In the last decades, microarray technology has spread, leading to a dramatic increase of publicly available datasets. The first statistical tools developed were focused on the identification of significant differentially expressed genes. Later, researchers moved toward the systematic integration of gene expression profiles with additional biological information, such as chromosomal location, ontological annotations or sequence features. The analysis of gene expression linked to physical location of genes on chromosomes allows the identification of transcriptionally imbalanced regions, while, Gene Set Analysis focuses on the detection of coordinated changes in transcriptional levels among sets of biologically related genes.In this field, meta-analysis offers the possibility to compare different studies, addressing the same biological question to fully exploit public gene expression datasets.a priori step before performing gene set analysis. The application of STEPath in individual studies produced gene set scores weighted by chromosomal activation. As a final step, we propose a way to compare these scores across different studies  on related biological issues. One complication with meta-analysis is batch effects, which occur because molecular measurements are affected by laboratory conditions, reagent lots and personnel differences. Major problems occur when batch effects are correlated with an outcome of interest and lead to incorrect conclusions. We evaluated the power of combining chromosome mapping and gene set enrichment analysis, performing the analysis on a dataset of leukaemia  and on a dataset of skeletal muscle diseases .We describe STEPath, a method that starts from gene expression profiles and integrates the analysis of imbalanced region as an In leukaemia, we identified the Hox gene set, a gene set closely related to the pathology that other algorithms of gene set analysis do not identify, while the meta-analysis approach on muscular disease discriminates between related pathologies and correlates similar ones from different studies.STEPath is a new method that integrates gene expression profiles, genomic co-expressed regions and the information about the biological function of genes. The usage of the STEPath-computed gene set scores overcomes batch effects in the meta-analysis approaches allowing the direct comparison of different pathologies and different studies on a gene set activation level. In the last decades, microarray technology has seen such an explosion of applications as to become a standard tool in biomedical research. It has allowed the discovery of many prognostic genome markers related to the development of pathologies -6. The sGiven the high-throughput nature of microarrays, statistical and bioinformatic methods were required to analyse such large amounts of data. Initial studies were focused on the identification of differentially expressed genes and their significance in many experimental designs (gene by gene approach). This analysis is time-consuming and sometimes ineffective because derived gene lists have to be interpreted, searching for patterns of genes that have similar function or are involved in particular processes . This apMeta-analysis may be broadly defined as the quantitative review and synthesis of the results of related but independent studies . DiffereTo improve microarray data analysis, the first tools developed were based on the integration of external genomic information such as gene location -19, ontoSeveral methods were devised to analyse gene expression as a function of physical location of genes on chromosomes. These approaches, collectively referred to as "chromosome mapping", were applied to microarray data of cancer studies. The studies identified regions with transcriptional imbalances that reflected large chromosomal aberrations typical of such pathologies. Examples of these applications are the Locally Adaptive statistical Procedure (LAP)  and the A more popular method for the integration of gene expression profiles with additional information is based on ontological and pathway annotations and is called Gene Set Analysis (GSA). This approach evaluates gene expression profiles among groups of related genes (gene sets), seeking coordinated changes in the expression levels of subsets of gene members. Usually, GSA has three main steps: a) computing associations of each expression pattern with a phenotype; b) computing enrichment scores for analysed gene sets; c) computing the global p-value and q-value for every tested gene set based on the appropriate permutation test. Several implementations of the GSA approach are now available, such as the algorithms developed by Subramanian  , Tian , that scores and integrates chromosomal region activation as an actories ,29, but We implemented a new gene expression analysis method that takes into account i) the activation or repression of genes in chromosome regions  and ii) H0, the region did not show differential expression; H1, the region is differentially expressed.To integrate physical locations of genes, STEPath measures the association of a gene expression profile with a phenotype  statistics ), rescalAfter the computation of chromosome profiles using significant regions, STEPath performs a gene set analysis using SAM statistics  smoothedUsing the smoothed statistic, an up- and down-regulation value for every gene set was calculated. As GSA relies on the quality of annotation and dimension of gene sets, it is possible that the signal of a small group of coordinated genes becomes lost in non-specific signals. If this could be an analysis limitation, we increased GSA power by adopting two approaches: a) we analysed the most-used database repositories for gene sets, and b) we extracted portions of pathways showing coordinated expression.a) We have compiled gene sets from Gene Ontology (GO) , Kyoto Eb) According to Efron  and lateH0, that the gene set shows the same pattern of association with the phenotype compared to the rest of the genes. The q-values were computed using the Benjamini Hochberg algorithm [Up- and down-regulation scores were independently tested for significance using a gene-based permutation approach. We tested the null hypothesis, lgorithm .In meta-analysis studies, step 3 primarily aims to determine if the results from one study are confirmed in other independent studies.Gup and Gdown, see Methods) and an associated q-value. Different pathologies can then be directly compared using the gene set summarization values produced for individual studies.For an individual study, the STEPath procedure (Step 1 plus Step 2) produces a list of gene sets with summarization values  gene).Raw expression data (CEL files) were downloaded from the GEO database (GEO series ID: GSE14062), processed using a gene-based custom Chip Definition File (CDF)  to betteThis dataset was used to analyse the performance of the main modules implemented in STEPath: chromosome mapping (Step 1) and gene set analysis (Step 2).Using our implementation, we were able to identify a spectrum of possible imbalanced regions across all chromosomes  the region containing the Homeobox genes (HOX cluster) on chromosome 7  [The choice between gene   and Sign (PCOT2) ) has bee (PCOT2)  and Goem (PCOT2)  papers.  (PCOT2)  and sigP (PCOT2) . STEPath (PCOT2) ,52.To evaluate the contribution of chromosome profile information, we performed our GSA procedure (STEPath) with and without chromosome profile correction. In both cases, STEPath was able to identify the HOX gene set probably due to a separate evaluation of up- and down-regulated genes. However, the integration of gene location with gene set analysis allows the combination of different levels of biological information (co-expressed/regulated genes) and helps to correctly identify disease-related genes, since they have a different position in a rank evaluation Table . To confWe applied STEPath in a meta-analysis approach involving expression datasets of limb girdle muscular dystrophies type 2A , type 2B  and type 2I (LGMD2I). We built a meta-dataset combining LGMD2A from two distinct datasets. The first was published by Bakay  , CTRLs vs LGMD2A (GSE11681), CTRLs vs LGMD2B (GSE3307) and CTRLs vs LGMD2I (GSE3307), where CTRLs are normal muscle controls from healthy donors , we chose GSE3307  as a reference study. The signature was composed of 70 gene sets: 55 Gene Ontology and 15 biological pathways that were used to build the gene set matrix (see Methods).Cluster analysis of the gene set matrix (derived from the main signature) shows that the two LGMD2As from different datasets are linked, and LGMD2B and LGMD2I segregate separately Figure .We then performed unsupervised cluster analysis  on gene Our approach overcomes the main limitations of gene expression meta-analysis and demonstrates that it is useful to reveal gene set signatures that discriminate different pathologies. In this way, we can evaluate the main signature discrimination/association power, projecting it into the second study Figure , point 3We extended the analysis performed for LGMDs including more variability with different skeletal muscular diseases  were downloaded from the GEO database  GEO ser.We compared STEPath meta-analysis results with a meta-analysis approach based on different GSA scores. Similarly for STEPath, we built a MGS matrix using scores derived by both sigPathway and GSEA (as implemented in limma). Clustering results of the MGS matrix from both sigPathway and GSEA failed to co-segregate the two LGMD2A datasets and the entire group of LGMDs Figure  and 4E.Gene set clusters were analysed focusing on both shared and peculiar pathology responses. Down-regulated gene sets show several clusters with the same expression level. These gene sets mainly refer to aerobic respiration, the production of ATP and mitochondria Figure . These rDysferlinopathy (LGMD2B) was characterized as dystrophies where dysferlin-deficient cells show abnormalities in vesicular trafficking . LGMD2B To search for discriminative gene sets among LGMD2A  and the other skeletal muscular diseases, we performed a template matching search . In FiguRecently, Beckman and Spencer  proposedhttp://www.r-project.org/, version 2.10.1), and they are freely available as R package at http://gefu.cribi.unipd.it/papers/stepath under the AGPL3 licence. The implementation depends on bioconductor [http://CRAN.R-project.org/package=samr).All functions to perform STEPath analysis are written in R (onductor  version onductor ) and samThe present implementation is microarray platform-independent and potentially could be applied to any kind of gene-phenotype association score .The algorithm we developed allows the analysis of gene expression data by integrating supplementary biological information to identify gene co-expression along the chromosomes and to perform a gene set analysis. The integration was initially tested on a leukaemia dataset, highlighting interesting imbalanced regions containing genes involved in ALL pathology: 1) MLL region on chromosome 11; 2) the region containing HOX gene cluster on chromosome 7; and 3) the region containing MEIS1 on chromosome 2. These regions are specifically enhanced by the STEPath algorithm and not by LAP or MACAT.The second integration was tested on gene expression datasets both from leukaemia and skeletal muscle diseases evidencing the importance of integration of the chromosome profiles in the gene set analysis. Integrating