TNF-α plays a role in angiogenesis and collagen synthesis, both essential in the wound healing process. There are concerns that pre-operative anti-TNF-α treatment may influence the surgical stress response and increase the risk of surgical complications. The aim of this study was to describe the surgical stress response in patients with inflammatory bowel disease (IBD) and to investigate whether the pre-operative administration of anti-tumor necrosis factor alpha (anti-TNF-α) agents modify the surgical stress response.This was a prospective, multi-center cohort pilot study. The primary outcome was the change in concentration of immunological biomarkers of the surgical stress response . Secondary outcome measures were changes in IL-8, IL-17A, C-reactive protein, white blood cells, cortisol, transferrin, ferritin, and D-Dimer in addition to 30 days’ post-operative complications and length of post-operative stay in the hospital (LOS).Forty-six patients with IBD undergoing major abdominal surgery were included, and 18 received anti-TNF- α treatment pre-operatively. Peak increase of most of the immunological biomarkers occurred 6 hours after surgical incision. Then the concentration decreased after 24 h followed by a plateau at 48 h. After adjusting for confounders including detectable blood concentrations, no difference in the concentrations of immunological, endocrinological or haematological biomarkers of stress was found between anti-TNF-α treated and anti-TNF-α naïve patients. No increase in post-operative complications or LOS was noticed in patients who received anti-TNF-α treatment.Anti-TNF-α did not affect surgical stress response in this pilot study. Withdrawal of anti-TNF-α drugs prior to surgical intervention in IBD patients might not be justified without measurement of drug concentration and drug antibodies.NCT01974869.Clinicaltrails.gov.:  Surgical injury induces a stress response with activation of endocrine, metabolic, and immunologic mediators aiming to restore hemostasis and induce tissue repair. The immunologic and inflammatory response to stress is regulated by cytokines produced in activated macrophages, fibroblasts, endothelial cells, and giant cells. Tissue injury and infection are sensed by a group of protein receptors called pattern recognition receptors (PRRs) that can be activated by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). A wide range of PRRs have been described, including membrane bound Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), or cytoplasmic receptors such as NOD (Nucleotide-binding oligomerisation domain)-like receptors (NLR). Following stimulation of these receptors, multiple downstream proteins will be activated leading to intracellular signaling pathways that culminate in activation (phosphorylation) of transcription factors such as NF-kB among others which in turn drive the production of a large array of both pro- and anti-inflammatory cytokines and chemokines , 2.The cytokines of special interest include interleukins (IL) and tumor necrosis factor-alpha (TNF-α).Anti-TNF-α agents  are antibodies directed against this key cytokine acting in two ways: First, by scavenging soluble TNF-α, thereby preventing activation of immune cells via TNF-a-receptors; and second, by “reverse signaling” acting on membrane bound TNF-α -receptors on monocytes and T-cells inducing apoptosis and inhibition of further cytokine release .Besides being an important component of the immune defense, TNF-α plays a role in angiogenesis , collageThe aim of this study was to describe the surgical stress response in patients with IBD undergoing surgical intervention and to investigate whether anti-TNF-α agents modify the surgical stress response.The null hypothesis was that pre-operative administration of anti-TNF-α agents within 12 weeks before surgery, have no significant effect on surgical stress response. To investigate this; a prospective, non-interventional multi-center pilot study was designed.The primary outcome measure was the difference in the plasma concentrations of the main immunological biomarkers of surgical stress response  between anti-TNF-α treated patients and anti-TNF-α naive.The secondary outcome measures were difference in the plasma concentrations of other biomarkers of surgical stress including IL-8, IL-17A, the ratio of TNF-α/ IL-10 and Il-6/IL10, cortisol, transferrin, ferritin, and D-Dimer in addition to 30-days, post-operative complications and length of hospital stay (LOS). Overall complication was defined as any deviation from the expected post-operative recovery. Intra-abdominal septic complications (IASC) were defined as overt anastomotic leakage, intra-abdominal abscess formation or enteric fistula. Superficial surgical site infection (SSI) was defined as clinically documented skin infection at the site of surgery with or without positive culture. Grade of complications were assessed using Clavian-Dindo classification of surgical complications.The choice of sampling intervals at six, 24 and 48 h after surgical incision was based on previous investigations –13. BiomInclusion criteria: adult patients with Crohn’s disease (CD) or ulcerative colitis (UC) who were scheduled to elective intestinal resection or terminal stoma closure in three