Veno-venous extracorporeal membrane oxygenation (vvECMO) can be a life-saving therapy in patients with severe acute lung failure refractory to conventional therapy. Nevertheless, vvECMO is a procedure associated with high costs and resource utilization. The aim of this study was to assess published models for prediction of mortality following vvECMO and optimize an alternative model.Established mortality risk scores were validated to assess their usefulness in 304 adult patients undergoing vvECMO for refractory lung failure at the University Medical Center Regensburg from 2008 to 2013. A parsimonious prediction model was developed based on variables available before ECMO initiation using logistic regression modelling. We then assessed whether addition of variables available one day after ECMO implementation enhanced mortality prediction. Models were internally validated and calibrated by bootstrapping (400 runs). Predictive ability, goodness-of-fit and model discrimination were compared across the different models.P = 0.03) and predictive ability . When classifying patients as lower (<40%) or higher (>80%) risk based on their predicted mortality, the pre-ECMO and day1-on-ECMO models had negative/positive predictive values of 76%/82% and 82%/81%, respectively.In the present study population, existing mortality prediction tools for vvECMO patients showed suboptimal performance. Evaluated before vvECMO initiation, a logistic prediction model comprising age, immunocompromised state, artificial minute ventilation, pre-ECMO serum lactate and hemoglobin concentrations showed best mortality prediction in our patients . Additional information about norepinephrine dosage, fraction of inspired oxygen, C-reactive protein and fibrinogen concentrations the first day following ECMO initiation further improved discrimination (AUC: 0.79, While pre-ECMO mortality prediction remains a challenge due to large patient heterogeneity, evaluation one day after ECMO initiation may improve the ability to separate lower- and higher-risk patients. Our findings support the clinical perception that chronic health condition, high comorbidity and reduced functional reserves are strongly related to survival during and following ECMO support. Renewed evaluation the first day after ECMO initiation may provide enhanced guidance for further handling of ECMO patients. Despite the usefulness of prediction models, thorough clinical evaluation should always represent the cornerstone in decision for ECMO. For more than 40 years, veno-venous extracorporeal membrane oxygenation (vvECMO) has been used in critically ill patients with reversible acute lung failure (ALF). Following major improvements in ECMO technology and safety, the survival benefit shown in the CESAR trial , the useObjective risk assessment is a valuable tool to aid the selection of therapeutic approach, to compare ECMO practice and success rates across different ECMO centers and to provide relatives with objective information about the patient's condition. Thus, there has been an increasing interest in identifying important prognostic factors for vvECMO. Published work includes two risk scores designed for pre-ECMO mortality prediction in patients with acute respiratory distress syndrome (ARDS), the ECMOnet score  and the A large study population, application of up-to-date statistical methods designed to avoid overfitting and use of thorough internal and external validation are crucial steps in the development of reproducible ECMO risk scores to be integrated in clinical practice. Based on prospectively collected data from an institution where different forms of ECMO are now performed more than 100 times per year, we validated the existing risk scores and compared their predictive ability with the more general Sequential Organ Failure Assessment (SOFA) score frequently used in the intensive care unit (ICU). Furthermore, in accordance with the hypothesis that ECMO outcome is largely dependent on the patient’s extra-pulmonary status and general health condition, our aim was to examine data regarding clinical characteristics, respiratory and laboratory parameters, to develop two novel risk scores: one designed for pre-ECMO mortality prediction and one including parameters measured one day after ECMO implantation. We compared the different pre-ECMO mortality prediction models and investigated whether day 1 parameters could enhance predictive ability.2/FiO2) <80 mmHg on a positive end-expiratory pressure of ≥16 cm H2O, or a refractory respiratory acidosis (pH <7.25), despite optimization of conservative therapy.From 1 January 2008 to 15 July 2013, vvECMO was initiated in 304 consecutive adults with ALF refractory to conventional therapeutic modalities at the University Medical Center Regensburg (UKR), Germany. The general indication for vvECMO was either a ratio of partial arterial pressure of oxygen/fraction of inspired oxygen  and femoro-femoral  cannula, respectively. Further ECMO settings and patient management followed an institutional protocol as described previously [The ECMO circuit consisted of a centrifugal pump and a coated polymethylpentene oxygenator. In 99% of the patients, cannulation was performed percutaneously with Seldinger’s technique. In 75%, single-lumen cannulas were implemented with blood-flow from the femoral vein and to the jugular 80%), subclavian (15%) or contralateral femoral (4%) vein. Nineteen percent and six percent of the patients received a single double-lumen jugulo-jugular , Stata , SigmaPlot , Minitab  and the ‘rms’, ‘Hmisc’, ‘xtable’ and ‘party’ packages of R statistical software .Data are given as median  for continuous variables, and as frequency (percentage) for categorical variables. The Mann Whitney U-test and χThe external scores were tested in the present patient population as described in the original publications  test. A simultaneous comparison of discrimination for all models (n = 241) was performed using the method for non-parametric correlated AUCs as proposed by DeLong et al.. The novClinical usefulness of the two novel models was evaluated by dividing the cohort into three groups based on predicted mortality risks: low (<40%), moderate (40% to 80%) and high (>80%)  died during mechanical support and 36 patients (12%) died after weaning from ECMO. The best outcome was noted in trauma patients 74%), followed by primary lung failure (66%), extra-pulmonary lung failure and other causes (both 50%). Before ECMO initiation, non-survivors were longer hospitalized, had higher serum lactate concentrations, lower hemoglobin concentrations and more frequently showed signs of additional organ dysfunctions . PaO%, followP = 0.67 and P = 0.25, respectively, Figure Both the ECMOnet and PRESERVE scores showed limited discrimination (AUC <0.70) and poor goodness-of-fit in our population Table . RecalibP = 0.50) and had a shrinkage factor of 0.83.The final pre-ECMO mortality prediction model contained five variables Table . A plot Comparison of pre-ECMO prediction models The mortality risk predicted by the external scores did not adequately fit with the observed mortality .P <0.001) and showed enhanced discrimination . In a separate nested analysis (n = 236), Model 2 (AUC: 0.794 (0.736 to 0.853)) also showed a better discriminative ability compared to the SOFA (AUC: 0.588 (0.512 to 0.665)), ECMOnet (AUC: 0.609 (0.536 to 0.683)) and PRESERVE (AUC: 0.699 (0.629 to 0.768)) scores calculated using day 1 data .Addition of day-1 parameters resulted in a predictive model based on eight variables Table . Model 2Classification of patients into three respective risk groups revealed good agreement between mean predicted and observed group mortality risks for both Models 1 and 2 Table .In this large study regarding mortality prediction in ALF patients with vvECMO support, we found that both the PRESERVE and ECMOnet score were suboptimal to predict mortality accurately in our population. Two novel risk models were developed showing improved predictive ability. The addition of parameters available the first day after ECMO initiation enhanced mortality prediction. The combination of predictors found to be associated with the most optimal risk prediction in this study add evidence to the hypothesis that high comorbidity and unresponsive respiratory failure are important determinants of mortality following vvECMO support.The SOFA score was designed for and has become well-integrated in clinical practice as an easily available bedside tool to evaluate organ failure/dysfunction over time in ICU patients. As recently described, a high pre-ECMO SOFA score has been associated with a higher mortality risk .The ECMOnet score is an additive score (range 0 to 10) based on baseline characteristics of 60 patients with severe ARDS due to suspected or confirmed H1N1-influenza virus infection . Recent The PRESERVE score was developed in a multicenter study comprising 140 patients with severe ARDS. The additive score (range 0 to 12) consists of eight predictors. To our knowledge, this study is the first to externally validate the score. Minimal differences in predictor distributions as well as similar overall mortality rates support the comparability of the original and validation study populations. There was a clear linear trend in the risk of mortality across increasing subgroups of PRESERVE score. However, the observed differences in the subgroup mortality rates and the reduction in PSEP may be related to overfitting from the original analysis . Again, Based on the confirmed usefulness of the established scores, we identified three strengths in our study which justify our attempt to develop a new risk model: First, the present study (n = 304) is the largest study investigating mortality prediction in ALF patients receiving vvECMO support. Second, the availability of a broad range of variables potentially related to ECMO outcome increases the chances of finding an improved combination of predictors for mortality following vvECMO. Third, the rigor of variable selection and the statistical methods applied have been designed to avoid overfitting the models to the study population -17.The final models described in the present study represent combinations of variables that contain the most useful information to predict ECMO mortality. The models are not designed to reflect causal relationships between single predictors and outcome, which would only be possible if all factors that influence mortality were known. The included predictors may not play causal roles themselves, but carry information from one or several other predictors and thus represent markers for other causal relationships.The composition of predictors in Models 1 and 2 underline the importance of the patient’s underlying health condition and regenerative capacity for prediction of ECMO outcome. Advanced age and chronic immunosuppression, both associated with reduced functional reserves, high comorbidity and reduced ability to recover, have been consistently associated with increased mortality ,9,18. In2 may bring about a vicious cycle with ventilator-induced lung injury [2 and norepinephrine on day 1 may indicate patients with unresponsive respiratory failure despite ECMO.The improved performance with Model 2 indicates that ECMO relieves patients from acute respiratory and hemodynamic stress and provides time to recover already within the first 24 hours. In patients with severe hypoxemia or high cardiac output, where ECMO may not improve gas exchange sufficiently to decrease aggressiveness of mechanical ventilation, continued invasive ventilation with high volume and FiOFurthermore, day 1 plasma concentrations of fibrinogen and C-reactive protein (CRP) were identified as important predictors of ECMO outcome. Surprisingly, low CRP was associated with increased risk of mortality. This might be related to the ability to activate the immune system in order to defend against on-going infection and trigger recovery mechanisms. Whereas survivors generally had higher CRP before and one day after ECMO implantation, they had lower CRP concentrations post-ECMO, leading to the hypothesis that survivors were able to quickly activate an effective immune response. The lower CRP in non-survivors, on the other hand, might indicate immune exhaustion or liver failure, with a failing attempt to bring about an effective response in order to establish control of their acute illness.The protective effect seen with higher fibrinogen concentrations may have several explanations. First, it may be another sign of an effective immune response, supported by the role of fibrinogen as an acute phase reactant. Given that both fibrinogen and CRP remained significant, they both provide information in addition to that from the other factor, even if the two may be somewhat correlated. Second, low fibrinogen levels are also associated with bleeding disorders, large-volume blood transfusions and disseminated intravascular coagulation, which are all associated with higher mortality. Fibrinogen may thus be an informative marker replacing the need for many individual markers and may contain information from several functions that affect outcome following ECMO support.All together, the