Indeed, it is established that the ubiquitously expressed JNK1 and JNK2 isoforms regulate energy expenditure and insulin resistance. However, the role of the neuron-specific isoform JNK3 is unclear. Here we demonstrate that JNK3 deficiency causes hyperphagia selectively in high fat diet (HFD)-fed mice. JNK3 deficiency in neurons that express the leptin receptor LEPRb was sufficient to cause HFD-dependent hyperphagia. Studies of sub-groups of leptin-responsive neurons demonstrated that JNK3 deficiency in AgRP neurons, but not POMC neurons, was sufficient to cause the hyperphagic response. These effects of JNK3 deficiency were associated with enhanced excitatory signaling by AgRP neurons in HFD-fed mice. JNK3 therefore provides a mechanism that contributes to homeostatic regulation of energy balance in response to metabolic stress.The cJun NHDOI:http://dx.doi.org/10.7554/eLife.10031.001 Consuming the right amount of food is important for health. Eating too little for a long time causes damage to organs, and overeating can cause harm as well, in the form of conditions such as obesity and type 2 diabetes. Several signaling molecules and brain regions are linked to controlling food consumption and ensuring the body receives the correct amount of nutrients to fuel its activities.Previous studies have found that two proteins called JNK1 and JNK2, which are found in most tissues of the body, can reduce how much energy cells use. This can trigger insulin resistance and fat accumulation, and so suggests that blocking the activity of these proteins may help to treat type 2 diabetes and obesity. However, the role of another JNK protein – JNK3, which is mostly found in the brain – was not known.Now, Vernia, Morel et al. have investigated the role of JNK3 in metabolism. It was found that JNK3 reduced the amount of food consumed by mice provided with a cafeteria (high fat) diet. Mice that lacked JNK3 ate far more food and gained more weight on a high fat diet than normal mice. However, JNK3 played no role in food consumption when mice were fed a standard chow diet. Treating normal mice with leptin – an appetite-suppressing hormone – caused them to lose weight, but did not affect mice that lacked JNK3.Examining the brains of the mice revealed that in normal mice, JNK3 in a specific sub-population of neurons decreases the production of proteins that promote eating. However, the proteins continued to be produced in mice that lacked JNK3, encouraging overeating.Overall, the results suggest that blocking the activity of all the JNK proteins will not help treat obesity and diabetes as shutting down JNK3 could encourage overeating. Therefore, future investigation into treatments for these conditions should focus on drugs that specifically target JNK1 and JNK2, and not JNK3.DOI:http://dx.doi.org/10.7554/eLife.10031.002 The regulation of energy balance (food consumption and energy expenditure) is important for health and survival. Sustained negative energy balance caused by cachexia and anorexia is associated with serious injury to multiple organ systems . SimilarIt is established that the arcuate nucleus (ARC) in the hypothalamus plays a key role in the regulation of energy balance . AgRP neAgRP and POMC neurons integrate signals from nutrients (e.g. glucose and fatty acids) and peripheral hormones  to mediate opposite actions regulating downstream neuroendocrine circuits linking internal and environmental stimuli with the coordinated control of homeostatic satiety . Thus, lThe anorexigenic hormone leptin plays a key role in the regulation of food consumption. Leptin can act directly on AgRP and POMC neurons, but leptin can also act on other neurons in several brain sub-regions, including mid-brain and brainstem nuclei . Control2-terminal kinase (JNK) signaling pathway regulates feeding behavior. Previous studies have established that the ubiquitously expressed JNK1 and JNK2 isoforms play an important role in the metabolic stress response of peripheral tissues (Mapk10 gene) plays a key role in the maintenance of energy balance during consumption of a high fat diet (HFD) by promoting leptin signaling. Mapk10 gene ablation studies identify AgRP neurons as a site of JNK3 function. JNK3 is therefore a key mediator of homeostatic regulation of energy balance in response to metabolic stress.The purpose of the study reported here was to test whether the cJun NH tissues . HoweverLeptin is an anorexigenic hormone. Indeed, treatment of chow-fed mice with leptin suppressed feeding behavior and caused decreased body mass . In contWe considered the possibility that a stress-activated MAP kinase pathway may contribute to the regulation of leptin signaling in HFD-fed mice. It is established that feeding a HFD causes activation of the ubiquitously expressed isoforms JNK1 and JNK2 in peripheral tissues, including liver, muscle, and adipose tissue . However-/-Mapk10 (JNK3-deficient) mice. We found that -/-Mapk10 mice gained similar body mass when fed a CD, but these mice gained significantly greater mass when fed a HFD compared with WT mice  or a HFD to wild-type (WT) mice or  WT mice . 1H-MRS ean mass . Indeed, WT mice . Microsc WT mice .Mapk10 gene ablation selectively increased consumption of a HFD, but not a CD , reduced glucose turnover, reduced whole body glycolysis, increased hepatic glucose production, and decreased hepatic insulin action compared with HFD-fed WT mice  & Cd68), increased expression of genes associated with M1-like macrophage polarization , and decreased expression of genes associated with M2-like macrophage polarization  in the adipose tissue of HFD-fed -/-Mapk10 mice compared with HFD-fed control mice  mice and Loxp/LoxPLeprb-cre Mapk10 (LepR∆J3) mice gained similar body mass when fed a CD. However, HFD-fed LepR∆J3 mice gained significant more body mass than LepRWT mice  LepRolerance , increasolerance , increasolerance , increasolerance , and incolerance . White aRWT mice . Moreoveteatosis .+ neuronal sub-population relevant to JNK3-regulated HFD feeding behavior, we examined Mapk10 gene ablation in selected neurons within the hypothalamus. Gene expression analysis demonstrated that JNK3 was required for HFD-induced regulation of Agrp and Npy, but not Pomc (Loxp/LoxPAgrp-cre Mapk10 (Agrp∆J3) mice and Loxp/LoxPPomc-cre Mapk10 (Pomc∆J3) mice. We found that JNK3 deficiency in POMC neurons of HFD-fed mice caused no significant changes in feeding behavior, glucose intolerance, blood glucose concentration, hypertrophy of white and brown adipocytes, and hepatic steatosis compared with control Pomc-cre (PomcWT) mice (Agrp-cre (AgrpWT) mice  mice . In contWT) mice . Metabolrol mice . Togethe-/-Mapk10 mice. This analysis demonstrated that JNK3 deficiency caused no change in mIPSC frequency or amplitude in CD-fed and HFD-fed mice  in neurons have been examined. This analysis demonstrated that compound JNK-deficiency caused markedly increased survival responses associated with increased autophagy  hyperphagia was found in mice with JNK3 deficiency in AgRP neurons, but not POMC neurons  mice do not exhibit altered feeding behavior  in WT mice would cause a small change in feeding behavior. However, the pro-apoptotic function of this activated Mapk8 allele  and cause insulin resistance in peripheral tissues indicates that drugs that block JNK signaling may be therapeutically beneficial for the treatment of pre-diabetes . However-/-Mapk10 mice previously (tm1(FLP1)DymGt(ROSA)26Sor/RainJ (tm2(cre)RckLepr/J mice (tm1(cre)LowlAgrp/J mice . Homologous recombination of the 3’ arm of the targeting vector was verified by PCR using the primers (3F: 5’-CTGAGTGACGTGTGGAG-3’ and 5R: 5’-TCATTGGGTTGGGATATTC-3’) followed by digestion with +XhoI . Cre-mediated recombination between the LoxP sites was detected by PCR using the primers 1F and 4R: 5’-GATTCTCCCTGTCTGAG-3’ . The Mapk10LoxP/LoxP mice were routinely genotyped by PCR using primers 1F and 2R .We established 1H-MRS . The mice were housed in a facility accredited by the American Association for Laboratory Animal Care (AALAC). The Institutional Animal Care and Use Committee (IACUC) of the University of Massachusetts and the University of Cincinnati approved all studies using animals.Male mice (8 wks old) were fed a chow diet  or a HFD  for 4 to 12 wks. Body weight was measured on a weekly basis and whole body fat and lean mass were non-invasively measured using The clamp studies were performed at the National Mouse Metabolic Phenotyping Center at the University of Massachusetts Medical School. A 2 hr hyperinsulinemic-euglycemic clamp was conducted using overnight fasted conscious mice with a primed and continuous infusion of human insulin , and 20% glucose was infused at variable rates to maintain euglycemia .The analysis was performed by the National Mouse Metabolic Phenotyping Centers at the University of Massachusetts Medical School and the University of Cincinnati. The mice were housed under controlled temperature and lighting with free access to food and water. The food/water intake, energy expenditure, respiratory exchange ratio, and physical activity were measured using metabolic cages .rd ventricle . Mice were monitored daily and allowed to recover for 1 week after surgery. Mice received either solvent  or Leptin (5 µg) in 2 µl delivered over 10 min. Leptin treatment by intraperitoneal (ip) injection was performed following 3 consecutive days of sham injection.Intracerebroventricular treatment with leptin was performed using mice with a cannula stereotaxically implanted into the 3Adipoq (Mm00456425_m1), Agrp (Mm00475829_g1), Arg1 (Mm00475988_m1), Ccl2 (Mm00441242_m1), Emr1 (F4/80) (Mm00802530_m1), Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), Mapk8 (Jnk1) (Mm00489514_m1), Mapk9 (Jnk2) (Mm00444231_m1), Mapk10 (Jnk3) (Mm00436518_m1), Mgl2 (Mm00460844_m1), Mrc1 (Mm00485148_m1), Mrc2 (Mm00485184_m1), Npy (Mm03048253_m1), Pomc (Mm00435874_m1), and Tnf (Mm00443258_m1). The relative mRNA expression was normalized by measurement of the amount of 18S RNA in each sample using Taqman© assays .Tissue isolated from mice starved overnight was used to isolate total RNA using the RNAeasy mini kit (Qiagen). Total RNA (500 ng) was converted into cDNA using the high capacity cDNA reverse transcription kit . The diluted cDNA was used for real-time quantitative PCR analysis using a Quantstudio PCR PCR machine (Life Technologies). TaqMan assays (Life Technologies) were used to quantify Blood glucose was measured with an Ascensia Breeze 2 glucometer . Adipokines and insulin in plasma were measured by multiplexed ELISA using a Luminex 200 machine .Glucose and insulin tolerance tests were performed by intraperitoneal injection of mice with glucose (1 g/kg) or insulin (1.5 U/kg) using methods described previously .Mice (8–12 week-old) were fasted overnight. Hypothalamic extracts were prepared using Triton lysis buffer . Extracts (30–50 µg of protein) were examined by immunoblot analysis by probing with antibodies to JNK3  and GAPDH . Activated JNK was isolated by immunoprecipitation with the mouse monoclonal p-JNK antibody G9  pre-bound to protein G Sepharose  and detected by immunoblot analysis by probing with an antibody to JNK3 . Immunocomplexes were detected by fluorescence using anti-mouse and anti-rabbit secondary IRDye antibodies  and quantitated using the Li-COR Imaging systemHistology was performed using tissue fixed in 10% formalin for 24 h, dehydrated, and embedded in paraffin. Sections (7 µm) were cut and stained using hematoxylin & eosin . Paraffin sections were stained with an antibody to F4/80  that was detected by incubation with anti-rabbit Ig conjugated to Alexa Fluor 488 (Life Technologies). DNA was detected by staining with DAPI (Life Technologies). Fluorescence was visualized using a Leica TCS SP2 confocal microscope equipped with a 405 nm diode laser .2, 5% CO2) high sucrose solution . Then, 300 µm thick coronal sections were cut with a Leica VT1000S Vibratome and incubated in oxygenated aCSF  at 34°C for 30 min. The slices were maintained and recorded at room temperature (20–24°C). The intracellular solution for voltage clamp recording contained the following: 140 mM CsCl, 1 mM BAPTA, 10 mM HEPES, 5 mM MgCl2, 5 mM Mg-ATP, and 0.3 mM Na2GTP, pH 7.35 and 290 mOsm.Brain slice preparations were performed using 8–10-weeks-old mice anaesthetized with isoflurane before decapitation and removal of the entire brain. The brains were immediately submerged in ice-cold, carbogen-saturated  were recorded in the presence of tetrodotoxin (1 µM) and picrotoxin (100 μM) in whole cell voltage clamp mode. To record miniature inhibitory postsynaptic currents (mIPSCs), the neurons were recorded in the presence of TTX and kynurenic acid (1 mM). The membrane potential was clamped at −60 mV. All recordings were made using a Multiclamp 700B amplifier, and data were filtered at 1.4 kHz and digitized at 20 kHz. Data was analyzed using Clampfit 10.2 and Origin Pro 8.6.Differences between groups were examined for statistical significance using the Student’s test or analysis of variance (ANOVA) with the Fisher’s test. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your submission has been favorably evaluated by Tony Hunter (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "JNK3 participates in an AgRP/NPY neuron circuit that suppresses feeding behavior in response to metabolic stress" for peer review at The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:All the reviewers agree that this manuscript does a fine job of demonstrating that JNK3 function is important for body weight regulation and that its function in AgRP-expressing neurons is particularly important. However, the mechanism(s) by which JNK3 acts within AgRP neurons is not sufficiently developed. In particular the deficiency in Lepr signaling seems unlikely.Essential revisions:eLife, the authors need to delineate the mechanism by which loss of JNK3 signaling in AgRP neurons promotes obesity. The reviewers have suggested several approaches to address this issue. Alternatively, the authors may decide that publishing the more descriptive part of the study  but not on a normal (control diet). The obesity is associated with elevated leptin, elevated insulin, glucose intolerance, insulin resistance, and many other parameters associated with obesity. The most important contribution is their systematic demonstration that the phenotype of global KO of the gene encoding JNK3 is replicated quite well by KO only in AgRP neurons. The authors go on to explore how loss of JNK3 function in AgRP neurons might lead to the hyperphagic obese phenotype, but those studies are less compelling.1) The authors show that leptin receptors can be activated (phosphorylated) in the JNK3 KO but the dose-response is apparently shifted – LepR is fully activated (phosphorylated) with 2.5 mg/kg leptin but only partially activated with 0.1 mg/kg . They attribute this hypo-sensitivity to a reduced level of Lepr mRNA . They show that phospho-cJun (transcription factor) normally binds to an AP1 site in the promoter of Lepr gene and hence the lack of phosphorylation of c-Jun could impair transcription of Lepr gene. There are two problems with this analysis: (a) The Lepr mRNA (rather than protein) measurements were made in hypothalamic samples, not in AgRP neurons where it is relevant. (b) Complete loss of Lepr in AgRP neurons has a relatively small effect on obesity and heterozygous Lepr db/+ mice  do not become more obese on a HFD than WT mice. Thus, it seems unlikely that reduced levels of LepR account for the obesity observed in this JNK3 KO model.before they become obese. The experiment in 2) Their data in Reviewer #2:Vernia and colleagues present an interesting paper in which the conclusion are based on a broad set of experiments and well justified. They show that the loss of the neuronally expressed JNK3 is associated with increased obesity due to increased food consumption. This is brought about by the elimination of JNK3 in leptin receptor expressing neurons. They narrow this down further by doing an AgRP-specific elimination and compare that to a POMC neuronal-specific elimination. They demonstrate hyperphagia to be present in only Agrp-specific nulls for JNK3. They go on to demonstrate that this is in fact due at least in part due to reduced leptin receptor expression on AgRP-neurons. All of this happens exclusively in the context of high fat diet exposure.These are interesting findings that should be of broad interest to the field. It is surprising to find JNK3 necessary for leptin receptor expression and to see it distinct from the role of JNK1/2 in the periphery where their lack results in improvement in the metabolic phenotype due to reduced inflammatory tone.There is very little to criticize here. The authors address an important question, find a surprising answer of broad interest and base their conclusion on a broad set of genetic data.lepR expression in AgRP neurons, and it does not lead to reduced lepR expression in POMC neurons?The only area that falls short is a further analysis of the signaling mechanisms in place. What other component of leptin signaling are affected by the lack of JNK3? The downstream analysis could be broader. Where does the cell specificity come in? How does JNK3 deficiency lead to reduce Reviewer #3:In this manuscript Vernia et al. show that JNK3 plays a role in inhibiting high-fat diet-induced weight gain and does so through actions at AgRP/NPY neurons, which may include bolstering leptin actions. Overall, the metabolic studies convincingly show that JNK3 deficiency increases HFD-induced weight gain, hyperphagia, adipose hypertrophy, glucose intolerance and insulin resistance. The logical set of cell-type-selective JNK3 deletions provides compelling evidence that JNK3 actions specifically in AgRP neurons are necessary to reduce HFD-induced weight gain. Thus, the overall conclusion is well supported by the data presented in The primary weakness in the work is the cursory attempt to provide some mechanistic insight . The mEPSC recordings add very little to the paper as is. It is important to know that mIPSCs don't also increase and how the deletion affects basal regulation of POMC neurons. In Figure 7, the overall JNK3 knockout was used and thus, it is not possible to determine if leptin receptor expression is also reduced in POMC cells or rather, if maybe POMC cells do not express JNK3 or require JNK3 to bolster leptin sensitivity. In my opinion, these last 2 experiments do not need to be included to support the conclusions made, but if they are included, they need to be done more thoroughly so that they can be better interpreted.JNK3 is the neuron-specific isoform of JNK and its activity is dynamically regulated rather than constitutive like the other 2 isoforms. Together with the compelling evidence provided here that JNK3 helps reduce high-fat diet-induced weight gain through actions in AgRP neurons, this work adds significant new insight into JNK3 function and the hypothalamic regulation of energy balance in the face of metabolic challenge.2-terminal kinase 3 in response to metabolic stress" for consideration by eLife. Your article has been evaluated by a Reviewing Editor and overseen by Tony Hunter as the Senior Editor.Thank you for submitting your work entitled "Excitatory transmission onto AgRP neurons is regulated by cJun NHNpy and Agrp expression and enhance feeding behavior makes sense, especially because the previous work from the Lowell lab  showing that removing NMDA receptors from AgRP neurons has the opposite effect. However, there are a few loose ends that need attention.In response to our reviews, the authors of this paper have now dropped data  suggesting that reduced leptin receptor expression was responsible for the obesity of JNK3-deficient mice in favor enhanced glutamatergic (mEPSC) signaling . The autMapk10 KO mice rather than mice lacking Mapk10 selectively in AgRP neurons; hence, it is not possible to make a strong conclusion that the enhanced excitatory transmission is due to lack of JNK3 in AgRP neurons.1) Unfortunately, the electrophysiology experiments were performed on 2) The authors suggest in the Discussion that NMDA signaling is enhanced, but their experiments do not distinguish NMDA from AMPA signaling.3) The hypothesis that links JNK3 deficiency and the presumptive enhanced NMDA signaling is not clearly explained. In the fifth paragraph of the Discussion the authors say that JIP proteins might be involved and refer to Kennedy et al. (2007). If I understand that paper correctly, it suggests that lack of JIP proteins influences JNK localization (activity) and prevents phosphorylation of NR2 subunit of NMDA receptor. If so, it seems like lack of JNK3 would impair (not enhance) NMDA function in the present studies. The authors admit that "further studies are required to identify the complete spectrum of JNK3 targets in AgRP neurons". I would argue that they have not identified any direct JNK3 targets in AgRP neurons.Essential revisions:At the very least, the authors need to discern whether AMPA or NMDA receptor signaling is altered in JNK3-deficient AgRP neurons and provide some data or reasonable hypothesis indicating how lack of JNK3 could lead to enhanced AMPA/NMDA signaling. Essential revisions:For publication in eLife, the authors need to delineate the mechanism by which loss of JNK3 signaling in AgRP neurons promotes obesity. The reviewers have suggested several approaches to address this issue. Alternatively, the authors may decide that publishing the more descriptive part of the study –5 in anoThank you for providing this opportunity to improve our manuscript. We have carefully considered the reviewers’ comments and we have thoroughly revised the manuscript by including new experiments to more fully establish our conclusions. Specifically, we demonstrate that JNK3 plays no role in the regulation of feeding behavior in chow-fed mice, but activated JNK3 in high fat diet-fed mice regulates excitatory transmission onto ARP/NPY neurons. Collectively, our data establish a new paradigm for the regulation of feeding behavior by metabolic stress.Reviewer #1: This paper provides evidence that lack of JNK3 expression in all cells, all neurons that express the leptin-receptor or just the sub-population of neurons that expresses AgRP leads to obesity when mice are reared on a high-fat diet (HFD) but not on a normal (control diet). The obesity is associated with elevated leptin, elevated insulin, glucose intolerance, insulin resistance, and many other parameters associated with obesity. The most important contribution is their systematic demonstration that the phenotype of global KO of the gene encoding JNK3 is replicated quite well by KO only in AgRP neurons. The authors go on to explore how loss of JNK3 function in AgRP neurons might lead to the hyperphagic obese phenotype, but those studies are less compelling. 1) The authors show that leptin receptors can be activated (phosphorylated) in the JNK3 KO but the dose-response is apparently shifted – LepR is fully activated (phosphorylated) with 2.5 mg/kg leptin but only partially activated with 0.1 mg/kg . They attribute this hypo-sensitivity to a reduced level of Lepr mRNA . They show that phospho-cJun (transcription factor) normally binds to an AP1 site in the promoter of Lepr gene and hence the lack of phosphorylation of c-Jun could impair transcription of Lepr gene. There are two problems with this analysis: (a) The Lepr mRNA (rather than protein) measurements were made in hypothalamic samples, not in AgRP neurons where it is relevant. (b) Complete loss of Lepr in AgRP neurons has a relatively small effect on obesity and heterozygous Lepr db/+ mice  do not become more obese on a HFD than WT mice. Thus, it seems unlikely that reduced levels of LepR account for the obesity observed in this JNK3 KO model.We agree. These data have been removed from the revised manuscript. Conclusions about leptin receptor expression have also been removed from the text. The mechanistic analysis in the revised manuscript focuses on the role of JNK3 to regulate excitatory transmission onto ARP/NPY neurons.2) Their data in We have revised the manuscript to address these important points:1H-MRS analysis on day 3 demonstrated no obesity phenotype (A) We performed a time course analysis using metabolic cages to examine when hyperphagia is detected in mice fed a high fat diet. This analysis demonstrates that hyperphagia is detected within 2 days of high fat diet consumption . 