Danish university hospitals during the study period (March 2014–May 2016). Open as well as laparoscopic approaches were included.Exclusion criteria: patients with pre-operative sepsis, acute intestinal obstruction, patients operated in acute setting (within 48 h of admission) and patients who had loop ileostomy take down without laparotomy or laparoscopy.Peripheral blood samples were taken before the induction of anesthesia, and six, 24 and 48 h after surgical incision. EDTA plasma and serum was separated by centrifugation, aliquoted and stored at − 80 °C until analysis.The concentration of anti-TNF-α biological compounds administered pre-operatively (drug concentration) was measured in peripheral blood at the day of surgery together with antibodies against the specific compound (anti-drug antibodies). Details of the method used explained in the laboratory homepage .Cortisol was measured by ELISA . IL-6, IL-10, IL-17A, and TNF-α were measured by a human high sensitive magnetic ProCartaPlex luminex kit . IL-8 and D-Dimer were measured using ProCartaPlex Human IL-8 simplex, ProCartaPlex Human D-Dimer simplex, and Human Basic kit . All samples were measured in duplicates according to the manufactures instructions, using the mean for statistical analyses. Plasma levels of CRP, transferrin, ferritin and D-dimer were measured using standard methods by the Department of Clinical Biochemistry, Copenhagen University Hospital, Amager and Hvidovre, Denmark.All the operations took place between 08:00 a.m. - 04.00 p.m. to avoid circadian rhythm as a confounder. General anesthesia was administered according to the standard practice of the anesthesia department in the participating hospitals. All patients received single prophylactic pre-operative antibiotics at the induction of anesthesia. The type and dose was determined by local standard of pre-operative care in the participating hospitals. Laparoscopic surgery and enhanced post-operative recovery principles were the standard procedures in the participating centers.Reference values for the changes in the biomarkers for surgical stress in IBD patients were not available at the time of the study to allow precise sample size calculations. Chalhoub et al. showed that 28 patients were needed to demonstrate a significant change in TNF-α concentration after moderately stressful surgery . MoreovePre-operative and peri-operative characteristics were compared between anti-TNF-α treated and anti-TNF-α naïve patients with chi-squared tests. Median, inter-quartile range (IQR), minimum and maximum were used to visualize changes in the concentration of biomarkers from baseline (before operation) to one of the post-operative follow-up time-points . The difference of the medians of the changes from baseline between anti-TNF-α treated and anti-TNF-α naïve patients was assessed using a bootstrap approach in which patients (retaining the up-to-four-measurements per patient) were sampled with replacement using 1000 bootstrap replicates. To reduce bias from confounding, the calculated medians were weighted by a propensity score, i.e. the inverse of the estimated probability of the received anti-TNF-α regimen conditional on potential confounders. These probabilities were estimated from a multivariable logistic regression model included: Harvey-Bradshaw index for disease severity, nutritional risk screening score, parenteral nutrition, previous IBD-related abdominal operations, steroid stress dose, pre-operative Dexamethasone, epidural analgesia, access to abdominal cavity (open versus laparoscopic), type of resection and disease classification in case of CD. These were pre-operative factors that were either significantly different between the two anti-TNF-α regimens, or deemed important determinants from clinical experience. Propensity scores were re-calculated within each bootstrap replicate.A permutation test, in which the four measurements for each patient were randomly redistributed over the time points in 1000 replicates to assess the null hypothesis of no development, was used to assess the statistical significance of the changes in the median concentration of biomarkers over time.p < 0.10.Logistic regression was used to investigate post-operative outcome where adjustment for confounding was done by stepwise backwards elimination, starting with a model including all pre- and peri-operative characteristics deemed clinically and/or statistically significant different between the two treatment groups. Variables were then removed one by one until all variables had All analyses, except for the bootstrap analyses, were done using IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp.2010. The bootstrap analyses were performed in the R environment for statistical computing version 3.1.2. A significance level of 5% was chosen.We used non-parametric, robust methods for inference, i.e. a non-parametric bootstrap on the difference of propensity score weighted medians. These methods are described in detail in the statistics section of the paper. The SAMPLE guidelines were not very applicable to the inferential methods we used. This has been referred to in the manuscript.Patients were identified using outpatient’s clinic records, operation lists and reports from IBD conferences. All