parameters from Model 2 enhanced discrimination between patients with high regenerative capacity and reversible disease from those with poor health condition, reduced functional reserves and intractable illness.This study illustrates the difficulties in creating a robust model for predicting mortality following vvECMO. Model 1 showed improved goodness-of-fit and somewhat improved discrimination; however, the discriminative ability was not significantly enhanced from the PRESERVE score. ECMO patients have a large heterogeneity in disease and health conditions, and behind ‘mortality of all causes’ there is a large diversity in underlying causality. Patients have different diagnoses, time courses, ages, comorbidities, different sites and types of infection, and different degrees of physiological dysfunction . TherefoP = 0.25, data not shown). Thus, it seems that a substantially larger study would be needed. Furthermore, any model aimed at predicting withdrawal of futile therapy has to be extremely accurate in order not to lead to erroneous decisions. From our present experience with 350 patients, we have observed previously unthought-of capacities of injured lungs to recover.It would be of interest to develop a prediction model for use later during the course of ECMO, for example on day 7. Such a model might aid in the decision to wean the patient from ECMO. However, due to smaller patient numbers caused by earlier weaning or death, a preliminary day 7 model for the remaining 193 patients (126 survivors and 67 non-survivors) showed wide CIs for the odds ratios and was not significantly better at predicting mortality than the day 1 model (In this study, different statistical approaches were explored and compared in order to derive the best and most robust prediction model. While previous models have based their variable selection on univariate analyses, we employed a method combining clinical experience and judgment with computer-based statistics. Nevertheless, the inhomogeneous study population makes overfitting a persisting challenge and the ability of a predictive model to estimate the prognosis of individual patients with high accuracy remains limited. The usefulness of a prediction model rather lies in the assistance it may provide for the discrimination between higher- and lower-risk patients in order to help identify potential candidates for ECMO support and give guidance for rational and ethical resource utilization ,11. We, Conventional rescue therapies, such as prone positioning and use of neuromuscular blockade, were not registered in all patients in the UKR database. A part of our study population dates back to 2008 when the advantages of prone positioning were not universally accepted and neuromuscular blockade was only rarely used in Germany. Hence, the quality of conventional treatment which may be associated with mortality irrespective of the other factors included in the model could not be evaluated in this study. In the calculation of the PRESERVE scores, all patients were assigned to ‘no prone positioning pre-ECMO.’ However, this may contribute to overestimating mortality risk with PRESERVE in our patient population. Furthermore, the PRESERVE score used mortality six months post-ICU discharge as the end-point, while the present study only followed patients up to hospital discharge. Mid- and long-term outcomes are important in ARDS patients receiving vvECMO support. Unfortunately, in the past, long-term outcomes of our patients and health-related quality of life were not reevaluated, as many patients were retrieved from distant hospitals after our team implanted ECMO as a rescue procedure to allow transport to our center. We plan to carry out a follow-up study to evaluate the validity of the presented models in the prediction of mid- and long-term mortality.Missing observations due to incomplete documentation for some laboratory and respiratory parameters reduced the effective sample size in the statistical analyses. Bias due to cohort selection or missing data in the presented analyses seems unlikely since the overall mortality rate was constant in all subsamples with complete information for the different parts of the study. Since validation through bootstrap methods is preferred over data splitting methods , we usedPre-existing tools for predicting outcome following vvECMO in patients with ALF have been of limited usefulness. The present study provides two novel risk models designed to help assist in defining treatment decision and further handling of ECMO. Markers of associated comorbidity, a poor pre-ECMO health condition and unresponsive respiratory failure were consistently associated with increased mortality. Mortality prediction improved the day following ECMO initiation, when ECMO has reduced acute stress and provided time to rest. Nevertheless, due to high variability among patients, predictive models can only act as supplementary tools and do not replace the role of clinical evaluation for indication and continuation of ECMO.• Established risk assessment tools for adult patients with severe acute lung failure on vvECMO are of limited usefulness.• Large patient heterogeneity makes generalized pre-ECMO mortality prediction a persisting challenge, despite use of robust statistical methods.• Addition of variables measured one day after ECMO initiation improves mortality prediction.• Risk prediction tools function as a useful supplement to guide clinical decision-making, although clinical evaluation remains the cornerstone of ECMO practice.aAvailable at: http://www.r-project.org. Accessed June 15, 2013.ALF: acute lung failure; ARDS: acute respiratory distress syndrome; AUC: area under receiver-operating characteristics curve; CI: confidence interval; CRP: C-reactive protein; FiO2: fraction of inspired oxygen; HL test: Hosmer-Lemeshow test; ICU: intensive care unit; LR test: likelihood ratio test; PaO2/FiO2: ratio of partial arterial pressure of oxygen/fraction of inspired oxygen; PRESERVE: PRedicting dEath for SEvere ARDS on VV-ECMO; PSEP: prognostic separation index; SOFA: Sequential Organ Failure Assessment; UKR: University Medical Center Regensburg; vvECMO: veno-venous extracorporeal membrane oxygenation.CS has been a consultant for Maquet Cardiopulmonary Care Germany. AP and TM received travel support from Maquet Cardiopulmonary Care Germany. TB is member of the advisory board of Novalung, Heilbronn Germany and he received honoraria. None of the other authors have a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.TBE made substantial contributions to the conception and design of the study, data analysis, statistical analyses and interpretation of data, and wrote and revised the manuscript. VV made substantial contributions to the conception and design of the study, statistical analyses and interpretation of data, and has been involved in drafting the manuscript and revising it critically for important intellectual content. AP and TM made substantial contributions to the conception and design of this study, acquisition and interpretation of data, and have been involved in drafting the manuscript and revising it critically for important intellectual content. AW made substantial contributions to the conception and design of the study, interpretation of data and has been involved in revising the manuscript critically for important intellectual content. ML, MF, CS and TB have made substantial contributions to the acquisition of data, interpretation of data and have been involved in revising the manuscript critically for important intellectual content. All authors read and approved the final manuscript.Detailed description of data collection and statistical methods.Click here for fileData on external scores. Information about the calculation of the SOFA, ECMOnet and PRESERVE scores validated in the study.Click here for fileVariable selection and modelling. An overview showing the three-step variable selection procedure used in order to derive two parsimonious models designed for predicting mortality in patients with severe lung failure before and one day after ECMO implementation, respectively.Click here for fileComparison of survivors and non-survivors. A detailed comparison between survivors and non-survivors with regard to baseline clinical and proce characteristics, as well as cardiorespiratory and laboratory parameters measured pre-ECMO and regularly throughout ECMO support.Click here for fileObserved versus predicted probabilities estimated by Model 1 and Model 2. A figure comparing the relationship between observed and predicted mortalities as well as model calibration for Models 1 and 2.Click here for file


Eosinophilic esophagitis (EoE) is an allergic disorder characterized by eosinophil-predominant esophageal inflammation, which can be ameliorated by food antigen restriction. Though recent studies suggest that changes in dietary composition may alter the distal gut microbiome, little is currently known about the impact of a restricted diet upon microbial communities of the oral and esophageal microenvironments in the context of EoE. We hypothesize that the oral and esophageal microbiomes of EoE patients are distinct from non-EoE controls, that these differences correspond to changes in esophageal inflammation, and that targeted therapeutic dietary intervention may influence community structure. Using 16S rRNA gene sequencing, we characterized the bacterial composition of the oral and esophageal microenvironments using oral swabs and esophageal biopsies from 35 non-EoE pediatric controls and compared this cohort to samples from 33 pediatric EoE subjects studied in a longitudinal fashion before and after defined dietary changes.Neisseria and Corynebacterium in the EoE cohort, and predominance of the Firmicutes in non-EoE control subjects. We detected a statistically significant difference between actively inflamed EoE biopsies and non-EoE controls. Overall, though targeted dietary intervention did not lead to significant differences in either oral or esophageal microbiota, reintroduction of highly allergenic foods led to enrichment in Ganulicatella and Campylobacter genera in the esophagus.Firmicutes were more abundant in esophageal samples compared to oral. Proportions of bacterial communities were significantly different comparing all EoE esophageal microbiota to non-EoE controls, with enrichment of Proteobacteria, including In conclusion, the esophageal microbiome in EoE is distinct from that of non-EoE controls, with maximal differences observed during active allergic inflammation.The online version of this article (doi:10.1186/s40168-015-0085-6) contains supplementary material, which is available to authorized users. Based upon these findings, we continued to include the six subjects with non-EoE esophagitis as part of the non-EoE control cohort in the subsequent analyses.Because a small number of esophageal samples in the non-EoE control cohort (six subjects) had histologic evidence of non-eosinophilic esophagitis, we performed a separate analysis to compare the six non-EoE control samples with esophagitis to the 25 non-EoE samples without esophageal inflammation. We did not detect a difference between subjects with and without inflammation (unweighted UniFrac versus non-EoE control (31 samples) and inactive EoE (20 samples) versus non-EoE control (31 samples) are shown in Figure Neisseria genus of the Proteobacteria was enriched in the EoE samples, as was Corynebacterium. A relative abundance plot showing the major genera enriched in active EoE samples compared to non-EoE controls is shown in Figure Streptococcus and Atopobium genera were consistently enriched in non-EoE control samples. Relative abundance plots for lineages enriched in non-EoE controls compared to active EoE are shown in Additional file We used the linear discriminant analysis effect size (LEfSe) test to determine which taxonomic groups were responsible for the changes observed with weighted UniFrac in EoE . Taxonom control 1 samples control  samples The SFED is an established treatment for EoE, with outcomes that compare favorably with both targeted elimination diets and an elemental diet. Following the establishment of a SFED, an EGD is typically performed to establish reduction in esophageal eosinophilia. Once endoscopic remission is established, groups of foods from the SFED are added back into the diet and surveillance EGD is performed several weeks after addition of the new food group. In our patient cohort, we included subjects before (time-point 1) and after (time-point 2) a clinically recommended diet change and hypothesized that dietary manipulation using foods from the SFED would not only lead to alterations in the esophageal inflammatory state but would also change esophageal microbial composition.P values: weighted UniFrac P = 0.220; unweighted UniFrac P = 0.450), although this could be explained by the small effective number of patients used in this specific analysis. Follow up analysis using LEfSe was carried out to determine if specific lineages were differentially represented following specific dietary changes in EoE [Granulicatella genus, Carnobacteriaceae family and the Campylobacter genus in the esophageal