A fundamental issue in molecular evolution is how to identify the evolutionary forces that determine the fate of duplicated genes. The dosage balance hypothesis has been invoked to explain gene duplication patterns at the genomic level under the premise that a dosage imbalance among protein-complex subunits or interacting partners is often deleterious. Here we examine this hypothesis by investigating the molecular basis of dosage sensitivity. We focus on the extent of protein wrapping, which indicates how strongly the structural integrity of a protein relies on its interactive context. From this perspective, we predict that the duplicates of a highly under-wrapped protein or protein subunit should (1) be more sensitive to dosage imbalance and be less likely to be retained and (2) be more likely to survive from a whole-genome duplication (WGD) than from a non-WGD because a WGD causes little or no dosage imbalance. Our under-wrapping analysis of more than 12,000 protein structures strongly supports these predictions and further reveals that the effect of dosage sensitivity on gene duplicability decreases with increasing organismal complexity. A gene duplication provides an extra gene copy that can be free to accumulate mutations and gain a new function. Therefore, gene duplication plays a very important role in evolution. However, the presence of an additional gene copy can sometimes be deleterious because it can lead to an excessive dosage relative to those of its interacting partners. This dosage imbalance effect in turn influences the fate of duplicated genes in evolution. Our study gives the first description to our knowledge of the molecular/structural basis for the dosage imbalance effect. We study the relationships between gene family size and extent of protein under-wrapping, a molecular quantifier of the reliance of the protein on binding partnerships to maintain structural integrity, indicative of the extent of structure protection from disruptive hydration. Using more than 12,000 protein three-dimensional structures from six organisms that range from bacteria to human, we show an inverse relationship between extent of protein under-wrapping and family size. That is, a duplication is unlikely to be tolerated if the protein is highly under-wrapped . We also show that the effect of dosage imbalance is more apparent in unicellular organisms but is buffered to some extent in higher eukaryotes. Gene duplication is a primary source for the emergence of new genes and increases genome complexity ,2. In reRecent advances in structural genomics and biophysics enable us to examine the dosage balance hypothesis in the light of the three-dimensional structure of proteins. In this regard, we focus on a specific attribute of protein structure, the so-called under-wrapping –17. Thisdehydrons [The under-wrapping parameter describes the solvent accessibility of the major determinants of protein structure: the backbone hydrogen bonds . Thus, iehydrons , represeehydrons ,15,17. TAs shown in m, the gene family size) for the corresponding gene. Interestingly, in all six organisms studied , we found a negative correlation between protein under-wrapping extent and gene duplicability  should decrease with the extent of hydrogen bond under-wrapping of the polypeptide encoded by the gene. To test this prediction, we compiled non-redundant proteins with PDB-reported structures, calculated the under-wrapping extent for each protein (subunit), and determined the duplicability  in humans, implying that most human proteins need binding partners to maintain the integrity of their functional structure. On the other hand, unicellular species appear to possess more autonomous protein folders (under-wrapping <35%), capable of operating without forming obligatory complexes [Our study reveals a universal negative effect of protein under-wrapping on gene duplicability in a variety of species, strongly supporting the dosage balance hypothesis. The decreasing tendency is most significant from omplexes . Howeverm = 2. We classified the yeast duplicates into two groups: one group from WGD and the other from non-WGD. By performing the analysis conditioned on the same m, the under-wrapping difference between the two groups should mainly be determined by the underlying duplication mechanisms. We found that the under-wrapping extent in WGD duplicates is significantly higher than that in non-WGD duplicates , implying that the dosage imbalance effect was indeed relaxed in the WGD. Again, we examined this trend in different functional categories and found that the WGD duplicates are consistently more under-wrapped than the non-WGD duplicates in each category (Duplicated genes can arise from either whole-genome duplication (WGD) or non-WGD  . In a WGcategory B.In higher eukaryotes, considerable amount of highly under-wrapped proteins are associated with highly duplicated genes, suggesting that complex organisms are less sensitive to the dosage imbalance effect. This can possibly be attributed to several factors. First, complex organisms may have more efficient systems to adjust gene expression levels . It has been shown that in cultured cells more than 60% human promoter polymorphisms cause more than two-fold differences in gene-expression level . Second,In summary, we have identified protein under-wrapping as a molecular basis of dosage sensitivity. An imbalance-generating duplication becomes less tolerable if the protein is severely under-wrapped and therefore requires substantial stabilizing interactions with other proteins. Indeed, the extent of under-wrapping in a protein can be used as an approximate predictor of the strength of the effect of dosage imbalance on gene duplicability. The prediction can be made more broadly and precisely in the future when more data on protein structures, especially on protein complexes, become available.E. coli, E. coli Genome and Proteome Database (http://genprotec.mbl.edu/) (GenProtEC); Saccharomyces cerevisiae, Saccharomyces Genome Database (http://www.yeastgenome.org/) (SGD1.01); Caenorhabditis elegans, WormBase (http://www.wormbase.org/) (WB170); Drosophila melanogaster, Berkeley Drosophila Genome Project (http://www.fruitfly.org/) (BDGP 4.3); Homo sapiens, Ensembl Genome Database (NCBI36); Arabidopsis thaliana, Arabidopsis Information Resource (http://www.arabidopsis.org/) (TIR7.0). Then, based on the GenProtEC family annotation, 4,485 E. coli genes were grouped into 2,901 gene families ; based on the Ensembl gene family annotation [We obtained gene information from the following sources: notation , 6,024 ynotation  with theet al. [E. coli genes, 476 yeast genes, 29 worm genes, 94 fly genes, 2,275 human genes and 168 thale cress genes, for which we have both gene duplicability and protein structural data.For each of the six organisms under study, we constructed a set of non-redundant genes with at least one PDB representative structure. From the reported structure we calculated the extent of protein under-wrapping by determining the ratio of the number of insufficiently wrapped hydrogen bonds (dehydrons) to the total number of backbone hydrogen bonds in the structure. The dehydron identification from reported protein structure follows the protocol detailed in Chen et al. . TogetheThe extent of hydrogen-bond wrapping, ρ, measures the number of non-polar groups contained within a desolvation domain defined as two intersecting balls of fixed radius (∼thickness of three water layers) centered at the α-carbons of the residues paired by the amide-carbonyl hydrogen bond . In thisThe under-wrapping variation of a protein generated by structural differences in reported PDB entries is less than 8.8%. This variability arises from the different structural adaptations (induced fits) adopted by the protein in different crystallized complexes or from differences between uncomplexed protein structure in solution (often determined by NMR) and crystal structure. To account for such differences, the under-wrapping extent for each gene is typically averaged over all its PDB representations (Text S1). We obtained per-gene-family under-wrapping distributions by averaging the under-wrapping values among members within a gene family whenever available.In this study, the wrapping computations involved more than 12,000 protein structures because a large fraction of the non-redundant proteins examined had various PDB representations with differences arising from the following sources: complexation diversity, level of structure resolution, NMR conformational diversity and high B-factors in the crystal (Text S1). The under-wrapping data obtained in our study are given in We obtained WGD gene duplicate pairs from Kellis et al. . We usedFigure S1(708 KB TIF)Click here for additional data file.Figure S2(654 KB TIF)Click here for additional data file.Table S1(112 KB XLS)Click here for additional data file.Table S2(69 KB XLS)Click here for additional data file.Table S3(19 KB XLS)Click here for additional data file.Table S4(27 KB XLS)Click here for additional data file.Table S5(309 KB XLS)Click here for additional data file.Table S6(33 KB XLS)Click here for additional data file.Text S1(44 KB DOC)Click here for additional data file.