patients who fulfilled the inclusion criteria accepted to participate in the project. One patient was excluded because blood samples were accidently discarded. This explorative study succeeded, thus, in recruiting 46 patients, of which 18 had one type or another of anti-TNF-α agent treatment within 3 months prior to surgery.Median age was  years, 25/46 of the patients were females (54.3%). Median body mass index was . In anti-TNF- α group, 4/18 (22.2%) had one or more co-morbidities compared to 7/28 (31.8%) in anti-TNF-α naïve. No difference in the mean duration of disease was found between anti-TNF-α treated patients (7.61 SD ± 7.82) years and anti-TNF-α naive (10.11 SD ± 9.15). Pre-and intra-operative patients’ characteristics are reported in Table p = 0.003). Anti-TNF-α treated patients were more likely to receive pre-operative parenteral nutritional support (p = 0.028). Moreover, anti-TNF-α treated patients with CD had a longer ileocecal/ileo-colic resected segment (mean 31.11 SD ± 35.51) cm versus 27.43 SD ± 18.83 p = 0.036) and were more likely to suffer from a stricturing CD phenotype (10/76.9% versus 8/42.1% p = 0.01). But this did not affect the results in multivariate analyses. Type of surgical incision and type of bowel resections were similar in the two groups.Anti-TNF-α naïve patients had higher rate of previous IBD surgeries compared to anti-TNF- α treated patients  except TNF-α, IL-17A and cortisol.Figures There was a tendency towards a higher concentration in most of the inflammatory biomarkers in patients treated with anti-TNF-α agents compared to the anti-TNF-α naive as shown in Fig. Sub-group analyses was done by selecting patients who underwent laparoscopic ileocecal/ileo-colic resections (12/46) to obtain a homogenous group of patients with same type of surgical procedure. Then by comparing anti-TNF-α treated and anti-TNF-α naïve in this sub-group showed no significant difference in surgical stress response (results not shown).No difference was found in adjusted analyses of overall complications between the two groups (27.8% versus 28.6%), superficial SSI (7.8% versus 7.1%), IASC (5.6% versus 7.1%) and re-admission rates (22.2% versus 25%). Mean LOS was 5.33 (± 2.57) for anti-TNF- α treated versus (6.25 ± 3.01) in the anti-TNF- α naive group. The difference was not statistically significant. The time interval between last dose of anti-TNF- α and the day of surgery did not affect the rate of complications. Nor did the type of anti-TNF- α agents used  and the lack of measurements of other influential factors in wound healing, for instance vascular endothelial growth factor (VEGF), granulocytes-macrophage colony stimulating factor (GM-CSF) and transforming growth factor (TGF-β). However, these factors are regulated by IL-6  and otheThis pilot study showed no difference in surgical stress response between anti-TNF-α treated and anti-TNF-α naïve patients. Withdrawal of anti-TNF-α drugs prior to surgical intervention in IBD patients might not be justified without measurement of drug concentration and drug antibodies. Further large sample prospective studies are needed.


Facts alone tend to be isolated in every narrowing niche of professionalism; views alone tend to degenerate into strong opinions nobody is interested in; and vision alone tends to become a nightmare if unsupported by facts and a beholder’s sharp view. In conjunction, they constitute the basic elements needed for a truly translational medicine, where findings from the bench may become part of a clinician’s view and where today’s clinical expertise may take a jump from a sleepwalker’ s routine to the disruptive insight of the visionary”Jan Gerris, Editorial, Facts Views Vis Obgyn, Vol 1, Nr 1“FV&V is a paragon of 21st century communication. In addition to new information, emphasis on discussion and the social interface makes it special while it strives to put related subspecialties in orbit around the star named reproduction.”Howard Jones, Editorial, Facts Views Vis ObGyn, Vol 3, Nr 2It’s always nice to look back and try to summarize what we achieved during these last 10 years of FV&V. We observed a broad input from all different fields including obstetrics, perinatal medicine, gynaecology and reproductive health. It was fascinating that many papers showed a lot of interest in the global perspective of our specialty. Structured reviews, strong opinion and viewpoint papers, excellent historical reviews, exactly what we were looking for from the very beginning.This could only be realized by an outstanding editorial board, a high number of faithful referees, the hard and dedicated work of the ‘Universa Press’ team, the scientific support of our universities and the financial input of the pharmaceutical industry.Thanks to our artistic reviewer, Koen Vanmechelen, our Journal got a different but very attractive look when compared to other scientific journals. The confrontation of art and science turned out to be unique: “One picture is worth ten thousand words” (Chinese proverb).In 2014, we succeeded to become PubMed-cited, the process was arduous and complex, but we finally got it. Thes was a major achievement in the history of our Journal. Nowadays the Journal is coverded by major indexing services such as BMJ Clinical Evidence, PMC , Pubmed, Web of Science, Google Scholar and Europe PMC.Besides the four regular issues published annually, we also published six Monographs. The first two Monographs on “Artificial Insemination” and “Social aspects of infertility care in developing countries” were published in 2010 and distributed to more than 8000 participants at the annual ESHRE meeting in Rome.rd Monograph “When training becomes fun for trainers and trainees” was published in 2012 and focused on medical education in obstetrics and gynaecology. It became a very attractive book with 13 papers, written by prominent colleagues from Europe and other parts of the world, describing a number of current issues and developments in training within our field. This Monograph was distributed to all participants of the EBCOG 2012 meeting in Tallinn, Estland.The 3th Monograph on “Biomedical infertility care in poor resources countries: Barriers, Access and Ethics” was also published in 2012 and distributed to more than 10000 participants at the annual ESHRE meeting in Istanbul, Turkey. This was the result of a successful collaboration between the ESHRE Special Task Force on “Developing countries and infertility”, the WHO, the University of Amsterdam and the Walking Egg non-profit organisation.The 4th Monograph “Preterm birth” was edited by Robert E. Garfield, Phoenix, USA, which turned out to become a very attractive book, with 12 papers from around the globe from some of the leading experts on preterm labor and birth. In the USA copies of the monograph were sent to all members of the Society for Maternal-Fetal Medicine and the Society for Gynecological Investigation.The 5On the occasion of a meeting in Cape Town, South-Africa, we published and distributed our 6th Monograph in bringing together the main actors of the Walking Egg Project and representatives of the interested centres and patient groups in Africa. The final objective of the Walking Egg Project is the implementation of infertility services in many developing countries preferably integrated in existing family planning and mother care services.Submissions: Out of 582 submissions, 310 papers were published, not taking into account the 40 editorials, 6 “A tribute to” papers and 12 “History of Medicine” contributions. It was our aim from the beginning to give a forum to “Views and Vision” papers. More than 20% of our publications (66/310) were “Opinion and Viewpoint” papers, and this shows exactly what we were standing for. Although PhD-summaries and Who’s Who papers don’t contribute to a reasonable Impact Factor, we are proud and happy to have published them.In From 2019 on, our Journal will be owned by the European Society for Gynaecological Endoscopy (ESGE) in cooporation with the Walking Egg non-profit organization. It will become the official Journal of the ESGE, this means that the major part of the papers will present the surgical aspects of endoscopic imaging and allied techniques. Another part of the Journal will still publish selected papers as we did before.We have completed 10 exciting years. On behalf of the editorial board and the publisher of “Facts Views Vis ObGyn”. I would like to thank all the authors and referees who contributed to the journal since 2009. It was an honour and a pleasure to be part of this exciting project. A bright and interesting future is waiting for us.We still count on our referees and our artistic reviewer, Koen Vanmechelen, who is responsible for the very attractive look of our journal. The confrontation of art and science will be continued.I look forward to a promising future.Editor-in-ChiefWillem Ombelet


Triticum aestivum L.) genotypes originating from 38 countries, representing more than a century of breeding and variety development. Visualisation of the pedigree enables illustration of the key developments in United Kingdom wheat breeding, highlights the wide genetic background of the UK wheat gene pool, and facilitates tracing the origin of beneficial alleles. A relatively high correlation between pedigree- and marker-based kinship coefficients was found, which validated the pedigree and enabled identification of errors in the pedigree or marker data. Using simulations with a combination of pedigree and genotype data, we found evidence for significant effects of selection by breeders. Within crosses, genotypes are often more closely related than expected by simulations to one of the parents, which indicates selection for favourable alleles during the breeding process. Selection across the pedigree was demonstrated on a subset of the pedigree in which 110 genotyped varieties released before the year 2000 were used to simulate the distribution of marker alleles of 45 genotyped varieties released after the year 2000, in the absence of selection. Allelic diversity in the 45 varieties was found to deviate significantly from the simulated distributions at a number of loci, indicating regions under selection over this period. The identification of one of these regions as coinciding with a strong yield component quantitative trait locus (QTL) highlights both the potential of the remaining loci as wheat breeding targets for further investigation, as well as the utility of this pedigree-based methodology to identify