two biological aspects in the STEPath algorithm , we have demonstrated that STEPath produces gene set expression scores that are suitable to directly compare different diseases and studies to perform meta-analyses.We applied STEPath and the meta-analysis approach to limb girdle muscular dystrophies (LGMDs), highlighting the co-segregation of two different studies of LGMD2A patients, and to a meta-dataset for inflammatory myopathies composed of both Affymetrix arrays and unpublished custom oligo arrays. Results of the study of inflammatory myopathies will be discussed in a separate paper.Step 1: The process to identify differentially expressed regions can be divided into four parts: I) computation of a per-gene statistic to measure differences in gene expression between two groups under investigation or in one group, if microarray experiments were performed in a competitive hybridization way, II) correction of the statistics based on the expression level of neighbourhood loci, III) identification of statistically differentially expressed regions by a permutational approach and IV) building of per-chromosome profile.I) In this study, we used the SAM t-statistic to measure the association of genes to the phenotype of the two conditions. All SAM analyses were computed using two-class unpaired comparisons between a disease state versus a reference condition based on 100 permutations via the samr R package .Eup) or inhibition (Edown) for every gene i considering the neighbour genes and the local gene density.II) This step computes the local index of global activation  that covers n = 2 up-regulated genes upstream TSSi and n = 2 up-regulated genes downstream TSSi. Given rupi, we can summarize the local gene expression contribution for up-regulated genes inside the region (S'iup), as described in equation 1:For any given gene U = {u ∈ rupi|Su ≥ 0}, and |U| denotes the cardinality of the set U.where up) as reported in equation 2:We can also define the local gene density of up-regulated genes :S'upc is the mean of summarization values for all designed rup in the chromosome c and ρupc is a per-chromosome estimation of the global up-regulated gene density. We defined per-chromosome global up-regulated gene density as the mean of all local densities for every up-regulated gene.where 'upi and S'upc is meant to rescale regional expression compared to the average situation in the chromosome c, while the ratio between ρupi and ρupc is meant to favour regions presenting genes more densely distributed than the mean local densities of the chromosome.The ratio between Si, we defined rdowni and the summarization value Edowni as described for up-regulated genes. In this case, U is defined as follows:In parallel, for any gene Eupi and Edowni. In particular, SAM statistics were randomly shuffled over gene positions. We applied procedure II) and III) to B = 100 of these permutations to compute E'upi,b and E'downi,b null distributions, where 1 ≤ b ≤ B . We computed p-values for every window centred in gene i as the probability that E'upi or E'downi exceed respectively the observed Eupi and Edowni over B permutations. We then corrected p-values for multiple testing error using the Benjamini Hochberg FDR control (preprocessCore r package [III) We adopted a permutational approach to identify significantly different  package ).rupi and rdowni for a given chromosome c. We used these dimensions because they are in accordance with the clustering scale dimensions found in mammalian genomes by Firneisz [Eup or Edown present in the sliding windows.IV) The final step was performed to produce a per-chromosome profile by scanning each chromosome gene by gene using a window of fixed length. For window size, we used the reference lengths for up- and down-windows defined as the average length of all Firneisz  and FarrFirneisz  .where Step 2: the GSA module can be divided into two parts: 1) computation of per-gene set scores and 2) identification of significant gene sets.SS. In particular, we performed the GSA computing up- and down-regulated gene contributions separately. Let the indices k, k = 1,...,K denotes the kth gene set, and i, i = 1,..,I denotes the ith gene. We defined an incidence matrix M with dimensions K × I, where Mk,i = 1 denotes the presence of gene i in the kth gene set, and Mk,i = 0 denotes the absence of gene i in the kth gene set. We computed a gene set up-regulation value Gupk for the kth gene set as the mean of SSi∈k ≥ 0 and similarly for down-regulated values (Gdownk).1) We implemented the measurement of gene set scores as the mean of the corrected SAM statistics, SS: SS'b. We applied B = 100 permutations on S, and for each permutation b, we smoothed S'b to compute SS'b (null hypothesis H0: the gene set shows the same pattern of association with the phenotype compared to the rest of genes). We applied the procedure described in 1) to compute Gup'b and Gdown'b null distributions for each gene set. P-values were then calculated for Gupk and Gdownk independently as the probability that Gup'k or Gdown'k exceed the observed gene set score Gupk or Gdownk over B = 100 permutations. P-values were finally corrected according to Benjamini Hochberg FDR control.2) To assess the significance of gene sets, we adopted a gene-based permutational scheme to compute null distributions of Step 3: step 3 compares different pathologies and different studies. The starting points are Gup and Gdown summarization values of the gene sets from step 2.Gup or Gdown exceeding the mean score of significant gene sets.A reference study was defined as the one with greater variability. From each pathology in the reference study, a gene set signature was extracted. It was defined as the significant gene sets  with either We called the union of the pathology signatures main gene set signature (MGS). Using the MGS, corresponding summarization values from all the pathologies in all studies  were extracted and a matrix (MGS matrix) was produced, where columns were the different pathologies, and rows were summarization values for every gene set on the signature. To make comparable values of each gene set signature among different pathologies, quantile normalization was applied. The normalized matrix provides a direct comparison of gene set activation and inhibition across pathologies and studies. We adopted this strategy because signatures identified in one study should be identified in independent studies for the same pathology; related pathologies from different studies should cluster together.We developed a gene-based custom Chip Definition File (CDF) by re-mapping probes of Affymetrix HGU133plus2 and HGU133A chips on the ENSEMBL gene database (ver 56). Gene-based custom CDFs were generated as follows: i) matching of ensemble gene sequences with all probes present in a given gene chip ; ii) filtering out of non-specific probes (probes that match more than one gene sequence); iii) grouping of remaining probes in meta-probe sets with at least 4 members; iv) discarding all probes not belonging to any meta-probe set defined in point iii) . None ofWe compiled a collection of gene sets using various public databases. We used gene sets from Gene Ontology (GO) , 204 KEGG pathways, 161 ByoCyc pathways, 102 Superarray pathways and 111 wiki pathways. Only gene sets with members in the CDF were used in the analysis.Multichip normalization was performed using RMA as implemented in the affy bioconductor package.The gene set expression matrix was normalized by quantile normalization as implemented in R package preprocessCore.All cluster analyses were performed using the Euclidean distance with complete linkage method. We used the Hierarchical Cluster Analysis (HCL) implemented in the TMeV suite (version 3.1) .PTM analysis was performed using the PTM function implemented in the TMeV suite and setting p-value threshold at 0.05.Limma GSEA was performed using the limma bioconductor R package.SigPathway analysis was performed using the sigpathway bioconductor R package.MACAT analysis was performed using the macat bioconductor R package.2; SAFE: Significance Analysis of Function and Expression, LGMDs: Limb Girdle Muscular Dystrophies; CTRL: Control; MAPE, Meta-Analysis of Pathway Enrichment; MGS: Main Gene set Signature; ATP: Adenosine triphosphate; GO_BP: Gene Ontology Biological Process; GO_MF: Gene Ontology Molecular Function; GO_CC: Gene Ontology Cellular Component; AGPL3: GNU Affero General Public License; FDR: False Discovery Rate; CDF: Chip Definition File; HCL: Hierarchical Cluster Analysis; TMeV: TIGR MultiExperiment Viewer; PTM: Pavlidis Template Matching.LAP: Locally Adaptive statistical Procedure; MACAT: MicroArray Chromosome Analysis Tool; AML: acute myeloid Leukaemia; ALL: acute lymphoblastic leukaemia; GSA: Gene Set Analysis; SAM: Statistical Analysis of Microarray; KEGG: Kyoto Encyclopedia of Genes and Genomes; GEO: Gene Expression Omnibus; RMA: Robust Multiarray Averaging; PAM: Predicted Analysis of Microarray; PCOT2: Principal Coordinates and Hotelling's TThe authors declare that they have no competing interests.PM implemented the STEPath algorithm, performed all of the statistical and bioinformatic analyses. DR, GS and CR participated in the design of the study, revised the manuscript and participated in the investigation of the significant gene sets and interpretation of the results. GL and SC conceived and supervised the study, wrote the manuscript, coordinated the work and the interpretation of the results. All authors read and approved the final version of the manuscript declaring that they have no potential conflicts of interests.Additional figures. Word document containing supplementary figures. The figures are provided one per page with a short description.Click here for fileAdditional tables. Excel spreadsheet file containing supplementary tables. External link to NCBI and Gene Card databases are provided for genes explored in the chromosome regions described in the text.Click here for fileLGMDs dataset data matrix. Text file containing data matrix for LGMD dataset gene set meta-analysis.Click here for fileMuscle disease dataset data matrix. Text file containing data matrix for muscular disease dataset gene set meta-analysis.Click here for file


