 36.20±7.86 39.00±9.53 2.54 0.08 Body weight (kg) 69.71±15.38 69.26±13.32 65.69±9.08 0.58 0.56 Body height (m) 1.59±0.06 1.59±0.04 1.60±0.05 0.06 0.93 BMI (kg/m 2 ) 27.31±5.6 27.10±4.6 25.66±3.71 0.73 0.48 One way analysis of variance, BAT: Body Awareness Therapy; AE: Aerobic Exercise In the statistical analysis that was performed, there was no difference found between the groups concerning age, body weight, body height and body mass index (BMI) (p>0.05) (Table 1). The distribution of headache frequency among the groups in last 3, last 6 and last 12 months was observed not to show a significant difference (p>0.05). It was medication/drug which was frequently used the method of treatment for the headache of the individuals who were incorporated in groups 1, 2 and 3 and there was no significant difference found in terms of the applied treatment methods for the headache between the groups (p>0.05) (Table 2). Table 2 The distribution of the difference between treatment methods which were applied for the headache previously and pain frequency of the people in last 3, 6 and 12 months. Pain times BAT Group AE Group Control Group χ 2 P n % n % n % Last 3 Months 1-7 times 10 50 9 45 5 25 8−14 times 4 20 6 30 8 40 15−30 times 4 20 4 20 3 15 31−160 times 2 10 1 5 3 15 6.26 0.61 180 and above - - - - 1 5 1 −7 times 3 15 2 10 1 5 8−14 times 6 30 7 35 5 25 15−30 times 5 25 6 30 7 35 5.73 0.67 Last 6 Months 31−160 times 6 30 4 20 4 20 180 and above - - 1 5 3 15 1−7 times 1 5 1 5 1 5 8−14 times 4 20 2 10 3 15 15−30 times 6 30 11 55 3 15 Last 12 Months 31−160 times 6 30 3 15 10 50 12.46 0.25 180 and above 3 15 3 15 3 15 Medicine 19 95 20 100 19 95 Treatment Methods Psychother apy 1 5 - - 1 5 1.03 0.59 Total 20 100 20 100 20 100 χ 2 : Chi-square test, BAT: Body Awareness Therapy, AE: Aerobic Exercise While in individuals in BAT group and in aerobic exercise group, there was a difference found in VAS, PDI and HIT values prior to and after the treatment (p<0.05); in individuals in control group, there was no difference found (p>0.05). While there was a difference found between PF, RP, VT, RE, MH, PCS, MCS values prior to and after the treatment of the individuals in BAT group; in individuals in aerobic exercise group, there was a difference found between PF, BP, VT, MH values (p<0.05). While there was a difference between MH values prior to and after the treatment of individuals in control group (p<0.05); there was no difference found between PF, RP, BP, GH, VT, SF, RE, PCS, MCS values (p>0.05) (Table 3). Table 3 The comparison of VAS, PDI, HIT and SF-36 values before and after treatment of people in each group Before Treatment X±SS After Treatment X±SS t p VAS 6.15±0.74 2.50±1.14 12.1 0.00* PDI 32.85±13.34 16.75±12.57 4.13 0.00* HIT 62.25±6.71 52.45±7.76 4.68 0.00* PF 46.05±8.38 52.31±5.1 -2.93 0.00* BAT GROUP RP 41.39±12.3 49.15±10.48 -2.21 0.03* BP 41.51±8.85 45.75±8.63 -1.61 0.12 GH 43.62±10.23 45.89±11.01 -1.14 0.26 VT 44.80±9.26 49.78±8.26 -2.38 0.02* SF 42.21±10.82 44.37±9.35 -0.69 0.49 RE 37.93±14.2 47.40±13.17 -3.21 0.00* MH 37.49±9.68 41.91±8.9 -2.51 0.02* PCS 43.76±7.44 48.74±7.88 -2.29 0.03* MCS 39.07±12.18 43.91±7.8 -2.57 0.01* VAS 6.1±1.02 3.00±1.28 8.42 0.00* PDI 32.33±10.34 19.94±12.59 4.63 0.00* HIT 63.83±3.8 51.50±7.03 6.32 0.00* PF 45.97±8.08 51.21±4.52 -2.70 0.01* RP 44.05±10.77 47.59±11.48 -0.90 0.37 BP 37.65±5.71 46.15±7.37 -3.93 0.00* GH 44.43±9.28 46.34±9.35 -0.87 0.39 VT 41.83±5.52 51.30±8.13 -4.41 0.00* AE GROUP SF 36.32±6.24 44.16±9.69 -2.81 0.01* RE 41.26±14.44 45.94±13.46 -0.92 0.36 MH 37.31±8.41 44.38±8.13 -2.84 0.01* PCS 43.07±8.7 47.70±5.52 -1.78 0.09 MCS 38.03±9.2 45.20±9.4 -2.10 0.05 VAS 5.90±0.71 5.65±0.74 2.03 0.05 PDI 27.10±9.97 26.10±10.39 0.65 0.52 HIT 59.95±7.71 59.30±6.89 0.49 0.62 PF 45.92±11.28 48.36±9.44 -1.22 0.23 RP 40.69±12.86 39.98±12.76 -0.37 0.71 BP 43.64±10.25 45.87±10.30 -1.57 0.13 GH 42.46±10.75 42.56±11.81 -0.05 0.95 VT 43.02±11.03 43.72±9.65 -0.27 0.78 CONTROL GROUP SF 37.87±10.03 40.85±11.54 -1.02 0.31 RE 37.41±13.71 41.10±14.58 -1.28 0.21 MH 35.45±10.57 38.86±9.74 -2.61 0.05* PCS 44.32±8.72 44.42±9.23 -0.07 0.94 MCS 36.37±11.58 39.61±12.34 -1.48 0.15 In the statistical analysis that was performed, there was no difference found between VAS, PDI and HIT values prior to the treatment among the groups (p>0.05). As to after the treatment, there was a difference found among the groups concerning VAS, PDI and HIT values (p<0.05). VAS and HIT values were found to be significantly low both in BAT group and in aerobic exercise group. As for the PDI values, they were found to be significantly low in BAT group. Among the groups, there was no variation found between PF, RP, BP, GH, VT, SF, RE, MH, PCS, MCS values prior to the treatment (p>0.05). As for after the treatment, there was a difference found among the groups concerning RP and VT values (p<0.05). RP values in BAT group and in aerobic exercise group were found to be significantly high, VT values in aerobic exercise group were found to be significantly high (Table 4). When the differentials of PF, RP, BP, GH, VT, SF, RE, MH, PCS, MCS among the groups prior to and after the treatment were taken, only the VT differential was found to be different for aerobic exercise group (p<0.05) (Table 4). Table 4 The comparison of VAS, PDI, HIT and SF-36 values among the groups before treatment and after treatment Group Before treatment After treatment X±SS F p X±SS F p VAS BAT Group 6.15±0.74 2.50±1.14 AE Group 6.10±1.02 0.49 0.61 3.00±1.28 49.14 0.00 * Control Group 5.90±0.71 5.65±0.74 * PDI BAT Group 32.85±13.34 16.75±12.57 * AE Group 31.70±10.1 1.46 0.24 19.94±12.59 3.19 0.04 * Control Group 27.10±9.97 26.10±10.39 HIT BAT Group 62.25±6.71 52.45±7.76 AE Group 61.00±3.72 0.67 0.51 51.50±7.03 6.72 0.00 * Control Group 59.95±7.71 59.30±6.89 * PF BAT Group 46.05±8.38 52.31±5.1 AE Group 45.94±7.65 0.001 0.999 51.21±4.52 1.79 0.17 Control Group 45.92±1.28 48.36±9.44 RP BAT Group 41.39±12.3 49.15±10.48 AE Group 43.86±11.18 0.375 0.689 47.59±11.48 3.53 0.03 * Control Group 40.69±12.86 39.98±12.76 BP BAT Group 41.51±8.85 45.75±8.63 AE Group 38.34±6.87 1.849 0.167 46.15±7.37 0.01 0.99 Control Group 43.64±10.25 45.87±10.3 GH BAT Group 43.62±10.23 0.106 0.899 45.89±10.01 0.71 0.49 AE Group 43.86±9.79 46.34±9.35 Control Group 42.46±10.75 42.56±11.81 VT BAT Group 44.80±9.26 49.78±8.26 AE Group 42.56±6.13 0.334 0.718 51.30±8.13 4.08 0.02 Control Group 43.02±11.31 43.72±9.65 SF BAT Group 42.21±10.82 44.37±9.35 AE Group 36.51±6.24 2.097 0.132 44.16±9.69 0.73 0.48 Control Group 37.87±10.03 40.85±11.54 RE BAT Group 37.93±14.2 47.40±13.17 AE Group 41.09±14.6 0.394 0.676 45.94±13.46 1.14 0.32 Control Group 37.41±13.71 41.10±14.58 MH BAT Group 37.49±9.68 41.91±8.9 AE Group 36.81±8.62 0.230 0.795 44.38±8.13 1.81 0.17 Control Group 35.45±10.57 38.86±9.74 PCS BAT Group 43.76±7.44 48.74±7.88 AE Group 43.21±8.54 0.090 0.914 47.70±5.52 1.67 0.19 Control Group 44.32±8.72 44.42±9.23 MCS BAT Group 39.07±12.18 43.91±7.8 AE Group 37.92±9.18 0.349 0.707 45.20±9.4 1.64 0.20 Control Group 36.17±11.58 39.61±12.34 * p<0,05, One way analysis of variance in each period, VAS: Pain severity scale, PDI: Pain Disability Index, HIT: Headache Impact Test, PF: Physical function, RP: Role-physical, BP: Body perception, GH: General health, VT: Vitality, SF: Social function, RE: Role-emotional, MH: Mental health, PCS: Physical health, MCS: General mental health The individuals in each of treatment and control groups who were incorporated in the study were those who had moderate degree pain; in these individuals, there was a statistically difference found concerning the average number of days which were noted in the pain diary. As the average number of days of pain in individuals who had mild and severe pain is concerned, statistically no difference was found among the individuals in each three groups (p>0.05). The average number of days which were noted in the pain diary in individuals who had moderate degree pain was found to be significantly high in control group (p<0.05). In the statistical analysis which was performed, there was a difference found between the groups in using medication when in pain according to the pain diary (p>0.05). Medication use was found to be significantly low in aerobic group (Table 5). Table 5 The comparison of average day values according to pain diary of people, among the groups BAT Group X±SS AE Group X±SS Control Group X±SS F p Mild 2.55±3.26 1.33±1.41 1.45±2.56 1.33 0,27 Pain Diary (Day) Intermedi ate 2.20±2.44 1.61±2.42 4.00±3.74 3.43 0,03 * Severe 0.50±0.88 0.38±0.69 1.15±1.53 2.67 0,07 Analgesics Use 0,96±0,22 0.65±0.48 * 1.10±0.44 6.43 0.00 * * p<0,05, One way analysis of variance in each period BODY.DISCUSSION: The pharmacological treatment methods used frequently in individuals who have a headache include; analgesics, antidepressants, myorelaxants and non-steroidal anti-inflammatory drugs (Furnal et al., 2008; Penacoba-Puente et al., 2008). In fact, it was medication which was frequently used as the method of treatment for a headache in participant individuals in our study. However, in recent years, due to cost-effective programs being in the forefront in healthcare and the problems arising from the side-effects of the medications, popularized the use of physiotherapy and rehabilitation practices and alternative treatment methods (Ertaş et al., 2012; Holroyd et al., 2001). In recent years, the number of studies which concentrate on different effects of different treatment methods on headache patients has increased. The effects of many of these treatment methods have not been completely proven yet (Penacoba-Puente et al., 2008; Bigal et al., 2009; Davis et al., 2008). Accordingly, the aim of our study is to research the effect of body awareness therapy (BAT) and aerobic exercises upon pain and quality of life in patients with a tension-type headache (TTH). The number of studies that examine the effectiveness of BAT and aerobic exercise in patients with TTH is quite a few. However, there are many studies available which research the effect of different exercise approaches upon pain (Fernandes-de-las-Penas, 2008). The effectiveness of aerobic exercises on migraine patients has been studied more (Dittrich et al., 2008; Köseoğlu et al., 2003; Busch et al., 2008; Varkey et al., 2009). In addition to this, relaxation methods have been used frequently in TTH treatment and their effectiveness in different patient groups has been proven. The effect of relaxation and cognitive behavior treatments, biofeedback practices are explained by physiopsychological mechanisms in the literature (Paiva et al., 1982). According to this, being nervous, stressed, operating with stiff muscles while working in daily life lead to pain especially in the stiff muscle and around it, sensitiveness, nervousness, fatigue and this can manifest as TTH in advanced stages. By relaxation training, to relax the stiff muscles of the individuals, to ensure the better feeling of them and them to complete the activity with less effort are aimed (Söderberg et al., 2011; Furnal et al., 2008). It has been proven that relaxation therapy reduces stress. Progressive relaxation focuses on respiration and relaxation of certain muscle groups. In a study which was performed, in 96% of patients with a headache, frequency of pain, duration of pain and severity of pain were reported to be declined after 10 sessions of relaxation therapy (Holroyd et al., 2001). Pamela D'Souza and her co-workers stated that relaxation techniques calm people with a headache and decrease the negative mood. The same study found that after relaxation training for 3 months, it reduces the headache frequency, headache-related disability and physical symptoms of the unconscious mind in patients when compared with control group (D'Souza et al., 2008). In our study, a significant decrease was found in pain frequency (pain diary), pain severity (VAS) and pain related disabilities (PDI and HIT) after the study in BAT applied individuals, which is consistent with the previous studies. Although there is not enough evidence to be efficient in systematic review studies about the effectiveness of physiotherapy practices, exercise and spinal manipulation practices in a headache in patients with TTH; there are studies that address the efficiency of exercise practices in patients with a headache (Osün et al., 2003; Fernandez-de-las-Penas et al., 2006). Although there are many physiologic effects of exercise practices, in patients with a headache the emphasis is laid on two mechanisms: decreasing peripheral sensitization and activation of descending inhibitory pathways. By aerobic exercise; power, durability, flexibility increase, activity level rises, sense of auto-control is enhanced, muscle relaxation is supported by providing the antidepressant effect. Consequently, the effect of pain lessens. The reason for that is the increase in beta-endorphin etc. values, prolactin and GH secretion and the reduction of pain by these hormones (Osün et al., 2003; Furnal et al., 2008; Köseoğlu et al., 2003). Koseoglu and his co-workers reported in their study that they examined the aerobic exercise and plasma beta-endorphin levels in patients with a migraine without aura, and found the beneficial effects of exercise on all migraine parameters. The increase in beta-endorphin levels is reported both after home exercise program and also after treadmill training. As a result, in this study, beneficial effects of the exercise was emphasized to exist especially in those patients with migraine without aura, whose basic beta-endorphin levels are low (Köseoğlu et al., 2003). In the collection study that was performed by Busch and Gaul, it was reported that there was a significant decrease in migraine attacks, pain severity, pain frequency and duration of pain, as a result of aerobic exercise programs applied on migraine patients. In the conclusion of the study, they emphasized that exercises which are done regularly for 2 or 3 times in a week might reduce the headache frequency (Busch et al., 2008). In our study also in agreement with other studies that were performed, aerobic exercise application was shown to reduce the severity of the headache in patients with TTH. Canady reported a reduction in headaches of patients, reduction in analgesic/painkiller use, and reduction in symptoms such as anxiety during treatment as a result of 8 weeks of aerobic exercise training which is done on patients with TTH and that exercise to be an effective treatment method in TTH treatment (Canady, 2002). In our study, a significant decrease was determined in pain frequency (pain diary), pain severity (VAS), pain related medicine use and pain related disabilities (PDI and HIT) of the patients with TTH on whom aerobic exercise was applied; and a significant increase in the parameters of quality of life such as physical function, body perception, vitality, social function and mental health were recorded. By finding the same conclusion with studies, we consider exercise to have an acute analgesic effect and by increasing endorphin production in the body; reducing the negative conditions such as fatigue, depression, anxiety. This is because exercise decreases the stress level and decreases the negative conditions arise in human body resulting from muscle tension and stress-related chemical substance secretion into blood circulation. It provides relaxation, increases blood and lymphatic circulation, provides more oxygen delivery to the tissues of the body and provides removal of toxins from the body more rapidly. Thus, it increases endorphin production in the body; decreases fatigue, depression, anxiety; enhances sleep and life quality. Likewise, it was discovered that exercise has an acute analgesic effect on healthy individuals (Guyton, 1996). Besides, the analgesic use rate was found to be decreased in those groups who received treatment in our study. In studies that examined the effect of BAT in different follow-up periods in those patients who had schizophrenia, chronic musculoskeletal system disease, psychosomatic problem fibromyalgia syndrome, non-specific musculoskeletal system diseases; it was reported that after BAT application, there was significant enhancement in the quality of motion, body perception, self-confidence, sexual relationship level, social communication, thinking capability, pain and psychogenic distress, quality of life parameters; that there were a decrease in stress-related symptoms, an increase in self-sufficiency, a decrease in depressive mood, a decrease in anxiety, better expression of their feelings and positive alteration in their lifestyle (Landsman et al., 2004; Hedlund et al., 2010). In comparative studies that were performed, BAT was found to be more effective than conventional treatment approaches (such as; massage, TENS, acupuncture, exercise training, mobilization) (Gard, 2005; Malmgren-olsson et al., 2011). Mannix and et al. found that the applied relaxation methods were to increase the quality of life of patients with TTH significantly compared to the control group (Mannix et al., 1999). Similarly, Söderberg and et al. also emphasized that by increasing the central nervous system-related symptoms affecting the well-being state (such as; satisfaction-happiness, self-managing, self-confidence, vitality-endurance, concentration, enthusiasm, sleep-night's sleep, quality of sleep), the relaxation techniques in patients with TTH increase the well-being state and increase the quality of life, therefore, the relaxation techniques which were to be used for the patients with TTH are quite important (Söderberg et al., 2011). In consistence with the literature, in those individuals on whom BAT was applied in our study, a significant decrease in pain frequency (pain diary), in pain severity (VAS) and in pain related disabilities (PDI and HIT) was found; an increase in the parameters of quality of life such as physical function, role-physical, vitality, role-emotional, mental health, physical health and general mental health was observed. The pain-related functional constraint related to pain severity in daily life decreased with a decrease of pain and with an increase in work and spare time activities, the life satisfaction and quality of life of the individual increased. Pain leads to a serious functional loss in daily life and reduces the quality of life dramatically. Therefore, to reduce pain in individuals with TTH and to bring it under control are quite important factors in increasing the quality of life (Stovner et al., 2007). There was no comparative study that was encountered in literature examining the effectiveness of BAT and aerobic exercise in patients with TTH. This study bears the qualification of being the first study on this subject that is performed. However, there are different studies researching the superiority of different treatment methods on each other in patients with TTH (Bigal et al., 2009; Davis et al., 2008). In our study, while it was seen that both treatment methods to be more successful when compared to the control group in decreasing the pain frequency, pain severity, duration of pain, pain-related disability, pain-related medication use and increasing the quality of life; aerobic exercise program and BAT were found to have similar effects in terms of decreasing the frequency and duration of pain and reducing the severity of pain-related disability, and BAT to be more effective in parameters of quality of life as; role-physical, role-emotional, general health and general mental health. On the other hand, aerobic exercises were observed to be more effective in pain-related medication use and the parameters of quality of life such as body perception and social function. BODY.CONCLUSION: We consider the approaches in which the active exercise programs to be applied to patients with TTH, and the BAT methods to be applied together, to be successful in decreasing the pain frequency, pain severity, duration of pain, pain-related medication use, pain-related disability; in increasing the quality of life and in reducing the TTH-related symptoms to minimum. As part of the active exercise program, even though the patient cannot exercise regularly, the patient should be kept away from physical inactivity by physical exercises as walking, jogging, cycling, and s/he should be encouraged about leading a constantly active life. This study bears the qualification of being the first study that examines the effectiveness of BAT and aerobic exercise programs in patients with TTH, and we are of the opinion that there will be more studies to be performed on this subject.

TITLE: Effect of a combination of genistein, polyunsaturated fatty acids and vitamins D3 and K1 on bone mineral density in postmenopausal women: a randomized, placebo-controlled, double-blind pilot study ABSTRACT.PURPOSE: Many postmenopausal women desire non-pharmaceutical alternatives to hormone therapy for protection against osteoporosis. Soybean isoflavones, especially genistein, are being studied for this purpose. This study examined the effects of synthetic genistein in combination with other potential bone-protective dietary molecules on bone mineral density (BMD) in early postmenopausal women. ABSTRACT.METHODS: In this 6-month double-blind pilot study, 70 subjects were randomized to receive daily either calcium only or the geniVidaTM bone blend (GBB), which consisted of genistein (30 mg/days), vitamin D3 (800 IU/days), vitamin K1 (150 μg/days) and polyunsaturated fatty acids (1 g polyunsaturated fatty acids as ethyl ester: eicosapentaenoic acid/docosahexaenoic acid ratio = ~2/1). Markers of bone resorption and formation and BMD at the femoral neck, lumbar spine, Ward's triangle, trochanter and intertrochanter, total hip and whole body were assessed. ABSTRACT.RESULTS: Subjects supplemented with the GBB (n = 30) maintained femoral neck BMD, whereas in the placebo group (n = 28), BMD significantly decreased (p = 0.007). There was also a significant difference (p < 0.05) in BMD between the groups at Ward's triangle in favor of the GBB group. Bone-specific alkaline phosphatase and N-telopeptide significantly increased in the GBB group in comparison with those in baseline and in the placebo group. The GBB was well tolerated, and there were no significant differences in adverse events between groups. ABSTRACT.CONCLUSIONS: The GBB may help to prevent osteoporosis and reduce fracture risk, at least at the hip, in postmenopausal women. Larger and longer-term clinical trials are warranted. BODY.INTRODUCTION: In response to declining estrogen levels, women can lose substantial amounts of bone mass in the decade following menopause, which markedly increases their fracture risk [1]. Until 2002, postmenopausal women were typically prescribed hormone therapy (HT) if they were considered to be at risk of developing osteoporosis. Although research from the Women's Health Initiative Trial confirmed that HT reduces postmenopausal bone loss and hip fracture risk [2], the results, along with findings from several other large-scale studies, have also raised safety concerns about the use of HT [3, 4]. These concerns have led to a dramatic decline in HT use [5] and the need to identify non-hormonal anti-osteoporotic agents. One widely studied non-pharmaceutical alternative for promoting postmenopausal bone health is isoflavones; among commonly consumed foods, they are found in physiologically relevant amounts only in the soybean and soyfoods. Isoflavones are present in soybeans almost exclusively as glycosides, and the three aglycone isoflavones genistein, daidzein and glycitein and their respective glycosides account for approximately 50, 40 and 10% of total isoflavone content, respectively [6]. Mean isoflavone intake in western countries is typically <3 mg/days [7], whereas in Japan, daily intake of approximately 30–50 mg can be achieved [8]. In general, isoflavones are considered to be phytoestrogens, because some members were shown to bind to transactivate estrogen receptors and to initiate gene expression [9]. Initial speculation about their efficacy was based on their estrogen-like properties and early research showing that the chemically synthesized isoflavone structurally derivatized drug, ipriflavone, exerted skeletal benefits [10]. The results of prospective epidemiologic studies conducted in Shanghai [11] and Singapore [12] seem to support the efficacy of isoflavones, as high soy intake in these studies was associated with approximately 30% reductions in fracture risk. However, the >25 clinical trials that have examined the effects of isoflavones on bone mineral density (BMD) have produced mixed results, although two out of three recently published meta-analyses found that soy isoflavones reduced bone loss at the lumbar spine [13–15]. However, only four studies, the 3-year Italian trial by Marini et al. [16], the 2- and 3-year US studies by Levis et al. [17] and Alekel et al. [18], respectively, and the 2-year Taiwanese study by Tai et al. [19] were more than 1 year in duration. In the study by Marini et al. [16], there were dramatic increases in postmenopausal spinal and hip BMD after genistein supplementation, whereas in the other three studies, there was little evidence that soy isoflavones produced skeletal benefits [17–19]. To substantially reduce the risk of a chronic disease such as osteoporosis, through lifestyle and dietary intervention, requires the adoption of a comprehensive approach. Evidence suggests that a combination of potentially bone-protective dietary agents working through different mechanisms is more likely to result in a substantial benefit than any single agent alone. For example, dietary protein is viewed as beneficial for bone when sufficient dietary calcium is consumed, but possibly harmful when it is not [20]. Also, vitamin D enhances the absorption of calcium and may have independent skeletal benefits [21]. There is also evidence that vitamin K is needed for γ-carboxylation of specific glutamic acids which converts 3 glutamic acid (Glu) residues in osteocalcin (OC) to γ-carboxyglutamic acid (Gla) [22, 23], an essential structural modification for the integration of osteocalcin into the bone matrix. Finally, supplementation with long-chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may reduce bone loss [24]. In mice, isoflavones and fish oil additively induced parameters of bone structure and increased bone mass synergistically in an ovariectomy-induced bone loss model [22]. It is reasonable to speculate that when a combination of agents is used, the amount of isoflavones required for efficacy may be reduced. Therefore, we decided to conduct a pilot study using geniVidaTM to determine whether a physiological dose of genistein (30 mg/days), when combined with other bioactives, will favorably impact bone health in postmenopausal women. This dose of genistein is in line with the mean intake of genistein among older Japanese following a traditional diet [8]. This pilot intervention study was conducted to generate data on the impact of a genistein bone blend (GBB) on bone loss in early postmenopausal women. Should efficacy be demonstrated, the data could be used as a basis for designing a larger and longer follow-up study. BODY.SUBJECTS AND METHODS.STUDY DESIGN AND TRIAL SUPPLEMENTATION: In this randomized, double-blind, placebo-controlled, 6-month pilot intervention study, the effect of a combination of genistein (30 mg/days), vitamin D3 (800 IU/days), vitamin K1 (150 μg/days) and polyunsaturated fatty acids 1 g (PUFAs) as ethyl ester: EPA/DHA ratio = ~2/1) on bone health in postmenopausal women was investigated. The protocol, informed consent form and advertisement for subjects were approved by the Creighton University institutional Review Board (IRB) (no. 06–14202). The study was conducted in accordance with Good Clinical Practice Guidelines from the International Conference of Harmonization and was registered at ClinicalTrial.gov (NCT 00698984). Genistein, vitamin K1 and D3 and PUFAs were manufactured by DSM Nutritional Products, Ltd. (Kaiseraugst, Switzerland), and capsule production (active and placebo) as well as packaging and labeling took place at Intergel Division, IVC Industries Inc., NJ (USA), under GMP requirements and control. One soft-gelatin geniVidaTM bone blend (GBB) capsule contained 15 mg genistein (geniVida 99.1% genistein), 500 mg PUFAs (ROPUFA® 75 n-3 Ethyl Ester), 75 μg vitamin K1 (99.7% phylloquinone) and 400 IU vitamin D3 (100% cholecalciferol) together with corn oil and bees wax. Placebo capsules contained only corn oil and bees wax. Two capsules were taken per day in the morning together with breakfast. In addition, one calcium carbonate tablet containing 500 mg elemental calcium (Ost-Cal 500, Goldine) was taken daily with either the placebo or GBB. Supplementation began on the day after the baseline visit (visit 1) and ended the morning before visit 3, 6 months after visit 1. BODY.SUBJECTS AND METHODS.STUDY DESIGN AND TRIAL SUPPLEMENTATION.RANDOMIZATION AND BLINDING: Four-block randomization was performed by an employee of DSM Nutritional Products Ltd., who was not involved in the study. The randomization list was provided to Intergel Division, IVC Industries Inc., for packaging and placement of capsules into bottles and bottle labeling. Bottles with randomization code numbers, which were shipped to Creighton University, were dispensed to study subjects in sequenced numbers. Unblinding occurred after all data management procedures were completed. Only emergency envelopes were located at the study site. The randomization code was kept locked at the safety management company (United BioSource Corporation (UBC), Geneva, Switzerland). Subject identification was written on the appropriate bottles and entered into the subject enrollment log along with the randomization code. BODY.SUBJECTS AND METHODS.STUDY DESIGN AND TRIAL SUPPLEMENTATION.COMPLIANCE: Study personnel dispensed supplements together with a personal diary in which the subjects documented the date and time of supplement use. Compliance was assessed on the basis of pill count. Subjects were considered compliant if 80% of the required number of pills was taken. Plasma genistein concentrations measured at baseline and at 3 and 6 months were used as a secondary confirmation of compliance but were not used as a basis for classifying subjects as compliant or non-compliant. BODY.SUBJECTS AND METHODS.STUDY DESIGN AND TRIAL SUPPLEMENTATION.PROCEDURES: Before the subjects were invited for the pre-study examination, a brief telephone screen was conducted to determine whether they were within the study age range, ≥1 year postmenopausal, and satisfied other inclusion/exclusion criteria. Candidates who successfully completed the telephone screening interview were scheduled for the pre-study examination. In the pre-study examination, which took place 1–2 months prior to study start, subject eligibility was again determined. Subjects signed an informed consent form prior to the pre-study examination being performed. After successful completion of the pre-study examination, volunteers were enrolled into the study, starting with a 2-week run-in period during which time they received the placebo. During the run-in period, the volunteers familiarized themselves with the study procedures and the dietary guidelines to which they were expected to adhere. If the subjects successfully completed the run-in, they were asked to sign a second informed consent for study participation as well as to allow disclosure of personal data. They were then randomly enrolled into either the placebo or GBB group and provided the appropriate capsules. Each subject had 5 visits (pre-study examination, start of run-in, visits 1, 2 and 3) and received 6 phone calls (screening and months 1, 2, 4 5 and follow-up) over a period of 7–9 months. They were instructed to maintain their normal diet and exercise routine. Telephone calls between visits allowed compliance to study protocol to be assessed. Fifteen days after the final visit, subjects were contacted by phone to identify any changes in health status since the final visit. At visits 1, 2 and 3, bone markers (see below), plasma genistein, 25-hydroxyvitamin D3 (25(OH)D) and phylloquinone concentrations, dietary records and physical activity levels were assessed, and diet counseling was conducted. BMD was measured at baseline and visit 3 and safety parameters at screening and visit 3. BODY.SUBJECTS AND METHODS.STUDY SUBJECTS, RECRUITMENT, SELECTION AND DISPOSITION: Subjects were recruited between January 2007 and April 2008 by the research team from the Creighton University Osteoporosis Research Center. Eligibility was based on inclusion/exclusion criteria determined by physical examination, medical history, electrocardiogram (ECG), mammogram, trans-vaginal ultrasound, BMD, clinical laboratory, serology and drug and thrombophilia screening. Inclusion criteria were healthy early postmenopausal women between the ages of 45 and 55 years, 1–3 years since the last spontaneous menstrual bleeding and follicle-stimulating hormone (FSH) and 17β-estradiol (E2) concentrations >75 IU/mL and <20 ng/L, respectively, natural menopause or total hysterectomy, and smoking <10 cigarettes/days. Exclusion criteria were T-score <–2.5 at total hip and spine (either or both), body mass index (BMI) >30 or <21, use of HT within the previous 6 months, use of any drug that might interfere with bone metabolism within the previous 12 months, extreme dietary habits, use of dietary supplements while on study except multi-vitamins, total genistein blood concentrations >100 ng/mL measured at pre-study examination, unexplained weight loss or weight gain of >5 kg in the 3 months prior to the study, history of liver or pancreatic diseases, cardiovascular disease, history of breast cancer, endometrial cancer or other malignancy except basal and squamous cell skin cancer, history of thromboembolism, any fractures within the past year except for fingers, toes and facial bones, susceptibility to fractures, endometrial thickness >6 mm, endometrial polyps, insulin-dependent diabetes mellitus, any condition that might interfere with the absorption of the investigational product, co-medications. A total of 70 women were enrolled and randomly assigned to supplementation groups (Fig. 1).Fig. 1Subject disposition BODY.SUBJECTS AND METHODS.MEASUREMENTS.BONE MINERAL DENSITY: BMD measurements at the femoral neck, lumbar spine, Ward's triangle, trochanter and intertrochanter, total hip and whole body were made by dual-energy X-ray absorptiometry (DXA) with a Hologic 4500 instrument (Hologic Inc. Waltham, MA). The densitometers in the Osteoporosis Research Center were operated from a core densitometry laboratory by certified radiological technicians. Densitometry scans were performed according to Osteoporosis Research Center Standard Operating Procedures based on Hologic training. To obtain DXA measurements, subjects were placed in the supine position on a padded table while a scanning arm passed back and forth over their entire body. Radiation exposure was trivial and judged to be acceptable by the Internal Review Board. The coefficient of variation of the Hologic 4500 at the Creighton University Osteoporosis Research Center is 1.1% at the spine and 1.3% at the hip. Measurements at baseline and 6 months were duplicated (with repositioning at each measurement), and the average of both measurements was used for statistical analyses. BODY.SUBJECTS AND METHODS.MEASUREMENTS.ANTHROPOMETRY, DIET AND PHYSICAL ACTIVITY: Body composition (fat mass and lean body mass) was calculated from DXA whole-body scans. Diets and physical activity were assessed via 3-day diaries. To complete the diet diary, subjects were asked to record everything they ate for 3 days (2 weekdays and 1 weekend day consecutive). The dietician reviewed with the subjects how to determine portion sizes. The 3-day diaries were analyzed by a dietician using The Food Processor Nutrition and Fitness Software (Version 7.8, 2001, ESHA Research, Salem, OR). Physical activity was assessed using a portion of the Paffenbarger activity questionnaire [25]. Subjects were asked to estimate the number of hours each day that they spent in various levels of physical activity. BODY.SUBJECTS AND METHODS.MEASUREMENTS.BIOLOGICAL SAMPLES: Bone markers: Plasma bone-specific alkaline phosphatase (BAP) was determined using a Beckman Coulter Access Immunoassay System by Chemiluminescent Immunoassay, and urinary N-telopeptide (Ntx) was determined by Immunochemical method suing Ortho Diagnostics Vitros Analysis. Total osteocalcin (OC) in plasma was determined by a solid-phase immuno-radiometric assay (Cis Bio International, France). Plasma concentration of undercarboxylated osteocalcin (ucOC) was determined by modification of the hydroxyapatite binding assay at Tufts University (Sarah Booth, Jean Mayer USDA Human Nutrition Research) [26]. Deoxypyridinoline (DPD) in urine was determined by enzyme immunoassay (EIA), and osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) were determined using an enzyme-linked immunoassay. Genistein: Ten milliliter of venous blood was drawn into tubes containing EDTA and centrifuged at 3,000 rpm for 10 min at 4 °C. Plasma was pipetted and aliquoted into two propylene tubes (1.5 mL each) and stored at −20 °C. Free and total (sum of unconjugated and conjugated) genistein was determined by LC/MS at DSM Nutritional Products Ltd., Kaiseraugst, Switzerland. Phylloquinone: Plasma vitamin K concentration was determined by reversed-phase high-performance liquid chromatography at Tufts University (Sarah Booth, Jean Mayer USDA Human Nutrition Research Center on Aging). 