microenvironment . Corresponding relative abundance plots for Granulicatella and Campylobacter in subjects with a restricted diet who later added an allergenic food as compared to non-EoE controls are shown in Figure A permanova test, using the weighted and unweighted UniFrac distances, was used to determine if dietary interventions could change the disease status and esophageal microbiome over time,. Dietary intervention did not have an appreciable global effect on the UniFrac distances  bacteria of the subjects  and pedisubjects  without ly males , whereas females , showingCorynebacterium and Neisseria of the phylum Proteobacteria were enriched in the setting of esophageal inflammation [Neisseria has been reported in previous studies of the esophagus [We observed differences in the esophageal microbiota when comparing samples from active EoE to non-EoE control subjects. Similar to previously published findings, members of the genera ammation ,34,35. Tsophagus ,36 and cSalmonella is increased in gut inflammation and proposed that inflammation promotes proliferation of Salmonella by production of compounds that Salmonella can use as terminal electron acceptors [Understanding the cause and effect relationship between disease state and the microbiome is a subject of great debate addressed in gnotobiotic studies. Baumler and colleagues found that cceptors ,38. In dcceptors , germ-frcceptors ,41. EoE Campylobacter and Granulicatella, following the addition of a food antigen in the SFED. Campylobacter species have been associated with inflammatory states of gastrointestinal tract including periodontal disease, Barrett’s esophagus, and IBD [Campylobacter jejuni had a greater association to the development of atopy [Although we were unable to detect global differences between subjects on an unrestricted diet and those that added or removed a highly allergenic food, our LEfSe analysis revealed a possible enrichment of two genera,  and IBD . This daof atopy . The speThe dietary additions and eliminations in this study were performed according to the clinical recommendations of the primary gastroenterology/allergy team and foods which were added or eliminated varied among EoE subjects. Although an initial goal of our study was to compare the microbiome of EoE subjects before and after an amino acid based elemental diet, the number of patients placed on an elemental diet at our institution and others has declined significantly due to the recently proven efficacy of the SFED ,8,43. ThStreptococcus, Prevotella, Neisseria, Haemophilus, Porphyromonas, Gemella, Rothia, Granulicatella, Fusobacterium, Actinomyces, Veillonella, and Aggregatibacter are predominant organisms in saliva of healthy subjects [Streptococcus, Fusobacterium, Neisseria, Haemophilus, and Prevotella in both sites [Streptococcus, Neisseria, and Prevotella were predominant organisms in both the oral and esophageal environments.Previous studies have characterized the oral microbiome in the absence of inflammation. The oral microbiomes of children and adults show a predominance of Firmicutes, Proteobacteria, Actinobacteria, and Fusobacteria . In othesubjects . Comparith sites . This isIn the current study, we aimed to study the correlation between these two microbial populations and determine whether the 16S rRNA gene tag analysis of the oral mucosa could serve as a biomarker of disease activity. By having a single sample per individual, we are able to avoid autocorrelation issues arising from having two samples from the same individual. The Mantel correlation and Procrustes values suggest a weak, albeit significant, correlation between the two datasets . We did not detect differences in the oral microbiome between active EoE and inactive EoE or non-EoE control samples, suggesting that in pediatric EoE, bacterial communities are stable and might not be altered by dietary modification. Thus, the data do not support use of oral samples in lieu of biopsies for EoE surveillance.Neisseria and Corynebacterium may be due to inflammation and not EoE itself. Our subjects were treated with a variety of dietary interventions. Although this study is underpowered to detected differences among the treatments, some possible effects were observed that can serve as hypotheses for future studies.We report distinctive microbiota in patients with active EoE compared to non-EoE controls. No significant differences were seen in inactive EoE samples compared to non-EoE controls. This difference suggests that the increase in Subjects enrolled in the study IRB# 10-007737) were undergoing diagnostic esophagogastroduodenoscopy (EGD) at The Children’s Hospital of Philadelphia (CHOP). Inclusion criteria included an age between 6 months and 21 years, ongoing proton pump inhibitor (PPI) use for at least 4 weeks prior to EGD, no antibiotic use for at least 4 weeks prior to EGD, and no other esophageal disease or chronic inflammatory diseases of the gastrointestinal (GI) tract. Non-EoE control subjects showed no histopathologic abnormalities in the esophagus or distal GI tract and were not previously diagnosed with EoE. Both newly diagnosed EoE subjects and subjects who had previously been diagnosed with EoE based on clinical guidelines [0-007737 All EoE subjects were further stratified as active (≥15 eosinophils/high-power field (hpf)) or inactive (<15 eosinophils/hpf) based on EoE diagnostic criteria . After wOral swabs and esophageal biopsies were separately stored in sterile Eppendorf tubes on dry ice, then transferred to a −80°C freezer until processing. Bacterial DNA extraction from oral swabs and esophageal biopsies was performed in a sterile tissue culture hood using the MoBio PowerSoil DNA Isolation Kit  and DNeasy Blood and Tissue Kit , respectively.For bacterial DNA isolation from oral swabs, sterile scissors were used to cut the swab tip into the MoBio Power-Bead tube, and DNA was isolated according to manufacturer’s instructions. For extraction of bacterial DNA from biopsies, samples were incubated in a lysozyme solution containing 20 mg/ml lysozyme in 180 μl of 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, and 1.2% Triton X-100, at 95°C for 5 min and 37°C for 60 min, followed by manufacturer’s instructions to complete DNA extraction. DNA was amplified by adding 10 μl of DNA to 40 μl of PCR mixture containing 5 μl of 10× PCR Buffer , 200 μM each dNTP, 50 pmol barcoded primer , and 2 uPooled DNA samples were sequenced using the Roche/454 Genome sequencer or Genome Sequencer Junior platforms . The sequences obtained were processed using the QIIME software package  using deN = 4), restricted diet (N = 13), elimination (N = 7), and reintroduction of an allergenic food (N = 10) were considered in order to reduce variability in the dataset. OTU differences across groups were analyzed using LEfSe [Global microbiome changes were analyzed using weighted and unweighted UniFrac distances across all samples. Statistical differences were assessed using the Adonis test implemented in the R package Vegan v2.0-10 . Adonis ng LEfSe . Only on


However, no changes were identified in the serum levels of tumor markers or HCC size following eltrombopag administration in the two cases. These HCCs were curatively treated by radiofrequency ablation without platelet transfusions or serious bleeding. Thus, short-term eltrombopag administration may not accelerate HCC proliferation and may be beneficial for invasive HCC treatment in cirrhotic patients with thrombocytopenia.Eltrombopag is an oral thrombopoietin (TPO) receptor agonist that increases platelet counts in patients with idiopathic thrombocytopenic purpura and in patients with liver cirrhosis. When cirrhotic patients with thrombocytopenia undergo elective invasive procedures, eltrombopag treatment reduces the requirement for platelet transfusions. However, TPO is known to have proliferative effects on hepatic progenitor cells and hepatic sinusoidal endothelial cells, which indicates that eltrombopag may accelerate tumor progression. Thus, the effect of eltrombopag on hepatocellular carcinoma (HCC) progression is an important issue. The current study describes two cases of HCC with cirrhosis-related thrombocytopenia. A two-week administration of eltrombopag increased platelet counts from 4.8 to 11.3×10 Thrombocytopenia is a common complication of liver cirrhosis and is an intractable issue when performing invasive procedures ,2. When Eltrombopag is an oral thrombopoietin (TPO)-receptor (R) agonist that stimulates megakaryocytes and thus, increases platelet counts in patients with idiopathic thrombocytopenic purpura . Since dTPO is known as a megakaryocyte growth and development factor that leads to megakaryocyte proliferation. In addition, TPO induces the proliferation of various cell types that express the TPO-R. In the liver, the TPO-R occurs in hepatic progenitor cells  and hepaThe current report presents two cases of HCC with cirrhosis-related thrombocytopenia. In these two cases, eltrombopag treatment increased platelet counts without any concomitant changes in the serum levels of tumor markers or HCC size. The HCCs were subsequently treated by curative radiofrequency ablation (RFA) without platelet transfusions or serious bleeding. Patient provided writted informed consent.In 2008, a 65-year-old male patient was referred to the Kurume University Hospital  for the treatment of hepatitis C virus (HCV)-related liver cirrhosis. The patient had a history of esophageal varices rupture (that occurred in 1993) and HCC, which was treated by transcatheter hepatic arterial chemolipiodolization with embolization in 2007.4 /μl) revealed severe thrombocytopenia.The patient was treated with ursodeoxycholic acid, glycyrrhizin and nutritional therapy, including branched-chain amino acid granules. However, in 2009, a regular abdominal ultrasound revealed a hypoechoic lesion with a maximum diameter of ~15 mm in Couinaud segment 5 of the liver. The lesion showed decreased contrast uptake in the hepatocyte phase as determined by gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (EOB)-enhanced magnetic resonance imaging (MRI) scan . Additio4 /μl during the administration of eltrombopag . No significant changes were observed in the serum levels of α-fetoprotein and des-γ-carboxy prothrombin (The risks and benefits of participation in the phase II study were explained as previously described . Writtenthrombin  or in ththrombin . At threThe effect of eltrombopag on the progression of malignant neoplasms is an important issue. The current report presents two cases of HCC with cirrhosis-related severe thrombocytopenia. The administration of eltrombopag increased the platelet count in these two cases without inducing significant changes in the serum levels of tumor markers or HCC size. These HCCs were successfully treated by RFA without platelet transfusions or serious bleeding. The observations from these cases may indicate that the short-term administration of eltrombopag does not affect the progression of HCC and may be useful when administered prior to invasive procedures in cirrhotic patients with thrombocytopenia.In the present study, eltrombopag treatment significantly increased platelet count, which subsequently resulted in successful RFA therapy and the avoidance of platelet transfusions. These beneficial observations are consistent with those of previous reports. Eltrombopag has previously been shown to improve thrombocytopenia and permit interferon therapy in patients with cirrhosis that is associated with HCV infection . More re3, which was avoided in the present study by close monitoring of the patients. Although the reason for the development of thrombosis remains unclear, von Willebrand factor levels have been found to increase as an adaptive mechanism in response to reduced platelet adhesion abilities in cirrhotic patients (In the current two cases, thrombosis did not develop during or following eltrombopag treatment. However, certain previous clinical trials have reported that invasive procedures increase the risk for the development of thrombosis, even in cirrhotic patients with thrombocytopenia ,5. The rpatients . Additiopatients ,11. Altepatients . Higher Hepatic progenitor cells and hepatic sinusoidal endothelial cells express TPO-R, in addition TPO induces the proliferation of these two types of cells ,8. FurthTPO-R expression has previously been found to be lower in hepatoma cell lines compared with expression in primary hepatocytes  and HCC In conclusion, the present study described two cases of HCC with cirrhosis-related thrombocytopenia. Eltrombopag treatment increased the platelet count in the two cases and resulted in successful RFA treatments without platelet transfusions or serious bleeding. Additionally, no changes were observed in the serum levels of tumor markers or HCC size. Although eltrombopag is not currently used for the treatment of thrombocytopenia in patients with chronic liver disease undergoing invasive procedures, the observations of the present study indicated that the short-term administration of eltrombopag may not accelerate the progression of HCC and may be beneficial for HCC treatment in cirrhotic patients with thrombocytopenia.


Depression is an adverse prognostic factor after an acute myocardial infarction (MI), and an increased propensity toward emotionally-driven myocardial ischemia may play a role. We aimed to examine the association between depressive symptoms and mental stress-induced myocardial ischemia in young survivors of an MI.We studied 98 patients (49 women and 49 men) age 38–60 years who were hospitalized for acute MI in the previous 6 months. Patients underwent myocardial perfusion imaging at rest, after mental stress (speech task), and after exercise or pharmacological stress. A summed difference score (SDS), obtained with observer-independent software, was used to quantify myocardial ischemia under both stress conditions. The Beck Depression Inventory-II (BDI-II) was used to measure depressive symptoms, which were analyzed as overall score, and as separate somatic and cognitive depressive symptom scores.There was a significant positive association between depressive symptoms and SDS with mental stress, denoting more ischemia. After adjustment for demographic and lifestyle factors, disease severity and medications, each incremental depressive symptom was associated with 0.14 points higher SDS. When somatic and cognitive depressive symptoms were examined separately, both somatic  and cognitive symptoms  were significantly associated with mental stress-induced ischemia. Depressive symptoms were not associated with ischemia induced by exercise or pharmacological stress.Among young post-MI patients, higher levels of both cognitive and somatic depressive symptoms are associated with a higher propensity to develop myocardial ischemia with mental stress, but not with physical  stress. Depression is a prevalent, debilitating, and underdiagnosed condition that affects approximately 20% of patients with recent myocardial infarction (MI), and is associated with increased risk of recurrent cardiac events The precise mechanisms through which depression may increase the risk for adverse cardiovascular outcomes after an acute MI are not known. Adverse behaviors, inflammation and abnormal autonomic function have been linked to both depression and recurrent CAD events, but whether these factors explain the increased risk associated with depression after MI is debatable We systematically tested a sample of young and middle-aged post-MI patients, a group with high burden of psychosocial distress Participants for this study were recruited from the pool of patients younger than 60 years of age admitted in the previous 6 months with a confirmed diagnosis of MI at Emory-affiliated hospitals as part of the Myocardial Infarction and Mental Stress Study (MIMS). Details of our recruitment strategy and number of eligible and excluded patients have been described Study subjects underwent three SPECT imaging scans, one with rest, one with mental stress and one with physical stress. The latter included predominantly exercise stress. However, 16 patients were unable to reach their target heart rate despite scoring ≥5 METs on the DASI and therefore underwent a pharmacological stress test. The two stress scans were performed on separate days, mostly within one week of each other , and the rest scan was performed on the first visit. The order of the two types of stress tests was counterbalanced. All testing was done after an overnight fast, and anti-ischemic medications were held for 24 hours prior to testing. Sociodemographic and psychosocial data were collected at the first visit prior to cardiac testing. At the end of the study protocol, medical records were abstracted for clinical information, including catheterization data. All participants signed a written informed consent. The study protocol and informed consent were approved by the Emory University Institutional Review Board.Initially, patients rested for 30 minutes in a quiet, dimly lit, temperature-controlled room. At the end of the resting period, mental stress was induced by a standardized public speaking task as previously described 99mTc sestamibi), at rest, after mental stress, and after physical  stress. Testing was done on a dedicated ultra-fast solid-state camera  without attenuation correction 99mTc] sestamibi according to body weight. The stress scan  followed at least 2 hours later and 30–45 mCi of [99mTc] sestamibi were administered, with a 3∶1 ratio of stress to rest radiotracer dose.Subjects underwent three SPECT myocardial perfusion imaging scans following injection of sestamibi radiolabelled with Technetium-99m  Information on sociodemographic factors was collected using standard questionnaires from population studies. A detailed medical history, including medication use, was obtained by a research nurse. Weight and height were used to calculate body mass index. Venous blood samples were drawn for the measurements of glucose and lipid profile after an overnight fast.Angiographic data were obtained from the coronary angiogram performed in conjunction with the index MI. CAD severity was quantified using the Gensini semi-quantitative angiographic scoring system Descriptive statistics were computed by comparing mean BDI-II scores according to levels of other study variables. Next, multivariate linear regression models were used to examine the association between depressive symptoms and stress perfusion scores adjusting for possible confounding factors. The SDS, which quantifies ischemia, was our main outcome of interest. Since the SDS for both mental and physical stress was highly skewed, while the SSS was approximately normally distributed, we used the SSS scores as dependent variables while adjusting for the rest score (SRS), yielding coefficients for depression identical to those from a model where the dependent variable is the difference score (SDS), adjusted for the SRS. In a series of cumulative hierarchical models, we adjusted for a set of factors that were considered a-priori as either possible confounding factors or mediators of the relationships under study. Adjustment factors included sociodemographic and lifestyle characteristics , medications , CAD severity , traditional CAD risk factors , and previous revascularization procedures (coronary artery bypass grafting and percutaneous coronary intervention).Between 2009 and 2012, 49 male and 49 female MI patients younger than 60 years were included in the study. The mean and median age was 50 years. Overall, 55% of the participants were African-American; 57% had at least high school education and 32% had an income below the poverty level Table 1.The BDI-II total scores were approximately normally distributed, with a mean of 11.2 . The mean somatic symptom score was 2.8 , and the mean cognitive symptom score was 8.4 . BDI-II total scores were higher among women, among patients with income below poverty and among current smokers Table 1.Three patients, 1 woman and 2 men, had missing myocardial perfusion data for both mental stress and exercise or pharmacological stress, and were excluded from the perfusion imaging analyses. The median SDS with mental stress was 2.0 , and the median SDS with physical stress was also 2.0 . The SDS with mental stress and physical stress were not correlated . Results were similar with no significant heterogeneity based on whether exercise stress or pharmacological stress was used.A higher BDI-II total score was associated with increased propensity for ischemia with mental stress. The graded fashion of this association was evident when the BDI-II total score was categorized according to quintiles Figure 1Next, depressive symptoms were analyzed as separate somatic and cognitive components Table 3.There were no significant differences in results according to sex, although the association between depressive symptoms and mental stress-induced ischemia appeared stronger among men. To rule out a possible nonlinear trend in the association between BDI-II and physical stress ischemia, we repeated the analysis using non-parametric regression using smoothing splines In this experimental study of young and middle-aged post-MI patients, we demonstrated a robust association between baseline depressive symptoms and inducible myocardial ischemia with mental stress, but not with exercise/pharmacological stress. We observed this association for both somatic and cognitive depressive symptoms. Given that mental stress-induced ischemia is a prognostic indicator in CAD patients 99mTc]sestamibi SPECT myocardial perfusion imaging to detect ischemia, which is a customary method for the detection of ischemia in contemporary clinical care.Only a small number of studies have examined the association between depressive symptoms and mental stress-induced myocardial ischemia, yielding inconsistent results. Jiang et al. Potential mechanisms that might be invoked to explain the association between depressive symptoms and mental stress-induced myocardial ischemia include downstream physiologic effects of stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis, which can be dysregulated in depression In our study, depressive symptoms were associated only with mental stress-induced myocardial ischemia and not with physical stress-induced ischemia. This finding differs from the study by Boyle et al. Our findings have substantial relevance for public health and clinical medicine. Depression affects approximately 20% of post-MI men and 40% of post-MI women below the age of 60 99mTc]sestamibi SPECT for the detection of myocardial ischemia, which has established sensitivity, specificity, and reproducibility to detect mental stress-induced myocardial ischemia 99mTc]sestamibi, once injected during mental stress, is trapped in the myocyte, thus providing a real-time “snapshot” of perfusion at the time of stress. It is likely that this feature enhances the accuracy of ischemia assessment during mental stress.The main limitation of our study is its small sample size, which may decrease statistical power. In addition, given that the study was restricted to young post-MI patients, our results may not be generalizable to other stable CAD patient populations or older age groups. However, we targeted an equal representation of women and men, and did not restrict study participation based on demographic factors other than age and sex. The other sociodemographic characteristics of our sample  are likely a result of the relatively young age and high proportion of women in our sample. Another limitation is lack of data linking mental stress ischemia to clinical outcomes. Nonetheless, a strength of our study is the use of a standardized mental stress protocol and a well-defined, relatively understudied patient population with an elevated prevalence of depressive symptoms. Furthermore, we used [In conclusion, depressive symptoms are associated with mental stress-induced myocardial ischemia in younger post MI patients. This association is robust even after multivariate adjustments for other risk factors and disease severity, and applies to both somatic and cognitive depressive symptoms. Since CAD patients who experience ischemia with mental stress are at elevated risk for new cardiac events and death