Coronary artery bypass grafting (CABG) on cardiopulmonary bypass (CBP) is associated with significant morbidity and mortality. In high-risk patients, doomed for reoperation the adverse effects of CBP may be more striking. We evaluated the results of reoperative CABG (redo-CABG) by either off-pump (OPCAB) or on-pump (ONCAB). Clinical endpoints were perioperative myocardial infarction, mortality, survival and as the most striking difference between prior studies the quality of life (QoL).We performed a prospective, non-randomized assessment for patients who underwent redo-CABG by redo-OPCAB (n = 40) or redo-ONCAB (n = 40) at our institution between January 2007 and December 2010. For evaluation of QoL the SF-36 health survey was used with self-administered assessment.During follow-up 37 of 40 patients were alive in the redo-OPCAB group versus 32 of 40 patients in the redo-ONCAB group (p < 0.05). The shorter operation time, less blood loss, fewer perioperative myocardial infarctions, the higher rate of totally arterial revascularisation and shorter intensive care stay were the significantly beneficial differences for patients in the redo-OPCAB group (p < 0.05). The 3-year survival rate was higher in the redo-OPCAB group with 81 ± 12% versus 63 ± 9%in the redo-ONCAB group. The quality of life survey did not reveal any significant differences between both groups.In conclusion, with our present retrospective study, we could demonstrate the safety and efficacy of the redo-OPCAB technique with even higher 3-year survival rate. Both techniques seem to have similar impact on the outcome of patients. Coronary artery bypass grafting (CABG) of high-risk patients with the use of the cardiopulmonary bypass (CPB) may be associated with deleterious effects, including the diffuse systemic inflammatory response, global myocardial ischemia and the risks of aortic manipulation ,2. TheseAs operative techniques evolve and survival after cardiac operations improves, the number of patients who have repeat sternotomy inevitably continues to increase. When coronary reoperation is necessary, the presence of a patent left internal mammary artery (LIMA) graft to the left anterior descending artery (LAD) creates specific risks, including the possibility of intraoperative injury to the graft and potential difficulties with myocardial protection ,9. ConveIn this study, we retrospectively analyzed our results of redo CABG either as redo-OPCAB or redo-ONCAB in non-randomized patients. The endpoints evaluated for the study were: in-hospital mortality, early mortality, late mortality, follow-up survival, hospital stay, intensive care stay, rate of totally arterial revascularization, incidence of perioperative myocardial infarction, perioperative stroke, major adverse cardiac events, postoperative renal failure, postoperative complications and transfusion of blood or blood products. Additionally, we explored the endpoints actual health status and quality of life (QoL). The recurrence of cardiac disorders like dyspnea according NYHA III and angina pectoris according CCS III were also evaluated. The results should clarify if clinical outcomes differ depending on the chosen operation for this selected high-risk patient group.The study protocol followed the Declaration of Helsinki and was approved by the Ethics and Research Committee of the Robert-Bosch-Hospital in Stuttgart Germany an academic training hospital of the Tuebingen University. Between January 2007 and December 2010 80 patients underwent reoperative CABG (redo-CABG) in the Departement for Cardiac- and Vascular Surgery of the Robert-Bosch-Hospital in Stuttgart Germany. A consecutive prospective series of n = 40 patients were operated off-pump (redo-OPCAB group) and n = 40 patients on-pump (redo-ONCAB group). A randomization which procedure is chosen for the patient was not applied. Data analysis indicate that in the beginning more patients were operated on-pump resulting in different follow-up times and that patients with renal insufficiency were operated preferentially off-pump   giving iPatients of the redo-OPCAB and redo-ONCAB group were analysed by comparing various preoperative, perioperative and postoperative variables Tables , 3 and 4In all enrolled patients the operations were performed by five staff surgeons, who are involved in our minimal invasive surgery program. These surgeons performed similar numbers of CABG but with different experience and learning curves regarding redo-OPCAB.The procedures were performed under general endotracheal anaesthesia with a regular single-lumen intubation. The patients were positioned on a bair hugger® mattress  in the operation room.Hemodynamic monitoring comprised of six-channel electrocardiography with ST-segment trend analysis and radial arterial pressure. Monitoring included regular continuous pulse oxymetry.TM monitor  were utilized for minimal-invasively hemodynamic monitoring. This system calculates cardiac output by analyzing arterial pressure waveform [In the redo-OPCAB group the FloTrac sensor and Vigileowaveform ,15. In tDuring the operation a transesophageal echocardiography (TEE)  was utilized for assessment of myocardial wall irregularities and unclear related walve pathologies especially in the Redo-ONCAB group if weaning of CPB was protracted.The anticoagulation regimen in the Redo-OPCAB group and Redo-ONCAB group differed. Intravenous heparin was applicated with a dose of 150 IU/kg body weight in the redo-OPCAB group, less than in the redo-ONCAB group with a dose of 300 IU/kg body prior to the institution of CPB to accomplish an activated coagulation time of greater than 400 sec. This different anticoagulation regimen, with lower heparin dose in the off-pump group derives from our clinical routine and is in accordance to data from literature  as cannuAfter median sternotomy the left internal mammary artery (LIMA) and or the right internal mammary artery (RIMA) were harvested by standard technique using hemoclips in a scletonizing fashion if not being utilized in the prior operation. In case of open graft vessels those were mobilized in their proximal segments. The other conduits  were harvested simultaneously using endoscopic vessel harvesting (EVH) . We used CTS OPCAB access platform and stabilizer  or Hercules Universal Stabilizer Arm™ with stabilizer  to stabilize the target coronary vessel. In the Redo-OPCAB group we always used intracoronary shunts  for anastomosis. In most cases, the left anterior descending coronary artery was the first coronary artery to be grafted. The vessels on the lateral and posterior wall were grafted later. However, the sequence of grafting was individualized for a particular patient, depending on the severity of the lesions in different coronary arteries and patient’s hemodynamics. Both the left anterior descending and right coronary arteries could be grafted without much displacement of the heart. For exposure of the circumflex and right coronary vessels pericardial traction sutures were used to pull the heart vertically. In most cases the pleurae were not opened. A vertical pericardiotomy was performed to herniate the heart to the right chest under the sternum. Other maneuvers such as the Trendelenburg position and tilting the table were performed as required. Inotropic agents were used when necessary during surgery. We used 8–0 Seralon polyamide suture  for the distal anastomosis and 6–0 for the proximal anastomosis.Conventional coronary artery bypass procedures were performed using standard CPB, which was established using ascending aortic and two-stage venous cannulation. An open extracorporeal circuit was used, which was identical in each patient and consisted of a membrane oxygenator, arterial filter and crystalloid priming. The LIMA and RIMA were harvested in a scelotonizing fashion if not being utilized in the prior operation. Open LIMA or RIMA in-situ graft vessels were mobilized in their proximal segments for the later cross-clamping during cardioplegia. The radial artery and saphenous vein were usually harvested utilizing endoscopic vessel harvesting equipment (EVH) . After instituting CPB the aorta and open LIMA or RIMA in-situ grafts were cross-clamped and the procedure was performed on an cardioplegic arrested heart. The patient was not actively cooled but temperature was allowed to drift as was usually between 32-34°C. Antegrade Brettschneider´s crystalloid cardioplegia (1500 ml) was used for myocardial protection . Cardioplegia was repeated if myocardial contractions occurred. Rewarming was initiated during completion of the last peripheral anastomosis. Reperfusion was performed with whole blood by declamping of the aorta and open LIMA or RIMA in-situ grafts. Reperfusion time was defined till the end of CPB.Routinely, all patients underwent preoperative clinical and non-invasive angiological examinations of the radial artery diameters  by the attending cardiovascular surgeon. Further a normal Allen’s test on the chosen side was compulsory. Radial arteries with immense atherosclerotic changes or luminal diameter less than 2 mm were excluded prior to harvesting. The saphenous vein was scanned additionally and was doomed as a graft if the luminal diameter was at least 3 mm. Duplex ultrasound was performed by the cardiovascular surgeon with a Siemens ACUSON Antares™ ultrasound system equipped with a VF13-5 transducer probe . A 2-D linear electronic probe at 7.0 MHz, pulse wave Doppler at 5.0 MHz and color Doppler at 5.0 MHz were used. The angle of the emitted Doppler ultrasound wave from the probe was adjusted to 60° to achieve the Doppler signal of the strongest intensity. The internal diameter of the vessel was measured using M-mode technique. Whenever feasible we favourized harvesting arterial grafts if not utilized in the past. Due to the prospective non-randomized character of this study the majority of the redo-ONCAB patients were operated in the first half of the observation period resulting in lesser utilization of arterial grafts in comparison to the redo-OPCAB patients. Further each patient was examined by coronary angiograpy assassing the viability of the native coronary arteries and the graft vessels preoperatively.Graft patency and bypass fow rates were analyzed intraoperatively using a handheld 2, 3 or 4 mm transit-time flowprobe connected to a MediStim VeriQ System . Postoperative ECG (electrocardiogram), serial samples of creatine kinase (CK), CK-MB, cardiac troponin I (cTnI) and after 2009 high-sensitive cardiac troponin I (hs cTnI) were determined every 6 h up to 48 h in any patient. Myocardial infarction was defined as either Q-wave in the postoperative ECG or an increase in the CK-MB enzyme fraction above 10% and simultaneous increase of cTnI of more than 10 ng/ml or hs cTnI of more than 100 ng/ml. An early postoperative control angiography was not recommended, except for patients with postoperative myocardial infarction suspected (patients with postoperative angina and increase of troponin I and CK-MB levels).Patients of both groups were transferred to the intensive care unit (ICU) and ventilation was kept till reaching a stabile haemodynamic situation. Usually, patients were weaned from ventilation as early as possible. On the next day, we transferred the patients to our intermediate care unit (IMC) for further surveying. We transferred the patients on postoperative day (POD) two our regular cardiovascular service till discharge from hospital.We aimed to assess the surveillance and quality of life of the operated patients by sending them self-administered questionnaires by mail. Usually, we sent them a cover letter, the SF-36 questionnaire, a self-administered questionnaire on angina pectoris and dyspnea and a stamped self-addressed return envelope. We instructed our patients to give information about their health situation of the last four weeks.The questionnaires consisted of two parts. Part one comprised items about the actual health situation and the recurrence of cardiac disorders like angina pectoris and dyspnea with necessary interventions in the follow-up period. The assessment of angina pectoris and dyspnea by self-administered questionnaire was considered valid in view of the very satisfactory agreement between the coding of the patient and medical coding (New York Heart Association (NYHA) and Canadian Cardiovascular Society classification (CCS)  for angiPart two, the SF-36 health survey is one of the most extensive standardized, self-administered, generic questionnaires for measuring both the physical and mental health of a patient ,13,19. IPatients medical-records were surveyed respectively the incidence of parameters like mortality peri- or postoperatively. The