25-hydroxyvitamin D3 (25(OH)D): Plasma 25(OH)D was measured by radioimmunoassay (RIA) at a certified clinical laboratory. EPA and DHA: Serum EPA and DHA were measured by GC-FID and HPLC–MS (Agilent Q-TOF 6,530 High resolution mass spectrometer) at DSM Nutritional Products Ltd., Kaiseraugst, Switzerland. Clinical blood chemistry and urine analysis: Plasma samples were used for the analysis of hormones (E2, parathyroid hormone, FSH, luteinizing hormone and thyroid stimulating hormone), lipids (total cholesterol, triglycerides, HDL, VLDL cholesterol, LDL cholesterol), hematology, HbA1c, coagulation factors, serology (hepatitis B and C) and thrombophilia screening. Urine electrolytes were determined by certified clinical laboratories according to clinically accepted standardized analytic protocols. Urine analysis (including microscopy) was performed in second spot urine. BODY.SUBJECTS AND METHODS.ADVERSE EVENT (AE) MONITORING: All AEs were reported in the case report form (CRF) and classified as mild, moderate or severe. In addition, all serious adverse events (SAEs) were reported to the Pharmacovigilance partner Contract Research Organization (CRO) UBC and the respective IRB. Subjects were instructed to contact the research nurses if they experienced an AE. Also, the nurses questioned the subjects at each visit about AEs. Medical records were obtained for any SAEs that involved medical follow-up. The research nurses followed each subject with an AE until the event was resolved. If treatment for AEs became necessary, the medication(s) were reported on the concomitant medication section of the CRF. AEs were presented in a frequency table by system organ class and intervention group. In addition, the nature, incidence, severity and cause for each AE were reported. BODY.SUBJECTS AND METHODS.DATA QUALITY ASSURANCES: Data quality was monitored by a professional monitor (RGB Consulting, Canada) and data management by the electronic data capture company (ClinIT, Germany). BODY.SUBJECTS AND METHODS.POWER ANALYSIS AND STATISTICAL ANALYSES.DETERMINATION OF SAMPLE SIZE: The primary health outcomes of this study were the effects of GBB on change in BMD at the lumbar spine and femoral neck. Sample size power calculations were based on the premise that the standard deviation is not greater than double the effect (as percent of baseline) of intervention (see references for evidence in support of this premise) [27–34]. As a result of these calculations, the sample size was determined to be 30 completers per group. Assuming a dropout rate of ~15%, the final sample size was determined to be 35 per group; with this sample size, the trial had 75% power to detect a statistically significant effect with a type I error equal to 10% (trend). BODY.SUBJECTS AND METHODS.POWER ANALYSIS AND STATISTICAL ANALYSES.STATISTICAL ANALYSIS: Differences between the GBB and placebo groups were examined using both an ANOVA model (independent t test with equal variance) and a covariance model (ANCOVA, including baseline values measured at visit 1 as the covariate). Variables measured at all three visits were analyzed with the general linear model repeated measures ANOVA. Differences were considered significant if p < 0.05. Safety data were evaluated by descriptive statistics, and statistically significant differences were determined by t tests (within the groups by paired t test). As appropriate, further exploratory analyses were performed (Pearson's correlations between concentrations of investigational products: genistein, phylloquinone, 25(OH)D and different efficacy and safety end-points), as well as stepwise regression analysis for determination of predictors of BMD change. BODY.RESULTS.DATA SET, SUBJECTS DEMOGRAPHICS AND SCREENING CHARACTERISTICS: Of the 70 subjects randomized, three withdrew before visit 2 and another three between visits 2 and 3. Therefore, 64 subjects were included in the per-protocol (PP) data set that does not take into consideration compliance. There were no statistically significant differences in baseline characteristics and demographics between groups with the exception that total cholesterol was significantly higher in the GBB group (Table 1). All subjects experienced natural menopause and had consumed no more than 2–3 dL of alcohol daily. About half of the subjects reported using multivitamins, but only one quarter reported taking them regularly. Six subjects were classified as non-compliant and were excluded from data analysis. Consequently, the PP analysis included 58 subjects (Fig. 1).Table 1Baseline characteristics and demographics at screening or baseline visitsParametergeniVidaTM bone blend n = 31Placebo n = 33All + n = 64Age (years)54.8 ± 2.554.7 ± 2.354.7 ± 2.4Body weight (kg)68.0 ± 9.271.1 ± 9.069.6 ± 9.2BMI (kg/m2)24.9 ± 2.825.9 ± 2.825.4 ± 2.9Fat mass (%)a38.0 ± 6.036.8 ± 6.137.5 ± 6.1Years since menopause (Y)2.2 ± 0.8 (n = 30)2.1 ± 0.8 (n = 32)2.1 ± 0.8 (n = 62)Hot flashes24/3127/3351/64Hot flashes since (Y)5.4 ± 5.8 (n = 24)8.1 ± 10.3 (n = 27)6.8 ± 8.5 (n = 51)T-score (lumbar spine)−0.58 ± 1.06−0.48 ± 1.33−0.53 ± 1.20T-score (hip)−0.52 ± 0.86−0.53 ± 0.84−0.52 ± 0.84Previous fractures more than 1 year before inclusion0/310/330/64Intake of concomitant medication24/3126/3350/64Intake of multivitaminsb15/3114/3329/64Smokers1/31 (max 10 cigarettes/day)0/331/64Systolic BP (mmHg)114 ± 13115 ± 11114 ± 12Diastolic BP (mmHg)72 ± 973 ± 873 ± 9Heart rate (bpm)68 ± 767 ± 867 ± 7Total cholesterol(mg/mL)223.0 ± 35.4*205.5 ± 29.0214.0 ± 33.2Estradiol (pg/mL)10.2 ± 2.110.4 ± 3.310.3 ± 2.8Follicle-stimulating hormone (mIU/mL)109.9 ± 47.4104.8 ± 33.7107.3 ± 40.7aat visit 1 (baseline); + data only include subjects who completed the studybAbout 25% of the subjects reported taking them regularly (~200 IU vitamin D and 150–300 mg Ca)* p < 0.5 compared to placeboReference ranges: Total cholesterol: <200 mg/mL BODY.RESULTS.BONE MINERAL DENSITY: At the 6-month time point, subjects supplemented with the GBB (n = 30) maintained femoral neck BMD, whereas in the placebo group (n = 28), BMD significantly decreased (p = 0.007, Fig. 2a), resulting in a 1.3% difference between groups (p < 0.05). Similarly, as shown in Fig. 2b, there was a significant difference in BMD between the two groups at Ward's triangle (+2.3 vs −1.1%, p < 0.05). The difference in femoral neck BMD between groups remained statistically significant in the intention to treat (ITT) analysis (p = 0.05); however, when baseline BMD was factored into the analysis, statistical significance was no longer quite achieved (p = 0.058). The BMD delta at Ward's triangle, even when considering baseline BMD, maintained statistical significance in the ITT analysis (p = 0.002).Fig. 2Change of bone mineral density (BMD; 6 months minus baseline) at femoral neck (a) and Ward's triangle (b) Lumbar spine BMD decreased slightly more in women given the placebo (−1.4%) than the GBB (−1.1%), but the difference between groups was not statistically significant (p = 0.55). Changes in BMD at the other sites measured (trochanter, intertrochanter, total hip and whole body) did not differ between groups (data not shown). BODY.RESULTS.BLOOD AND URINE ANALYTES: At both the 3- and 6-month time points, both BAP and Ntx significantly increased in the GBB group in comparison with those in baseline and in the placebo group (Table 2). There were no other significant changes in bone markers (OC, ucOC, DPD, OPG and RANKL) nor did urinary calcium and phosphorus excretion differ between groups (data not shown). As expected, in comparison with baseline, total plasma genistein concentrations and EPA and DHA increased significantly in the GBB group at 3 and 6 months, whereas there was no change in the placebo group (Table 2). Although plasma phylloquinone concentrations increased from baseline to 3 months in the GBB group, at 6 months, values were lower than at baseline, but none of these changes were statistically significant. Although baseline plasma 25(OH)D concentrations were in the optimal range for both groups, suggesting that the subjects were generally fit [35], we observed an increase at 3 months in the GBB group that was maintained to the end of the study (Fig. 3a; Table 2). The plasma 25(OH)D remained stable in the placebo group. Parathyroid hormone (PTH) significantly decreased in the GBB group reflecting the increased plasma 25(OH)D concentration (Table 3).Table 2Blood analytes at different time points (PP considering compliance), mean (SD) and repeated measures ANOVA statisticsParametergeniVida bone blend (n = 30)Placebo (n = 28)p = GBB versus PlcBSL3 months (V2)6 months (V3)BSL3 months (V2)6 months (V3)BSL/V2BSL/V3BAP (μg/L)14.68 (4.64)15.17 (4.38)15.06 (4.87)15.94 (3.96)14.52 (3.03)14.71 (3.93)0.0020.054Ntx/crt (nM/mM crt)40.73 (11.75)41.33 (12.15)45.23 (14.17)44.46 (13.01)44.04 (12.54)41.46 (12.01)ns0.024Genistein tot (ng/mL)2.1 (9.7)96.1 (88.1)129.8 (179.7)4.8 (14.5)1.5 (4.4)2.6 (5.6)<0.001<0.000125(OH)D (nM/L)74.9 (23.0)90.2 (16.5)91.1 (17.8)76.3 (22.2)80.7 (23.178.1 (18.8)0.0090.009Phylloquinone (nmol/L)2.2 (3.1)2.6 (2.9)2.1 (2.1)1.5 (2.2)1.6 (1.3)1.2 (0.9)0.07nsEPA (μg/100μL)2.87 (1.06)Not determined7.16 (2.86)**4.17 (4.28)Not determined4.09 (4.05)na0.003*DHA (μg/100μL)7.29 (2.71)Not determined11.17 (4.16)**9.35 (5.20)Not determined8.34 (5.59)na<0.05*BSL Baseline, V2: 3-month visit, V3: 6-month visit, Reference range: BAP: 0–22.4 μg/L, ns nonsignificant, na not applicable, 25(OH)D 25-hydroxyvitamin D3, EPA Eicosapentaenoic acid (C20:5n-3), DHA Docosahexaenoic acid (C22:6n-3)* Significant at V3 versus placebo** Significant versus BSL (p < 0.0001)Fig. 325(OH)VitD concentration course with statistical significance between GBB and placebo (a) and correlation to PTH (b)Table 3Blood analytes at different time points (PP, safety population), mean (SD)ParametergeniVida bone blend (n = 31)p = V3 versus scraPlacebo (n = 33)p = V3 versus scrascr p = GBB versus PlcbV3 p = GBB versus PlcbScreening6 months (V3)Screening6 months (V3)TC (mg/mL)223.0 (35.4)207.8 (28.5)p = 0.014205.5 (29.0)195.9 (25.7)p = 0.002p = 0.033nsLDL-C (mg/mL)130.3 (33.0)122.4 (31.7)ns118.0 (26.7)115.1 (24.2)nsnsnsHDL (mg/mL)72.3 (15.9)67.7 (16.1)p = 0.01468.8 (16.3)62.1 (13.4)<0.0001nsnsEstradiol(pg/mL)10.2 (2.1)11.5 (3.9)p = 0.04410.4 (3.3)10.7 (2.3)nsnsnsPTH (pg/mL)42.4 (13.8)38.0 (11.4)p = 0.01537.8 (13.3)37.8 (14.5)nsnsnsScr screening, V3: 6-month visit, Reference ranges: TC: <200 mg/mL; LDL-C: <100 mg/mL; HDL: 40–59 mg/mL; Estradiol: <20 pg/mL; PTH: 12–88 pg/mL, ns nonsignificant, TC total cholesterol, PTH parathyroid hormoneaPaired t testbUnpaired t test The season of the year during which subjects were enrolled, which could reflect differences in sun exposure and therefore endogenous vitamin D synthesis, had no effect on the change in BMD at any bone site. Stepwise regression analysis revealed that baseline BMD was the strongest predictor of BMD after 6-month supplementation; baseline 25(OH)D did not further predict BMD. The second most predictive factor determining BMD was GBB independent of the season when subjects were enrolled. This observation suggests that supplementation was sufficient to counterbalance seasonal variations in vitamin D concentrations. In addition, there was a significant relationship between plasma 25(OH)D and PTH at screening/baseline and visit 3 (pooled data: Fig. 3b). There were no statistically significant correlations at any of the three time points between any blood analytes and BMD or bone markers. BODY.RESULTS.ANTHROPOMETRY AND DIETARY INTAKE: There were no significant changes in body weight, BMI and body fat mass in either group when comparing baseline with final values. Similarly, there were no changes in macro- or micronutrient intakes (Table 1). Average daily intake of vitamin D3, calcium and n-3 fatty acids ranged from 100 to 140 IU, 700 to 800 mg and 700 mg—4 g (1–6% from total fat intake), respectively. According to the dietary assessment, isoflavone intake was zero. BODY.RESULTS.SAFETY: Laboratory findings: Clinical chemistry, hematology, lipids, coagulation factors, hormones and urine analysis (including microscopy) were analyzed in all subjects who completed the full dosage regimen (n = 64). Total cholesterol and LDL-C were above the normal reference range at screening in both groups (Table 3), and HDL levels were higher than the medium reference range. Although some single determinations slightly deviated from normal values, most laboratory parameters were well within the normal reference range. Of note, E2 increased in the GBB group by 12.7% (p = 0.044), but remained within the normal range (Table 3). Endometrial Thickness: At no time point did endometrial thickness (ET) differ between the groups. However, ET significantly decreased in the GBB group between screening and study end (2.3–1.8 mm, p = 0.007), whereas there was no change in the placebo group (2.2–2.3 mm, p = 0.62) and no statistical significant difference observed between GBB and placebo at study end. ECG and vital signs: There were no statistically significant changes in vital signs or ECG recordings during the course of the study in any subject, and in no case did any change raise clinical concern. BODY.RESULTS.TOLERABILITY: Summary of adverse events: There were no significant differences in AEs between groups. Of the total of 59 AEs that occurred in 35 subjects, 37 were reported in 20 subjects in the placebo group and 22 in 15 subjects in the GBB group. One AE in each of three individuals led to their withdrawal from the study, and 51% of AEs led to treatment. None of the AEs were classified as severe, 66.1% were considered mild and 33.9% as moderate. All of the AEs except one were considered to be unrelated to the trial supplement (98%). One moderately severe AE (abdominal pain) was judged as probably related to the GBB supplement. After withdrawal from the trial during the second month, this symptom resolved. Approximately 80% of the AEs resolved at study completion. AEs were distributed over a wide range of system organ classes (SOC); however, most of the AEs fell into the SOC of "Infections and infestations" and "Reproductive system and breast disorders." The most frequently reported AE was vaginal hemorrhage (vaginal bleeding and spotting); 5 AEs of this type occurred in 4 subjects in the placebo group compared to one each in 2 subjects in the GBB group. Serious Adverse Event (SAE): There were three cases of incidental findings on the final vaginal ultrasounds, two in one subject in the placebo group and one in the intervention group. These SAEs were documented as Unanticipated Events and unrelated to the effects of the GBB by the investigator who reported to the IRB. BODY.DISCUSSION: The results of this pilot study show that GBB can reduce bone loss in early postmenopausal women. In the placebo group, women lost 1.2 and 1.1% BMD at the femoral neck and Ward's triangle, respectively, whereas women in the GBB group gained 0.1 and 2.3% at these sites, respectively. The bone maker results were unexpectedly inconsistent. We observed increases in both BAP and Ntx at both the 3- and 6-month time points in the GBB group, whereas no changes were noted in the placebo group. This was remarkable since another bone formation marker (OC) and one bone resorption marker (DPD) remained unchanged. In general, the bone turnover marker BAP is decreased during bone-sparing osteoporosis therapies, for example, bisphosphonates [36], hormone therapies [37] or selective estrogen receptor modulars (SERMs). Interestingly, only the more sophisticated therapy, using intermittent application of a recombinant human PTH (1–34) that can reverse bone loss, is also accompanied by increased BAP levels [38]. We speculate that the increased bone turnover is linked to genistein initiating bone formation [39], which was evident after just 3 months. Bone formation is coupled to a parallel bone resorption response, which is observed by the increased Ntx level after 6 months. Therefore, it is unclear why the bone formation marker OC and the resorption marker DPD apparently remained unchanged in the treatment group. An increase in OC and a reduction in DPD were observed in the genistein supplementation study of Morabito et al. [29] using a daily 54 mg dose. Interestingly, when using 200 μg/days vitamin K1 alone for 6 weeks, Bügel et al. [40] did not observe any effect on bone turnover markers (total osteocalcin, BAP, Ntx and DPD). Nevertheless, the vitamin K status markers were improved after supplementation. The serum ucOC/cOC ratio was significantly decreased. Interestingly, Schurgers et al. [41] could improve the serum carboxylated/undercarboxylated osteocalcin ratio with even a lower dose of 100 μg vitamin K1. Our study included healthy subjects that were taking a balanced diet on the top of the GBB supplementation. Obviously, the 150 μg vitamin K1 had no measurable impact on their K status. As expected, plasma levels of genistein, EPA and DHA and 25(OH)D increased in the active group. That there were significant effects at the femoral neck and Ward's Triangle is especially notable because the sample size for this study was calculated to detect a trend, not necessarily statistically significant effects. Therefore, the results indicate that a follow-up study requires a sample size of only 70 subjects to confirm the effects of the GBB at these two bone sites. However, the results generated from this pilot study also indicate that to detect a statistically significant effect (p < 0.05) at the lumbar spine would require a sample size of 900 subjects. The findings of the present pilot study are consistent with the majority of the published positive isoflavone supplementation trials that also struggled with the limitations of small sample size (<50 subjects/group) and short duration (≤1 years) [13–15]. Intriguingly, the four large isoflavone studies that lasted longer than 2 years resulted in ambiguous outcomes [16–19]. In the study by Marini et al. [16], there were dramatic increases in postmenopausal spinal and hip BMD in response to 54 mg/days genistein in aglycone format. On the other hand, Levis et al. and Alekel et al. [17, 18] that used mixtures of isoflavones in glycoside format observed only modest or no effect at all. Both groups used high doses of isoflavones, 80 and 120, and 200 mg/days, respectively. The contrasting results trigger speculations that the different application formats, aglycone vs glycoside, could be responsible for the results due to different bioavailability [42–44]. However, most recent research now casts doubts on this hypothesis. Tai et al. [19] administered 300 mg isoflavones in aglycone format and yet did not observe skeletal benefits. An alternative explanation is that genistein by itself functions differently than genistein in combination with daidzein and glycitein. Currently, there is little evidence to support this theory, however. Another possibility could be that 80 mg and higher doses of genistein are not beneficial; consequently, the bone-sparing effect of genistein is negated. The in vitro data of Dang et al. [45] point in this direction. They have shown that isoflavones stimulate osteogenesis at low concentrations and inhibit osteogenesis at high concentrations in osteoblasts and osteoprogenitor cells [45], which could explain the observed discrepancy. Although the current results are consistent with some previous trials showing genistein exerts skeletal benefits, there are important differences. For example, during the first year of the 3-year trial by Marini et al. [16], women in the genistein group gained 2.4% BMD at the femoral neck, whereas women in the placebo group lost approximately this much bone. There were also marked increases in spinal BMD in response to genistein. The more pronounced effect observed in that trial could be because the subjects were osteopenic and as such had much lower baseline BMD (e.g., femoral neck BMD, 0.0667 vs 0.740) than women in the current trial). However, if the women in the placebo group in the current trial continued to lose BMD at their 6-month rate over the course of 1 year, their bone loss would have essentially matched the loss in the Italian study. Therefore, differences in baseline BMD may not have contributed to differences between the two studies. An alternative and more straightforward explanation is that the lower dose used in the current study (30 vs 54 mg/days) accounts for the less robust results. Animal data suggest this could be the case [46]. Differences in dose may also explain why the current study did not show effects at the spine whereas the study by Marini et al. [16] did. We believe that a longer duration trial is required to see significant effect of the GBB on spinal BMD. It is notable that GBB supplementation had a greater effect on Ward's triangle than it did on the femoral neck. Bone loss at Ward's triangle in the placebo group was similar to that of the femoral neck, whereas there was a 2.3% increase in the GBB group. Ward's triangle was not measured in the study by Marini et al. [16]. However, in an earlier study from this group, the effect of genistein on Ward's triangle was slightly greater than it was on the femoral neck. Ward's triangle is not a true anatomic area but is generated by the DXA scan as the area having the lowest BMD in the femoral head. Interestingly, a small study by Yoshihashi et al. [47] found that in men, the only DXA BMD measurement that was sensitive for detecting osteoporosis was Ward's triangle. Among the women in the current study, the DXA BMD at Ward's triangle and the femoral neck were equally sensitive in detecting changes in BMD. Because a combination of ingredients was used, it is not possible to determine to what extent the individual components contained in the GBB contributed to the observed skeletal benefits. Combined calcium and vitamin D supplementation has been shown to reduce fracture risk in postmenopausal women to a greater extent than supplementation with either agent alone [48]. In the current study, the baseline plasma 25(OH)D concentrations were in the normal range. As expected, only GBB supplementation further increased the 25(OH)D level. Each group was supplemented with calcium (500 mg), which when added to their dietary intake brought their total calcium intake to >1,200 mg/days. Interestingly, despite the rather small study sample size, it was still possible to detect an inverse relationship between plasma vitamin D and PTH levels (Fig. 3b), which is consistent with previously published data [21, 49]. In 2006, Cockayne et al. [50] reviewed the fracture risk reduction after vitamin K supplementation in a meta-analysis. They showed that phylloquinone and menaquinone-4 reduced bone loss and fracture risk. A more recent review by Iwamoto et al. [22] also concluded that vitamin K supplementation reduces fracture risk. However, most trials included in this analysis used far higher doses than the amount contained within the GBB [51]. Furthermore, according to these authors, vitamin K works via mechanisms other than by increasing BMD and affecting bone turnover. Thus, even if the inclusion of vitamin K enhanced the ability of the GBB to reduce fracture, the benefits may have not been detected in the health outcomes analyzed. The fact that there was no change in the undercarboxylated/carboxylated osteocalcin ratio also argues against vitamin K contributing to the increases in BMD that were observed. Finally, in regard to omega-3 fatty acids, the evidence that they exert skeletal benefits is intriguing but still quite speculative [51–53]. Nevertheless, in ovariectomized rats, Krammer [54] found that genistein (15 mg/kg bw) and n-3 PUFA (5% by weight) independently increased femoral BMD over an 8-week period and the combined effect was greater than the effect of either agent alone. Our study has taken advantage of this finding and used nutritional levels of PUFA and genistein in combination with vitamin D and K in the GBB supplementation mixture. The results suggest that genistein supplementation at levels that are compatible with Asian isoflavone intake exerts favorable effects on bone health although long-term trials are needed to confirm efficacy. The GBB was well tolerated by the subjects as there were few AE and no SAE attributable to the intervention. Plasma levels of E2 did increase although modestly so and they remained within the normal range, which is generally consistent with the literature showing that isoflavones have little effect on estrogen levels [55]. Furthermore, and more importantly, there were no effects of the GBB on endometrial thickness. In fact, endometrial thickness decreased over time in the GBB group although the difference in final values between groups was not significant. Estrogen markedly stimulates endometrial tissue and increases risk of endometrial cancer [56]. In the previously mentioned 3-y trial by Marini et al. [16], genistein (54 mg/d) had no effect on endometrial thickness in postmenopausal women. In conclusion, the results of this pilot study suggest that the use of the physiological relevant dose of genistein in combination with EPA and DHA and vitamins D3 and K1 (GBB) may help to prevent osteoporosis and may reduce fracture risk, at least at the hip, in postmenopausal women. The results are also reassuring about the safety of this product. However, additional research and especially longer-term clinical trials are needed before definitive conclusions can be made.

TITLE: Efficacy and Safety of a Traditional Herbal Medicine, ABSTRACT: Hochu-ekki-to is a traditional herbal (Kampo) medicine that has been shown to be effective for patients with Kikyo (delicate, easily fatigable, or hypersensitive) constitution. Previous case reports have suggested that this herbal drug was effective for a certain subgroup of patients with atopic dermatitis (AD). We aimed to evaluate the efficacy and safety of Hochu-ekki-to in the long-term management of Kikyo patients with AD. In this multicenter, double blind, randomized, placebo-controlled study, 91 Kikyo patients with AD were enrolled. Kikyo condition was evaluated by a questionnaire scoring system. All patients continued their ordinary treatments (topical steroids, topical tacrolimus, emollients or oral antihistamines) before and after their protocol entry. Hochu-ekki-to or placebo was orally administered twice daily for 24 weeks. The skin severity scores, total equivalent amount (TEA) of topical agents used for AD treatment, prominent efficacy (cases with skin severity score = 0 at the end of the study) rate and aggravated rate (more than 50% increase of TEA of topical agents from the beginning of the study) were monitored and evaluated. Seventy-seven out of 91 enrolled patients completed the 24-week treatment course (Hochu-ekki-to: n = 37, placebo: n = 40). The TEA of topical agents (steroids and/or tacrolimus) was significantly (P < 0.05) lower in the Hochu-ekki-to group than in the placebo group, although the overall skin severity scores were not statistically different. The prominent efficacy rate was 19% (7 of 37) in the Hochu-ekki-to group and 5% (2 of 40) in the placebo group (P = 0.06). The aggravated rate was significantly (P < 0.05) lower in the Hochu-ekki-to group (3%; 1 of 37) than in the placebo group (18%; 7 of 39). Only mild adverse events such as nausea and diarrhea were noted in both groups without statistical difference. This placebo-controlled study demonstrates that Hochu-ekki-to is a useful adjunct to conventional treatments for AD patients with Kikyo constitution. Use of Hochu-ekki-to significantly reduces the dose of topical steroids and/or tacrolimus used for AD treatment without aggravating AD. BODY.INTRODUCTION: Atopic dermatitis (AD) is a common, chronic, relapsing eczematous skin disease with severe pruritus (1–3). The incidence of AD appears to be increasing worldwide (4,5), among which the percentage of adolescent- and adult-type AD cases have also been increasing (6,7). The precise pathogenesis of AD remains obscure and appears complex. Topical steroids, topical tacrolimus, emollients and oral antihistamines are used as the first-line treatments in standard therapeutic guidelines for AD (2,3,8). However, long-term application of topical steroids could induce local adverse effects such as skin atrophy and telangiectasia in a substantial number of patients. Topical tacrolimus is a potent calcineurin inhibitor that does not exhibit the hormonal adverse effects associated with steroid therapy (9,10). The major adverse effect in using topical tacrolimus is sensation of skin burning or irritation after application (11). In addition, the Food and Drug Administration (FDA) has issued a public health advisory to inform healthcare professionals and patients of a potential cancer risk by the use of topical calcineurin inhibitors (http://www.fda.gov/bbs/topics/ANSWERS/2005/ANS01343.html). This concern is based on animal studies, case reports with a small number of patients, or knowledge of the drug's action mechanism as immunosuppressant. According to the FDA statement, it may take human studies of 10 years or longer to determine if the use of topical calcineurin inhibitors is actually linked to cancer development in human. These adverse effects and emotional fear of long-term use of topical steroids and/or tacrolimus have caused topical steroid/tacrolimus phobia in substantial number of patients with AD worldwide who would like to avoid these topical agents if possible (12). Moreover, clinical experience has shown that some of AD patients are really refractory to these conventional treatments, and indeed current AD therapeutic guidelines recommend further intensive treatments such as ultraviolet phototherapy or oral cyclosporine for such patients (8). In Japan, an alternative approach has been pursued to treat these grave and/or refractory AD patients with traditional herbal medicine such as Saiko-seikan-to, Shohu-san, Oren-gedoku-to, Byakko-ka-ninjin-to, Tokaku-joki-to, Unkei-to, Hochu-ekki-to and so on (13–33). These prescriptions comprise several elemental herbs, combined use of which is thought to help increase the treatment effects and to diminish adverse reactions of each individual herb. Among these prescriptions, formula like Tokaku-joki-to are mainly aimed at eliminating disease factors, whereas Hochu-ekki-to is aimed at correcting abnormal homeostasis of the body. Each prescription is selected individually according to the pharmacological features and the constitution of each patient. Hochu-ekki-to is composed of hot water extracts from 10 species of herbal plants and is used for patients with Kikyo constitution. Kikyo, or deficiency of Ki, is defined as delicate, easily fatigable, or hypersensitive constitution typically associated with poor gastrointestinal functions, anorexia and night sweats (34). In experiments using animal models, orally administered Hochu-ekki-to exhibits various immunopharmacological effects, especially anti-allergic properties. They include suppression of serum IgE level and eosinophil infiltration and improvement of dermatitis through controlling Th1/Th2 balance possibly by inducing interferon γ production from intraepithelial lymphocytes (35–42). In addition, Hochu-ekki-to helps to correct leukocytopenia of mice treated with anti-cancer agents and augments the resistance against bacterial infections (43,44). These results suggest that Hochu-ekki-to may be applicable to the treatment of AD patients who have Kikyo constitution. Although accumulating reports have shown clinical efficacy of Hochu-ekki-to for AD patients (13,15,17,20,21,25,27,29–33), no placebo-controlled study has previously been conducted. In this double-blind, placebo-controlled, randomized clinical trial, we address a question of whether Hochu-ekki-to has beneficial effects for Kikyo patients with AD who have been treated with conventional modalities. BODY.METHODS.HOCHU-EKKI-TO: Hochu-ekki-to fine granules of 7.5 g contain hot water extract (6.4 g) from 10 species of medicinal plants including Ginseng radix (4.0 g), Atractylodis rhizoma (4.0 g), Astragali radix (4.0 g), Angelicae radix (3.0 g), Zizyphi fructus (2.0 g), Bupleuri radix (2.0 g), Glycyrrhizae radix (1.5 g), Zingiberis rhizoma (0.5 g), Cimicifugae rhizoma (1.0 g) and Aurantii nobilis Pericarpium (2.0 g). BODY.METHODS.ASSESSMENT OF : Kikyo condition was evaluated by a questionnaire scoring system. As shown in Table 1, the scoring questionnaire consisted of one 'must have' major sign (10 points) and 10 minor signs (2 points each). The patients who had the major sign and earned 18 points or more using the questionnaire were determined as Kikyo constitution. Table 1.Questionnaire scoring system for Kikyo constitutionItemsSigns and conditionsScoresMajor sign (must-have)Easy fatigability or lack of perseverance10Susceptibility to infectionsSusceptible to cold2Delayed recovery from cold2Vulnerable to other infectious diseases (herpes virus etc)2Susceptible to suppuration2AnorexiaRecent very little eating2Appetite loss2Easily-becoming full stomach2Nahrungsverweigerung2Digestive symptomDiarrhea (laxity)2OthersEasy drowsiness especially after meals2Total scores30Patients who have the major sign and earn 18 points or more in this questionnaire scoring system are determined as Kikyo constitution. BODY.METHODS.STUDY POPULATION: Patients (20–40 years of age) with Kikyo constitution who fulfilled the diagnostic criteria of Japanese Dermatological Association for AD were eligible for this study. All the 91 AD patients enrolled had been treated with topical steroids (mild, strong, or very strong rank) and/or topical tacrolimus for more than 4 weeks prior to the study, and were expected to continue the same therapeutic regimen after the initiation of the study. Patients were not eligible for the study if they had been treated with only weak topical steroids (without stronger topical steroids or tacrolimus), strongest topical steroids, systemic steroids, oral suplatast tosilate, allergen desensitization therapy, or any other herbal medicines for <4 weeks prior to the study. The study was approved by the responsible ethics committee and was performed in accordance with the Declaration of Helsinki and with Good Clinical Practice. An informed witnessed consent was obtained from all the patients. BODY.METHODS.STUDY PROTOCOLS: The study was performed in a multicenter, double blind, randomized, placebo-controlled parallel-group design. The patient number was randomly assigned a treatment code of either Hochu-ekki-to or placebo using a block size of 10 (5 per each group) by an independent controller of the investigators. This code was concealed from the investigators. During trial period, patients were randomized to receive twice daily either Hochu-ekki-to fine granules (Kracie Co., Ltd, Tokyo, Japan) or its inactive placebo which were indistinguishable by its appearance, odor and savor. The daily doses were 7.5 g. All patients continued their ordinary treatments such as topical steroids (other than strongest class), topical tacrolimus, emollients or oral anti-histamines. The skin severity score, amounts of topical agents, adverse effects and laboratory examination including serum IgE, lactate dehydrogenase (LDH) or eosinophil counts were monitored at pre (0-week)-, mid (12-week)- and post (24-week)-treatment. The prominent efficacy (skin severity score = 0 at the end of the study) rate and the aggravated rate (more than 50% increase of amounts of topical agents from the beginning of the study) were also evaluated. BODY.METHODS.ASSESSMENT OF SKIN SEVERITY SCORES: The skin severity scores of AD patients were assessed using the scoring system by the Atopic Dermatitis Severity Evaluation Committee of Japanese Dermatological Association (issued 2001). The skin severity scores were composed of eruption intensity score and affected skin area score. The body surface was divided into five sites, namely head and neck, anterior trunk, posterior trunk, upper extremities and lower extremities. The eruption intensity and affected skin area were evaluated and scored. Eruption intensity scores were evaluated using three eruption items: (i) erythema/acute papules, (ii) oozing/crusts and excoriation and (iii) lichenification/chronic papules and nodules) in the severest area scored from 0 to 3 points (0 = absent, 1 = mild, 2 = moderate, or 3 = severe) for each item in each body site. The affected skin area was also evaluated and scored in each body site as 0, 1, 2, 3 points when affected skin area was absent, less than one-third, one-third to two-third, more than two-third of each body site, respectively. Thus, the skin severity score ranges from 0 to 60 points. BODY.METHODS.ASSESSMENT OF THE DOSAGE OF TOPICAL STEROIDS AND TOPICAL TACROLIMUS: At each patient visit, the actual amounts used of topical steroids and/or topical tacrolimus were measured by weighing the returned ointment tubes from examinees. The amount of topical agents (steroids and tacrolimus) per day was expressed as total equivalent amount (TEA) (gram arbitrary unit; gau) by multiplying potency equivalent factors as follows; weak rank steroids = × 1, mild rank steroids = × 2, strong rank steroids = × 4, very strong rank steroids = × 8, tacrolimus = × 4. Percent change of TEA to that of the beginning of the study was calculated for each patient at 12th and 24th week of treatment using the following formula; BODY.METHODS.STATISTICAL ANALYSIS: Statistical analysis was performed using SAS statistical software (version 8.02; SAS Institute, Cary, NC) under the Windows XP operating system. Data were expressed as the means ± standard deviation (SD) or standard error (SE). A P-value of <0.05 was considered to indicate statistical significance. Treatment efficacy was analyzed by comparing the difference between Hochu-ekki-to and placebo control groups using unpaired Student's t-test or Fisher's exact test. BODY.RESULTS.PATIENT ENROLLMENT AND/OR EXCLUSION: In total, 91 patients were enrolled and randomized in this trial from February to November 2002. Seven out of the initial 91 enrolled patients were excluded from subsequent analysis for the following reasons; agreement acquisition violation (n = 4), eligibility violations (n = 2) and failure to take trial medicine at all (n = 1). Thus the number of patients who actually received medication (hereafter termed 'full analysis set: FAS' group) and who were analyzed was 84 (Hochu-ekki-to: n = 40, placebo: n = 44). Seventy-seven out of the 84 patients completed the 24-week treatment course (Fig. 1), including a patient in whom TAE of topical agents could not be assessed because of the insufficient descriptions on the item. There were no statistically significant differences between Hochu-ekki-to- and control groups in terms of age, sex, physique, duration of AD morbidity, incidence of past- or present history of other allergic or non-allergic diseases, Kikyo score, skin severity score, initial TAE of topical agents per day (Supplementary data 1). Two out of the 7 dropped-out cases during the trial (One each in placebo and Hochu-ekki-to group) were excluded because of a significant aggravation of skin eruption and occurrence of headache, respectively. Others were found unfit for further analyses (dismissal of medication etc). Figure 1.The chart of case enrollment and exclusion. Among the 91 enrolled AD patients, 84 patients were medicated and 77 out of the 84 patients completed full term (for 24 weeks) of trial. BODY.RESULTS.CLINICAL EFFICACY OF : The overall skin severity score gradually decreased as examination went on and was slightly lower in Hochu-ekki-to group (closed circle) than in placebo group (open circle) at 24th week (Fig. 2). The TEA of topical agents gradually increased in the placebo group during the trial period, whereas such increase of TEA was minimal to unchanged in the Hochu-ekki-to group. The percent change of TEA at 24th week was significantly (P < 0.05) lower in the Hochu-ekki-to group (closed circle) than in the placebo control group (open circle) (Fig. 3). Figure 2.The time course change of skin severity score during examination. Skin severity scores were assessed at pre-, mid (12-week)- and post (24-week)-treatment in Hochu-ekki-to- (closed circle) and placebo group (open circle). Data were expressed as the mean ± SD. There is no significant difference between Hochu-ekki-to- and placebo groups. Figure 3.The time course change of equivalent dosage of topical agent during examination. The percent changes of TEA of topical agents were assessed at pre-, mid (12-week)- and post (24-week)-treatment in Hochu-ekki-to- (closed circle) and placebo group (open circle). Data were expressed as the mean ± SE. The TEA of topical agents gradually increased in the placebo group as trial went, while such increase was minimal to unchanged in the Hochu-ekki-to group. *P < 0.05. Since Hochu-ekki-to was thought to be a slow-acting herbal medicine and the present trial was relatively long-term, we wondered if a striking beneficial effect was observed in a certain population of patients by long-term use of Hochu-ekki-to. Therefore, we analyzed a prominent efficacy rate, the rate of patients whose skin severity score became 0 at the end of the study. The prominent efficacy rate was indeed moderately higher in the Hochu-ekki-to group (19%; 7 of 37) than in the placebo group (5%; 2 of 40), although there was not a significant difference (P = 0.06). Furthermore, the aggravated rate, defined as ratio of patients whose TEA had increased more than 50% at 24 weeks from the beginning of the study, was significantly lower in the Hochu-ekki-to group (3%; 1 of 37) than in the placebo group (18%; 7 of 39) (P < 0.05). There was no significant difference in the serum IgE, LDH, or eosinophil counts in peripheral blood in both groups (data not shown). BODY.RESULTS.ADVERSE EVENTS AND ABNORMAL LABORATORY FINDINGS: The adverse events, including those of unclear causality with treatment using tested agents, were observed in 13 of 40 patients (32.5%, total 33 events) in the Hochu-ekki-to group