MicroRNAs (miRNAs) are a set of small non-coding RNAs serving as important negative gene regulators. In animals, miRNAs turn down protein translation by binding to the 3' UTR regions of target genes with imperfect complementary pairing. The identification of microRNA targets has become one of the major challenges of miRNA research. Bioinformatics investigations on miRNA target have resulted in a number of target prediction tools. Although these tools are capable of predicting hundreds of targets for a given miRNA, many of them suffer from high false positive rates, indicating the need for a post-processing filter for the predicted targets. Once trained with experimentally validated true and false targets, machine learning methods appear to be ideal approaches to distinguish the true targets from the false ones.k-gram frequencies in the seed, the non-seed and the entire regions. Informative features are selected based on their discriminating abilities. Prediction accuracies are assessed using 10-fold cross-validation experiments. Our system achieves AUC (area under the ROC curve) of 0.86, sensitivity of 83.59%, and specificity of 73.68%. More importantly, the system correctly predicts majority of the false positive miRNA:target interactions (28 out of 38). The possibility of over-fitting due to the relatively small negative sample set has also been investigated using a set of non-validated and randomly selected targets (from miRBase).We present a miRNA target filtering system named MiRTif (miRNA:target interaction filter). The system is a support vector machine (SVM) classifier trained with 195 positive and 38 negative miRNA:target interaction pairs, all experimentally validated. Each miRNA:target interaction pair is divided into a seed and a non-seed region. The encoded feature vector contains various .MiRTif is designed as a post-processing filter that takes miRNA:target interactions predicted by other target prediction softwares such as TargetScanS, PicTar and miRanda as inputs, and determines how likely the given interaction is a real or a pseudo one. MiRTif can be accessed from  MicroRNAs (miRNAs) are small RNA molecules of about 22 nucleotides that are negative regulators of protein expression. Studies have shown that these small RNAs are involved in the regulation of a variety of biological processes, including developmental timing, cell death, cell proliferation, haematopoiesis and patterning of nervous systems . MiRNAs Since 2003 many computational approaches have been developed to predict miRNA targets, some of them which are publicly available ,13. MostIn spite of its progress, computational prediction of miRNA targets is still unreliable partly due to the lack of experimentally validated targets . A recenk-gram [.Here we present a machine-learning algorithm based on support vector machine (SVM) that can be used as a post-processing software for filtering the targets predicted by other miRNA prediction tools. The prediction system is trained with the experimentally supported animal miRNA targets found in TarBase . Each mik-gram  frequenck-gram ,24, and k-gram . Higher k-gram ,11,26,27The prediction accuracies of the ten-fold cross-validation are listed in Table C. elegans [Current miRNA target prediction methods are mostly based on similar characteristics of base pairing derived from lin-4 and let-7, and their genetically validated mRNA targets in  elegans -32. Despk-gram frequencies in the interacting miRNA:target pairs (see Methods). Features are ranked according to their ability to discriminate positive and negative samples (the F score), and only those with high discriminating abilities are retained for SVM training. These features are hence termed "informative features". Table F scores. See Methods for the calculation of F scores.The feature space used in current SVM classification system includes various k-gram code in the non-seed region makes some contribution to the discriminating ability of the same k-gram code in the entire region; otherwise we would have observed a high F score for this k-gram code in the seed region. This observation hints the potential importance of the k-gram frequencies in the non-seed region in discriminating positive and negative miRNA:target interaction pairs. In fact, among the 100 most informative features, non-seed region features account for 43, entire region features account for 44, whereas seed region features only account for 13. It again shows that many of the highly informative features lie within the non-seed region.It is worth noting that almost all the 25 most informative features in Table To study the potential over-fitting problem due to the relatively small number of training samples (195 positives and 38 negatives), we took non-validated targets from miRBase [MiRTif should not be considered as a general tool for miRNA target prediction. It is rather a post-processing filter for the miRNA:target interactions predicted by other seed-sensitive computational methods. Because MiRTif is trained on validated miRNA:target duplexes, both positive and negative training duplexes possess the known miRNA:target binding properties such as the strong seed complementarity and high binding energy. A random duplex (e.g. generated using BLAST) without prescreening by those seed-sensitive programs is not the ideal candidates for using MiRTif.Machine learning techniques such as Naïve Bayes and SVM have been previously applied to the miRNA target prediction problem -20. UnliThe lack of validated negative miRNA:target interaction remains a problem for machine learning-based target prediction approach. For example, the training set of the ensemble algorithm  containsIn our study, the validated miRNA:target interactions and the associated binding patterns were retrieved from TarBase, which obtained their information from respective original research papers. Although the duplexes were generated by different prediction tools, many of the duplexes have at least their seed regions experimentally validated. In comparison with miTarget, where the binding patterns were re-computed using RNAfold  by the aOne of selection criteria employed by many miRNA target prediction algorithms such as TargetScanS , PicTar The current collection of experimentally validated miRNA:target interaction pairs is still far from being sufficiently comprehensive to give an accurate representation of the target site diversity. Problems with limited dataset can be observed in Table ab initio target prediction software programs, PicTar, miRBase and TargetScan. Here the example miRNA is hsa-miR-224. This miRNA is the most significantly up-regulated miRNAs in hepatocellular carcinoma patients [To demonstrate how MiRTif is applied in real world, Table patients . The MiRpatients . We beliWe built an SVM classifier serving as a post-processing filter for miRNA target prediction. The system, MiRTif, takes the targets predicted by other target prediction tools as the input and reports the SVM scores indicting the likelihood of them being true miRNA targets. Tested with the experimentally validated miRNA targets found in TarBase using ten-fold cross validation, the obtained high AUC measurement (0.86) indicates that true positives are indeed ranked higher than false ones. With more biologically validated miRNA:target duplexes in the future, the accuracy increase of knowledge-based, machine-learning approaches such as MiRTif could be anticipated.C. elegans, human, mouse, rat and zebrafish were extracted. Duplicated entries and entries with incomplete binding diagrams were removed, resulting in 195 positive and 21 negative miRNA:target duplexes. As the number of negative samples is small, we went on predicting the binding patterns of those known negative interactions without duplexes being reported in the original papers. There are 15 negative miRNA:target interactions from two papers [. For the nine negative interactions from the work of Robins [.Known miRNA:target interactions were downloaded from the TarBase version 3.0 . Translao papers ,36 that o papers  have thef Robins  to predik-gram is known as a subsequence of length k. For the case of miRNA:target duplex, we define a k-gram as a duplex fragment of length k. Therefore, a 1-gram contains 5 different pairing types, which we call them "1-gram codes". Similarly, 2-gram and 3-gram contains 52 and 53 codes, respectively. For each miRNA:target interaction, we compute the 1-gram, 2-gram and 3-gram frequencies in the seed, non-seed and entire duplex, giving 3 × 5 + 3 × 52 + 3 × 53 = 15 + 75 + 375 = 465 features. The 2-gram and 3-gram codes are counted from the 5' end of the miRNAs. For example, in Figure For a given miRNA:target duplex, the gaps at both ends are firstly removed, if any. The duplex is then partitioned into two parts, with the first part covers nucleotides 1–9 from the 5' end of the miRNA, and the second part covers the rest of the duplex. These two parts are named "seed" and "non-seed" regions, respectively  was first introduced by Vapnik ,24. It hH. Instead, mapping is done implicitly by defining a kernel function ϕ is the mapping to the space H. A detailed description of the mathematics behind SVM can be found in an article by Burges [light (version 6.01) created by Joachims [ and is free for academic use.The SVM algorithm requires the solving of a quadratic optimization problem. To simplify the problem, SVM does not explicitly map the feature vectors of all the samples to the space y Burges . In the Joachims . The pacγ > 0. A grid search over the values of the parameters c, j and γ was performed. Parameter c controls the trade-off between training error and margin, j is the cost factor by which training errors on positive samples  outweigh errors on negative samples , and γ controls width of the RBF kernel. For each parameter combination, we measured AUC after 10-fold cross-validation. A receiver operating characteristic (ROC) curve is a plot of true positives as a function of false negatives [c = 300, j = 0.14 and γ = 0.1. The performance of the final SVM classification is also measured by the quantity of true positives (TP), true negatives (TN), false positives (FP), false negatives (FN), sensitivity (SE), specificity (SP) and overall accuracy Q. The definition of sensitivity, specificity and overall accuracy are listed below:The training of SVM requires selection of several hyper-parameters, whose values determine the function that SVM optimizes and hence have a crucial impact on the performance of the trained SVM classifiers. In this work, the optimal hyper-parameter set was selected by a 10-fold cross-validation on the entire dataset. This approach has been shown to be a robust method for hyper-parameter tuning . We chosegatives . An AUC The authors declare that they have no competing interests.YY collected the raw data, designed and implemented the cross validation experiments. YPW and YY implemented the web server. YY, YPW and KBL were all involved in analyzing the validation results. YY and KBL prepared the manuscript. KBL initialized and supervised the whole project. All authors read and approved the final manuscript.


