Magnetic resonance imaging (MRI) is often used to diagnose and monitor treatment effects of juvenile spondyloarthropathy (SpA). Our objective was to describe MRI findings in juvenile SpA and determine predictors of active sacroiliitis and response to treatment.Children who had MRI of the sacroiliac (SI) joints and were referred to the pediatric rheumatology clinic from 2009 to 2012 were retrospectively studied. The clinical parameters, laboratory studies and findings on MRI were collected and a composite score ratio (CR) was calculated for both SI joints on each MRI study based on a semi-quantitative scale that included evaluation of bone marrow edema (BME), synovial enhancement (SE), and erosions (ER). The findings on MRI were correlated with clinical and laboratory values.50 subjects who underwent 76 MRI for suspected or known SpA were included in the study. Sacroiliitis was seen in 48 MRIs in 32 subjects. Of the subjects with sacroiliitis, mean age ± standard deviation was 13.7 ± 2.6 years, 71% were male and 41% were HLA B27 positive. SE without BME was seen in 31% cases of sacroiliitis. In subjects with sacroiliitis, 79% also had hip arthritis and 41% had enthesitis of the pelvic region on MRI. In 38% of subjects with sacroiliitis, physical exam was not indicative of sacroiliitis or hip arthritis. Longitudinal data were available for 13 subjects. Sacroiliitis on MRI improved in 9 subjects with the greatest improvement in MRI composite score ratio after initiation of etanercept therapy. CR improvement was due to improvement of BME and SE components, while the ER score remained the same or worsened in all but 1 subject.History, physical exam or laboratory data may not predict sacroiliitis in children. Magnetic resonance imaging plays a valuable role in the initial evaluation and later treatment monitoring of children with spondyloarthropathy. Synovial enhancement is significantly reduced after treatment, and unlike adults, synovial enhancement may be detected without accompanying bone marrow edema, which suggests gadolinium contrast may be an important component in the assessment of children with spondyloarthropathy. Spondyloarthopathy (SpA) is a type of arthritis with unique features of axial joint disease and abnormal new bone formation,2. SacroRadiography only detects structural damage that occurs late in the disease. Magnetic resonance imaging (MRI) will detect earlier stages of inflammation and lesions of sacroiliitis that are occult on radiography. Bone maThe aims of our study are to describe MRI findings of juvenile SpA, determine predictors of active sacroiliitis from the history, physical exam, and laboratory findings, and describe treatment effects of sacroiliitis as depicted on MRI.This was a retrospective chart and imaging review of children age 5 to 21 years who were seen in the Pediatric Rheumatology clinics from 2009 to 2012. All children had an MRI of the SI joints ordered by either the pediatric rheumatologist or referring provider for evaluation of the SI joints for suspected SpA. Longitudinal data were collected from subsequent MRIs performed in subjects with a diagnosis of sacroiliitis. Sacroiliitis was defined by the presence on the first MRI examination of synovial enhancement, bone marrow edema, and/or erosions (Please see a detailed description of the MRI scoring system below).Data were collected by chart review on electronic and paper medical records including demographics  and medication history. Race and ethnicity were collected by self-report. Race categories included: African American, Asian, American Indian, Pacific Islander, White, or Other; ethnicity included Hispanic or non-Hispanic. Since this was a retrospective study, the history and physical exam were not performed on the same date as the MRI scan. The history and physical exam were collected from the pediatric rheumatology clinic visit nearest the MRI scan date within 6 months of MRI scan date (prior of after scan date); however treatment changes could not be made within the time period between the clinic visit and MRI scan date.Data collected from the subject’s history included complaints of back pain, hip pain, and morning stiffness. The history was collected from a review of systems checklist on the patient intake form and extrapolated from the recorded history of the treating rheumatologist. Elements of the physical exam collected included active hip arthritis defined as pain on range of motion (POM) and limited range of motion (LOM), LOM only or POM only of the hips, tenderness to palpation over SI joints, and tenderness over the SI joint on pelvic compression with moderate pressure, and anterior spinal flexion measured by modified pediatric Schober’s exam,10. The Laboratory data collected included white blood cell count (WBC), platelet count (Plt), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and human leukocyte antigen B27 (HLA-B27). Laboratory data had to be within 12 weeks of MRI scan date, and no treatment changes could be made within the time frame between the laboratory collection date and MRI scan date. Abnormal inflammatory markers were defined as: WBC greater than 15 × 10/L, PlateMRI examinations were performed at either 1.5 Tesla with a Signa Twinspeed or 3 Tesla Discovery 750HD scanner  with patients placed in supine position. The following pulse sequences were obtained in an oblique coronal orientation dedicated to the SI joints: short tau inversion recovery (STIR), T1-weighted, and T1-weighted fat saturated pre- and post-gadolinium (intravenous Gd-DTPA 0.1 mmol/kg body weight) contrast enhanced images. The slice thickness was 4-5 mm; field of view = 20 cm; matrix = 512 × 512 for the T1-weighted sequence and 256 × 256 for the STIR and fat saturated T1-weighted sequences.MR images were evaluated by a board-certified pediatric radiologist with an extra-year of fellowship training in musculoskeletal radiology and six years of post-fellowship experience. A subset of 22 MRI exams were evaluated independently by another board-certified pediatric radiologist with an additional year of fellowship training in abdominal and pelvic imaging and three years of post-fellowship experience in the interpretation of imaging examinations of pediatric bones and joints.The MRI findings of sacroiliitis were defined as described by Rudwaleit et. al. and included the presence of synovial enhancement, bone marrow edema, and/or erosions. MRI exaPredictors of MRI-diagnosed sacroiliitis were determined using Fisher’s exact for categorical variables  and two-sample t-tests for continuous variables . Inter-reader agreement for MRI scoring was evaluated using Kendall’s tau and Kappa statistics. Treatment effects on MRI scores in longitudinal data were evaluated using non-parametric analysis with Wilcoxon matched pairs signed rank test. All statistical tests were performed with STATA 12.0 software for Windows .Fifty subjects were included in the study and their demographics are summarized in Table 70% of subjects complained of back pain, 48% hip pain, and 78% morning stiffness. Modified pediatric Schober’s exam was abnormal in 32% of subjects, tenderness over SI joint on palpation or pelvic compression in 30%, and hip arthritis by physical exam in 34%.Seventy-six MRIs were performed in 50 subjects, and 48 MRIs showed sacroiliitis in 32 subjects. ER was seen in 48 exams in 28 subjects, BME in 44 exams in 30 subjects, and SE in 42 exams in 29 subjects. The CR ranged from 0 to 2.67. The inter-reader agreement of CR and individual BME, SE, ER scoring on the subset of exams was poor with 27-54% agreement (kappa = 0.1-0.3). However, inter-reader agreement on presence or absence of active sacroiliitis showed substantial agreement of 81.8% with kappa = 0.6 . Due to the low kappa we then re-evaluated the images and used consensus to make a final determination for the scores for each patient.Of the 32 subjects with MRI-diagnosed sacroiliitis, 71% were male . Mean age was 13.7 ± 2.6 years (range: 8.2-20.8 years), and majority were White (65.6%) followed by Hispanics (18.8%) and African Americans (6.3%) . Two subjects with sacroiliitis also had facet arthritis on MRI; one was a 14.7 year old HLA-B27 (+) Caucasian male, and the second a 14.4 year old HLA-B27 (-) African American female. Two subjects had partial fusion of the sacroiliac joint; one was a 16.8 year old HLA-B27 (+) Hispanic male, and the second a 19.3 year old HLA-B27 (+) Caucasian female.We evaluated the history, physical exam, and laboratory tests at the clinic visit associated with MRI (+) sacroiliitis. Platelet count  sacroiliitis, 75% reported back pain, 56% hip pain, and 78% morning stiffness at their associated clinic visit. Only 1 subject denied all 3 symptoms. Four subjects (13%) had a normal physical exam . No subjects were asymptomatic with a normal physical exam on their initial MRI demonstrating (+) sacroiliitis.The hip joints could be evaluated on 51 of 76 MRI exams in 41 subjects. Hip arthritis was found in 21 subjects on 23 MRI exams (45.1%) and associated with hip pain in 79.2% of exams. Concomitant sacroiliitis was seen in 87% of MRI in 90% of subjects. Using MRI as the gold standard, physical exam consistent with hip arthritis  had sensitivity and specificity of 52.4% (CI = 30.3-73.6%) % and 64.3% (CI = 44.1-80.7%), respectively. When the presence of LOM or POM or both was used to diagnose hip arthritis, sensitivity was 87% (CI 65.3-96.6%) % and specificity 42.9% (CI 25-63.6%).Enthesitis in the pelvic region was seen in 22.4% of all MRI exams and in 31.3% of MRI demonstrating sacroiliitis. Enthesitis was always associated with either sacroiliitis (n = 11 subjects) or MRI (+) hip arthritis (n = 2 = subjects).Fourteen of the 32 subjects with sacroiliitis had at least 1 subsequent MRI to monitor disease activity with a mean follow up period of 2.3 years (range: 0.3-7.0 years). The CR improvement was due to improvement of the SE component, while the ER score remained the same or worsened in all but 1 subject. The BME