and in 12 of 44 patients (27.3%, total 20 events) in the placebo group. All the adverse events were moderate symptoms such as nausea and diarrhea etc or slight increase or decrease of laboratory data (Table 2). Table 2.Adverse effectsThe number of cases with adverse effectsAdverse effects (the number of cases if not one), including those of unclear causality with treatment using tested agentsHochu-ekki-to group13/40 (32.5%)Symptoms: nausea (2), diarrhea (2), stomach discomfort (2), enlarged feeling of abdomen, epigastralgia, anorexia, loose stools, right hypochondrium pain, malaise, dizziness, headache, light-headed feeling, rhinitis, acne pustulosa, feverish thirstiness, dental caries.Laboratory data: eosinophilia (3), GPT elevation, IgE elevation, BUN decline, serum K elevation.Placebo-control group12/44 (27.3%)Symptoms: ovarian disorder (2), diarrhea, epigastric discomfort, anorexia, malaise, hives, insomnia, feverish limbs.Laboratory data: eosinophilia (4), LDH elevation (2), GOT elevation (2), γ-GTP elevation, serum total protein decline, hemoglobin decline. BODY.DISCUSSION: The present study demonstrates that 24-week oral administration of Hochu-ekki-to has a substantial benefit over placebo in the treatment of Kikyo patients with AD. The administration of Hochu-ekki-to significantly reduces dosage of topical steroids and/or tacrolimus, compared with placebo, although there was no significant difference in the mean skin severity scores between both groups. These results indicate that the patients in the placebo group need significantly more topical steroids and/or tacrolimus in order to control their skin conditions. In keeping with this notion, the post-treatment prominent efficacy rate tends to be higher in the Hochu-ekki-to group than in the placebo group. In contrast, the aggravated rate was significantly lower in the Hochu-ekki-to group than in the placebo group. It was surprising that 19% of patients in the Hochu-ekki-to group were devoid of skin eruption after the 24-week treatment. Considering the long-term history of the majority of the 7 'eruption-free' patients (10–34 years, mean ± SD: 21.9 ± 8.0), this marked improvement would indicate the beneficial effects of Hochu-ekki-to, as 'spontaneous' healing was not likely to take place in those severe AD patients during the course of trial. Hochu-ekki-to, however, did not seem to be effective for all the AD patients in this trial. Individual difference of intestinal flora has been proposed to be one of the major reasons for such disparity of the efficiency of Kampo therapy. It is because active components of orally administered herbal drugs are known to be assimilated through the intestinal mucosa under the influence of the intestinal flora (45). Although we generally advise patients to choose traditional Japanese diet in usual Kampo treatment as intestinal flora can be affected by daily diet, we have avoided intensive intervention during this trial period, assuming that such intervention could be a considerable stress for certain patients and could significantly influence their clinical course of skin symptoms by increasing itch sensation etc. The clinical action of Hochu-ekki-to appears to be rather mild and limited; however, the present study clearly demonstrates an add-on beneficial effect of this herbal drug over the conventional treatments. Kampo herbal drugs are broadly classified into two categories, immediate acting- and slow acting ones. Since Hochu-ekki-to is considered to work in the latter manner, supporting the inactive Kikyo body to become warm and active. Thus, it needs to be administered for a long time to be shown effective. A long-term (for 24 weeks) administration was adopted in our protocol design because of the relatively slow acting profile of Hochu-ekki-to. The significant clinical benefits indeed appeared at 24th week rather than 12th week, as shown by the reduction of TEA of topical agents and of the reduced aggravated rates. Kampo herbal drugs are usually prescribed according to patient's Sho (constitution/condition) such as Yin (negativity) and Yang (positivity) or Kyo (deficiency) and Jitsu (fullness) and to target components to treat such as Ki (energy, spirit and function), Ketsu (blood and organs) and Sui (fluid), all of which are considered to be basic components constituting human body. Hochu-ekki-to is recommended to be used for Kikyo condition, a state of functional deficiency. In our trial, only AD patients with Kikyo constitution were selected eligible by using the questionnaire scoring system. This selection may favorably contribute to carve in relief of the clinical significance of Hochu-ekki-to over placebo, thus it remains to be tested if the herbal drug has such beneficial effect on AD patients without Kikyo constitution. Although considerable attention has been paid on traditional herbal medicines as a treatment option for AD (13–33), there are only a few reports examining their efficacy in a randomized, double blind manner, as reviewed by Armstrong and Ernst (46). Sheehan et al. (47–49) have reported the usefulness of the composite herbs of Zemaphyte® in a randomized, double blind cross-over trial, where it was shown that the Zemaphyte's composite herbs possess anti-inflammatory and anti-congestive function (48). However, a subsequent trial by Fung et al. (50) failed to confirm its superiority over placebo. Although efficacies of herbal medicines are now being recognized even outside the Asian countries (51), there has been no report of double-blind, randomized and placebo-controlled trial of herbal drugs as treatment for AD other than Zemaphyte® before the present study. In conclusion, our results indicate that Hochu-ekki-to is a useful adjunct to conventional treatments for Kikyo patients with AD. We contend that it can reduce the dosage of topical steroids and tacrolimus without aggravating the clinical course of AD. BODY.SUPPLEMENTARY DATA: Supplementary data are available at eCAM online. BODY.SUPPLEMENTARY MATERIAL: [Supplementary Data]

TITLE: Oral ibandronate reduces the risk of skeletal complications in breast cancer patients with metastatic bone disease: results from two randomised, placebo-controlled phase III studies ABSTRACT: BODY: Intravenous (i.v.) bisphosphonates are the standard of care for patients with metastatic bone disease, with proven efficacy in reducing skeletal complications (Hortobagyi et al, 1998; Theriault et al, 1999; Hillner et al, 2000; Lipton et al, 2000; Rosen et al, 2001; Pavlakis and Stockler, 2002; Body et al, 2003c). Yet, for many patients, i.v. bisphosphonate therapy is less than ideal. The risk of infusion-related adverse events (AEs) and the possibility of renal toxicity adds to the treatment burden already faced by patients with advanced cancer. The need for frequent hospital visits and lengthy infusion duration (2 h for pamidronate) can make treatment cumbersome and inconvenient for the patient, particularly as a long-term therapy. Bisphosphonates administered via the oral route would allow the convenience of self-administration at home. However, oral clodronate has been shown to be less effective than i.v. pamidronate at reducing the risk of skeletal-related events (SREs) (Pavlakis and Stockler, 2002), and its use can be associated with unpleasant gastrointestinal (GI) AEs, especially diarrhoea (Kristensen et al, 1999; Powles et al, 2002). Owing to its relatively low potency (Green et al, 1994), high doses are required and at least two large tablets that are difficult for some patients to swallow have to be taken daily (Paterson et al, 1993; Robertson et al, 1995). These factors, coupled with a recommended 1-h prefood fasting period, may affect patient adherence to treatment. Ibandronate, a highly potent, third-generation aminobisphosphonate, has been developed in both i.v. and oral formulations for the management of metastatic bone disease. As reported elsewhere, the i.v. formulation of ibandronate (6 mg infused every 3–4 weeks) has been shown to reduce significantly the risk of skeletal complications, alleviate bone pain and improve quality of life in patients with metastatic breast cancer, in the absence of renal safety concerns (Body et al, 2003a, 2003b; Diel et al, 2003; Tripathy et al, 2003). This paper presents the results of a pooled analysis of two phase III clinical trials that assessed the efficacy and safety of oral ibandronate (50 mg day−1) in the treatment of women with breast cancer and bone metastases. BODY.PATIENTS AND METHODS.STUDY POPULATION: Two randomised, parallel-group, double-blind, placebo-controlled studies were conducted by centres across Europe and other countries, including Australia and the United States of America. Women with histologically confirmed breast cancer and radiologically confirmed bone metastases were recruited into the studies. Patients were required to have a WHO performance status of 0, 1 or 2, be at least 18 years of age, and to provide written informed consent. The exclusion criteria included previous treatment with bisphosphonates or gallium nitrate within the last 6 months, life expectancy <60 weeks, hypercalcaemia (serum calcium, albumin corrected, ⩾2.7 mmol l−1), hypocalcaemia (serum calcium, albumin corrected, ⩽2.0 mmol l−1), severely impaired renal function (serum creatinine >3.0 mg dl−1), Paget's disease of the bone, primary hyperparathyroidism, known liver/brain metastases, receiving a high-dose chemotherapy (i.e. dose intensity >3 times standard therapy), having a medical history of aspirin-sensitive asthma, or receiving treatment with aminoglycoside antibiotics within 4 weeks prior to the start of study medication. In the two studies, patients were randomised to treatment with oral ibandronate 20 mg, 50 mg or placebo once daily for up to 96 weeks. Only the ibandronate 50 mg data (vs placebo) are reported in this pooled analysis, as 50 mg will be the recommended dose for clinical use. Patients were instructed to take one tablet in the morning 1 h before breakfast with a glass of water, but not with milk, milk products or calcium tablets. To assess compliance with therapy, patients were required to return their oral medication to the investigator every 12 weeks for checking. Concomitant treatments were allowed during the study, except those specified as the exclusion criteria. BODY.PATIENTS AND METHODS.EFFICACY AND SAFETY ASSESSMENTS: Efficacy and safety data from the two studies were pooled for analysis, as predefined in the study protocols. Fractures, bone pain, analgesic consumption, episodes of radiotherapy and surgical interventions were assessed at 4-weekly clinic visits. Urine samples were collected at weeks 4, 12, 24, 48, 72 and 96 for the assessment of c-telopeptide (CTx), a marker of bone turnover, using CrosslapsTM CTx assay. AEs were recorded continuously throughout the study. To allow for the possibility of nonrandom withdrawal from study groups, postwithdrawal follow-up (PWFU) efficacy and safety data (i.e. for the period from study withdrawal until death or the last scheduled study visit) were also collected. Postwithdrawal follow-up data collection were discontinued when treatment with another bisphosphonate began. BODY.PATIENTS AND METHODS.ANALYSIS OF EFFICACY: The primary efficacy parameter was the skeletal morbidity period rate (SMPR) defined as the number of 12-week periods with new skeletal complications, divided by the total observation time. Skeletal complications included vertebral fractures, pathological nonvertebral fractures, radiotherapy for bone complications (uncontrolled bone pain or impending fractures) and surgery for bone complications (fractures or impending fractures). To allow for the time spent in the study, SMPR was calculated using a revised event ratio method, as follows (Scott et al, 2003): As prespecified in the data analysis plan, analyses of the primary end point excluded data collected in the first 12-week period. Exclusion of early events avoids the loss of power associated with events occurring too early to have been prevented by bisphosphonates. It was anticipated that effects on bone events of ibandronate vs placebo would begin to appear 6–8 weeks after drug initiation. The first 12 weeks were selected for exclusion as study visits were on a 3-month basis. Supportive analyses of the SMPR included the mean number of skeletal events per patient, the mean number of 12-week measurement periods with events per patient, the percentage of patients with skeletal events and time to first new bone event. A multivariate Poisson's regression analysis was performed to assess the risk of developing a skeletal event over the entire 96 weeks of treatment, while controlling for any differences in baseline characteristics between the oral ibandronate 50 mg group and the placebo group. The input variables for the Poisson's regression analysis were country, age, estrogen/progesterone receptor status, performance status, time from breast cancer and metastatic bone disease diagnoses to study initiation, extraosseous metastases, prior hormone and chemotherapy, pathological fractures at baseline, pain score, analgesic score and baseline laboratory measures (e.g. haemoglobin, alkaline phosphatase, aspartate transaminase, white blood cell counts). BODY.PATIENTS AND METHODS.STATISTICS: The global null hypothesis (no difference in SMPR between ibandronate and placebo) was tested at the two-sided α-level of 5% using the nonparametric Jonckheere–Terpstra method (Terpstra, 1952; Jonckheere, 1954). If the global hypothesis was rejected, pairwise comparisons between treatments were performed using the Wilcoxon's rank-sum method, maintaining an overall two-sided α-level of 5% and following a closed-test procedure. The trial was designed such that the statistical analysis of the study was powered for the composite end point SMPR, but not for the components of the composite. Efficacy analyses were conducted on the intent-to-treat (ITT) population (all patients randomised) and included PWFU data. Evaluation of safety was based on all randomised patients who had received at least one dose of study drug and had at least one follow-up assessment. BODY.PATIENTS AND METHODS.ETHICS: The study was performed in accordance with the principles of the Declaration of Helsinki, the Guidelines on Good Clinical Practice and local medicines legislation in place at the time of study initiation. BODY.RESULTS.PATIENTS: A total of 564 patients were randomised to treatment with oral ibandronate 50 mg (n=287) or placebo (n=277) and were included in the ITT analysis. Patient demographics and baseline characteristics are shown in Table 1 Table 1Patient demographics and baseline characteristics Placebo (n=277)Ibandronate, 50 mg (n=287)Age (years) Median (range)56 (26–87)57 (27–92) Race, Caucasian (%)9493Median time from diagnosis to first drug intake (years)3.873.44Median time from bone metastases diagnosis to study entry (years)0.480.46 Performance status (%) WHO grade 0 or 18584 WHO grade 21516 Mean pain score1.131.33Mean analgesic score0.981.09Prior fractures at baseline (%)4352. Although the study groups were generally comparable at baseline, the oral ibandronate 50 mg group contained a higher percentage of patients receiving ongoing cytotoxic therapy, with a higher mean bone pain score and a higher percentage of patients with pre-existing fractures than in the placebo group (differences between groups nonsignificant). The percentage of patients completing the 96-week treatment period was 42% in the ibandronate group and 38% in the placebo group. The median time on study (from randomisation to study end) was 79 weeks with ibandronate compared with 69 weeks with placebo (NS). The most frequent reasons for withdrawal were malignancy progression (affecting 12% of patients receiving ibandronate vs 19% of patients receiving placebo), death (15 vs 12%) and other AEs (10 vs 12%). BODY.RESULTS.EFFICACY: The mean SMPR for all new bone events was significantly reduced with oral ibandronate 50 mg compared with placebo (P=0.004) (Figure 1Figure 1Summary of the mean SMPR, weighted for observation time (*P<0.05, **P<0.01 and ***P<0.001).). Analysis of the individual components revealed that this effect was due primarily to significant reductions in bone events requiring radiotherapy (P<0.001) or surgery (P=0.037) (Figure 1). There was no significant difference in the number of skeletal fractures with ibandronate compared with placebo (P=0.195). When bone events occurring during the first 12-week period were included in the SMPR calculation, the impact of ibandronate on the incidence of skeletal events was reduced, but remained significant for overall SMPR and for events requiring radiotherapy (Table 2 Table 2Supportive analysis of SMPR, including events occurring during the first 12 weeks of treatmentMean SMPRPlaceboIbandronate, 50 mgAll new bone events1.150.99, P=0.041*Vertebral fractures0.520.49, P=0.145*Nonvertebral fractures0.520.51, P=0.330*Need for radiotherapy0.980.80, P<0.004*Need for surgery0.440.40, P=0.098*SMPR=skeletal morbidity period rate.*Wilcoxon's rank-sum test, pairwise comparisons vs placebo.). Supportive analyses of new bone events demonstrated that the mean number of events and the mean number of measurement periods with events per patient were significantly reduced in the ibandronate group compared with placebo (P=0.008 and 0.015, respectively, Table 3 Table 3Supportive analyses of new bone events PlaceboIbandronate, 50 mgNo. of events per patient1.851.15, P=0.008*No. of 12-week periods with events per patient0.990.71, P=0.015*% of patients with events52.245.3, P=0.122**Wilcoxon's rank-sum test, pairwise comparisons vs placebo.). The median time to first bone event was 90.3 weeks with oral ibandronate 50 mg and 64.9 weeks with placebo (P=0.089). Multivariate Poisson's regression analysis showed that the risk reduction for a skeletal event in the ibandronate 50 mg group was significantly lower than in the placebo group (hazard ratio 0.62, 95% CI=0.48, 0.79, P<0.0001), translating to a 38% risk reduction for ibandronate vs placebo. Patients receiving oral ibandronate 50 mg showed a significant decrease from baseline in the bone marker urinary CTx over the 96-week study period compared with placebo (median change −77.3% and +11.0%, P<0.001) (Figure 2Figure 2Change in urinary CTx during study period (*P<0.001).). BODY.RESULTS.SAFETY: A total of 563 patients were included in the safety analysis. As would be expected with skeletal metastases due to advanced cancer, almost all patients reported AEs during the course of the study. The percentage of patients experiencing any AE was similar between the oral ibandronate 50 mg and placebo groups (94.4 vs 95.3%). The most frequently recorded AE was malignancy progression (affecting 67.5 and 70.8% of patients, respectively). There was a slightly higher incidence of drug-related AEs with ibandronate (26.6%) than with placebo (17.7%), primarily due to more reports of hypocalcaemia in the ibandronate group (Table 4 Table 4Treatment-related AEs (reported by ⩾2% of patients in any treatment group)AEsPlacebo (n (%))Ibandronate, 50 mg (n (%))Abdominal pain2 (0.7%)6 (2.1%)Dyspepsia13 (4.7%)20 (7.0)Nausea4 (1.4%)10 (3.5%)Oesophagitis2 (0.7%)6 (2.1)Hypocalcaemia14 (5.1%)27 (9.4%)AEs=adverse events.), a side effect associated with the use of any bisphosphonate. Serious AEs that were considered to be drug related were experienced by 1.0% of patients receiving ibandronate, compared with 1.4% of patients in the placebo group. The incidence of mild treatment-related upper GI AEs (dyspepsia, nausea and oesophagitis) was slightly higher in the oral ibandronate 50 mg group compared with placebo (Table 4). The incidence of drug-related upper GI AEs known to be associated with oral bisphosphonate administration was similar in the placebo and 50 mg oral ibandronate groups (Table 4). Only two serious upper GI AEs (duodenal ulcer haemorrhage and nausea, each recorded by one patient) were considered related to ibandronate treatment. The incidence of renal AEs was comparable between ibandronate (5.2%) and placebo (4.7%), and there were no reports of serious AEs (renal failure) in the active treatment group. During the course of the study, 20% of patients (n=57) in the ibandronate 50 mg group and 15% of patients (n=42) in the placebo group died as a result of an AE. Death was most commonly due to malignancy progression, and no deaths were considered to be related to study treatment. BODY.DISCUSSION: The primary efficacy measure used in these two phase III trials of oral ibandronate was the SMPR, defined as the number of 12-week periods with new bone events, weighted for observation time. By assessing 12-week time periods where all complications are considered as a single occurrence, the SMPR avoids multiple counting of events, and therefore represents a conservative measure of efficacy. Clinical trials of other bisphosphonates in patients with metastatic bone disease have used the SRE or skeletal morbidity rate to assess the impact of treatment on skeletal complications. By counting all occurrences of new bone events, these measures may overestimate the effect of treatment, as many skeletal events (e.g. radiotherapy, fracture and bone surgery) are likely to be related in many cases. The pooled results of the two oral trials demonstrated that ibandronate 50 mg once daily effectively reduces the incidence of new bone events in women with breast cancer and bone metastases. A statistically significant clinical benefit was observed for overall SMPR compared with placebo. This effect was maintained when skeletal events occurring in the first 12-week treatment period (including prescheduled radiotherapy events, which may have reduced the observed effect of active treatment) was included in the analysis. Ibandronate also significantly improved the need for bone radiotherapy and the need for bone surgery, both of which are considered to be highly clinically relevant indicators of disease outcomes. As the study was not powered to detect statistical significance on individual components of the SMPR, these results strongly support the clinical impact of ibandronate on the occurrence of new bone events. The results for fractures did not reach statistical significance in contrast to the data for i.v. ibandronate (Body et al, 2003a), and the patients had overall less fractures in the oral studies. Since a large meta-analysis of clinical trials has shown that bisphosphonates significantly decrease skeletal morbidity including fractures and need for radiotherapy (Ross et al, 2003), the trend that was observed for oral ibandronate will need to be confirmed in larger studies. The percentage reduction in SMPR with oral ibandronate vs placebo in this pooled analysis (19%) was comparable to that observed in a clinical trial of i.v. ibandronate 6 mg (20%), which had a similar study design and was also conducted in patients with metastatic breast cancer (Body et al, 2003a, 2003c). The Poisson's regression analysis conducted on the pooled data set and the results of the i.v. trial also revealed comparable reductions in the risk of SREs with oral and i.v. ibandronate compared with placebo (hazard ratio 0.62, P=0.001 and hazard ratio 0.60, P=0.0033, respectively) (Body et al, 2003a, 2003c). Comparisons between these trials are cautious, as patients in the i.v. study had received a diagnosis of metastatic bone disease approximately 10 months earlier prior to study entry than patients in the oral trials, indicating that they had more severe disease. However, patients in the trials were similar in terms of their clinical presentation (age, baseline fracture incidence, performance status and bone-pain level), suggesting that the oral and i.v. formulations had broadly similar efficacy. Supporting this, oral and i.v. ibandronate were shown to have similar effects on secondary efficacy end points, with bone pain significantly reduced and maintained below baseline over 2 years of treatment, and significantly less deterioration in quality of life compared with placebo (Body et al, 2003b; Tripathy et al, 2003). This would be expected given that a daily oral 50 mg dose and an i.v. 6 mg given every 3–4 weeks provide the same bone surface exposure to ibandronate (Leyland-Jones, in press). Direct comparisons between bisphosphonates are difficult because of differences in study methodology. A comparative trial is currently examining the effects of oral ibandronate and i.v. zoledronate in patients with metastatic bone disease due to breast cancer. Long-term drug safety and tolerability is an important consideration in the selection of treatment for skeletal metastases, due to the high disease-related morbidity burden and the side effects associated with systemic cancer therapy. Oral ibandronate 50 mg day−1 for 2 years of treatment was well tolerated in these trials, with an AE profile quite similar to placebo. As demonstrated for i.v. ibandronate (Body et al, 2003a; Lyubimova et al, 2003), oral ibandronate was not associated with renal AEs. This contrasts with the enhanced risk of renal AEs reported with i.v. zoledronate and pamidronate in a phase III trial (Rosen et al, 2001). With its benign renal safety profile, oral ibandronate may be used in patients with existing renal impairment. In addition, the results suggest that serum creatinine monitoring can be made, depending on the assessment of the individual patient, at the clinician's discretion. The associated reductions in renal monitoring time and costs could help to relieve the burden of bisphosphonate care on nursing staff and hospital budgets (Body, 2003). As well as efficacy and safety, the availability of oral ibandronate could offer improved treatment flexibility for physicians and convenience for patients. Oral ibandronate may be prescribed alongside other oral agents (particularly hormonal treatment) for at-home dosing (e.g. when hospital care is not being received). Patients would no longer have to spend time travelling to and from the hospital solely for bisphosphonate infusion, allowing them to maintain their lifestyle without unnecessary disruption. The dosing regimen of oral ibandronate is convenient for patients. Adequate adherence is important in real-life situations, where dosing instructions are not closely monitored, unlike in clinical trials. In conclusion, oral ibandronate 50 mg is an effective, well-tolerated and convenient treatment for the skeletal complications of metastatic bone disease.

TITLE: Efficacy of a Web-Based Guided Recommendation Service for a Curated List of Readily Available Mental Health and Well-Being Mobile Apps for Young People: Randomized Controlled Trial ABSTRACT.BACKGROUND: Mental disorders are highly prevalent for the people who are aged between 16 and 25 years and can permanently disrupt the development of these individuals. Easily available mobile health (mHealth) apps for mobile phones have great potential for the prevention and early intervention of mental disorders in young adults, but interventions are required that can help individuals to both identify high-quality mobile apps and use them to change health and lifestyle behavior. ABSTRACT.OBJECTIVES: The study aimed to assess the efficacy of a Web-based self-guided app recommendation service ("The Toolbox") in improving the well-being of young Australians aged between 16 and 25 years. The intervention was developed in collaboration with young adults and consists of a curated list of 46 readily available health and well-being apps, assessed and rated by professionals and young people. Participants are guided by an interactive quiz and subsequently receive recommendations for particular apps to download and use based on their personal goals. ABSTRACT.METHODS: The study was a waitlist, parallel-arm, randomized controlled trial. Our primary outcome measure was change in well-being as measured by the Mental Health Continuum-Short Form (MHC-SF). We also employed ecological momentary assessments (EMAs) to track mood, energy, rest, and sleep. Participants were recruited from the general Australian population, via several Web-based and community strategies. The study was conducted through a Web-based platform consisting of a landing Web page and capabilities to administer study measures at different time points. Web-based measurements were self-assessed at baseline and 4 weeks, and EMAs were collected repeatedly at regular weekly intervals or ad hoc when participants interacted with the study platform. Primary outcomes were analyzed using linear mixed-models and intention-to-treat (ITT) analysis. ABSTRACT.RESULTS: A total of 387 participants completed baseline scores and were randomized into the trial. Results demonstrated no significant effect of "The Toolbox" intervention on participant well-being at 4 weeks compared with the control group (P=.66). There were also no significant differences between the intervention and control groups at 4 weeks on any of the subscales of the MHC-SF (psychological: P=.95, social: P=.42, emotional: P=.95). Repeat engagement with the study platform resulted in a significant difference in mood, energy, rest, and sleep trajectories between intervention and control groups as measured by EMAs (P<.01). ABSTRACT.CONCLUSIONS: This was the first study to assess the effectiveness of a Web-based well-being intervention in a sample of young adults. The design of the intervention utilized expert rating of existing apps and end-user codesign approaches resulting in an app recommendation service. Our finding suggests that recommended readily available mental health and well-being apps may not lead to improvements in the well-being of a nonclinical sample of young people, but might halt a decline in mood, energy, rest, and sleep. ABSTRACT.TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12614000710628; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=366145 (Archived by WebCite at http://www.webcitation.org/ 6pWDsnKme) BODY.INTRODUCTION.BACKGROUND: In Australia, clinical mental disorders are highly prevalent among young adults aged between 16 and 25 years. Approximately one in four suffer from at least one diagnosable mental disorder in the past 12 months [1], and mental disorders account for a quarter of the burden of disease in this age group [2]. With similar prevalence rates globally (eg, in Europe, Africa, the United States, and Asia) adolescent mental health is an international public health challenge [3]. Adolescence is a crucial developmental stage for the individual and disruption to mental health during this stage can have far-reaching effects, and whose full personal and socioeconomic impact often only becomes apparent at a later stage in life [3]. Thus, effective, engaging, and easy to disseminate strategies that reduce the multiplicative impact of these risk factors within young people are needed. BODY.INTRODUCTION.MHEALTH APPS: Although technology mediated mental health interventions have frequently been praised for their potential and ease of access, previous research into one-size-fits-all intervention has demonstrated that these interventions have limited appeal and that they have failed to gain traction within health care [4]. It is possible that this limitation can be overcome by delivering interventions using mediums and resources young people are already interacting with and that are tailored to their circumstances. Mobile health (mHealth) apps have great potential for the prevention and early intervention of many physical and mental health problems. To date, there are approximately 165,000 health apps available for Android and iOS mobile phones and tablet devices, approximately 10% of which are mobile apps for mental health problems [5]. However, it has been shown that many easily available mHealth apps are of dubious quality and do not follow evidence-based principles [5]. For example, research into currently available apps for individuals with bipolar disorder found that the majority were not developed in line with best practice clinical guidelines or self-management principles currently used in the treatment of bipolar disorder. Most apps also did not contain source citations or privacy policies, making it difficult for users to assess app quality [6]. An evaluation of mobile apps for mindfulness highlighted that many apps often claim to be for a particular purpose or provide a particular intervention when they in fact do not [7]. Those mobile mental health apps that are based on evidence-based principles and have demonstrated efficacy often have been developed as part of research studies and are not available publically [8]. This suggests that there is a gap between evidence-based research and readily available existing mHealth interventions in the open market. Therefore, to utilize the public health potential of these existing mHealth apps, it is important to identify those apps of high quality and guide individuals in finding interventions that meet their need and likely work. BODY.INTRODUCTION.RATING APPS: With regard to identifying high quality apps, most people use app store ratings as a marker for app quality, as indicated by the correlation between app user ratings and their popularity on the marketplace [9]. However, these ratings mainly reflect subjective experiences from a usability and aesthetics perspective, and not whether the apps are designed with appropriate strategies necessary to improve health outcomes [10]. Obtaining more objective evaluations of mobile apps with regard to their quality is hampered by the fact that even with regard to simple criteria, such as the degree of personalization, the funding source of related research, or data import and export capabilities, interrater reliability is poor [11]. In an attempt to overcome the limitations of user ratings for mobile apps, Stoyanov et al [10] created the mobile app rating scale (MARS), a questionnaire to assess the quality of health apps on the domains engagement, functionality, aesthetics, information, and subjective quality. Whereas this scale is a more objective marker of app quality than app store ratings, it is also resource intensive and requires thorough assessment. As a result, application of this approach on a large scale is still in its infancy due to the large volume of mHealth apps. With regard to identifying effective apps, research suggests that theory-driven health interventions, that is, interventions employing evidence-based behavior change techniques (BCTs) are more effective than interventions that are not theory-driven [12]. The implementation of such strategies within mobile apps is influenced by how users interact with mobile apps [13]. Mobile phone apps are generally optimized to the way people engage with such phones and as such tend to implement only some strategies with functionalities that are brief and easy to use. As a result, a single app alone is unlikely to contain all necessary strategies for mental health, and furthermore identical strategies might be implemented in different apps with varying aesthetics and ease of use. Thus, characterizing strategies implemented within an app will be a crucial step to identify effective apps. To ensure that ethical values in health care are met, this characterization process could be facilitated by clinicians or researchers by reviewing the scientific literature, searching apps stores, reviewing app descriptions and reviews, and piloting the app themselves [14]. However, it is important to recognize that individuals are less likely to engage with interventions that implement effective strategies but poor aesthetics and usability, thus resulting in ineffective outcomes [15,16]. One way of overcoming these challenges is to create a repository of high-quality mHealth apps and guide users in the process of identifying effective and engaging ones. The "Beacon" website is one such resource developed in Australia that categorizes, reviews, and rates websites and mobile apps for mental and physical disorders [17]. Recent findings suggest participants are willing to use several apps when they are recommended a range of custom-selected apps with different behavioral strategies [13]. The challenges of this approach have been highlighted in the UK's National Health Service's attempt of creating a curated app repository for patients with chronic health conditions. Many of the apps were found to transmit sensitive data without the knowledge of the user [18] or provide clinically questionable advice [19] that resulted in the app library to be closed. In addition, the majority of apps are generally identified and downloaded by users directly from apps stores and the keywords people use when searching for specific health apps do not necessarily yield the most appropriate or effective app [20]. Instead they might be reflective of these words appearing in place like app name, text used in description of the apps combined with the user rated popularity of the apps, none of which alone are markers of quality. Developing a nuanced understanding of search patterns for mental health and well-being problems will be crucial to facilitate access to apps of high quality. BODY.INTRODUCTION.IMPACT ASSESSMENT: Methods to measure the impact of app usage may require different approaches, given the way people tend to interact with apps, usually for short periods of time, on a regular or irregular basis. An alternative to traditional questionnaire-based measures of mental health and well-being, ecological momentary assessment (EMA) [21] may be a suitable means of detecting short-term changes with regard to parameters, such as mood or sleep on a day-to-day basis. In this paper, we report on findings from a waitlist randomized controlled trial (ACTRN 12614000710628) [22] which was designed to test the efficacy of a guided recommendation service for readily available mobile mental health apps for young people aged 16-25 years. Whereas our primary outcome measure was the well-validated Mental Health Continuum-Short Form (MHC-SF), we employed ecological momentary assessments to track mood, sleep, and energy. BODY.METHODS.OVERVIEW: We conducted a Web-based parallel-arm randomized controlled trial comparing "The Toolbox," a guided app recommendation service, to a waiting list control group. Web-based measurements were assessed at baseline and 4 weeks, and ecological momentary assessments were collected repeatedly at regular weekly intervals or ad hoc when participants interacted with the study platform. Details of study design, intervention and control conditions, outcome measures, and sample sizes are reported extensively in the previously published study protocol [22]. A brief overview of the study is outlined in the following section. The study received ethical approval by the Social and Behavioural Research Ethics Committee of Flinders University (registration number 64780) and is registered in the Australian New Zealand Clinical Trials Registry (ACTRN: 12614000710628). It also gained ethical approval for recruitment by the Department of Education and Child Development of South Australia (DECD CS/14/511-23). BODY.METHODS.RECRUITMENT: Participants were recruited from the general young adult (16-25 years) population across Australia, with access to a computer or mobile phone and the Web. Preexisting mental health conditions were not considered as exclusion conditions for this study. Several Web-based and community strategies, either paid or unpaid, were utilized for recruitment. The recruitment message was formulated around overall well-being and health (and not on illnesses): Examples of such messages included: Want to improve your energy and fitness? Find out what your wellbeing looks like and use apps to achieve your goals. Better health & fitness: Monitor your wellbeing, set goals, & access health & fitness apps. Summer fun taking its toll? Track your wellbeing & download