The persistent lack of evidence on causal mechanisms between social capital and health threatens the credibility of the social capital—health association. The present study aims to address this ongoing problem by investigating whether health behaviours  mediate the prospective relation between workplace reciprocity and future sickness absence.A cohort of 24,402 Belgian employees was followed up during 12 months for sickness absence. Workplace reciprocity was measured with four indicators—colleague help, colleague interest, supervisor help, and supervisor concern. Three types of multilevel mediation models were applied.Overall, workplace reciprocity negatively related to high sickness absence (≥ 10 days) mainly independently from health behaviours. Uniquely, colleague interest positively related to smoking  and smoking in turn, positively related to sickness absence . No behavioural pathways could be identified between company-level reciprocity and sickness absence, and company-level health-related behaviours did not mediate the relation between company-level reciprocity and individual sickness absence.These results suggest that both social capital and health behaviours are relevant for employee health, but health behaviours seem not to be the underlying explanatory mechanism between workplace reciprocity and health. It is widely recognized that beyond the individual, social environments such as neighbourhoods and workplaces may promote or constrain the practice of healthy lifestyles that ultimately lead to health and illness . For theThe elastic definition of social capital  tolerateProspective multilevel studies investigating the effect of social capital on health are limited  and haveIn line with Choi et al.  the presXij), health behaviour (Mij), and sickness absence (Yij) variables are conceptualized at the first or individual level of the data. For example, this type of mediation conceptualizes smoking uniquely as individual risk behaviour and tests the individual dimension of the workplace reciprocity model [Following Krull and MacKinnon , we formty model . The secBELSTRESS is a large epidemiological cohort study on job stress, cardiovascular risk factors and sickness absence ,37. BaseThe measurement of reciprocity as a cognitive aspect of social capital was based on the classical measure of Kawachi et al.  but addiWe analyzed three different health behaviours: smoking, alcohol consumption and leisure time physical activity . SmokersIn collaboration with the personnel administration departments of the participating companies, information on absenteeism was gathered during a follow-up period of 12 months. Because the recording of sickness absence in Belgium is strictly ruled, requiring medical certification, we may assume that the sickness absence registration was highly accurate. Since former research clearly demonstrated a relationship between social capital and health ,25 and dThe data source was indicated by a binary variable . Age was categorized into three groups , with persons aged 30 to 39 years as the reference category. Educational level was assessed by the highest level of completed education based on a 6-point rating scale . Higher education refers to both vocational courses (only bachelor) and academic courses (bachelor and master). University is more specific and refers to the academic master degree. Occupation was assessed according to the International Standard Classification of Occupations code  and clasTo examine the several behavioural pathways between reciprocity defined both at employee- and workplace level and future high sickness absence, three types of mediation models were estimated in two steps. Mplus 7.11  was usedXi)→(Mi)→(Yi). Three fit indices were used to evaluate the models: (1) the chi-square value of model fit; (2) the Root Mean Square Error of Approximation (RMSEA) of the model; and (3) the Comparative Fit Index (CFI) [Recursive path analysis was used as it enables dealing with complex social variables and their interrelations. The starting point for our model trimming approach was a complex just-identified model that tests the theory that health behaviours  explain the prospective relation between individual-level reciprocity and high sickness absence. Therefore, the model included direct and indirect paths from all individual-level reciprocity variables to the sickness absence outcome via the different health behaviour variables. Bootstrapping was used to compute bootstrap standard errors for the indirect effects . The modex (CFI) . Values The implementation of mediational pathways is not straightforward within the conventional approach to multilevel modeling . Buildin2 = 194.70, df = 1, p < 0.001).2 = 14.262, df (8), p = 0.075; CFI = 0.998; RMSEA = .006). Only reciprocity variables that refer to the supervisor were significantly and negatively associated with high sickness absence. Supervisor concern  was the strongest protective factor for sickness absence: for one unit of increase on supervisor concern the odds of sickness absence decreases with 5.6%. The decrease in the odds of sickness absence for supervisor help  was 3.1%. Nevertheless, only colleague interest indirectly related to high sickness absence via smoking behaviour: colleague interest positively related to smoking  and smoking in turn, positively related to high sickness absence . The specific indirect effect of the behavioural smoking pathway was statistically significant at the 0.05 level , and company-level health-related behaviours did not mediate the relation between company-level reciprocity and individual high sickness absence (2→2→1) .The present study demonstrated a relationship between workplace reciprocity and high sickness absence so that higher levels of supervisor reciprocity related to lower future sickness absence. Even though objective follow-up data improve causal inference, without a clear understanding of the underlying mechanisms, it remains a black-box operation. To address this, three types of mediation were tested for multiple health behaviours  in the relation between workplace reciprocity and future high sickness absence. Overall, workplace reciprocity related to sickness absence independently from health behaviours. The finding is in line with Oksanen et al.  who founFurthermore, the multilevel path models (2→1→1 & 2→2→1) showed that individual sickness absence was mainly driven by individual factors. Differences in sickness absence between companies were not attributable to either compositional or contextual differences in reciprocity [The systematic assessment of several (multilevel) multiple mediation models was a major strength of the present study. Our path analysis approach has advantages over the basic mediational analysis framework which involves a simple three variable system . A path However, several limitations of this study are noteworthy. First, although sickness absence was measured during a follow-up period of 12 months, reversed causality is still possible. Second, no objective baseline sickness absence information was available. Third, sickness absence duration, which contains no information about the frequency of absence, was used. However, we performed some additional analysis for sickness absence frequency (≥ 3 episodes). The results only differed from the reported sickness absence duration (≥ 10 days) findings such that high levels of company-level colleague interest decreased sickness absence frequency . Fourth, the study assessed health behaviours with self-reports, which can cause recall, reporting and response bias. Non-response and misclassification are likely to influence different health behaviours to differing degrees. For example, self-reported current smoking is probably more accurate than self-reported alcohol use . Last, wThis study adds to previous research by showing that workplace reciprocity relates to future sickness absence, however mainly independent from health behaviours. Our findings therefore suggest that both these factors may be relevant in terms of employee health, but arguing that health behaviours are the underlying explanatory mechanism between workplace reciprocity and health seems to be a bridge too far.


We present the case of a 42-year-old woman with metastatic synovial sarcoma of parotid origin, treated definitively with chemoradiation, who subsequently developed oligometastatic disease limited to the lungs. She underwent multiple left and right lung wedge resections and left lower lobectomy, followed by right lower lobe stereotactic ablative radiotherapy (SABR), 54 Gy in three fractions to a right lower lobe lesion abutting the chest wall. Two years later, she was treated with cryoablation for a separate right upper lobe nodule abutting the chest wall. Two months later, she presented with acute shortness of breath, pleuritic chest pain, decreased peripheral blood O2 saturation, and productive cough. A computed tomography (CT) scan demonstrated severe chest wall necrosis in the area of recent cryoablation that, in retrospect, also received a significant radiation dose from her prior SABR. This case demonstrates that clinicians should exercise caution in using cryoablation when treating lung tumors abutting a previously irradiated chest wall.Note: Drs. Loo and Shah contributed equally as co-senior authors. As systemic cancer therapies continue to improve, oncologists are seeing more patients with oligometastatic or oligoprogressive disease, where metastatic disease is well controlled except for in discrete locations. For these cases, we have become more comfortable offering focal therapies, such as surgery, stereotactic ablative radiotherapy (SABR), and/or cryoablation. However, the risk of multimodal local tumor therapy is not fully understood in these scenarios. It is also not always well understood how much time and distance is safe between different treatment modalities. Here, we describe a case of a patient who developed severe chest wall necrosis after cryoablation in the setting of prior SABR to a nearby chest wall-abutting nodule. Informed consent was obtained from the patient for this study.A 40-year-old female with a synovial sarcoma of parotid origin developed lung-only oligometastases 17 months after completing her original course of definitive therapy. She underwent chemotherapy, bilateral lung wedge resections, and a left lower lobectomy. Six years after the diagnosis, she presented to our clinic for consideration of stereotactic ablative radiotherapy (SABR) of a right lower lobe oligometastasis abutting the lateral sixth rib. The lesion was round, well-defined, and abutted the right chest wall next to the sixth and seventh ribs Figure . We feltTwo years after SABR, she developed progression of a separate oligometastatic right upper lung nodule. This had been present two years earlier Figure , origina2 89%, requiring intubation. A CT post-procedure showed extensive hemorrhage involving all lobes of the right lung. The patient recovered after a short ICU stay and was discharged home in stable condition on 1.5-2 L nasal cannula oxygen.During the procedure, the patient developed hypoxia with SpOTwo months later, the patient was admitted to the hospital with four days of fever, productive cough, shortness of breath, and right-sided pleuritic chest pain. A CT demonstrated a large cavitary lesion, extending from the right peripheral lung through the adjacent right lateral chest wall at the level of ribs three, four, and five into the musculature, with adjacent lobar consolidation Figure .We monitored the patient closely on telemetry, and administered broad-spectrum antibiotics, nasal cannula oxygen, nebulizers, and chest physical therapy. Subsequent CT scans showed the persistence of the large right lung cavitary lesion extending through the right lateral chest wall, but an improvement of right-sided lobar consolidation. Her respiratory symptoms improved, she became afebrile and was discharged from the hospital in stable condition after a five-day inpatient stay, with her previous baseline requirement of 1.5-2 L nasal cannula oxygen. We considered surgical repair of the large chest wall defect, but with substantial concerns about impaired healing due to prior radiotherapy and cryotherapy. She declined surgery in favor of supportive care. Her respiratory symptoms ultimately worsened, and she died six months later. Studies have shown that SABR of lung tumors near the chest wall can be associated with toxicity, such as chest wall pain and rib fractures -2. HowevSimilarly, severe chest wall toxicity after cryoablation, even of tumors abutting the chest wall, is extremely rare . CryoablIn our patient’s case, since we treated a right lower lobe oligometastasis directly abutting the chest wall with SABR 54 Gy, we elected to use a complementary modality to treat a separate right upper lobe nodule two years later. The new nodule was treated with cryoablation, which was followed by severe chest wall toxicity, including massive soft tissue necrosis. In retrospect, although the right upper lobe lesion treated with cryotherapy was originally far outside the treatment field of the right lower lobe lesion treated with SABR, over time, post-SABR fibrosis and volume loss led to the right upper lobe lesion moving inferiorly and abutting the region of the chest wall within the prior radiation field at the level of ribs five and six Figure . We hypoThere is no standard set of dosimetric or cryotherapy constraints when using these complementary modalities. Thus, it is difficult to predict whether a patient might experience a severe toxicity as our patient unfortunately did. However, it is well-known that radiotherapy impairs wound healing. This case illustrates that we should exercise caution when performing cryoablation in the setting of prior SABR for lesions abutting the chest wall. We cannot exclude the possibility that cryoablation overlapping with less intensive palliative radiotherapy doses could also be toxic.We present here a report of severe chest wall necrosis following cryoablation of a nodule abutting a portion of chest wall that received a significant dose of radiation from a prior course of stereotactic ablative radiotherapy to a separate lesion. We recommend caution when combining these two modalities for treatment of pulmonary tumors abutting the chest wall.