component did improve but was not statistically significant . Improvement in composite score was due to improvements in BME and SE score components while the ER score component remained the same or worsened in all 9 subjects. HLA-B27 was present in 6 subjects and 8 subjects were male. Eight subjects were taking NSAIDs, 4 were taking etanercept (ETN) only, 3 were taking ETN + MTX, 1 was taking methotrexate (MTX) only, and 1 was taking sulfasalazine when CR decreased. ETN (including ETN + MTX) had the greatest mean% CR decrease of 38% compared to MTX only (25%), and sulfasalazine (20%). Interestingly, the 3 subjects on ETN + MTX had a lower mean %CR decrease (26%) compared to the 4 subjects on ETN only (47%).Of the 5 subjects with worsened or stable composite score ratios, mean increase was 0.58 points (mean percent worsening = 18.9%). Worsening was due to worsening in all 3 components, greatest in the BME component (mean BME increase = 0.6 points). Erosion scores remained the same or worsened in all 5 subjects. HLA-B27 was present in 2 subjects, and 2 subjects were male. Two subjects were taking ETN + MTX, 2 NSAIDs only, and 1 MTX only when their composite score ratios worsened. Of the 2 subjects taking ETN + MTX, one subject was a 15 year-old HLA-B27 (-) female with bilateral sacroiliitis who had been on ETN for 3 months, and the second subject was a 14 year HLA-B27 (+) female with bilateral sacroiliitis who had been on ETN for 12 months.Examining the history and physical exam during follow up MRI, 50% denied back pain, 44% hip pain, and 50% morning stiffness, and 31% denied all 3 symptoms despite persistent active sacroiliitis. On physical exam, modified pediatric Schober’s and hip and SI joint exams were normal in 8 subjects (50%) despite ongoing inflammation. Five subjects (31%) were asymptomatic and had a normal physical exam despite ongoing active sacroiliitis.This work examines potential roles for MRI for children with SpA when placed in the context of their clinical history including physical examination, laboratory values, and treatment. Although this study has a relatively small cohort of 50 subjects with 32 subjects having sacroiliitis identified on MRI, we have found several important results and can draw some important conclusions. Synovial enhancement appears to be an important predictor of active inflammation and treatment effect.The demographics of juvenile SpA differ from ankylosing spondylitis (AS). The male:female ratio was 2.6:1, which is significantly less than previously reported 7:1 ratio in juvenile AS. This raPredicting sacroiliitis in children from history, physical exam, or laboratory results remains a challenge. In our study, only elevated platelet count and MRI (+) hip arthritis predicted sacroiliitis. It is unclear if platelet count is a meaningful predictor, since other inflammatory markers  were not predictive of sacroiliitis. Finding that hip arthritis as a predictor of sacroiliitis is consistent with previous studies in children with ERA or SpA which report hip arthritis as a predictor of sacroiliitis,19. UnfoAt the initial diagnosis of MRI (+) sacroiliitis, almost all patients were symptomatic with back pain, hip pain, or morning stiffness (97%) or had an abnormal physical exam (87%). However, on follow up MRI of previously diagnosed MRI (+) sacroiliitis, symptoms and physical exam were often normal despite ongoing inflammation. Of subjects with persistently active sacroiliitis on subsequent MRI, 50% had a normal physical exam, and 31% were asymptomatic with a normal physical exam. Therefore, children with previously diagnosed sacroiliitis and on therapy can still have active inflammation without findings on physical exam or history. This suggests that physical exam and history should not be the sole guides for altering the therapy for sacroiliitis in children.The MRI characteristics of sacroiliitis in children demonstrated unique features specific to children compared to sacroiliitis in adults. In the majority of cases of MRI (+) sacroiliitis, all 3 components of the MRI score  were positive. Interestingly, there were 15 (31.3%) cases of sacroiliitis where BME was not seen concomitantly with SE, and 80% of these cases had erosions. In adult spondyloarthropathy, SE is rarely seen without BME in cases of active sacroiliitis; therefore, non-contrast imaging for evaluating sacroiliitis has been advocated,21. The In children, hip arthritis and pelvic enthesitis are often seen concomitantly with sacroiliitis on MRI. There was a high prevalence of MRI (+) hip arthritis (79%) in children with MRI (+) sacroiliitis, even without signs of hip arthritis on history or physical exam. This suggests that in children at risk of SpA, SI and hip joint MRI should be performed concomitantly in order to evaluate for the extent of the disease. Pelvic enthesitis diagnosed by MRI may be a specific finding in SpA. Pelvic enthesitis was seen in 31% of MRI (+) sacroiliitis and always associated with either sacroiliitis or hip arthritis on MRI. This is similar to findings in adult spondyloarthropathy where pelvic enthesitis is frequently associated with active sacroiliitis.In subjects with follow up MRI exams, we were able to evaluate response to treatment. 69% of subjects had an improvement of their MRI composite score ratio, and the largest absolute and percent CR decrease  occurred with initiating etanercept therapy. Improvements in MRI composite score ratio was seen in inflammatory components  whereas erosions remained stable or worsened in all cases except one.We observed no significant difference in the pre- and post-treatment groups for BME score. This may be due to several factors. The pre- and post-treatment trend for the BME score was variable; although there was improvement in the mean BME score, several patients actually showed an increase BME score after treatment. BME is a reactive phenomenon that may also be seen when secondary osteoarthritis or mechanical changes about a joint are present-25. ThesThis is a retrospective study with a small number of subjects especially subjects with longitudinal follow up. Many clinical variables were not recorded for all subjects, and useful information such as inflammatory character of the back pain, family history of HLA-B27 related disease, uveitis, improvement in pain/stiffness after therapy, etc. were not consistently available in the clinical records. The indication for requesting an MRI by the ordering physician was not clearly recorded in the medical record and may have varied among providers. The scoring system used in this study has yet to be validated. Several scoring systems have been utilized to evaluate sacroiliitis on MRI in the adult population. The scoring system implemented in this study was a modification from a study previously described in adults. We needWe observed a high percentage of erosions and synovial enhancement in our study cohort than has been reported in other studies focused on adults,28. In aMRI of the sacroiliac joints is often useful in the workup or ongoing evaluation of pediatric patients who are suspected or have SpA. The imaging findings of SpA may be present in children when the physical examination is negative and changes in synovial enhancement may reflect treatment effects. Also, the MRI findings are different in children than adults. Unlike adults, synovial enhancement without bone marrow edema is often present and this supports the use of gadolinium for contrast enhanced imaging to detect and characterize SpA in pediatric patients.MRI: Magnetic resonance imaging; SI: Sacroiliac; BME: Bone marrow edema; SE: Synovial enhancement; ER: Erosions; HLA: Human leukocyte antigen; UCSF: University of California-San Francisco; MRI: Magnetic resonance imaging; POM: Pain on range of motion; LOM: Limited range of motion; WBC: White blood cell count; ESR: Erythrocyte sedimentation rate; CI: Confidence interval; CRP: C-reactive protein; CR: Composite score ratio; SD: Standard deviation; NSAIDs: Non-steroidal anti-inflammatory drugs; ETN: Etanercept; MTX: Methotrexate; SpA: Spondyloarthropathy; AS: Ankylosing spondylitis; ERA: Enthesitis-related arthritis.Clara Lin, MD: none. John MacKenzie, MD: none. Jesse Coutier, MD: none. Jeff Gu: none. Diana Milojevic, MD: none. The authors declare that they have no competing interests.CL conceived of the study, participated in its design, data acquisition, data analysis, and data interpretation, coordinated the study, and drafted the manuscript. JM participated in the design, interpreted all radiographic imaging, and critical review of manuscript for intellectual content. JC assisted in the interpretation of radiographic imaging. JG assisted with data acquisition, study coordination, and study design. DM conceived of the study, participated in its design and data interpretation, and critical review of manuscript for intellectual content. All authors read and approved the final manuscript.CL is an Assistant Professor of Pediatric Rheumatology at Children’s Hospital Colorado. JM is a board certified pediatric radiologist with one with an extra-year of fellowship training in musculoskeletal radiology and six years of post-fellowship experience. His research focuses on developing imaging techniques for the detection and treatment monitoring of autoimmune diseases of the bones and joints. JM is an Assistant Professor in Residence and Chief of Pediatric Radiology at the University of California, San Francisco. JC is a board-certified pediatric radiologist with an additional year of fellowship training in abdominal and pelvic imaging and three years of post-fellowship experience in the interpretation of imaging examinations of pediatric bones and joints. He is an Assistant Clinical Professor in Pediatric Radiology at the University of California, San Francisco. His research interests include technique optimization in pediatric body MR applications. DM is Chief of Pediatric Rheumatology at the Floating Hospital for Children at Tufts Medical Center.