important breeding targets in other crops. Further evidence for selection was found as greater linkage disequilibrium (LD) for observed versus simulated genotypes within all chromosomes. This difference was greater at shorter genetic distances, indicating that breeder selections have conserved beneficial linkage blocks. Collectively, this work highlights the benefits of generating detailed pedigree resources for crop species. The wheat pedigree database developed here represents a valuable community resource and will be updated as new varieties are released at https://www.niab.com/pages/id/501/UK_Wheat_varieties_Pedigree.Information on crop pedigrees can be used to help maximise genetic gain in crop breeding and allow efficient management of genetic resources. We present a pedigree resource of 2,657 wheat ( Breeding activities undertaken in the world’s most important crop species have resulted in large increases in yield potential over the last century. Bread wheat is a key crop for both human and animal nutrition worldwide. To help inform future breeding and research activities, we have developed a pedigree resource of over 2,600 bread wheat accessions, originating from 38 countries and representing more than a century of breeding and variety development. Pedigree-based relationships between lines are largely confirmed by genetic marker data. By combining the genetic and pedigree data sets, we are able to identify genetic signatures of selection across the pedigree, identifying genomic regions selected for via modern breeding activities. The resource developed here will serve as an evolving platform to inform and manage wheat genetic diversity in breeding programmes around the world and highlights the utility of developing and exploiting similar resources in other crop species. Triticum aestivum L.) pedigree would be a valuable resource for the wheat research and breeding communities. Its development would allow inheritance and origins of beneficial genes and alleles to be tracked through the pedigree to identify sources of traits and genetic variation for research and efficient exploitation. For example, as new races of pathogens evolve to overcome variety resistances [1 hybrid varieties [Information of variety pedigree  can be used by breeders to prioritise crosses between highly performing parents whilst maintaining genetic diversity in the offspring for selection. However, development of varieties by commercial breeding companies in recent decades may have resulted in knowledge fragmentation and duplication of resources . We propistances , sourcesistances . Within istances . Crossesistances  and alsoarieties –8. Inforarieties .Studies of smaller scale pedigrees of crop varieties have often compared calculations of kinship between varieties based on pedigree or genetic marker data. However, these studies commonly used low marker numbers and found low correlations between the two methods for estimating kinship –12. Suchhttp://wheatpedigree.net/), this pedigree information is only available on a ‘per accession’ basis. Few other pedigree resources are available in wheat. Here, we present a large-scale pedigree of United Kingdom (UK) wheat varieties and their ancestors, available in a format suitable for visualisation in software such as Helium  [Large-scale pedigree databases have been developed as research tools in other crops, including oats  and riceand, UK) , Pedigreand, UK) , and Pedand, UK) , represeand, UK) –19. We shttps://www.niab.com/pages/id/501/UK_Wheat_varieties_Pedigree), allowing visualisation of the pedigree structure  was found between the two equivalent kinship matrices. Notable, nonrandom deviations from this relationship were found, which facilitated identification of erroneous pedigree information, errors in genotyping, or evidence of strong selection within crosses. Detailed investigation of marker kinships between varieties and their immediate ancestors and descendants revealed clearly erroneous information in either the pedigree or seed source used for genotyping for 40 varieties. When these varieties were removed, an improved correlation of 0.68 was found between marker- and pedigree-based kinships. The removal of five additional closely related varieties with information on only one parent improved this correlation to 0.71. Detection of pedigree and/or seed errors is exemplified in To account most effectively for multiple generations of inbreeding used in wheat variety development, kinship calculations were made using an augmented pedigree, including seven intermediate generations of selfing from parents to progeny for each accession with parent information. Using the 15,852 entries within the augmented pedigree, 121,391,571 pairwise comparisons of kinship were calculated; 29% of these comparisons were between entries without known common ancestors in the pedigree that gave a kinship of zero and were omitted from further analysis. Pedigree-based kinship coefficients were compared with a subset of 454 varieties for which genotypic data  were available. For these, kinship between different varieties based on pedigree varied from 4.7 × 10p ≤ 0.001 significance threshold of 0.54 (Evidence for selection by breeders within family crosses was found using a combination of