Apis mellifera) and bumblebees (Bombus spec.), revealing – among other taxa – host-specific lactic acid bacteria  that are not found in solitary bees. Here, we characterized the microbiota of three Australian stingless bee species (Apidae: Meliponini) of two phylogenetically distant genera (Tetragonula and Austroplebeia). Besides common plant bacteria, we find LAB in all three species, showing that LAB are shared by honeybees, bumblebees and stingless bees across geographical regions. However, while LAB of the honeybee-associated Firm4–5 clusters were present in Tetragonula, they were lacking in Austroplebeia. Instead, we found a novel clade of likely host-specific LAB in all three Australian stingless bee species which forms a sister clade to a large cluster of Halictidae-associated lactobacilli. Our findings indicate both a phylogenetic and geographical signal of host-specific LAB in stingless bees and highlight stingless bees as an interesting group to investigate the evolutionary history of the bee-LAB association.Bacterial symbionts of insects have received increasing attention due to their prominent role in nutrient acquisition and defense. In social bees, symbiotic bacteria can maintain colony homeostasis and fitness, and the loss or alteration of the bacterial community may be associated with the ongoing bee decline observed worldwide. However, analyses of microbiota associated with bees have been largely confined to the social honeybees ( Mutualistic interactions are widespread in the animal and plant kingdoms and have left their footprints in the evolutionary history of many organisms. One of the most common groups of mutualists associated with multicellular organisms are bacteria, which are particularly prevalent across insects Bees represent an ecologically and economically important group of insects due to their functional role as pollinators in most ecosystems. Lately, they have declined in both abundance and species richness Bombus terrestris, gamma- and betaproteobacterial symbionts convey resistance against an intestinal parasitic protozoan (Crithidia bombi) that negatively impacts the bees' fecundity Bacillus strains, actinomycetes, as well as some fungal associates have been isolated from pollen, honey and nest building material and are thought to protect bee colonies and/or enhance their growth Lactobacillus and Bifidobacterium, have been described for several bee species, including honeybees (Apis mellifera) Xylocopa, halictids, In this context, the microbial community associated with social bees has received considerable attention, and previous studies found a consistent core microbiota across honeybees and bumblebees The microbiota of all social and solitary bees analyzed so far includes widespread LAB that are not host-specific. These LAB are closely related to flower-inhabiting, fructophilic lactobacilli or other lactobacilli found in the environment, which are likely obtained by bees when foraging at flowering plants While the microbial community of honeybees has received considerable attention, much less is known about the microbiota associated with stingless bees (Apidae: Meliponini) but see . StingleAustroplebeia australis, Tetragonula carbonaria, and Tetragonula hockingsii), with particular focus on the bee-associated LAB. According to the hypothesis that host-specific LAB are maintained by obligate colony fissioning , we assume that LAB associated with the Firm3-5 clusters occur across all Meliponini. While the home ranges of the three species overlap broadly Austroplebeia (comprising five species) is endemic to Australia and Papua New Guinea and genetically more closely related to stingless bee lineages from the ancient African clade, whereas the closest relatives of the genus Tetragonula  are found across Southeast Asia Tetragonula collecting Here, we characterized the microbial community of three sympatric Australian stingless bee species  in March 2011 and 2012. All colonies had access to the same resource environment and faced the same ecological conditions, with a mixed rainforest and eucalypt forest as well as gardens included in their foraging range (approximately 500 m radius of the hive).Tetragonula carbonaria, four colonies of Austroplebeia australis and one colony of Tetragonula hockingsii, a species closely related to T. carbonariaSpecimens were collected from five colonies of Taq polymerases (Qiagen). Sequencing extended from Gray28F, using a Roche 454 FLX instrument with Titanium reagents and procedures described at Molecular Research LP . Quality control and analysis of 454 reads was done in QIIME DNA was extracted from six individual worker bees of all ten colonies sampled, respectively, using the MasterPure DNA Purification Kit (Epicentre Technologies) according to the manufacturer's instructions. For each colony, a pooled DNA sample was sent to an external service provider  for bTEFAP with 16S rRNA primers Gray28F (5′-GAGTTTGATCNTGGCTCA-3′) and Gray519R (5′-GTNTTACNGCGGCKGCTG -3′) Lactobacillus sp. as the first BLASTn hit were selected (20 OTUs). The representative sequences for these OTUs were trimmed to 350 bp in order to remove potential low-quality ends that were not detected by the preceding quality-trimming steps (see above). The trimmed reads were combined with the Lactobacillus sequences used in McFrederick et al. For phylogenetic analysis, all OTUs with A. australis, five colonies of T. carbonaria, and one colony of T. hockingsii. In total, 139,771 reads were obtained , 126,919 of which passed quality filtering and chimera screening . Rarefaction analyses indicate that the microbiota associated with the individual colonies was exhaustively sampled, with the possible exception of the T. carbonaria colony H89 , we characterized the microbial communities associated with worker bees from four colonies of lony H89 . Based olony H89 .Ralstonia  was consistently present across all colonies of the three bee species, two OTUs related to the genus Pantoea  were detected in six of the ten colonies across the three species, and one OTU associated with the family Acetobacteriaceae  occurred in all six Tetragonula colonies, but not in A. australis  as well as Beta-, Gamma-, and Alpha-Proteobacteria were the dominant taxa across colonies, but their relative abundance varied considerably within and between species . One OTUustralis . With thLactobacillus. Phylogenetic analyses including 620 additional lactobacilli and outgroup sequences revealed the placement of the OTUs in four clusters  fell within the Firm5 cluster comprising honeybee- and stingless bee-associated LAB. (iii) Five OTUs only found in the two Tetragonula species formed the sister clade to the Firm5 cluster. And (iv) a monophyletic group of nine OTUs that were present in all three stingless bee species investigated in this study formed the sister clade to a large cluster of Halictidae-associated LAB.Among the 241 Meliponini-associated OTUs reported in this study, 20 were identified by RDP classification and BLASTn searches as members of the genus clusters . (i) FivWhile the microbial community of honeybees has been thoroughly investigated, the microbiota associated with stingless bees (Apidae: Meliponini) has only been addressed in a single comparative study with honeybees conducted by Vasquez et al. Ralstonia , which are known as common pathogens Pantoea , which is a genus commonly found on plant roots, leaves Tetragonula colonies  is often found in floral nectar and in the environment of bees  as well as Beta-, Gamma-, and Alpha-Proteobacteria as the dominant bacterial taxa in all three stingless bee species. Samples from all ten colonies contained bacteria related to the genus Tetragonula species contained LAB that are closely related to other stingless bees and honeybee associated LAB, i.e. belong to the host-specific Firm4 and Firm5 clusters. Interestingly, neither our study nor the study of Vasquez et al. A. australis indicates that they are not present in all corbiculate bee species that propagate through colony fissioning as suggested by McFederick et al. Lactic acid bacteria (LAB) were present in nine out of ten colonies, but only the two A. australis and the two Tetragonula species. Despite its phylogenetic affiliation with the Lactobacillus buchneri group, this monophyletic group  may represent a novel host-specific clade of bee-associated LAB.In addition to the Firm4–5 clusters, we identified a novel cluster of LAB that is closely related to Halictidae-associated LAB in both A. australis having secondarily lost the Firm4–5 cluster. This agrees with earlier studies detecting LAB of the Firm4–5 cluster in other stingless bees of the genera Trigona, Melipona, and MeliponulaAustroplebeia with the African genus LisotrigonaConsidering the occurrence of host-specific LAB (particularly Firm4–5) across corbiculate bees, their presence in stingless bees appears to be the ancestral state, with Our analysis of the microbiota associated with three Australian stingless bee species shows that the LAB community associated with stingless bees resembles that associated with honeybees, but lacks LAB of the highly host-specific Firm3 cluster and instead comprises an additional clade of likely host-specific LAB that form a sister clade to a large cluster of Halictidae-associated lactobacilli. This finding suggests that LAB are of similar ecological importance to stingless bees as they are to other corbiculate bees, but that their composition depends on the phylogenetic background and geographic region of their hosts. Therefore, stingless bees represent interesting organisms for understanding the evolutionary history of the bee-LAB association.Figure S1Rarefaction analysis with the sequencing data for 10 colonies belonging to three different species of Australian stingless bees. Different numbers denote different bee colonies. Aaus  =  Austroplebeia australis, Tcar  =  Tetragonula carbonaria, Thoc  =  Tetragonula hockingsii.(TIF)Click here for additional data file.Table S1Abundance of 241 operational taxonomic units (OTUs) across ten colonies of three different Australian stingless bee species . Taxonomic assignment was done with the RDP classifier based on the representative sequences for the OTUs. OTUs associated with the genus Lactobacillus (BLASTn results) are highlighted in bold print.(XLSX)Click here for additional data file.Data S1Representative sequences for 241 OTUs identified across ten colonies of three different Australian stingless bee species. Each sequence is identified by the OTU number, the species and colony it was found in , and a unique sequence identifier. If an OTU was detected in multiple colonies, only one colony/species is indicated.(FASTA)Click here for additional data file.Data S2Alignment of 656 lactobacilli and outgroup sequences used for the phylogenetic analyses of Meliponini-associated lactic acid bacteria. The sequences obtained in this study were combined with the Lactobacillus sequences used in McFrederick et al. (FASTA)Click here for additional data file.