Purpose. To quantitatively evaluate growth plates around the knees in adolescent soccer players utilizing the diffusion-weighted MR imaging (DWI). Methods. The knees and adjacent growth plates of eleven 14-year-old male soccer players were evaluated by MRI before (end of season's summer break) and after two months of intense soccer training. MRI evaluation was conducted in coronal plane by PD-FSE and DWI. All images were screened for any major pathological changes. Later, central growth plate surface area (CGPSA) was measured and the apparent diffusion coefficient (ADC) values were calculated in two most central coronal slices divided into four regions: distal femur medial (DFM), distal femur lateral (DFL), proximal tibia medial (PTM), and proximal tibia lateral (PTL). Results. No gross pathology was diagnosed on MRI. CGPSA was not significantly reduced: DFM 278 versus 272, DFL 265 versus 261, PTM 193 versus 192, and PTL 214 versus 210. ADC decrease was statistically significant only for PTM: DFM 1.27 versus 1.22, DFL 1.37 versus 1.34, PTM 1.13 versus 1.03 (p = 0.003), and PTL 1.28 versus 1.22. Conclusions. DWI measurements indicate increased cellularity in growth plates around knees in footballers most prominent in PTM after intense training. No detectable differences on a standard PD-FSE sequence were observed. Soccer is the most popular and widely played sport worldwide, especially among children and adolescents . The plaThe study was designed as a 4-month single-centre case series. The protocol was approved by the National Medical Ethics Committee (number 86/02/13). The work was conducted in accordance with the Declaration of Helsinki (1964).The study group comprised eleven asymptomatic junior members U14 of the soccer club NK Maribor, Slovenia, who had a history of soccer training for a minimum of 3 years. Informed consent was obtained from all individual participants and their parents. The following exclusion criteria were set: history of lower limb surgery or any serious knee injury, musculoskeletal abnormalities or systemic disease with a possible impact on joints, or presence of metallic foreign bodies that would had prevented MRI examination. Players' general data and physical examination  were acquired upon inclusion. Clinical measurements of lower limb mechanical alignment were conducted in a weight-bearing position with a calliper, similarly as previously described , 21. DemThe initial examinations and baseline MRIs were performed at the end of “summer soccer vacation,” which had lasted from mid-May 2014 till mid-June 2014. During this one-month period the players were not involved in any systematic soccer training. They were allowed to conduct only low intensity running, swimming, and cycling. No cutting and pivoting sports were performed during this period. The players were asked to fill in daily activities diary. After this quiet period, the players were enrolled into preparations for the new season. They followed an intense practice routine of circuit training workouts, aerobic and anaerobic exercises with and without the ball, high intensity running (sprinting and jogging), plyometric and isometric exercises, and soccer training skills . They were also involved in the preparation matches. Participants were active in the training process between 1.5 and 3.5 hours daily, 6 days a week. The second MRI was conducted after two months of such training, within 24 hours after their last sporting activity.b = 0 s/mm2) and the other with diffusion weighting (b = 400 s/mm2) with the inferior-superior direction of gradient orientation. FOV included the distal femur and proximal tibia in a coronal plane thereby including the distal femoral and proximal tibial metaphysis  MRI scanner using eight-channel transmit-receive knee coil. Both knees were scanned in 7 players and only dominant leg was scanned in 4 players due to limited parent consent. MRI examinations were first performed in the proton-density fast spin-echo (PD-FSE) sequence with eighteen fat-suppressed slices in the coronal plane: TR (repetition time) = 2080 ms, TE (echo time) = 11 ms, ETL = 6, ST (slice thickness) = 3 mm, spacing = 0.3 mm, FOV (field of view) = 18 cm, and matrix = 384 × 384 . This putaphysis .All MRI analyses and measurements were performed by consensus of a musculoskeletal radiologist with 10 years of experience (MR) and an orthopaedic surgeon (ZK) being blind to the study group and the examination time. They were performed 2 months after the last MRI to minimize bias resulting from the consensus reading.Coronal fat-suppressed PD-FSE images were first analysed for any gross pathology in and around the knee. Screening for BME was then performed. BME was defined as an area of visually detected clearly increased signal intensity in the bone marrow at least on two consecutive PD-FSE images, measuring between 0.5 cm² and 1.5 cm². Larger homogenous areas of slightly increased signal intensities, found predominantly in the diaphysis of femur and tibia, were not considered as BME, since they represent areas of active red marrow, which may be normally present in this age group .b values, apparent diffusion coefficient (ADC) maps were calculated. This was followed by the calculation of ADC values (mm/s × 10−3) for each region with the subsequent average of both slices. Also, the area of each region was recorded and the average was calculated from both consecutive slices representing the central growth plate surface area (CGPSA) value (mm2).For DWI analyses, the region of interest (ROI) encircled the border of the growth plates of femur and tibia in two consecutive most central coronal slices. At each slice, ROIs were divided into medial and lateral half  yielding CGPSA and the ADC in each of the growth plate regions were compared by Student's t-test for paired samples with a statistical significance set to P < 0.05. The computer software IBM SPSS Statistics, Version 20, was used. Post hoc power analysis of ADC preactivity to postactivity values in the proximal tibia medial growth plate (PTM) showed an effect size of 0.80 and a statistical power of 0.95 .All the data are presented as mean values with SD. Preactivity to postactivity values ofp = 0.003); and PTL 1.28 versus 1.22  to mechanical overload of proximal medial tibial physis . The susb value) is determined by the amplitude of the gradient pulses, as well as by their duration and spacing. Two DWI acquisitions with different b values enable calculation of ADC map. Higher ADC values correspond to elevated diffusion in the extracellular space; the motion of water is less restricted [−3 mm2/s at 37°C) [−3 mm2/s) [The histological evaluation of growth plates in humans is limited to the material retrieved at the surgical procedure, epiphysiodesis, which cannot be used for systematic studies of sporting population. Conventional radiography, CT, and MRI had been used in the assessment of growth plates injuries in the past , 25, 26.stricted . In areastricted . One of at 37°C) , but hig3 mm2/s) , 17. Dec3 mm2/s) , 28. Con3 mm2/s) . PreservBME signal is an unspecific MRI finding showing increased water content in the bone marrow accompanying fracture, stress reaction, bone contusion, inflammation, or tumour. Therefore the correlation with other imaging and clinical findings is necessary. The few areas of BME, found in three cases, most probably represent an unspecific stress reaction that was previously already described by Soder et al. in their study of asymptomatic adolescent soccer players . Preservb value  is susceptible to influences related to perfusion and T2. However we were cautious not to lose signal also in the growth plates with higher b values, being aware of biexponential behaviour of the signal intensity decay observed with increasing b values [The presented study has the following limitations. The study group was rather small therefore necessitating the confirmation of the results by a prospective study on a larger number of sporting and nonsporting adolescent subjects. To minimize bias resulting from consensus analyses by the MSK radiologist and the orthopaedic surgeon, MRI analyses were performed 2 months after the last MRI. Still, this might represent another study limitation. DWI with low b values , in partThe presented study confirmed the feasibility of DWI in the evaluation of growth plates. Quantitative DWI measurements indicate increased cellularity in the medial part of the proximal tibial growth plate around the knee linked to intense soccer training in asymptomatic adolescent players. This suggests an asymmetric growth plate involvement that may consequently lead to bow-leg deformity. No detectable differences on a standard PD-FSE sequence were observed. Having a quantitative imaging tool for growth plates evaluation is important to delineate harmful sporting activities and to avoid or modify them accordingly to prevent long-term impacts on the growing skeleton.


Serum 25-hydroxyvitamin D [25(OH)D], the major circulating form of vitamin D used for evaluating the vitamin D status of patients, has been associated with survival in a variety of cancers with conflicting evidence. We aimed to investigate this association in newly diagnosed advanced non-small-cell lung cancer (NSCLC) patients.This was a consecutive cohort of 359 newly diagnosed stages III-IV NSCLC patients who underwent a baseline serum 25(OH)D evaluation prior to receiving any treatment at our institution between January 2008 and December 2010. We used the vitamin D categories of “deficient (<20 ng/ml)” and “not deficient (> = 20 ng/ml)”. Cox regression was used to evaluate the prognostic significance of serum 25(OH)D after adjusting for relevant confounders.p = 0.06). Season of diagnosis, performance status, smoking status and hospital location significantly predicted vitamin D status. On univariate Cox analysis, gender, stage of disease, hospital location, histologic subtype, subjective global assessment (SGA), performance status, smoking status, body mass index and serum albumin were significantly associated with survival . On multivariate Cox analysis, six variables demonstrated statistically significant associations with survival: stage of disease, hospital location, histologic subtype, SGA, smoking status and serum albumin . Serum vitamin D, which was borderline significant in univariate analysis, lost its significance in multivariate analysis.Mean age at diagnosis was 57.4 years. Of the 359 patients, 151 (42.1 %) were deficient in vitamin D at the time of diagnosis. The median survival in deficient and not deficient cohorts was 11.7 and 12.8 months respectively D and survival in our patients. While several studies have demonstrated a positive association between serum vitamin D and survival in multiple cancer types including gastric , colorecSpecific to non-small cell lung cancer (NSCLC), there have been 7 published studies evaluating the relationship between serum 25(OH)D and survival with 3 of them demonstrating positive association, 3 null association and 1 negative association. A study by Zhou et al. conducted in 447 patients with early-stage NSCLC, found higher levels of vitamin D to be associated with improved survival particularly among stage IB-IIB patients . A NorweGiven this variability in findings on the relationship between serum vitamin D and survival in NSCLC, additional studies with large sample sizes are needed to better understand the direction and strength of this association. We investigated this association in a large homogenous group of newly diagnosed advanced NSCLC patients treated at a national network of oncology hospitals.A consecutive series of 359 newly diagnosed stages III–IV NSCLC patients treated at three Cancer Treatment Centers of America® (CTCA) hospitals  between January 2008 and December 2010 was evaluated. We included a consecutive case series of patients to minimize the probability of selection bias. The present study was conducted according to the guidelines laid down in the Declaration of Helsinki and was approved by the Midwestern Regional Medical Center Institutional Review Board (IRB) at Cancer Treatment Centers of America®. The IRB waived the need for informed consent because there was no direct patient contact in this study. This study involved collection of existing data from patient records in such a manner that subjects could not be identified, directly or through identifiers linked to the subjects.Serum samples were collected within 30 days of first visit at our hospital, and prior to initiation of anticancer therapy. Serum was collected at the laboratory, packed in coolpacks and sent to the Laboratory Corporation of America  where a chemiluminescence immune assay  was used to measure 25(OH)D. Serum samples were incubated with antivitamin-D coated microparticles and isoluminol derivative-conjugated 25(OH)D before measurement of chemiluminescent signals. Analysis was completed within 48 h of collection. The DiaSorin Liasion 25(OH)D assay has been clinically validated to be comparable in accuracy and precision to the radioimmunoassay (RIA). This method uses the same particles used in the DiaSorin RIA technique. Studies have found this to be a rapid, accurate, and precise tool for the measurement of serum 25(OH)D .Serum 25(OH)D was the primary independent variable of interest. We used the categories “deficient (<20 ng/ml)”, and “not deficient (> = 20 ng/ml)” in accordance with previously published research in this area , 14. A cThe primary endpoint was patient survival and was defined as the time interval between the date of first serum vitamin D assessment and the date of death from any cause or the date of last contact/last known to be alive. Patients were followed prospectively until December 2014. The