However, the impact of induced blood flow changes on oxygenation is not well understood. We used oxygenation-sensitive CMR to assess the relations between myocardial oxygenation and coronary sinus blood oxygen saturation . Both SvO2 and CMR signal intensity were exponentially related to coronary blood flow, with SvO2 approaching 87%.During administration of acetylcholine and adenosine, CMR signal intensity correlated linearly with simultaneously measured SvO2 and is exponentially related to vasodilator-induced increases of blood flow. Oxygenation-sensitive CMR may be useful to assess ischemia and microvascular function in patients. Its clinical utility should be evaluated.Myocardial oxygenation as assessed with oxygenation-sensitive CMR imaging is linearly related to SvO Myocardial oxygenation, reflecting the balance or imbalance between oxygen demand and supply, is an important diagnostic target in various clinical settings, but may2*-sensitive, Blood-Oxygen-Level-Dependent Cardiovascular MR (BOLD-CMR) uses the paramagnetic properties of deoxygenated hemoglobin as an endogenous contrast mechanism and is thus oxygenation-dependent[2* measurement and T2*-mapping techniques have been shown to have high BOLD sensitivity, they had limited clinical use thus far because of long acquisition times and relatively low signal-to-noise ratios. Additionally, magnetic field inhomogeneities, blood flow and cardiac motion may all impair image quality.Tdependent. In oxygdependent. Such sedependent and simidependent-10. Alth2* mapping.Recently, BOLD-sensitive, steady-state free precession (SSFP) techniques with much more consistent image quality have been introduced -13 and ain vivo.We hypothesized that SSFP BOLD-CMR can accurately and consistently detect changes of myocardial oxygenation We used a canine model with selective intracoronary vasodilator infusion. In order to cover the full range of physiological flow changes we applied graded infusions of the endothelium-dependent vasodilator, acetylcholine, as well as the endothelium-independent vasodilator, adenosine.Seven mongrel dogs (weight 15 to 25 kg) were studied; all experiments were conducted in accordance with the most recent policies and "Guide to the Care and Use of Experimental Animals" by the Canadian Council on Animal Care. The local animal care and use board approved the study protocol and sample size.®-18 Hi-Flow, Boston Scientific Ltd., Cork, Ireland) was introduced into the LCX through a diagnostic coronary catheter . The tip of the infusion catheter was placed a few millimeters proximal to the flow probe while ensuring that there were no visible side branches located between the infusion catheter and flow probe. In addition, a 4-F balloon catheter  was introduced into the coronary sinus (CS) for blood sampling. Blood gases were analyzed using a portable analyzer . All procedures including the CMR scan were performed in adjacent rooms with the dogs being placed on an MR-compatible cradle that allowed for a quick and easy transport to and from the MR system.Under general anesthesia, a midline sternotomy was performed and a 2-mm MR-compatible flow probe  placed around the proximal left circumflex (LCX) coronary artery. Under fluoroscopic control, a 2.7-F infusion catheter  with a 6-element phased-array coil resting on the chest and another below the spine. After acquiring localizer planes and performing manual regional shimming, BOLD-CMR was performed in a single mid-ventricular short-axis view at baseline (BL 1-3) and during intracoronary vasodilator infusion into the LCX. Acetylcholine (ACh) was infused in three increasing doses as previously described [®, Bayer, Germany) for accurately identifying the LCX perfusion territory and confirming the correct position of the intracoronary catheter.All CMR scans were performed in a clinical 1.5-T MRI system  using a mono-polar readout. K-space lines from the different echoes were assigned to different images, and the resulting 8 images were used to generate a T2* map. Other typical parameters for the multi-echo GRE sequence were: FOV = 225 × 400 mm; matrix size = 109 × 256; in-plane resolution = 2.1 × 1.6 mm; slice thickness = 10 mm; flip angle = 20°; TR = 120 ms; and typical breath-hold duration, 16 s. Reproducibility was assessed by 20 repeated baseline SSFP BOLD-CMR acquisitions in each of three subjects.SSFP BOLD-CMR was performed with a Tescribed . Scan pa42, CIRCLE Cardiovascular Imaging Inc., Calgary, Canada). A region of interest (ROI) was drawn around the LCX perfusion territory, which was clearly identified on the perfusion images as contrast was injected from the infusion catheter placed in the LCX . Intra-arterial blood pressure was monitored continuously and the rate-pressure product (RPP) was calculated from heart rate and systolic pressure.t-tests with Bonferroni adjustments . Correlation analysis was done  by linear regression analysis or by regression analysis using a 3-parameter, exponential-rise equation that asymptotically approached a maximum.Continuous variables with normal distribution were summarized as mean ± standard error of the mean. Means at each level of drug infusion were analyzed with repeated measures ANOVA and post-hoc comparisons, using paired Sample-size calculation was performed aiming for the detection of a 200% increase in flow, which was considered to be relevant in assessment of myocardial perfusion reserve, with 80% power and 95% significance and based on results of previous studies with a similar experimental setting,17.2* data was included in the analysis.The drug-infusion protocol was completed and the perfusion territory of the LCX was identified in all 7 animals . Using the 3-parameter, exponential-rise equation, SvO2 correlated with LCX blood flow  with SvO2approaching 87%  accurately, and with a precision that promises to make the technique clinically useful.Our in vivo study to compare oxygenation-sensitive CMR using a state-of-the-art SSFP sequence against simultaneous coronary venous blood sampling. Similar studies have been done using T2* mapping for assessing blood oxygenation[2* mapping was more sensitive to oxygenation changes but showed more variability. The fact that BOLD CMR shows a linear correlation to coronary sinus oxygen saturation is in agreement with the expected proportionality between the BOLD effect and the absolute tissue content of deoxygenated hemoglobin[To our knowledge, this is the first ygenation. Our resemoglobin,19,20. Iemoglobin.2 and LCX flow can be described using a 3-parameter, exponential-rise equation. With constant myocardial oxygen consumption , coronary sinus SvO2 approached an asymptotic value (87%) as the LCX was over-perfused with near-saturated (97%) arterial blood under the influence of coronary vasodilators (ACh and Ade).As shown in Figure 2 was observed for ACh- and Ade-induced flow changes . In our protocol, we included the inferolateral wall, which is particularly susceptible for off-resonance artifacts in BOLD-sensitive CMR[2* and SvO2 changes. We believe that this difference might be explained by T2 and T1 interference in T2-prepared SSFP imaging since the acquisition follows a T2-preparared SSFP imaging since the acquisition follows a T2-preparation period followed by signal readout during which T1 recovery is also expected to occur. Finally, in our method, the relatively short breath hold of less than 15 seconds is an advantage.The effects of ACh and Ade on coronary flow shown in this study are within the range of previously reported studies,17,24. Fitive CMR. They usBesides its apparent utility for clinical research, BOLD-CMR appears particularly useful for the clinical assessment of coronary perfusion reserve as used for the detection of relevant coronary artery stenoses. Since BOLD-CMR provides a biomarker (Hb oxygenation) instead of surrogate markers, it may overcome many limitations of currently used diagnostic tools. Furthermore, it can be applied as part of a comprehensive CMR study including cardiac morphology, function, coronary anatomy, ischemia, and tissue pathology.2 should reflect the degree of oxygenation of the LCX myocardium that we evaluated by BOLD SI. We cannot rule out the possibility that there was some admixture by blood draining from areas other than the LCX-perfused myocardium. However, the LCX is the dominant coronary artery in the dog and its course parallels that of the coronary sinus. Furthermore, as pharmacological vasodilatation increased LCX flow, SvO2 approached 87% -- only 10% below arterial saturation -- suggesting that LCX drainage was overwhelmingly dominant.The critical element in our experimental design is that SvO2*-sensitive SSFP BOLD sequence allows for non-invasive assessment of changes of myocardial oxygenation in vivo; specifically changes in BOLD signal are impressively proportional to changes in coronary sinus oxygen saturation. Further studies to assess the feasibility and accuracy of this technique in clinical settings are warranted.Oxygenation-sensitive CMR using a T2: Venous oxygen saturationBOLD: Blood-Oxygen-Level-Dependent; CMR: Cardiovascular Magnetic Resonance; FOV: Field of view; Fr or F: French; GRE-EPI: GRadient Echo-Echo Planar Imaging; LCX: Left circumflex coronary artery; MRI: Magnetic resonance imaging; Px: Pixel; SSFP: Steady-state-free-precession; SvOHusky Energy Research Program for the Early Detection of Cardiovascular Risk. During the experiments, Matthias Vöhringer was supported by a research scholarship from Robert-Bosch-Foundation, Germany and was a Canadian Institute of Health Research (CIHR) strategic training fellow in TORCH , Rohan Dharmakumar was supported by American Heart Association (SDG 0735099N) and National Institutes of Health (HL 091989), and Jordin Green was a full-time employee with Siemens Canada Limited; Matthias Friedrich is a scientific advisor and stockholder of Circle Cardiovascular Imaging Inc.The research was supported by the MV has performed the invasive experiments, collected the data, performed the evaluation and was significantly involved in writing the manuscript. JAF was responsible for the experimental setup of the experiments and in gathering and documenting the data. She participated in the scanning procedures and was significantly involved in writing the manuscript. JDG has modified the CMR sequences for our study. He was significantly involved in all scanning procedures and in the preparation of the manuscript. RD developed the CMR sequence and was involved in designing the CMR experiments and in writing the manuscript. JJW was involved in evaluation of the data related to analyses of coronary blood flow and significantly involved in preparing the figures. JVT was significantly involved in designing the experiments and the protocol and in writing the manuscript. MGF was responsible for the overall concept and was significantly involved in the study and protocol design. He was also significantly involved in writing the manuscript.