1H-MRS henotype . These dB) We performed a pair-feeding study using high fat diet-fed WT and JNK3-deficient mice. This analysis demonstrates that no obesity phenotype was detected when the WT and JNK3-deficient mice consumed the same amount of food . These dC) We have not performed in vivo recording and calcium imaging of AgRP neurons, as requested; we believe that such studies are beyond the scope of the present manuscript. We did examine cFos staining, but no differences in the number of cFos-positive AgRP neurons were detected between WT and JNK3 KO mice. We have expanded the electrophysiological analysis of AgRP neurons from chow diet-fed mice and high fat diet-fed mice in the revised manuscript . This anReviewer #2: […] There is very little to criticize here. The authors address an important question, find a surprising answer of broad interest and base their conclusion on a broad set of genetic data. The only area that falls short is a further analysis of the signaling mechanisms in place. What other component of leptin signaling are affected by the lack of JNK3? The downstream analysis could be broader. Where does the cell specificity come in? How does JNK3 deficiency lead to reduce lepR expression in AgRP neurons, and it does not lead to reduced lepR expression in POMC neurons?Based on the comments of the other reviewers, we have deleted the section of the original manuscript on LepRb expression. In the revised manuscript, we have focused the data presentation and the central conclusions of our study on the finding that excitatory transmission onto AgRP neurons is regulated by the JNK3 signaling pathway in HFD-fed mice. This observation provides a mechanism for the HFD-specific hyperphagia of JNK3 KO mice. Further studies will be required to identify direct molecular targets of JNK3 signaling; this analysis is beyond the scope of the present study.Reviewer #3: […] The primary weakness in the work is the cursory attempt to provide some mechanistic insight . The mEPSC recordings add very little to the paper as is. It is important to know that mIPSCs don't also increase and how the deletion affects basal regulation of POMC neurons. In Figure 7, the overall JNK3 knockout was used and thus, it is not possible to determine if leptin receptor expression is also reduced in POMC cells or rather, if maybe POMC cells do not express JNK3 or require JNK3 to bolster leptin sensitivity. In my opinion, these last 2 experiments do not need to be included to support the conclusions made, but if they are included, they need to be done more thoroughly so that they can be better interpreted.We agree that the leptin receptor expression data do not strengthen our study. Based on this criticism and the comments of reviewer #1, we have deleted these data from the revised manuscript. We have revised the manuscript to include new data to document mIPSCs  and we hJNK3 is the neuron-specific isoform of JNK and its activity is dynamically regulated rather than constitutive like the other 2 isoforms. Together with the compelling evidence provided here that JNK3 helps reduce high-fat diet-induced weight gain through actions in AgRP neurons, this work adds significant new insight into JNK3 function and the hypothalamic regulation of energy balance in the face of metabolic challenge.Thank you for these comments.In response to our reviews, the authors of this paper have now dropped data  suggesting that reduced leptin receptor expression was responsible for the obesity of JNK3-deficient mice in favor enhanced glutamatergic (mEPSC) signaling . The aut1) Unfortunately, the electrophysiology experiments were performed on Mapk10 KO mice rather than mice lacking Mapk10 selectively in AgRP neurons; hence, it is not possible to make a strong conclusion that the enhanced excitatory transmission is due to lack of JNK3 in AgRP neurons.2) The authors suggest in the Discussion that NMDA signaling is enhanced, but their experiments do not distinguish NMDA from AMPA signaling.3) The hypothesis that links JNK3 deficiency and the presumptive enhanced NMDA signaling is not clearly explained. In the fifth paragraph of the Discussion the authors say that JIP proteins might be involved and refer to Kennedy et al. (2007). If I understand that paper correctly, it suggests that lack of JIP proteins influences JNK localization (activity) and prevents phosphorylation of NR2 subunit of NMDA receptor. If so, it seems like lack of JNK3 would impair (not enhance) NMDA function in the present studies. The authors admit that "further studies are required to identify the complete spectrum of JNK3 targets in AgRP neurons". I would argue that they have not identified any direct JNK3 targets in AgRP neurons.Our focus on JNK3 function in AgRP neurons is based on the findings that:Agrp and Npy expression, but not Pomc expression, in the response to leptin administration (1) JNK3-deficiency causes changes in stration  or feedistration ;2) mEPSC amplitudes are increased in AgRP neurons of JNK3-deficient mice ; and3) JNK3-deficiency in AgRP neurons is sufficient to cause hyperphagy on a HFD .+ Mapk10LoxP/LoxP NPY-GFPAgRP-cre mice would be interesting, but these mice are not available in our colony; we consider these studies to be beyond the scope of the present study.Together, these data provide strong evidence for a function of JNK3 in AgRP neurons. We agree that electrophysiological studies of These data represent a key finding that provides a mechanism for JNK3 function mediated by the regulation of AMPA responses. It is established that AMPA receptor phosphorylation by JNK controls AMPA receptor trafficking and function .We apologize for the poor presentation of the conclusions of our study in the Discussion section of our manuscript where we suggested that NMDA receptors may be regulated (based on the two papers we cited) without adequately addressing the role of AMPA receptors. We have revised the manuscript to make our presentation clearer. Moreover, we have included new data in the manuscript  that demAlthough an altered AMPA response in AgRP neurons contributes to the JNK3 phenotype, we cannot exclude the possibility that there is also an altered NMDA response. We feel that dissecting the specific contributions of AMPA and NMDA receptors, while interesting, is outside the scope of this study. In addition, while our recordings show alterations in mEPSC amplitudes, further studies are needed to identify the full spectrum of JNK3 targets that could potentially result in this observation. Such targets may include the JNK scaffold protein JIP1  and JIP2 , but we consider studies of these scaffold proteins to be beyond the scope of the present manuscript.Essential revisions:At the very least, the authors need to discern whether AMPA or NMDA receptor signaling is altered in JNK3-deficient AgRP neurons and provide some data or reasonable hypothesis indicating how lack of JNK3 could lead to enhanced AMPA/NMDA signaling.We agree – this is an important point. We did not make conclusions about AMPA vs. NMDA currents in the previous version of this manuscript. We have therefore revised the manuscript to include new data documenting that the mEPSC currents that we detect are mediated by AMPA receptor responses, not NMDA receptor responses . These d


FRZB as a heart failure marker. Moreover, calcium handling and contractile function genes , potential arrhythmia-related genes , and cytoskeleton and cellular organization-related genes  were among the most deregulated genes in rTOF ventricles. To our knowledge, this is the first comprehensive report on global gene expression profiling in the heart of a long-term swine model of repaired TOF.Surgical repair of Tetralogy of Fallot (TOF) is highly successful but may be complicated in adulthood by arrhythmias, sudden death, and right ventricular or biventricular dysfunction. To better understand the molecular and cellular mechanisms of these delayed cardiac events, a chronic animal model of postoperative TOF was studied using microarrays to perform cardiac transcriptomic studies. The experimental study included 12 piglets (7 rTOF and 5 controls) that underwent surgery at age 2 months and were further studied after 23 (+/- 1) weeks of postoperative recovery. Two distinct regions (endocardium and epicardium) from both ventricles were analyzed. Expression levels from each localization were compared in order to decipher mechanisms and signaling pathways leading to ventricular dysfunction and arrhythmias in surgically repaired TOF. Several genes were confirmed to participate in ventricular remodeling and cardiac failure and some new candidate genes were described. In particular, these data pointed out  Tetralogy of Fallot (TOF) is the most frequent cyanotic congenital heart disease in humans with an incidence of 1/3600 live births ,3,4. UnlIn order to better understand the implication of prolonged dyssynchrony, we established and characterized a chronic large animal model that mimicked essential parameters of postoperative TOF . This moMoreover, in the past decade, the completion of Sus Scrofa genome sequencing  led to the development of commercially available pig cDNA microarrays that could be used to detect changes in swine genes expression.In the present study, we aimed at deciphering the sequence of molecular events and pathways leading to the progression of ventricular dysfunction and arrhythmias, by studying gene expression profiles of repaired Fallot (rTOF) pigs’ hearts compared to control animals (sham-operated), in four different localizations of the heart (epicardium and endocardium of right and left ventricles), by a whole genome approach. Some transcripts were selected to verify the accuracy and reproducibility of the microarray data by real-time qRT-PCR.The experimental protocol followed the European rules for animal experimentation (European legislation 2010/63/UE—2010) which was implemented under French legislation from February 2013 and following which all animal experimental protocols, including the present study, at our Institution were reviewed and approved by the local Ethics Committee "Comité d'Ethique en Expérimentation Animale de Bordeaux—CEEA50. The experimental protocols were in compliance with the Guiding Principles in the Use and Care of Animals published by the National Institutes of Health .The experimental study included 12 piglets (7 rTOF and 5 controls), aged three months and weighing less than 12 kg, at the time of surgery. The animal model was established as previously described . BrieflyAfter 23 (+/- 1) weeks of postoperative recovery, cardiac function was assessed in anesthetized pigs  by cardiac magnetic resonance in a Siemens Magnetom Avanto 1.5T MRI scanner . Animals were euthanized, their hearts excised and washed in ice-cold cardioplegic solution to remove any residual blood.Myocardial samples were dissected out and immediately frozen into liquid nitrogen. Biopsies were then stored at -80°C until RNA extraction. Total RNA was extracted from tissues using QIAzol reagent . RNA was purified and DNase treated using the QIAGEN RNeasy Kit. RNA purity and integrity, were assessed both by spectrophotometry  and nanoelectrophoresis .cDNA was synthesized from 200 ng of total-RNA using the direct cDNA Labeling System. Aminoallyl-cRNA was synthesized from cDNA using the Superscript Indirect cDNA Labeling System. The cRNA was purified using RNeasy QIAGEN RNeasy Kit.Labeling and hybridization of the cRNA was performed with Agilent Whole Porcine Genome Oligo (4 × 44 K) Microarrays (one-color platform), according to the manufacturer’s protocols. The slides were scanned and analyzed using the histogram method with default settings in an Agilent G2565C Microarray Scanner System with SureScan Technology.Among the 43,603 probes present on the Porcine 44K Agilent microarray (V2), only 15,458 of these were annotated with HUGO Gene Nomenclature Committee (HGNC) gene symbols , corresphttp://david.abcc.ncifcrf.gov/)[http://www.pantherdb.org/pathway/) and Ingenuity Pathway Analysis Hierarchical clustering of the significantly differentially expressed (DE) genes from microarray data was carried out by the Genespring software (Agilent Technologies), with a p-value corrected by Benjamini Hochberg False Discovery Rate (FDR). Different softwares were used to interpret the biological functions and canonical pathways of the gene lists: Visualization and Integrated Discovery Online platform (crf.gov/), using GIn order to confirm the reliability of the expression profile from the microarray analyses, the expression level of some genes was assessed by real-time RT-qPCR.HPRT1 and GUSB. This allowed quantification of the target gene in one sample relative to that in another  using the “2−ΔΔCt method” of calculating fold changes in gene expression.Sequences for primers were obtained from Ensembl Genome Browser. Primers were designed using Primer designing tool (NCBI) and synthesized at Sigma Aldrich. 