apps for mind+body. Web-based paid advertisements were placed on Facebook, Twitter, YouTube, and Google AdWords from November 19, 2014 to March 12, 2015. The keywords for the advertisements were defined in collaboration with a reference group representing the target population to ensure their validity and relevance. Examples of keywords included fitness, stress, relationships, balance, and goals. A total of 12 advertisements were placed across the previously mentioned platforms, with an average duration of 21 days per advertisement. The paid strategy also included recruitment through a recruitment agency for clinical trials. The agency referred individuals in the target demographic to the study website over a period of 2 months (July 8, 2015 to September 2, 2015). In addition to paid advertisements, links to the study site were provided to 39 organizations and educational institutions frequently visited by young people from different backgrounds in Australia (most notably the partner organizations of the Young and Well Cooperative Research Centre) to integrate into their websites and promote via their social media channels (Facebook, Twitter). Community-based organizations such as schools, universities, sporting clubs, and local councils in one rural region of South Australia were approached and asked to help promote the study. Promotional packages comprising a video, information, and instructions on how to access the Online Wellbeing Centre (OWC) were distributed to 32 institutions and community contacts and presented to potential participants. Participants with informed consent and aged between 16 and 25 years were included, and parental consent was obtained if participants were recruited from community organizations and were below 18 years old. Using unique links, data was collected to objectively identify the recruitment source for each participant. The yield per strategy and characteristics of participants between channels are reported elsewhere [23]. BODY.METHODS.PROCEDURES: The study was conducted through an OWC platform consisting of a landing Web page and capabilities to manage consent, sign up, randomize, administer study measures at different time points, monitor engagement, and provide feedback to users in a meaningful graphic display. Study advertisements linked participants to the landing page of the OWC which contained a brief overview of the study, detailed participant information sheet, and a Web-based consent form. After completing the Web-based consent form, participants completed a registration form. The OWC software randomized the participants and sent them their login details, either via email or SMS (short message service). Participants logged into the OWC to complete study measurements and if they were assigned to the intervention arm, a link to the intervention website (The Toolbox) was accessible through the OWC immediately. Participants in the control group accessed "The Toolbox" 4 weeks after registration. During the study period, participants from both the intervention and control arm received weekly SMS or email prompts at a time chosen by them during registration, encouraging them to log in to the OWC. The prompts contained a unique link which when clicked logged them in and took them to a page to complete their EMAs. After completing these assessments, they were directed to the OWC homepage, which contained "The Toolbox" access link for the intervention group, and generic well-being advice for the control group. They also received prompts to log in to the OWC to complete study measures at 4 weeks or until they completed. BODY.METHODS.INTERVENTION: The intervention was a personalized app recommendation service called "The Toolbox," available through the ReachOut.com website [24]. The content and structure of the Web-based intervention was determined by young adults' perspectives on well-being and expectations with Web-based interventions. End-user studies were conducted with Australian young adults to investigate how young adults conceptualize well-being. Data were collected via user experience workshops with young people aged 15-21 years. Key findings from the workshop influenced the structure and content of the developed Web-based intervention. The workshops with young people were analyzed, resulting in a nuanced understanding of young adults' conceptualization of health and wellness, and an empirical knowledge of concrete behaviors and actions in their daily lives that they associate with well-being. Data from the workshops were subsequently synthesized into 27 key action areas or goals, and categorized into 6 overarching key themes: health and fitness (15 apps), being independent (8 apps), relationships and helping others (3 apps), thoughts and emotions (18 apps), and dealing with tough times (14 apps). Of these apps, 31 were available for free, 12 apps were paid with costs of up to Aus $6.49, and 3 apps either offered a free lite version or were available for free on one of the platforms, but not on the other. The process of selecting apps to populate "The Toolbox" consisted of two stages. First, a contextual review of available apps was conducted, followed by a review of these apps according to the MARS [10] by professionals and young people. For the contextual review, a list of key search terms was created (see Multimedia Appendix 1), which was drawn from a conceptual well-being model of promoting resilience and flourishing developed for the Young and Well Cooperative Research Centre, as well as qualitative input gained in workshops with young people. These terms were then used to conduct an audit of existing well-being mobile apps available on Google Play and the Apple App Store in late 2013. Only apps that (1) appeared in the first 200 search results, (2) were under Aus $5, (3) were available for Android or iOS, and (4) were deemed appropriate for 13-25 year olds, were included in the rating process. During the rating process, irrelevant apps were removed as well as those not meeting minimum functionality and aesthetic requirements. Remaining apps were rated by researchers using the MARS for both effectiveness and usability and only the highest scoring apps were selected for additional rating by a mental health expert. Apps that contained valid information and were deemed not harmful for young people were selected for "The Toolbox" and additionally rated on the MARS by at least two young people. The final curated list of 46 readily available apps, categorized according to the 27 goals, were put together into a Web resource called "The Toolbox," with an average of 3.62 (SD 3.05) apps per goal (see Multimedia Appendix 2). "The Toolbox" is a responsive website hosted by Reachout.com. Participants first choose the areas they want to focus on, guided by an interactive quiz and subsequently receive recommendations for particular apps to download and use based on their preferences (see Figure 1). For each recommended app, additional information is provided, including the MARS score and reviews by both health professionals and end users on what they liked and did not like, along with costs and links to download from the app store (see Figure 2). Participants assigned to the intervention arm upon completion of their baseline measures were displayed a Web link which gave them immediate access to "The Toolbox." Over the 4-week study period, participants were sent weekly reminders (via email or SMS) advising them to visit "The Toolbox" at least once, take the quiz, and use the recommended apps. The use of "The Toolbox" website and the recommended third-party apps constitutes the intervention in this study. Participants were aware at all times that the researchers assumed no responsibility for the content and/or functionality of these apps. Figure 1Flow of "The Toolbox" website, including home, well-being category selection, goals selection, and recommended apps pages. BODY.METHODS.CONTROL: Participants in the control group were advised that they were on a waiting list for 4 weeks before they would be given a link to access "The Toolbox." At 4 weeks after completing the baseline measures, participants were provided access to "The Toolbox" via the OWC. BODY.METHODS.MEASURES: The primary outcome was a self-reported measure of well-being assessed using the 14-item MHC-SF that measures subjective psychological, emotional, and social well-being. Secondary outcomes were EMAs of 3 questions: How are you feeling today? How is your energy level today? How well did you sleep last night? (see Figure 3). Participants completed primary outcome measures on the Web at baseline and 4 weeks. The EMAs were completed each time participants logged into the OWC during the study period. The log file from the Web app during the trial period was gathered to derive engagement with the study platform. The EMAs of participants were obtained for up to 6 months' postcompletion of trial. Figure 2Example of information related to each app available on "The Toolbox" website, including app overview, link to download, user and professional ratings, and app reviews. Figure 3Text message and ecological momentary assessment (EMA). BODY.METHODS.STATISTICAL ANALYSIS: Differences in attributes between groups (Table 1) and in attrition versus not (Table 2) were assessed using chi-squared tests, t test or Wilcoxon rank-sum tests as appropriate. The primary analysis was based on intention to treat, and missing values from all randomized participants were imputed with 50 samples redrawn from the original data. The primary outcome was analyzed using linear regression (Table 3) with well-being score at 4 weeks as the dependent variable. The independent variables were well-being, measured at baseline, and group assignment. A multivariable intention to treat linear regression sensitivity analysis was also conducted, as well as the same analysis, using observed data only. To investigate how the momentary assessment of mood, sleep, rest, and energy were influenced by the intervention (Table 4), the trajectories of momentary assessment measures were examined using random effects mixed modeling. The independent variables were group assignment, engagement with the study platform (coded as number of logins), the product term of group assignment, and engagement, and potential confounders were age, gender, prior application use (coded as a binary variable), baseline energy, baseline mood, and whether or not an app was downloaded. Subject was entered into the model as a random effect to account for correlated readings within an individual. Differences between groups were assessed using interaction terms. Similar multivariable linear regression analyses were conducted with the MHC-SF subscales as outcomes, with covariates listed as before. To examine whether engagement with the study platform was associated with changes in the EMA measures, a linear regression model was run with postintervention EMA measures as the outcome. All models contained an additional term representing the number of logins. For energy, mood, and rest, the other covariates were listed before in the sensitivity analyses. For sleep the other covariates were baseline sleep and group assignment due to the small number of observations. For these regressions, postintervention measurements for EMAs were taken as the measurement that occurred between 30 and 45 days, with the earliest one after 30 days. All results are reported with 95% CI and P values. A P value <.05 (2-tailed) was taken to be significant. All analyses were performed using Stata version 13.1 (StataCorp). BODY.RESULTS.FLOW OF PARTICIPANTS: Figure 4 shows the flow of participants. A total of 476 people were consented and signed up on the Web. Of these, 387 completed baseline scores and were randomized into the control (n=195) and active (n=192) arm of the trial. BODY.RESULTS.FLOW OF PARTICIPANTS.PARTICIPANT CHARACTERISTICS: The demographic characteristics and baseline scores are shown in Table 1. Table 1 Baseline characteristics. Participant characteristics Control Intervention Total Statistics P value Age in years, median (interquartile range) 23 (20-25) 23 (20-25) 23 (20-25) z =0.67 .51 Female gender, n (%) 152 (78.4) 143 (75.3) 295 (76.8) χ 2 1 =0.5 .47 Prior app usage, n (%) 72 (36.9) 65 (33.9) 137 (35.4) MHC-SF a , median (interquartile range) 41 (28-51) 39 (27-51) 40 (27.5-51) t 358 =0.16 .44 Subscale: emotional, median (interquartile range) 10 (7-12) 10 (8-12) 10 (7-12) z =0.24 .81 Subscale: social, median (interquartile range) 13 (7-17) 12 (6-17) 12 (6-17) z =0.83 .41 Subscale: psychological, median (interquartile range) 18 (11-22) 18 (11-23) 18 (11-23) z =0.03 .98 EMA b “rest,” median (interquartile range) 50 (36-65) 50 (35-64) 50 (36-54) z =0.03 .98 EMA “mood,” median (interquartile range) 50 (40-70) 50 (40-70) 50 (40-70) z =0.03 .98 EMA “energy,” median (interquartile range) 50 (40-60) 50 (30-60) 50 (40-60) z =0.03 .98 a MHC-SF: Mental Health Continuum-Short Form. b EMA: ecological momentary assessment. Figure 4Consolidated Standards of Reporting Trials (CONSORT) flow diagram of study participants during study enrolment, allocation, follow-up, and analysis. BODY.RESULTS.ATTRITION: Attrition measured as the failure to respond to the primary outcome measurements at 4 weeks postrandomization was 45.1% in the control group (88/195) versus 55.2% in the active group (106/192), P=.047. In the control group, the mean baseline MHC-SF for those who responded at 4 weeks, versus those who did not was 40.2 versus 37.6 (P=.25). In the intervention group, the mean baseline MHC-SF for those who responded at 4 weeks, versus those who did not was 36.7 versus 42.3 (P=.12). A comparison of characteristics between completers (ie, those that reported data on the primary outcome at 4 weeks) and noncompleters revealed no significant differences across all demographics at baseline, apart from mood as assessed through EMA (Table 2). Table 2 Baseline characteristics for those who provided data at four weeks versus not. Participant characteristics a Noncompleters Completers Statistics P value Age in years, median (interquartile range) 23 (20-25) 23 (20-25) z =0.05 .96 Female gender, n (%) 14 (77.0) 148 (76.7) χ 2 1 =0.0 .95 Prior app usage, n (%) 69 (35.6) 68 (35.2) t 358 =0.50 .94 MHC-SF a , median (interquartile range) 41 (27-50) 40 (28-52) t 358 =0.50 .78 Subscale: emotional, median (interquartile range) 10 (7-12) 10 (8-12) z =0.82 .41 Subscale: social, median (interquartile range) 12 (6-17) 12 (7-17) z =0.49 .62 Subscale: psychological, median (interquartile range) 19 (11-22) 18 (12-23) z =0.14 .89 EMA b “rest,” median (interquartile range) 50 (35.5-63) 50 (36-65) z =0.18 .86 EMA “mood,” median (interquartile range) 50 (40-70) 60 (50-70) z =2.13 .03 EMA “energy,” median (interquartile range) 50 (30-60) 50 (40-60) z =0.66 .51 EMA “sleep,” median (interquartile range) 420 (375-480) 435 (360-525) z =0.22 .85 a MHC-SF: Mental Health Continuum-Short Form. b EMA: ecological momentary assessment. BODY.RESULTS.PRIMARY ANALYSIS: The mean (SD) observed MHC-SF scores at 4 weeks for the control and active groups were 38.6 (SD 15.4) and 42.0 (SD 16.8), respectively. In the primary intention-to-treat (ITT) analysis, those in the intervention group experienced an improvement of 0.63 (95% CI −2.26 to 3.53) in MHC-SF score relative to the control group, but this was not significant, P=.66. In multivariable sensitivity intention to treat analysis the difference was almost identical, 0.64 (95% CI −2.27 to 3.54), P=.66. In a further multivariate sensitivity analysis with observed data only, there was also no difference between groups, 1.10 (95% CI −1.68 to 3.89), P=.44. In a completers only analysis there was also no difference between groups in MHC-SF scores 1.17 (95% CI −1.98 to 3.53), P=.40. BODY.RESULTS.SUBSCALES OF MENTAL HEALTH CONTINUUM-SHORT FORM (MHC-SF): There were no significant differences between groups in any of the subdomain scores, psychological 0.57 (95% CI −0.67 to 1.81), P=.95, social 0.46 (95% CI −0.68 to 1.59), P=.42, and emotional −0.02 (95% CI −0.72 to 0.68), P=.95. BODY.RESULTS.ANALYSES OF ECOLOGICAL MOMENTARY ASSESSMENTS: For all EMA measures, the control group decreased significantly per login in contrast to the active group which showed no significant change in scores over time. Thus, the difference between groups per login was also significant (Table 4). Table 3 Multivariable intention to treat analyses (adjusted for group assignment, age, gender, prior app use, energy, mood, and whether or not an app has been downloaded). Outcome Measure Value beta (95% CI) P value MHC-SF a .64 (−2.27 to 3.54) .66 Psychological .57 (−0.67 to 1.81) .95 Social .46 (−0.68 to 1.59) .42 Emotional −.02 (−0.72 to 0.68) .95 a MHC-SF: Mental Health Continuum-Short Form. Table 4 Changes per login in ecological momentary assessments (adjusted for group assignment, age, gender, prior app use, whether or not an application has been downloaded). Ecological Momentary Assessments Control beta (95% CI), P value Intervention beta (95% CI), P value Difference between groups beta (95% CI), P value Mood −.25 (−0.40 to −0.11) , P =.001 .15 (−0.04 to 0.33), P =.13 .40 (0.16-0.63), P =.001 Energy −.20 (−0.33 to −0.07), P =.003 .11 (−0.06 to 0.29), P =.19 .31 (0.10-0.52), P =.004 Rest −.19 (−0.36 to −0.08), P =.001 .12 (−0.04 to 0.29), P =.15 .31 (0.11-0.52), P =.002 Sleep −2.34 (−3.16 to −1.52), P <.001 −.46 (−1.66 to 0.74), P =.15 1.88 (0.43-3.34), P <.001 BODY.RESULTS.ENGAGEMENT-RESPONSE ANALYSIS: There was no evidence to suggest that a beneficial effect was associated with the number of logins in any of the measures, mood 0.15 (95% CI −0.58 to 0.87), P=.69, energy 0.08 (95% CI −0.56 to 0.72), P=.81, rest −0.13 (95% CI −0.91 to 0.64), P=.73, and sleep −5.29 (95% CI −11.96 to 1.54), P=.12. BODY.DISCUSSION.PRINCIPAL FINDINGS: The aim of this study was to assess the efficacy of "The Toolbox" Web-based well-being intervention in a young adult population. Results from the randomized controlled trial demonstrated no significant benefit on well-being (as assessed using the MHC-SF) at 4 weeks compared with the control group. There were no significant differences between the active and control groups at 4 weeks on any of the subscales of the MHC-SF either. The trial results also suggest that the impact of receiving weekly texts and the opportunity to monitor and visualize sleep, mood, and energy led to repeated logins in both control and active groups. In addition, participants in the control group reported a significant decline in mood, energy, rest, and sleep as assessed with EMAs with an increasing number of logins, whereas the intervention group showed no change. Thus, repeat engagement with the intervention might halt decline in mood, energy, rest, and sleep, without resulting in significant changes in well-being as assessed by the MHC-SF at fixed points. It remains unclear as to whether this interaction can be attributed to using "The Toolbox" intervention or using the study platform and its repeated assessments. The magnitude of change in the control group was very small, which may explain why no change in well-being as measured by the MHC-SF was observed. BODY.DISCUSSION.COMPARISON WITH PREVIOUS WORK: The lack of effect on MHC-SF well-being scores observed in this intervention are comparable with results from similar Web-based intervention trials, conducted in older (mean age 43.2 years) [25] and under 16-years-old school-based samples [26]. Across both studies, positive benefits were shown in depression scales but benefits assessed using mental well-being scales themselves were minuscule and nonsignificant both immediately after the intervention and at follow-up. Compared with these studies, the intervention in our study was targeted at a general young adult sample (mean age 23 years) with a broad inclusion criterion that did not exclude participants based on symptom screening, which closely resembles the real-world setting of intervention delivery through the Reachout.com website. Considerably more females than males participated in this study, which is in concordance with the majority of research into mental health and well-being interventions. In part, the higher proportion may be attributed to the higher prevalence of mental disorders in females in this age group [27]. However, differences in help-seeking behavior between males and females likely account for the majority of this difference [28]. In our study, instead of administering depression scales, we assessed symptoms of depression at multiple time points through momentary assessments. Despite the differences in type of assessment, we detected comparable benefits on depression as evident by significant improvement in mood trajectories in the intervention group. One plausible explanation from these findings is that modest improvements detected in mental well-being might actually be a reduction in depression symptoms that have collinearity with mental well-being [29]. This raises questions about sensitivity of mental well-being scales to detect change and if Web-based interventions can change mental well-being as an independent construct in the absence of mental illness. Compared with past studies, the intervention in this study is unstructured, in the form of a collection of curated list of mobile app resources accessible through a self-guided hub, as well as monitoring tools to engage participants and provide feedback irrespective of app use. Disseminating a curated list of mental health and well-being apps for depression and anxiety alone has been recently demonstrated to yield better app uptake [13]; however, ours is the first study to investigate effectiveness of such an intervention. In order to ensure optimal app recommendations, different components and strategies within apps that serve as active intervention ingredients must thus be identified [30] and aligned with end user needs. The intervention in this study included a broad range of curated apps (n=46) with an algorithm that assigned these apps to one of the 27 action areas used in the app selection quiz, that were identified based on young adults' conceptualization of well-being through codesign activities. Since the spread of apps were not uniform across all action areas, the collection of apps might not have been optimal to be suitable and effective for all individuals. We also observed a slightly higher baseline symptomology in participants dropping out from the intervention, which might be caused by the mismatch between apps and individual health circumstances that were not factored in the matching algorithm. The use of specific apps over a 1-month period may not have been sufficient to induce significant changes in well-being as measured by a global mental health scale, such as the MHC-SF. Instead, our results suggest that app usage may affect momentary moods and behaviors more easily measured by EMAs. It may be that EMAs provide a more accurate measure of the day-to-day impact of app usage. To date, there are few studies of well-being interventions utilizing momentary assessments as outcome measures, although their superiority to traditional questionnaire measures has recently been reported [31]. BODY.DISCUSSION.LIMITATIONS: There are several limitations to our study. There was a high attrition rate of almost 50% subject randomized, which is not unusual in Web-based interventions. However, the results of the primary analysis were consistent with the sensitivity analyses. Interestingly, we found a higher rate of attrition in the intervention group compared with the control group, a finding which was also reported by Bolier et al [25]. The reason for the greater attrition rate in the intervention group is unclear and, along with many factors associated with the high attrition rates in Web-based interventions, requires further study [32]. Given that intervention and study dropout are often linked in Web-based interventions, it is possible that overall attrition was higher in the intervention group because participants immediately had access to the intervention and thus had no incentive to participate in the 4-week assessment. Alternatively, it is possible that the content, functionality, and aesthetics of some apps may have changed during the short time from when they were added to "The Toolbox" to when they were accessed by participants, as is common on mobile app marketplaces [33], thus not meeting, or differing significantly from their expectations. All of the recommended apps remained available on the app stores for the duration of the study. Due to the nature of the intervention, it was not possible to quantify the download and use of the apps recommended by "The Toolbox." Thus, there was no direct measure of intervention adherence. This was accounted for by using ITT analysis; however, ultimately it was not possible to determine whether the lack of effect on well-being was due to nonadherence or due to a lack of effectiveness of "The Toolbox" and its recommended apps. This is an inherent problem when studying the effectiveness of third-party intervention that can only be overcome by retaining full ownership of the intervention tool, as was the case in Lattie et al [13]. The study was also limited by a relatively heterogeneous sample of participants recruited using varying strategies, although this could be both a strength and limitation as it replicates app recommendation interventions in real-life setting. The other major limitation was the lack of longitudinal follow-up data; however, it is unlikely longitudinal effects would be found when no benefits were observed at follow-up (4 weeks), which is when most changes are expected. BODY.DISCUSSION.CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH: Whereas there are several randomized controlled trials of the efficacy of Web-based services to improve mental health, previous studies have been conducted in adults with symptoms of anxiety and depression [25,34,35]. In comparison, this was the first study to assess the effectiveness of a well-being intervention designed to recommend the use of readily available mobile apps in a sample of young adults. The design of the intervention utilized expert rating of existing apps and end-user codesign approaches, resulting in an app recommendation service. Our findings cast doubt on the effectiveness of mobile apps for well-being and mental health in a nonclinical population of young adults. This intervention included a self-guided optimal selection of apps. Further work could focus on developing algorithms to automate the process of determining optimal apps for an individual, taking into account active ingredients in apps, personal characteristics, engagement, and needs. Intervening with the right combination of quality apps is critical to realizing benefits of over 30,000 mental health related apps available in the app store. The instruments used for assessing mental well-being in this study may not have been sensitive enough to detect change caused by app usage. Future research should focus on refining the construct so that it is sensitive to change even when symptoms of depression or mental illness are absent. In addition, consideration should be given to the measurement of the specific behaviors targeted by particular apps as well as to overall constructs, such as well-being.

TITLE: Reduction of Nasal Bone Fracture using Ultrasound Imaging during Surgery ABSTRACT.BACKGROUND: Most nasal bone fractures are corrected using non-invasive methods. Often, patients are dissatisfied with surgical outcomes following such closed approach. In this study, we compare surgical outcomes following blind closed reduction to that of ultrasound-guided reduction. ABSTRACT.METHODS: A single-institutional prospective study was performed for all nasal fracture patients (n=28) presenting between May 2013 and November 2013. Upon research consent, patients were randomly assigned to either the control group (n=14, blind reduction) or the experimental group (n=14, ultrasound-guided reduction). Surgical outcomes were evaluated using preoperative and 3-month postoperative X-ray images by two independent surgeons. Patient satisfaction was evaluated using a questionnaire survey. ABSTRACT.RESULTS: The experimental group consisted of 4 patients with Plane I fracture and 10 patients with Plane II fracture. The control group consisted of 3 patients with Plane I fracture and 11 patients with Plane II fracture. The mean surgical outcomes score and the mean patient dissatisfaction score were found not to differ between the experimental and the control group in Plane I fracture (p=0.755, 0.578, respectively). In a subgroup analysis consisting of Plane II fractures only, surgeons graded outcomes for ultrasound-guided reduction higher than that for the control group (p=0.007). Likewise, among the Plane II fracture patients, those who underwent ultrasound-guided reduction were less dissatisfied than those who underwent blind reduction (p=0.043). ABSTRACT.CONCLUSION: Our study result suggests that ultrasound-guided closed reduction is superior to blind closed reduction in those patients with Plane II nasal fractures. BODY.INTRODUCTION: Facial fractures are common presentations in the field of plastic surgery. Among these, nasal bone fractures occur most frequently and thus represent a significant portion of the plastic surgery practice [12]. Management of nasal bone fractures can be largely divided into open and closed reductions. Of the two, closed reduction is the treatment of choice in most cases because of the relatively short operation time, minimal scars, tissue damage, and shorter recovery time. However, the closed approach does not allow direct visualization of the fractured site and can lead to incomplete or failed reduction. Postoperative outcomes are difficult to predict for this reason [134]. Reductions can be visualized intraoperatively using portable X-ray, ultrasound, and C-arm fluoroscopy. Among these methods, we investigated whether the use of intraoperative ultrasound is associated with improved outcomes following closed nasal bone reduction [1345]. BODY.METHODS: A single-institutional prospective study was performed for nasal fracture patients (n=28) presenting between May 2013 and November 2013. Upon research consent, patients were randomly assigned to either the control group (blind reduction, n=14) or the experimental group (ultrasound guided reduction, n=14). Patients were excluded for Plane III fractures in the Stranc classification, as these patient would require open reduction. Reduction operation was usually performed 7 days after trauma, under general anesthesia. For the control group, the closed reduction was performed after evaluating the fracture with palpation and the necessity of reduction using the conventional manner, that is, only by the surgeon's sense of touch. For the experimental group, the nasal bone was reduced while visualizing the fracture in longitudinal and transverse views (Fig. 1) using a 2D ultrasound probe (Accuvix V10, 8.0-MHz linear probe, L5-13IS, Samsung, Seoul, Korea). All operations were performed by a single surgeon. After each reduction, the nasal cavities were packed with Merocel (Medtronic, Minnesota, USA) for 5 days. Patients were asked to use a Denver nasal splint (JMEDICS, Cundinamarca, Colombia) for 2 weeks and, afterwards, to use the splint at night-time for an additional 2 weeks. Postoperative outcomes were evaluated by two surgeons who are highly experienced in facial trauma surgery and not involved in the care of the patients in this study. Each of the two surgeons evaluated the preoperative and 3-month postoperative X-ray (Figs. 2, 3) images as well as clinical photographs (Figs. 4, 5). Reduction was evaluated for nasal bone deviation, bony hump, and for other types of displacement. Each of these criteria was evaluated between 1 ("poor") to 4 ("excellent"), and the mean score between the two rater was used for analysis. The interrater and intrarater reliabilities were assessed using intraclass coefficient. Intra-class coefficients between 0.60 and 0.80 were considered substantial; coefficients greater than 0.80 were excellent [6]. In addition, patient dissatisfaction was evaluated using a questionnaire survey, which focused on aesthetic and functional aspects at 3 months after the reduction operation. For each survey, a patient answered questions to five aesthetic items and five functional items, all of which referred to post-nasal reduction complications commonly described in the literature [7]. The aesthetic items included complaints regarding the nose shape and consequential stress, the disruption of daily life and interpersonal relationship, and the feeling of depression. The functional items included swelling, nasal congestion, dyspnea, insomnia, and exertional dyspnea. For each time, patients assigned a score between 0 (very satisfied) to 4 (very dissatisfied) (Table 1). Within each study group (control and experimental), the results were analyzed for Plane I and II in the Stranc classification [4]. In order to verify the statistical significance, p-values were obtained with a 95% confidence interval by Mann–Whitney test using IBM SPSS ver. 19 (IBM Inc., Armonk, NY, USA). BODY.RESULTS: The experimental group consisted of 4 patients with Plane I fracture and 10 patients with Plane II fracture. The control group consisted of 3 patients with Plane I fracture and 11 patients with Plane II fracture. In patients with Plane I fractures, the postoperative outcome scores were 3.6 points for ultrasound-assisted reduction and 3.5 points for blind reduction. In patients with Plane II fractures, the postoperative outcome scores were 3.3 points for ultrasound-assisted reduction and 2.5 points for blind reduction. The intrarater and interrater reliabilities were 96.1% (95% confidence interval, 0.916 to 0.982) and 93.8% (0.867 to 0.971), respectively (Table 2). On the questionnaire survey, patients who had Plane I nasal fractures reported a combined score of 6.0 points in the control group and 5.5 points in the experimental group. Patients with Plane II fractures reported a combined score of 9.1 points in the control group and 6.9 points in the experimental group (Table 3). Specific to Plane II fracture subgroups, patients indicated the highest amount of dissatisfaction, aesthetically, for nasal shape (1.6 points for the control group and 1.5 points for the experimental group), whereas disruption of work was cause for the least amount of dissatisfaction (0.7 for the control group and 0.4 for the experimental group). As far as functional outcome was concerned, patients expressed highest amount of dissatisfaction for breathing difficulty during exercise (1.3 for the experimental group and 1.5 for the control group) (Table 4). Statistical analyses did not discover any significant difference between blind reduction and ultrasound-assisted reduction for patients with minor fractures (Plane I). However, ultrasound-assisted reduction was associated with higher levels of satisfaction among those patients with moderate degrees of nasal fracture (Plane II). BODY.DISCUSSION: Nasal bone fractures are the most common among facial fractures, and reduction of these fractures are delayed until the edema has subsided. Because of this, nasal fractures are usually addressed at about 7 days after the injury [7]. Fractures can be reduced via closed or open approaches, but most fractures are reduced in closed fashion because of the advantages over open reduction such as simplicity of operation, low risk of infection, and lack of postoperative scars. The disadvantage of closed reduction is the difficulty in accurately reducing the nasal bones as it is based on the surgeon's sense of touch. Because direct visualization is not possible, closed reduction poses the risk of undercorrection or overcorrection of the fracture. This difficulty can result in increased number of reduction attempts, which is associated with the risk of edema, hemorrhage, inflammation, and nasal synechiae [4]. Particularly in severe fractures (i.e., Plane II or III fractures), soft-tissue edema is significant enough to obscure palpation of the fractured segment. In addition, complex fracture lines it make accurate reduction that much more difficult when compared with minor fractures. If for such reason, reduction is attempted several more times, soft tissues in the vicinity of fractured site may be damaged, which increases the chance of complications such as edema, epistaxis, or inflammation. Chen et al. [3] suggested that closed reduction of traumatic nasal bone fracture was 14%–62% and that 9% of such cases needed reoperation. Han et al. [4] noted that the most appropriate time to judge the adequacy of reduction for facial bone fractures is during the operation, while it is still possible to adjust the degree of reduction. It is possible to view the fractured site indirectly, at the time of operation, using portable X-ray, C-arm fluoroscopy, or ultrasonography. Portable X-ray requires re-positioning of the patient to obtain the desired perspective. C-arm fluoroscopy carries the risks of irradiation, and its accuracy was found to be lower than that of ultrasound in evaluating the nasal fracture [58]. Ultrasound is a favorable imaging modality to evaluate the fracture site during closed reduction of a nasal fracture because of the thinness of nasal skin and mucosal layer under the skin [8]. In a previous study, Abu-Samra et al. [9]. had compared the usefulness of ultrasound in closed reduction of nasal fractures. The authors compared evaluation of the nasal fracture between ultrasound and plain radiography and found that ultrasound was superior in diagnosing the nasal bone fracture, as the ultrasound-guided method was 100% sensitive while plain radiography was only 59% sensitive. However, the patient-reported satisfaction revealed no significant outcomes difference between those treated with ultrasound-assisted closed reduction and those treated with conventional closed reduction. The authors argued that the lack of significant difference was most likely due to the fact that patients who underwent the reduction of a nasal bone fracture as reconstruction had lower aesthetic demands than those who underwent surgery for cosmetic purpose [9]. However, their study had not distinguished this comparison according to the extent of fracture. In the present study, we investigated outcomes following ultrasound-guided closed reduction to overcome the limitations of blind approach. In those patients with Plane I fractures, patient-survey scores were not statistically different between the groups, as the average of combined scores were 5.5 points with ultrasound and 6.0 points without ultrasound (p=0.578). Among patients with Plane II fractures, however, patients who underwent blind reduction reported a significantly higher level of dissatisfaction (9.1 points) when compared to that of the ultrasound-assisted closed reduction (6.9 points) (p=0.043). Potential reasons for ultrasound-guided closed reductions increasing patient satisfaction for Plane II fractures are as follows. Fracture patterns are complex and edema is severe in Plane II fractures, which makes it difficult to determine the exact fracture patterns solely with palpation. Ultrasound imaing is helpful in such cases as it allows for a more accurate reduction while decreasing the number of attempts at reduction, which can minimize the damage to soft tissues including the mucous membrane. In addition, ultrasound allows for intraoperative identification of undercorrection and overcorrection of fracture displacement. In this context, our study result suggests that ultrasound-guided closed reduction is superior to blind closed reduction in those patients with Plane II nasal fractures. Future studies should focus on this subset group of nasal fracture patients.