mortality in this period was defined as in-hospital mortality. Death occurring until 30 days postoperatively was defined as early and after this period as late mortality.Like in the preceding section mentioned the questionnaires consisted of two parts. Part one comprised items about the actual health situation and the recurrence of cardiac disorders like dyspnea according NYHA III and angina pectoris according CCS III with necessary coronary interventions in the follow-up period. If the questionnaires were not sent back after one month patients were telephoned or the family physician was contacted. In addition, patient medical-records were studied if during the last hospital stay MACCE (Major Adverse Cardiac and Cerebrovascular Events) occurred. Dyspnea according NYHA III, angina pectoris according CCS III, MACCE, in-hospital mortality, early and late mortality occurring during the hospital-stay and follow-up were defined as a censored event.Later Kaplan-Meier analysis and Cox proportional hazards models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI), with adjustments for baseline patient and disease characteristics.Survival analyses consisted of two parts. Part one was cumulative survival with the subgroup 3-year survival. The censored event was defined as death occurring as in-hospital mortality, early and late mortality during follow-up. Part two was the assessment of composite event-free survival with subgroup 3-year survival. The censored event was defined as dyspnea according NYHA III, angina pectoris according CCS III, MACCE, recent coronary intervention, in-hospital mortality, early and late mortality occurring during the hospital-stay and follow-up.Values were expressed as mean ± standard deviation. Continuous data were analyzed using the Wilcoxon´s rank test. The Kaplan–Meier method was used to analyse cumulative survival, 3-year survival and freedom from events . The log rank test was used to compare the survival and freedom from events between redo-OPCAB and redo-ONCAB. Probability values (P) of less than 0.05 were considered significant. Generally we presented the exact P-value. Statistical analysis was performed using the JMP program .The patients preoperative demographic data did not differ significantly (p < 0.05) between the redo-OPCAB and redo-ONCAB groups Table . Both grThere were significant (p < 0.05) differences in the redo-OPCAB und redo-ONCAB groups. The shorter operation time, more use of arterial grafts, and less blood loss were the significant differences in the redo-OPCAB group Table . No signPatients in the redo-OPCAB group differed significantly (p < 0.05) with shorter intensive care stay, lower CK, Troponin and LDH expression. No significant difference existed for the incidence of postoperative major cardiac adverse effects, impaired wound healing and in-hospital mortality Table .The groups significantly differed in the operation time   . Although not significant, slightly more patients undergoing redo-ONCAB were ventilated longer  .The Kaplan–Meier analyses are depicted in Figure The hazard ratio was 2.99 with a 95% CI  of ratio 0.78–5.42.The Kaplan–Meier analyses are presented in Figure The hazard ratio was 1.32 with a 95% CI of ratio 0.54–3.21.The redo-OPCAB and redo-ONCAB groups were evaluated on the basis of eight categories according to the SF-36 health survey , off-pump coronary artery bypass grafting (OPCAB) has gained broader acceptance since the last decade. Nevertheless conventional non-beating heart on-pump CABG is still the standard technique used in coronary artery surgery in many institutions. Complications in relation to this technique are due to the release of inflammatory mediators, the use of cardioplegia, aortic cross clamping and hypothermia . Blood cOPCAB technique was introduced to avoid such complications. Despite its efficiency and safety over conventional CPB, the technique was criticized by many investigators regarding completeness of myocardial revascularization, graft patency and long term results. Coronary artery bypass grafting on the beating heart is a more demanding procedure than conventional CABG. It is more difficult in terms of technical skills and requires more attention to the surgeon and the anaesthesiologist. Many authors ,21,22 haConsidering the above mentioned data, we have meanwhile established the OPCAB technique for coronary revascularization as our standard technique. Nowadays we perform at least more than 90% of primary coronary revascularizations by OPCAB (not yet published data). In the cases of redo coronary revascularisations we achieved similar rates utilizing OPCAB (not yet published data). Therefore our presented data contains a significantly difference in regard of the postoperative follow-up period in the redo-OPCAB and redo-ONCAB group. The explanation for this circumstance is that in the second half of the observation period most of the redo coronary revascularisations were performed by redo-OPCAB. As in the first half of the observation period more single saphenic vein grafts were anastomosed in the second half the trend was towards total arterial revascularization with sequential anastomosis. This may be the result of the maturation process or completing the learning curve and changes of staff surgeons with different experience and learning curves regarding redo-OPCAB. Despite of the referred drawbacks of the OPCAB technique , we had The present study reports clinical outcomes and quality of life surveillance in 69 patients undergoing redo coronary revascularization either by redo-OPCAB or redo-ONCAB. After prior review of the appropriate literature we could not find any study investigating the quality of life of these highly selective patients group in such an extent. Our study aims to clarify if both procedures differ at the endpoints mortality, transfusion requirements, perioperative myocardial infarction, inotropic drug support, major adverse effects, hospital stay, quality of life and recurrence of cardiac disorders. Considering our study population with a mean Euroscore (logistic) around 23, it is justified to declare them as high risk patients. To our knowledge, it is unique and different from published data to include particularly the high risk patients in such a study ,27. In tIn contrast to the above presented data, the evaluation of the SF-36 health survey  did not We want to address few limitations of our present study. The study comprises, in comparison to the referred data high-risk patients undergoing coronary redo revascularisation. Both groups differ in the follow-up period. The limitations of our study include the prospective single-institution methodology being not a randomized clinical trial. Further limitations are the small number of patients, the inability to conduct a reliable multivariate analysis of predictor variables of late mortality due to the small number of patients with the index event.In conclusion, with our present retrospective study, we could confirm the safety and efficacy of both surgical procedures of redo-CABG, with better results for the redo-OPCAB technique. Nevertheless in this era where the use of multivessel coronary angioplasty is extensively employed the therapeutic option of redo-CABG, tendencially declining, should not be ignored in our clinical routine. Our future effort will be to perform a double-blinded randomized prospective study to gain more information and to clarify which technique is superior in this selective patient group especially in regard to the quality of life.Written informed consent was obtained from the patient for publication of this report and any accompanying images.The authors declare that they have no competing interests.UE carried out the patient evaluation and participated in the study design and coordination. UE performed the statistical analysis and created the manuscript. UE and ER were engaged in the data evaluation process. UA, NR, MM, S-G S and FUF carried out the redo coronary revascularisations by either method. FUF conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Lumbar spinal stenosis is one of the most common causes of low back pain among older adults and can cause significant disability. Despite its prevalence, treatment of spinal stenosis symptoms remains controversial. Epidural steroid injections are used with increasing frequency as a less invasive, potentially safer, and more cost-effective treatment than surgery. However, there is a lack of data to judge the effectiveness and safety of epidural steroid injections for spinal stenosis. We describe our prospective, double-blind, randomized controlled trial that tests the hypothesis that epidural injections with steroids plus local anesthetic are more effective than epidural injections of local anesthetic alone in improving pain and function among older adults with lumbar spinal stenosis.We will recruit up to 400 patients with lumbar central canal spinal stenosis from at least 9 clinical sites over 2 years. Patients with spinal instability who require surgical fusion, a history of prior lumbar surgery, or prior epidural steroid injection within the past 6 months are excluded. Participants are randomly assigned to receive either ESI with local anesthetic or the control intervention . Subjects receive up to 2 injections prior to the primary endpoint at 6 weeks, at which time they may choose to crossover to the other intervention.Participants complete validated, standardized measures of pain, functional disability, and health-related quality of life at baseline and at 3 weeks, 6 weeks, and 3, 6, and 12 months after randomization. The primary outcomes are Roland-Morris Disability Questionnaire and a numerical rating scale measure of pain intensity at 6 weeks. In order to better understand their safety, we also measure cortisol, HbA1c, fasting blood glucose, weight, and blood pressure at baseline, and at 3 and 6 weeks post-injection. We also obtain data on resource utilization and costs to assess cost-effectiveness of epidural steroid injection.This study is the first multi-center, double-blind RCT to evaluate the effectiveness of epidural steroid injections in improving pain and function among older adults with lumbar spinal stenosis. The study will also yield data on the safety and cost-effectiveness of this procedure for older adults.NCT01238536Clinicaltrials.gov  Lumbar spinal stenosis is one of the most common causes of low back pain among older adults and can result insignificant disability . The cauDespite the prevalence of symptomatic lumbar spinal stenosis among older adults, treatment remains controversial, with limited comparative effectiveness evidence. Common treatments include conservative measures such as non-steroidal anti-inflammatory drugs (NSAIDS), activity modification, and physical therapy, as well as more invasive treatments such as epidural steroid injections (ESI) and surgery . AlthougA few cohort studies suggest that ESI may provide short-term pain relief ,8. HowevOnly one RCT of fluoroscopically-guided ESI compared to injections with local anesthetic alone has been published . This stThe overall objective of this multi-center, double-blind RCT is to evaluate the effectiveness of ESI plus local anesthetic versus local anesthetic alone in improving pain and function among older adults with back pain and lumbar spinal stenosis. We hypothesize that patients randomized to receive ESI with local anesthetic will show significantly greater improvement in