pedigree and genetic marker data. Out of a set of 109 ‘simplex families’ with SNP data (in which a simplex family represents two parents and their one progeny), the most closely related parent to the progeny within each family shared a median proportion of genome of 0.57, with 77/109 to a greater extent than expected based on 1,000 simulations with gene dropping at the  of 0.54 . The varP value of 0.001), compared to only 0.1% showing higher diversity than five standard deviations of the simulated distribution found from 100 gene dropping simulations , gene dropping simulations compared observed- and simulated-allelic diversity measures for 45 derived varieties released after the year 2000 . Averageulations ; Table 12 fell to 0.15 was 25.9 cM in the observed data, compared to 11.2 cM for the simulated data , defined by the International Union for the Protection of New Varieties of Plants (UPOV) as when ‘(i) it is predominantly derived from the initial variety, or from a variety that is itself predominantly derived from the initial variety, while retaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety, (ii) it is clearly distinguishable from the initial variety and (iii) except for the differences which result from the act of derivation, it conforms to the initial variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety’ . We founVRN-A1, VRN-B1, VRN-D1 . This is expected, because these loci of major phenotypic effect are already fixed in the materials and time spans used here to investigate selection. However, at the genetic resolution currently available, the major yield QTL on 7AL (Qyld.csdh.7AL) is thought to be principally due to increased grain number per ear [We demonstrated selection over multiple generations by comparing observed and simulated genotypes in a subset of the pedigree in which 110 genotyped varieties released before the year 2000 were used to predict the distribution of marker alleles in the absence of selection of 45 varieties released after the year 2000. Amongst these 45 varieties, significant deviations from the expected simulated allelic diversity were used as an indication of breeders’ selection. When directional selection is in favour of one allelic variant, allelic diversity is ultimately reduced. Increased diversity could indicate selection for a more equal balance of alleles in the population, which may be the case if different alleles are favoured in contrasting wheat classes, such as for yield or quality. Alternatively, it could result from a transient polymorphism resulting from a rare allele, which has increased in frequency under selection but has yet to be fixed. Our results indicate that whilst average allelic diversity across all markers was similar to expected from simulations, a small number of lower than expected values were found for individual makers. This gives an indication of the location of genomic regions consistently under selection during the development of varieties released in the 21st century. The regions identified at the stringent significance thresholds used here are not localised to the known major flowering time loci iewed by ) or the  per ear –41 appro per ear  and SNP It has been suggested that progress in wheat breeding has in part been a result of assemblage of beneficial linked epistatic gene interactions . This is1, provided the parents are homozygous, but we may detect distortion after several subsequent generations of selfing (or doubled haploid production) with accompanying selection. Because we do not have large numbers of simplex families, tests for distortion at individual loci would have low power. However, we have found extensive distortion in favour of one of the parents in many crosses.Pedigree-based tests for selection are more common in animals and humans than in plants. Our test for selection within families is essentially the same as the transmission disequilibrium test –49, whicLarkin and colleagues  detectedhttps://www.niab.com/pages/id/501/UK_Wheat_varieties_Pedigree, where it will be periodically updated to incorporate newly released varieties. It is anticipated that further engagement with the wheat breeding community will enable correction of errors in historic pedigrees, as well as provision of pedigree data that is not yet publicly available, thus augmenting the utility of the resource. Whilst the pedigree focuses on UK wheat varieties, significant historic crossover with varieties originating from other countries will facilitate interlinking of fragmented wheat pedigrees maintained in other countries or breeding companies and extend the utility of the resource to the wheat breeding community across the world. Future analysis of the current pedigree will focus on identifying selection on haplotypes.We present a pedigree resource for wheat varieties released in the UK up to 2017. The resource is available at We present a comprehensive wheat pedigree as a resource for the wheat research and breeding community. Recently developed software enables visualisation and navigation of the pedigree, as well as highlights historically important varieties and the diverse origins of elite UK wheat varieties. Comparison of kinship coefficients calculated using the pedigree, as well as genetic markers, validated the