1μg of RNA was reversed transcribed using a cDNA Reverse Transcription kit (Life Technologies) according to the manufacturer’s protocol. Quantitative PCR was performed in a 25 μL reaction volume , a volume of 10 μM upstream and downstream primers respectively, and added ddH2O to 25 μL) on the BIO-RAD C 100 Touch Thermal Cycler / CFX96 Real time System. Real-time PCR conditions were as follows: 3 min at 95.0°C, 40 cycles of denaturation at 95°C for 30 s followed by 30 s annealing and elongation at 60°C. Efficiency of primer pairs was previously evaluated. Melting curves were obtained at the end of each run to confirm a single PCR product. All samples were run in triplicate. Non-template controls were included in each run to exclude contamination and nonspecific amplification. Expression levels of samples were normalized by using a normalization factor calculated by the software CFX Manager (BIO-RAD). This normalization factor was calculated based on RT-qPCR results for two selected reference genes, Upon cardiac magnetic resonance examination at 23 ± 1 weeks, Fallot pigs presented with significantly decreased RV ejection fraction and increased RV end-diastolic volume compared to controls indicating RV dysfunction and RV dilation respectively.We first performed microarray experiments on myocardial biopsies from the right ventricle (endocardium and epicardium separately), because we expected more changes in this particular region with an animal model of right ventricular dysfunction. We observed the biggest list of statistically significant DE genes in the endocardium of the right ventricle and focused on the exploration of these genes. We then explored the left ventricle (endocardium and epicardium).Out of the 43,603 probes represented on the Sus Scrofa microarray (Agilent), 153 probes were significantly different in rTOF pigs versus controls, after preprocessing and statistical analysis  . These 1The biological processes depicting genes that were deregulated in rTOF hearts are shown in To investigate the interconnectivity of the differentially regulated genes with other gene products, pathways, and biological processes, molecular networks were formed with IPA software. These networks include genes from our transcriptomic data and their interactions with genes that are biologically relevant to the pathway, coding for hub molecules not altered in our experiments. The top 4 networks among the genes most differentially expressed are represented in FRZB), calcium handling and contractile function-related genes—Sarcolipin (SLN), Alpha cardiac actin (ACTC1), Calsequestrin (CASQ1), Troponin (TNNT1), Myotilin (MYOT), Phospholipase C Z(PLCZ), Phospholipase C D4 (PLCD4), electrophysiology and arrhythmia-related genes—Myosin 5B (MYO5B), Four and a half LIM domains protein 1 (FHL1C), Collagen type VIII (COL8A1), Potassium voltage-gated channel subfamily A member 6 (KCNA6) and Cytoskeleton/Cellular organization-related genes—Xin actin-binding repeat-containing protein 2 (XIRP2), Nexin (SERPINE2)  .FHL1C and MYOT. This could be due to different isoforms not tested by qPCR (9 targets deregulated in microarray for each of these two genes).Representative genes were selected for validation via qRT-PCR, using the same tissue samples used in the microarray. For these fourteen genes, the fold changes obtained via microarray analysis demonstrated substantial agreement with the fold change values determined via qRT-PCR  thereby confirming the microarray data . Two genBNP (Brain Natriuretic Peptide) as a heart failure marker, SERCA2A2 , known to be down regulated in failing hearts, and KCNA5 coding for Kv1.5 which seems to interact with MYO5B and FHL1C (found deregulated in our microarray data) , however one of the 7 rTOF animals had a much stronger expression of BNP compared to others (We aimed to explore expression changes in candidate genes: ay data) . We obseo others . This rTPerforming the same experiments and statistical analyses on the three other localizations , we found a marked heterogeneity of regional gene expression. Target genes were globally differentially expressed in the same way as in the RV endocardium, between the rTOF and the controls, but with no statistical significant p-value (p>0.05). FRZB, PLCZ, TNNT1, XIRP2 and ACTC1 , cell adhesion and reshaping in response to external stimuli, angiogenesis. Molecular networks were also formed with IPA software involving the lipid metabolism that may reflect the changes in energy metabolism associated with ventricular hypertrophy. In this network, ERK-PKC-MAPK are central hub molecules. The role of the pathway MAPK/ERK/PKC in cardiac hyperthrophy has been well established ,10.Differentially expressed genes identified by microarrays were further validated using RTqPCR. We confirmed the level of transcript abundance of fourteen genes of interest using RT-qPCR, and the Fold Changes were concordant in most cases. Among the most de-regulated genes in the endocardium of right ventricle, we identified some genes of special interest.FRZB gene codes for a secreted frizzled-related protein B expressed in ventricular myocardium and involved in the Wnt/β-catenin signaling cascade affecting cell proliferation signals and endocardial cushion morphogenesis [The BNP (Brain Natriuretic Peptide) is a cardiokine that also belongs to the fetal cardiac gene program and is used as a biomarker of cardiac hypertrophy and heart failure in humans because it is secreted in response to myocardial stretching. As expected, its expression tended to be upregulated among our rTOF pigs. The ogenesis . This geogenesis . Its oveogenesis . These oATP2A2), is a key regulator of intracellular Ca2+ trafficking, pumping it back to Sarcoplasmic Reticulum during myocardial relaxation. It is also a member of the fetal cardiac gene program which is reactivated with cardiac hypertrophy. This protein tended to be down-regulated in the RV of the rTOF pigs compared to controls (even if the difference was not significant), as it was observed in human RV endomyocardial biopsies at early stage of the Fallot disease (in immature cardiomyocytes with hypertrophy and hypoxia from children aged 15 to 29 months) [ATP2A2 is typical of heart failure, and was also recently described in a rat model of right heart failure [ATP2A2 in the cardiomyocytes of hypoxia-induced hypertrophic RV [The SERCA2a protein (encoded by  months) . This doophic RV ,17.SLN coding for sarcolipin also belongs to the Ca2+ regulatory protein family. It is predominantly expressed in the cardiac atria in humans, however there are chamber specific and species specific differences in its expression [SLN in our model represents a new finding compared to the study by Wu et al. that did not find any deregulation of this gene at the RNA and protein level in human cardiomyocytes, and compared to other studies studying SLN under several pathological conditions [in vivo measurements of cardiac function showed a significant decrease of +dP/dt and–dP/dt with ventricular hypertrophy. The inhibitory effect of SLN on these models was reversed by a βadrenergic agonist, isoproterenol, which restored cardiac contractility [pression . SLN andpression . The incnditions ,19. Intenditions . Mouse mactility .PLN (data not shown) could argue for a possible independent role of SLN in inhibiting SERCA2a, as previously hypothesized [The absence of concomittant deregulation of thesized .ACTC1, coding for a cytoskeletal protein or alpha cardiac actin, was the most significantly downregulated gene in our set and was already described as associated with the pathogenesis of dilated cardiomyopathy and heart failure in several expression studies [ACTC1 were found in familial Atrial septal Defect and reduced expression of ACTC1 was observed in different congenital heart diseases in humans including Tetralogy of Fallot [ studies . Autosomf Fallot . Our datCASQ1 was found significantly up regulated in the rTOFs, however this isoform is predominantly expressed in the skeletal muscle in humans and pigs (CASQ2 being the main cardiac isoform).Phospholipases C D4 and Z were found upregulated in the rTOF pigs confirming the link of phospholipase C with PKC / Frizzled / Wnt signaling pathway .KCNA5 gene) and control channel recycling in rat cardiomyocytes [KCNA5 gene was also significantly upregulated in the right ventricles of our rTOF pigs. This gene, mainly expressed in the atrium, mediates IKur and contributes to atrial repolarization. It has also been shown to be expressed in the human left ventricle [Myosin 5B coding for the unconventional myosin motor VB  is overexpressed in our model. It is a molecular motor that was recently found to regulate the cell surface trafficking of ion channels, such as Kv1.5  is significantly expressed in human cardiac fibroblasts [KCNA6 expression we observed in our animal model may reflect fibroblast proliferation.roblasts  while itroblasts . Thus, tXIRP2 (overexpressed in rTOF pigs) belongs to the evolutionarily conserved, muscle specific, actin-binding Xin gene family. This protein (mXinβ) has a role in the intercalated disc maturation and post-natal heart growth in mice [in vitro and in vivo models of hypertrophy and heart failure at early stage [ in mice . This gely stage . XIRP2 aly stage ,35.COL8A1 was found significantly activated in our rTOF group as it was already observed in the cardiac RV of a mouse model of pulmonary artery clipping [Interestingly, clipping . This coclipping  but coulTNNT1, FRZB, ACTC1, PLCZ and XIRP2), in all four locations, led to hypothesize the existence of a molecular “gradient” of expression progressing from the RV to the LV of rTOF pigs. Mechanical stress could be transmitted from RV to LV through molecular and cellular alterations mediated by transcriptomic changes of certain genes. The nature and extent of gene expression vary with time. Indeed, distinct transcriptional changes were observed at different time-points after pulmonary artery clipping surgery in a mouse model [Transcriptional responses in the left ventricle in our set of experiments followed the same tendency than for the right ventricle, but were much less strong. Comparison of the expression levels of different genes  was significantly increased in Fallot pigs compared to Control indicating RV dilatation. C: RV ejection fraction (RVEF) was significantly lower in Fallot pigs than Controls highlighting RV dysfunction. Control N = 4, Fallot N = 6, * P < 0.05; *** P< 0.001.(TIF)Click here for additional data file.S2 FigRelative expression (RT-qPCR) of BNP gene in samples from the RV endocardium of controls and rTOF hearts. Transcript expression is normalized to the reference genes HPRT1 and GUSB.(TIF)Click here for additional data file.S3 Fig50μg of total protein were loaded for TNNT1 Western blot analysis. Quantification of band intensity was calculated after normalization for total protein loaded (p = 0.08).(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.