pain and function compared to patients randomized to receive local anesthetic only. We will also examine whether ESI is differentially effective in improving pain and function for different patient subgroups . Finally, we will compare resource use and costs in the two treatment groups as well as incremental cost-effectiveness and incremental cost utility from both payer and societal perspectives.The Lumbar Epidural steroid injections for Spinal Stenosis (LESS) study is a double-blind RCT of ESI plus local anesthetic injections versus local anesthetic injections for the treatment of pain associated with lumbar spinal stenosis in older adults. Patients, study investigators, and the research staff collecting data will remain blinded to patient treatment allocation.The patient follow-up has three distinct phases--from randomization and baseline injection to the primary endpoint at six weeks, from optional crossover at six weeks until nine weeks, and the follow-up period from nine weeks to one year after randomization  project. The project involves building a registry designed to capture data from patients enrolled in three large integrated health care systems in the United States. The BOLD registry will provide a mechanism for collecting outcomes data on a large number of patients with back pain and will serve as a portal for entry into studies such as the LESS trial. We will also be recruiting patients from several academic medical centers. Each participating clinical site obtained local Institutional Review Board (IRB) approval prior to subject recruitment. Patients with spinal stenosis referred for an ESI are asked if they would like to participate in the LESS trial. Prior to recruitment, the treating physician determines whether the patient is an appropriate candidate for ESI based on history, physical examination and imaging studies. The physician introduces the study at the end of this consultation if the patient is deemed an appropriate candidate for ESI. Each potential participant is invited to view an educational video that describes the study and provides detailed information about the procedures and implications of participation in the study. Patients provide written informed consent when enrolling in the study.Study inclusion and exclusion criteria are listed in Table Patients with spinal stenosis who are being referred for an ESI as part of their treatment plan are invited to participate in this study by the treating physician. Interested patients are then screened for eligibility by the research coordinator at the site. If eligible, the potential participant is asked to participate in the study and to complete the informed consent process. A study physician reviews each potential participant's imaging studies to determine the eligibility criterion of presence of central spinal canal stenosis and to categorize the stenosis as mild, moderate or severe.Baseline questionnaires assess demographic, clinical, and disease-history information and include patient self-report measures of pain, function, fear avoidance , and paiOur primary outcomes are measures of function and pain. These include the Roland-Morris Disability Questionnaire, a back pain-specific functional status questionnaire adapted from the generic Sickness Impact Profile (SIP). It consists of 24 yes/no items, which represent common dysfunctions in daily activities experienced by patients with low back pain. The other primary outcome measure is pain recorded on an 11-point numerical rating scale(NRS) measuring average leg and back pain (separately) in the past week. The primary outcome measures are completed at 3 days, 14 days, 3 weeks, 6 weeks, and 3, 6 and 12 months post-randomization.Secondary outcome measures include the Brief Pain Inventory (BPI) Interference scale , the PatAt baseline and at three weeks and six weeks post-injection, we draw blood to measure HbA1c and fasting blood glucose and morning blood cortisol levels to monitor for hyperglycemia and adrenal suppression following steroid administration. Because patients are blinded to treatment assignment, these are checked in all patients regardless of treatment received. In addition, at each in-person visit, we check blood pressure and weight because these can also be affected by steroid administration.Other measures include patient-completed resource utilization questionnaires to collect information on prescription and over-the-counter medication use, medical visits outside the health plan, and other patient burdens and costs during four 3-week periods throughout the 12-month study. Resource use and costs for utilization within each health system are available through the electronic health information systems .Treatment assignments are stored centrally in a secure database at the study data coordinating center in Seattle, Washington. For each recruitment site, we utilize permuted-block randomization to achieve roughly balanced groups. We use random blocks of size 4, 6, 8, or 10 that are not divulged to the research assistants involved in recruitment in order to avoid bias in the recruitment process. Two pre-filled opaque syringes are obtained for every study procedure, one filled with the steroid and the other with the local anesthetic. The randomized treatment assignment is obtained via a password-protected study website by the clinical nurse or assistant not involved with subsequent data collection indicating which syringe should be labeled 'inject' or 'discard'. The clinical nurse or assistant confirms the use of the opaque syringe marked 'inject' by the physician performing the injection. The physician, the research staff conducting follow-up interviews, and the patient are therefore all blinded to treatment received.The procedure is performed in a fluoroscopy suite under strict aseptic conditions. The patient is placed prone on the procedure table. Standard fluoroscopy is used for localization of the spinal/vertebral level to be treated and needle placement for the injection. Fluoroscopic target identification and needle tip entry into the targeted anatomic space is done using previously described procedural techniques . The skiEpidural steroid injectate is 1-3 ml of 0.25-1% lidocaine followed by 1.5-3 ml of 40 mg/ml triamcinolone (i.e. 60-120 mg triamcinolone) in an opaque syringe. The choice of which steroid to use is at the discretion of the treating physician based on his or her usual clinical practice. Betamethasone (6-12 mg), dexamethasone (8-10 mg) or methylprednisolone (60-120 mg) may also be used. The needles are then removed and the patient taken to the recovery area.The control procedure is identical to the ESI except that the epidural injectate is 1-3 cc of .25%-1% lidocaine followed by 1.5-3 cc of .25%-1% lidocaine in an opaque syringe. The total volume of injectate is the same for the ESI and the control injection to avoid differential treatment effects based on volume.Every effort is made to maintain blinding of the patients and evaluators during the course of the trial. All baseline and follow up interviews are conducted by a study research assistant who remains blinded to the randomized assignment of the patient throughout the study. The research assistant also completes assessments of blinding at all follow up time points. All datasets have the randomization code masked until all pre-specified analyses are conducted to ensure that the research team remains unaware of patient assignment. Patients are asked to guess which treatment they actually received at 2 weeks, 5 weeks, and 3 months after the initially randomized injection  and to provide a reason for and confidence in treatment guesses other than "don't know". Specific instances of un-blinding will be recorded and the study statistician will monitor treatment guesses to diagnose systematic problems with the blinding procedure or over the course of conducting the study. Using Fisher's exact test, we will formally compare treatment guesses between arms 2 weeks after randomization when the study results are reported.We are utilizing "intent-to-treat"  analysesOur primary analysis will evaluate the effect of ESI + local anesthetic compared to local anesthetic alone using repeated measures analysis of covariance (ANCOVA). Each ANCOVA model will adjust for the respective outcomes measured at baseline and a dummy variable indicating study recruitment site. From the ANCOVA models, we will report on the means and standard deviations of six-week outcome measures for each group and an adjusted estimate of the mean difference (95% CI) between groups. We will adjust the primary analysis for important baseline characteristics that, despite randomization, differ between groups and are associated with the outcomes.We will conduct two subgroup analyses to assess evidence for treatment effect on the primary outcome measures within categories defined by race and injection approach . We do not hypothesize that the treatment effect will be larger or smaller in one subgroup compared to another, only that there is a significant treatment effect within each subgroup.We will assess secondary outcomes using ANCOVA models for continuous measures and logistic regression models for binary measures. These analyses will include assessment of group differences in clinically meaningful changes in pain and function .Secondary analyses will consider the complete time profile for the primary outcome measures of pain and function through 12 months using methods appropriate for the analysis of repeated measures. Crossover and missing data will complicate analysis of follow-up data. Long-term outcomes will be evaluated using linear mixed effects models for repeated measures in both intent-to-treat and as-treated analyses. We will tabulate crossover rates by randomized treatment arm and compare the rates using Chi-squared tests and logistic regression as appropriate. To assess the impact of crossover on the overall evaluation of ESI, we will examine longitudinal models of outcome that incorporate a time-varying covariate indicating when treatment occurred.http://www.euroqol.org. We will estimate incremental cost-effectiveness ratios for six weeks, six months and one year using advanced sensitivity analyses to adjust parameter ranges for uncertainty in clinical, cost, or effectiveness variables [The economic evaluation will include an assessment of health care utilization, costs, and cost-effectiveness for the steroid group compared to those receiving local anesthetic alone in the intention-to-treat sample. The co-primary economic outcomes will be incremental per participant average costs compared to per participant Roland Morris Disability Questionnaire improvement and incremental cost per quality-adjusted life year (QALY) gained from both a payer and societal perspective . The QALariables . The imp® [The primary economic research question is whether treatment with steroid combined with anesthetic will be safer and more effective than treatment with local anesthetic alone, at a differential cost within willingness-to-pay thresholds of U.S. payers, using an incremental cost per QALY gained approach. The one-year societal perspective analysis will include direct and indirect costs, using standardized payer-based reimbursement amounts . In summarizing overall costs, we will include costs reported by participants via resource use questionnaires, in addition to the sum of estimated reimbursed amounts associated with care used. Within-health-system resource utilization will be assessed using clinical, pharmacy and administrative data from the participating health plans' electronic systems. We will use the health plan data to assess medical utilization events  and the pharmacy data to assess filled prescriptions. Average unit prices for resource use  will be applied to participants' health system utilization using a reimbursement database containing payer expenditures within each health system. These unit cost estimates  will come from an independent, private insurance claims data warehouse, i.e., Marketscan® .