Transendothelial permeability (Kps) and fractional plasma volume (fPV) were calculated in each subunit. An avascular and necrotic region, an intermediate zone and a well-vascularised periphery were reliably identified. During untreated tumour growth, the identified sub-regions did not substantially change their enhancement pattern. Treatment with SU6668 induced major changes at tumour periphery where a significant increase of Kps and fPV was observed with respect to control tumours. Histology revealed a sub-capsular layer composed of hyper-dense viable tumour cells in the periphery of untreated tumours. The rim of viable neoplastic cells was reduced in treated tumours, and replaced by loose connective tissue characterised by numerous vessels, which explains the observed hyper-enhancement. The present data show a peripheral abnormal development of cancer-associated stroma, indicative of an adaptive response to anti-angiogenic treatment.Dynamic contrast-enhanced  magnetic resonance imaging, performed during 2 weeks of daily administration of an inhibitor of tyrosine kinase receptors (SU6668) in an HT-29 colon carcinoma model, revealed the onset of a hyper-enhancing rim, not observed in untreated tumours. To account for tissue heterogeneity in the quantitative analysis, we segmented tumours into three subunits automatically identified by cluster analysis of the enhancement curves using a  Tumours possess an extraordinary plasticity that enables them to adapt to drastic microenvironmental changes; this leads to the undesirable development of therapy resistance, recurrence and metastatic process. In recent years, epithelial–stromal interaction in carcinomas is emerging as a crucial factor, which could be involved in their adaptive plasticity . For exaThe development of resistance to anti-angiogenic therapies is a clinically important paradigm of tumour plasticity. Clinical studies over the past two decades have demonstrated that treatment with tyrosine kinase pathway inhibitors results in a fleeting period of clinical benefit, after which the disease restart to progress. Four distinct adaptive mechanisms are currently thought to be at the basis of the transitory effects of anti-angiogenic drug : (1) actβ and FGFR1.In the present study we provide evidence of an additional mechanism, that is the development of an aberrant vascular supporting stroma at tumour periphery, following a prolonged anti-angiogenic treatment. For this purpose, we performed a time evolution study in an experimental colon carcinoma model by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to assess tumour vascular responses during 2 weeks of treatment with SU6668, a small molecule inhibitor of the angiogenic receptor tyrosine kinases, VEGFR-2 (Flk-1/KDR), PDGFR-The investigation complied with national legislation about the care and use of laboratory animals.nu/nu mice. Treated animals (n=7) underwent daily administration of SU6668  for 14 days; control animals (n=5) received Cremophor-based vehicle. SU6668, a small molecule multi-tyrosine kinase inhibitor, is an analogue of sunitinib, developed in Sugen  and then clinically discontinued because sunitinib was preferred due to a better response.HT-29 human colon carcinoma fragments were implanted s.c. in the flanks of 12 athymic 3. The acquisition time for a single scan was 104 s; a dynamic scan of 24 images was acquired with 30-s time intervals between each image . The 30-s time interval allowed to avoid overheating of the gradient insert. Pre-contrast T1 values were measured using an inversion recovery snapshot Flash technique. The contrast agent was injected in bolus during the time between the first and the second scan. The plasma kinetics of contrast medium was determined ex vivo. From DCE-MRI data, transendothelial permeability (Kps) and fractional plasma volume (fPV) were calculated on a pixel-by-pixel basis as in Dynamic contrast-enhanced magnetic resonance imaging was performed at days 0, 7 and 14 on both treated and untreated animals. Gd-DTPA-albumin was used as contrast agent according to the protocol described in k-means cluster algorithm  to the enhancement curves, Enh(t), defined by: t) and SI(0) are the signal intensity values at time t and 0 respectively. The L1 metric =Σi ∣xi−yi∣ for vectors x and y) was selected as the ‘distance’ function for all partitioning steps of the clustering algorithm. Although both from MRI and from histological examination two main tumour compartments were identified (an avascular core and a well-vascularised periphery), a third cluster was considered in the k-means clusterisation algorithm, to take into account pixels having intermediate vascularisation and/or possible partial volume effects.To provide quantitative analysis taking into account of tumour heterogeneity, an automated operator-independent method, based on cluster analysis, was developed to identify sub-regions inside the tumour. A volume of interest (VOI) was manually drawn to cover the whole tumour. Each VOI was then segmented into three different compartments by applying a For each of the three identified subunits mean Kps, fPV and volume were calculated; for the whole VOI mean and Kps and fPV were calculated. Non-parametric test (Wilcoxon) was then applied on the obtained values to statistically compare treated and untreated tumours.μm thick slices were cut and stained with H&E.At day 14 mice were killed and their tumours were excised for histological examination. Two animals belonging to the treated group were killed at day 7. After fixation in zinc fixative for 6 h, tumours were cut in half on a plane corresponding to that used for the MR images. After embedding in paraffin, 5 The combined fPV/Kps maps clearly showed the architecture of tumour vasculature  and 2. TP<0.05) after 7 days of therapy than the values obtained in the corresponding control tumours (Cluster analysis allowed identification of sub-regions inside the tumour , with a  tumours . The samIn untreated tumours the peripheral zone, which surrounds central semi-necrotic areas, proved to be densely populated by viable neoplastic cells . After 7in vivo biomarker to evaluate the efficacy of angiogenesis inhibitors and other cancer treatments (k-means, closely related to PCA (Dynamic contrast-enhanced magnetic resonance imaging is now being used as an d to PCA , can be To develop an approach independent from any pharmacokinetics model, we have directly analysed the enhancement curves instead of using calculated Kps/fPV values. However, as the scanner gain could change between examinations, the signal intensity values were normalised to pre-contrast values. The successive evaluation of pharmacokinetic parameters on the obtained clusters, and in particular on the peripheral sub-region, proved to be more sensitive to the alteration induced by anti-angiogenic therapy than the analysis performed on the whole tumour VOI. Our results raise the hypothesis that cancer-associated stroma is involved in the ability of carcinomas to adapt to anti-angiogenic therapy. Prolonged SU6668 administration promoted abnormal development of the stromal compartment at the periphery of the treated tumours: this rim appeared significantly more perfused with respect to control tumours, consistently with the well-recognised role of stroma in tumour vasculature organisation. Our findings may suggest a different and more comprehensive mechanism of resistance to anti-angiogenic therapies, which encompasses those already described by The observations at days 7 and 14 complement previous findings . The app


Background and purpose Few studies have compared the long-term survival of cemented primary total hip arthroplasties (THAs), and several prostheses have been used without adequate knowledge of their endurance. We studied long-term outcome based on data in the Norwegian Arthroplasty Register.Patients and methods The 10 most used prosthesis brands in 62,305 primary Palacos or Simplex cemented THAs reported to the Register from 1987 through 2007 were included. Survival analyses with revision as endpoint (for any cause or for aseptic loosening) were performed using Kaplan-Meier and multiple Cox regression with time-dependent covariates. Revision rate ratios (RRs) were estimated for the follow-up intervals: 0–5, 6–10, and > 10 years.Results 5 prosthesis brands (cup/stem combinations)  were investigated with 0–20 year follow-up (inserted 1987–1997). After 18 years, 11% (95% CI: 10.6–12.1) were revised for any cause and 8.4% (7.7–9.1) for aseptic loosening. Beyond 10 years of follow-up, the Charnley cup had a lower revision rate due to aseptic loosening than Exeter (RR = 1.8) and Spectron (RR = 2.4) cups. For stems, beyond 10 years we did not find statistically significant differences comparing Charnley with Titan, ITH, and SP stems, but the Exeter stem had better results (RR = 0.5). 10 prosthesis brands  were investigated with 0–10 years of follow-up (inserted from 1998 through 2007). The Charnley cup had a lower revision rate due to aseptic loosening than all cups except the IP. Beyond 5 years follow-up, the Reflection All-Poly cup had a 14 times higher revision rate. For stems, beyond 5 years the Spectron-EF (RR = 6.1) and Titan (RR = 5.5) stems had higher revision rates due to aseptic loosening than Charnley. The analyses also showed a marked improvement in Charnley results between the periods 1987–1997 and 1998–2007.Interpretation We observed clinically important differences between cemented prosthesis brands and identified inferior results for previously largely undocumented prostheses, including the commonly used prosthesis combination Reflection All-Poly/Spectron-EF. The results were, however, satisfactory according to international standards. A systematic review of the literature concerning outcome and clinical effectiveness of prostheses used for primary total hip arthroplasty (THA) showed that among the many cemented prostheses in use in Norway in 2000, only the Charnley and the Lubinus IP prosthesis had been reported with results beyond 15 years of follow-up . SeveralThe NAR was established September 15, 1987 . IndividBy December 31, 2007, 110,882 primary THAs had been reported to the Register, 8,094 of which (7.3%) were revised by 2007. Only THAs with cemented acetabular and femoral components were eligible for inclusion in the present study (78%). Furthermore, THAs with unknown information on prosthesis brand or cement brand (n = 114), or on whether the components were cemented with two different cement brands , were excluded. An exclusion criterion was also that the prosthesis components should be cemented with either a Palacos type cement , or a Simplex cement  . Furthermore, only the 10 most common prosthesis brand combinations were studied, totaling 62,305 THAs .Survival analyses used revision of either cup or stem, revision of cup, or revision of stem as endpoints. Separate analyses were performed for revisions for any cause and revisions due to aseptic loosening. Information on deaths or emigrations was retrieved from Statistics Norway, Oslo, until December 31, 2007. The survival times of implants in patients who had died or emigrated without revision of the prosthesis were censored at the date of death or emigration. Survival times of unrevised prostheses were otherwise censored at the end of the study on December 31, 2007. Use of prosthesis brands changed throughout the study period . To ensuOverall revision percentages were estimated using the Kaplan-Meier method. Median follow-up was calculated for each prosthesis brand following the reversed Kaplan-Meier method . Cox regTo investigate whether the adjusted log RRs changed with year of operation, we fitted an extended Cox model based on generalized additive models for survival data with penalized splines . This smAll p-values less than 0.05 were considered statistically significant. The statistical software programs S-Plus 7  and SPSS version 15.0  were used.During the period 1987–2007, 4 of the 10 prosthesis brand combinations (cup/stem) constituted 85% of the operations . Only 3 5 of the 10 prosthesis cup/stem combinations had been used in more than 250 operations during the years 1987–1997 , totalinAll 10 prosthesis brand combinations had been used in more than 250 operations during this period , totalinIn this study, the Exeter was the most common prosthesis in 2007. With revision due to aseptic loosening of the cup as endpoint and the Exeter as reference (results not shown in tables), in the 6–10-year time interval we observed better results for Charnley , and inferior results for Reflection All-Poly  and Elite  cups. With revision due to aseptic loosening of the stem as endpoint and the same time interval, we observed inferior results for Spectron-EF  and Titan  stems compared to the Exeter. The other brands of stem did not perform statistically significantly different from the Exeter.Although it was used in over 1,500 operations and being the third most used prosthesis in 2007, the Contemporary/Exeter combination could not be evaluated due to the short follow-up time (median 1.3 years).Extended Cox regression analyses showed a reduction in the risk of revision due to aseptic loosening since about 1995 (p for linearity = 0.003) . HoweverOverall survival for THAs inserted early (1987–1997) and late (1998–2007) showed short-term and long-term results that were satisfactory according to international standards . HoweverThe study was based on information given to NAR by orthopedic surgeons since 1987. Ideally, the quality of prostheses should be evaluated in high-powered randomized clinical trials, but for practical and economic reasons  such triBefore 1998, the Charnley cup did well compared to others while the Charnley stem had inferior results—at least with short-term follow-up. Most Charnley cups were OGEE-flanged, which has been shown to give good cementation . The 22.Overall, the revision rates due to aseptic loosening were similar for Charnley and Exeter, but the long-term risk of aseptic loosening was higher for Exeter cups and lower for Exeter stems. This could be influenced by head size , sterilization procedures, the quality of polyethylene, design, surgical techniques, or a combination of these. This finding is corroborated by other studies showing excellent 12-year results for the Exeter stem, but not so for the Exeter cup . InferioConcern has been raised about the use of titanium stems in cemented prostheses . Our stuThe Spectron cup initially had better results than the Charnley cup, but this changed with longer follow-up—probably due to the 32-mm heads used with the Spectron cup . SatisfaWe observed low revision rates for the IP/SP prosthesis (90% SP II stems). This is in accordance with those reported for the SP II prosthesis from other register-based studies with a 10-year revision rate due to aseptic loosening of 4.3% . HoweverThe Reflection All-Poly cup and the cobalt-chrome Spectron-EF stem was the most common prosthesis combination in 2007 (with no restrictions regarding cement brand and including cups with highly crosslinked polyethylene). Compared with either the Charnley or the Exeter prosthesis, both the cup and the stem had higher revision rates. The 2007 annual report from the Swedish Hip Arthroplasty Register showed a revision rate (for any cause) at 10 years for this prosthesis combination of 8% , which iWe could not find any published reports for the Kronos/Titan combination either, or for the Kronos cup. Our results should be interpreted with caution, as the Kronos/Titan combination was used mainly in one hospital.Inferior results were noted for the Elite cup in combination with the Titan stem, as compared to either Charnley or Exeter. The Elite cup has the same manufacturer as the Charnley, and is the same except that it can be used with larger heads (98% with 28-mm heads). The finding for the Elite cup is in accordance with the concern raised by With a median follow-up time of 1.3 years, the Contemporary/Exeter combination had the shortest follow-up. Even though the combination was one of the most commonly used prostheses in 2007, it could not be evaluated, and as far as we know no results have been reported from other studies.We observed clinically important differences between cemented prosthesis brands and identified inferior results for previously largely undocumented prostheses, including the commonly used prosthesis combination Reflection All-Poly/Spectron-EF. The study has further demonstrated the importance of long-term follow-up, as several of the prostheses with low short-term revision rates did not perform as well with longer follow-up. However, although we observed variation in prosthesis-specific survival, the overall results were satisfactory according to international standards.