Kaplan-Meier method was used to calculate survival. The log rank test statistic was used to evaluate the equality of survival distributions across the 2 serum 25(OH)D groups. Clinical, demographic and serum 25(OH)D variables were evaluated using univariate Cox proportional hazards models to determine which parameters showed individual prognostic value for survival. Multivariate Cox proportional hazards models were then performed to evaluate the independent prognostic significance of all variables that were evaluated in univariate analysis. We adjusted for the following variables in the multivariate analysis: age, gender, CTCA hospital, stage of disease, Eastern Cooperative Oncology Group (ECOG) performance status, body mass index (BMI), season of diagnosis, serum albumin, smoking status, histologic subtype, and nutritional status as measured using Subjective Global Assessment (SGA). Season of diagnosis was defined as winter: December-February; spring: March-May; summer: June-August, or fall: September-November. The effect of serum 25(OH)D and other variables on patient survival was expressed as hazard ratios (HRs) with 95 % confidence intervals (CIs).Cox regression with time-invariant covariates assumes that the ratio of hazards for any two groups remains constant in proportion over time. We checked this proportional hazards (PH) assumption using a combination of graphical and statistical testing procedures. First, we examined log-minus-log plots for categorical predictors. As a second approach, we ran an extended Cox model with time-dependent covariates for continuous predictors. Finally, a goodness-of-fit testing approach based on Schoenfeld residuals was used to evaluate the PH assumption.Finally, to assess the possible influence of sample bias on the results, as well as to investigate the stability of the model coefficients, we performed a bias-corrected and accelerated (BCa) bootstrap resampling procedure. We generated 1000 samples, each the same size as the original data set, by random selection with replacement. Cox regression was then run separately on these 1000 samples to obtain robust estimates of the standard errors of coefficients, and hence the p values and 95 % BCa CIs of the model coefficients .No formal sample size calculations were conducted for this analysis. All reported P values are from two-sided tests. All statistical analyses utilized SPSS version 20.0 .Table p <0.05). Patients who presented to us in the summer and fall months were less likely to be deficient in vitamin D compared to those who presented in winter and spring. The mean (standard deviation [sd]) serum vitamin D levels were 24.7 (11.8), 31.2 (20.8), 22.3 (10.4) and 23.5 (14.1) nanograms per milliliter (ng/ml) for summer, fall, winter and spring months respectively. Patients with ECOG performance scores of 0–1 were less likely to be deficient in vitamin D compared to those with scores 2–4. Current smoking status was associated with a greater prevalence of vitamin D deficiency compared to past or no smoking history. Finally, patients diagnosed at our Philadelphia hospital had a lower prevalence of vitamin D deficiency compared to patients diagnosed at Zion and Tulsa hospitals. In addition, patients with vitamin D deficiency had lower serum albumin levels compared to those with non-deficient serum vitamin D levels, the finding being borderline significant (p = 0.06). Similarly, well-nourished patients had a lower prevalence of vitamin D deficiency compared to malnourished patients, the finding being borderline significant (p = 0.07).Table Table p = 0.04. The Philadelphia cohort (66.7 %) also had a significantly greater proportion of well-nourished patients compared to the Tulsa cohort (45 %); p = 0.04. Further, the Philadelphia cohort (24 %) had a significantly smaller proportion of current smokers compared to the Zion (42.3 %) and Tulsa (47.5 %) cohorts; p = 0.002. Finally, the mean baseline serum albumin in the Philadelphia cohort (4.1 grams per deciliter [g/dl]) was significantly higher compared to the Zion (3.4 g/dl) and Tulsa (3.6 g/dl) cohorts; p <0.001. There were no systematic differences among the 3 hospitals with regard to age, stage at diagnosis, BMI and ECOG performance status.To further understand the differences in survival across the 3 hospitals, we evaluated the distribution of baseline clinical and demographic characteristics stratified by the hospital (detailed results not shown in the interest of space). The Philadelphia cohort (66 %) had a significantly greater proportion of females compared to the Zion (47.6 %) and Tulsa (42.5 %) cohorts; The systematic differences among the 3 hospitals with regard to gender, nutritional status, smoking status and serum albumin might, in part, explain the observed differences in median survival.p = 0.008) and every 1 g/dl increase in serum albumin was associated with a 56 % reduction in mortality hazard . In the full model, all variables tested in the univariate analysis were evaluated simultaneously in the same model. Six variables demonstrated statistically significant associations with survival: stage of disease, CTCA hospital, histologic subtype, SGA, smoking status and serum albumin. Every 1 g/dl increase in serum albumin was associated with a 54 % reduction in mortality hazard . In the final model, only those 6 variables that were statistically significant in the full model were evaluated together. All of them excepting histologic subtype were found to be statistically significant.Table To account for potential sampling bias and further investigate the stability of the classical multivariate Cox model reported in Table We investigated the association between serum 25(OH)D and survival in newly diagnosed stages III-IV NSCLC patients. The findings of our study add to the growing body of literature on the potential association between serum vitamin D and survival in NSCLC.Consistent with the findings published by Heist et al. , Anic etTwo predictors demonstrated borderline significance in their association with serum vitamin D levels: serum albumin and nutritional status. Patients with vitamin D deficiency had lower serum albumin levels compared to those with non-deficient serum vitamin D levels, and well-nourished patients had a lower prevalence of vitamin D deficiency compared to malnourished patients; both findings consistent with our recently reported research in prostate cancer .In contrast with previously published research , 28–30, In vitro and animal studies have shown that vitamin D has antiproliferative, antimetastasis, and antiangiogenesis activities in lung cancer and may modulate the immune function of lung epithelial cells , 33–35. There are some limitations of our study that are worth acknowledging. This is an association study which cannot prove causality. Reverse causality (the effect of cancer on serum vitamin D levels) is always a possibility in observational studies and cannot be ruled out with certainty . PotentiThere are some strengths of our study. We examined a homogeneous patient population of newly diagnosed advanced NSCLC which minimizes potential confounding by tumor stage and prior treatment history. We measured serum vitamin D at disease diagnosis prior to receiving any treatment which eliminates the possibility of treatment and lifestyle changes affecting serum vitamin D levels after diagnosis. We had a large sample size of histologically confirmed NSCLC cases. By using a consecutive case series of all eligible patients seen at our institution during a fixed time period, we minimized the possibility of selection bias in our study. Lastly, we adjusted for a wide range of potential clinical and demographic confounders thereby minimizing the possibility of residual confounding. That being said, the possibility of residual confounding can never be completely ruled out in observational studies.In conclusion, we did not find any significant association between serum 25(OH)D and survival in newly diagnosed stages III-IV NSCLC patients. This finding needs further exploration in future prospective studies of larger sample sizes across all stages of NSCLC.


The specimens of each group were randomly assigned to two subgroups to test two different adhesive systems. A three-step/etch-and-rinse adhesive system (Optibond FL) and a two-steps/self-etch adhesive system (Optibond XTR) were used. Composite buildups were performed with a nanohybrid composite (Herculite XTR). All specimens were submitted to thermocycling ageing (10000 cycles). The specimens were sectioned in 1 mm2 sticks. Microtensile bond strength tests were measured. Nonparametric statistical analyses were performed due to nonnormality of data. Optibond XTR on irradiated and nonirradiated teeth did not show any significant differences. However, Optibond FL bond strength was more effective on nonirradiated teeth than on irradiated teeth. Within the limitations of an in vitro study, it can be concluded that radiotherapy had a significant detrimental effect on bond strength to human dentin. However, it seems that adhesive choice could be adapted to the substrata. According to the present study, the two-steps/self-etch (Optibond XTR) adhesive system tested could be more effective on irradiated dentin compared to three-steps/etch-and-rinse adhesive system (Optibond FL).The aim of this study was to assess the effect of radiotherapy on bond efficiency of two different adhesive systems using tensile bond strength test. Twenty extracted teeth after radiotherapy and twenty nonirradiated extracted teeth were used. The irradiation was applied  Hyposalivation which is induced by irradiation , 2, dietWhile there is lack of data published on this topic, evidences suggest a conservative approach using adhesive restorations . Havemanμm), loss of prismatic structure, and bacterial colonization associated with the obliteration of the dentinal tubules and odontoblastic process atrophy  and adhesive system (XTR/FL) leading to 4 subgroups and (ii) a random factor (tooth) nested in each subgroup: 10 teeth per subgroup with one to four replicates per tooth. The conditions for the application of statistical treatment were carefully verified. The effect of the tooth factor on the explained variable , between the two irradiated subgroups (I:XTR versus I:FL), and for each adhesive system: (NI:XTR versus I:XTR) and (NI:FL versus I:FL). Correction for multiple comparisons was performed to maintain the family-wise error rate at the significant level of 5%. For 4 pairwise comparisons, Bonferroni correction gave a significant level of 2-tailed single test equal to 0.05/4, that is, 0.0125. Data were reported as mean ± SD per subgroup. Statistics were performed using the R language, version 3.1.2 available on the https://cran.r-project.org/ website. Package nlme was used to perform mixed linear model.In case of no tooth effect on p < 10−3). No effect of factor tooth was significant in each subgroup using Kruskal-Wallis test with p values ranging from 0.46 (I:XTR) to 0.84 (I:FL).Three teeth and one tooth out of 10 were missing in NI:XTR and I:FL subgroups, respectively. Linear mixed model was not appropriate because of the nonnormality of the normalized residuals  and in I:FL subgroup 11.3 ± 2.8 MPa (p = 0.97 > 0.0125). On nonirradiated dentin, they did not show any significant difference on bond strength with μTBS in NI:XTR subgroup equal to 14.5 ± 4.8 MPa and in NI:FL subgroup 16.4 ± 6.2 MPa (p = 0.42 > 0.0125).On irradiated dentin, both adhesive systems (XTR and FL) did not show any significant difference with p = 0.0009 < 0.0125). μTBS was observed 1.5 times higher in nonirradiated subgroup in case of FL (33% decrease from nonirradiated to irradiated subgroups). On the other hand, no statistical differences were found for XTR adhesive system (p = 0.040 > 0.0125) with μTBS observed 1.2 times higher in nonirradiated subgroup (16% of decrease from nonirradiated to irradiated subgroups).Regarding FL groups, the value was significantly different between nonirradiated and irradiated dentin  in irradiated dentin for FL subgroups are consistent with literature. It is reported that the ionizing radiations may have an effect on the collagen fibers of dentinal tubules [Several studies involving the two adhesive systems used in this work have been performed and have shown similar results , 49, 50. tubules , 52. Mor tubules –56.μTBS (16%) in irradiated dentin could be due to the chemical connections between the carboxylic or the phosphate groups of functional monomers and the phases of dissolved hydroxyapatite. These chemical connections would contribute to a better cohesion of the infiltrated resin after polymerization and, probably, in better resistance in the hydrolysis of this zone [With the XTR adhesive system, the weak decrease of his zone . in vivo and, then, underwent adverse effects like hyposalivation.The results are in agreement with those of Naves et al. and S. Yadav and H. Yadav , 57. NevThe changes resulting from the irradiation on the hardness, the crystalline structure or the collagen matrix, seem to influence the adhesive agents bond strength to dentin. The dental substrate might have experienced radiation effects that could compromise bonding ability by impairing hybrid layer formation. in vitro study, it appears that, regarding the type of adhesive system, radiotherapy may affect the microtensile bond strength of composite restorations on irradiated dentin. Therefore, it is advisable for a clinician to restore all cavities before radiotherapy and initiate caries prevention modalities in patients undergoing radiation therapy.Under the limitations of thisFurther studies are needed to help the practitioner to adapt the choice of the adhesive system after radiotherapy of head and neck.