Intrapartum-related events are the third leading cause of childhood mortality worldwide and result in one million neurodisabled survivors each year. Infants exposed to a perinatal insult typically present with neonatal encephalopathy (NE). The contribution of pure hypoxia-ischaemia (HI) to NE has been debated; over the last decade, the sensitising effect of inflammation in the aetiology of NE and neurodisability is recognised. Therapeutic hypothermia is standard care for NE in high-income countries; however, its benefit in encephalopathic babies with sepsis or in those born following chorioamnionitis is unclear. It is now recognised that the phases of brain injury extend into a tertiary phase, which lasts for weeks to years after the initial insult and opens up new possibilities for therapy.There has been a recent focus on understanding endogenous neuroprotection and how to boost it or to supplement its effectors therapeutically once damage to the brain has occurred as in NE. In this review, we focus on strategies that can augment the body's own endogenous neuroprotection. We discuss in particular remote ischaemic postconditioning whereby endogenous brain tolerance can be activated through hypoxia/reperfusion stimuli started immediately after the index hypoxic-ischaemic insult. Therapeutic hypothermia, melatonin, erythropoietin and cannabinoids are examples of ways we can supplement the endogenous response to HI to obtain its full neuroprotective potential. Achieving the correct balance of interventions at the correct time in relation to the nature and stage of injury will be a significant challenge in the next decade. Intrapartum-related insults at full term such as hypoxia-ischaemia (HI) are the third leading cause of global child deaths.5NE is a descriptive term for neurological dysfunction in the newborn infant, manifested by symptoms including difficulty with initiating and maintaining respiration, depression of tone and reflexes, subnormal level of consciousness, poor feeding and seizures.The clinical signs of NE progress after a latent period of hours to days. This timing of the evolution of clinical signs is thought to reflect brain energy levels and the cascade of neurochemical processes responsible for brain injury. These are summarised in 2+ concentrations arise through several mechanisms, including overactivation of glutamate receptors ), other channels and transporters, or through release from internal stores through physical damage to mitochondria and endoplasmic reticulum2+ triggers many downstream neurotoxic cascades. As well as generating an osmotic gradient that leads to oedema and lysis of cells, Ca2+ activates nitric oxide synthase, which in turn generates high levels of the toxic reactive oxygen species nitric oxide (NO•). At high concentrations, NO• reacts with superoxide (O•−) to produce peroxynitrite (ONOO−), which damages mitochondria via peroxidation and nitrosylation of membrane lipids. Consequently, mitochondrial dysfunction and membrane depolarisation develop with further release of O•− and decline in endogenous anti-oxidants such as glutathione. Ca2+ also triggers the activation of cytosolic phospholipases, which increase eicosanoid release leading to inflammation.During the acute hypoxic-ischaemic insult, some cells undergo primary cell death, the magnitude of which will depend on the severity and duration of HI. In the absence of substrates , the neuron's supply of high-energy metabolites such as ATP falls below a critical threshold. The Na+/K+ ATP-dependent pump begins to fail, neuronal depolarisation occurs and the synaptic cleft floods with glutamate, which activates the N-methyl-D-aspartate (NMDA) receptor. Toxic cytoplasmic CaAfter reperfusion, the initial hypoxia-induced cytotoxic oedema and accumulation of excitatory amino acids partially resolve in 30–60 min, with apparent recovery of cerebral oxidative metabolism. It is thought that the neurotoxic cascade is largely inhibited during the latent phase, when there is endogenous inhibition of oxidative metabolism and increased tissue oxygenation.1431P) MRS have demonstrated the deterioration in cerebral oxidative metabolism 6–24 h after HI (termed secondary energy failure)  . DespiteThis secondary phase is marked by the onset of seizures, secondary cytotoxic oedema, accumulation of cytokines and mitochondrial failure . MitochoThere is evidence that active pathological processes occur for weeks, months and years after a hypoxic-ischaemic insult; this has been termed tertiary brain injury.Brain damage and lasting functional impairment after NE are the results of a balance between injurious mechanisms  and endogenous protection . Optimal therapy will demand exploitation of multiple pathways that prevent brain cell death and promote repair.We discuss five interventions whose actions include the augmentation of the endogenous neuroprotective response. Birth asphyxiated babies have an endogenous cooling response;http://www.nice.org.uk/guidance/ipg347), therapeutic hypothermia is now standard clinical care for moderate-to-severe NE in the UK and high-income countries.5For over 50 years, it has been known that babies with birth depression have an endogenous cooling response.et alet al2 consumption and CO2 production, reduced loss of high-energy phosphates during HI and during secondary cerebral energy failure, reduced excitotoxicity, reduced reactive oxygen species production, protein synthesis preservation, decreased oedema, modulation of the inflammatory cascade and a change in pro-apoptotic and anti-apoptotic signalling.34–36Pathways underpinning hypothermic neuroprotection are covered in detail in recent reviews by Wassink In intensive care settings, clinical trials have included whole body cooling with core temperature reduced to 33.5°C for 72 h40There is clear evidence that therapeutic hypothermia as a therapy for moderate-to-severe NE reduces adverse outcome  at 18 months of age Recently, therapeutic hypothermia has been shown to be ineffective and even harmful in the presence of infection/inflammation in adult clinical studies.et al,Brain injury and hypothermia both alter immune responsiveness. Following HI, a bidirectional communication between the injured brain and the peripheral innate and adaptive immune system regulates the progression of both ischaemic pathology and tissue repair . Use of a remote limb makes RIPostC a feasible clinical treatment strategy for NE.Ischaemic RIPostC has been shown to protect the adult and neonatal brain in rodent models of stroke. The protective mechanisms of RIPostC are incompletely understood, but are thought to involve three intimately inter-related pathways initiated by the release of a number of endogenous autocoids  from the ischaemic skeletal muscle. These pathways are (i) the neuronal pathway; (ii) the humoral pathway and (iii) the systemic response .64 AnimaIn brief, the neuronal pathway describes the autocoid-mediated stimulation of local afferent nerves that effect remote protection via efferent nerves, including the autonomic nervous system.69Following remote ischaemic stimulus, these three pathways converge in the brain to increase cerebral blood flow, attenuate neuroinflammation and at a cellular level to activate pro-survival signalling cascades, including genetic and epigenetic modulation. Ultimately these processes protect mitochondrial integrity, reduce energy demands, increase cell survival and promote repair mechanismsIn neonatal75In our large animal (piglet) model of perinatal asphyxia, we found that four 10 min cycles of ischaemia/reperfusion of both lower limbs, starting immediately following resuscitation, provided protection in the white matter,77RIPostC has been explored in clinical settings for conditions, including cardiac disease and stroke.81Clinical trials are needed to establish whether RIPostC is safe and protective in NE. It will be important to address the safety and reproducibility of inducing intermittent limb ischaemia, with and without concomitant cooling therapy. There remain difficult hurdles such as the dose–response of RIPostC , the therapeutic time windows and the precise protective mechanisms.82Melatonin is a naturally occurring neuroendocrine molecule secreted in response to environmental light–dark cycles. Melatonin is both lipophilic and hydrophilic. It easily crosses biological membranes and acts via receptor-dependent and receptor-independent processes to modulate cell signalling and gene expression.Endogenous melatonin is integral to normal neurodevelopment and protects the developing brain from injury.Acting via specific cell membrane and nuclear receptors, melatonin achieves powerful neuroprotective effect via anti-oxidant, anti-apoptotic and anti-inflammatory processes116–121Further, melatonin's wide-ranging immune-modulating propertiesMelatonin is an extremely safe neurotherapeutic. No study of antenatal or postnatal melatonin treatment has shown any serious side effects,132Endocannabinoids are emerging as a potential neurotherapeutic for NE. The endocannabinoid system is a neuromodulatory system that participates in a wide range of physiological processes in mammals.145Endocannabinoids modulate the intensity and extension of neurotoxic processes168http://www.clinicaltrials.gov). A previous phase III randomised controlled trial of 861 adult patients given dexanabinol as a neuroprotectant following traumatic brain injury showed that dexanabinol was safe but not efficacious.The main established clinical uses of cannabinoids are for chronic pain, for muscle spasms and as an appetite stimulant.et alReported side effects of cannabinoids have all been mild and transient, including sedation, anxiety, dizziness and nausea.Epo is a pleiotropic cytokine with multiple roles in addition to that of a haemopoietic growth factor. As with melatonin and cannabinoids, the role of Epo in normal brain development and neuroprotection is becoming clear  for treatment of NE found no deaths and only one infant with moderate–severe disability at age 2 years.Perinatal HI leading to NE sets up a cascade of processes that lead to an evolving brain injury, which includes a latent phase, secondary energy failure phase and tertiary brain injury phase. Coexisting infection/inflammation may exacerbate this injury. The brain mounts a potent, though only partially successful, defensive response against many of the deleterious secondary mechanisms of injury. Therapies that augment the endogenous neuroprotective response such as RIPostC are attractive but need further study to define optimal protocols. Part of the endogenous neuroprotective response is lowering of the core body temperature as well as increased melatonin, Epo and cannabinoid levels. Augmenting these endogenous responses have shown protection in preclinical studies and therapeutic hypothermia is now a routine therapy for moderate-to-severe NE. Clinical trials are now ongoing for Epo-augmented hypothermia. We anticipate that future newborn brain protection will comprise a tailored combination of therapies; the challenge will be to ensure the timing and dose of each neuroprotectant are appropriate for the phase of injury to ensure optimal and lasting protection.