Elevated cardiac troponin I (cTnI) is frequently observed in patients with severe sepsis and septic shock. However, the mechanisms underlying cTnI release in these patients are still unknown. To date no data regarding coagulation disturbances as a possible mechanism for cTnI release during sepsis are available.Consecutive patients with systemic inflammatory response syndrome (SIRS), sepsis or septic shock without evidence of an acute coronary syndrome were analyzed. Coagulation parameters (clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), α-angle) were assessed in native whole blood samples, and using specific activators to evaluate the extrinsic and intrinsic as well as the fibrin component of the coagulation pathway with the use of rotational thrombelastometry (ROTEM).Thirty-eight patients were included and 22 (58%) were cTnI-positive. Baseline characteristics between TnI-positive and -negative patients were similar. The CT, CFT, MCF and the α-angle were similar between the groups with trends towards shorter CT in the extrinsic and fibrin activation.We found no differences in coagulation parameters analyzed with rotational thrombelastometry between cTnI-positive and -negative patients with SIRS, severe sepsis, and septic shock. These findings suggest that pathophysiological mechanisms other than thrombus-associated myocardial damage might play a major role, including reversible myocardial membrane leakage and/or cytokine mediated apoptosis in these patients. Elevated concentrations of cardiac troponin I (cTnI) are frequently observed in patients with severe sepsis and septic shock even in the absence of an acute coronary syndrome (ACS) All patients, or in case of unconsciousness, their closest relatives, signed a written informed consent. The study was approved by the ethics comittee of the Kanton St. Gallen, Switzerland.Thirty-eight consecutive patients referred to our medical intensive care unit and fulfilling criteria for severe sepsis, septic shock or SIRS were studied. Definitions for sepsis, septic shock, and SIRS correspond to the criteria of the consensus conference of the American College of Chest Physician and Society of Critical Care Medicine A 12-lead ECG was obtained at study inclusion and after 12 and 24 hours. The simplified Acute Physiology Score II (SAPS-II) was used to describe/assess disease severity at study entry Blood was obtained from a radial arterial line and cTnI was measured at the time of study inclusion and after 12, 24, 48, 96, 192 and 288 hours and analyzed from the serum on a day by day basis with a second-generation assay . Two consecutive cTnI values >0.5 µg/l were considered “cTnI-positive”. In addition, creatine phosphokinase (CK), CK-MB activity and C-reactive protein (CRP) were assessed. Blood samples for hemostasis analysis were taken at study inclusion, and after 12, 24 and 48 hours and analyzed using rotational thrombelastometry . The method and the parameters of thrombelastometry have been described in detail previously All coagulation parameters were measured in whole blood samples without addition of specific reagents (NATEM), and using specific activators to evaluate the extrinsic (EXTEM) and intrinsic (INTEM) pathway of the coagulation system. In addition, by blocking platelets the fibrin component of the coagulation (FIBTEM) was analyzed separately. Thus, through analysis of whole blood samples, thrombelastometry assesses cellular and plasmatic components simultaneously, allowing the plasmatic coagulation system to interact with cellular components, and therefore provides complete information during the process of clot initiation, formation and stabilization Categorical data are presented as numbers (percentages) and continuous data as mean ± standard deviation or median values  as appropriate. Fisher's exact test was used to compare categorical variables, and Mann Whitney-U tests were used to compare continuous data. A p-value <0.05 was considered statistically significant. Statistical calculations were performed using the statistical package SPSS 16 for Mac (SPSS Inc. USA).Demographic data of the thirty-eight included patients are presented in Median values of LVEF, laboratory parameters such as CRP, creatinine, and CK, supportive therapy by means of mechanical ventilation, renal replacement therapy, and the use of catecholamines did not differ between cTnI-positive and -negative patients. In addition, disease severity indicated by SAPS-II at study inclusion was similar in cTnI-positive and -negative patients .No differences in the coagulation parameters CT, CFT, MCF and the α-angle analyzed with the use of rotational thrombelastometry were found between cTnI-positive and -negative patients. In EXTEM and FIBTEM trends towards shorter CT were found .Twenty-two (58%) of all study patients were cTnI-positive. Median cTnI level in cTnI-positive patients was 1.7 µg/l (0.8–4.6). Among the sixteen cTnI-positive survivors, significant flow limiting CAD could be excluded by dobutamine stress-echocardiography after recovery from disease in seven patients (44%), and by coronary angiography in one patient (6%). In the remaining eight cTnI-positive survivors, one had a normal coronary angiogram performed one day before study inclusion, and in two patients a recent (<6 months) non-invasive cardiac exercise testing  did not reveal significant CAD. Thus, in 69% (11/16) of cTnI-positive survivors, significant CAD could be ruled out. Ten patients of the entire study population (26%), 6/22 (27%) cTnI-positive and 4/16 (25%) cTnI-negative patients had died. Autopsy was performed in seven patients; thereof four patients (57%) were cTnI-positive. Of those three had normal coronary arteries, and one patient had CAD with a thrombotic obstruction of the left anterior descending artery and cardiomyocyte necrosis representing an acute myocardial infarction. In three cTnI-negative patients, no flow-limiting CAD was found at autopsy. Overall we could exclude relevant CAD in 64% (14/22) of cTnI-positive patients. Mortality assessed 28 days after study inclusion did not differ between cTnI-positive and -negative patients.We hypothesized that cTnI elevation in SIRS and septic patients might be a consequence of microvascular flow disturbance provoked by a hypercoagulable state resulting in cardiomyocyte damage or death. Therefore, we prospectively investigated clotting parameters in 38 patients with SIRS and sepsis with the use of rotational thrombelastometry. This is to our knowledge the first study investigating the coagulation system in SIRS and septic patients with respect to their cTnI-status. However, no differences in clotting parameters as assessed by rotational thrombelastometry between cTnI-positive and -negative patients were found. Furthermore we ruled out relevant CAD as an alternative cause for cTnI elevation in the majority of the cTnI-positive patients.It has been shown that thrombelastographic variables reflect additional information on the hemostatic process compared to common coagulation laboratory parameters, such as fibrinogen or activated partial thromboplastin time How can our findings be interpreted in context to previous studies? Several decades ago Parillo et al. described a circulating myocardial depressant substance in septic patients who showed reversible depression of LVEF This hypothesis is supported by our previous clinical study in septic patients showing significantly elevated tumor-necrosis-factor α (TNFα) and IL-6 in troponin-positive as compared to -negative patients The complexity of the human coagulation system is difficult to assess by a single laboratory analysis, and other changes of the coagulation system not detected by thrombelastometry might play a role in this setting. As this is an exploratory study we cannot exclude that other components of the coagulation system  might be important with regard to troponin positivity. Classical thrombelastography is rather insensitive to platelet function. Despite the increased robustness of the ROTEM assay, we cannot exclude that ROTEM analysis assessed from arterial blood specimens is not sensitive enough to detect minor coagulation differences in cardiac microcirculation between troponin positive and negative patients.The small sample size is a limitation to our study. We did not explore the impact of disease severity (e.g. from SIRS to septic shock) on the coagulation system between cTnI-positive and –negative patients due to the limited sample size. Given the trends towards shorter CT times in EXTEM and FIBTEM, one could speculate that the results may have been different had more patients been included. However, we are aware of the difficulties to assess the function of the coagulation system “ex vivo”.In a relatively small group of patients with SIRS, sepsis, and septic shock we found no differences in coagulation parameters analyzed with rotational thromboelastometry between cTnI-positive and -negative patients. According to the results of the present study, other mechanisms than thrombus-associated myocardial damage might play a major role in cTnI-positive patients with severe sepsis and septic shock. Clinical and experimental studies suggest that cytokines, especially TNFα, IL-1β and IL-6 appear to play a pivotal role in mediating the hemodynamic effects and the release of cardiac troponin in patients with severe sepsis and septic shock. Proposed mechanisms are cytokine-mediated reversible myocardial membrane leakage and/or apoptosis.