In utero exposure to EDCs may affect disease propensity through epigenetic mechanisms.There is increasing concern that early-life exposure to endocrine-disrupting chemicals (EDCs) can influence the risk of disease development. Phthalates and phenols are two classes of suspected EDCs that are used in a variety of everyday consumer products, including plastics, epoxy resins, and cosmetics. The objective of this study was to determine whether prenatal exposure to multiple EDCs is associated with changes in miRNA expression of human placenta, and whether miRNA alterations are associated with birth outcomes.Our study was restricted to a total of 179 women co-enrolled in the Harvard Epigenetic Birth Cohort and the Predictors of Preeclampsia Study. We analyzed associations between first-trimester urine concentrations of 8 phenols and 11 phthalate metabolites and expression of 29 candidate miRNAs in placenta by qRT-PCR.p < 0.05). By assessing gene ontology enrichment, we determined the potential mRNA targets of these microRNAs predicted in silico were associated with several biological pathways, including the regulation of protein serine/threonine kinase activity. Four gene ontology biological processes were enriched among genes significantly correlated with the expression of miRNAs associated with EDC burden.For three miRNAs—miR-142-3p, miR15a-5p, and miR-185—we detected associations between Σphthalates or Σphenols on expression levels (Overall, these results suggest that prenatal phenol and phthalate exposure is associated with altered miRNA expression in placenta, suggesting a potential mechanism of EDC toxicity in humans.http://dx.doi.org/10.1289/ehp.1408409LaRocca J, Binder AM, McElrath TF, Michels KB. 2016. First-trimester urine concentrations of phthalate metabolites and phenols and placenta miRNA expression in a cohort of U.S. women. Environ Health Perspect 124:380–387;  Prior epidemiologic studies have indicated that unfavorable gestational conditions or exposures such as preeclampsia, maternal obesity, gestational diabetes, alcohol consumption, and smoking can influence epigenetic profiles in placenta and potentially impact risk for adverse health outcomes . There iin vitro . MicroRNAs (miRNAs) are noncoding RNAs that are approximately 22 nucleotides in length. In animals, miRNAs can regulate gene expression post-transcriptionally by imperfect complementarity with a target mRNA, thereby inhibiting protein synthesis . Concentin vitro . Severalin vitro .In vivo and in vitro studies have revealed that exposure to bisphenol A (BPA) can alter miRNA expression  and the Predictors of Preeclampsia Study (POPS) at the Brigham and Women’s Hospital in Boston, Massachusetts. Data and biospecimens for the HEBC were collected from June 2007 through June 2009; the cohort includes 1,941 mother–child dyads  in 2007. Urine samples were gathered at the first prenatal visit (< 16 weeks gestation), and at four additional visits during pregnancy. Additional information about this cohort has been published elsewhere  were collected in polypropylene urine cups and frozen at –80°C. Before biomarker analysis, samples were defrosted at 4°C overnight. After another round of vortexing, samples were aliquoted to 1.6-mL polypropylene tubes and refrozen at –80°C. For each participant, one tube was shipped on dry ice overnight to the Centers for Disease Control and Prevention (CDC) for measurement of urinary concentrations of phthalate metabolites and phenols. To adjust for urine dilution, specific gravity (SG) was measured at Brigham and Women’s Hospital. SG was measured using a handheld refractomer , which was calibrated with deionized water before each measurement. We adjusted for urine dilution using SG, rather than urinary creatinine, which is likely altered by stage of pregnancy . Using the mirVANA RNA Isolation Kit , miRNA-containing RNA was isolated according to the manufacturer’s protocol.miRNA selection. We performed a pilot project to identify miRNAs to be differentially expressed across a subset of 48 samples. The 48 samples represented the highest and lowest quintiles of exposure for phthalates and phenols. The pilot study analyzed 86 miRNAs among the 48-sample subset using Qiagen miFinder PCR Arrays . We chose to validate candidate miRNAs that were statistically significantly associated with additive phenol and phthalate concentration groups. The miRNAs analyzed in this study are listed in Supplemental Material, Table S2. miRNAs were measured using specially designed Qiagen PCR Arrays, which have extremely high reproducibility, thereby eliminating the need for technical triplicates. Example triplicate reproducibility for the miRNAs assessed in this project is shown in Supplemental Material, Figure S2. For the 4 samples with microRNAs measured in triplicate, between 71% and 84% of the expression measurements had a coefficient of variation (CV) < 1. In one sample, reproducibility was lower (CV > 5) for 2 miRNAs (miR-125b-5p and miR-30a-5p), but the reproducibility of these measurements was strong for all other samples (CV < 1). The relative distribution of phthalates and phenols was similar for both the pilot project and the main study . The range of metabolite values in the pilot captured the range of values measured in the entire study. The reproducibility of miRNA expression between the pilot study and the main study is shown in Supplemental Material, Figure S4, demonstrating consistency. The correlation in expression values measured in the pilot and main study ranged from moderate to strong (r = 0.43 to 0.82), with approximately 50% having an r ≥ 0.6. Two miRNAs showed poor correlation between the two study stages, miR-128 and miR-155-5p. Additionally, these two miRNAs had two samples that were outliers. The sensitivity to the inclusion of these possibly influential points was assessed in the final models. The full study analyzed 29 miRNAs chosen from the pilot project that were found to be differentially expressed across a subset of 48 samples.Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). All RNA quality was assessed using the nanodrop ND-1000 (NanoDrop), and all RNA used had passing 260/280 values, which was defined as ≥ 1.8. Using the miScript II RT Kit (Qiagen), 250 ng cDNA was reverse-transcribed from RNA according to the manufacturer’s instructions. The PCR reaction condition was as follows: incubated at 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min. The qRT-PCR was performed using the miScript SYBR® Green PCR Kit (Qiagen) and custom designed miScript PCR arrays (Qiagen) according to manufacturer’s instructions on a Life Technologies 7900HT qPCR machine at the Harvard Medical School ICCB Screening Facility with reverse transcription controls. The qRT-PCR cycling conditions were as follows: 95°C for 15 min, and 40 cycles of 94°C for 15 sec, 55°C for 30 sec, and 70°C for 30 sec. All qRT-PCR data were normalized using the average of two small nucleolar RNAs, SNORD61 and SNORD95. Delta Ct (ΔCt) was defined as the expression difference between the target miRNA and the average of the two normalizing nucleolar RNAs: ΔCt = Ctnormalizing RNA – CtmirRNA.Statistical analysis. Phthalate body burden was defined by four classifications: Σphthalates , high molecular weight (HMW), low molecular weight (LMW), and DEHP [di(2-ethylhexyl) phthalate] metabolites. Phenol body burden was defined by three classifications: Σphenols , paraben, and non-paraben. MicroRNAs were modeled as a function of each log-transformed EDC body burden measurement, adjusting for maternal age, maternal ethnicity, and self-reported maternal smoking (yes/no), and infant sex. Effect modification by infant sex was assessed by incorporating an interaction term between EDC level and infant sex into our models. In the case of a significant interaction, determined by a Wald test, the association between EDCs and miRNA levels was reported separately for male and female infants. If infant sex was not a significant modifier, the association was reported for male and female infants together. Although we have previously observed that individual phthalate and phenol biomarker concentrations are significantly correlated with one another in their respective chemical groups, we have also observed that phthalate body burden is not strongly correlated with phenol body burden in first-trimester urines  was used to predict targets for miRNA of interest  of individuals with excellent RNA quality. Genome-wide expression was assessed on the Affymetrix GeneChip® Human Gene 2.0 ST Array at the Microarray Core Facility at the Dana-Farber Cancer Institute in Boston. Signal intensities were processed before analysis using the Affymetrix Expression Consule Software (Affymetrix), which included robust multichip analysis (RMA) background correction, quantile normalization, and gene-level summarization of expression using median polish73,74. For each miRNA significantly associated with EDC burden, we assessed the Spearman correlation with all RefSeq genes, using a conservative Bonferroni correction to identify significant correlations accounting for multiple testing . GO ontology enrichment among the genes correlated with miRNA levels was analyzed similarly to the enrichment among in silico predicted targets.MicroRNA targets were predicted interest . Target interest . These sng terms , 2010. APhthalate and phenol measurements. Characteristics of the study population, including maternal age, ethnicity, smoking, and body mass index of 179 pregnant women–newborn dyads are listed in Associations between grouped phthalate and phenol measurements and miRNA expression in placenta. Two miRNAs were significantly associated with additive phenols, and miR-185 was associated with ΣLMW (p < 0.05) with one of the measured phenols in our adjusted models . Most of these miRNAs were either positively associated with BPA levels or negatively associated with BP-3.ith ΣLMW . Levels n-butyl phthalate), which have a significant moderate positive correlation with MEP (p < 0.05) with at least one of the individual phthalates . A majority of these miRNAs were associated with at least MCOP .We also modeled miRNA expression as a function of each additive phthalate group .Predicted mRNA targets and potentially affected pathways. In silico target prediction software was used among mRNA (messenger RNA) targets of the three miRNAs that were significantly associated with phenol or phthalate levels . We investigated biological process enrichment among the genes that had at least four databases linking them to one of the three miRNAs. q = 0.07) enrichment for bicarbonate transport after adjusting for multiple testing  PDFClick here for additional data file.