® data will be used for unit cost estimates.The secondary economic question is whether steroid injections represent good value for money as evidenced by non-QALY incremental cost-effectiveness ratios . Other consequences (outcomes) will be described in relation to cost or use profiles . MarketscanDuring four, three-week time periods over the course of the twelve month study , participants will complete Resource Utilization Questionnaires to record information related to out-of-pocket (out-of-system) costs, time related to seeking back pain treatment, daily use of prescription and over-the-counter medication use, and additional back therapies, along with use or purchases of other back-care products. We will describe the participant-reported data by randomized groups. Additional descriptive analyses will include assessing treatment group differences in dis-aggregated EQ-5D items representing activities of daily living .® data during several years. Costs will be estimated in 2013 US$, the year the study will conclude. As is standard practice in cost-effectiveness analyses of 12 months or less, no discounting of costs or effects will be performed for the 12-month study. Sensitivity analyses will be conducted by testing a 20% range versus base case values for cost parameters and effectiveness measures, as well as using probabilistic sensitivity analysis approaches [The effectiveness measures for the cost-effectiveness analysis will be obtained directly from the primary statistical analysis. The cost-effectiveness assessment will use a decision-tree framework to quantitatively assess the costs and effects for the randomized groups. Short-term cost-effectiveness outcomes will use the intention-to-treat approach, while longer-term, one-year outcomes will use empirical approaches to estimate EQ-5D and Roland Morris Disability Questionnaire outcomes in relation to cost. The assessment will allow for different resource use patterns and test ranges of assumptions for treatments and procedures. Empirical data from LESS and from external sources which included the relevant outcome measures ,29 will proaches . AdditioOur target is to enroll up to 400 patients with half randomized to ESI + local anesthetic and half to local anesthetic alone. We determined overall sample size based on the primary outcome (Roland Morris Disability Questionnaire) with two subgroup analyses . We will use ANCOVA as the primary method of analysis with the following parameters: 85% follow-up at 6 weeks; two-sided Type 1 error rate of α = 0.05; and adjustment for recruitment site and the outcome measured at baseline. For the purpose of sample size estimation, we conservatively assume six-week Roland Morris Disability Questionnaire scores will have a standard deviation of 7.5 for each group and a correlation between baseline scores and six-week scores of 0.3. For the combined (n = 400) assessment of ESI + local anesthetic vs. local anesthetic alone, the LESS study has 83% power to detect between-group mean differences as small as 2.25 points on the Roland Morris Disability Questionnaire.within each subgroup; we did not power the study for the hypothesis of a larger treatment effect within one subgroup compared to another (e.g. an interaction). We adjusted the overall two-sided Type 1 error rate of α = 0.05 using a Bonferroni correction for two tests of the data. Using ANCOVA methods, a sample size of n = 200 (100 per group) provides 83% power to detect between-group mean differences of 3.25 points on Roland Morris Disability Questionnaire.Subgroup analyses were powered to detect clinically meaning differences between treatments While formal interim analyses of treatment effect are not planned for this study, we will generate blinded variance estimates for the primary outcomes measured at 6 weeks after a minimum of 100 patients have been enrolled. Conservative estimates of outcome variance and 6-week retention utilized in the initial design of the study yielded an overall upper-bound sample size of n = 400. Adaptive sample size re-estimation will be conducted to provide more precise overall sample size requirements given the observed variance and retention rate, while maintaining the overall Type 1 error rate and power for the stated effect sizes .As there has been scant research focusing on ESI in older adults with pain due to spinal stenosis, little is known about treatment risks and benefits for this population. Although it has been demonstrated that surgery can be effective for severe spinal stenosis, there are many elderly patients for whom surgery carries substantial risk. There is a need for safer, less-invasive treatment options for older adults with the hope of reducing functional disability and promoting independence. Although ESI is a promising treatment alternative and is widely used in clinical practice, its effectiveness is unknown. Compared to young and middle-age adults, a much higher percentage of older adults have co-morbid conditions such as diabetes that may increase the risks due to systemic absorption of corticosteroids.As in most clinical trials, we anticipate that recruitment may be a challenge. Patients suffering from pain may be reluctant to delay what they might consider to be definitive therapy. We have developed strategies to increase our chances of successful recruitment including the use of a patient-education video, which was a successful strategy in our prior trials . We haveThe results of this study should provide compelling evidence as to whether or not epidural steroid injections are effective in improving function and pain in the elderly with lumbar spinal stenosis. The LESS trial will provide high-quality data for shorter-term outcomes (six weeks) and longer-term (one-year) outcomes. The patient-reported outcomes, the electronic medical and pharmacy utilization records, and supporting economic data providing reimbursed amounts for resources used will enable a robust evaluation of the comparative benefits and harms associated with epidural steroid injections with anesthetic versus local anesthetic alone.ANCOVA: Analysis of co-variance; BOLD: Back pain Outcomes using Longitudinal Data; BPI: Brief Pain Inventory Interference; CEA: Cost effectiveness analysis; ESI: Epidural steroid injection; GAD-7: Generalized Anxiety Disorder measure; LESS: Lumbar Epidural steroid injections for Spinal Stenosis; PHQ-8: Patient Health Questionnaire (PHQ-8); SSSQ: Swiss Spinal Stenosis Questionnaire (SSSQ); NRS: Numerical rating scale; QALY: Quality adjusted life year.The authors declare that they have no competing interests.All authors contributed to development of the research protocol, manuscript writing and editing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/13/48/prepub


The efficacy of chemotherapeutic drugs has traditionally been evaluated by their ability to exhibit a direct cytotoxic effect on malignant cells. Recently, it has been reported that certain of these drugs can also enhance the antitumor efficacy and immunotherapy response through their capacity to modulate innate and adaptive immunity . Our res+ B cells were preincubated with chemotherapeutic drugs over night. Next day these cells were used in the FASCIA assay [+ T cells. The proliferative response was analyzed with FACS, by identifying lymphoblasts as CD3+/CD4+/CD45RO+/HLA-DR+ cells. Further analysis of several B-cell surface markers revealed that the co-stimulatory molecule CD86 was upregulated by some drugs. The mRNA expression of CD86 and associated regulatory genes were also analyzed by QT-PCR.Human CD19IA assay , to evalIncubation of B cells with Doxorubicin increased the proliferative response in the functional FASCIA assay, while several other drugs tested caused suppression. Further experiments revealed this effect to be due to an increased expression of the co-stimulatory molecule CD86, and inhibition experiments with the addition of anti-CD86 antibody reversed the observed finding.+ B cells leads to an increased expression of CD86, and increased antigen presenting function in the FASCIA assay. Gained knowledge of the immunomodulatory properties of chemotherapeutic drugs will permit the development of drug dose and time schedules which allow for recovery of immune function, and may possibly lead to augmented anti-tumor responses.We have demonstrated that Doxorubicin treatment of human CD19


A large number of HIV-1 infections in Africa occur in married couples. The predominant direction of intracouple transmission and the principal external origins of infection remain important issues of debate.env gp41 sequences from both partners. Standardized interview data were used to deduce the direction as well as the external sources of the intracouple transmissions.We investigated HIV-1 transmission in 46 HIV-1 concordant positive couples from Dakar, Senegal. Intracouple transmission was confirmed by maximum-likelihood phylogenetic analysis and pairwise distance comparisons of HIV-1 Conservative molecular analyses showed linked viruses in 34 (74%) couples, unlinked viruses in 6 (13%) couples, and indeterminate results for 6 (13%) couples. The interview data corresponded completely with the molecular analyses: all linked couples reported internal transmission and all unlinked couples reported external sources of infection. The majority of linked couples (93%) reported the husband as internal source of infection. These husbands most frequently (82%) reported an occasional sexual relationship as external source of infection. Pairwise comparisons of the CD4 count, antiretroviral therapy status, and the proportion of gp41 ambiguous base pairs within transmission pairs correlated with the reported order of infection events.In this suburban Senegalese population, a majority of HIV-1 concordant couples showed linked HIV-1 transmission with the husband as likely index partner. Our data emphasize the risk of married women for acquiring HIV-1 as a result of the occasional sexual relationships of their husbands. The HIV-1 epidemic in Sub-Saharan Africa is mainly driven by heterosexual transmission A related topic under debate involves the role played by concurrent sexual relationships, i.e. having several sexual partners at the same time, in the African HIV-1 epidemic. It was postulated that a high frequency of concurrent sexual relationships results in an interlocking sexual network that could greatly facilitate the spread of HIV-1 env gp41 sequences from both partners. Standardized interview data from both partners reporting separately on the history of their HIV-1 infection were then used to analyze the direction of the intracouple transmission as well as the external sources of the infection. In this way, we found that a majority of HIV-1 concordant couples harbored linked HIV-1 sequences with the husband as the likely index partner in these couples.Most studies of HIV-1 transmission in African married