Fludarabine, is one of the most active single agents in the treatment of chronic lymphocytic leukemia (CLL). Over time, however, virtually all CLL patients become fludarabine-refractory. To elucidate whether microRNAs are involved in the development of fludarabine resistance, we analyzed the expression of 723 human miRNAs before and 5-days after fludarabine mono-therapy in 17 CLL patients which were classified as responder or refractory to fludarabine treatment based on NCI criteria.By comparing the expression profiles of these two groups of patients, we identified a microRNA signature able to distinguish refractory from sensitive CLLs. The expression of some microRNAs was also able to predict fludarabine resistance of 12 independent CLL patients. Among the identified microRNAs, miR-148a, miR-222 and miR-21 exhibited a significantly higher expression in non-responder patients either before and after fludarabine treatment. After performing messenger RNA expression profile of the same patients, the activation of p53-responsive genes was detected in fludarabine responsive cases only, therefore suggesting a possible mechanism linked to microRNA deregulation in non-responder patients. Importantly, inhibition of miR-21 and miR-222 by anti-miRNA oligonucleotides induced a significant increase in caspase activity in fludarabine-treated p53-mutant MEG-01 cells, suggesting that miR-21 and miR-222 up-regulation may be involved in the establishment of fludarabine resistance.This is the first report that reveals the existence of a microRNA profile that differentiate refractory and sensitive CLLs, either before and after fludarabine mono-therapy. A p53 dysfunctional pathway emerged in refractory CLLs and could contribute in explaining the observed miRNA profile. Moreover, this work indicates that specific microRNAs can be used to predict fludarabine resistance and may potentially be used as therapeutic targets, therefore establishing an important starting point for future studies. Chronic Lymphocytic Leukemia (CLL) is the most common hematologic neoplasia in the Western world and is characterized by a clonal expansion of CD5+ B-cells. The disease may have a long and indolent course, not requiring treatment for years, or it may rapidly progress. Initially most patients respond to purine nucleoside analogs, which today represent pivotal agents for first and second-line treatment . HoweverMicroRNAs (miRNAs) have been involved in the regulation of many physiological and pathological processes, through their wide action as post-transcriptional gene expression modulators ,9. In CLRecently, Zenz and colleagues found that fludarabine-refractory CLLs were characterized more frequently (53%) by lower levels of miR-34a than sensitive CLLs (9%) . MiR-34aFludarabine is an active agent in CLL and, it is frequently used nowadays in combination regimens in first and second line treatment, especially in younger and fit patients. Fludarabine as single agent produces better response rates than chlorambucil and represents a reasonable choice as single agent in frail or elderly patients . BecauseTo investigate the possible involvement of miRNAs in fludarabine resistance, we analyzed the miRNA expression profile of 17 CLL patients, before and after 5 days of treatment with fludarabine as single agent. Based on NCI criteria, 9 of the patients exhibited a clinical response (CR), i.e. they attained either complete or partial response, while 8 patients were resistant to the treatment and were classified as non responders (NR). Characteristics of the enrolled patients are shown in Table Table S1 and Additional file Table S2). A hierarchical clustering algorithm, performed using the list of 37 miRNAs, correctly classified the pre versus post fludarabine samples  able to assess the expression levels of 723 human miRNAs (MiRBASE release 10.1). First, we identified the miRNAs induced or repressed by fludarabine treatment by comparing post CLLs to the pre-treatment samples . We compared pre and post samples in the entire groups or in the NR and CR sub-groups separately, which revealed 97, 67 and 60 differentially expressed miRNAs, respectively. Among these, using Venn Diagram intersection, we identified 37 miRNAs that were differentially expressed either in the CR cohort and in the NR cohort of samples .We validated the microarray results for miR-21, miR-222 and miR-148a by quantitative reverse-transcription PCR (RT-qPCR) on the same group of 16 post samples, adding also 3 new samples. We chose these miRNAs considering the extent of their modulation and because they are differentially expressed both before and after the treatment. The expression level of every miRNA in every post CLL was assayed in triplicate and normalized on U6 RNA expression levels. Microarray results were confirmed by RT-qPCR in every group of CLLs  might be able to predict the efficacy of fludarabine treatment and might therefore become useful in directing patients therapy, we collected a new cohort of 12 patients (test set). These patients were treated by fludarabine as single agent because of disease progression. Samples were collected immediately before the treatment and the miRNA expression was assayed by RT-qPCR. In this new cohort, results were even more clear than in the microarray training set of samples Figure . PatientTo better elucidate the molecular context responsible for the differential expression of miRNAs in NR versus CR patients, we performed a mRNA expression profile on a subgroup of the samples. Experiments data were obtained for pre and post fludarabine treatment of 14 patients , 7 of them were CRs and 7 NRs. We hybridized the samples on a human mRNA platform (Agilent Technologies) able to assess the expression levels 41,000 human transcripts. We performed an analysis aimed at identifying the genes induced or repressed by the pharmacologic treatment, with a particular interest in genes modulated only in CRs or NRs. To this end, we normalized the expression of every gene in each post sample to the expression of the same gene in the matched pre sample. We identified the mRNAs that exhibited a significant differential modulation  after fludarabine treatment, separately analyzing refractory and sensitive CLLs (data not shown). Then, we identified the most significant pathways modulated by fludarabine through a Pathway Enrichment analysis of the two gene lists. Interestingly, the p53 pathway emerged as the most significantly induced pathway in the cohort of CR patients Table , while tThe above results suggest that an abnormal miRNA expression may be involved in the generation of fludarabine resistance. Among the various miRNAs differentially expressed between NR versus CR patients, miR-21, miR-222 and miR-148a emerged as significantly up-regulated in two different cohorts of refractory patients, both before and after fludarabine administration. MiR-21 has a well-established role in protection from apoptosis and drug-response modulation ,24. We tin vivo.We analyzed the expression profile of 29 CLL patients who were treated by fludarabine, a pivotal drug in the modern treatment of CLL ,26. To bOur results revealed that many miRNAs are modulated by fludarabine treatment, as shown by the 37 miRNAs that are modulated following the treatment in both CR and NR patients, while 23 and 30 miRNAs were differentially modulated only in CRs or NRs, respectively, suggesting that these two cohorts of patients show signs of differences in the execution of molecular pathways modulated by fludarabine.et al., in fact 3 patients with the 17p deletion involved in this study displayed no miR-34a expression (data not shown).Indeed, in an effort to elucidate the mechanisms underlying the differential expression of these miRNAs, we analyzed the molecular pathways modulated by fludarabine in sensitive and refractory CLLs that emerged by mRNA expression analysis. The discovery that many p53 pathway genes  were activated in CRs but not in NRs provides further support to the notion that fludarabine treatment of patients with CLL induces a p53-dependent gene expression response  and thatBy comparing the miRNA expression profile of sensitive and refractory CLLs, we have been able to identify expression signatures that could correctly classify more than 80% of the CR vs NR cases, either before or after treatment. Interestingly, after validation by quantitative PCR of the most significant miRNAs  in an independent population, the prediction of response to therapy for pre-therapy samples was impressive: a predictive score based on miRNA expression levels reached an overall accuracy of 100%. This information may have important clinical implications because it might spare subsequent treatment in patients identified as resistant during disease progression.MiRNAs aberrant expression may not only represent a phenomenon able to reveal an underlying molecular defect, such as p53 dysfunction, but it may also represent one of the possible mechanisms responsible for fludarabine resistance. In this study, we found that some miRNAs, namely miR-222, miR-148a and miR-21 were differentially expressed between NR and CR CLLs both before and after fludarabine treatment and were all up-regulated in the NR group. These findings, not previously described in CLL, appears to be truly significant.Indeed, miR-222 can target the cell growth suppressive cyclin-dependent kinase inhibitors p27 and p57 and the c-KIT receptor (source: Tarbase) thus promoting cell cycle progression ,32. AmonMiR-21 up-regulation appears to be an important mechanism of malignant transformation, since it was found up-regulated in several types of neoplasia ; it accHere, we report that inhibition of miR-222 and miR-21 in the MEG-01 human cells using an anti-miRNA oligonucleotide increases susceptibility to fludarabine, as shown by the significant increase in caspase activity. Our findings suggest a direct role of miR-222 and miR-21 in conferring resistance to fludarabine chemotherapy independently from the p53 pathway, which is dysfunctional in this human cell line .Less clear is the role of miR-148a. MiR-148a is up-regulated in NR CLL patients either before and after fludarabine treatment and it was identified also as resistance predictor in pre CLLs. However, its mechanism of action is not clear, as only two gene targets are presently experimentally validated. One is the isoform 1 of DNA methyltransferase 3b (DNMT3b1)  and the This study revealed that a characteristic miRNA expression profile, as assessed before and soon after start of treatment, can discriminate between fludarabine sensitive and fludarabine resistant CLLs. We confirm the importance of a defective p53 pathway in conferring fludarabine resistance. This mechanism may be relevant in affecting miRNA expression; however, we suggest that an aberrant miRNA expression, i.e. up-regulation of miR-21, miR-148a and miR-222 in NRs, may be important in establishing resistance independently from p53, protecting cells from apoptosis. Indeed, inhibition of miR-21 and miR-222 may increase sensitivity to fludarabine in vitro in a p53-mutant cell line. The differential expression of a small number of miRNAs can have an effect on the capability to respond to fludarabine in CLL patients