Oldenlandia diffusa (OD) is commonly used with various diseases such as cancer, arthritis, and autoimmune disease. Liver cirrhosis is a predominant risk factor for hepatocellular carcinoma (HCC). Here, we show that the therapeutic effect of OD, which was investigated both in vitro and chemically, induced HCC model. OD significantly enhanced apoptosis and antiproliferative activity and reduced migration ability of HCC cells. In vivo, OD was treated twice a day for 28 days after confirmed HCC model through 2-[18F]-fluoro-2-deoxy-D-glucose (18F-FDG) imaging. The survival in OD treated groups was shown to have a greater therapeutic effect than the control group. 28 days after OD treatment, OD treated groups resulted in a significant reduction in tumor number, size, 18F-FDG uptake, and serum levels such as alanine transaminase, aspartate transaminase, and alkaline phosphate compared to the control group. Also, proliferated cells in tumor sites by OD were reduced compared to the control group. Furthermore, several rats in OD treated group survived over 60 days and liver morphology of these rats showed the difference between tumor mass and normal tissue. These results suggest that OD may have antiproliferative activity, inhibition of metastasis, and apoptotic effects in chemically induced HCC model and can have the potential use for clinical application as anticancer drug of the herbal extract. Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer death worldwide , 2. HCC  in vitro and in vivo using natural herb extract [ Oldenlandia diffusa (OD) has intrigued interest because of its anticancer and chemoprevention effect on various cancer and normal cells [A number of studies have reported the beneficial effect of herbal medicine in diverse diseases such as central nervous system and cancer. Also, many herbal drugs and prescriptions have in fact been used clinically for the treatment of cancer and autoimmune diseases. Several studies reported anticancer activity extract –10. Receal cells , 12.OD is a well-known medicinal plant used in Korean and Chinese herbal medicine for the treatment of hepatitis, tonsillitis, urethral infection, and malignant tumors of the liver and lung. A number of studies have reported multiple biological activities of the OD such as antitumor, chemopreventive, anti-inflammatory, antioxidant, and proapoptotic effect , 14. HowIn this study, we investigated the ability of OD enhanced anticancer effect in apoptotic cell death, inhibition of proliferation, and migration of HCC cell lines. In addition, we were able to demonstrate OD enhanced antitumor effects in regulation of hepatic function, glucose metabolism, and metastasis in chemically induced liver cancer model.The herbal sample of OD Roxb was purchased from an herbal market in Gyeongdong. OD was extracted using reflux extraction equipment in hot water for an hour and concentrated using a rotary evaporator before lyophilization. OA and UA were purchased from Sigma-Aldrich . The compounds were dissolved in DMSO and the final concentration of DMSO in samples was 0.15%.http://www.atcc.org). The cells were maintained in Dulbecco's Modified Eagle's Medium . The huh7 and hepG2 cells were supplemented with antibiotics and 10% of fetal bovine serum . Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.Liver cancer cell lines such as huh7 and hepG2 were obtained from the American Type Culture Collection  or 24-well plates (2 × 104 cells/well) in medium containing 10% FBS and were incubated for 24 h. Subsequently, cells were treated with medium containing OA (0–100 μg/mL), UA (0–100 μg/mL), and OD (0–500 mg/mL) at 37°C for 24 h. The untreated cells in medium containing 0.15% DMSO were used as 100% viability controls. The cells with CCK-8 were incubated for 2 h and were then measured at 450 nm with a spectrophotometer .Cell viability was measured using a Cell Counting Kit-8  allowing sensitive colorimetric assay by using highly water-soluble tetrazolium salt that is reduced by dehydrogenases in living cells to give a colored product (formazan). The cells were seeded in 96- (5 × 105 cells/well) and maintained in DMEM with 10% FBS. The monolayer cells were scraped by using the tip of a 200 μL micropipette, creating linear streaks 2 mm apart. Cells were washed twice with medium to remove nonadherent cells and were further incubated in fresh medium with 10% FBS containing OD (200 mg/mL), OA (100 μg/mL), and UA (50 μg/mL) for 48 h. Wound closure or cell migration was photographed at 0, 24, and 48 h after wounding, using an inverted microscope equipped with digital camera . The images were analyzed into the Meta-Morph imaging software . For statistical analysis, at least three independent scratch wound experiments were used for calculations.To investigate the effect of OD, OA, and UA on the migration of HCC cells, a scratch-induced migration assay was performed. The cells were seeded in 6-well plates (1 × 10http://dx.doi.org/10.1155/2015/501508). RT-PCR was conducted as described previously [Total RNA was extracted from cancer cells using RNeasy Mini Kit . cDNA was synthesized with total RNA and oligo(dT) primer  using Omniscript RT kit (Qiagen) for real time polymerase chain reaction (RT-PCR). The cDNA samples were diluted 20-fold with water and were used as templates for RT-PCR with 2× SYBR Green Master Mix and oligonucleotide primers. Quantitative RT-PCR was performed using the MyiQ Single-Color RT-PCR Detection System . Details of the primers are described in the Supporting Information (Supplementary Table 1) , (2) the second group was fed with OD , and (3) the third group was fed with OD . To assess the survival of the OD treated animals, rats were randomized  after last DEN injection and were treated with OD twice a day for 28 days. We recorded starting from day 0 after OD treatment. Survival was followed for a maximum of 60 days.Animals were assigned randomly to one of the following three major groups: (1) one group of rats was treated with distilled water as control group (18F-FDG (1.1 ± 0.04 mCi) was injected intravenously into the caudal vein. After 30 min, the rats were taken using a PET/CT scanner and body temperature was kept at 37°C with a heating pad on the scanner bed.PET/CT imaging and data analysis were performed as previously reported , 25. Ima18F-FDG uptake, regions of interest (ROIs) in the lesioned areas were identified in images of the coronal and horizontal sections. Each ROI value was defined on the coronal tomograms that showed the highest uptake to be in the middle of the tumor and calculated as the averaged nCi/cc after calibration of both liver of insulated areas and muscle in the same images. We expressed the standard uptake value (SUV) in ROIs values.To assess the changes of To test liver function, we examined the serum levels of alkaline phosphatase , aspartate transaminase , and alanine transaminase  28 days after OD treatment. Briefly, the blood samples were collected before sacrifice and centrifuged at 3000 rpm for 20 min. The supernatant was analyzed to quantify the concentration in strict accordance with the manufacturer's protocols.n = 5, for each group) were deeply anesthetized with 15% urethane and sacrificed by decapitation. The whole liver tissue was sliced into 2 mm thickness. The tumors were counted by macroscopic examination of the liver through two independent investigators according to the criteria.Four weeks after the last DEN injection, all group rats  at 4°C overnight. Subsequently, slides were incubated in Alexa 546-conjugated goat anti-mouse IgM . The slides were counterstained with 4′,6-diamidino-2-phenylindole  and detected using a Zeiss LSM510 confocal microscope .28 days after OD treatment, animals from each group were sacrificed for histological examination. The tissue was cut coronally at a thickness of 4  t-test. Probability values less than 0.05 were considered statistically significant. Also, the statistical analysis of survival was carried out using a log-rank test.All data are presented as mean values ± standard deviation from at least 3 independent experiments. Statistical differences between different test conditions were determined using Student's in vitro experiments. We treated HCC cells with OD for 72 h and assessed the number of viable cells. We showed that OD has time dependent growth inhibitory effect  and hepG2 (OD: 37.4% ± 4%) compared to the FBS treatment . Further18F-FDG has long been demonstrated to be a marker of glucose metabolism in cirrhosis and tumor. The specific areas of liver identified with increased uptake of 18F-FDG in PET imaging  were significantly lower compared to the control group . Also, tTo investigate the inhibitory effect of OD on the proliferation in tumor cells, we performed immunostaining in PCNA, which is a cell proliferation marker. In OD group, PCNA-positive cells were significantly decreased compared to the control group . Also, w in vitro using a natural herb extract [Herbal medicine has been used in Asian countries and numerous studies have reported anticancer activity extract , 26. OD, extract , 27, 28 α receptor, plays a critical role in normal physiology as well as the pathology of many human diseases including cancer, inflammation, and autoimmune disease. Also, CXCR4 correlates with the histological grade and invasive capacity of tumor cells, as well as with tumor cell survival [This study demonstrated that OD induced apoptosis by caspase-3 pathway in HCC cells. Also, OD suppressed proliferation of HCC cells compared to untreated control cells. These results were consistent with previous findings of anticancer effect in breast cancer cells . Since Hsurvival . Our ressurvival , 32. OA  in vivo model using the chemically induced liver cancer model. We employed a DEN-induced cancer model which can establish HCC and liver cirrhosis simultaneously as the malignant tumor progresses from liver cirrhosis. This model may provide similarity between experimental and human HCC and be a better scheme for studying human HCC than the implanted HCC model with cirrhosis. The survival in vivo experiment revealed that OD enhances survival rate compared to the control group. Also, 30% of rats in OD treated group survived more than 50 days. 18F-FDG can provide a useful indicator of tumor growth and metastasis because 18F-FDG accumulates in tumor mass with increased glucose metabolism [18F-FDG imaging can provide additional information including tumor development and secondary tumor growth. In a previous study, we have reported that we analyzed 18F-FDG imaging using the PET/CT time-dependently in DEN-induced liver cancer model. We analyzed 18F-FDG imaging at day 0 and day 28. The uptake of 18F-FDG of liver in OD treated group was lower in certain areas compared to the control group. Furthermore, OD can change hepatic function related factors including ALT, AST, and ALP on serum level in HCC model. Serum components such as ALT, AST, and ALP are markers of hepatic function and their increases in blood indicate liver damage by the cytotoxic effects of DEN [We investigated the anticancer effect of OD intabolism . Also, 1s of DEN , 35.Our results show that OD may regulate glucose metabolism in tumors through enhancing hepatic function. Also, these results show similar pattern as reduction of the number categorized by tumor size, liver/body ratio, and cell proliferation in tumors. We investigated liver morphology, tumor number, and size of rats that survived more than 60 days after OD treatment. We observed the large tumor in two sites. Except for tumor site, the rest of the liver tissue was similar in morphology to normal liver, despite the chemically induced HCC model generating numerous tumors and cirrhosis in the liver . This is in vivo.In conclusion, this study demonstrated that OD treatment has anticancer effects in inhibiting the growth of HCC cell in chemically induced liver cancer model. Also, OD can regulate the hepatic function and glucose metabolism. Furthermore, our results have shown that OA and UA derived from OD extracts act as the bioactive components of the anticancer effects. Further studies are needed to identify the detailed mechanisms involved in the therapeutic effect of ODThe Supplementary Material contains primer sequences of real time-polymerase chain reaction in the expression of migration related receptor including CXCR1, CXCR2, and CXCR4. (Supplementary Table 1). Also, the principal component in oldenlandia diffusa such as oleanolic acid and ursolic acid showed anticancer effect including cytotoxicity, antiproliferation activity , and migration ability  in hepatocellular carcinoma cells.