pedigree and allowed identification and correction of pedigree and genotyping errors. In conjunction with pedigree and genotypic data, gene dropping simulations demonstrated significant effects of selection within crosses as well as over multiple generations of the pedigree, modulating allelic diversity and conserving LD. These analyses identify the genomic regions controlling putative wheat breeding targets and serve as a model for the identification of genomic regions controlling breeding targets in other crops. The resource developed here will serve as an evolving platform to inform and manage wheat genetic diversity in breeding programmes in the UK and around the world and highlights the need to develop and maintain similar resources in other crop species.http://genbank.vurv.cz/ewdb/), associated information with commercial varieties released in the UK , textbooks [http://www.niab.com/pages/id/326/Resources/. Pedigrees were visualised using Helium version 1.18.03.15 [Pedigree information was sourced from publicly available breeders’ records, genebank passport information  for the 454 accessions that were in common with the pedigree. An IBS approach was chosen for simplicity and ease of comparison with the pedigree estimates, because the assumption that alleles are drawn from a random global population—and the subsequent estimation of allele frequencies—is problematic for calculating genomic relationship matrices in the complexly structured population of inbred lines found in the pedigree. The 26,018 available polymorphic Illumina iSelect SNPs were thinned to remove closely linked markers by removal of one of each pair of markers with an absolute correlation >0.75 to minimise the effect of the very high levels of marker clustering observed in cereal crops [Kinship coefficients based on genetic markers were calculated based on identity by state (IBS) allele-sharing implemented in R/EMMA , the proportion of alleles inherited from each parent was calculated. To estimate the variation in this measurement in the absence of selection, Genedrop  simulation software (as described in ) was useetic map . Seven gTo investigate selection effects over multiple generations, a subset pedigree was made that included 110 founding accessions with genotype data that were released before the year 2000. This date was chosen as the approximate median value of the date of release of the varieties with available genotype data. From these genotyped founders released prior to 2000, 207 descendants could be identified, which do not include ancestry outside of the founder gene pool. Forty-five of the descendants were released after the year 2000 and had genotypic data. One hundred simulations were carried out for this pedigree subset using Genedrop software with the 1,821 polymorphic SNPs available with genetic map positions sourced from [pi is the frequency of the ith allele.From the 45 descendants, estimates of allelic diversity were calculated for each biallelic marker locus for both observed and simulated genotype data. Diversity  was calc2) were calculated for all 311,655 pairwise comparisons among 790 markers across the genome for observed and simulated data to estimate LD. These markers were a subset of the 1,821 outlined above with a minor allele frequency >0.2. For each chromosome, the simulated and observed LD decay curve was modelled using a loess curve fit, with span smoothing parameter set to 0.75. The fitted curve was used to estimate the genetic distance at which LD fell to 0.15 for each chromosome in the observed and simulated data sets. The fitted curve was additionally used to estimate the average LD at different genetic distances along the chromosomes.Squared correlation coefficients .To anchor genetic markers to the physical map, DNA sequences associated with selected genetic markers were used as queries for BLASTn   searchesS1 Fig(PNG)Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S3 Fig(PNG)Click here for additional data file.S4 FigPoints in red indicate kinship comparisons involving the varieties ‘Cyber’ and ‘Maris-Ensign’, for which the kinship and pedigree relationship estimates place the varieties in different major wheat populations (‘winter’ versus ‘spring’).(TIF)Click here for additional data file.S5 FigNode size is proportional to number of direct offspring in the subset.(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S1 TableAdditional information (year of release and country of origin) is also presented in Helium format.(XLSX)Click here for additional data file.S2 Table2 equals 0.15 from fitted loess curves for observed and simulated genotypes. Summary statistics shown are per chromosome, per subgenome, and across all chromosomes. LD, linkage disequilibrium.Expressed as distance (cM) when linkage R(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Background: The purpose of this study was to investigate cytochrome P450-7B1 (CYP7B1) in the human and rat testes to regulate 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activity. We hypothesized that dehydroepiandrosterone (DHEA) and its product 7α-hydroxydehydroepiandrosterone (7αOHD) after catalysis of CYP7B1 played a critical role in driving the direction of 11β-HSD1, because 7αOHD is an alternative substrate for 11β-HSD1.Methods: We examined the influence of DHEA and 7αOHD on 11β-HSD1 activities in both intact Leydig cells and microsomes using radioactive substrates and identified the location of CYP7B1 in Leydig cells using immunohistochemical staining, Western blot, and qPCR.Results: We found that DHEA stimulated 11β-HSD1 oxidase activity in intact cells (EC50 = 0.97 ± 0.11 μM) and inhibited its reductase activity (IC50 = 1.04 ± 0.06 μM). In microsomes, DHEA was a competitive inhibitor of the reductase activity. The 11β-HSD1 oxidase activity in intact cells was inhibited by 7αOHD (IC50 = 1.18 ± 0.12 μM), and the reductase activity was enhanced (EC50 = 0.7 ± 0.04 μM). 