Firmicutes and Verrucomicrobia with increased Bacteroidetes and Proteobacteria compared with the HSCR group. In contrast, the fecal fungi composition of the HAEC group showed marked reduction in diversity with increased Candida sp., and reduced Malassezia and Saccharomyces sp. compared with the HSCR group. The most striking finding within the HAEC group is that the Candida genus segregated into “high burden” patients with 97.8% C. albicans and 2.2% C. tropicalis compared with “low burden” patients 26.8% C. albicans and 73% C. tropicalis. Interestingly even the low burden HAEC group had altered Candida community structure with just two species compared to more diverse Candida populations in the HSCR patients. This is the first study to identify Candida sp. as potentially playing a role in HAEC either as expanded commensal species as a consequence of enterocolitis (or treatment), or possibly as pathobioants contributing to the pathogenesis of HAEC. These findings suggest a dysbiosis in the gut microbial ecosystem of HAEC patients, such that there may be dominance of fungi and bacteria predisposing patients to development of HAEC.Development of potentially life-threatening enterocolitis is the most frequent complication in children with Hirschsprung disease (HSCR), even after definitive corrective surgery. Intestinal microbiota likely contribute to the etiology of enterocolitis, so the aim of this study was to compare the fecal bacterial and fungal communities of children who developed Hirschsprung-associated enterocolitis (HAEC) with HSCR patients who had never had enterocolitis. Eighteen Hirschsprung patients who had completed definitive surgery were enrolled: 9 had a history of HAEC and 9 did not. Fecal DNA was isolated and 16S and ITS-1 regions sequenced using Next Generation Sequencing and data analysis for species identification. The HAEC group bacterial composition showed a modest reduction in  Congenital aganglionic megacolon, more commonly known as Hirschsprung disease (HSCR), was first described by Harald Hirschsprung in 1887 . Even toClostridium difficile and rotavirus have been implicated as causative agents of HAEC, however neither were consistently present in patients HAEC [Bifidobacteria and Lactobacilli genera were decreased in HSCR patients who developed HAEC, compared with those who did not develop HAEC [et al. recently reported characterization of the colonic bacterial microbiome of 4 infants with HSCR, two of which had HAEC [While many etiologies have been proposed for HAEC, the underlying biological mechanisms are poorly understood. A microbial role in the development of HAEC has been suspected since it was first described over 50 years, although at present, no specific organisms have been identified . Both Clnts HAEC –6. With lop HAEC , suggesthad HAEC . Fecal shad HAEC .et al. showed that by manipulating the commensal colonic fungal population in Dectin-1 knockout mice, the severity of colitis could be either improved or worsened [Recently, the intestinal fungal microbiome (or “mycobiome”) has been recognized to coexist with bacteria. Iliev worsened . These fHerein, we studied 18 HSCR patients from four centers, all of whom had completed definitive surgery to treat HSCR. We compared the colonic bacterial and fungal populations in 9 children with HSCR alone, with 9 children who had at least one episode of HAEC.Inclusion criteria for enrollment were children less than 18 years of age with histopathological diagnosis of Hirschsprung disease who had completed definitive pull-through surgery. Exclusion criteria included presence of intestinal diversion or active HAEC at the time of stool collection, colonic pseudoobstruction and intestinal neuronal dysplasia. This research was approved by the Cedars-Sinai Medical Center IRB (Protocol# 00020809) as a multi-center study, and has been conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from a parent by the attending surgeons, research fellows, or research nurses at each site. Children were enrolled by four member institutions of the HAEC Collaborative Research Group (HCRG): Cedars-Sinai Medical Center, Los Angeles, California; Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden; Children’s Hospital Los Angeles, Los Angeles, California; Children’s Hospital of Oakland, Oakland, California. We enrolled 20 children with HSCR, 10 never had a history of enterocolitis and 10 had a history of at least one episode of HAEC based on HAEC scoring system described by Pastor et al . DetaileTwo subjects were excluded from analysis: subject 03–0003 was excluded because he had a diverting ileostomy ; and 02–0039 was excluded because he had an active HAEC episode at the time of stool collection. Subject 04–0005 was excluded from fungal microbiome analysis only due to failure to pass quality control after sequencing, although bacterial microbiome analysis was completed.Within one week of enrollment in the study, stool was collected and frozen at -80°C within 24 hours of collection. All samples were kept frozen and shipped to the coordinating site (CSMC) where all of the samples were prepared for bacterial and fungal DNA. Fecal sμL PCR reactions using 20 ng of fecal DNA with 25 cycles using high-fidelity Phusion Polymerase  at 52.7°C annealing using with degenerate 8F (AGAGTTTGATCMTGGCTCAG) and R357 (CTGCTGCCTYCCGTA) primers. Fungal ITS-1 amplicons were generated in 20 μL PCR reactions using 20 ng of fecal DNA with 35 cycles using Phusion Polymerase at 56.1°C annealing using ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS2R (GCTGCGTTCTTCATCGATGC) primers yielded sufficient amplification of ITS targets. All PCR reactions were purified using Agencourt AmPure Magnetic Beads (Beckman), resuspended in 20 μL of nuclease-free water and quantified using a Qubit fluorometer .Bacterial 16S rRNA gene amplicons spanning variable regions one to four (V1–4) were generated in 20 Paired-end adapters with unique indexes were ligated to 100 ng of 16S amplicons and used to generate Ion Torrent sequencing libraries using the Ion Xpress Library Kit . Illumina paired-end adapters with unique indexes were ligated to 100 ng of ITS-1 amplicons using a modified TruSeq DNA Sample Preparation  where adapters and PCR primers were diluted 1:10 to accommodate lower input of amplicon mass for both 16S and ITS-1 preparations. Library enrichment was performed with 10 cycles of PCR and purified using Agencourt Ampure Magnetic Beads (Beckman). All libraries were subjected to quality control using qPCR, DNA 1000 Bioanalyzer (Agilent), and Qubit  to validate and quantitate library construction then pooled at equimolar concentrations.Pooled libraries were assayed on Agilent Bioanalyzer  to check final sizing and to check for small fragments as well as KAPA Biosciences qPCR for quantitation. 16S samples were multiplexed and sequenced on the Ion Torrent PGM on a 318 chip with 400bp chemistry. For ITS-1 sequencing final diluted pool was amplified on to a Single End flow cell using clonal bridge amplification on the MiSeq. 250 single-end sequencing-by-synthesis was performed using the MiSeq Illumina sequencer . The sequence data will be submitted to the NIH Sequencing Read Archive with the submission number: SRP051546.Ion Torrent reads shorter than 200bp, or not containing the designed 16S primers (>2nt mismatches) were discarded. 300bp sequences of remaining high-quality reads were aligned to the Greengenes reference database (February 2011 release) using BLAST v2.2.22 in QIIME v1.5 wrapper with an FASTQ data was de-multiplexed and filtered through a stringent quality control procedure to ensure that only high-quality sequences were analyzed further. To identify fungal species, the filtered reads were aligned with the Findley ITS Database using BLThe original OTU table was randomly subsampled (rarefied) to create a series of subsampled OTU tables. Alpha diversity was calculated in QIIME on each sample using the OTU table and Shannon indices were collated into a single file and the number of species identified for both bacteria and fungi for each sample versus the depth of subsampling was plotted.Candida albicans (ATCC 90028) was obtained from the American Type Culture Collection . Fungi were cultured in aerobic conditions on Sabouraud Dextrose Broth  for overnight at 37°C. The cultured cells were harvested for DNA preparation using the QIAmp DNA Stool Mini Kit .Fungal reference strain Candida albicansTTTATCAACTTGTCACACCAGA (Forward) and ATCCCGCCTTACCACTACCG (Reverse); [μL), and 4ul of reverse primer (3 pmol/ μL). The PCR protocol was modified from Iliev et al[C. albicans in fecal specimens was determined by using a standard curve generated by Candida albicans (ATCC 90028) DNA with 10-fold serial dilution from 102 ng to 10–4 ng against the threshold cycle C(t), and normalized to the amount of total fecal DNA being used. The qPCR results are reported as C. albicans cell numbers per ng fecal DNA, in which the C. albicans cell numbers were calculated from cell counts per nanogram DNA of reference Candida albicans (ATCC 90028) where 3x10-4ng DNA represents one cell.Quantitative PCR was performed on DNA isolated from human stool using SYBR Green Kit (Bio-Rad). Specific primer pairs for everse); . In a 20iev et al: InitialStudent’s t test, unpaired, two-tailed, 95% confidence interval. Data are presented as means ± SEM, unless otherwise stated. Statistical analysis was performed using GraphPad Prism 5 software .Both OTU sequence numbers and percentages between groups were compared using Each group of subjects meeting inclusion criteria consisted of 8 males and 1 female. The median age of all children was 2.7 years (range 5 months to 8 years); the median age of the HSCR group was 2.3 years and the HAEC group was 3.5 years  dominated the bacterial microbiota in most samples  and HSCR (37550 ± 11210) (P<0.0001) and HAEC “low burden” patients (18910 ± 10830) (P = 0.001), respectively, but not between HSCR and HAEC “low burden” patients. In the HAEC group, the species composition of “high burden” patients showed 97.8% was C. albicans and only 2.2% C. tropicalis compared with “low burden” patients 26.8% C. albicans and 73% C. tropicalis [Further, this is the largest study to date comparing the bacterial microbiome composition of children with HSCR to those who had a history of HAEC that demonstrated modest but potentially important differences. The HAEC group showed reduced abundance of the phyla ase (IBD)–21, and ase (IBD),23. ThesC. albicans may be either a commensal species that is expanded as a consequence of enterocolitis (or treatment), or the intriguing possibility that C. albicans is a pathobioant that may contribute to the pathogenesis of HAEC. While the mechanism leading to the C. albicans expansion is unclear, it opens up the possibility of an underlying defect in the gut innate immunity of Hirschsprung patients that may predispose these patients to developing HAEC, as has been demonstrated in severe sub-type of UC patients [Our findings also raise the possibility that there may be a subset of children with HAEC in whom patients . DespiteOne limitation of this study is a lack of long-term antibiotic history (prior to 2 months), which may influence the structure of mycobiome. This may confound the interpretion of these results especially in HAEC patients who may have received antibacterials to treat HAEC remotely compared with HSCR patients who have not developed HAEC. Another limitation of this study is the limited sample size of each group.In summary, we recognize that the microbiota differences between HSCR and HAEC groups may be caused by treatment; may be caused by HAEC; and may, or may not, actually contribute to HAEC. Nevertheless, we believe that these changes are part of the phenotype of these patients and warrant further study. In the future, an expanded study with more patients is needed to confirm findings, as well as development of animal models to further investigate mechanisms by which the bacterial and fungal communities play a role leading to enterocolitis in the unique milieu of Hirschsprung disease.S1 FigRarefaction curves showing the Shannon diversity index change with increasing sequencing depth show that the bacterial (top) and fungal (bottom) sequencing of samples from HSCR patients (left) and HAEC patients (right) reached saturated plateau phase. The plateau in each curve estimates the minimum number of sequences necessary to capture diversity.(TIF)Click here for additional data file.S2 FigA,16S rRNA gene sequence of fecal bacteria of nine HSCR patients and nine HAEC patients. The pie charts show average relative abundance of 11 major genera and subdominant genera (summarized as “All others”). B, histograms demonstrating the genera level bacterial composition of individual subjects with HSCR and HAEC. Individual subject numbers are labeled on the X axis and expressed as relative OTU abundance per each subject. Colors were assigned for each of the 11 major genera with the scheme at the right side.(TIFF)Click here for additional data file.S1 TableThe 16S and ITS sequence numbers analyzed, including mean.(DOCX)Click here for additional data file.S2 Tablet-test.Relative OTU abundance of 16S rRNA gene sequences of fecal bacteria of nine HSCR patients and nine HAEC patients. Comparisons between HSCR and HAEC groups for each genus was performed using (DOCX)Click here for additional data file.