The study aimed to assess serum RANKL:OPG ratio, Dkk-1 and deposition of calcium in aortic valve in relation to the presence of concomitant coronary atherosclerosis in patients with symptomatic calcified aortic stenosis (CAS).OPG, soluble RANKL and Dkk-1 were measured in 218 consecutive patients who were undergoing cardiac catheterization because of symptomatic CAS. Values of studied compounds were compared between patients without (Group A) and with (Group B) coronary atherosclerosis. Computed tomography derived Agatston score was assessed by using 256-slice CT.. RANKL/OPG Ratios (mean (95% C.I.)) were: 20.04  in Group A and 12.69  in Group B, resp., p = 0.018). After adjustment, the difference in RANKL:OPG ratios was no longer significant. Multivariable regression underscored the significance of difference in Dkk-1 (pafter adjustement = 0.020). Group A patients had significantly higher Dkk-1, significantly higher deposition of calcium in aortic valve and were symptomatic in significantly younger age (p < 0.001) as compared to group B patients: Agatston score (mean (95% C.I.)) 4069.9  and 2413.5 , p = 0.007.Presence of coronary atherosclerosis was related to significantly (p = 0.007) higher OPG and to significantly (p = 0.004) lower Dkk-1. Coronary atherosclerosis was also associated with a trend towards a decrease of RANKLDkk-1 and deposition of calcium in aortic valve differ significantly in relation to the presence/absence of coronary atherosclerosis in patients with symptomatic CAS. A positive association was found between Dkk-1 and calcium load in aortic valve in patients with symptomatic CAS and angiographically normal coronary arteries. Every 50 of life ,2.It has been recognized, that the most significant predictor of clinical progression of calcified aortic valve disease is the load of calcium in aortic valve . The proDegenerative aortic valve disease shares many features with atherosclerosis. The initial plaque of aortic stenosis is similar to that seen in coronary artery disease . HoweverThe research was carried out according to the principles of the Declaration of Helsinki. Patients gave informed consent and the Ethics committee of University Hospital Kralovske Vinohrady in Prague (Czech Rep.) approved the study.2 were excluded. Patients gave informed consent and the local ethics committee approved the study. Baseline characteristics of study population are presented in Table Study group consisted of 218 consecutive patients who were undergoing cardiac catheterization being considered for aortic valve replacement for symptomatic CAS in a tertiary care institution [Blood samples were collected during cardiac catheterization. After standard processing, serum was stored at −70°C until assayed. Samples were assayed with OPG ELISA kit , sRANKL ELISA kit  and Dkk-1 ELISA kit , respectively, according to the manufacturer’s protocol. Limits of detection were: 0.1 pmol/l of OPG, 0.4 pmol/l of sRANKL, and 0.01 ng/ml of Dkk-1.Values of circulating OPG, sRANKL, Dkk-1 were compared between patients without (Group A) and with (Group B) concomitant coronary atherosclerosis (coronary artery stenosis ≥ 50%). Patients in group A had angiographically normal coronary arteries and without coronary calcification.Computed tomography (CT) derived Agatston score was assessed by using 256-slice Brilliance iCT  Figure . Scans wt-test and multiple linear regressions, which were used to adjust for potential confounders. Categorical data are presented as absolute frequencies and percentages and were analyzed using Fisher’s exact test and the proportional odds model. A statistical analysis was performed by statistical software Stata, release 9.2 . All statistical tests were evaluated at a significance level of 0.05.Continuous data are presented as arithmetic and geometric means for normally and log-normally distributed variables, respectively, and 95% confidence intervals are used to describe the amount of uncertainty associated with estimate of a population mean. For the analysis, the asymmetrically distributed dependent variables were log-transformed. The comparison of groups was based on Student’s . RANKL:OPG Ratio (mean (95% C.I.)) was 20.04  in Group A and 12.69  in Group B, respectively, p = 0.018).Presence of coronary atherosclerosis was related to significantly higher OPG and to significantly lower Dkk-1 in comparison to presence of normal coronary arteries in patients with CAS Table . Concomiafter adjustement = 0.020).After adjustment for variables that significantly influenced levels of OPG  and RANKL , the difference in RANKL:OPG ratio between Group A and Group B was no longer significant Table . ConversPatients with CAS and without coronary atherosclerosis had significantly higher deposition of calcium in aortic valve and were symptomatic in significantly younger age as compared to patients with CAS and with concomitant coronary atherosclerosis Table : AgatstoActive osteochondrogenic differentiation and signaling were demonstrated in aortic valves in CAS . Load ofThe OPG/RANKL/RANK cytokine axis appears as the final effectors of most of the osteotropic factors already identified. The system consists of the transmembrane protein RANK, its ligand (RANKL), and the soluble receptor OPG. RANKL, a protein expressed on the osteoblast cell membrane, binds to RANK, a receptor located on the osteoclast membrane. This cell-to-cell interaction initiates a cascade of events resulting in activation and differentiation of osteoclasts. OPG is a soluble decoy receptor that binds to RANKL, to limit activation of RANK . RegulatBoth pathways, RANKL/RANK/OPG and Wnt, appear to be activated by atherogenic factors . There iWe suggest that the main finding of this study is that elevation of serum Dkk-1 was associated with a significant increase of load of calcium in aortic valve. Rosenhek and colleagues  demonstrin vivo measurements confirmed a paradox of simultaneous osteolysis and ectopic calcification in the same animal [Clinical observational studies showed that aortic valve calcification is inversely correlated with low bone tissue mineral density ,18. Diree animal . This eve animal . RANKL –e animal . It was e animal . It was e animal ,24. Dkk-e animal . Monocloe animal . Our resCirculating Dkk-1 and calcium deposition in aortic valve differ significantly in relation to the presence of coronary atherosclerosis in patients with symptomatic CAS. A positive association was found between serum Dkk-1 signaling and calcium load in aortic valve in patients with symptomatic CAS and angiographically normal coronary arteries.