TITLE: Efficacy of local drug delivery of ABSTRACT.CONTEXT:: Periodontitis is an inflammatory disease of microbial origin. Locally delivered antimicrobials reduce subgingival flora. Achyranthes aspera gel has antimicrobial, antioxidant, anti-inflammatory, and immunostimulant effects. ABSTRACT.AIMS:: To evaluate the efficacy of local drug delivery of A. aspera gel in the management of chronic periodontitis. ABSTRACT.MATERIALS AND METHODS:: Thirty patients with chronic periodontitis were considered in the study and categorized into two equal groups (Group A: scaling and root planing (SRP) with A. aspera gel, Group B: SRP with placebo gel). Patients were enlisted from the Department of Periodontics, Mamata Dental College and Hospital. The clinical parameters (gingival index, bleeding on probing, probing pocket depth, and clinical attachment level) were recorded at baseline and 3 months. ABSTRACT.STATISTICAL ANALYSIS USED:: All the obtained data were sent for statistical analyses using SPSS version 18. ABSTRACT.RESULTS:: The periodontitis and the Achyranthes were statistically analyzed. A comparison of clinical parameters for test group and control group from baseline to 3 months was done using paired t-test. Intergroup comparison for both the groups was done using independent sample t-test. ABSTRACT.CONCLUSIONS:: A. aspera gel when delivered locally along with SRP showed a beneficial effect. A. aspera gel as a non-surgical local drug delivery system proved to be without any side effects in the management of periodontitis. A. aspera gel has strong anti-inflammatory effects in addition to its antioxidant activity. BODY.INTRODUCTION: In the 21st century, herbal plants are gaining its place back by substituting modern drugs all over the world.[1] This drastic changeover is due to the irreversible damage caused by modern drugs.[2] The advantage of herbal plants over modern drugs is that they usually consist of several phytochemical components, including flavonoids and alkaloid compounds.[3] Achyranthes aspera is an important herbal remedy used most commonly in allied medicine,[4] as it has various pharmacological properties such as antimicrobial, analgesic, antipyretic, anti-inflammatory, immunostimulant, antioxidant, antifertility, hypoglycemic, diuretic, hypolipidemic, cardiac stimulant, antihypertensive, antinoiceptive, prothyrodic, antispasmodic, and hepatoprotective properties.[5] Periodontitis is an array of inflammatory diseases of microbial origin mainly composed of Gram-negative anaerobic bacteria. The etiopathogensis of these diseases influences the interaction between bacterial agents, environment, and the host defense mechanisms to the microorganisms.[6] The development of "specific plaque hypothesis" has led to newer treatment modalities which aim at suppressing or eliminating the specific periodontal pathogens. Putative pathogens causing periodontal diseases are sensitive to a variety of antiseptics and antibiotics.[7] The existing treatment approaches in the management of periodontal diseases aim at targeting specific micro-organisms and thereby help in the modification of the destructive host defence mechanism. The success of conventional mechanical periodontal treatments depends on the day-to-day patient's oral hygiene maintenance and supplement use of systemic antimicrobial agents helpful in treating recurrent periodontal pockets.[8] BODY.MATERIALS AND METHODS: A total of thirty patients were enlisted from the Department of Periodontics, Mamata Dental College and Hospital, Khammam, with clearance from ethical committee and followed by informed consents from all the patients who participated in the study. Healthy patients diagnosed with chronic periodontitis (probing pocket depths [PPDs] ≥5 mm) and individuals who can maintain satisfactory oral hygiene after the initial treatment were included in the study and patients diagnosed with aggressive periodontitis, pregnant and lactating females, smokers, and patients under antibiotic therapy or who underwent periodontal therapy in the last 6 months were excluded from the study. A stent was manufactured to measure the periodontal probe depth before and after the treatment. It is designed to reach the same position before and after the treatment, thus avoiding errors during treatment protocol. The study patients were categorized into two groups as follows: Group A (test): 15 patients who were treated with scaling and root planing (SRP) followed by subgingival application of A. aspera gelGroup B (control): 15 patients who were treated with only SRP. Followed by a complete supragingival scaling, proper oral hygiene instructions were given to the individuals and customized stents were designed for the region where the plant preparation has to be delivered for the standardization of the values throughout the study. The following clinical parameters such as gingival index (GI), PPD, and clinical attachment level (CAL) were assessed at baseline before SRP and local drug delivery of A. aspera gel in Group A and before SRP alone in Group B and then again at 3 months after the treatment. BODY.MATERIALS AND METHODS.PREPARATION OF : A. aspera powder was obtained from the pure root extract of the plant and an aqueous-based gel was prepared, which consists of the following proportions: A. aspera powder 4 g, gelatin 12 g, glycerin (wetting agent) 0.2 ml, peppermint oil (flavoring agent) 0.1 ml, sodium saccharine (sweetening agent) 0.1 ml, and purified water qs 100 ml. All the ingredients were homogenized using a homogenizer to obtain the gel [Figure 1a and b]. Figure 1(a) Achyranthes aspera powder extracted from the root of the plant (b) an aqueous-based gel preparation BODY.MATERIALS AND METHODS.LOCAL DRUG DELIVERY: Following a complete SRP and irrigation with saline in both the groups, A. aspera gel was locally delivered into the periodontal pockets in Group A patients with the help of a blunt cannula of syringe. Patients were advised not to chew hard, sticky foods and brushing or use of interdental aids was also avoided at the drug-delivered site for a week [Figure 2a and b]. Figure 2(a)The periodontal pockets measured using a periodontal probe and designed stent placement (b) Achyranthes aspera gel was locally delivered into the periodontal pockets with the help of a blunt cannula of syringe BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: All the required data were obtained and sent for statistical analyses using SPSS version 18.0 IBM SPSS (IBM Inc. Chicago). A statistically significant P < 0.05 was considered. A comparison of clinical parameters for the test and control groups from baseline to 3 months was done using paired t-test [Tables 1 and 2]. Intergroup comparison for both the groups was done using independent sample t-test [Table 3]. Table 1 Means of probing pocket depth, clinical attachment level, and gingival index at baseline and 3 months for both the test and control groups Table 2 Intergroup comparison of pocket depth, clinical attachment level, and gingival index at baseline and 3 months between the test and control groups Table 3 Clinical parameter differences in both the test and control groups from baseline to 3 months BODY.RESULTS: The clinical parameters such as GI, probing depth (PD), and CAL after statistical analysis showed a significant reduction from baseline to 3 months. The mean GIs in the test and control groups at baseline were 2.60 and 2.57, respectively, and after 3 months were 1.35 and 2.32, respectively, which showed statistically significant results (P < 0.001). The mean PDs in the test and control groups at baseline were 5.16 and 4.44, respectively, and after 3 months were 2.92 and 4.10, respectively, which showed statistically significant results (P < 0.001). The mean CALs in the test and control groups at baseline were 1.03 and 0.61, respectively, and after 3 months were 0.30 and 0.41, respectively, which showed statistically significant results (P < 0.001) [Table 1]. The intergroup comparison between the groups results showed that the mean GIs in the test and control groups at baseline were 2.60 and 2.57, respectively, which were nonsignificant, and after 3 months were 1.35 and 2.32, respectively, which showed statistically significant results (P < 0.001). The mean PDs in the test and control groups at baseline were 5.16 and 4.44, respectively, and after 3 months were 2.92 and 4.10, respectively, which showed statistically significant results (P < 0.001). The mean CALs in the test and control groups at baseline were 1.03 and 0.61, respectively, which showed statistically significant results (P < 0.045) and after 3 months were 0.30 and 0.41, respectively, which were statistically nonsignificant [Table 2]. The clinical parameter difference in both the test and control groups from baseline to 3 months showed that the mean GIs in the test and control groups were 1.25 and 0.25, respectively, the mean PDs in the test and control groups were 2.24 and 0.34, respectively, and the mean CALs in the test and control groups were 0.73 and 0.20, respectively, which showed statistically significant results (P < 0.001) [Table 3]. BODY.DISCUSSION: According to several studies, mechanical debridement alone cannot completely eliminate all periodontopathogenic bacteria from the subgingival environment. In cases with the presence of bacteria in inaccessible areas such as furcation area, root concavities, and deep periodontal pockets, these local delivery of antimicrobial agents can work effectively.[9] The major advantage of using local drug delivery at the target site includes achieving high intrasulcular concentration.[10] Various herbal agents have been used for the management of periodontal diseases. As per our knowledge, none of the clinical studies were reported in the literature which evaluated the efficacy of the local drug delivery of A. aspera plant extract for the management of periodontal diseases.[11] A. aspera is a medicinal plant and many traditional healers used it for the management of fever, asthma, hypertension, dysentery, diabetes, cough, bronchitis, and rheumatism.[12] As per Ayurveda, it is bitter, pungent, heating, and laxative and used for the management of vomiting, bronchitis, heart disease, piles, itching abdominal pains, dyspepsia, dysentery, and blood diseases. Roots of A. aspera contain alkaloids, flavonoids, saponins, steroids, terpenoids, and ecdysterone.[13] The success of any periodontal treatment is measured using two important clinical parameter values such as decrease in PPD and gain in CAL. In the present study, PPD was decreased in both the groups, where Group A (test) showed a decrease from 5.96 to 2.92 mm and Group B (control) from 4.44 to 4.10 mm at the end of 3 months. Comparing PPD in both the groups revealed statistically significant results. CAL evaluated in both the groups showed a gain from 1.03 to 0.30 mm in Group A and from 0.61 to 0.41 mm in Group B. On comparison, Group A showed a greater gain in CAL after 3 months. The variation in the clinical parameters of the present study are due to the soft-tissue wall changes caused by the resolution of gingival inflammation.[14] The antioxidant property of A. aspera results in terminating the radical chain reaction by inhibiting free-radical quenching and high levels of phenolic compounds in the ethyl acetate extracts which may have partly contributed to the antioxidant activity.[15] Anand et al.[13] reported that extracts of the plant showed potent antioxidant activities. Edwin et al.[16] reported the antioxidant properties of the ethanolic and aqueous extracts of plant, which were assessed using 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and superoxide scavenging activity. Kumar et al.[17] reported the analgesic activity of the hydroalcoholic extract from roots and leaves of A. aspera in adult male albino rats, using doses of 200 and 400 mg/kg. The maximum analgesic activity was seen at a dose of 400 mg/kg. Kumar et al.[18] reported the anti-inflammatory activity of alcoholic extract from roots of A. aspera in Wister rats. Phosphate-buffered saline extract of A. aspera had displayed immunostimulant action as stated by the promotion of T-lymphocyte proliferative responses.[19] Therapeutic approaches with herbal medicine are often alternative due to lack of data with meticulous clinical trial evidence. It is recommended that more researches should be undertaken.[20] BODY.CONCLUSIONS: A. aspera gel as a nonsurgical local drug delivery system in periodontal disease patients after thorough SRP has shown acceptable results with good prognosis without any side effects. A. aspera gel has various pharmacological properties with strong anti-inflammatory effects and its antioxidant activity. However, further studies with large sample size are required to evaluate the efficacy of A. aspera gel in the management of periodontal disease patients. BODY.CONCLUSIONS.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONCLUSIONS.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Effects of Vitamin D Intake on FEV1 and COPD Exacerbation: A Randomized Clinical Trial Study ABSTRACT.AIM:: This study aimed to evaluate the effects of vitamin D intake on COPD exacerbation and FEV1 in the patients with severe and very severe COPD. ABSTRACT.METHODS:: This double blind placebo control randomized clinical trial study was done in the Ashayer university hospital in Khorramabad in 2012. Eighty eight patients with severe and very severe COPD were randomly selected from those who recoursed to the internal medicine clinic of Ashayer hospital. They were randomly allocated to case and placebo group. The patients received routine treatment for COPD. Along with the routine treatment, placebo group received 100,000 IU of oral vitamin D per month, for 6 months. Data was analyzed using SPSS computer software, paired t-test, independent t-test, non parametric t-test and Pearson correlation coefficients. ABSTRACT.RESULTS:: In each group, there were 44 patients. After the intervention, there were significant differences in FEV1 and the number of COPD exacerbation between the case and control group patients. Also, after the study, in the case group, FEV1 was increased and the number of COPD exacerbation was decreased significantly. ABSTRACT.CONCLUSION:: Vitamin D intake decreased COPD exacerbation and improved FEV1 in the patients with severe and very severe COPD. It is suggested that baseline serum vitamin D levels will recorded in similar studies and the effect of vitamin D intake will evaluated regarding the baseline serum vitamin D levels. BODY.1. INTRODUCTION: Chronic Obstructive Pulmonary Disease (COPD) is a chronic disease that causes persistent airflow obstruction. The airflow obstruction in this disease is generally progressive (MacNee et al., 2005). COPD has two clinical phases (stable phase and exacerbation phase), both of which are associated with inflammation (Barbu et al., 2011). Smoking, passive smoking, reactivity of airways, occupational factors and air pollution are the risk factors of COPD (Reilly et al., 2004). Independent risk factors for COPD are male gender, advanced age, low socioeconomic status, occupational exposure and cigarette smoking (Caballero et al., 2008). Based on the World Health Organization estimation, COPD will be the third cause of mortality in the world in 2020 (Murray et al., 1997). Ninety percent of COPD deaths occur in low and middle income countries (Murray et al., 1997). In European countries, depending on the age of participants, the methods used and the location, the prevalence of COPD ranged from 2.1% to 26.1% (Atsou et al., 2011). It was 8.9% in India (from 6.2% to 13.5%; based on spirometry) (Afonso et al., 2011), 3.02% in the Netherlands (in a population-based study including subjects ≥ 40) (8), 17.4% in Copenhagen (aged 35 years or older) (Fabricius et al., 2011) and 3.7% in Abu Dhabi (in 40-80 year old subjects) (Al Zaabi et al., 2011). Nowadays, the attention to nonskeletal effects of vitamin D has been increased (Kunisaki et al., 2011). An association between pulmonary function and serum vitamin D levels has been reported in some studies. It has been reported that vitamin D deficiency correlates with severity of COPD (Janssens et al., 2010). Also, in some studies, it has been declared that COPD patients had a raised risk for vitamin D deficiency (Persson et al., 2012, Zhang et al., 2012). Likewise, in one study, it has been stated that total vitamin D intake was negatively associated with COPD (Shaheen et al., 2011). According to previous studies, the effect of levels vitamin D is controversial on COPD exacerbation and FEV1. This study aimed to evaluate the effect of vitamin D intake on COPD exacerbation and FEV1 in the patients with severe and very severe COPD. BODY.2. METHOD: This double-blind, placebo-controlled, randomized clinical trial was done in Ashayer university hospital in Khorramabad in 2012. The Ethics and Research Committee of Lorestan University of Medical Sciences approved this study. Furthermore, we obtained signed informed consents from all the participants. This study has been recorded in Iranian Registry of Clinical Trials at www.irct.ir as a clinical trial (IRCT2012071810332N1). Eighty-eight patients with severe and very severe COPD were randomly selected from those who recoursed to the internal medicine clinic of Ashayer hospital. Severe and very severe COPD were defined as Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines (Donaldson et al., 2002). The selected patients were allocated to the study and placebo groups by simple random sampling method. The patients in both the groups received the routine treatment for COPD. Along with the routine treatment, the study group received 100,000 IU of oral vitamin D per month, for 6 months. In contrast, the placebo group received oral placebo for 6 months. Before the study, forced expiratory volume in 1 second (FEV1) was determined, and the number of COPD exacerbations during the last 6 months was recorded in the both groups. After 6 months of treatment, FEV1 was determined, and the number of COPD exacerbations during the study span was evaluated in the both groups. The patients received a telephone call every 2 months to assess respiratory symptoms consistent with a COPD exacerbation. The definition of COPD exacerbation was either the presence of 2 or more of these major symptoms (increase in sputum purulence, sputum volume or dyspnea) or any of major symptoms accompanied by any of minor symptoms (increase in nasal discharge, wheeze, sore throat, cough or fever) for at least two consecutive days (Donaldson et al., 2002). BODY.2. METHOD.2.1 STATISTICS: The data was analyzed using the SPSS computer software and paired t-test, independent t-test, non-parametric t-test and Pearson correlation coefficients. The P-values<0.05 were considered statistically significant. BODY.3. RESULTS: In each group, there were 44 patients, 30 of whom (68.2%) were male. There were no significant differences between age distribution, cigarette smoking and addiction in the study and placebo groups (Table 1). Table 1 Age distribution, cigarette smoking and addiction in case and placebo groups Case group Number (%) placebo group Number (%) p value Age (Year) <45 2 (4.5%) 1(2.3%) 0.83 45-60 15 (34.1%) 16 (36.4%) >60 27 (61.4%) 27 (61.4%) Cigarette smoking (cigarette per month) None 11 (25%) 13 (29.5%) 0.22 < 50 26 (59.1%) 23 (52.3%) > 50 7 (15.9%) 6 (13.6%) Addiction None 17 (38.6%) 18 (40.9%) 0.53 Oral use 9 (20.4%) 8 (18.2%) Inhalation use 16 (36.4%) 18 (40.9%) Injection use 2 (4.5%) 0 The mean of FEV1 and COPD exacerbations according to sex, age distribution, cigarette smoking and addiction, before and after the study, are shown in Table 2 and Table 3. Table 2 The mean of FEV1 according to sex, age distribution, cigarette smoking and addiction, before and after the study Case placebo Before After p value Before After p value Sex Male 33.6±7.7 51.2±5.9 0.0001 33.6±4.6 33.1±9.7 0.44 Female 36.8±10 52.5±14.6 0.009 35±8.6 34.2±4.6 0.32 p value 0.3 0.76 - 0.17 0.59 - Age (Year) <45 39±1.4 55±4.2 - 35±0 32 - 45-60 36.8±8 51±13.9 0.004 35.6±9.4 34.2±8.4 0.062 >60 33.1±8.9 51.7±6.4 0.0001 34.5±9.2 34.8±7.7 0.13 p value 0.3 0.85 - 0.88 0.53 - Cigarettesmoking (cigarette/month) None 35.2±8.4 49.3±15.6 0.047 38.7±9.3 34.1±6.7 0.018 < 50 39.5±3.6 55.7±5.1 <0.0001 38.7±4.4 35.7±5.4 0.011 > 50 33.07±9.1 51.5±6.4 <0.0001 32.1±9.2 31.1±8.7 0.32 p value 0.19 0.38 - 0.063 0.31 - Addiction None 37±8.6 51.1±13.3 0.003 38.3±6.2 34.4±6.1 0.001 Oral use 37.1±7.6 54.6±5.1 <0.0001 36.1±11.7 30±9.2 0.015 Inhalation use 32.1±7.8 51.6±6.7 <0.0001 32.6±9.7 30.2±8 0.078 Injection use 34±9.8 42.5±3.5 - - - - p value 0.187 0.42 - 0.164 0.23 - Table 3 The mean of COPD exacerbation according to sex, age distribution, cigarette smoking and addiction, before and after the study Case Control Before After p value Before After p value Sex Male 18.8±3.5 9.8±1.3 0.0001 19.3±4.2 19.6±3.9 0.056 Female 16.3±2.4 9.3±1.3 0.0023 18.1±2.5 17.7±2.5 0.73 p value 0.095 0.25 - 0.23 0.053 - Age (Year) <45 19±1.4 10 - 18±0 18 - 45-60 16.8±3.3 9.5±1.1 0.0001 18.8±1.4 17.8±1.6 0.79 >60 18.2±2.8 9.7±1.5 <0.0001 19.4±2.9 19.7±2.9 0.64 p value 0.28 0.82 - 0.66 0.069 - Cigarette smoking (cigarette/month) None 17.1±1.9 8.8±1.3 <0.0001 17.3±2.6 17.1±2.3 0.65 < 50 18.5±2.2 9.8±1.4 <0.0001 19.2±3.3 19.2±2.8 0.38 > 50 19.1±3.4 10±1.2 <0.0001 19.1±4.4 19.4±4.1 0.89 p value 0.185 0.038 - 0.42 0.2 - Addiction None 16.1±2.8 9.2±1.2 <0.0001 17.5±3.1 17.8±3.5 0.42 Oral use 20.4±4.7 10±1.8 <0.0001 20±3.7 20.2±3.9 0.56 Inhalation use 18.9±1.7 10±1.1 <0.0001 19.2±4.4 19.2±3.4 0.99 Injection use 15±2.1 10±1.4 - - - - p value 0.023 0.35 - 0.24 0.24 - Before the study, there were no significant differences in FEV1 and the number of COPD exacerbations between the study and placebo group patients. But, after the study, there were significant differences in FEV1 and the number of COPD exacerbations between the study and placebo group patients. Also, after the study, in the study group, FEV1 was increased and the number of COPD exacerbations was decreased significantly (Table 4). Table 4 FEV1 and the number of COPD exacerbation in the case and placebo groups Case Control p value FEV1 (M±SD) Before 34.6±8.5 34.4±9.2 0.89 After 51.6±9.4 31.9±7.6 <0.001 p value <0.001 0.53 - COPD exacerbation (M±SD) Before 18.02±3.3 18.7±3.8 0.38 After 9.7±1.3 18.8±3.6 <0.001 p value <0.001 0.83 - BODY.4. DISCUSSION: COPD is a chronic and common disease. COPD can cause severe complications. Afonso et al., reported that 26% and 2.8% of the patients with very severe COPD and non-COPD patients had died after 1 year of follow-up in the Netherlands (Afonso et al., 2011). An association between pulmonary function and serum vitamin D levels has been reported in some studies. It has been reported that vitamin D deficiency correlates with the severity of COPD (Janssens et al., 2010). Also, it has been reported that a significant relation between FEV1 and serum 25-hydroxy vitamin D levels (Azargoon et al., 2011). However, in a study, baseline 25-hydroxy vitamin D levels were not predictive of acute exacerbation in patients with severe COPD (Kunisaki et al., 2012). But the relationship between vitamin D and COPD has been reported in some studies. Also, it has been stated that total vitamin D intake was negatively associated with COPD (Shaheen et al., 2011). Regarding these results, vitamin D intake can be beneficial in COPD patients. This study aimed to evaluate the effect of vitamin D intake on COPD exacerbation and FEV1 in the patients with severe and very severe COPD. According to our knowledge, the effect of vitamin D on FEV1 and COPD exacerbations has been studied in few studies. In this study, vitamin D intake improved COPD exacerbations and FEV1 in the patients with severe and very severe COPD. Hornikx et al., reported that 100.000 IU of vitamin D per month for one year had improved maximal oxygen uptake and inspiratory muscle strength significantly in the COPD subjects who had followed a rehabilitation program (Hornikx et al., 2012). These findings can vindicate the results of our study. In a similar study, Lehouck et al., compared the effects of vitamin D and placebo on FEV1 and exacerbation rate in the patients with moderate to very severe COPD (Lehouck et al., 2012). In their study, each patient received 100,000 IU of vitamin D every 4 weeks for 1 year. In contrast to our results, they reported that this dose of vitamin D had not improved FEV1 and exacerbation rate. This difference may be due to the difference between baseline serum vitamin D levels in these studies. Although serum vitamin D level was not determined in our study, some studies have stated that vitamin D deficiency is prevalent in Iran. Studies suggest that Vitamin D increase production IL-10, an antiinflammatory cytokine involved in the pathogenesis of asthma, from T cells, increase production IL-37, antimicrobial peptide, regulate matrix metalloproteinases (MMP), shifting theTh1 and Th2 balance and reducing inflammation (Xystrakis et al., 2006, De Smet et al., 2005; Finklea et al., 2011). Taken together vitamin D intake (100,000 IU per 4 weeks for 6 months) improved COPD exacerbation and FEV1 in the patients with severe and very severe COPD significantly. It is suggested that baseline serum vitamin D levels be recorded in similar studies and the effect of vitamin D intake be evaluated regarding the baseline serum vitamin D levels.

TITLE: Stenting versus aggressive medical therapy for intracranial arterial stenosis: more harm than good ABSTRACT.EXPANDED ABSTRACT.CITATION: Chimowitz MI, Lynn MJ, Derdeyn CP, Turan TN, Fiorella D, Lane BF, Janis LS, Lutsep HL, Barnwell SL, Waters MF, Hoh BL, Hourihane JM, Levy EI, Alexandrov AV, Harrigan MR, Chiu D, Klucznik RP, Clark JM, McDougall CG, Johnson MD, Pride GL Jr, Torbey MT, Zaidat OO, Rumboldt Z, Cloft HJ, for the SAMMPRIS Trial Investigators. N Engl J Med 2011, 365:993-1003. PubMed PMID: 21899409. This is available on http://www.pubmed.gov. ABSTRACT.EXPANDED ABSTRACT.BACKGROUND: Atherosclerotic intracranial arterial stenosis is an important cause of stroke that is increasingly being treated with percutaneous transluminal angioplasty and stenting (PTAS) to prevent recurrent stroke. However, PTAS has not been compared with medical management in a randomized trial. ABSTRACT.EXPANDED ABSTRACT.METHODS: Objective: To determine whether intracranial stenting (using the Wingspan self-expanding nitinol stent, Boston Scientific) and intensive medical therapy is superior to intensive medical therapy alone for preventing stroke in recently symptomatic patients with severe intracranial atherosclerotic stenosis. Design: Phase III, multi-center, randomized, open label, clinical trial. Setting: 50 sites in the US Subjects: Patients who had a recent transient ischemic attack or stroke attributed to stenosis of 70 to 99% of the diameter of a major intracranial artery. Intervention: Eligible patients were randomized to receive either aggressive medical medical management alone or aggressive medical management plus PTAS with the use of the Wingspan stent system. Outcomes: The primary end point was stroke or death within 30 days after enrollment or after a revascularization procedure for the qualifying lesion during the follow-up period or stroke in the territory of the qualifying artery beyond 30 days. Patients in the medical arm who undergo angioplasty for recurrent TIAs (i.e. crossovers) and who have a stroke or death within 30 days will also meet this endpoint. ABSTRACT.EXPANDED ABSTRACT.RESULTS: Of the 451 patients who underwent randomization, 227 were assigned to the medical management group and 224 to the PTAS group. The 30-day rate of stroke or death was 14.7% in the PTAS group (nonfatal stroke, 12.5%; fatal stroke, 2.2%) and 5.8% in the medical-management group (nonfatal stroke, 5.3%; non-stroke-related death, 0.4%) (P = 0.002). Beyond 30 days, stroke in the same territory occurred in 13 patients in each group. The probability of the occurrence of a primary end-point event over time differed significantly between the two treatment groups (P = 0.009), with 1-year rates of the primary end point of 20.0% in the PTAS group and 12.2% in the medical-management group. ABSTRACT.EXPANDED ABSTRACT.CONCLUSIONS: In patients with intracranial arterial stenosis, aggressive medical management was superior to PTAS with the use of the Wingspan stent system, both because the risk of early stroke after PTAS was high and because the risk of stroke with aggressive medical therapy alone was lower than expected. BODY.COMMENTARY: Stroke is a major public health problem as it is a leading cause of death and long-term disability in the United States. There are approximately 795,000 incident strokes each year, resulting in more than 140,000 deaths annually [1]. In 2010, the cost of stroke care was estimated at $74 billion, with a mean lifetime cost of $140,000 per patient [2]. Atherosclerotic disease of the major intracranial arteries is a frequent cause of ischemic stroke with annual risk as high as 24% [3]. Patients with severe intracranial arterial stenosis (70% to 99%) are at high risk of stroke in the territory of the stenotic artery [4], which is potentially amenable to intracranial angioplasty or stenting. Current primary prevention strategies include a combination of life style modification (smoking cessation, dietary intervention, weight loss, and exercise), antihypertensive medications, antithrombotic therapy, and statins [5]. Recommended secondary prevention includes a combination of medical therapy and revascularization [6]. Tremendous advances have been made in cerebral revascularization techniques in recent years, which include percutaneous transluminal angioplasty (PTA) alone, PTA with stenting (PTAS) using balloon-mounted coronary stents, and extracranial-intracranial surgical bypass. However, unlike extracranial carotid disease, the long term efficacy and safety of revascularization strategies of intracranial atherosclerotic disease have not been well established. Several retrospective case series suggest that PTA alone is a safe and effective treatment strategy for intracranial atherosclerotic disease [7]. However, PTA has several technical drawbacks including immediate elastic recoil of the artery and high restenosis rates [8]. Surgical bypass was shown to be inferior to medical therapy for middle cerebral artery stenosis [9]. While some studies reported that PTAS with balloon-mounted coronary stents was safe and effective [10], others showed high rates of periprocedural morbidity (23%) and mortality (5%) [11]. The Wingspan, a flexible, self-expanding, is the first stent designed specifically for the treatment of symptomatic intracranial atherosclerotic disease. The high flexibility of these stents makes them suitable for treatment of lesions of the distal internal carotid artery and middle cerebral artery that are either inaccessible or difficult to reach with a balloon-mounted coronary stent. The Stenting and Aggressive Medical Management for Preventing Recurrent stroke in Intracranial Stenosis (SAMMPRIS) trial is the first prospective randomized trial in patients' with symptomatic intracranial atherosclerotic disease to compare the Wingspan stent and aggressive medical therapy with aggressive medical therapy alone. Aggressive medical therapy included aspirin 325 mg per day for entire follow-up, clopidogrel 75 mg per day for 90 days after enrollment, and aggressive risk factor management primarily targeting blood pressure <140/90 mmHg (<130/80 if diabetic) and LDL <70 mg/dl. However, the Data Safety Monitoring Board halted the trial after enrollment of 451 (59%) of the planned 764 participants because of the significant stroke and mortality rates observed in the stent arm. The SAMMPRIS is a well-conducted, multicenter, controlled trial that has provided strong scientific evidence of the superiority of medical therapy to PTAS. The two-fold increased risk of stroke with stent clearly indicates that stenting should not be used for stroke prevention in high-risk individuals. Of particular concern is the significant number of periprocedural intracranial hemorrhages. Unlike coronary vasculature, intracranial vessels are more challenging to stent because they are difficult to navigate due to their very tortuous course and sharp angles, and those vessels are delicate and more susceptible to injury due to lack of supporting connective tissue. Based on the current study, the authors recommend adding clopidogrel to aspirin for the first 90 days following stroke in patients with high grade intracranial stenosis. However, it has to be pointed out that the control arm does not represent the current standard of care for stroke prevention as addition of clopidogrel to aspirin has been shown to increase the risk of hemorrhage [12]. The main reason for adding clopidogrel to aspirin in the control arm of SAMMPRIS trial was to balance the antithrombotic regimen in order to establish the superiority of the stent. The study did not include an aspirin monotherapy arm to compare the result of aspirin monotherapy with dual antiplatelet (aspirin and clopidogrel) therapy. Therefore it is unclear whether a combination of aspirin and clopidogrel is superior to aspirin monotherapy, combination of aspirin and extended-release dipyridamole, and clopidogrel monotherapy, all of which are acceptable options for secondary prevention after ischemic stroke or TIA [6]. Two ongoing trials, Vitesse Intracranial Stent Study for Ischemic Therapy (VISSIT), which is comparing the use of the balloon-mounted stents to aspirin and clopidogrel, and Interventional Management of Stroke III (IMS III) trial, which is evaluating different revascularization strategies including clot retrieval devices, should hopefully provide more insight towards optimal strategy for stroke prevention. BODY.RECOMMENDATION: Treatment with the Wingspan stent system for patients with intracranial stenosis carries substantial risk of death and stroke and should be abandoned. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests.

TITLE: Treatment of Postoperative Pain in Pediatric Operations: Comparing the Efficiency of Bupivacaine, Bupivacaine-Dexmedetomidine and Bupivacaine-Fentanyl for Caudal Block ABSTRACT.BACKGROUND: Caudal analgesia is a common method for postoperative pain management in pediatric patients. Additive agents such as opioids and α2 agonists have been used to enhance the analgesic effects of local anesthetics for caudal block. ABSTRACT.OBJECTIVES: The aim of this study was to compare the additive effects of dexmedetomidine and fentanyl on bupivacaine-induced caudal analgesia in pediatric patients who had undergone elective inguinal hernia repair. ABSTRACT.METHODS: This randomized, double-blind clinical trial included children aged 1 - 5 years who were divided into three groups: the bupivacaine group (Group B) received 0.25% bupivacaine (1 ml/kg), the bupivacaine-dexmedetomidine group (Group BD) received 0.25% bupivacaine (1 mL/kg) plus 2 μg/kg dexmedetomidine, and the bupivacaine-fentanyl group (Group BF) received 0.25% bupivacaine (1 mL/kg) plus 2 μg/kg fentanyl. The hemodynamic variables (heart rate, systolic blood pressure, respiratory rate, and peripheral arterial oxygen saturation) were measured perioperatively. Pain, sedation and motor block scores and adverse events (nausea and vomiting, pruritis, hypotension, bradycardia, urinary retention and respiratory depression) were documented at 30 and 60 minutes, and the 1st, 2nd, 4th, 6th, 12th and 24th hours after the operation. The other recordings include the duration of surgery and analgesic requirement. ABSTRACT.RESULTS: A total of 61 patients were analyzed. The lowest pain scores were found in the BD group at all time points (P < 0.001). The sedation scores were higher in the BD group than in the other two groups at all time points (P < 0.001). No motor block was observed after the operation. Only three patients required analgesic administration 2 to 6 hours after the operation in group B. No side effects were observed in any of the groups, and there was no significant difference in the duration of surgery among the three groups. ABSTRACT.CONCLUSIONS: The results show that the analgesic and sedative effects were better when dexmedetomidine was added to bupivacaine than when fentanyl was added or bupivacaine alone was administered in the pediatric population studied here that underwent elective inguinal hernia repair. BODY.1. BACKGROUND: If acute pain is left untreated or not treated properly, it can progress to chronic pain. There is a large amount of evidence for the use of multi-modal approaches to counteract pain in the pediatric population (1). Caudal analgesia has been widely used as a pain management modality in a variety of pediatric operations (2). Bupivacaine is a long-acting reliable local anesthetic agent that is used as a caudal analgesic, but different auxiliary agents need to be co-administered to improve its analgesic efficient (3, 4). Single shots of a combination of local anesthetics, such as ketamine, midazolam, neostigmine, adrenaline, opioids, clonidine and, recently, dexmedetomidine, have been used (5). Opioids are effective analgesic agents, but they are associated with respiratory depression, itching, urinary bladder dysfunction, nausea and vomiting (6). Clonidine and dexmedetomidine are both α2 adrenergic receptor agonists, but dexmedetomidine has eight times stronger receptor affinity than clonidine. Dexmedetomidine has sedative, hypnotic, anxiolytic, analgesic, anesthetic-sparing and sympatholytic effects and does not have any adverse effects on respiratory or cardiovascular functions (7). BODY.2. OBJECTIVES: The aim of this study was to compare the postoperative analgesic effects of bupivacaine alone with the additive effects of fentanyl and dexmedetomidine on bupivacaine-induced caudal analgesia. BODY.3. METHODS: This randomized, double-blind clinical trial included children aged 1 to 5 years who were admitted to the operating room for elective inguinal hernia repair. According to the tenets of the Declaration of Helsinki, the written informed consent of their parents was obtained prior to the intervention. One-sided analysis of variance of the mean values from a previous study was used to calculate the sample size for the present study (8), with α = 0.05, β = 0.1, σ = √MSE = 1/1, μ1 = 2.5, μ2 = 2.5, and μ3 = 1.25. The calculated λ resulted in a non-central distribution with χ2 equal to 12.66 and the number of cases in each group (Δ) equal to 16. The number of patients who were likely to drop out was calculated to be 20 in each group (NCSS software). The study was a double-blind randomized one (Random Allocation Software, version 1.0.0). A total of 81 patients were enrolled in the study and 20 were excluded (the flow chart for patient selection is shown in Figure 1). Children with cardiopulmonary congenital anomalies, a history of drug allergy to the drugs used, contraindications for caudal block, and ASA class > I were excluded. Figure 1.CONSORT Flow Diagram No premedication was administered to the children. Anesthesia was induced through inhalation of 50% oxygen/50% nitrous oxide and sevoflurane (7% every four successive breaths). This was followed by intravenous administration of atracurium (0.5 mg/kg) and endotracheal intubation. The child was placed in the lateral position, and the caudal area was prepared and sterilized. A 23-gauge needle was inserted in the sacral hiatus area by an experienced anesthesiologist. The children were randomly assigned to one of the following groups: the bupivacaine group (Group B), in which 1 mL/kg of 0.25% bupivacaine was administered (Marcaine Spinal 0.5% Heavy, AstraZeneca, Horizon Place, 600 Capability Green, Luton, Bedfordshire, LU1 3LU); the bupivacaine-dexmedetomidine group (Group BD), in which 1 mL/kg of 0.25% bupivacaine and 2 μg/kg dexmedetomidine were administered (Precedex®; Hospira, Lake Forest, Illinois, USA); and the bupivacaine-fentanyl group (Group BF), in which 1 mL/kg of 0.25% bupivacaine and 2 μg/kg fentanyl were administered (fentanyl citrate; Caspian Tamin Pharmaceutical Co., Rasht-Iran). All solutions were prepared from 0.5% bupivacaine to which distilled water was added to prepare the 0.25% solution. Anesthesia was maintained with sevoflurane (MAC 2.0% - 2.6%, age adjusted) throughout the operation. Data were collected by an anesthesiology resident, who was blinded to the type of caudal analgesic solution administered. The hemodynamic indices (heart rate (HR) and systolic blood pressure) were maintained in the 20% pre-induction range. If the value of the indices exceeded this range, the analgesics administered were considered to be insufficient and an intravenous dose of 1 μg/kg fentanyl was administered. Moreover, the patient was excluded from the study. The hemodynamic variables (HR, systolic blood pressure (SBP), respiratory rate, and peripheral arterial oxygen saturation) were measured before and every 5, 10 and 15 minutes after the analgesics were administered; every 5 minutes during the operation; and every 15 minutes in the recovery room. Ringer's solution was administered at a dosage of 4 - 6 mL × kg-1 × h-1 perioperatively. On completion of the surgery and reversal of the effects of the neuromuscular blocking agents, the patients were evaluated in the recovery room and surgical ward (for 24 hours) by the anesthesiology resident in charge of the study. The scores for pain, sedation and motor block were determined at 30 and 60 minutes and the 1st, 2nd, 4th, 6th, 12th and 24th h after the operation. The Persian version of the face pain scale-revised (FPS-R) tool, scored on a scale of 0, 2, 4, 6, 8, and 10, was used to measure pain (9). If the pain score was equal to or more than 4, a rescue analgesic agent (meperidine, 1 mg/kg) was administered. The level of sedation was measured at the same time points as mentioned above with the University of Michigan sedation scale (UMSS) (0 = awake and conscious, 1 = mild sedation, 2 = moderate sedation, 3 = deep sedation, 4 = no response) (10). The level of motor block was assessed by the modified Bromage score (MBS) with 0 = free movement, 1 = only able to bend the knees and move the feet, 2 = unable to bend the knees but moves feet, and 3 = unable to move the feet (11). The patients were also assessed at the above time points for adverse events such as nausea and vomiting, pruritis, hypotension (SBP < 70 + age × 2), bradycardia (HR < 100/min for 1-year-old patients, and HR < 95/minutes for 2- to 5-year-old patients), urinary retention (inability to voluntarily empty the bladder for more than 12 hours, with urine volume greater than that expected for the patient age ((age in years + 2) × 30 cc), or a palpably distended bladder) and respiratory depression (respiratory rate less than 10). The postoperative recordings included the duration of surgery, time of first analgesia administration, and occurrence and treatment of postoperative nausea and vomiting (PONV) and pruritus. PONV and pruritis were treated by intravenous administration of 0.1 mg/kg ondansetron in the form of a slow bolus injection. BODY.3. METHODS.3.1. STATISTICAL ANALYSIS: The Kolmogorov-Smirnov Z test was used to analyze the equality of the distributive functions of the variables. A one-way analysis of variance (ANOVA) and a generalized linear model repeated-measure ANOVA were used to analyze the parametric variables. For non-parametric variables (pain sedation scores and duration of surgery), the Kruskal-Wallis test, Friedman Test and chi-square test were applied. Later, Bonferroni post-hoc analysis was used to analyze the difference between groups. P values < 0.05 were considered to indicate statistical significance. BODY.3. METHODS.3.2. ETHICS STATEMENT: This study was approved by the deputy for research and medical ethics committee of Hormozgan University of Medical Sciences (4-HEC-93-7-8) and registered with the Iranian registry of clinical trials (IRCT2015110318091N6). BODY.4. RESULTS: A total of 61 patients were analyzed, of whom 56 (91.8%) were boys and 5 (8.2%) were girls. However, no significant relationship was found between gender and the groups in the study. The other demographic data (age, height and weight) were not significantly different between the groups (Table 1). Table 1. Demographic Data a , b Group B Group BD Group BF Degree of Freedom P Value ASA I/II 18/2 0/20 0/21 - 0.12 No. of boys 20 18 18 2 0.23 No. of girls 0 2 3 Age, mo 24 36 36 46 0.21 Height, cm 84.05 ± 13.61 92.80 ± 12.82 87.00 ± 10.97 2 0.09 Weight, kg 10.80 ± 3.03 12.15 ± 2.34 12.95 ± 3.77 3 0.09 Duration of surgery, min 33.70 ± 12.98 33.00 ± 11.96 41.65 ± 11.79 F test 8.685 0.054 a Values are expressed as mean ± SD. b Group B, Bupivacaine group; Group BD, Bupivacaine-dexmedetomidine; Group BF, Bupivacaine-fentanyl. BODY.4. RESULTS.4.1. PAIN: There was no significant difference in the mean postoperative pain scores between different time points in each group. However, the mean pain scores were significantly different between the three groups at all time points (P < 0.001), with the subsequent Bonferroni test indicating a significant difference between group B and BD (P < 0.001) and between group B and BF (P < 0.001). The lowest mean scores were observed in the BD group. In the 1st, 2nd and 4th h after the operation, the mean pain scores were significantly lower in the BD group than in the BF group (P < 0.001). However, in the following hours (6th, 12th and 24th), the mean pain scores in the BD group were lower but not significantly different from those in the BF group (Figure 2). Figure 2.Pain Scores of the Three Groups (Bupivacaine, Bupivacaine-Dexmedetomidine and Bupivacaine-Fentanyl) After the Operation (Recovery and Ward Period) BODY.4. RESULTS.4.2. SEDATION: There was a significant difference in the mean sedation scores between different postoperative time points in each group. There was also a significant difference between the groups with regard to the mean sedation scores (P < 0.001) at all time points, with the subsequent post-hoc analysis revealing a significant difference in the mean sedation scores between group B and BD (P < 0.001), group B and BF (0.003 ≤ P ≤ 0.049) and group BD and BF (< 0.001 ≤ P ≤ 0.034). The lowest scores were observed in group B (0.35 ± 0.49) and the highest were observed in group BD (1.65 ± 0.67), whereas the scores in the BF group were intermediate (0.95 ± 0.22) at the 30-min recovery time. The mean sedation scores were not significantly different between group BD and BF only at the postoperative 60-min, 1-h and 24-h time points (Figure 3). Figure 3.Sedation Scores of the Three Groups (Bupivacaine, Bupivacaine-Dexmedetomidine and Bupivacaine-Fentanyl) After the Operation (Recovery and Ward Period) BODY.4. RESULTS.4.3. MOTOR BLOCK: Motor block was not detected in any of the patients after the operation. BODY.4. RESULTS.4.4. ANALGESIC REQUIREMENT: The incidence of pain was generally low in patients of both groups, except for three patients aged between 12 and 19 months in the bupivacaine group: two 1-year-old children and one 1.5-year-old child. In the 1-year-old children, the first episode of pain occurred at 6 hours after caudal block initiation, and in the 1.5-year-old child, it occurred at 2 hours after caudal block initiation. BODY.4. RESULTS.4.5. SIDE EFFECTS: No side effects, such as nausea and vomiting, respiratory depression, pruritis, bradycardia, hypotension, and urinary retention, occurred in any of the study groups. BODY.4. RESULTS.4.6. DURATION OF SURGERY: The duration of surgery was longer in the BF group, but it was not significantly different from that in the other groups (Table 1). BODY.5. DISCUSSION: The use of caudal analgesics is popular in pediatric operations (12); however, a single shot of one analgesic agent alone may not be sufficient to induce prolonged analgesia, so many multiple modalities have been introduced. The use of caudal catheters in children may affect postoperative mobility or carry the risk of an infection (13). Therefore, it would be beneficial to use a multimodal method with a block solution that has prolonged analgesic effects and does not have any adverse effects on the perioperative vital conditions of the patient. The aim of our study was to determine the effect of adding other analgesic agents (dexmedetomidine and fentanyl) to a bupivacaine solution for caudal block. The results revealed a significant difference in the efficacy of the drug solution containing additives and bupivacaine alone. An ideal sedative agent is a one that has the least cardiovascular (e.g., hypotension) and respiratory (e.g., apnea) side effects. In this study, the effects of administering dexmedetomidine and fentanyl as additional sedatives along with bupivacaine were studied. Dexmedetomidine has a dual effect as an α2 adrenergic agonist and an α1 adrenergic antagonist that acts on the arterial vascular system (7). α2 adrenergic stimulation of the brain and spinal cord is associated with sedative, analgesic, anxiolytic and sympatholytic effects. Unlike other sedative agents, the effects of dexmedetomidine can be easily reversed with slight stimulation and no untoward effects on respiratory functions. Further, it has been reported that dexmedetomidine does not cause respiratory depression even at high doses (7). Fentanyl belongs to the same group of opioids as phenyl piperidine; it is a potent short-acting opioid that affects different opioid receptors and may have dose-related side effects such as respiratory depression, pruritis and nausea and vomiting (6). The analgesic effect of α2 agonists is unique as they act on peripheral tissues as well as the brain, brainstem and spinal cord. The locus coeruleus is a pivotal supraspinal site of action for α2-adrenergic and opioid agents. The effect of these agents on the spinal cord is brought about via activation of the descending medullospinal noradrenergic pathway and presynaptic ganglionic block, which attenuates spinal sympathetic outflow (14). In a study by Shukla et al. the postoperative analgesic effects of 1 mL/kg 0.25% ropivacaine + 2 μg/kg clonidine were compared to those of 1 mL/kg 0.25% ropivacaine + 1 μg/kg fentanyl: the co-administration of fentanyl was associated with significantly more complications such as respiratory depression, vomiting, and bradycardia (15). Opioids such as fentanyl can migrate through the cerebral spinal fluid to reach chemo-receptors in the brain stem. In particular, in the case of lipophilic opioids such as fentanyl and sufentanil, early respiratory depression may occur during the first 30 minutes after injection and may last for 2 hours (6). In our study, the overall pain scores were in the lower range. The pain scores were generally lower with dexmedetomidine throughout the study, but the difference compared to the other treatments was only significant in the early phase of the postoperative period. Despite this, the results did not definitively indicate the superiority of dexmedetomidine over fentanyl in preventing postoperative pain. A study by Gaitini et al. showed that administration of 1 μg/kg fentanyl with 2% lidocaine for caudal epidural block was not beneficial for preventing postoperative pain after circumcision in children (16). However, in our study, the bupivacaine-fentanyl group had significantly better analgesia scores than the bupivacaine only group. This may be related to the difference in the type of local anesthetic used as well as the higher dosage of fentanyl (2 μg/kg) used in our research. In our study, pain relief was observed earlier in the postoperative course in the BD group than in the BF group: this is probably because caudal application of dexmedetomidine had better analgesic effects than caudal application of fentanyl. The pharmacokinetics of bupivacaine after induction of caudal anesthesia in children administered 2.5 mg/kg of bupivacaine has been studied by Mazoit et al.: the serum levels were found to be in the range of 0.5 - 1.9 μg/mL, with the peak plasma levels observed 10 - 60 minutes after administration (17). However, the peak levels of caudal dexmedetomidine have not been defined yet. After intravenous administration of dexmedetomidine, the onset time of anesthesia is at 15 minutes after administration, and the peak concentration is attained in approximately an hour under continuous infusion. Further, the terminal half-life of dexmedetomidine is 2 - 3 hours (18). In a study by Koroglu et al. the onset of sedation after intravenous administration of dexmedetomidine was observed at 19 minutes (19). In a study by She et al. the effective onset times for caudally applied 0.20% levobupivacaine with 2 μg/kg dexmedetomidine were 9.91 min (8.55 - 11.28, 50% confidence interval) and 16.39 min (13.32 - 19.46, 95% confidence interval). The mean duration of analgesia in these children was 19.6 hours (range, 8 - 24 hours) (20). The duration of analgesia reported by them is similar to the values in this study, but we did not measure the onset time because it was during the operation. However, we did measure the peak effect time during the recovery phase, which has not been reported by any other study. Addition of dexmedetomidine along with levobupivacaine prolongs the duration of analgesia during caudal block in children (20). Epidural dexmedetomidine inhibits the propagation of C-fiber impulses, which affects the hyperpolarization of postsynaptic dorsal neurons. The synergistic effect of dexmedetomidine and local anesthetics produces a blocking effect in the Aδ and C fibers, which lowers the absorption of local anesthetics and hampers the sympathetic system, leaving the cholinergic system uninhibited. Although dexmedetomidine is involved in these complex mechanisms, the concentration of the local anesthetic administered may have an important effect on the pharmacokinetics of the analgesic (20). The average duration of anesthesia was 58 minutes in the BD group, and the end point of anesthesia in this group coincided with the peak effect of dexmedetomidine. However, the pain scores decreased till the 6th hour of ward stay or 7 hours after the end of the operation. If 20 min are added to account for the time required for anesthesia and block initiation, it means that the analgesic effect of causal dexmedetomidine peaked at 440 minutes in the recovery period. In agreement with our findings, a number of other studies have shown that administering dexmedetomidine with bupivacaine has better analgesic and sedative effects than administering bupivacaine alone (21-23), and one study has also shown that administering fentanyl with bupivacaine has better analgesic and sedative effects than bupivacaine alone (24). In one study, 0.25% bupivacaine (1 ml kg-1) with either 2 μg kg-1 dexmedetomidine or clonidine was used in pediatric lower abdominal surgery. Even though dexmedetomidine has eight times (25) more affinity for α2 receptors than clonidine, no difference was observed in the efficiency of postoperative analgesia between these two agonists. However, the analgesic profile was significantly better with these additives than with bupivacaine alone (26). In our study, the overall analgesia and sedation scores were generally less than 2, with the best sedation scores found in the BD group: the level of sedation decreased significantly in descending order of the BD, BF and B group. In supporting to these findings improved sedation and pain scores with dexmedetomidine have also been shown with intratechal administration of BD too (27). Motor block was not observed in any of the patients in our study. However, it has been shown that clonidine, which is also an α2 agonist, has the ability to increase the time period and intensity of the motor block when it is intrathecally administered with bupivacaine for cesarean section (28). This is believed to be the result of α2 adrenergic stimulation, which may augment the local anesthetic properties first by enhancing potassium efflux from neuronal A-delta and C-type fibers and then resulting in a depressed action potential; thus, the increase in the vasoconstrictory effect of α2 agonists decreases the absorption of local anesthetic agents from the blocked area (29, 30). The motor block could be related to the type of agents used. For instance, in one study, administration of 0.1% ropivacaine (1 ml kg-1) and clonidine (2 μg kg-1) did not induce a motor block (31). This may be due to the dilution effect on the local anesthetic agent in the caudal epidural space. In agreement with the results of our study, the study by She et al. also reported that the postoperative motor block was not augmented by the addition of dexmedetomidine (20). Further, in a recent study, intrathecal administration of 0.5% bupivacaine (2.5 mL) + 10 μg dexmedetomidine was associated with significantly more efficient analgesia and motor block than intrathecal administration of 0.5% bupivacaine (5 mL) + 25 μg fentanyl (0.5 mL) (8). We expected to observe respiratory depression in the fentanyl-bupivacaine group, but respiratory depression was not observed during the postoperative period in any of our study groups. Similarly, it has been reported that the respiratory indices are similar in the BD group and B group (21, 26, 32). In a study by Rathmell et al. it was found that caudal administration of BF did not have respiratory depressive effects (33). Thus, the findings of these studies are in agreement with those of the present study (33). No hypotension or bradycardia was observed in our patients. In contrast, Al-Zaben et al. reported a 6.67% incidence of bradycardia in their study, which may be related to the dose of the drugs used and/or the type of surgery performed (22). Wu et al. have also reported that using dexmedetomidine as an adjuvant for inducing neuroaxial anesthesia can cause bradycardia without profound hypotension (34). However, in agreement with our present findings, a number of other studies (21, 24) have also reported that postoperative vomiting and nausea were not observed. Similar to the results of the studies by Umarani et al. and Raval et al. (23, 35), in our study, urinary retention was not observed. However, the Al-Zaben et al. study (22) reported a 3.44% incidence of urinary retention. For caudal block, administration of 1 to 2 μg/kg of dexmedetomidine with bupivacaine prolongs the duration of analgesia without significant side effects and also reduces the onset time of sensory-motor block, the total dose of analgesics required and the chances of postoperative shivering. Delaying motor regression and need of first rescue analgesic. Prolonging the duration of sensory block and postoperative analgesia (36) as in our results. BODY.5. DISCUSSION.5.1. CONCLUSIONS: The results of our study show that administration of bupivacaine-dexmedetomidine was more beneficial than administration of bupivacaine-fentanyl or bupivacaine alone with regard to inducing analgesia and sedation in the group of 1- to 5-year-old children examined after elective inguinal hernia repair. No side effects, such as motor block, hypotension, bradycardia, pruritis, urinary retention and nausea and vomiting, were observed in our study groups. BODY.5. DISCUSSION.5.2. LIMITATIONS: The patients were admitted to different wards in the children's hospital, which may have caused a bias in their postoperative follow-up and evaluation findings. Moreover, we did not anticipate or record the onset time of analgesia after the application of the caudal block. BODY.5. DISCUSSION.5.3. RECOMMENDATIONS: Although a number of studies have investigated the effects of caudal block with bupivacaine and dexmedetomidine, none of these studies ran comparisons with a third group (like the bupivacaine-fentanyl group in this study).