In this study, respiratory indirect plethysmography (RIP), electromyography (EMG) of the transversus abdominis and scalene muscles and indirect calorimetry are used to achieve an integrated picture of the changes in the respiratory system after exposure to 10 cmH2O of continuous positive airway pressure (CPAP).To investigate the changes in breathing pattern, respiratory muscle mechanics and work of breathing induced by 10 cmH2O CPAP. RIP was used to evaluate the contribution of the rib cage (RC) and abdominal (AB) compartments to the inspired tidal volume, and to allow measurement of the continuous degree of asynchrony between the thoracic and abdominal movements. EMG activity of the accessory respiratory muscles  and measurements of oxygen consumption (V02) were recorded during both ZEEP and CPAP settings.Fourteen young, healthy individuals participated in the study, assessed in the semi-recumbent position initially at ZEEP and then at 10 cmHTidal volume increased from ZEEP (738 ± 208 ml) to CPAP  with RIP revealing that this increase was mainly due to RC rather than AB contribution. No significant thoraco-abdominal asynchrony was observed in the CPAP setting. During the first twenty breaths during CPAP the difference between mean tidal volume inspired (862 ± 235 ml) and mean tidal volume expired (831 ± 875 ml) was 31 ml, and the volume trapped was approximately 600 ml. This volume trapped was accommodated during the first four CPAP breaths, after which mean tidal volume inspired and mean tidal volume expired did not differ significantly. EMG recordings indicated increased inspiratory activity of the scalene muscles and increased expiratory activity of the transversus abdominis during CPAP, leading to an increase in oxygen consumption from 278 ± 40 ml/min to 322 ± 52 ml/min  measured with indirect calorimetry.2O of CPAP induces acute overdistention. This overdistension activates the accessory respiratory muscles and increases the work of breathing. The increased inspiratory scalene muscle activity during CPAP explains the increased RC contribution to tidal volume, whereas the expiratory transversus abdominis recruitment probably defends the lung from overinflation by pulling back the diaphragm towards the initial FRC position. We can assume that in patients with acute respiratory failure not tolerating various levels of CPAP, CPAP induces over-distension and increases the work of breathing.In normal individuals 10cm H


The crypts of the intestinal epithelium house the stem cells that ensure the continual renewal of the epithelial cells that line the intestinal tract. Crypt number increases by a process called crypt fission, the division of a single crypt into two daughter crypts. Fission drives normal tissue growth and maintenance. Correspondingly, it becomes less frequent in adulthood. Importantly, fission is reactivated to drive adenoma growth. The mechanisms governing fission are poorly understood. However, only by knowing how normal fission operates can cancer-associated changes be elucidated. We studied normal fission in tissue in three dimensions using high-resolution imaging and used intestinal organoids to identify underlying mechanisms. We discovered that both the number and relative position of Paneth cells and Lgr5+ cells are important for fission. Furthermore, the higher stiffness and increased adhesion of Paneth cells are involved in determining the site of fission. Formation of a cluster of Lgr5+ cells between at least two Paneth-cell-rich domains establishes the site for the upward invagination that initiates fission. Crypt fission—a process responsible for normal intestinal growth and for the formation of adenomas —is governed by differential adhesion, stiffness, and proliferation of Lgr5+ cells and Paneth cells in the intestinal stem cell niche. The intestinal tract undergoes many changes during development, and after birth it has to significantly elongate and widen in order to increase the surface area for absorption. Crypt fission is a key process in intestinal tissue expansion and is also involved in adenoma growth. Despite the importance of crypt fission, the mechanisms controlling it are poorly understood. Understanding how crypt fission is regulated in normal tissue can help us to determine how the process changes in cancer. Here, we describe cellular behaviour during crypt fission. We identify a specific cellular arrangement in the intestinal stem cell niche that is associated with crypt fission and reveals insights into the mechanisms controlling crypt fission. There are two different cell types at the crypt base, Lgr5+ and Paneth cells, which play distinct roles in this process. We find that both their location and differences between them in proliferation, stiffness, and adhesion are important for fission. Based on our data, we propose a model in which stiffer and more adhesive Paneth cells are necessary to shape the crypt base and establish where fission occurs, whereas softer Lgr5+ cells allow shape changes and proliferation to expand newly formed crypts. Our model is an important step in understanding how crypt fission is initiated in normal tissue and provides a framework to understand how the process changes in tumorigenesis. ApcMin/+ mice and in familial adenomatous polyposis (FAP) patients are initiated by and expand through crypt fission ). Both growth conditions caused altered crypt morphology: crypts in ENR-CV were longer, while crypts in ENR-CD were shorter and rounder , S9 Fig.asurably , S5 Dataasurably , S4 Dataasurably , S4 DataNew branches in organoids are reported to form at the location of Paneth cells; therefore, we examined whether mislocalising Paneth cells could affect crypt fission. Eph/Ephrin signalling is important for the normal positioning of Paneth cells , and inhExperimental data from organoids suggests a role for both Paneth and Lgr5+ cells during fission. However, our data that increasing the number of Paneth cells reduced fission contradicts our observation that new branches form where Paneth cells are located. To examine this in more detail, we utilised computational modelling of connected epithelial layers to investigate the role of Paneth cells in fission. Paneth cells were modelled to be 4.5 times stiffer than neighbouring cells , but theCrypt fission is at the core of normal growth and maintenance of intestinal tissue. Importantly, the normal decrease in fission that occurs with ageing is reversed in early cancer, and aberrant fission in adult tissue is associated with adenoma growth. This study aims to determine how fission works normally so that mechanisms involved in fission in adenoma growth can be identified more readily. We found that the initiation of fission was marked by a cluster of Lgr5+ cells in the middle of the crypt base. Paneth cells appeared only lateral to this region and marked the bases of prospective daughter crypts. We discovered that differential adhesion of Paneth and neighbouring Lgr5+ cells to the basement membrane via β4 Integrin is likely involved in fission. This conclusion was supported by our finding that blocking β4 Integrin inhibits fission in intestinal organoids.To identify how fission changes in cancer, we first had to establish what constitutes normal fission in situ. We found symmetric and asymmetric fission with similar frequency in healthy tissue, contrary to a recent report that identified “asymmetric budding” as the prevalent form of crypt fission . These dFission incidence in different regions of the gut correlates with mitotic rate . HoweverOne question that remains is how the Lgr5+ cluster forms. Our computer model provided some clues. First, it showed that the greater stiffness of Paneth cells deforms the crypt wall , consistOnce the Lgr5+ cluster has formed, it creates a softer area in the crypt base. This area deforms in response to forces exerted by neighbouring crypts and results in the upward buckling event that initiates the formation of new crypt walls. Expansion of these walls is likely achieved by proliferation of the Lgr5+ cells in the cluster, as suggested by the mitotic cells we observed in the bifurcation of fissioning crypts . One conSox9flox/flox mice, which are depleted of Paneth cells, have wider and deeper crypts ).(TIF)Click here for additional data file.S3 FigFixed human tissue stained against Hoechst (blue), Lysozyme (red), and Phalloidin (green). Paneth cells are excluded from the region underneath the bifurcation in fissioning crypts in human tissue.(TIF)Click here for additional data file.S4 FigApcMin/+ mice and (C) ApcMin/Min organoids were stained to visualise F-actin , Lysozyme (white), and β4 Integrin (red). Lines in middle panels mark the boundaries between Paneth cells and their neighbours. Signal intensity of β4 Integrin is not elevated in Paneth cells in polyps  or in ApcMin/Min organoids  compared to neighbouring cells . (D′) In organoids, the ratio of β4 Integrin signal intensity is more variable and not significantly different from wild-type . Underlying data for panels B, B′, D, and D′ can be found in (A) Polyps from ng cells . (B′) In(TIF)Click here for additional data file.S5 FigProjection of a colonic crypt sectioned and stained for F-actin , Muc2 (white), and β4 Integrin (red) shows that Muc2-positive cells at the crypt base have more β4 Integrin on their basal surfaces than their neighbouring cells. Arrows in the top right panel point to CBCs neighbouring Muc2+ cells. White lines in the top left panel indicate the boundaries between Muc2+ cells and their neighbours.(TIF)Click here for additional data file.S6 FigWild-type small intestinal crypts were sectioned and stained to visualise F-actin , β4 Integrin (green), and secretory cells . White lines in the left panel indicate the boundary between secretory cells and their neighbouring cells. Both types of secretory cell have higher levels of β4 Integrin on their basal surfaces than neighbouring cells.(TIF)Click here for additional data file.S7 FigCrypts stained to visualise nuclei  and F-actin  show that daughter crypts in situ (A) elongate parallel to each other. When fissioning crypts are removed from tissue prior to fixing (B), daughter crypts adopt a more splayed conformation. In organoids (C), daughter crypts elongate at a 90°–180° angle. These observations suggest daughter crypts are kept parallel by physical constraint from surrounding tissue; when this physical constraint is released, daughter crypts adopt a more relaxed conformation. In organoids, daughter crypts elongate at larger angles due to the lack of constraint.(TIF)Click here for additional data file.S8 Figp = 0.7263, t test, n = 26 and 59 Paneth cells for organoids and tissue, respectively). Underlying data can be found in The levels of β4 Integrin on Paneth cells was measured relative to that on neighbouring CBCs . In both(TIF)Click here for additional data file.S9 Figt test, and significance is compared to ENR control. Underlying data for panels A–C can be found in Correlation between crypt length and Paneth cell numbers  from org(TIF)Click here for additional data file.S10 FigThe length of crypts from organoids grown in ENR (A), ENR-CV (B), and ENR-CD (C) were measured and the number of Paneth cells (A′-C′) determined by counting . Fission(TIF)Click here for additional data file.S11 Figp = 0.2480, t test, n = 26 for ENR and 34 for Eph-treated). Underlying data can be found in The abundance of β4 Integrin on Paneth cells was measured relative to that on neighbouring CBCs . In both(TIF)Click here for additional data file.S12 Figp/μs = 4.5). Error bars in (A) represent the standard deviation in cell area for Lgr5+ cells (blue) and Paneth cells (red). Underlying data for panels A and B can be found in (A) Average cell area and (B) the relative cell area ratio (Paneth cell area ÷ Lgr5+ cell area) for varying Paneth cell numbers in the epithelial layer model . Here, P(TIF)Click here for additional data file.S13 Fig Optical sections of a fissioning crypt stained against Hoechst (cyan) and Phalloidin (red). Mitotic cells were observed underneath the bifurcation, apparent by condensation of DNA (arrow). Imaris-rendered surfaces showing crypt lumen (red), crypt wall (transparent yellow), and PH3+ nuclei (green spots) from the bottom (C) and side (D) of a fissioning crypt.  Optical sections from tissue stained against PH3 (red) and Hoechst (cyan) reveal mitotic cells can be present both above and below the bifurcation in fissioning crypts.(TIF)Click here for additional data file.S1 TablePercentage of crypts undergoing fission in all regions of the gut from mice aged 2, 3, 4, 5, 6, and 10 wk see also . Percent(TIF)Click here for additional data file.