couples used prospectively followed cohorts of HIV-1 discordant couples. These studies may suffer from inclusion bias and changes in sexual behavior due to counseling and follow up. Studies of couples with an already HIV-1 concordant status do not have these problems, yet to date few studies have analyzed the patterns of HIV-1 transmission using such cohorts. In the present study, we set out to unravel intracouple HIV-1 transmission in a cohort of HIV-1 concordant positive couples from Dakar, Senegal. Couples with genetically linked viruses were first selected by phylogenetic analysis of HIV-1 From May 2005 until February 2009, HIV patients consulting at the outpatient clinic of the Centre Hospitalier Universitaire Fann in Dakar, Senegal were invited to participate with their partners in a follow up study investigating correlates of protection against HIV. During the screening visit, participants donated blood samples for HIV testing and they were interviewed by a social assistant about socio-demographics and sexual behavior. Participants who tested HIV positive received individual counseling during which they were advised to disclose their status to their partner, and they were enrolled for clinical follow up with access to antiretroviral therapy if needed. Couples with an HIV discordant status were invited for follow-up every four months for a period of up to three years. Couples with an HIV concordant positive status were invited for one additional visit to the clinic four months after the screening visit. During every visit, the participants donated blood samples for laboratory testing and they were interviewed about socio-demographics and sexual behavior. All participants received safe sex counseling during every visit. The present study investigates intracouple transmission of all enrolled HIV-1 concordant couples. HIV-1 concordant couples with confirmed intracouple transmission will be used along with HIV-1 discordant couples in future studies of HIV protective immunity.The study was approved by the Internal Review Board of the Institute of Tropical Medicine , the Ethical Committee of the University Hospital of Antwerp , and the Ethical Committee of the Senegalese Ministry of Health . All subjects gave written informed consent before enrolment.env gp41 was amplified from genomic DNA using previously described primers Whole blood was drawn in EDTA tubes (Becton Dickinson). Plasma and peripheral blood mononuclear cells (PBMC) were separated by gradient centrifugation, separated into aliquots, and stored at −80°C. HIV-1/2 status was evaluated in plasma by current serological testing combining enzyme linked immunosorbent assays (ELISAs) and Western blotting. CD4 counts were determined in whole blood using a FACScan flow cytometer (Becton Dickinson). HIV-1 viral load was quantified in plasma by the Amplicor HIV-1 Monitor assay version 1.5 (Roche). Genomic DNA was extracted from PBMC using a QIAamp DNA blood mini kit (Qiagen). A PCR fragment spanning approximately 560 bp of HIV-1 http://www.hiv.lanl.gov). Distances between all possible pairs of sequences of the same HIV-1 subtype but from different couples  were then selected to estimate the within-subtype variability of unlinked gp41 sequences of our study population. The average value of this normal distribution minus two times the standard deviation  was taken as the lower cut-off value for unlinked sequences, for which a value of 0.04 was obtained. Consequently, two unlinked sequences have a 97.5% chance of having a pairwise distance value above this value. Thus, in addition to the phylogenetic criteria, couples' sequences were considered to be genetically linked if their pairwise distance value was below 0.04.A maximum-likelihood phylogenetic tree incorporating gp41 sequences from all individuals was constructed using MEGA5 software Male and female partners were interviewed separately about socio-demographics and sexual behavior. Interviews were done by a trained and experienced social assistant who performed oral interviews based on a standardized questionnaire. The social assistant assured that the participants understood all questions, if needed by using different formulations. The social assistant performed the first level of quality control by checking inconsistencies in the reported information during the interview. Importantly, this was done independently for every participant and for every interview; the social assistant did not check for inconsistencies between screening and follow-up interviews or between partners of the same couple. Interview forms were entered into a database twice by two different researchers and checked electronically for errors. Socio-demographic and behavioral data used in this study were extracted from the screening interview. Data not expected to vary in time  were checked for consistency with data from the follow-up interview; this is the second level of quality control.Male and female partners of HIV-1 concordant couples were interviewed about the date of their first positive HIV test, their antiretroviral therapy status and start date (if applicable), and their suspected source of HIV infection. Epidemiological analyses of intracouple transmission and its direction were primarily based on the suspected sources of HIV infection as reported by both partners. Couples for whom information on the suspected source of infection was not available for both partners were excluded from these analyses. We considered couples to report internal HIV transmission if (i) one partner named the other partner as suspected source of infection while the other partner named an outside source and (ii) the reported dates of first positive HIV test and, if applicable, start of antiretroviral therapy did not contradict the reported order of infection events. We considered couples to report external HIV transmission if both partners named an outside source of infection.Characteristics of male versus female and index versus recipient partners in HIV-1 concordant couples were compared by using non-parametric Mann-Whitney U tests for independent continuous data, Wilcoxon signed rank tests for paired continuous data, or McNemar tests for paired proportional data. Statistical analyses were performed with SPSS version 16.0.The numbers of couples that were enrolled and analyzed in the different parts of the study are shown in We first set out to determine intracouple transmission by conducting a molecular analysis of a 520 bp fragment of the gp41 gene. Gp41 amplification failed for both partners in 3 couples despite repeated PCR attempts with different primer sets. This resulted in a total of 88 gp41 sequences representing 46 HIV-1 concordant couples that were used for the calculation of pairwise genetic distances and the construction of a maximum-likelihood phylogenetic tree . The majNext, we investigated HIV-1 transmission by analyzing the interview data. Forty three of 49 couples (88%) reported a suspected source of HIV infection for both partners . Six couThen, we compared the molecular and epidemiological data for 41 HIV-1 concordant couples with both data available. The molecular and epidemiological data corresponded completely . All couFor 30 HIV-1 concordant couples with confirmed intracouple transmission by molecular as well as epidemiological methods, we analyzed the reported sources of infection and the direction of the intracouple transmission . Twenty Finally, for the 30 confirmed HIV-1 transmission pairs, we compared the reported order of infection events with a paired index/recipient analysis of clinical parameters of HIV infection . Index pIn this study, we provide conservative molecular evidence for internal HIV-1 transmission in a large proportion of Senegalese HIV-1 concordant couples. The majority of these couples reported the male partner as the source of the intracouple transmission, who in turn most frequently reported to have acquired HIV through an occasional sexual relationship. These data suggest that HIV-1 is frequently transmitted within married couples in Senegal with the husband as the most likely index partner. Our findings are in line with previous reports showing that married women in Africa have a high risk for acquiring HIV as a result of the extramarital relationships of their husbands The high proportion of linked transmissions (74%) in our cohort is in agreement with previous studies of HIV-1 transmission in African married couples. Two large HIV prevention trials in cohorts of HIV-1 discordant couples found genetically linked transmissions in 72% and 76% of the cases, respectively Our data are not in agreement with recent meta-analyses of HIV-1 discordant couple cohorts estimating large proportions of wife-to-husband transmissions in African married couples An important limitation of HIV-1 concordant couple studies however is that they have to rely on interview data to determine the direction of the intracouple transmission. To date, the order of infection events cannot easily be deduced from viral sequence information env gp120 V2–V3 region makes it ideal for studying recent transmission events. On the other hand, the low genetic variability of HIV-1 pol makes it more suitable for establishing older transmissions. Assuming that our Senegalese cohort of HIV-1 concordant couples contained recent as well as older HIV-1 transmissions, we decided to use env gp41, a gene with a genetic variability in between that of env gp120 and pol. Previous studies successfully used gp41 in HIV-1 subtyping and linkage analyses of similarly diverse datasets gag or pol genes or the use of clonal or deep sequencing techniques could help establish genetic linkage in these couples, but this was beyond the scope of this study.The application of phylogenetic analysis to confirm HIV-1 transmission events is well established, and it is frequently used for providing evidence in legal cases Phylogenetic analyses cannot exclude that two linked sequences are connected through a third intermediate sequence not included in the analysis. This possibility could frequently occur in populations of e.g. men who have sex with men where promiscuous behavior has been shown to result in large clusters of linked viruses with uncertainty of the origins and directionality of the underlying transmissions The majority of the couples enrolled in our study lived in the Dakar metropolitan area, and they reported a wide range of occupations and levels of education characteristic of those of an African suburban population. Therefore, we should be careful with extrapolating the observed patterns of HIV-1 transmission in this study population to populations living in more rural areas or in other African countries. Future studies should investigate HIV-1 transmission in HIV-1 concordant couple cohorts enrolled from other African populations with different socio-epidemiological factors.In summary, we found that a majority of HIV-1 concordant couples in Dakar, Senegal showed genetically linked HIV-1 transmission with the husband as likely index partner. Our data emphasize the risk of married women for acquiring HIV-1 as a result of the occasional sexual relationships of their husbands. Understanding the origins and dynamics of HIV-1 infection in married couples in Africa will be crucial for the future planning of successful HIV prevention programs.Table S1Epidemiological analyses of HIV-1 transmission for 49 HIV-1 concordant couples.(DOC)Click here for additional data file.