TITLE: Antihistamines in the treatment of pruritus in psoriasis ABSTRACT.AIM: To evaluate the efficacy of antihistamines in reducing pruritus in psoriasis, 61 patients were randomized to be treated for 1 week with clemastine (n = 20), levocetirizine (n = 21) or placebo (n = 20). ABSTRACT.MATERIAL AND METHODS: All patients received the same routine antipsoriatic treatment. Itch intensity was assessed with VAS and the Itch Questionnaire, and hand movements during sleep were counted with an accelerometer. ABSTRACT.RESULTS: There was a statistically significant decrease in mean VAS scoring in clemastine and levocetirizine groups (p < 0.001), but not in the placebo group. Questionnaire scoring decreased significantly during the study in all study groups, with the greatest improvement noted in the clemastine group. The number of wrist movements during sleep did not differ significantly between groups. ABSTRACT.CONCLUSIONS: Antihistamines of the first and second generations seem to be effective in reducing itch in patients with psoriasis, albeit the antipruritic effect is rather moderate. These observations need to be confirmed on larger patient groups. BODY.INTRODUCTION: Psoriasis is one of the most common chronic inflammatory skin diseases, which affects approximately 1–2% of the world general population. The etiopathogenesis of psoriasis has not been yet fully understood, but genetic predisposition, hyperproliferation of keratinocytes, vascular alterations in the skin, upregulation of cytokines, immunological disturbances as well as environmental factors are thought to play an important role in its development [1, 2]. Several studies have clearly shown that pruritus affects about 70% to 90% of patients with plaque type psoriasis and is often described as the most burdensome symptom of the disease [2–7]. Although the mean severity of itch seems to be lower than in other highly pruritic skin conditions, such as atopic dermatitis or lichen planus, it has been documented that majority of patients with psoriasis consider pruritus to have a negative effect on their quality of life (QoL) [3, 8]. Importantly, there is still no effective anti-pruritic treatment proven for psoriasis as the pathogenesis of itch in this disease has not been fully elucidated. Although histamine has been believed not to play a crucial role in the pathogenesis of psoriatic pruritus, antihistamines (especially the first-generation ones with additional sedative properties) are often used in daily clinical practice to reduce psoriatic itch. It has been suggested that a potential anti-pruritic effect, if any, is related to the sedative effect of the first-generation antihistamines and that selective histamine blockade does not improve itch, but this effect has not been studied well so far. BODY.AIM: Therefore, the current investigator-initiated study has been designed as a double-blinded, randomized and placebo-controlled trial to evaluate the potential antipruritic effectiveness of antihistamines of both, the first and second generations, in the treatment of itch in psoriasis patients. BODY.MATERIAL AND METHODS.PATIENT CHARACTERISTICS: The study was approved by the Ethics Committee of Wroclaw Medical University (agreement No. 485/2012). All patients gave their informed consent prior to inclusion in the study. A total of 61 adult patients with plaque type psoriasis and concomitant pruritus hospitalized in the Department of Dermatology of the Regional Specialist Hospital in Wroclaw were enrolled in the study. The study group consisted of 24 males (39.3%) and 37 females (60.7%) aged from 19 to 86 years (mean age: 54.4 ±15.0 years). All patients were randomly divided into one of the following three groups: group 1 – patients were given clemastine twice daily – one tablet orally (2 × 1 mg) for 6 days; group 2 – patients were given levocetirizine once daily – one tablet in the evening (1 × 5 mg) and one placebo tablet in the morning for 6 days; group 3 (control group) – patients were given placebo twice daily – one tablet for 6 days (to ensure blindness of the study all tablets were put in the starch petal by the hospital pharmacist and were given to the patients by a physician not involved in the study assessments). Twenty (32.8%) patients were randomly assigned into group 1, 21 (34.4%) patients into group 2 and 20 (32.8%) patients into group 3. Detailed characteristics of the enrolled patients are shown in Table 1. Table 1 Clinical characteristics of study patients with psoriasis Parameter Clemastine group Levocetirizine group Placebo group P -value Number of patients 20 21 20 – Age [years]: 0.97  Mean ± SD 55.1 ±14.2 53.9 ±13.9 54.2 ±17.3  (Range) (19–76) (32–80) (25–86) Gender, n (%): 0.8  Female 13 (65.0) 13 (62.0) 11 (55.0)  Male 7 (35.0) 8 (38.0) 9 (45.0) Duration of psoriasis [years]: 0.87  Mean ± SD 20.2 ±16.4 21.4 ±14.8 22.8 ±16.4  (Range) (1–58) (0.3–46) (1–49) Number of patients with psoriatic arthritis, n (%) 2 (10.0) 2 (9.5) 1 (5.0) 0.82 Positive family history of psoriasis, n (%) 9 (45.0) 6 (28.6) 9 (45.0) 0.46 BMI [kg/m 2 ]: 0.62  Mean ± SD 27.3 ±5.3 28.8 ±5.5 27.6 ±4.8  (Range) (18.4–37.4) (19.5–39.8) (20.1–36.5) PASI (Baseline): 0.44  Mean ± SD 12.5 ±5.3 13.1 ±6.6 14.9 ±5.5  (Range) (6.4–27.6) (4.6–28.8) (6.0–28.4) PASI (EoS): 0.42  Mean ± SD 7.1 ±4.6 7.0 ±4.5 8.6 ±3.4  (Range) (2.7–21.0) (2.3–16.5) (3.2–14.8) BSA (Baseline) (%): 0.45  Mean ± SD 13.6 ±6.2 14.5 ±9.6 17.1 ±10.2  (Range) (6–27) (6–45) (5–42) BSA (EoS): 0.45  Mean ± SD 13.4 ±6.3 14.1 ±9.5 16.8 ±9.6  (Range) (6–27) (6–43) (5–42) BMI – body mass index, EoS – end of study, SD – standard deviation. BODY.MATERIAL AND METHODS.INCLUSION AND EXCLUSION CRITERIA: Adult patients (aged 18 years old or older) with plaque type psoriasis and concomitant pruritus were enrolled into the study. The diagnosis of plaque type psoriasis had to be made at least 3 months prior to the enrollment. Patients with a non-plaque form of psoriasis (e.g. erythrodermic, guttate or pustular psoriasis) were excluded. Any potential patient who met any of the following criteria was also excluded from the study: NYHA III or NYHA IV heart failure, itch that could be induced by other dermatological or systemic diseases, receiving any medications which are known to cause pruritus or have an antipruritic effect. Women who were pregnant or breast-feeding as well as patients with contraindications to antihistamines were also excluded. Patients who were enrolled into the study were not allowed to receive any antihistamines, phototherapy or systemic treatment of psoriasis within 2 weeks prior to randomization. BODY.MATERIAL AND METHODS.STUDY DESIGN: The study was designed as a double-blinded, randomized and placebo-controlled trial. All patients underwent a careful anamnesis and physical examination in order to collect demographic and clinical data. Patients were examined on the first day of admission before any anti-psoriatic and antipruritic treatment was initiated. All patients received the same routine treatment of psoriatic skin lesions (topical treatment with keratolytics followed by anthralin and UVB 311 nm phototherapy). The whole study period was 7 days for each patient. Such a short period was chosen for two reasons: all patients received antipsoriatic treatment as in-patients and many of them already demonstrated a significant improvement of skin lesions after 1 week's stay at the hospital and subsequently, marked pruritus reduction. Secondly, because of administrative reasons some patients could not stay longer in the hospital and in order not to have a high drop-off rate, the study period had to be kept short. At baseline (day 1) and at the end of the study (day 7), the severity of psoriasis with Psoriasis Area and Severity Index (PASI) and body surface area (BSA) [9–12] and quality of life according to Dermatology Life Quality Index (DLQI) [13, 14] were assessed. Patients were asked to assess pruritus intensity over the last 24 h according to the 10-point visual analogue scale (VAS) [15, 16], and the Itch Questionnaire which was used successfully in our previous studies on pruritus [17]. Within next days of the study (day 2 through day 6) patients only assessed itch intensity using VAS diary. In addition, an accelerometer ActiSleepPlus (ActiGraph, FL, USA, 850.332.7900) was worn on the wrist of the dominant hand of the subject for 6 consecutive nights to measure the hand movements during sleep. This method, called actigraphy, was previously used to assess pruritus in different skin disorders [18, 19]. The digital accelerometer output was exported to software for analysis on day 7. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: All data were analyzed statistically with Statistica 10.0 (Statsoft, Krakow, Poland). Means, standard deviations (SD), median values and frequencies were calculated. The differences between the groups of patients were analyzed using the Student's t test for independent variables, Mann-Whitney U test, analysis of variance (ANOVA) and multiple regression analysis, where appropriate. Correlations between analyzed parameters were verified by using Spearman's rank correlation test (ρ – correlation coefficient). χ2 test was used to determine whether there was a significant difference between the expected and observed frequencies in one or more categories. The results were considered statistically significant, if p-value was less than 0.05. BODY.RESULTS: The baseline PASI was similar in all study subgroups (clemastine: 12.5 ±5.3 points, levocetirizine: 13.1 ±6.6 points and placebo: 14.9 ±5.5 points, p = 0.44) and reduced significantly in all patients: PASI for the clemastine subgroup at the end of the study was 7.1 ±4.6 points, for the levocetirizine subgroup: 7.0 ±4.5 points and for the placebo subgroup: 8.6 ±3.4 points, p = 0.42) (Table 1). The BSA values were also similar in all subgroups at baseline (p = 0.45), however, BSA did not reduce significantly during the study period (for details see Table 1). The mean itch intensity before treatment was 4.6 ±2.8 points according to the VAS scale and 11.9 ±4.5 points according to the Itch Questionnaire. Most of the patients suffered from mild (0–3 points) and moderate (3–7 points) pruritus: 36.1% and 41%, respectively. Very severe itch (more than 9 points in VAS scale) was reported by 6.6% of the patients (one patient in the clemastine group and 3 patients in the levocetirizine group) (p = 0.11). Regarding the studied subgroups, the mean intensity of pruritus according to the VAS before treatment was 5.6 ±2.7 points in the clemastine group, 4.7 ±3.2 points in the levocetirizine group and 3.6 ±1.9 points in the placebo group (p = 0.07). A statistically significant decrease in mean VAS scoring was observed in clemastine and levocetirizine groups (p < 0.001), but not in the placebo group. The greatest improvement of VAS was seen in the clemastine group by 2.6 ±3.1 points comparing to levocetirizine (by 2.0 ±2.9 points, p = 0.56) and placebo (by 0.5 ±2.3 points, p = 0.01) groups. The difference between levocetirizine and placebo subgroups was also significant (p = 0.03). The relative improvement for each study subgroups is demonstrated in Figure 1. Figure 1Relative change of the visual analog scoring in the treated groups Mean Itch Questionnaire values before treatment and on day 7 were similar in all subgroups (clemastine group: 13.3 ±4.7 points, levocetirizine group: 12.2 ±4.6 points, placebo group: 10.1 ±3.9 points, p = 0.09). Itch questionnaire scoring decreased significantly during the study in all groups. The greatest decrease was seen in the clemastine group with a change of 4.5 ±3.3 points (p < 0.0001). The improvement of itch questionnaire scoring in the levocetirizine group was 2.2 ±3.4 points and 1.9 ±3.5 points in the placebo group (p < 0.01 and 0.04, respectively) (Figure 2). Figure 2Relative change of Itch Questionnaire scoring in the treated groups The number of wrist movements during sleep did not differ significantly between groups within all analyzed nights. There was no significant difference in the number of wrist movements during sleep over the following nights when compared to the baseline night in any of the three groups (for the clemastine group p = 0.3, for the levocetirizine group p = 0.09, for the placebo group p = 0.84). In addition, no significant differences regarding the duration of the nocturnal movement activity in the studied subgroup of patients were found. Mean DLQI values before treatment and on day 7 did not differ significantly between groups (p = 0.11 and p = 0.38, respectively). A statistically significant decrease in DLQI scoring was observed on day 7 when compared to the baseline values in all groups. The greatest improvement was seen in the clemastine group (9.7 ±5.6 points, p < 0.0001) followed by the levocetirizine group: 6.6 ±6.2 points (p < 0.0001) and the placebo group: 3.3 ±3.3 points (p < 0.001) (Table 2). Table 2 Scoring of pruritus and quality of life in studied patients Parameter Clemastine group Levocetirizine group Placebo group P -value VAS (Baseline): 0.07  Mean ± SD 5.6 ±2.7 4.7 ±3.2 3.3 ±1.8  (Range) (0.9–9.9) (0.8–9.4) (1.0–6.8) VAS (EoS): 0.93  Mean ± SD 3.0 ±2.9 2.7 ±3.0 2.8 ±1.9  (Range) (0.1–8.9) (0–9.4) (0.4–7.0) IQ (Baseline): 0.08  Mean ± SD 12.2 ±4.6 13.3 ±4.7 10.1 ±3.9  (Range) (4–22) (4–19) (4–15) IQ (EoS): 0.48  Mean ± SD 8.5 ±4.5 10.0 ±5.4 8.2 ±4.3  (Range) (4–17) (3–22) (3–20) DLQI (Baseline): 0.12  Mean ± SD 14.8 ±7.9 14.0 ±6.3 10.3 ±6.6  (Range) (4–30) (2–27) (3–21) DLQI (EoS): 0.38  Mean ± SD 5.1 ±5.0 7.4 ±6.0 7.1 ±5.4  (Range) (1–20) (1–27) (1–17) EoS – end of study, IQ – Itch Questionnaire, SD – standard deviation. BODY.DISCUSSION: Pruritus affects approximately 70–90% of psoriatic patients. Treating itch in patients with psoriasis is challenging because its pathogenesis is still not completely understood and there are no evidence-based guidelines to make reasonable therapeutic decisions. However, several studies documented the clinical manifestation of pruritus in psoriasis. It is more common among patients with plaque-type psoriasis than among subjects with erythrodermic, guttate or pustular psoriasis [3, 18]. The published data show that the intensity of itch in psoriasis reflected by the VAS scale is scored between 3.7 and 6.4 [3, 4, 7, 20–23]. In our study the initial mean itch intensity was within this range, i.e. 4.6 ±2.8 points. Interestingly, very severe itch (more than 9 points in the VAS scale) was reported by only 6.6% of the patients while in other published studies it ranged between 18 and 33% [3, 4, 24]. However, the methodology and sample size of these studies were different and thus the direct comparison is difficult to be made. As mentioned above, the pathogenesis of pruritus in psoriasis is still not fully elucidated. Currently most scientists indicate that it could be related to disturbed innervation and dysregulated expression of neuropeptides in the skin [25], whereas histamine, an important itch mediator in allergic diseases, especially in urticaria, was not considered as a relevant pruritogen in psoriasis. Wiśnicka et al. [22] found no correlation between pruritus intensity and histamine plasma levels in psoriasis, as well as no difference in histamine plasma levels between pruritic and non-pruritic subjects. However, the plasma level of histamine does not necessarily have to reflect its content in the skin. The role of mast cells, which are major histamine producers in humans, is being still intensively studied. Harvima et al. [26] found that psoriasis is characterized by an increased number of mast cells in the upper dermis and in epidermis while Schubert and Christophers [27] observed that degranulated mast cells are seen at the very early stages of psoriatic inflammation in the skin. Using microdialysis technique Petersen et al. [28] found that the histamine level was increased in the involved psoriasis skin compared to the normal skin in the controls and uninvolved psoriatic skin. They treated 16 patients with high-dose ranitidine for 6 months observing PASI reduction and a decrease in histamine concentration in psoriatic skin lesions without any significant reduction in mast cell number. Furthermore, Gschwandtner et al. [29] found that the most recently described histamine H(4) receptor is highly expressed on plasmacytoid dendritic cells (pDC) in psoriasis and that histamine influences cytokine production and migration of pDC. In addition, Mommert et al. [30] showed that stimulation by histamine or H4 receptor agonist increases production of IL-17 by Th17 cells, which is thought to play a crucial role in the pathogenesis of psoriasis. These observations suggested that skin mast cells in active psoriasis might be functionally hyperreactive and that histamine may be involved in the pathogenesis of psoriasis. Based on our own observations, antihistamines are commonly used to treat itch in psoriatic patients in clinical practice, mainly the first-generation ones due to their additional sedative effect, despite they have never been shown effective in any controlled study. Other authors also mentioned antihistamine prescriptions. Prignano et al. [24] pointed new antihistamines as being effective in only 13% of psoriatic patients with pruritus and only for a short time. Similarly, Amatya et al. [3] found that some psoriatics used antihistamines but all complained about very short effectiveness of these drugs. In turn, Yosipowitch et al. [4] showed that antihistamines as antipruritic treatment were used by 45% of patients, among whom hydroxyzine was the most often used drug. An antipruritic effect was short in the majority of patients (84%), while 16% reported lack of any itch reduction. In the light of the above-mentioned studies, we have found our results quite surprising. At the beginning, we expected that antihistamines would not improve itch in plaque-type psoriasis and any potential effect would concern only sedative drugs of first-generation antihistamines. However, we have found a statistically significant decrease in mean VAS scoring not only in the clemastine group but also in the levocetirizine group. A significant improvement was seen in the clemastine group on the second day and in the levocetirizine group on the third day. Patients in the placebo group did not achieve any significant improvement of itch during the whole treatment period of 7 days (p = 0.48). Itch intensity according to the Itch Questionnaire decreased significantly in all treatment groups, but again the greatest improvement was seen in the clemastine group followed by the levocetirizine group and the placebo group. The effect in the placebo group might be attributed to the antipsoriatic treatment given to all patients, however, a significantly larger itch reduction in the other groups might indicate the possible additional effect of histamine blockade. Lack of change of wrist movements during the entire period of study requires further investigation, but in our opinion, this method is still not well validated for itch intensity measurements and a number of co-factors might influence the final results achieved with such instruments. Similar observations come from other studies which used actigraphy as a possibly objective method of scratching behavior assessment. Murray and Rees [31], in their study including 117 patients with atopic dermatitis, psoriasis, cholestasis and idiopathic itch, found no correlation between VAS scoring and actigraphy measurements. In the study of Bender et al. [32], scratching measured by accelerometers did not correlate with results of subjective methods of evaluating pruritus. Bringhurst et al. [18] made similar observations – there was no relationship between VAS scoring in adults with pruritus and actigraphy results. In contrast, they found a statistically significant correlation in the pediatric population (r = 0.4, p = 0.049). Bringhurst et al. [18] suggested that the difference between adults and children could be due to the fact that among adults (especially in elderly) insomnia and concomitant diseases are more common and they can influence their night activity regardless of pruritus. However, in another large randomized study with 336 children with eczema (aged 6 months to 16 years) conducted by Wootton et al. [33], actigraphy did not correlate well with disease severity or quality of life when used as an objective outcome measure and was not responsive to change over time. They noticed, just like we did in our study, the problem in distinguishing between eczema-related and eczema-nonrelated movements (so-called restless movements) which could be related to other factors like nightmares, anxiety, concomitant diseases or high temperature in the room at night. Although actigraphy seems to be a very promising method of objective itch assessing, further work is needed to establish improved ways of analyzing obtained data. The first steps were made by Feuerstein et al. [34] who used k-means cluster analysis to differentiate scratching from walking and restless sleep, which are potential confounds for nighttime scratching. This work indicated that the features described there can be used to develop a classifier that discriminates scratch from other activities. Whether indeed such algorithms would be of help will be shown in the future. BODY.CONCLUSIONS: Pruritus in psoriasis is a frequent and burdensome symptom of the disease and its treatment remains challenging. Studies on itch pathogenesis as well as randomized, placebo-controlled studies on new and old drugs with a potential antipruritic effect are highly needed. BODY.CONFLICT OF INTEREST: The authors declare no conflict of interest

TITLE: The Effect of Midazolam on Decreasing the Duration of Intussusception Hydrostatic Reduction in Children ABSTRACT.BACKGROUND AND OBJECTIVE:: The use of sedative medications may be effective in the success and facilitation of the intussusception non-surgical treatment. Therefore, the purpose of this study was to examine the effect of midazolam on decreasing the duration of intussusception hydrostatic reduction in children. ABSTRACT.MATERIALS AND METHODS:: In a double-blind clinical trial, 32 children were diagnosed with ileocolic intussusception based on sonographic findings, were studied and randomly divided into two groups. After obtaining written informed consent from the parents, 5 minutes before reduction, an intravenous Midazolam at the concentration of 1.0 mg/kg (up to 3 mg) was infused, and then barium reduction was performed under fluoroscopy guideline. In the control group, sterile water was injected as placebo and the remaining reduction steps were performed compared with the experiment group. ABSTRACT.RESULTS:: Of 16 patients that received Midazolam, 15 patients demonstrated successful reduction; and of 16 patients that received distilled water, only 11 patients showed successful reduction (P=0.07). The mean duration of a successful reduction in the Midazolam group and placebo was 34.8±11.35 and 32.73±19.2 min, respectively (P=0.733). ABSTRACT.CONCLUSIONS:: The use of Midazolam as a benzodiazepine with known sedative and muscle relaxant effects can increase the success rate of enema reduction in intussusception. BODY.1. INTRODUCTION: Intussusception is the most common cause of childhood intestinal obstruction and its standard treatment, in most cases, is the reduction by two non-surgical methods using air or liquid pressure (1-3). If nonsurgical treatment fails or does not have any indication, open surgery may be necessary. The use of an intravenous spasmolysis with glucagon in facilitating the reduction in intussusception has been investigated in several studies and different results have been obtained, so that in some studies, it had a significant effect on easier intussusception reduction, but it had no effects on others (4, 5). Also, the reduction in the operating room under general anesthesia has been investigated in several studies, from which, different results, ranging from no effect to increased success rate have been obtained. It seems that the use of lower doses of sedative medications is effective in the success rate and facilitating the non-surgical treatment of intussusception. However, the frequent use of sedative medications in all radiology departments, including this center, is not common. This may increase the rate of unnecessary surgical treatment of intussusception (6, 7). Although among the sedative medications, oral chloral hydrate and diazepam have been used for non-invasive radiological procedures, including the intussusception hydrostatic reduction, and different results have been obtained, no study concerning the use of Midazolam as a sedative in the process of reduction has been performed so far. Midazolam is not only a medication with sedative and anti-anxiety effects that will increase child's cooperation, but also is a muscle relaxant which can have effects similar to the spasmolytic agents, such as glucagon. On the other hand, it can improve amnesia and restore child's memory from stressful procedures such as Intussusception reduction. This effect makes it superior to diazepam and proves the necessity of such a study. BODY.2. MATERIALS AND METHODS: In a double-blind clinical trial, 6-months to 4-year children referred to Amirkola Hospital who had a definite diagnosis of ileocolic intussusception based on sonographic findings, were examined. Exclusion criteria included the presence of an underlying pathologic factor (Lead point) for Intussusception, passage of more than 48 hours of the onset of symptoms, the presence of signs of peritonitis or bowel perforation, dehydration, lethargy, history of a known gastrointestinal disease such as celiac disease or cystic fibrosis, history of a chronic hypoxic pulmonary disease, cyanotic heart disease, history of previous intussusception and the history of allergy to benzodiazepines. After obtaining written informed consent from the parents (Clinical Trial Registry Number: IRCT138904264395N1), 32 eligible patients were randomly assigned (every second one) to the study and control groups. Five minutes before reduction, 5 mg per mil of intravenous Midazolam, at the concentration of 0.1mg, which was determined based on body weight and was prepared by Elixir Company (up to 3 mg) was infused to the patients of the study group under supervision of an anesthesiologist. Then, reduction with barium under fluoroscopy guidelines was applied to the patients. Before administration of intravenous Midazolam, the vital signs including heart rate, level of consciousness, respiration and blood pressure of patients were monitored and their arterial oxygen saturation were measured and recorded using a pulse oximeter. During the reduction operation by pulse oximeter, cardiac rhythm and arterial oxygen levels of patients were measured and duration of the reduction, the success of reduction, and patient's cooperation during the process were recorded in a checklist for each patient. Reduction refers to a condition, in which the entry of barium into ileal loops is viewed in the graph. Failure refers to the stoppage of barium column, at least for 15 minutes, in a part of the colon. After performing the required assessments and reaching a stable condition, patients were transferred to the recovery room, and evaluated again by sonography (ultrasound) to check if the symptoms of intussusception are remained. For an additional precaution, emergency resuscitation equipment, including oxygen ambu bag and intubation devices were provided to the experiment group that received Midazolam; and flumazenil was used as an antagonist of Midazolam as needed. After successful reduction, the patients in the experiment group were observed for one hour in the recovery room. During this period, vital signs were monitored and pulse oximetry was performed as well. After full recovery from anesthesia, and ensuring that there were no complications, they were discharged from the unit of radiology and were transferred to the relevant unit. In case the operation failed (and, in some cases, after observing the patient for about 12 hours), they would be transferred to the operating room with the consent of the surgeon and anesthesiologist. In the control group, after taking IV line, ampule of sterile water was injected as placebo and the remaining reduction steps were performed in contrast to the experiment group. For data analysis, Statistical Package of the Social Sciences (SPSS), version 16, was used. For comparison of quantitative and quantitative variables, T-test and chi-square tests were performed, respectively. The Fisher exact test was used when necessary. A P value of less than 0.05 was considered significant. BODY.3. RESULTS: Generally, 32 patients were studied, of which 17 were male and 15 were female. The average age of patients was 12.32±21.16 months (The youngest patient aged 8 months and the oldest one aged 48 months). 16 patients received Midazolam before reduction and 16 patients received placebo in the course of reduction. Background characteristics of patients are listed in Table 1. Table 1 Background characteristics of patients in both groups Of 16 patients receiving Midazolam before reduction in intussusception, 15 (93.8%) patients demonstrated successful reduction. Moreover, among 16 patients receiving distilled water before reduction in intussusception, 11 (86.8%) patients showed successful reduction. The success rate in both groups showed a significant relationship with onset of symptoms (P=1 for less than 10 hours, and P=0.041 for more than 10 hours) (table 2). As far as the duration of the procedure in the successful reduction was concerned, the mean duration in the group receiving Midazolam, before reduction in intussusception was 11.35±34.8 minutes and in the group receiving distilled water prior to intussusception reduction was 19.2± 32.73 minutes. This difference in duration was not statistically significant (P=0.733). Table 2 The success rate in both groups according to onset of symptoms The mean cardiac rhythm during the reduction process in patients receiving Midazolam was 6.68±123.713 minutes and in the patients receiving distilled water was 23.34±130.73 minutes (P=0.432). There was the possibility of performing the tapping procedure only on 12 patients (75%) receiving Midazolam, while among patients who received distilled water it was possible to do this procedure only on 5 (31.3%) patients. This difference was statistically significant (P<0.001). In addition, there was the possibility of manipulation only on 12 (75%) patients receiving Midazolam, while among patients who received distilled water, it was possible to perform manipulation only on 3 (18.8%) patients. This difference was statistically significant (P<0.001). BODY.4. DISCUSSION: Intussusception has been always one of the most common causes of acute intestinal obstruction in children. If less invasive treatments such as reduction with enema are performed at the right time, cases leading to surgery and specific complications associated with it can be reduced (8). In the present study, the role of Midazolam as a sedative medication for more effective reduction in intussusception in children was examined. Also, all patients received Midazolam before intussusception reduction through barium rectal, except for one patient, who had successful reduction. However, in the group that did not receive Midazolam, 5 patients had not successful reduction. Although in our study, the rate of reduction between the two groups was not statistically significant, given the small sample size in our study, the lack of statistical significance can be attributed to this reduction. Previous studies show different rates of the success of enema reduction. In a study in Tehran, Ghavami Adel et al. reported that the success rate of air enema in intussusception reduction is 71.4% (9). In their study, Daneman et al., also reported the success rate of 85 to 90 percent for non-surgical reductions (10). A similar success rate was also reported in other studies (11,12). In a study by Franken et al., conducted to investigate the role of intravenous glucagon in hydrostatic reduction, the success rate of 53 % was achieved in both groups (4). Given the success rate of about 85 percent in most previous studies, our study showed that the success rate of reduction can be increased through the use of Midazolam before intravenous reduction. However, in the study conducted by Franken et al., it was shown that glucagon did not influence the success rate significantly, but Midazolam which was first examined in our study, was associated with the success rate increased to 93.8% in the group receiving Midazolam. In the present study, the average reduction time in the group receiving Midazolam was 34.8±11.35 minutes and in the group receiving distilled water was 32.37±19.2 minute. Therefore, the reduction times did not significantly differ. In the study conducted by Franken et al., the use of intravenous glucagon as a relaxant for smooth muscles of the intestinal wall had no effect on the reduction time (4). In the present study, the mean duration of the procedure in the group receiving distilled water was slightly less than the group receiving Midazolam which perhaps reflects the carelessness in recording the exact duration of procedure. However, it seems that the use of Midazolam did not influence the duration of the procedure. Of course, ignoring other variables involved in the study, such as the skills of all the care team responsible for the reduction and characteristics of the patients can also be partly add to the total procedure time. Further studies with adequate sample sizes for the study of the effect of sedative medications on duration of intussusception reduction are recommended. Patient's cooperation in the intussusception reduction process is very important to get the best outcome. Due to their young age and fear of therapeutic interventions and the nature of the disease that causes restlessness, children do not have good cooperation during reduction. Therefore, the use of sedative medications can be effective in increasing children's involvement in the treatment process. In the present study, the opportunity to carry out two maneuvers of tapping and manipulation was used to examine the effectiveness of sedative medications. Tapping and manipulation were possible to be performed on 75% of patients who received Midazolam before reduction, whereas tapping and manipulation were possible to be performed only on 31.33% and 18.8% of the cases in the group receiving distilled water. BODY.5. CONCLUSION: The results of this study showed that the use of Midazolam significantly increases the cooperation and sedation levels in patients compared with the control group. It also showed that the use of Midazolam as a benzodiazepine with the known sedative and muscle relaxant effects can increase the success rate of enema reduction in intussusception in children. However, the use of Midazolam had no effect on the duration reduction. Further studies with greater sample sizes are recommended to evaluate factors affecting the duration of reduction.