Reconstruction of large bone defects after resection of malignant musculoskeletal tumors is a significant challenge in orthopedic surgery. Extracorporeal autogenous irradiated bone grafting is a treatment option for bone reconstruction. However, nonunion often occurs because the osteogenic capacity is lost by irradiation. In the present study, we established an autogenous irradiated bone graft model in the rat femur to assess whether osteogenic matrix cell sheets improve osteogenesis of the irradiated bone. Osteogenic matrix cell sheets were prepared from bone marrow-derived stromal cells and co-transplanted with irradiated bone. X-ray images at 4 weeks after transplantation showed bridging callus formation around the irradiated bone. Micro-computed tomography images at 12 weeks postoperatively showed abundant callus formation in the whole circumference of the irradiated bone. Histology showed bone union between the irradiated bone and host femur. Mechanical testing showed that the failure force at the irradiated bone site was significantly higher than in the control group. Our study indicates that osteogenic matrix cell sheet transplantation might be a powerful method to facilitate osteogenesis in irradiated bones, which may become a treatment option for reconstruction of bone defects after resection of malignant musculoskeletal tumors. Reconstruction of large bone defects after malignant musculoskeletal tumor resection is a significant challenge in orthopedic surgery. Several procedures have been attempted for reconstruction of such bone defects, including conventional autogenous bone grafting , vasculaRecently, intraoperative extracorporeal autogenous irradiated devitalized bone grafting has become a treatment option for reconstruction of bone defects after malignant musculoskeletal tumor resection , 7. SurgWe previously reported a cell transplantation method in which bone marrow-derived stromal cells (BMSCs) are cultured and lifted as a cell sheet structure. The cell sheet has an osteogenic potential and is therefore designated as an osteogenic matrix cell sheet , 10. OstIn the present study, we established bone defects in the femurs of rats, which were reconstructed using autogenous irradiated bone grafts together with transplantation of an osteogenic matrix cell sheet. The aim of the present study was to evaluate whether the osteogenic matrix cell sheet enhances bone union in such a model.The experimental procedures were approved by the Animal Experimental Review Board of Nara Medical University. Fisher 344 rats were purchased from Japan SLC . Seven- and 12-week-old male rats were used to prepare bone marrow cells and establish the autogenous bone graft model, respectively.2 culture flasks  containing 15 mL minimal essential medium  with 15% fetal bovine serum  and antibiotics . The cells were cultured in an incubator at 37°C with 5% CO2. At confluency, the primary cultured cells were trypsinized from the flasks using trypsin/EDTA .The method for BMSC preparation has been reported previously , 11. Bri4 cells/cm2 in 10 cm dishes  containing medium with 10 nM dexamethasone  and ascorbic acid phosphate  and cultured to confluency (approximately 12–14 days). The cells were rinsed twice with phosphate-buffered saline , and then the osteogenic matrix cell sheet was lifted by a scraper , which was inserted into the intramedullary femur shaft from the distal femur condyle in a retrograde fashion. Subsequently, two osteogenic matrix cell sheets were wrapped around the grafted bone without suturing (sheet group). The transplanted osteogenic matrix cell sheets filled the gap between the grafted bone and host bone . The conX-ray images were taken under anesthesia at 4, 8, and 12 weeks postoperatively to evaluate bridging bone formation around the grafted devitalized bone. Rats were sacrificed and right hind limbs were harvested at 12 weeks postoperatively . After rTwo of the harvested femurs in each group were dissected from the surrounding muscle, fixed in 10% formalin, and embedded in methyl methacrylate resin. For histology, the femurs were cut longitudinally and subjected to von Kossa and Villanueva-Goldner staining. In von Kossa staining, calcified bone is represented as black. In Villanueva-Goldner staining, calcified bone is represented as green and an osteoid is represented as red.Eight of the harvested femurs in each group were applied to mechanical testing (three-point bending test) using a universal testing machine equipped with a computer for data acquisition . As shown in U test. A value of P < 0.01 was considered to be statistically significant.Statistically significant differences in the mechanical test analyses were determined by the Mann-Whitney X-ray images taken at 4 weeks after transplantation showed obvious callus formation around the grafted bone in the sheet group. Callus formation had developed during the experimental period and bridging bone formation with the host bone was observed at 12 weeks postoperatively, resulting in bone union. In contrast, the control group showed no bridging callus formation around the grafted bone even at 12 weeks .Micro-CT images recorded at 12 weeks postoperatively showed bridging bone formation around the grafted bone in the sheet group. In contrast, the control group exhibited no bridging bone formation around the grafted bone even at 12 weeks , which wRepresentative histological sections of the harvested femur showed new bone formation around the grafted bone in the sheet group. In Villanueva-Goldner stained sections, grafted bone with empty lacunae and the host femur were united by the newly formed bone. Although there was no obvious remodeling at 12 weeks postoperatively, the grafted bone appeared to be stabilized by the newly formed bone in the gap between grafted and host bones as well as around the whole circumference of the graft. In contrast, no bone formation was observed around the grafted bone in the control group. Soft tissue interposition was observed between grafted and host bones, which was consistent with the nonunion findings .In the three-point bending test, the median failure forces at the site of the junction between the grafted bone and host femur were 101.2 N (41.7–233.5) and 14.2 N (0−70.3) in the sheet and control groups, respectively.P = 0.004) (The failure force in the sheet group was significantly higher than that in the control group (= 0.004) .The present study clearly demonstrated that osteogenic matrix cell sheets can facilitate the osteogenesis of autogenous irradiated devitalized bone grafts. By combining the devitalized bone graft with an osteogenic matrix cell sheet, bone union was achieved at 12 weeks postoperatively. Although remodeling of the bone graft was not observed until 12 weeks, we observed bridging bone formation over the grafted bone between both ends of the autogenous bone, resulting in stability between the grafted bone and host bones. Mechanical testing revealed significantly higher strength in the sheet group, indicating that transplantation of devitalized bone grafts with osteogenic matrix cell sheets can be useful for bone reconstruction after musculoskeletal tumor resection.In clinical cases of bone defects after musculoskeletal tumor resection, intraoperative extracorporeal autogenous irradiated bone grafting is one of the bone reconstruction methods. The method consists of a wide en bloc resection of the tumor, curettage of the tumor, extracorporeal irradiation with 50 Gy, and reintroduction of the irradiated bone into the host with fixation devices. The problem with this reconstruction method is that the osteogenic capacity is lost by irradiation and nonunion or delayed union often occurs . Therefo in vitro culture in osteoinductive medium containing Dex, Asap, and β-glycerophosphate [ de novo formed bone in the transplantation model of osteogenic matrix cell sheets using the sex-determining region Y (Sry) gene as a marker of donor cells. Sry was detected in the de novo formed bone, indicating that the cells in the transplanted osteogenic matrix cell sheet without a scaffold could survive at the fracture site and differentiate into osteogenic lineage cells in vivo [BMSCs have a self-renewal potential and can differentiate into osteoblasts, adipocytes, chondrocytes, neurons, and myogenic cells , 13, 15.hosphate –18. BMSChosphate , 19, 20.hosphate . Our cel in vivo . de novo formed bone in the junction. This union may be achieved by the fact that the osteogenic matrix cell sheets are pulpy and able to enter the gaps, that is, bone fracture sites, nonunion sites, and bone-grafted sites. Cell sheet transplantation prevented interposition of soft tissue into the gaps between the graft and host bones. Subsequently, bone tissue was formed in the gap as well as around the whole circumference of the graft. Therefore, the present study indicates that transplantation of an osteogenic matrix cell sheet provides an osteoinductive matrix as well as osteogenic cells, resulting in facilitation of osteogenesis.We used irradiated devitalized bone as a graft in the present study. Therefore, the outcome of the graft and host bones might easily be nonunion because of the interposition of soft tissue in the junctions between them. However, we observed bridging bone formation over the grafted bone between both ends of the host bones as well asMoreover, the osteogenic matrix cell sheet produces angiogenic factors, such as vascular endothelial growth factor A (VEGF-A), thereby promoting vessel sprouting from the existing vasculature and blood flow around the devitalized bone. Osteogenic matrix cell sheet transplantation can be applied to not only bone fractures but also nonunion and necrotic bone sites. Therefore, we believe that cell sheet transplantation can contribute to bone reconstruction in cases involving not only nonunion but also irradiated bone grafts.The present study has some limitations. First, we used an irradiated bone model for our analyses. Clinically, intraoperative extracorporeal bone devitalized with liquid nitrogen is also used for reconstruction of bone defects after resection of malignant musculoskeletal tumors, because devitalization by liquid nitrogen can preserve the cartilage matrix and sufficient biomechanical strength –23. HereOur study clearly indicates that osteogenic matrix cell sheet transplantation facilitates osteogenesis in irradiated devitalized bone grafts. Therefore, transplantation of autogenous irradiated bone grafts with osteogenic matrix cell sheets may become a treatment option for reconstruction of bone defects after resection of malignant musculoskeletal tumors.