and can be used for response prediction. Therefore our data may help identify those elderly patients unlikely to respond to fludarabine used as single agent and may assist in the definition of the induction treatment in younger patients, given the availability of non cross-resistant drugs, such as bendamustine . This inSeventeen patients (training set) plus 12 patients (test set) followed at our centre and treated with fludarabine as single agent  were enrolled in this study after obtaining informed consent. The training set patients received fludarabine as first line treatment in 4 cases and as second line treatment after chlorambucil in 13 cases. All the patients had typical morphologic features  and a high immunophenotypic score , consistent with an unequivocal diagnosis of CLL. Cytogenetic and fluorescence in situ hybridization studies were performed as previously described  as part For miRNA/mRNA expression studies, peripheral blood (PB) lymphocytes were separated to a >97% as previously described . In eachMiRNA expression was investigated using the Agilent Human miRNA microarray v.2 . This microarray consists of 60-mer DNA probes synthesized in situ and contains 15,000 features which represent 723 human miRNAs, sourced from the Sanger miRBASE database (Release 10.1). mRNA expression was detected using thee Agilent whole human genome oligo microarray . This microarray consists of 60-mer DNA probes synthesized in situ, which represent 41,000 unique human transcripts. About 500 ngs of total RNA were employed in each experiment. RNA labeling and hybridization were performed in accordance to manufacturer's indications. Agilent scanner and the Feature Extraction 10.5 software (Agilent Technologies) were used to obtain the microarray raw-data.Microarray results were analyzed by using the GeneSpring GX 10 software (Agilent Technologies). Data transformation was applied to set all the negative raw values at 1.0, followed by a Quantile normalization and a log2 transformation. Filters on gene expression were used to keep only the miRNAs/mRNA expressed in at least one sample (flagged as P). Then, samples were grouped in accordance to treatment (pre and post fludarabine) and response to treatment (NR and CR). Differentially expressed genes were selected as having a n-fold expression difference between their geometrical mean among the groups of interest and a statistically significant p-value (<0.05) by Welch t-test statistic. For gene expression statistical analysis, the Benjamini and Hoechberg correction for false positives reduction was used. Differentially expressed genes were employed in Cluster Analysis, using the Manhattan correlation as a measure of similarity and the complete linkage rule for genes and samples clusterization. The identification of mRNAs modulated after fludarabine, in matched samples, was done using the filter on 0-10th percentile in at least 75% of patients, for the miRNAs down-modulated by treatment, and a filter on 90-100th percentile in at least 75% of patients, for the miRNAs up-modulated by treatment. All Microarray data were submitted to ArrayExpress, accession number to be received.The Pathway Enrichment Analysis was performed using GeneSpring GX tools, based on KEGG, BioCarta, Biopax, Cellmap and NCI-Nature databases.-ΔCt (Comparative Ct) method as implemented by Biorad Genex macro for excel program. Significance in qRT-PCR results was determined by t-test. Box plot graphs were created using Microsoft Excel program.Quantitative Real time RT-PCR (qRT-PCR). Mature miRNAs expression was evaluated by Taqman MiRNA assays (Applied Biosystem) specific for miR-21, miR-222, miR-148a and RNU6B as reference gene according to the manufacturer's protocol. Briefly, 5 ng of total RNA was reverse transcribed using the specific looped primer; real time quantitative PCR was conducted using the standard Taqman MiRNA assay protocol on a Biorad-Chromo4 thermal cycler. Each sample was analyzed in triplicate. The level of miRNA was measured using Ct (threshold cycle). The amount of target, normalized on U6 RNA amount, was calculated using 2For miR-21, miR-222 and miR-148a, a CR vs NR threshold value was determined by calculating and selecting the first integer number above the 98th percentile of expression values (as described in quantitative RT-PCR method) distribution in CR group. For each miRNA, a score of 0 was assigned when expression was below the threshold and a score of 1 when above. The sum of these scores for the three miRNAs was used to assign patients to one of two classes (CR or NR): for scores 0 to 1, the patient was predicted as CR; for scores 2 to 3, the patient was predicted as NR.MEG01 (ATCC number CRL-2021), cell line was cultured with RPMI-1640 medium with 10% fetal bovine serum and 0.1% gentamicin. LNA knockdown probe anti-miR-2 and Scramble-miR control were from Exiqon, Negative control, anti-miR-148a and anti-miR-222 were from Ambion. Transfection of oligos was carried out with lipofectamine 2000 (Invitrogen) and Optimem reduced serum medium (Invitrogen) in accordance with manufacturer's procedure.Caspase 3/7 activity assay was performed on MEG01 cell line. Cells were cultured in 96-well plates the day before LNA-anti-miR-21, anti-miR-222, anti-miR-148a and control transfections (50 nmol). After 24 hours from transfection, cells were treated with fludarabine at the concentrations of 0 and 1 μM for additional 24 hours. The assay was performed in accordance with manufacturer's protocol . Each experiment was performed in triplicate.CLL: Chronic Lymphocytic Leukemia; miR or miRNA: microRNA; LNA: Locked nucleic acid; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; CR: clinical response ; NR: not responder.The authors declare that they have no competing interests.MF performed the research, analyzed data and wrote the paper, BZ performed microarray experiments, LR, FC, MC and CDA contributed patient samples and data, AV, LL and ES performed the research, AG performed bioinformatics analysis, AC and MN designed the research and wrote the paper. All authors critically reviewed and edited the paper.Table S1. MicroRNAs modulated by fludarabine in sensitive and refractory CLL patientsClick here for fileTable S2. MicroRNAs modulated by fludarabine in either sensitive or refractory CLL patientsClick here for fileFigure S1. Quantitative RT-PCR validation for miR-222, miR148a and miR-21 in CLL patients. MiRNAs expression in Not Responder (NR) and Complete Responder (CR) patients, after fludarabine treatment, was quantified using TaqMan Real-time RT-PCR. Every expression data was normalized on endogenous U6 RNA levels by 2-ΔCt method. Each sample was analyzed in triplicate. Data are displayed using vertical scatter plot (GraphPad v.5), bars represent means ± SEM. Two-tailed t-test was used to determine the p-values.Click here for file


Increasing lines of evidence show that activation of Kupffer cells plays a pivotal role in initiation and progression of alcoholic liver disease. Kupffer cell activation is evoked by endotoxin , which leads to rapid elevation of intracellular calcium accompanied by release of a variety of mediators including cytokines, eicosanoids and reactive radical species. Among them, TNF-alpha is a critical factor in the pathogenesis of alcoholic liver disease.Thalidomide  was initially used as a sedative and antiemetic during pregnancy but was withdrawn from the market due to its teratogenic effects. Before it was banned, thalidomide was recognized to reduce dramatically symptoms associated with erythema nodosum leprosum, a complication of Hansen's disease. Subsequently, it was shown that thalidomide suppresses TNF-alpha production by macrophages and other cell types such as activated T cells ,2, whichAccordingly, the purpose of this study was to determine whether thalidomide could prevent alcohol-induced liver injury. Here we report that thalidomide prevents liver damage caused by chronic ethanol exposure through not only suppression of TNF-alpha production but abrogation of Kupffer cell sensitization to LPS.In this study, a model of alcoholic liver injury based on the sensitization of Kupffer cells, in which rats are given ethanol (5 g/kg body wt) once every 24 hours . Gut per2+]i was measured using a microspectrofluorometer with fura-2, and TNF-alpha in the culture media was measured using an enzyme-linked immunosorbent assay (ELISA) kit .Kupffer cells were isolated by collagenase digestion and [Cat test as appropriate. p < 0.05 was selected prior to the study to reflect significance.All results were expressed as mean – S.E.M. Statistical differences between means were determined using analysis of variance (ANOVA) or ANOVA on ranks or Bonferroni's post-hoc test or Student's Ethanol administration once every 24 h for 8 weeks caused pronounced pathological changes in the liver lobule, including steatosis, necrosis and inflammation in the liver, confirming our earlier results. In marked contrast, these pathological changes were prevented almost completely by coadministration of thalidomide (5 mg/kg). Likewise, ALT was increased to 91 – 7 IU/L in the 8-week ethanol group, which was three times higher than the values in the control, non-treated rats  was administered i.v. and liver histology were evaluated 24 hours later (data not shown). LPS caused focal necrosis with neutrophil infiltration in the control liver. In the group treated with thalidomide for 4 weeks, liver histology revealed only slight infiltration of neutrophils, but lacking evidence of necrosis. This result agrees with serum transaminase levels, which were slightly lower in the thalidomide-only group. In the 4-week ethanol group, LPS injection resulted in marked aggravation of these pathological parameters. Moreover, steatosis was prominent in this group. Treatment with thalidomide dramatically improved liver injury, which exhibited near normal histology. The increased ALT levels observed 24 hours after LPS injection in the 4-week ethanol group was blocked completely by thalidomide. Similar results were obtained with AST values . Liver from control rats expressed the 55 kD CD14. Thalidomide did not change the amount of CD14 protein; however, the band was far more intense in liver from rats treated with ethanol chronically. Furthermore, the ethanol-induced increase in CD14 protein levels was abrogated completely with co-administration of thalidomide for 4 weeks.As reported previously, Kupffer cell sensitization to LPS is caused by LPS. Therefore, we determined the effect of thalidomide on gut-permeability Table . ThalidoAfter addition of LPS (100 ng/ml) to Kupffer cells isolated from the untreated, control rats, the amount of TNF-alpha mRNA increased and reached a maximal value after 1 hour, followed by a gradual decline over 2 hours (data not shown). Inclusion of thalidomide (5 micromolar) in the culture media did not alter the peak level of TNF-alpha mRNA occurring at 1 hour after LPS. However, 3 hours after LPS addition, the amount of TNF-alpha mRNA in Kupffer cells treated with thalidomide was 30% lower than that of control Kupffer cells, indicating that thalidomide accelerated decrease in TNF-alpha mRNA Table . Thalidoin vitro (data not shown). Treatment with TNF-alpha (10 ng/ml) for 24 