Retirement from work involves significant lifestyle changes and may represent an opportunity to promote healthier eating patterns in later life. However, the effectiveness of dietary interventions during this period has not been evaluated.We undertook a systematic review of dietary interventions among adults of retirement transition age (54 to 70 years). Twelve electronic databases were searched for randomized controlled trials evaluating the promotion of a healthy dietary pattern, or its constituent food groups, with three or more months of follow-up and reporting intake of specific food groups. Random-effects models were used to determine the pooled effect sizes. Subgroup analysis and meta-regression were used to assess sources of heterogeneity.P <0.00001), with similar results in the short-to-medium  and long-term  and for body mass index (BMI) stratification. Interventions produced slightly higher intakes of fruit (mean 54.0 g/day) than of vegetables (mean 44.6 g/day), and significant increases in fish  and decreases in meat intake .Out of 9,048 publications identified, 68 publications reporting 24 studies fulfilled inclusion criteria. Twenty-two studies, characterized by predominantly overweight and obese participants, were included in the meta-analysis. Overall, interventions increased fruit and vegetable (F&V) intake by 87.5 g/day (Increases in F&V intakes were positively associated with the number of participant intervention contacts. Dietary interventions delivered during the retirement transition are therefore effective, sustainable in the longer term and likely to be of public health significance. Increased life expectancy has resulted in the rapid growth in the proportion of the oldest old (>85 years), particularly in developed nations. These tAdopting healthy dietary patterns can reduce morbidity and mortality risk. For example, the so-called ‘Mediterranean’ diet (MD), characterized by higher intake of vegetables, fruit, legumes, cereals and fish, lower intake of meats and dairy products and moderate intake of red wine, is a dietary pattern that is associated with lower risks of all-cause mortality, death from cardiovascular disease (CVD) and cancers, age-related diseases including Parkinson’s and Alzheimer’s diseases, obesity and weight gain-9. MultiLife events represent windows of opportunity in which behavior change interventions may be more effective. RetiremThis systematic review and meta-analysis aimed to assess the effectiveness of dietary interventions that promote a health dietary pattern such as the MD, or any of its component food groups, among adults in the retirement transition age range, with dietary behavior change as the primary outcome. We also aimed to identify characteristics of effective interventions.Our systematic review was conducted according to Cochrane and the In April 2013, 12 electronic databases were searched systematically from inception: Medline, Embase, PsycInfo, Scopus, Web of Science, CINAHL, ASSIA, Cochrane Database of Systematic Reviews, CAB Abstracts, Conference Papers Index, WorldCat Dissertations database and Index to Theses. Reference lists of identified publications and previously published related systematic reviews were hand searched to identify other studies potentially eligible for inclusion.The search strategy involved combining words from the following three concepts: 1) diet; 2) randomized controlled trials (RCTs); 3) people in the retirement transition or of a relevant age. Prior to searching, reviewers carried out an extensive exercise to identify relevant terms. The search terms were translated into a search strategy using a combination of index terms and keywords, which was refined iteratively in response to emerging results. Highly sensitive search filters for identifying RCTs were used in Medline and Embase. The finOnly RCTs, including cluster-RCTs, were included. To identify interventions carried out in populations with standards of living similar to the UK, only publications from most developed countries in the United Nations Index, with anGiven the heterogeneity in age at retirement, here we defined the retirement transition period as age 54 to 70 years and sought RCTs of adult participants with sample mean or median ages between 54 and 70 years. Studies involving non-institutionalized adults with or without health risk factors  were included.We searched for interventions promoting healthy dietary patterns such as the MD, as well as interventions promoting any of its component food groups ; legumes or pulses; nuts and seeds; unrefined cereals; olive oil; fish; moderate consumption of wine; low consumption of meat and meat products). Studies investigating lifestyle interventions including other components, such as physical activity, were included only if the effects of the diet component were reported independently. To assess sustained impacts on behavior change, only interventions with a follow-up of more than three months were included.Studies promoting other dietary patterns , laboratory feeding trials (not intended to assess behavior change), studies on change in a specific macro/micro nutrient , studies promoting pre-fabricated diet foods or meal-replacement drinks, and studies testing dietary supplements  were excluded.Our primary outcome was dietary change defined as: change in consumption of one or more components of the MD; improved MD adherence at follow-up as assessed by established MD-scores; and quaTwo reviewers independently assessed publications for eligibility. The decision to include studies was hierarchical and made initially on the basis of the study title and abstract; when a study could not be excluded with certainty at this stage, the full-text was obtained for evaluation. A standardized, pre-piloted form was used to extract data from the included studies for assessment of study quality and evidence synthesis. Discrepancies between reviewers were resolved through discussion with a third reviewer and a consensus approach was used. Extracted information included: study design ; participants characteristics ; description of measurement methods; outcome measures (dietary intake); and information to assess the risk of bias. Study quality was assessed using the Cochrane risk of bias tool. Two revP-values. A random effects model accounting for inter-study variation was used, thereby minimizing potential bias due to methodological differences between studies. Multiple dietary intervention arms from three studies were included in the meta-analysis. As suggested by Higgins et al.[Review Manager  and Stata  were used to pool and analyze results from the individual studies. Pooled results are reported as mean differences with 95% CIs and with two-sided s et al., excessiI2 statistic[I2 were calculated using Higgins et al.’s method[I2 was >50%, the degree of heterogeneity was considered high. We performed subgroup analysis to investigate the effects of body mass index (BMI) categories , with asymmetry assessed formally with Egger’s regression test.The searches yielded 9,048 publications and results of the screening process are described in Figure −2 at baseline thus including a significant proportion of overweight and obese participants, while mean BMI was 23.6 in one study, and 7 studies did not report BMI . The mean ages of the samples in these studies ranged from 54 to 67 years. Four studies recruited women only and two studies men only. Out of 24 studies included in the overall meta-analysis, 14 studies involving 53,987 participants reported a mean BMI ≥25 kg.mMost studies used food frequency questionnaires (FFQs) or F&V screener questionnaires to assess self-reported food intake; one study employed a dietary history method.Four studies  promoting the MD,28-30 weNone of the studies satisfied all of the criteria of the quality assessment tool. However, included studies provided an adequate description of methods and randomization procedures, and the average retention rate for the 22 RCTs was 90 ± 10% for all studies, with 5 studies reporting retention rates <80%,31-34. NP = 0.394)  with no significant differences when stratifying studies by type of intervention  and 44.6 g per day  for fruits and vegetables, respectively    . Responses to interventions were unrelated to level of F&V intake at baseline . Our meta-analysis of 22 published RCTs involving 63,189 participants demonstrated a moderate but consistently significant (mean 87.5 g/day) increase in F&V consumption in both the short-to-medium term (4 to 12 months) and the longer term (>12 months). The increase in F&V intake observed in these RCTs was positively associated with the number of contacts with participants during the interventions. The increase in F&V consumption was similar in studies involving predominantly overweight or obese participants and in those with and without additional health risk factors. Interventions were slightly more effective in increasing fruit intake (mean 54.0 g/day) than in increasing vegetable intake (mean 44.6 g/day). We found that interventions based on the MD et score-52 are wet score, the laret score.et al.[et al.[et al.[et al.[In a previous analysis of studies recruiting mainly younger adults, Brunner et al. reportedl.[et al. found grl.[et al. found thl.[et al., the pre.[et al. reviewedInterestingly, in our analyses, interventions showed no significantly different effect on dietary change in those studies investigating healthy participants than in studies where participants had health risk factors. This suggests that the presence of these risk factors  did not enhance or reduce participants’ responses to the dietary interventions. Interventions delivered indirectly  were only slightly less effective than those delivered face-to-face; this finding is supported by recent reports on the effectiveness of telephone-based interventions on physical activity and diet,60.We observed a modest but significant increase in fish consumption, equivalent to an increase of 50 g or 0.35 portions of fish per week,28,35,36per se reported similar effect sizes . The well-recognized limitations of all widely used dietary reporting tools may be amplified when attempting to assess responses to interventions. This is because, by necessity, study participants are aware of the expected dietary behavior and because repeated measurements are burdensome[Among the limitations of the present analysis are the high levels of heterogeneity among the studies and hence our findings should be treated with caution. We did subgroup analysis to explore a number of potential sources of heterogeneity; type of intervention, study design, ethnicity, sex, geographic origin of studies and mode of delivery of interventions were identified as sources of heterogeneity. It is of note that the intervention studies included in this review were consistently, and significantly, successful in enhancing F&V intake. Subgroup analysis of 16 studies involving both men and women  showed a mean increase in F&V intake of 64.3 g/day with acceptable heterogeneity levels. In addition, interventions promoting the Mediterranean diet and those interventions which promoted F&V intake rdensome, which mrdensome,64 may oper se (we found no such studies), we investigated the effectiveness of interventions targeting groups of retirement transition age that is, 54 to 70 years.The decision to operationalize the retirement transition as a mean or median age in the range 54 to 70 years was based on the wide variability in age at retirement. In the The findings of this meta-analysis are striking and show a consistently positive effect of interventions on dietary behavior with respect to F&V consumption across the diversity of RCTs. In addition, the overall good quality of the studies, including low attrition rates, provide confidence in the robustness of the findings.The results of this review have important public health implications. Our findings are in line with a recent report from the U.S. Preventive Services Task Force (USPSTF) recommendation statements on behavioral counseling to promote a healthful diet and physical activity in adults without pre-existing cardiovascular disease (CVD) or its risk factors,70. The et al.[P <0.0001), independent of age, systolic blood pressure, blood cholesterol, cigarette smoking habits, diabetes and supplement use[P for trend = 0.03)[P <0.0001), and diastolic blood pressure [Lock et al. estimatement use. More re = 0.03). In addi = 0.02). Recent  = 0.02),52,75-78 = 0.02).In conclusion, this systematic review of RCTs has demonstrated the effectiveness of dietary interventions in increasing F&V and fish intake among adults in the retirement transition age range. Despite the heterogeneity of intervention modalities, F&V intake was increased by approximately 87 g/day and was sustained in the longer term (>12 months). The increase in F&V intake in these RCTs was associated positively with the number of contacts with participants during the intervention indicating that more intensive interventions may offer advantages, although differential cost-effectiveness has not been assessed. These results provide evidence to support the development of interventions to improve dietary behavior at this life-stage to promote health and well-being and to reduce the risk of age-related disease and associated costs to society.BMI: Body Mass Index; EPIC: European Prospective Investigation of Cancer; FFQ: food frequency questionnaires; F&V: fruits and vegetables; MD: Mediterranean Diet; RCTs: randomized controlled trials; USPSTF: U.S. Preventive Services Task Force.The authors declared that they have no competing interests.JCM, MW, PJM and FFS conceived the study. JL, NH, PJM, TDM, AA, LE LR, FFS, MW and JCM designed the study, while PJM, TDM, AA, LR, FFS, MW and JCM oversaw its implementation. LE performed database searches. JL coordinated and performed study selection, data extraction and quality assessment. JL, MW and JCM planned and interpreted, and JL conducted the meta-analyses and meta-regression. JL and JCM wrote the first draft. All authors reviewed the study findings, and read and approved the final version before submission.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/12/60/prepubTable S1. Characteristics of RCTs included in systematic review of effectiveness of dietary interventions among adults of retirement age. Table S2. Features of dietary interventions among adults of retirement age included in the systematic review. Figure S1. RCTs reporting overall fruit and vegetable intakes by body mass index among people of retirement age. Figure S2. RCTs reporting fruit intakes by body mass index among people of retirement age. Figure S3. RCTs reporting vegetable intakes by body mass index among people of retirement age. Figure S4. RCTs reporting fish intake among people of retirement age. Figure S5. RCTs reporting meat intake among people of retirement age. Figure S6. Meta-regression analysis of effects of length of follow-up on fruit and vegetable intake. Slope = 0.72, Q = 0.91, d.f. = 1, P = 0.34. The circle size reflects the weight that a study obtained in the meta-regression. Figure S7. Meta-regression analysis of effects of study sample size on fruit and vegetable intake. Slope = 0.002; Q = 0.13, d.f. = 1, P = 0.72. The circle size reflects the weight that a study obtained in the meta-regression. Figure S8. Meta-regression analysis of effects of study retention rate on fruit and vegetable intake. Slope = 0.77; Q = 0.39, d.f. = 1, P = 0.53. The circle size reflects the weight that a study obtained in the meta-regression. Figure S9. Meta-regression analysis of effects of baseline F&V intakes on fruit and vegetable intake. Slope = 0.14; Q = 1.30, d.f. = 1, P = 0.25. The circle size reflects the weight that a study obtained in the meta-regression. Figure S10. Prisma checklist.Search Strategy for systematic review of effectiveness of dietary interventions among adults of retirement age: database searched - Ovid MEDLINE(R) (1950 to April Week 3 2013). Click here for file