TITLE: Effect of a Mindfulness Training Program on the Impulsivity and Aggression Levels of Adolescents with Behavioral Problems in the Classroom ABSTRACT: Objective: The aim of the present study was to analyze the effects of a mindfulness training psycho-educative program on impulsivity and aggression levels in a sample of high school students. Methods: A randomized controlled trial with pre-test–post-test measurements was applied to an experimental group and a control group (waiting list). The Barratt Impulsivity Scale (BIS-11) Patton et al. (1995) and the Aggression Questionnaire (Buss and Perry, 1992) were used. Results: Statistical analyses showed a significant decrease in the levels of impulsivity and aggressiveness in the experimental group compared with the control group. These results have important implications for improving the level of academic engagement and self-efficacy of students and for reducing school failure. Conclusion: This is one of the first studies showing the effectiveness of mindfulness training at reducing impulsive and aggressive behaviors in the classroom. The efficacy of mindfulness-based programs is emphasized. BODY.INTRODUCTION: Aggressive behavior in both children and adolescents is considered a complex phenomenon that involves multiple factors and manifests in a variety of forms. Among the variables related to this phenomenon are personal traits (e.g., psychopathy, neuroticism, impulsivity, search for sensations), socio-emotional (lack of empathy, self-esteem, personal values), and cognitive variables (e.g., maladaptive schemas and dysfunctional thoughts) (Condon et al., 2013; Estévez et al., 2016; Orue et al., 2016; Pellerone et al., 2016). Diverse sources and causes of impulsivity and aggressiveness in students are discussed in the literature, including parental style and insecure attachment, peer pressure (Estévez et al., 2016), conflictual relations with the teachers (Settanni et al., 2015), lack of emotional self-control, especially of negative affectivity (Peters et al., 2015; Sanger and Dorjee, 2015), emotional avoidance (Mestre et al., 2012), low compassion (Morley et al., 2016), and even neuroanatomical (Thijssen et al., 2015), among others. Students with aggressive behavior show deficiency in emotional self-control and empathy, features pertaining to the emotional intelligence trait. Therefore, these students will face more difficulties to deal with social situations and their incapacity to adequately managing their emotions may lead them to behave in aggressive ways before uncertain situations (Mestre et al., 2012; Inglés et al., 2014). Impulsivity is a risk factor associated with reactive aggression and antisocial behavior during adolescence (Orue et al., 2016). In particular, impulsive motor behavior appears to be the factor that seems to discriminate better between aggressive and non-aggressive adolescents (Oberle et al., 2011; Andreu et al., 2013). Thus, impulsive adolescents without sufficient emotional control and no ability to delay gratification are driven by the emotional momentum and little or inadequate forethought. Furthermore, impulsivity and aggressiveness are related, on the one hand, to maladaptive or risk-taking behaviors such as substance abuse or sexual promiscuity (Paydary et al., 2016) and, on the other hand, to mental disorders, attention-deficit hyperactivity disorder, reading problems, and poor academic results (Fix and Fix, 2013; Nelson et al., 2015). School related tasks require the ability to use and regulate emotions in order to increase concentration, develop intrinsic motivation, and control impulsive thinking and hostility. Adolescents with high levels of aggression use non-productive coping strategies to a greater extent, whereas less aggressive adolescents mainly focus on strategies aimed at solving problems and relating to others in more adaptive way (Samper et al., 2008; Mestre et al., 2012). In Spain, the percentage of school failure or dropout (i.e., those students who leave the educational system) during the 2012–2013 course was 23.5% (Eurostat, 2014), the double of the European Union percentage (11.9%) for the same period and higher than in other countries, such as the United States and China. Some regions in Spain even reached 29.8% (Veas et al., 2016). The considerable percentage of students that fail school or underachieve can be related, in part, to personal problems, including anxiety, depression, and behavioral problems such as impulsivity and aggression (Broderick and Metz, 2009). Furthermore, school failure is related to alcohol consumption. A study conducted by Goldberg-Looney et al. (2016) found that academic problems explained 5.1% of the variance in adolescents' alcohol use in a sample of 567 adolescents in Spain. Impulsivity and risk-taking behavior increase from childhood to adolescence (Oberle et al., 2011; Mestre et al., 2012). The high rate of school failure, along with the increase of emotional disorders, related to stress, anxiety, and aggressive behavior found in Secondary Education and High School students, and even among university students (Amutio and Smith, 2008; Inglés et al., 2014), requires the implementation of psycho-educational programs and emotional self-regulation strategies aimed at activating students' internal resources, including self-efficacy in order to promote the improvement of interpersonal relationships and academic performance (León, 2008; Amutio et al., 2015a; Gouda et al., 2016), and reducing the risk of school failure. All of the above also implies the need for providing special training to teachers (Kaspereen, 2012; Kemeny et al., 2012; Gouda et al., 2016). The number of programs to assess and prevent aggressive behavior in adolescence is scarce. In Spain there is a program directed to prevent different types of school violence and aggressiveness, including bullying and cyberbullying (Cyberprogram 2.0; Garaigordobil et al., 2015; Garaigordobil and Martínez-Valderrey, 2016). It consists of different activities to develop coping strategies and other transversal goals, such as developing interpersonal skills (empathy, active listening, social skills, constructive conflict resolution, etc). Additional techniques to reduce aggressiveness include emotional education, improving self-control and problem-solving skills, especially with adolescents showing impulsivity and reactive aggression (Orue et al., 2016). Another type of intervention, whose effectiveness has been proven are mindfulness techniques. Mindfulness-based interventions have been associated with numerous beneficial outcomes in emotional regulation, including decreased anxiety (Amutio et al., 2015b), depression (Condon et al., 2013), and anger expression reduction (Fix and Fix, 2013; Zenner et al., 2014; Gouda et al., 2016). In the last decade, the practice of mindfulness has proven effective to the development of healthier habits and the generation of better classroom climate (Schonert-Reichl and Lawlor, 2010; López-González et al., 2016), which, in turn, have led to improvements on students' performance (Franco et al., 2011; Wisner, 2013; López-González and Oriol, 2016). Consequently, a range of mindfulness programs is taking place nowadays in schools as, for example, the Mindfulness Based Wellness Education (MBWE) of Toronto University, the Mindfulness in Schools Project (MISP) in England, the Inner Kids Program, Cultivating Awareness and Resilience in Education (CARE) and Stress Management and Relaxation Techniques (SMART) in the USA. In Spain, it is worth noting the TREVA Program (López-González et al., 2016), Aulas Felices (Arguís, 2014), and the Meditación Fluir Program (Franco et al., 2011). One definition of mindfulness is to pay attention in a particular way, on purpose, in the present moment, and non-judgmentally (Kabat-Zinn, 2009). Furthermore, mindfulness implies observing the thoughts and emotional reactions that occur at each moment by distancing from them (decentering), that is, not reacting before their presence in the automatic usual way (Krishnakumar and Robinson, 2015; Peters et al., 2015), thus, breaking the thinking-feeling-acting typical pattern. Thereby, and through continuous practice, students learn to concentrate on the task they are performing, without allowing their minds to digress or get distracted. This provides students with a new perspective that facilitates reflection and learning. Currently, extensive data support the use of mindfulness in the achievement of greater levels of relaxation, well-being, and improvement of academic performance (Beauchemin et al., 2008; León, 2008; Schonert-Reichl and Lawlor, 2010; Franco et al., 2011; Choi et al., 2012; Amutio et al., 2015c). In addition, recent neurodevelopmental findings show that mindfulness and social-emotional learning programs implemented in regular school curricula improve executive functions in children and adolescents in terms of inhibitory control, enabling them to manage excessive levels of negative emotions that interfere with academic performance (Davidson et al., 2012; Sanger and Dorjee, 2015). Studies conducted on both clinical and non-clinical samples (Zenner et al., 2014) include children and adolescents with attention deficit hyperactivity disorder (van de Weijer-Bergsma et al., 2012; Van der Oord et al., 2012; Cardoso-Moreno et al., 2015), anxiety (Beauchemin et al., 2008), hostility (Sibinga et al., 2011), and externalizing disorders, such as impulsivity (Bögels et al., 2008), as well as adolescents at risk (Bluth et al., 2016). In spite of these findings, meditation treatment effects among youth are relatively unknown (Black et al., 2009). Currently, controlled studies measuring the impact of mindfulness training on reducing impulsivity and hostility levels of adolescents in the classroom barely exist. Moreover, little is known about the association of mindfulness with decreased emotional reactivity and improved impulse-control, especially in adolescents. Among the few studies conducted, we highlight those of Oberle et al. (2011) and Fishbein et al. (2016). These studies consisted of an intervention in adolescents with high-risk behaviors and are among the first ones assessing the effectiveness of a mindfulness-training program to reduce impulsivity and aggression levels of adolescents in the classroom. Given the current situation, the aim of this study is to prove the effect of a mindfulness training psycho-educational program applied to a group of adolescents with behavioral problems in the classroom on their impulsivity and aggression levels, assuming the following hypothesis: H1:Those adolescents with behavioral problems in the classroom and participating in a mindfulness training psycho-educational program will experience a significant decrease in their impulsivity levels compared to the group of adolescents with behavioral problems in the classroom that were not part of the intervention program.H2:Those adolescents with behavioral problems in the classroom and participating in a mindfulness training psycho-educational program will experience a significant decrease in their aggression levels compared to the group of adolescents with behavioral problems in the classroom that were not part of the intervention program. BODY.MATERIALS AND METHODS.PARTICIPANTS: Twenty seven students with ages from 12 to 19 years (Mean = 15.85; Standard deviation = 2.38), who were attending a public high school center located in the province of Granada participated in this study. In this sample, 59% of the participants were boys and 41% girls. The control group was made up of 14 individuals (57% boys and 43% girls), while the 13 individuals remaining were sent to the experimental group (62% boys and 38% girls). BODY.MATERIALS AND METHODS.INSTRUMENTS.BARRATT IMPULSIVITY SCALE (BIS-11) (: This questionnaire is composed of 30 items grouped in three impulsivity sub-scales: –Cognitive impulsivity (8 items): tendency to make quick decisions.–Motor impulsivity (10 items): propensity to act solely on the spur of the moment, without thinking of the consequences.–Non-planned impulsivity (12 items): indicates lack of planning of future actions. Each item comprises four Likert-type answer options: rarely/never, occasionally, often, and almost always/always. The score of each sub-scale is calculated by adding up the partial scores obtained in each item. The total score is the sum of all the items. The Spanish version of this scale, created by Oquendo et al. (2001) was administered. The internal consistency of the different scales used in the study sample was obtained using Cronbach's alpha, which presented values ranging from 0.77 to 0.92. BODY.MATERIALS AND METHODS.INSTRUMENTS.AGGRESSION QUESTIONNAIRE (AQ) (: This instrument is used to measure aggressiveness. In this study, the Spanish version created by Rodríguez et al. (2002) was used. The questionnaire is composed of 29 items with five Likert-type answer options (1 = very few times, 5 = lots of times) that form the following scales: –Physical aggressiveness (9 items): it refers to physical behaviors that hurt or harm other people.–Verbal aggressiveness (5 items): it is related to verbal behaviors that hurt or harm other people.–Hostility (8 items): assesses the cognitive aspects of aggression.–Anger (7 items): assesses the emotional and affective aspects of aggression. Regarding the reliability coefficients for the studied sample, these range from 0.72 in the verbal aggression scale to 0.85 in the physical aggression scale. BODY.MATERIALS AND METHODS.PROCEDURE: Firstly, to obtain the sample, an interview with the principal, the head of studies and the head of the counseling department of the high school center was conducted in order to explain the study objectives and to ask permission and collaboration for the application of the questionnaires. Subsequently, 27 students that had been sent more than five times to the counseling room during the first term of the school year due to misbehavior in the classroom were selected as the sample. All parents provided informed consent. The study was approved by the Committee of Bioethics of the University of Almería, Spain. The registered data for each of the instruments was alphanumerically coded, ensuring confidentiality and anonymity, in order to comply with the Personal Data Protection Act by the Ethics Committee for Research related to Human Beings (CEISH). International ethical guidelines for studies with human subjects described in the Nuremberg Code and in the Declaration of Helsinki were applied (Kim, 2012). Students were randomly assigned to the control (n = 14) and experimental groups (n = 13), controlling for sex and grade to avoid the interference of these variables in the results. Once the sample was obtained, pre-test measurements for the different dimensions of the impulsivity and aggressiveness variables were obtained by asking the participants to individually complete the questionnaires. Subsequently, the intervention program was applied to the experimental group over 10 weekly sessions that took place during the counseling hours of the students. This intervention program consisted in the learning and daily practice of a mindfulness technique named Meditación Fluir for 15 min (Franco et al., 2011, 2014). The principal goal of this practice is attempting neither to control thoughts, sensations or feeling nor altering or change them by new ones, but the contrary, i.e., let them free to come and go, and accepting any personal sensation and feeling that may arise spontaneously. Therefore, the essence of this technique is being aware of what happens in the mind and body in a passive way, without exerting any effort on modifying or changing the situation, perceiving things as they truly are and as they occur in every moment. Thus, this mindfulness practice enables the capacity to observe thoughts and other mental activity without getting involved in it—i.e., without analyzing, judging, or evaluating it—, breaking people's habit of being carried away and dragged by automatic and uncontrolled thoughts. In other words, students become aware of the presence of thoughts during the practice, but do not reflect upon their content or truthfulness, realizing that thoughts and sensations change at every moment and are constantly flowing. Consequently, through this technique, students understand from experience that thoughts continuously appear and disappear in a constant flow. In this way, students learn to be present, open and balanced against any mental or emotional phenomenon or process. Another aspect of the mindfulness program that was learned and put into practice during the 10 sessions was the performance of body-scan exercises (Kabat-Zinn, 2009). Body-scan is a technique in which attention is orderly and systematically paid to different body parts, subsequently expanding consciousness to the whole body, thereby achieving holistic awareness of the body without making value judgments nor trying to change or eliminate anything (e.g., proprioceptive and interoceptive sensations, mental reactions, etc.), that is, being always present. Once mindfulness training was completed in the experimental group, post-test measurements for the different dimensions of the impulsivity and aggressiveness variables were obtained by the same method used in the pre-test stage. Once the study, was completed the mindfulness-based training program was delivered to the control group. BODY.MATERIALS AND METHODS.DESIGN AND DATA ANALYSIS: A quasi-experimental comparison group pre-test–post-test design with experimental and control groups was used to analyze the effects of the mindfulness training program (independent variable) on the different dimensions of the impulsivity and aggressiveness variables (dependent variables). The existence of statistically significant differences between the mean scores of the control and experimental group in the different dimensions of impulsivity and aggression in each stage of the study was proved by means of the non-parametric statistical test Mann–Whitney U for independent samples, since data did not adjust to the normal probability distribution. Next, non-parametric statistical test Wilcoxon for comparing related samples was used in order to prove the existence of statistically significant differences between the mean scores of the different dimensions of impulsivity and aggression in each stage of the study, for both the control and experimental groups. Finally, Cohen's d and the percentage of change in the pre-test–post-test scores were used to determine the magnitude of the change experienced after the intervention program in the different impulsivity and aggression dimensions in the experimental group. All the statistical analyses were computed using the SPSS 22.0 package. BODY.RESULTS: Firstly, variable means and standard deviations corresponding to the control and experimental group in each stage of the study were calculated (Table 1). Table 1 Pre-test and post-test means and standard deviations corresponding to the different impulsivity and aggressiveness dimensions for the control and experimental groups. Pre-test Post-test Control Experimental Control Experimental Variable M SD M SD M SD M SD Impulsivity Cognitive 22.98 4.84 23.48 4.79 23.45 4.28 18.93 3.95 Motor 28.81 5.17 27.94 4.93 27.53 5.34 24.36 4.56 Non-planning 32.43 6.93 31.07 6.44 31.91 5.52 27.91 5.87 Total 84.22 11.04 82.49 10.27 82.89 11.04 71.2 9.16 Aggressiveness Physical 27.21 5.28 28.07 5.49 26.87 4.51 24.17 4.14 Verbal 15.45 3.75 16.04 4.11 16.09 4.68 12.12 3.84 Hostility 25.18 5.30 23.95 4.88 26.36 3.98 19.36 4.01 Anger 23.04 5.53 23.89 6.04 24.11 6.87 20.03 5.31 Mann-Whitney U test for independent samples on the pre-test scores revealed no statistically significant differences between the control and experimental pre-test mean scores in the study variables. Contrarily, statistically significant differences did appear between the control and experimental groups at post-test in all the dimensions of impulsivity and aggressiveness (Table 2). Table 2 Mann-Whitney U test for independent samples of the pre-test and post-test differences between the control and experimental groups for the different dimensions of impulsivity and aggressiveness. Pre-test Post-test Variable z p z p Impulsivity Cognitive 0.345 0.733 3.81 0.006 ∗∗ Motor 0.423 0.637 2.38 0.031 ∗ Non-planned 0.516 0.475 3.31 0.014 ∗ Total 0.741 0.309 3.68 0.008 ∗∗ Aggressiveness Physical 0.629 0.543 3.16 0.019 ∗ Verbal 1.02 0.248 2.74 0.026 ∗ Hostility 693 0.494 3.39 0.013 ∗ Anger 0.759 0.328 3.94 0.005 ∗∗∗ ∗∗∗ p = 0.005; ∗∗ p < 0.01; ∗ p < 0.05. After conducting Wilcoxon test for related samples on the experimental group scores, statistically significant differences were observed when comparing the pre-test and post-test scores in all the dimensions of impulsivity and aggressiveness. No significant differences were found in such variables after the pre-test and post-test comparisons for the control group (Table 3). Table 3 Wilcoxon test for related samples of the pre-test and post-test differences between the control and experimental groups for the different dimensions of impulsivity and aggressiveness. Control Experimental Variable z p z p Impulsivity Cognitive 0.928 0.375 -4.36 0.002 ∗∗∗ Motor -0.829 0.244 -2.29 0.038 ∗ Non-planned -0.468 0.614 -3.17 0.019 ∗ Total -0.679 0.438 -3.43 0.012 ∗ Aggressiveness Physical -0.619 0.456 -3.97 0.005 ∗∗ Verbal 0.731 0.314 -2.46 0.033 ∗ Hostility 0.132 0.842 -3.16 0.019 ∗ Anger 0.607 0.557 -4.44 0.003 ∗∗ ∗∗∗ p < 0.005; ∗∗ p = 0.005; ∗ p < 0.05. With the purpose of assessing the magnitude of the change occurred in the experimental group between pre-test and post-test scores, Cohen's d (1988) was used, with values above 1.5, between 1.5 and 1, and between 1 and 0.5 indicating very important, important and medium changes, respectively. Cohen's d showed the existence of very important changes in the cognitive impulsivity, total impulsivity and hostility dimensions, and medium to high changes in the other dimensions of impulsivity and aggressiveness (Table 4). Table 4 Cohen’s d and pretest-post-test percentage change in the experimental group for the different dimensions of impulsivity and aggressiveness. Variable d Pre-Post % Pre-Post Impulsivity Cognitive 1.04 -19.38 Motor 0.753 -12.81 Non-planned 0.514 -10.17 Total 1.16 -13.69 Aggressiveness Physical 0.803 -13.91 Verbal 0.995 -24.44 Hostility 1.03 -19.17 Anger 0.679 -16.16 Finally, the percentage of change between pre-test and post-test scores in the experimental groups for the different impulsivity and aggressiveness dimensions was calculated. Table 4 shows reductions of approximately from 24% in the verbal aggression dimension and to around 10% in the non-planned impulsivity dimension. BODY.DISCUSSION: As a result of the application of the Fluir meditation technique (Franco et al., 2011) during 10 weeks, significant reductions in all the dimensions of impulsivity and aggressiveness levels occurred in the experimental group composed of high school students, thus, confirming the hypotheses. The obtained results are in line with the findings of other studies (Oberle et al., 2011; Fishbein et al., 2016). There may be different explanations for the obtained results, namely decrease in rumination (Borders et al., 2010; Peters et al., 2015; Orue et al., 2016), reduction in hostile affect, including frustration and anger feelings (Kemeny et al., 2012; Krishnakumar and Robinson, 2015) and increase in self-control before stressors (Broderick and Metz, 2009; Yusainy and Lawrence, 2014), amongst others. In this way, the capacity to regulate attention and emotion are forms of self-regulation that support dispositions conducive to learning and maintaining positive social relationships (Flook et al., 2015). Aggressiveness is a complex phenomenon involving multiple factors, including psychosocial. Aggressive behavior is seen by some adolescents as a strategy to avoid future victimization or rejection, while for others it is interpreted as an opportunity to achieve the desired popularity among peers (Estévez et al., 2014). Our data confirm the efficacy of the Meditación Fluir-mindfulness technique. However, integrative psycho-educational intervention programs oriented to promote emotional education and in values within the school centers (Peña and Canga, 2009; Sanger and Dorjee, 2015) are needed in order to reduce the high rates of violent behavior in adolescence, improve classroom climate and diminish the risk of school failure. The development of the ability to delay gratification, along with an adequate emotional self-regulation that includes rumination control and a negative assessment of the consequences of using aggression, would be key elements to be addressed by psycho-educational programs directed to adolescent population in school and community environments. The practice of mindfulness can help students focus on the present, thus, reducing obsessive ruminations and enhancing the experience of positive emotions, as well as diminishing the probability of involvement in impulsive behaviors, which, in general, tend to aggravate the same emotional problems that want to be alleviated or solved by the use of aggression (Fix and Fix, 2013; Amutio et al., 2015a). In turn, this training allows recognizing the first signs of aggressive impulses in such a way that they are more likely to be inhibited by using the skills developed through the practice of mindfulness (i.e., acceptance and equanimity). Additionally, mindfulness has also been linked to increased compassion for both the self and others, which may foster a great sense of interconnectedness and facilitate the response to potential conflict with non-aggressive approaches (Condon et al., 2013). There are hardly any studies in Spain on the influence of mindfulness in aggression or impulsivity in adolescents, since a majority are focused on stress or anxiety and are mainly directed to the adult population. Although research on mindfulness, especially with children and adolescents, is still in relatively early stages, an increasing number of studies have shown the potential benefits of mindfulness practices to students' physical health and psychosocial well-being, enhancing academic performance and diminishing the risk of school failure (Franco et al., 2011, 2014; Amutio et al., 2015a; López-González et al., 2016). This is a very important concern given that school failure in adolescence is a strong predictor of other high-risk behaviors, including delinquency, substance abuse and adolescent pregnancy (Stratton, 2006; Morley et al., 2016). More generally, a negative attitude toward school is, in fact, a risk factor for aggressive behaviors (Estévez et al., 2014). Conversely, school connectedness, engagement, academic achievement, and academic enjoyment may protect against alcohol use, suspension, lower educational aspirations, and poor academic performance (Goldberg-Looney et al., 2016). BODY.CONCLUSION: The main limitation of this study is the reduced size of the sample. Further studies need to be carried out with bigger samples in order to determine specific causal mechanisms (e.g., changes in prefrontal brain structures, decrease in ruminations, emotional self-regulation, development of compassion) of the observed effects. However, this is one of the first studies showing the effectiveness of a mindfulness program for reducing impulsive and aggressive behaviors in the classroom. In an era of budget cuts, this type of group psycho-educational programs implies a considerable optimization of the economic and social resources invested on education in order to improve academic performance and decrease school failure. BODY.AUTHOR CONTRIBUTIONS: CF: Data collection and analyses. AA: Data interpretation, introduction, and discussion. LLG: Bibliography, literature review, and corrections. XO: Literature review and procedure. CMT: Literature review and article revision. 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 trigger point dry needling for plantar heel pain: a randomized controlled trial ABSTRACT: BODY.BACKGROUND: Plantar heel pain (plantar fasciitis) can be managed with myofascial trigger point dry needling of myofascial trigger points, however there is only poor quality evidence supporting its use. Therefore, we aimed to evaluate the effectiveness of trigger point dry needling for plantar heel pain. BODY.METHODS: 84 participants with plantar heel pain were randomized to real or sham trigger point dry needling. The intervention consisted of one treatment per week for six weeks. Participants were followed for 12 weeks. Primary outcome measures included 'first-step pain' measured with a Visual Analogue Scale and foot pain measured with the pain subscale of the Foot Health Status Questionnaire. The primary end-point for predicting the effectiveness of dry needling for plantar heel pain was six weeks. BODY.RESULTS: At the primary end-point, significant effects favored real dry needling over sham dry needling for pain (adjusted mean difference: VAS first-step pain -14.4 mm, 95% CI -23.5 to -5.2, p=0.002; FHSQ foot pain 10.0 points, 95% CI 1.0 to 19.1, p=0.029), although the between-group difference was lower than the minimal important difference. The frequency of minor transitory adverse events was significantly greater in the real dry needling group (70 real dry needling appointments [32%] compared with only 1 sham dry needling appointment [<1%]). BODY.CONCLUSION: We found that dry needling provided statistically significant improvements in plantar heel pain, but the magnitude of this effect should be considered against the frequency of minor transitory adverse events.