Noble metallic nanoparticles have prominent optical local-field enhancement and light trapping properties in the visible light region resulting from surface plasmon resonances.We investigate the optical spectral properties and the surface-enhanced Raman spectroscopy of two-dimensional distinctive continuous ultrathin gold nanofilms. Experimental results show that the one- or two-layer nanofilm obviously increases absorbance in PEDOT:PSS and P3HT:PCBM layers and the gold nanofilm acquires high Raman-enhancing capability.The fabricated novel structure of the continuous ultrathin gold nanofilms possesses high surface plasmon resonance properties and boasts a high surface-enhanced Raman scattering (SERS) enhancement factor, which can be a robust and cost-efficient SERS substrate. Interestingly, owing to the distinctive morphology and high light transmittance, the peculiar nanofilm can be used in multilayer photovoltaic devices to trap light without affecting the physical thickness of solar photovoltaic absorber layers and yielding new options for solar cell design. Noble metal nanoparticles and nanofilms with strong localized surface plasmon resonances (LSPRs) have attracted great interests in fields such as nanoscale photonics, biological sensing ,2, surfa61-butyric acid methyl ester (P3HT:PCBM) on ITO glass substrates, and the SERS measurements of molecules adsorbed on gold nanocrystals deposited on ITO glass substrates. Our results suggest that the continuous ultrathin nanofilm can obviously enhance visible-range absorption in the active layer of solar cells and obtain an ultrasensitive SERS-active coating.We have recently reported a relatively simple approach in fabricating uniform gold nanocrystal-embedded nanofilms via a conventional magnetron sputtering method. In this method, one can more conveniently assemble noble metals with precise gap control in the sub-10-nm regime  than anyOur approach is based on the formation of Au nanofilms on the buffer layer surface of PEDOT:PSS or on ITO glass utilizing magnetron sputtering deposition of metal atoms. The ITO-coated glass substrate was first cleaned with detergent, then ultrasonicated in acetone and isopropyl alcohol for further cleaning, and subsequently dried in a vacuum oven at 80°C for 3 h. PEDOT:PSS films with thicknesses of 30 nm are prepared via spin coating on top of the ITO glass and cured at 130°C for 10 min in air. On top of the freshly prepared PEDOT:PSS layer, metallic gold are sputtered by magnetron sputtering in an electrical current of 0.38 A, vacuum of 0.15 Pa, Ar flux of 25 sccm, and discharge of 1 s. The ITO/Au nanofilm is fabricated in an identical magnetron sputtering manner.The SERS measurements were performed at room temperature on a confocal Raman spectrometer with the 514-nm laser focused to a diameter of 1 μm. The incident power was 0.55 mW, and the accumulation time was 10 s.Figure The localized absorption characteristic of Au films is highly sensitive to the surrounding medium, particle size, surface structure, and shape. The ultrathin Au nanofilm on the ITO glass substrate exhibits an ultraviolet–visible (UV–vis) optical spectrum in Figure Plasmonic enhancement of the P3HT:PCBM bulk heterojunction system is demonstrated in a spin-cast device with an incorporated ultrathin gold nanofilm thickness of 2 nm or so. Figure Figure To compare the impact of continuous ultrathin gold nanofilms on the absorption of visible light, plasmonic enhancement of the P3HT:PCBM bulk heterojunction system is demonstrated in a spin-cast device with an incorporated continuous ultrathin gold nanofilm thicknesses of 2 nm or so which are chosen to be sufficiently thin to limit the amount of light absorbed before reaching the active layer. The nanofilm incorporated with gold in the active P3HT:PCBM layer is shown to have significantly greater absorbance enhancement than the nanofilm without gold in the entire excitation spectral range in Figure For the ITO/Au film/PEDOT:PSS/Au film/P3HT:PCBM and ITO/PEDOT:PSS/Au film/PEDOT:PSS/Au film/P3HT:PCBM structures, the plasmon resonance is located at a wavelength range of 350 to 1,000 nm. The plasmonic peak better overlaps the P3HT:PCBM absorption band. These enhancements concerning light absorption in the visible region can be explained by the surface plasmon polariton resonance of metallic nanoparticles in the gold nanofilm. When metallic nanoparticles are in close proximity, their plasmon resonances couple with each other and generate a light-scattering spectrum that depends strongly on the interparticle distance. The two-dimensional distinctive ultrathin continuous gold nanofilms can be used as subwavelength antennas in which the plasmonic near-field is coupled to the organic semiconductor, increasing its effective absorption cross section. A corrugated metallic film on the back surface of the P3HT:PCBM photosensitive layer can couple light into surface plasmon polariton (SPP) modes supported at the metal/P3HT:PCBM organic semiconductor interface as well as guided modes in the organic semiconductor slab. Both the shape and size of metal nanoparticles are key factors in determining the coupling efficiency. The two-layer ultrathin nanofilm increases the nanoparticle density; according to the Mie theory, the extinction coefficient is proportional to the nanoparticle density. Consequently, optical local-field enhancement of the two-layer continuous ultrathin gold nanofilm is stronger than that of the one-layer ultrathin continuous gold nanofilm. Figure −3 to 10−6 M are shown in Figure To investigate the performance for electromagnetic enhancement, SERS spectroscopic measurements were carried out using Rhodamine 6G, a well-characterized test molecule. Spectra obtained from Rhodamine 6G molecules at a concentration of 10In conclusion, we have produced continuous ultrathin gold nanofilms with high local-field enhancement effect and a high SERS activity. Spectral analysis suggests that the prominent light absorption in organic photosensitive materials and the high SERS activity arise from the near-field effect of localized surface plasmons of nanoparticles. Owing to their distinctive morphology and high light transmittance, continuous ultrathin gold nanofilms can be used in multilayer organic solar cells to trap light without affecting the physical thickness of solar photovoltaic absorber layers and yielding new options for solar cell design. Further work is needed to research two-dimensional distinctive continuous gold nanofilms that are utilized to trap light in solar cells which may be suitable for application to the high photoelectric conversion efficiency of organic solar cells.The authors declare that they have no competing interests.L-DW carried out the design and the characterization of ultrathin gold films, performed the ultrathin gold nanofilm surface plasmon resonance analysis, and drafted the manuscript. R-ZL participated in the fabrication of gold films. S-QZ participated in the SERS measurement. TZ, X-YZ, Q-LW, and XL read the manuscript and contributed to its improvement. All authors read and approved the final manuscript.