hours resulted in enhancement of CD14 expression in Kupffer cells. Inclusion of thalidomide (5 micromolar) in the culture media reduced CD14 expression to a level comparable to the control.To explore the mechanism by which thalidomide abrogated Kupffer cell sensitization to LPS caused by chronic ethanol, we determined the effect of TNF-alpha on CD14 expression in Kupffer cells obtained from normal rats LPS-induced TNF-alpha production by isolated Kupffer cells was compared between groups cultured for 24 hours in the presence or absence of TNF-alpha (10 ng/ml) prior to LPS challenge. Kupffer cells pretreated with TNF-alpha produced about 30% more TNF-alpha in response to LPS (100 ng/ml) than the control cells cultured in the absence of TNF-alpha Table . ThalidoThe results of this study showed that thalidomide prevented liver injury caused by chronic ethanol treatment Table . To inveTNF-alpha has a pleiotropic cytokine and regulatory mechanisms of TNF-alpha production in macrophages and its intracellular signaling have been studied extensively . It was in vitro.This scenario is supported by the facts that pretreatment with TNF-alpha led to an increased TNF-alpha synthesis in response to LPS and enhancement of CD14 expression in Kupffer cells, and that inclusion of thalidomide in the culture media inhibited this TNF-alpha-induced autocrine activation of TNF-alpha synthesis Table . FurtherThe precise mechanisms of the thalidomide actions remain to be pursued, however, the results of this study suggest that thalidomide prevents liver injury caused by chronic ethanol consumption through destabilization of TNF-alpha mRNA and abolishing Kupffer cell sensitization to LPS. Though currently its side effects preclude the use of thalidomide in clinical settings and require rigors of the drug distribution program and prescription decision, efforts to synthesize thalidomide analogues lacking teratogenic effects but having more potent efficacy are underway . Given t


CDAD led to significantly worse outcomes in these patients. Clostridium difficile–associated disease (CDAD), particularly in CDAD-endemic settings. We conducted a retrospective cohort study of nonsurgical inpatients admitted for >48 hours in 2003 . The adjusted hazard ratios for readmission  and deaths within 180 days  were significantly different among CDAD case-patients and noncase patients. In a propensity score matched-pairs analysis that used a nested subset of the cohort (N = 706), attributable length of stay attributable to CDAD was 2.8 days, attributable readmission at 180 days was 19.3%, and attributable death at 180 days was 5.7%. CDAD patients were significantly more likely than controls to be discharged to a long-term-care facility or outside hospital. Even in a nonoutbreak setting, CDAD had a statistically significant negative impact on patient illness and death, and the impact of CDAD persisted beyond hospital discharge.Data are limited on the attributable outcomes of  Clostridium difficile–associated disease (CDAD) is an increasingly common cause of hospital-associated diarrhea , diagnosis and procedure codes , a 1,250-bed, tertiary-care academic hospital in St. Louis, Missouri. Eligibility was limited to nonsurgical patients admitted for re codes ; inpatieFor each patient, a modified APACHE II Acute Physiology Score (APS) was calculated to adjust for severity of illness . The microbiology laboratory only performs toxin tests on unformed stool, so all patients with a positive result for toxin were considered case-patients. Both community-onset and hospital-onset CDAD case-patients were included in the analyses.CDAD case-patients were defined as inpatients with positive Analyses were performed on the full cohort and a nested case–control population. The first component was a retrospective cohort. For CDAD patients, the admission date when the patient’s CDAD was first identified was used as the index admission. For noncases with >1 admission during the study period, 1 admission was randomly selected as the index admission. The nested case–control population consisted of propensity score matched cases and controls from patients identified in the cohort.Survival was defined as the number of days from the index hospital admission until death. Survival was censored at 180 days. Time to readmission was calculated as the number of days between the index hospitalization discharge date and the date of the subsequent admission to BJH, if applicable. Days until readmission were censored at death or 180 days, whichever occurred first.2, and Mann-Whitney U tests were used to compare characteristics of patients with and without CDAD. Time-to-event methods were used to estimate the effect of CDAD on 180-day survival and time-to-readmission. Patients who died during the index hospitalization were excluded from the time-to-readmission analysis. Kaplan-Meier analysis was used to evaluate the unadjusted relationships between CDAD and time-to-event outcomes. Cox proportional hazards regression was used to estimate unadjusted and adjusted hazard ratios and 95% confidence intervals (CIs). All variables with biologic plausibility or p<0.15 in the univariate analysis were considered in the multivariable Cox regression analysis by using backward stepwise selection. Variables were sequentially removed from the final model, starting with the variable most weakly associated with the outcome. The significance of individual covariates was determined by using a Wald statistic of p<0.05. The proportional hazards assumption was verified by assessing the parallel nature of curves in log-log plots.Fisher exact, χThe second component of this study was a propensity score matched-pairs analysis of outcomes attributable to CDAD. This study design complemented the cohort by enabling analyses that could not be conducted in the entire cohort, specifically hospital discharge status, attributable length of stay, attributable time-to-readmission, and attributable death. Hospital discharge status could not be analyzed for the entire cohort because manual review of medical records was required to determine the discharge location of each patient. The large size of the cohort prohibited this analysis. In addition, survival and time-to-readmission estimates generated in the cohort analysis were validated in the matched-pairs analysis.<0.05 in univariate analysis or biologic plausibility were included in the full logistic regression model. CDAD case-patients and controls were matched by a 1:1 ratio that used the nearest-neighbor method within calipers of 0.015 standard deviations  and SAS version 9.1 . The Washington University Human Studies Committee approved this project.All tests were 2-tailed, and pAmong 18,050 nonsurgical inpatients admitted during the 1-year study period, 390 had CDAD and 17,660 did not. Selected patient characteristics of the cohort are summarized in Of 17,492 patients alive at the index hospitalization discharge, 4,207 (24%) were readmitted to BJH within 180 days. Fifty-two percent of CDAD patients were readmitted within 180 days versus 23% of noncases (log-rank p<0.001). Univariate and multivariable Cox regression results for time to readmission are presented in By 180 days after hospital admission, 149 (38%) of 390 CDAD case-patients and 2,150 (12%) 17,660 noncase-patients had died. In the Kaplan-Meier analysis, the mortality rate was significantly higher for CDAD case-patients than noncases (log rank p<0.001) . UnadjusThe propensity score matched-pairs analysis included 353 CDAD cases and 353 controls (N = 706). There were no significant differences between the matched cases and controls after correcting for multiple testing with the Bonferroni procedure. Thirty-seven CDAD case-patients were dropped because a nearest-neighbor control was not available. Unmatched CDAD patients had significantly higher modified APS , longer median length of stay , and higher percentage of deaths at 180 days  than matched case-patients.In the matched-pairs analysis, median length of stay was 9.6 days for CDAD patients compared with 5.8 days for controls, and the increased attributable length of stay for CDAD patients was 2.8 days (Wilcoxon signed-rank p<0.001). Among the 706 patients in the matched-pairs analysis, 445 (63%) were discharged to home and 188 (27%) were discharged to a long-term-care facility. Only 7 (1%) patients were discharged to an outside hospital; therefore, these patients were combined with patients discharged to a long-term-care facility in the analysis. CDAD patients were significantly more likely than controls to be discharged to a long-term-care facility or outside hospital .Among 290 matched-pairs with both patient and control alive at index hospitalization discharge, 148 CDAD patients were readmitted to BJH within 180 days compared with 92 controls, for an attributable readmission of 19.3% (11.4%–27.2%). In the Kaplan-Meier and Cox model analyses, CDAD patients were significantly more likely than controls to be readmitted to the hospital within 180 days , Table 4By 180 days after hospital admission, 127 CDAD patients died compared with 107 controls, for an attributable mortality of 5.7% (95% CI –1.3%–12.6%). Although CDAD case-patients were no more likely than controls to die within 60 days of hospital admission, a divergence in survival between CDAD case-patients and controls began 60 days after hospital admission , Table 4C. difficile that had the binary toxin and tcdC deletion  patients had been discharged from the hospital. This finding is consistent with those of 2 recent nested matched case–control studies in nonoutbreak settings, in which no significant excess deaths were reported after 30 days  were not represented in the dataset. The absence of these patients, as well as the 37 unmatched case-patients, may have resulted in estimates of attributable length of stay and death that are biased low.Data on attributable outcomes associated with CDAD are scarce. As previously mentioned, some data on attributable mortality and length of stay exist; however, these findings are limited by lack of adequate controls, small sample size, or outbreak settings. Our study provided detailed analysis on the effect of CDAD on time-to-readmission. Another key strength of this study is the combination of 2 analytical methods: Cox proportional hazards regression in the primary cohort and propensity score matched-pairs analysis. Mortality and time-to-readmission analyses, which were conducted in both the cohort and matched-pairs populations, had remarkably similar results. The results of this study suggest that endemic CDAD can lead to significantly poorer patient outcomes, including increased hospital length of stay, death, risk for admission to a long-term-care facility, and risk for hospital readmission. Even when the most severe CDAD cases are not considered, the detrimental effect of CDAD on patient health appears to extend beyond hospital discharge. Although prospective validation of these findings is needed, proper allocation of healthcare resources toward prevention of this infection is necessary to prevent further illness and death attributable to CDAD.Details on ICD-9-CM Codes and Creation of the Propensity Score forClostridium difficile-associated disease