TITLE: Prolonged-release fampridine and walking and balance in MS: randomised controlled MOBILE trial ABSTRACT.BACKGROUND:: Mobility impairment is a common disability in MS and negatively impacts patients' lives. ABSTRACT.OBJECTIVE:: Evaluate the effect of prolonged-release (PR) fampridine (extended-release dalfampridine in the United States) on self-assessed walking disability, dynamic/static balance and safety in patients with MS. ABSTRACT.METHODS:: MOBILE was a randomised, double-blind, exploratory, placebo-controlled trial. Patients with progressive/relapsing-remitting MS and Expanded Disability Status Scale score of 4.0–7.0 were treated with PR-fampridine or placebo twice daily for 24 weeks. Efficacy endpoints included change from baseline in the 12-item MS Walking Scale (MSWS-12), Timed Up and Go (TUG) test and Berg Balance Scale (BBS). ABSTRACT.RESULTS:: 132 patients were randomised at 24 sites in six countries. PR-fampridine therapy resulted in greater median improvements from baseline in MSWS-12 score, TUG speed and BBS total score versus placebo over 24 weeks. A higher proportion of patients receiving PR-fampridine versus placebo experienced significant improvements at MSWS-12 improvement thresholds ⩾7 (p = 0.0275), ⩾8 (p = 0.0153) and ⩾9 points (p = 0.0088) and TUG speed thresholds ⩾10% (p = 0.0021) and ⩾15% (p = 0.0262). PR-fampridine was well tolerated. ABSTRACT.CONCLUSIONS:: PR-fampridine therapy resulted in early and sustained improvements in broad measures of walking and balance over six months. BODY.INTRODUCTION: Walking impairment is common in MS,1,2 even early in disease,3 and maintaining mobility is a high priority among patients with MS.4,5 Prolonged-release (PR) fampridine tablets (known as sustained/modified-release fampridine in some countries and extended-release dalfampridine in the United States) are approved for improving walking in MS based on results from two pivotal trials6,7 that demonstrated consistent improvements in walking speed on the Timed 25-Foot Walk8 and on patients' self-assessment of ambulatory disability on the 12-item MS Walking Scale (MSWS-12).9 Walking is a complex activity influenced by many variables, including impairments in balance,10–12 which are frequent among patients with MS13 and correlate with slower walking speed.10 Furthermore, balance impairment is a strong predictor for perceived difficulty in mobility in MS.14 This highlights the need for studies that assess effects of PR-fampridine on balance. PR-fampridine improved standing balance in a small (n = 8) 14-week study;15 however, randomised, placebo-controlled studies assessing the effect of PR-fampridine on balance have not previously been performed. The Timed Up and Go (TUG) test16,17 and Berg Balance Scale (BBS)17,18 measure mobility/balance and have demonstrated high reliability in patients with MS.17,18 The objective of MOBILE was to explore the effect of PR-fampridine on endpoints related to patients' self-assessed walking disability and dynamic/static balance, assessed using the BBS and TUG, as well as to subjective impression of well-being and patients' impression of change in walking. BODY.METHODS.STANDARD PROTOCOL APPROVALS, REGISTRATIONS AND PATIENT CONSENTS: This trial was conducted in compliance with the Declaration of Helsinki, the International Conference on Harmonisation Good Clinical Practice guidelines, the European Union Clinical Trials Directive and local regulatory requirements. Approval for the trial protocol and all amendments was obtained from local ethics committees. Written informed consent was obtained from each patient before any evaluations were conducted for eligibility. The trial is registered on ClinicalTrials.gov (identifier NCT01597297) and the European Union Clinical Trials Register (EudraCT number 2012-000368-90). BODY.METHODS.TRIAL DESIGN: MOBILE was a randomised, double-blind, exploratory, multi-centre, placebo-controlled trial in patients with MS (electronic Supplementary Materials, Figure 1). Patients were screened for eligibility during a 14-day screening period. Eligible patients were randomised (1:1) to receive PR-fampridine 10 mg tablets or matching placebo twice daily every 12 hours for 24 weeks. Scheduled visits took place at screening, day 1 and weeks 2, 4, 8, 12, 16, 20 and 24. A post-dosing follow-up visit was conducted two weeks after the end of treatment. Randomisation was determined by a centralised system on day 1 of the trial and treatment assignments were made through an Interactive Web Response System. All patients and trial staff, including the principal investigator, were blinded to patient treatment assignments. Blinding was achieved by using a matched placebo. Treatment kits were prepared centrally and labelled with unique numbers, which were used to maintain the blind when drug supplies were dispensed. BODY.METHODS.PATIENTS AND TRIAL TREATMENTS: Eligible patients were male or female, aged 18–70 years with an Expanded Disability Status Scale score of 4.0–7.0 and diagnosis of primary-progressive MS, secondary-progressive MS, progressive-relapsing MS or relapsing-remitting MS per revised McDonald criteria19,20 of ⩾3 months' duration. Most stable concomitant therapies for treatment of MS were permitted. Key exclusion criteria included: treatment with 4-aminopyridine or 3,4-diaminopyridine in any formulation ⩽30 days before screening; known allergy to pyridine-containing substances; any history of seizure, epilepsy or other convulsive disorder; renal impairment (creatinine clearance <80 ml/min); onset of MS exacerbation ⩽60 days before screening; and a body mass index ⩾40 kg/m2. BODY.METHODS.OUTCOME MEASURES AND CLINICAL ASSESSMENTS: Outcome measures were assessed at screening, day 1 and weeks 2, 4, 8, 12, 16, 20 and 24 (on-treatment period) and at the week 26 follow-up visit (except where noted otherwise). Because this was an exploratory study, there were no pre-specified primary/secondary endpoints and no formal statistical hypothesis testing was planned. Walking ability was assessed using the MSWS-129 and the seven-item Patient Global Impression of Change (PGIC) scale.21 The MSWS-12 is a 12-item questionnaire that asks patients to rate limitations of their mobility owing to MS during the preceding two weeks on a five-point scale (from 1 = not at all to 5 = extremely). Total score ranges from 1–60 and was transformed to a scale of 0–100; reduced score indicates improvement in walking. The PGIC is a global assessment of the patient's impression of how the study drug affected their overall walking during the preceding seven days. The PGIC was assessed at weeks 2, 4, 8, 12, 16, 20 and 24, and scored on a seven-point scale (from 1 = very much worse to 7 = very much improved). Mobility and dynamic balance were assessed using the TUG test,16,17 which measures the time/speed it takes for a patient to stand up from a seated position, walk three metres out, turn around and walk back and return to the seated position. Increased speed on the TUG indicates improvement in mobility and dynamic balance. Static and dynamic balance were measured using the BBS,17,18 which is comprised of 14 balance-related tasks scored from 0 (unable to perform) to 4 (able to perform independently). The BBS is the sum of scores across these tasks and ranges from 0 (poor balance) to 56 (good balance). Positive change on the BBS indicates improvement in balance. The TUG and BBS, which have demonstrated high reliability in patients with MS,17,18 were chosen to further assess the impact of PR-fampridine treatment on mobility, specifically on static and dynamic balance. Balance has not been previously evaluated with PR-fampridine treatment in a randomised, placebo-controlled trial but may impact patients' walking ability and mobility. The impact of MS on the patient and quality of life (QoL) were assessed using the 29-item Multiple Sclerosis Impact Scale (MSIS-29) physical subscale (PHYS)22 and the EuroQoL-5 Dimensions 5-level (EQ-5D-5L).23 The self-administered MSIS-29 questionnaire contains a 20-item PHYS and a nine-item psychological subscale. For a particular visit, the MSIS-29 PHYS score was calculated by summing the 20 items and transforming the score to a scale with a range of 0 (no impact of MS) to 100 (extreme impact of MS). Negative change on the MSIS-29 PHYS indicates improvement in physical health. The EQ-5D-5L is a generic instrument comprised of five questions and a visual analogue scale (VAS). A utility score is derived from the five questions addressing mobility, self-care, usual activities, pain/discomfort and anxiety/depression, with responses/scores ranging from 1 (no problem) to 5 (severe problem). The VAS score ranges from 0 (worst imagined health state) to 100 (best imagined health state). The EQ-5D was assessed at day 1 and weeks 4, 8, 12, 16, 20 and 24, and a positive change in both the utility and VAS scores indicates an improvement in health state. Safety and tolerability were assessed by monitoring adverse events (AEs), serious AEs (SAEs) and concomitant medications and by performing physical examination, vital sign measurements and 12-lead electrocardiograms. BODY.METHODS.STATISTICAL ANALYSES: A sample size of 120 patients (60 patients per group) was chosen based on bootstrapping that sampled data from a previous study6 to create 1000 datasets of 60 patients each, and repeated for sample sizes of 70 and 80 patients. For each sample, the proportion of patients with a six-point change or more in MSWS-1224 score was calculated, and the percentage of samples that fell within an interval of the actual percentage in the study was calculated to explore whether increasing the sample size increased the precision. Using the sample size of 60 patients for both treatment groups, 84% of samples were within 8% of the true proportion and increasing the size did not appreciably increase precision. The intent-to-treat population consisted of all patients who were randomised, received at least one dose of treatment and had at least one post-baseline assessment for a given parameter. Efficacy analyses were based on an intent-to-treat analysis with missing data imputed by the last observation carried forward method when at least one post-baseline value was available. Baseline values were not carried forward and were defined as the mean over the screening and baseline visits. Mean changes in each efficacy endpoint, except for the PGIC, were calculated over 24 weeks using the last observation carried forward values to calculate the average. Because the PGIC was designed to assess change since the previous visit, week 2 was the only time point that was valid for between-group comparisons of treatment effect. This was an exploratory study, and descriptive statistics were used to summarise endpoints. No formal statistical hypothesis testing was planned. Based on the distribution of changes from baseline across patients, all by-visit analyses were summarised using median change from baseline on the outcome. Median changes were presented with corresponding non-parametric 95% confidence intervals. Mean changes from baseline on the MSWS-12 were categorised using the following thresholds: a less than one-point improvement/no change/worsening and then increasing thresholds of improvement from ⩾1 point to ⩾10 points in one-point increments. These categories were summarised as the number/percentage of patients meeting each threshold. Mean percentage changes from baseline in TUG speed were categorised using the following thresholds: ⩽0% (worsening/no change), >0% (any improvement) and then increasing thresholds of improvement from ⩾10% to ⩾40% in 5% increments. These categories were summarised as the number/percentage of patients meeting each threshold. In order to characterise magnitude of the observed treatment effect relative to variance in the outcome measures, post hoc statistical testing compared multiple thresholds of improvement between treatment groups for MSWS-12 and TUG using a logistic regression adjusted for baseline. Multiple MSWS-12 thresholds of change were examined based on ranges previously identified as clinically meaningful (⩾4–6 points based on a 100-point scale) over three months.24 A post hoc analysis using the chi-square test compared the percentage of patients in each treatment group who reported any improvement in PGIC after two weeks of treatment. To assess magnitude of treatment effect over time, changes from baseline to each visit also were summarised for each efficacy endpoint, except for PGIC. For missing data in MSWS-12, a visit in which ⩾50% of the component questions were answered but at least one question was missing, scores from unanswered questions were imputed using the respondent-specific mean score. MSWS-12 score was considered missing for a visit in which ⩾50% of the questions were unanswered. For the TUG test, two trials of the TUG were conducted at each visit and speed for any particular visit was calculated as the average for trials 1 and 2. If either trial was missing, then speed from the completed trial was used. For the BBS, if at least two questions were missing at a visit, the score was set to missing. If two or fewer questions were missing, these were imputed using the respondent-specific mean score. For MSIS-29 PHYS, if a patient had missing data for <10 of the 20 items, the mean of the non-missing items was used to impute the missing items. If ⩾10 items were missing, then the PHYS score was set to missing. For EQ-5D-5L, no imputation was used for missing values for the five summary scores or for VAS. A summary utility index value was calculated for patients with non-missing data for each of the five questions at a visit. The crosswalk method23 was used to map EQ-5D-5L to the EQ-5D 3-level United Kingdom value set, because value sets for EQ-5D-5L are still under development. Utility index value ranges from −0.594 (worst health state) to 1.000 (best health state). BODY.RESULTS: A total of 132 patients were randomised at 24 sites in Belgium, Canada, Italy, the Netherlands, Sweden and the United Kingdom. The first patient was treated on 30 August 2012 and the trial ended on 8 August 2013. All randomised patients were treated and included in the analysis: 64 were treated with placebo and 68 with PR-fampridine. In each group, 81% of patients completed treatment and 19% of patients discontinued treatment (Figure 1). AE was the most common reason for discontinuation and one patient in each treatment group discontinued owing to lack of efficacy (Figure 1). Patient characteristics were comparable between treatment groups at baseline (Table 1). Figure 1.MOBILE patient disposition. AE: adverse event; CrCl: creatinine clearance; ITT: intent-to-treat; PR: prolonged-release. Table 1. Baseline characteristics. Characteristic Placebo ( n = 64) PR-fampridine ( n = 68) Total ( N = 132) Mean age, years 49.8 49.8 49.8 Female, n (%) 33 (52) 38 (56) 71 (54) Mean (SD) body mass index, kg/m 2 26.5 (6.2) 26.8 (4.9) 26.6 (5.6) Mean (median) time since first MS diagnosis, years 12.4 (12.0), n = 63 10.9 (9.5) 11.6 (10.0), n = 131 Disease course, n (%)  RRMS 20 (31) 24 (35) 44 (33)  SPMS 37 (58) 31 (46) 68 (52)  PPMS 6 (9) 12 (18) 18 (14)  PRMS 1 (2) 1 (1) 2 (2) Mean (median) time since most recent relapse, years 3.3 (2.4), n = 56 4.2 (3.5), n = 56 3.8 (2.8), n = 112 Mean (median) EDSS score 5.9 (6.0) 5.6 (6.0) 5.7 (6.0) Outcome measures: median (mean) [min, max]  MSWS-12 score 81.3 (75.9) [8.3, 100.0] 75.0 (71.7) [25.0, 100.0] ND  TUG speed, m/s 0.32 (0.34) [0.0, 0.8], n = 63 0.38 (0.38) [0.1, 0.7] ND  BBS score 41.0 (39.3) [5.0, 56.0], n = 63 43.8 (40.9) [6.5, 56.0] ND  MSIS-29 PHYS score 57.5 (53.0) [13.1, 91.9] 50.0 (50.9) [8.1, 100.0] ND  EQ-5D-5L utility index score 0.547 (0.509) [–0.19, 1.00] 0.584 (0.540) [0.04, 0.85] ND  EQ-5D-5L VAS 60.0 (59.1) [4.0, 90.0], n = 63 60.0 (61.6) [25.0, 90.0] ND BBS: Berg Balance Scale; EDSS: Expanded Disability Status Scale; EQ-5D-5L: EuroQoL-5 Dimension 5-level; MSIS-29: 29-item Multiple Sclerosis Impact Scale; MSWS-12: 12-item Multiple Sclerosis Walking Scale; ND: not determined; PHYS: physical subscale; PPMS: primary-progressive multiple sclerosis; PR: prolonged-release; PRMS: progressive-relapsing multiple sclerosis; RRMS: relapsing-remitting multiple sclerosis; SPMS: secondary-progressive multiple sclerosis; TUG: Timed Up and Go; VAS: visual analogue scale. PR-fampridine therapy resulted in greater median improvements from baseline in TUG speed, BBS total score and MSWS-12 score compared with placebo during the 24-week treatment period (Figure 2(a), (c) and (d)). After treatment discontinuation at week 24, improvements declined and approached zero by the week 26 follow-up visit in the PR-fampridine group. Figure 2.Median changes from baseline and corresponding 95% confidence intervals in efficacy measures by study visit. Outcome measures: (a) 12-item Multiple Sclerosis Walking Scale (MSWS-12); (b) 29-item Multiple Sclerosis Impact Scale (MSIS-29) physical subscale (PHYS); (c) Timed Up and Go (TUG) test; and (d) Berg Balance Scale (BBS) were assessed at baseline (mean over screening and day 1) and weeks 2, 4, 8, 12, 16, 20, 24 and week 26 (off-treatment visit; not assessed for MSIS-29). Error bars denote non-parametric 95% confidence interval for the median change at each visit. PR: prolonged-release. When changes from baseline were summarised as a change at pre-specified thresholds of improvement, a higher proportion of patients receiving PR-fampridine versus placebo met each threshold of improvement in the TUG test (>0% and from ⩾10% to ⩾40%), with statistically significant differences at thresholds ⩾10% (p = 0.0021) and ⩾15% (p = 0.0262; Figure 3). Similar results were observed for the MSWS-12; a higher proportion of patients receiving PR-fampridine versus placebo met each threshold of improvement in MSWS-12 (⩾1 to ⩾10 points) with statistically significant differences at thresholds ⩾7 (p = 0.0275), ⩾8 (p = 0.0153) and ⩾9 (p = 0.0088; Figure 3). Figure 3.Cumulative percentage of patients with mean improvement in 12-item Multiple Sclerosis Walking Scale (MSWS-12) and Timed Up and Go (TUG) speed over 24 weeks. MSWS-12 (upper panel): cumulative percentage of patients with increasing levels of improvement on the MSWS-12 over the on-treatment period (weeks 2–24) across multiple thresholds (thresholds ⩽–1 to ⩽–10 represent improvements in ⩾1 to ⩾10 points). TUG speed (lower panel): cumulative percentage of patients with average percent increase from baseline in TUG speed over the on-treatment period (weeks 2–24) across multiple thresholds. PR-fampridine versus placebo: *p = 0.0275; **p = 0.0153; ***p = 0.0088; ****p = 0.0021; *****p = 0.0262. PR: prolonged-release. In a post hoc analysis, a significantly greater proportion of patients (n (%)) treated with PR-fampridine (31 (46%)) versus placebo (16 (26%)) also reported improvement on PGIC at the week 2 visit (p = 0.023). PR-fampridine therapy also resulted in greater improvements from baseline in MSIS-29 PHYS score versus placebo (Figure 2(b)). These improvements were apparent throughout the 24-week trial duration. However, no apparent differences between treatment groups were observed in EQ-5D-5L results; median treatment difference (95% confidence interval) for PR-fampridine versus placebo for EQ-5D-5L VAS was 0.00 (–4.17, 4.67) and for utility index was 0.00 (–0.04, 0.04). The proportion of patients with any AE was similar in placebo-treated patients (49 (77%)) versus patients receiving PR-fampridine (51 (75%)). Nasopharyngitis and urinary tract infections (UTIs) were the most frequently reported AEs in patients treated with PR-fampridine and placebo, respectively (Table 2). Incidence of falls was higher in patients receiving placebo versus PR-fampridine, whereas incidences of balance disorder, gait disturbance and dizziness were higher in patients receiving PR-fampridine. A lower proportion of patients receiving PR-fampridine (3%) versus placebo (8%) reported SAEs. In the PR-fampridine group, SAEs were considered unrelated to treatment and included moderate MS relapse in one patient and moderate MS relapse and severe paraparaesis (worsened MS symptoms) in the second patient. No seizures were reported during treatment. Table 2. Incidence of treatment-emergent AEs reported for ⩾5% of patients. n (%) Placebo ( n = 64) PR-fampridine ( n = 68) No. of patients with AE 49 (77) 51 (75) No. of patients with SAE 5 (8) 2 (3) AEs  Nasopharyngitis 9 (14) 11 (16)  Urinary tract infection 12 (19) 6 (9)  Back pain 3 (5) 6 (9)  Balance disorder 1 (2) 6 (9)  Headache 5 (8) 5 (7)  Gait disturbance 2 (3) 5 (7)  Fall 8 (13) 4 (6)  Arthralgia 4 (6) 4 (6)  Fatigue 4 (6) 4 (6)  Nausea 3 (5) 4 (6)  Dizziness 1 (2) 4 (6)  MS relapse 3 (5) 3 (4)  Influenza 4 (6) 2 (3)  Muscle spasms 3 (5) 1 (1) AE: adverse event; PR: prolonged-release; SAE: serious adverse event. BODY.DISCUSSION: In this trial, PR-fampridine therapy resulted in greater/sustained improvements in mobility/balance over the six-month trial duration compared with placebo as measured by the TUG test and BBS. Benefits were seen as early as two weeks after treatment initiation and were maintained throughout the trial. Upon discontinuation of PR-fampridine therapy, improvements in outcome measures reversed and approached pre-treatment levels within two weeks. Early benefits also were seen in walking ability measured by MSWS-12, and although MSWS-12 score fluctuated between weeks 4 and 12, it stabilised after week 16 and benefits were maintained to the end of the study in patients receiving PR-fampridine therapy. In addition to greater median improvements in TUG speed and MSWS-12 score, a higher cumulative proportion of patients receiving PR-fampridine versus placebo met each threshold of improvement in TUG speed (>0% and ⩾10% to ⩾40%) and MSWS-12 score (⩾1 to ⩾10 points), including thresholds in a range (⩾4 to ⩾6 points) previously identified as clinically meaningful for MSWS-12 with different data sources.24 The MOBILE study was the first randomised, placebo-controlled study that assessed the effect of PR-fampridine on dynamic and static balance. Walking is a complex activity10,11 and individual patients with MS may improve in one or more domains, such as speed and/or balance. The findings of MOBILE suggested that the previous definition of PR-fampridine responder that was based on walking speed alone6,7 may be too narrowly defined and may not identify some patients who received treatment benefits associated with improved balance. PR-fampridine therapy also demonstrated improvements on MSIS-29 PHYS, a patient-reported measure of the physical impact of MS, but no clear differences between treatment groups were observed in EQ-5D-5L VAS and utility index results over time. The discrepancy observed in this trial between the MSIS-29 and EQ-5D-5L may be related to the insensitivity of generic measures to assess MS-related changes in QoL. The MSIS-29 PHYS results in this trial supported those observed in the ENABLE study, in which PR-fampridine therapy demonstrated significant/clinically meaningful improvements on QoL/health state, measured by a broad range of MS-specific and generic patient-reported endpoints, over 48 weeks in >600 patients who received PR-fampridine.25 Safety findings from MOBILE were consistent with the known safety profile of PR-fampridine,6,7,26 with the exception that incidence of UTIs was higher in placebo-treated patients compared with patients receiving PR-fampridine. This is in contrast to that observed in the pivotal studies,6,7 in which incidence of UTIs was higher for the PR-fampridine group. In the current study, UTIs were confirmed by culture. In contrast, the pivotal trials did not require confirmation of UTIs, which may have resulted in an overestimation of the rate of UTIs. No seizures were observed in MOBILE and although more patients receiving PR-fampridine reported balance disorders, gait disturbance and dizziness versus placebo, the incidence of falls was higher in the placebo group. Balance disorders, gait disturbance and dizziness are broad, non-specific AEs, and increases in these events may not result in an increase in falls, as was the case in the MOBILE study. Furthermore, the improvement in TUG and BBS could have manifested in fewer falls among the patients treated with PR-fampridine. The findings of MOBILE confirm and expand findings of previous controlled studies with a longer treatment period, geographically different study population and broader range of objective and patient-reported measures of mobility and balance to assess different domains of walking.6,7 MOBILE was exploratory in design and its findings require confirmation in a prospective trial that is underway.27 Nevertheless, the results of MOBILE support that PR-fampridine therapy provides significant and sustained improvements in walking characteristics beyond walking speed; in particular, PR-fampridine therapy resulted in early and sustained benefits on measures of dynamic and static balance and mobility as well as patient-reported walking disability. Overall, these findings provided additional support for the potential of PR-fampridine to result in clinically meaningful improvements in walking quality/ambulatory function in patients with MS with walking disability. BODY.SUPPLEMENTARY MATERIAL: Supplementary material Supplementary material

TITLE: Psycho-Biological Changes with Add on Yoga Nidra in Patients with Menstrual Disorders: a Randomized Clinical Trial ABSTRACT: Introduction: Menstrual disorders are common problems among women in the reproductive age group. Yuga interventions may decrease the physical and psychological problems related to menstrual disorders. The present study was aimed to assess the effect of Yoga Nidra on psychological problems in patients with menstrual disorders. Methods: A total number of 100 women recruited from the department of obstetrics and gynecology and were then randomly allocated into two groups: a) intervention received yogic intervention and medication for 6 month, and b) control group received no yogic intervention and they only received prescribed medication). Psychological General Well-Being Index (PGWBI) and hormonal profile were assessed at the time of before and after six months on both groups. Results: The mean score of anxiety, depression, positive well-being, general health, and vitality scores, as well as hormonal levels, in posttest were significantly different in intervention group as compared with pretest. But there was no significant difference in control group. Conclusion: Yoga Nidra can be a successful therapy to overcome the psychiatric morbidity associated with menstrual irregularities. Therefore, Yogic relaxation training (Yoga Nidra) could be prescribed as an adjunct to conventional drug therapy for menstrual dysfunction. BODY.INTRODUCTION: Menstrual disorders are common problems faced by women, which contributes to physical and psychological problems. Most luteal phases marked by menstrual bleed occurring between 21-35th day with 3-10 days of bleeding and 30-40 ml of blood loss on an average. Menstrual dysfunction is common problem in the population of reproductive age group. The affected women have high rates of amenorrhea (9%) and menstrual irregularity (33%). The psychological factors including stress and deprivation were found to be associated with menstrual irregularities.1 Approximately 19% of women aged 18-55 years reported experiencing menstruation-related problems during menstrual cycle throughout their reproductive life. The problems include fatigue, pain, vomiting, anxiety, and depression. Fluctuations in hormones that regulate menstruation, such as estrogen and progesterone, can affect appetite, digestion, and energy levels, which all of which affect the mood. Therefore, menstrual disorders are common disorders, which contributes to physical and psychological problems. Either single or combinations of medications are available for the patients with menstrual disorders, e.g. medroxy progesterone, norethistrone, ethinyl estrodiol, levonorgestrol, tranexemic acid, and ethamsylate. Todays, non-pharmacological and complementary approach is proposed for treatment of these complications. Practice of Yoga is simplest method of relaxation which helps to improve the physical and psychological status. Previous study shows that there was significant alteration of autonomic functions and psychological status by using yoga. Moreover, regular practice of yoga has positive effects on menstrual cycle and psychological well-being probably by balancing neuro-endocrinal axis.2 Another study proposed that, yoga reduced the severity and duration of primary dysmenorrhea. Author also suggests that yoga is a safe and simple approach for primary dysmenorrhea.3 The significant effect of relaxation technique was also observed in subjects of premenstrual syndrome, in which the stress is considerably reduced by relaxation.4,5 Previous studies also reports the significant effects of the yoga (especially Yoga Nidra) on the menstrual disorders.6-10 Practice of Yoga Nidra is a simple method of relaxation which is practiced in the flat lying position of shavasana (lying on the back, the arms and legs are spread at about 45 degrees), and followings the spoken instruction of yoga therapist. Yoga Nidra, which is derived from the tantras, is a powerful and wonderful technique. This technique is not only useful for physical or mental relaxation but also for preparing the mind for yogic discipline.5It concerns mainly with Pratyahara (the fifth state of Astanga Yoga which involves withdrawal of senses) and Dharana (concentration). It is to be understood that ordinary sleep is not relaxation and tensions cannot always be resolved completely in ordinary sleep. Yoga Nidra is qualitatively different relaxation. During the practice of Yoga Nidra the consciousness is at different levels. It is a 'sleep' where all the burdens are thrown off to attain more blissful state of awareness, a relaxation much more intense than ordinary sleep. When one practices Yoga Nidra, it opens the deeper phases of the mind.11 Regular and proper practice of Asanas and Pranayamas have been proposed beneficial for all women. It may be useful for women who suffer from menstrual disorders. Asanas can be defined as a physical Yoga posture or position that is designed to help the body and enhance the body's functions. Yoga exercises is useful method by creating strength and endurance, improving circulation and energy flow, cleansing organs and other systems, and expanding muscles and joints. Pranayama is a Sanskrit (ancient Indian language) word meaning "extension of the prana or breath" or "extension of the life force". The word is composed from two Sanskrit words: prana meaning life force (noted particularly as the breath), and ayama, to extend or draw out. (Not "restrain, or control" as is often translated from yam instead of ayama). Therefore, Asanas and Pranayamas are helpful in preserving the general health and reducing the minor complaints regarding menstruation by improving the vigor and strength of the body.12However, it should be noted that, yogic practices are not advisable during menstruation for 3-5 days. Yoga helps correcting and balancing the functioning of the endocrine system, toning up nervous system by relaxing the body and mind, and reducing psychological problems of the patients.11 In the present study we want to examine the effect of Yoga Nidra on the menstrual disorders and psychological problem associated with menstruation. BODY.MATERIALS AND METHODS: The study was a randomized controlled trial among women with menstrual irregularities. The study was conducted in Lucknw, Uttar Pradesh (India) from 2008 to 2013. The study was granted by institutional ethics committee and written informed consent was obtained from all participants. Sample size of the study was calculated with the help of PS Power and Sample Size Calculations (version 2.1.30; William D. Dupont and Walton D. Plummer, Vanderbilt University, USA), by considering type I error of 0.05 and power of 80%, the expected difference in the experimental and control mean was 1.8 with standard deviation of 4.35 and ratio between intervention and control group was considered.13 A total of 100 subjects were included and were randomly assigned into two groups including intervention and control group. The intervention group consisted of 50 subjects who received both Yoga Nidra apart from regular medications and the control group consisted of 50 subjects who received only medications. Subjects were randomly assigned to each group. For this, we used computer-generated randomization table in the study. Small paper chits written either case or control were placed in opaque, sequentially numbered envelopes prepared by a biostatistician who was blind to the participants. Women suffering from menstrual irregularities diagnosed by senior consultants were recruited from the Department of Obstetrics and Gynecology, King George's Medical University U.P Lucknow. Participants aged between 18 to 45 years with current menstrual irregularities (pathological amenorrhea, dysmenorrhoea, oligomenorrhoea, polymenorrhoea, hypomenorrhoea, menorrhagia, and metrorrhagia) for more than 6 months were included. Interested individuals were initially screened for eligibility by senior consultants. According to inclusion and exclusion criteria, eligible subjects were invited for yoga classes. Conservatively diagnosed patients who are consenting were recruited. Women having known gynecological neoplasm, requiring surgery, having Pelvic inflammatory disease (PID), and pregnant women are excluded from the study. The anthropometric measurements and history of pervious treatments such as physical therapy and yoga therapy were inquired and recorded. Either single or combination of medications e.g. Medroxy progesterone, norethistrone, ethinyl estrodiol, levonorgestrol, tranexemic acid, ethamsylate were prescribed to the patients by the consultants as and when required. Baseline assessments of all the subjects were carried out. Psychological assessment employing Psychological General Well-being Index (PGWBI) and biochemical parameters including thyroid-stimulating hormone (TSH), follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, progesterone, estradiol, testosterone, and DHEA-S (dehydroepiandrostone sulfate) was conducted before and after six months of Yoga Nidra intervention. Psychological General Well-being Index (PGWBI)14 consists of 22 self-administered items, rated on a 6-point scale so the score varies between 22 to 132, which assess psychological and general well-being of respondents in six health related quality of life (HRQoL) domains: anxiety, depression, positive well-being, self-control, general health and vitality. The higher scores indicate better quality of life. Anthropometric measurements were also recorded. Height was measured with the participants standing without shoesand was recorded to the nearest of 0.5 cm. Weight was measuredusing a digital scale, with the participants wearing light cloths, and was recorded to the nearest 100 grams. Five milliliters of peripheral fasting blood was collected during 2nd or 3rd days of menstrual cycle (follicular phase) from all the subjects before yogic intervention for baseline investigations and after six months. For estimation of hormonal profile, serum was separated by centrifuge machine (3500-4000 rotations/minute) at room temperature. The following kits were used for the estimation of the parameters studied – TSH ELISA by RFCL Ltd. SIDCUL, Haridwar Uttarakhand, FSH, LH, Prolactin ELISA by Dialab Ges. M.B.H., Estradiol, Progesterone, Testosterone by Syntron Bioresearch, Inc., and DHEA-S ELISA by Adaltis Italia S.P.A. The laboratory tests were performed in the Department of Pathology, King George's Medical University U.P Lucknow. External quality assurance service to the laboratory was provided by the Bio RAD and internal assurance was also procured. Elisa reader was used for measuring the variables taken in the study. Yoga Nidra intervention: the Yoga Nidra intervention was originally developed by Swami Satyananda Saraswati, School of Yoga, Munger, Bihar, India.11 Yoga instructor was selected by an expert panel for this study. The yoga classes for the patients consisted of 30 to 35 minute /day session, five days in week where Yoga Nidra taught by a trained Yoga Therapist at the Department of Physiology King George's Medical University, Lucknow, Uttar Pradesh for first three months. Thereafter the patients were advised to continue the same at their homes for next three months; the yoga lasted for 6 months in the intervention group. Also, a diary provided to them for noting the schedule of home based practice was verified. The details were cross checked with attendant accompanying. Statistical analysis was done using GraphPad InStat version 3.05 software Inc. year 2000. Paired t-test was used to determine the mean difference scores of all the parameters of the subjects at baseline and after six months in both intervention and control groups. The differences in pre and post intervention scores were used for the analysis. This was done to take into account the imbalances, if any, at the baseline characteristics of the subjects. The independent t-test was used to compare the differences in scores between the groups (yoga vs. non yoga group).15,16 BODY.RESULTS: Out of 100 subjects, 5 subjects from intervention group and 8 subjects from control group were considered drop outs. Out of 13 drop outs, 8 patients could not follow the time schedule and 5 were not willing to continue as research subjects after one week. Out of 50 subjects from the intervention group, 45 subjects attended Yoga Nidra sessions till end (completed at least 80% classes of Yoga Nidra). One of the patients who dropped out in later part of the study reported non-restorative sleep after first four to five days of initiating the Yoga Nidra practices. Another lady who discontinued found it difficult to find time for the practicing Yoga Nidra. The numbers of subjects completing the study were 45 and 42 in intervention and control groups, respectively. The Mean (SD) age of women was 26.57 (7.45) years in the intervention group, and 25.52 (6.27) year for control group. Table 1 and 2 shows baseline and post intervention PGWBI scores and hormonal profile of both intervention and control groups. The mean score of anxiety, depression, positive well-being, general health, and vitality scores in posttest were significantly different in intervention group as compared with pretest (P<0.05) (Table 1). But there were no significant differences in control group. Table 2 depicts the significant differences in The mean of hormonal level including Thyroid-Stimulating Hormone, Follicle-Stimulating Hormone, Luteinizing Hormone, Prolactin within intervention group was significantly different in posttest as compared with posttest (P<0.05). But there was no significant differences in control group (P>0.05) (Table 2). Table 3 and 4 depicts comparison of mean differences in PGWBI scores and hormonal profile. Significant improvement in domains of anxiety (P<0.01), depression (P<0.02), positive well-being (P<0.01), general health (P<0.04) and vitality (P<0.02) in intervention group was noted after six months of yogic intervention when compared to control group (Table 3). There was no statistical difference in domain of self-control in intervention group when compared control group (Table 3). We found significant changes in thyroid-stimulating hormone (P<0.02), follicle-stimulating hormone (P<0.01), luteinizing hormone (P<0.001) and prolactin (P<0.03) in intervention group as compared to control group (Table 4). Table 1 Mean of scores of PGWBI (psychological general well-being schedule index) at baseline and after six months in intervention and control groups (paired t-test) Intervention group, (N=45) Control group, (N=42) Baseline Post-test t P Baseline Post-test t P Anxiety 14.1 (3.35) 16.2 (3.30) 3.24 0.02 14.3 (4.15) 14.9 (4.60) 1.74 0.5 Depression 9.25 (2.11) 10.7 (1.30) 2.56 0.02 9.35 (2.91) 9.85 (2.53) 1.61 0.1 Positive well-being 11.3 (2.43) 12.8 (1.63) 2.65 0.01 11.2 (1.93) 11.37 (3.48) 1.21 0.3 Self-control 9.0 (2.40) 9.1 (2.19) 2.50 0.07 9.28 (2.71) 9.98 (2.61) 1.32 0.3 General health 9.08 (2.87) 10.1 (2.81) 2.61 0.04 9.37 (3.02) 9.98 (2.71) 1.11 0.2 Vitality 11.2 (2.81) 12.3 (2.54) 2.11 0.03 10.9 (2.53) 10.88 (3.21) 1.52 0.2 Table 2 Mean of hormonal profile at baseline and after six months in intervention and control groups (paired t-test) Intervention group, (N=45) Control group,(N=42) Baseline Post-test t p Baseline Post- test t P TSH (µlU/ml) 3.80 (2.03) 2.36 (1.82) 5.93 0.001 3.42 (2.27) 3.17 (2.00) 1.20 0.3 FSH (µlU/ml) 17.03 (7.72) 16.16 (6.88) 5.02 0.01 16.16 (7.55) 16.04 (4.96) 1.71 0.4 LH (mlU/ml) 19.23 (10.47) 17.40 (8.70) 9.37 0.002 18.99 (8.18) 18.14 (7.91) 1.51 0.2 Prolactin (ng/ml) 16.42 (6.86) 15.59 (6.37) 8.37 0.01 15.94 (5.63) 15.28 (6.91) 1.72 0.6 Progesterone (ng/ml) 7.27 (7.17) 7.82 (7.26) 9.51 0.08 8.35 (7.83) 8.95 (7.51) 1.71 0.4 Estradiol (Pg/ml) 112.56 (73.80) 113.8 (66.94) 23.64 0.14 113.36 (69.06) 113.54 (68.99) 1.34 0.4 Testosterone (ng/ml) 1.71 (1.28) 1.55 (1.13) 4.42 0.26 2.90 (1.75) 2.23 (1.68) 1.42 0.1 DHEA-S (µg/ml) 1.79 (0.81) 1.43 (0.81) 4.01 0.35 1.92 (1.02) 1.74 (1.03) 1.65 0.6 TSH: Thyroid-stimulating Hormone, FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone and DHEA-S: Dehydroepiandrostone Sulfate Table 3 Comparison of mean differences in PGWBI between intervention and control groups (independent sample t-test) Intervention group (N=45) Control group (N=42) t p Anxiety 0.75 (0.42) 0.57 (0.22) 2.30 0.01 Depression 0.78 (0.45) 0.43 (0.22) 2.12 0.02 Positive well-being 0.85 (0.33) 0.62 (0.40) 2.16 0.01 Self-control 0.55 (0.22) 0.64 (0.33) 0.18 0.7 General health 0.90 (0.33) 0.67 (0.22) 1.68 0.04 Vitality 0.52 (0.11) 0.31(0.18) 1.18 0.02 Table 4 Comparison of hormonal profile (student’s independent sample t-test) between intervention and control groups Variables Intervention group (N= 45) Control group (N=42) P TSH (µlU/ml) 0.54 (0.61) 0.18 (0.97) 0.02 FSH (mlU/ml) 1.72 (2.27) 0.75 (0.48) 0.01 LH (mlU/ml) 1.46 (1.21) 0.52 (2.17) 0.001 Prolactin (ng/ml) 1.66 (1.32) 1.34 (1.35) 0.03 Progesterone (ng/ml) 1.39 (1.34) 1.41 (1.39) 0.09 Estradiol (Pg/ml) 2.04 (1.78) 2.33 (1.49) 0.30 Testosterone (ng/ml) 0.52 ( 0.67) 0.67 (1.07) 0.24 DHEA-S (µg/ml) 0.28 ( 0.67) 0.15 (0.37) 0.27 TSH: Thyroid-stimulating hormone, FSH: Follicle stimulating hormone, LH: Luteinizing hormone and DHEA-S: Dehydroepiandrostone Sulfate BODY.DISCUSSION: The present study revealed significant improvement in anxiety scores after six months of intervention with Yoga Nidra in intervention group in comparison to control group. Previous studies have also shown significant reduction in the trait anxiety scores following meditation17 and breathing exercises.18 Studies have also reported reduction in state anxiety following muscle relaxation techniques and listening to music.19 During anxiety, there is an increased response of hypothalamus and heightened sympathetic activity. Yoga Nidra appears to regulate hypothalamus, in a way resulting in decreased sympathetic (excitatory) nervous activity and increased parasympathetic (inhibitory) function.11 In the intervention group which practiced Yoga Nidra for six months, there was significant decrease in their degree of depressive symptoms (according to the psychological general wellbeing Index). Other studies also have shown that those with depression could be benefited from Sudarshan Kriya and related practices.20 Previous studies have also shown that using yoga interventions in other conditions (cancer survivors, self-reported emotional distress), found to be effective in depressive and mood symptoms, as well as anxiety and physical well-being.21 The results showed that there was a significant improvement in positive wellbeing, general health and vitality in intervention group. Yoga Nidra is believed to balance psychic and vital energies within the psychic channels (Nadis) of the energy framework underlying the physical body. Free flow of these energies is considered to be the basis of optimal physical and mental health. Findings from other studies also is in line with present study.22-24 However, in domain of self-control there was no statistically significant difference, but in intervention group there was more improvement in this parameter when compared to the control group. However, previous studies have found significant improvement in self-esteem with Yogic exercises.25 Furthermore, the results showed that Yoga Nidra intervention program decreased depressed mood, feelings of guilt, insomnia, genital problems, tension, fear and anxious mood which are symptoms included as items of PGWBI. Subjects experienced better control over their feelings. Previous studies have also shown the beneficial effects of one week of yoga program in self rated fear, anxiety, sadness and disturbed sleep in tsunami survivors of the Andaman Islands. There was a significant decrease in self rated fear, anxiety, sadness and disturbed sleep, respiratory and heart rate was also significantly improved.26 There was significant change in TSH, FSH, LH, and Prolactin levels in intervention group as compared to the control group. Previous studies found that long term practice of yoga lead to decreased TSH, growth hormone, and prolactin imbalances significantly. Metabolic effects of meditation (Yoga Nidra) includes a decreased adrenocortical activity, long term decreased cortisol secretion and lesser thyroid stimulating hormone (TSH) abnormalities.27 Metabolic effects of meditation (Yoga Nidra) includes a decreased adrenocortical activity, long term decreased cortisol secretion and decreased thyroid stimulating hormone (TSH). Imbalances in the hormonal profile also predispose women to depression, especially in relation to pituitary, thyroid and reproductive hormones. The practice of hatha yoga and Asanas has been found to be extremely effective in rectifying the situation.12 Menstruation is dependent on the proper functioning of the chain made up of hypothalamus- pituitary-ovary and uterus. Pituitary hormones, follicle stimulating hormone (FSH), luteinizing hormones (LH), prolactin and thyroid hormones are required for normal development of ova and need to be investigated in cases of chronic anovulation oligomenorhea and amenorrhea.28 It was inferred that after Yoga Nidra practice, patients acquired relief in heavy bleeding and irregular menstrual periods. As other research project, our study has some limitations. The main limitation was that amenorrhea, dysmenorrhea, oligomenorrhea, polymenorrhea, menorrhagia, metrorrhagia, and hypo menorrhea were included in the study all together. The sample size was not large enough to analyze the hypomenorrhic group and hypermenorrhic subgroups separately. The information of the menstrual cycle was based on the participant's self-reports not based on ultrasound scans; this is also another limitation of the study. In future this study could be repeated in other populations with large number of patients. Further, some other yogic practices may be tried and compared with the present ones. BODY.CONCLUSION: Yoga Nidra can be an effective practice to overcome the psychiatric morbidity associated with menstrual irregularities apart from bringing the hormonal profile towards normalcy. Therefore, Yogic relaxation training (Yoga Nidra) could be prescribed as an adjunct to conventional drug therapy for menstrual dysfunction. BODY.ACKNOWLEDGMENTS: We are very thankful to entire yoga team and faculty of department of physiology, department of geriatric mental health, department of obstetrics & gynecology, and King George's Medical University U.P Lucknow. The financial support was received from department of AYUSH, ministry of health and family welfare, government of India. BODY.ETHICAL ISSUES: None to be declared. BODY.CONFLICT OF INTEREST: The authors declare no conflict of interest in this study‏.