The molecular determinants of chronic thromboembolic pulmonary hypertension (CTEPH) and idiopathic pulmonary arterial hypertension (iPAH) remain poorly understood. The receptor for advanced glycation endproducts (RAGE) and its ligands: HMGB1 and S100A9 are involved in inflammatory disorders. We sought to investigate the role of the RAGE axis in patients with CTEPH undergoing pulmonary endarterectomy (PEA), iPAH undergoing lung transplantation (LuTX). The high pulmonary vascular resistance in CTEPH/iPAH results in pressure overload of the right ventricle. We compared sRAGE measurements to that of patients with aortic valve stenosis (AVS) – pressure overload of the left ventricle.We enrolled patients with CTEPH(26), iPAH(15), AVS(15) and volunteers(33). Immunohistochemistry with antibodies to RAGE and HMGB1 was performed on PEA specimens and lung tissues. We employed enzyme-linked immunosorbent assays to determine the concentrations of sRAGE, esRAGE, HMGB1 and S100A9 in serum of volunteers and patients with CTEPH, iPAH, AVS before and after PEA, LuTX and aortic valve replacement (AVR).+vimentin+CD34−), recanalizing vessel-like structures of distal myofibrotic tissues and endothelium of neointima. RAGE was differentially expressed in prototypical Heath Edwards lesions in iPAH. We found significantly increased serum concentrations of sRAGE, esRAGE and HMGB1 in CTEPH. In iPAH, sRAGE and esRAGE were significantly higher than in controls. Serum concentrations of sRAGE were significantly elevated in iPAH(p<0.001) and CTEPH(p = 0.001) compared to AVS. Serum sRAGE was significantly higher in iPAH compared to CTEPH(p = 0.042) and significantly reduced in AVS compared to controls(p = 0.001). There were no significant differences in sRAGE serum concentrations before and after surgical therapy for CTEPH, iPAH or AVS.In endarterectomised tissues from patients with CTEPH RAGE and HMGB1 were identified in myofibroblasts (α-SMAOur data suggest a role for the RAGE pathway in the pathophysiology of CTEPH and iPAH. PEA improves the local control of disease but may not influence the systemic inflammatory mechanisms in CTEPH patients through the RAGE pathway. The European Society of Cardiology (ESC) and European Respiratory Society (ERS) have classified these conditions into six groups. Pulmonary arterial hypertension  can be the result of a wide array of underlying diseases. The entity idiopathic pulmonary arterial hypertension  is used if no underlying causative disease can be diagnosed. The increase in pulmonary vascular resistance (PVR) is related to different mechanisms, including vasoconstriction, proliferative and obstructive remodeling of the pulmonary vessel wall, inflammation and thrombosis. The pathology of idiopathic pulmonary arterial hypertension affects the small distal pulmonary arteries (PAs) with a diameter less than 500 µm. Typical findings are hypertrophy of the media, intimal proliferative and fibrotic changes, thickening of the adventitia with perivascular inflammatory infiltrates, complex and thrombotic lesions Pulmonary hypertension (PH) is currently defined as a hemodynamic and pathophysiological condition with a mean pulmonary artery pressure  is remodeled central and proximal PAs. Organized thrombotic formations build the luminal lining of the PA vessel wall replacing physiological intima As described above, iPAH and CTEPH are progressive diseases of the distal (iPAH) and proximal (CTEPH) pulmonary vessels leading to increased PVR and PAP. In the further course of these diseases right ventricular dysfunction and ultimately right ventricular failure is the leading cause of death. The prognosis of patients is associated to right ventricular performance measures, such as cardiac index and right atrial pressure. Right heart failure is caused by pressure overload of the right ventricle. The increase in wall stress leads to increased wall thickness by muscular hypertrophy (increase in cell size by addition of sarcomeres). However, the chronic exposure to high RV pressures results in RV dilation with a decrease in contractile forces. Pathological inflammatory responses, oxidative stress and humoral responses may further promote right heart failure The overexpression of cytokine cascades may contribute to the progression of heart failure - “cytokine hypothesis” for heart failure The Receptor for Advanced Glycation Endproducts (RAGE), a transmembrane receptor, is a member of the immunoglobulin superfamily of receptors that interacts with different ligands. Since its first characterization in 1992 where a high basal expression in the lung was shown The extracellular soluble form of the receptor (sRAGE) can be detected in serum of patients The RAGE ligand HMGB1 is a non-histone chromosomal protein which functions as a DNA chaperone. The molecule is composed of two homologous DNA binding domains and an acidic tail. Different binding domains for its receptors: RAGE, Toll-like receptor 4 (TLR4) and a p53 transactivation domain have been identified. Once HMGB1 is released from the cell it acts as a signaling molecule, namely a damage-associated molecular pattern molecule (DAMP) There is a myriad of studies  implicating a role for RAGE and its ligands in the pathogenesis of vascular disease. Most of these studies have investigated the systemic vessels. Studies in mice with diabetic atherosclerosis showed that treatment with murine sRAGE suppressed the development of accelerated diabetic atherosclerosis in a dose-dependent manner in vivo animal models of monocrotaline- and Sugen-induced PAH was recently described The hypothesis that RAGE could be a key player in pulmonary hypertension was inferred from evidenced based reviews of the scientific literature 2)-induced PH treatment with sRAGE was protective against increases in RV pressure but did not affect distal pulmonary vascular remodeling. In vitro the administration of sRAGE modulated vasoreactivity of intralobar pulmonary arteries from hypobaric hypoxic mice and further enhanced hypoxia-induced proliferation of Chinese hamster lung fibroblasts In a mouse model of hypobaric hypoxia  in iPAH patients undergoing lung transplantation. Moreover, we hypothesized that systemic inflammatory changes pertaining to RAGE and RAGE ligands  can be measured in patients with CTEPH and iPAH. We compared systemic measurements of CTEPH and iPAH patients to those of patients with aortic valve stenosis (AVS). We aimed to filter out changes specific to PH, a disease characterized by pressure overload of the right ventricle in comparison to a disease that inflicts pressure overload on the left ventricle, such as AVS. Lastly, we sought to investigate the effects of surgical therapy, such as PEA, LuTX and AVR on systemic inflammation.Ethical approval was obtained from the Medical University Vienna review board on human research. Written informed consent was obtained from all patients and volunteers participating in this study.PH is defined as an increase in mean pulmonary arterial pressure (PAP) ≥25 mmHg at rest as assessed by right heart catheterization iPAH is a clinical condition characterized by the presence of precapillary PH in the absence of other causes of precapillary PH We prospectively enrolled 26 patients with CTEPH undergoing PEA, 15 patients with iPAH undergoing lung transplantation, 15 patients with severe aortic stenosis undergoing aortic valve replacement and 33 healthy control subjects between 2010 and 2014. PEA and LuTX surgery were carried out at the department of thoracic surgery, Medical University Vienna, aortic valve replacement (AVR) surgery at the department of cardiac surgery, University Debrecen. The diagnosis of CTEPH and indication for PEA surgery was established by teams of specialists in the diagnosis and treatment of patients with pulmonary hypertension, CTEPH and lung transplantation in every case. All patients with CTEPH were classified to have type 2 disease, intimal thickening and fibrosis proximal to the segmental arteries, according to the intraoperative classification system None of the control subjects studied had any evidence or suspicion of any form of pulmonary hypertension, autoimmune disease, malignancies or infectious conditions at the time of entry into this study. None of the volunteers received anticoagulants, prostaglandins, immunosuppressant therapy or any other type of prescribed medication.Fresh tissues  were harvested at the time of PEA (from patients with CTEPH), lung transplantation  and video-assisted thoracoscopic surgery for recurrent primary spontaneous pneumothorax . Histological diagnoses and classification of iPAH in this study was routinely performed at the clinical institute of pathology at the Medical University Vienna.Serum samples were centrifuged within 60 minutes of collection and stored at −80°C until analysis. In CTEPH the first results from serum analysis were obtained in 26 patients compared to 33 controls . For theFormaldehyde-fixed and paraffin-embedded human PEA specimens, lung tissues and PAs were prepared according to routine protocols of the clinical institute of pathology. Briefly, sections 2 µm in thickness, were baked for 1 hour at 55°C, deparaffinized in three xylenes and rehydrated in ethanol as follows: 2×100%, 1×95%, 1×90%, and 1×70%, followed by PBS. Antigen retrieval was performed by boiling slides at 600 watt (3×5 min) in a microwave oven using citrate buffer at pH 6.0 . Endogenous peroxidase activity was blocked by applying hydrogen peroxide 0.3%. Sections were incubated with 2% bovine serum albumin or blocking serum of the same species as the biotinylated secondary antibody to deplete unspecific protein-protein interactions. Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG  or monoclonal mouse anti-human HMGB1 IgG2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies . Immunoreactivity was amplified using biotin-avidin peroxidase conjugates . 3,3′-diaminobenzidine was used as chromogen . Counterstaining was performed using Mayer’s hematoxylin. Slides were dehydrated with ethanol: 1×95% for 1 min, 1×100% for 6 min and cleared in n-Butanol before mounting .1  and monoclonal rabbit anti-human vimentin IgG . Heat pre-treatment was conducted in Ultra cell conditioner number 1 buffer . Color was developed with Ultraview Universal Detection DAB-kit . Immunohistochemical staining for RAGE and HMGB1 was reproduced with the described automated system.Immunohistochemistry for representative markers of hematopoietic precursor cells CD34, the intermediate filament vimentin and smooth muscle α-actin (α-SMA) were performed on adjacent sections of PEA specimens using the automated Ventana Benchmark platform  according to routine protocols of the clinical institute of pathology. Sections were stained with monoclonal mouse anti-human α-SMA , monoclonal mouse anti-human CD34 IgGOmission of primary antibody served as negative control. Hematoxylin and eosin (H&E) staining was performed according to routine protocols. Analysis and image documentation was done with Axio Imager 2 microscope and AxioVision software .Specimens of explanted lungs from patients undergoing lung transplantation for iPAH were harvested at the time of transplantation. Sections were stained for hematoxylin and eosin (H&E) and Elastica van Gieson (EvG) to visualize elastin and analyzed according to a modified Heath Edwards classification system changes in small pulmonary arteries. Grade 1 is characterized by extension of muscle cells into distal arterioles and thickening of the media of muscular arteries. Grade 2 is defined as hypertrophy of the media with intimal proliferation in small muscular arteries. Grade 3 shows progressive fibrous vascular occlusion and concentric intimal fibrosis. Grade 4 is characterized by progressive arterial dilatation with plexiform lesions, grade 5 by chronic dilatation with fibrosis of intima and media, prominent plexiform and angiomatoid lesions and pulmonary hemosiderosis.Analysis of immunoreactivity was performed by two observers blinded to the type of antibodies used for staining. Two to four slides per patient were assessed. We assigned a score from 0 to 3 to assess staining intensity for RAGE or HMGB1 cytoplasmic or nuclear expression in PEA specimens, PA and small PA in lungs of patients with pneumothorax, iPAH and COPD .To test the hypothesis that RAGE and HMGB1 are involved systemically in patients with pulmonary hypertension, we employed enzyme-linked immunosorbent assays (ELISA) for the detection of sRAGE, esRAGE, S100A9 and HMGB1 in serum of patients with CTEPH, iPAH, AVS and healthy volunteers. All ELISA tests were performed according to the manufacturers’ instructions: sRAGE , esRAGE , S100A9  and HMGB1 . Researchers performing the assays and data analyses were blinded to the groups associated with each sample.We performed an observational study with longitudinal  and cross-sectional design (e.g. serum sRAGE concentration in CTEPH/iPAH compared to controls). Statistical analysis of data was performed using SPSS software . Data were reported as mean (median) ± standard deviation (and standard error mean) in tables and as mean ± standard error mean in the abstract and results section. The concentrations of proteins in serum of patients with CTEPH, iPAH and AVS were compared to those of healthy volunteers using independent Student’s t test or One-way ANOVA for normal (Gaussian) distributions. Kruskal-Wallis rank test or Mann-Whitney U test was used to evaluate non-normal distributions. Post hoc comparisons were computed with the Tukey correction. The paired t-test was applied to before and after measurements made on the same group of subjects, such as sRAGE serum concentrations before and after PEA. Pearson’s χ2 test for independence was used for analysis of categorical data, such as sex differences. Spearman’s rank correlation test was used to assess possible correlations of sRAGE and mean pulmonary artery pressure. The level of statistical significance was set at <0.05 .Diseased central, lobar and segmental PAs in CTEPH patients undergoing PEA showed thromboembolic material incorporated into the remodeled vessel wall in the form of intimal thickening and formation of neointima. The macroscopic aspect of a representative PEA specimen is shown . Hematox+ and HMGB1+ cells in endarterectomized tissues. We found that RAGE+ and HMGB1+ cells were also expressing the intermediate filament vimentin and α-SMA. CD34, a representative marker of hematopoietic precursor cells, did not correlate with the expression pattern seen for RAGE+ HMGB1+ vimentin+ α-SMA+ cells. The expression of CD34 expressing cells can rather be described as sporadic nests in 30.0% of tissues, a homogeneous distribution throughout any of the specimens was not found  and smooth muscle cells of the media (64.8%) of regular main PAs. Vessel-like structures in distal areas of endarterectomized tissues showed RAGE and HMGB1 expression. Neointima  covering the organized thromboembolic material of diseased PAs displayed RAGE and HMGB1 expression . These sIn order to quantify the expression of RAGE in PA changes prototypical for PH we employed immunohistochemical analysis for the detection of RAGE in lung tissue specimens of patients with iPAH, COPD and pneumothorax . PrototyBasic demographic and hemodynamic data of patients with CTEPH and volunteers are detailed in Basic demographic and hemodynamic data of patients with iPAH and volunteers are detailed in We wanted to test the hypothesis that sRAGE serum concentrations are only elevated in diseases of the pulmonary circulation leading to pressure overload of the right ventricle compared to disease leading to pressure overload of the left ventricle, such as AVS. Basic demographic and hemodynamic data of patients with CTEPH undergoing PEA, iPAH undergoing lung transplantation, AVS undergoing AVR and volunteers are detailed in ANOVA analysis of sRAGE concentrations from patients with CTEPH, iPAH, AVS and healthy volunteers revealed significant differences (p<0.001). Post-hoc comparisons showed significantly higher serum concentrations of sRAGE in patients with iPAH (p<0.001) and CTEPH (p = 0.001) compared to AVS . Separate analysis of patients with CTEPH and iPAH showed significantly higher serum sRAGE concentrations in patients with iPAH compared to CTEPH (independent samples t-test: p = 0.042). Serum sRAGE concentrations were significantly reduced in patients with AVS compared to controls (p = 0.001).d to AVS . FurtherThere were no significant differences in sRAGE serum concentrations before and after surgical therapy for CTEPH, iPAH or AVS. Basic demographic and hemodynamic data are detailed in For patients undergoing PEA, early (10 days after PEA) and late (one year after PEA) postoperative serum samples were available: There was no difference in sRAGE concentrations when measurements were stratified between early and late: before and early after PEA (n = 12): : 804.9±214.7 vs. 877.6±167.1, p = 0.767; in comparison to all patients (n = 21) together: as above: p = 0.724.+CD133+Flk-1+) were identified in neointima of proximal thromboembolic material as well as distal regions  in vivo animal models of monocrotaline- and Sugen-induced PAH. Immunofluorescence staining revealed a correlation of RAGE protein expression with disease severity in patients with PAH mean, we chose to investigate RAGE expression in prototypical vessel changes according to the modified Heath and Edwards classification that was routinely applied during the diagnostic workup of lungs with iPAH at our institution. We found a greater staining intensity in endothelial cells as well as smooth muscle cells of higher Heath Edwards grades.The results of this study describe for the first time the expression of RAGE and HMGB1 in myofibroblasts of patients with CTEPH. Prior studies have shown the majority of cells in endarterectomised tissues from patients with CTEPH to be myofibroblasts mean. In C57BL6/J mice exposed to CH-induced PH a statistically not significant nearly two-fold increase in RAGE mRNA was observed. Also of interest are the observations in RAGE knockout (RAGE−/−) compared to wild-type mice exposed to CH: there was the same increase in right ventricular (RV) systolic pressure, but decreased RV hypertrophy in RAGE−/− mice. In the same model RAGE−/− mice neither showed significantly different vascular changes nor did the levels of mouse endothelin −1 (ET-1) or mouse soluble intracellular adhesion molecule 1 (sICAM-1) differ −/− mice is puzzling and does not parallel our data – stronger expression of RAGE in higher Heath Edwards stages - or the above described data by Meloche et al - 6-fold upregulation of RAGE in PASMCs of patients with PAH Recently, a role of the damage-associated molecular pattern molecule (DAMP) HMGB1 was shown to contribute to PH via a TLR4-dependent mechanism in a mouse model of CH-induced PH. In patients with iPAH extra-nuclear HMGB1 in pulmonary vascular lesions was identified. Increased concentrations of serum HMGB1 correlated with PAPThe migration of human pulmonary artery endothelial cells (huPAEC) in vitro could be inhibited by HMGB1 via TLR4 and IRF3-dependent mechanisms In patients with chronic heart failure and impaired left ventricular function activation of the immune system as measured by increased levels of proinflammatory cytokines is associated with poor prognosis Our results on patients with AVS are in line with a previous study that showed that plasma sRAGE levels were significantly lower in patients with AVS than in controls and independently associated with the risk for AVS. In that study there was an inverse correlation with age, cholesterol levels and coronary calcification In lung transplant recipients elevated plasma sRAGE concentrations measured four hours after reperfusion of the lung allograft were associated with longer duration of mechanical ventilation and longer intensive care unit length of stay Multivariate logistic regression analysis revealed plasma sRAGE concentrations immediately after cardiopulmonary bypass surgery to be an independent predictor for postoperative acute lung injury after cardiac surgery in children mean5) Current standard preoperative evaluation of PEA candidates is unreliable in predicting patients at risk for persistent pulmonary hypertension because of surgically inaccessible thromboembolic material or coexistent small vessel disease which are major reasons for poor outcome As sRAGE serum concentrations did not correlate with their corresponding pulmonary artery pressures in this study we can only hypothesize about an on/off-phenomenon of chronic inflammation in iPAH and CTEPH patients. The current information raises new questions. What pathophysiologic threshold has to be reached to turn on chronic inflammation through the RAGE axis? Is the RAGE axis involved in the primary events of remodeling of the thromboembolic material into the PA vessel wall or is it turned on at later stages of CTEPH and iPAH? Could RAGE blockade terminate chronic inflammation in these diseases and be of clinical value in patients as an adjunct to current therapies?We are not suggesting that our absolute concentration values can be used to make any judgments about the diagnosis of, for example CTEPH. While comparative results (e.g. control vs. CTEPH) gained during one experiment could be repeated in separate ELISA experiments, the absolute values for the individual serum samples vary in our experienced hands with the recommended additional reagents from the manufacturer. So we never compare absolute values from samples measured with the RAGE Duoset from different experiments. The intraassay coefficient of variation was 2.3%. We run control serum samples on each ELISA. The RAGE Duoset has quite some interassay variability. The commercially available ELISA is sold for research use only and not for diagnostic purposes. We don’t see this interassay variability with the other ELISA assays used in this manuscript.Soluble RAGE was measured in serum/plasma of other pulmonary diseases with different methods. In stable COPD patients plasma sRAGE was significantly lower compared to healthy control subjects: 400.2 pg/ml vs. 783.3 pg/ml, p<0.001; measured by ELISA, R&D systems, Minneapolis, MN, USA In summary, we have shown the expression of RAGE and HMGB1 in myofibroblasts of endarterectomised tissues from patients with CTEPH and increased expression of RAGE in prototypical lesions in lung of patients with iPAH. Our immunohistochemical results were corroborated by alterations in the serum concentration of soluble RAGE variants and HMGB1. The results may have substantial implications for diagnosis and/or treatment of patients with pulmonary hypertension. PEA improves the local control of disease with the resultant decrease in pulmonary artery pressure but may not influence the systemic inflammatory mechanisms in CTEPH patients through the RAGE pathway. A more detailed understanding of the RAGE-HMGB1 axis and related molecules in diseases associated with pulmonary hypertension is needed and warrants future study.