7αOHD was a competitive inhibitor of 11β-HSD1 oxidase. CYP7B1 was present in rat Leydig cells, as shown by immunohistochemistry, Western blotting, and qPCR analysis.Conclusion: Our results are consistent with a conclusion that DHEA in the circulation driving 11β-HSD1 toward an oxidase in Leydig cells mainly through inhibiting the reductase of the enzyme, while 7αOHD  drives the enzyme toward the opposite direction. There are two isoforms: type I (11β-HSD1) and type II (11β-HSD2) . 11β-HSDd testis , 6. 11β-nd colon .+/NADPH ratio in the smooth endoplasmic reticulum (SER). 11β-HSD1 is a membrane binding enzyme with cofactor-binding domain facing SER lumen (+/NADPH with hexose-6-phosphate dehydrogenase (H6PDH), which stays in SER lumen  (+ as cofactor into 7keto-DHEA (7KD) and NADPH  –22. Howend NADPH . We hypo3H] Corticosterone (3H-CORT), specific activity 40 Ci/mmol, was purchased from Dupont-New England Nuclear . 11-dehydrocorticosterone (3H-DHC) was prepared from labeled 3H-CORT as described earlier . -CORT . 11β-HSD1 reductase activity assay tubes contained 25 nM DHC spiked with 60,000 dpm [3H]-DHC. 2.5 × 104 intact ALCs were added to start the reaction and the reaction was maintained in 34°C and 75 rpm shaking water bath for 30 min. The reactions were stopped by adding 2 ml ice-cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin layer plates in chloroform and methanol , and the radioactivity was measured using a scanning radiometer . The percentage conversion of CORT to DHC and DHC to CORT was calculated by dividing the radioactive counts identified as DHC  by the total counts associated with CORT plus DHC.11β-HSD1 oxidase and reductase in intact ALCs were performed as previously described .11β-HSD13H]-CORT and 0.2 mM NADP+ for oxidase or 25 nM [3H]-DHC and 0.2 mM NADPH for reductase activity in presence of different concentrations of DHEA or 7αOHD. For testing mode of action, microsomes were incubated with 2 nM to 10 μM [3H]-CORT and 0.2 mM NADP+ for oxidase or 2 nM to 10 μM [3H]-DHC and 0.2 mM NADPH for reductase activity. 1.5 μg of ALC microsomes were added to initiate the reaction. The reactions were stopped by adding 2 ml ice-cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin layer plates in chloroform and methanol , and the radioactivity was measured using a scanning radiometer . The percentage conversion of CORT to DHC and DHC to CORT was calculated by dividing the radioactive counts identified as DHC  by the total counts associated with CORT plus DHC.11β-HSD1 oxidase and reductase in microsomes were performed as previously described . For meaad hoc DUNCAN multiple comparisons testing to identify significant differences between groups when three and more groups were calculated, or by the student t-test when two groups were calculated. All data are expressed as means SEM. Differences were regarded as significant at *P < 0.05 or **P < 0.01 or ***P < 0.001.Experiments were repeated three to four times. Data were subjected to analysis by one-way ANOVA followed by 50 value of DHEA of inhibiting 11β-HSD1 reductase activity was 1.04 μM (11β-HSD1 reductase converts DHC into CORT. We measured 11β-HSD1 reductase activity in intact ALCs in presence of DHEA (10 nM−10 μM). DHEA inhibited 11β-HSD1 reductase activity with the lowest observable effective level (LOEL) of 100 nM and it lowered 11β-HSD1 reductase activity to 12% of control at 10 μM . The IC5 1.04 μM . This in50 value of DHEA of stimulating 11β-HSD1 oxidase activity was 0.97 μM (11β-HSD1 oxidase converts CORT into DHC. We measured 11β-HSD1 oxidase activity in intact ALCs in presence of DHEA (10 nM-10 μM). DHEA stimulated 11β-HSD1 oxidase activity with LOEL of 100 nM . DHEA in 0.97 μM . We calc 0.97 μM  and foun 0.97 μM . DHEA in50 value of DHEA of inhibiting 11β-HSD1 reductase activity in ALC microsomes was 1.29 μM . DHEA decreased ALC microsomal 11β-HSD1 reductase activity with LOEL of 100 nM and it lowered 11β-HSD1 reductase activity to 25% of control at 10 μM . The IC5 1.29 μM . This is11β-HSD1 oxidase activity was measured in ALC microsomes in presence of DHEA (10 nM-10 μM). DEHA did not affect microsomal 11β-HSD1 oxidase activity. Actually, DHEA also inhibited 11β-HSD1 oxidase activity at 10 μM . AlthougCyp7b1 expression in Leydig cells was developmentally relevant. Cyp7b1 mRNA levels detected by qPCR in templates prepared from rat testis on postnatal days 2–90 did not vary significantly (Cyp7b1 mRNA was 3-folds more abundant in ILCs and 50-folds more abundant in ALCs than that in PLCs (Cyp7b1 mRNA levels were enriched by 33-folds in ALCs when compared to age-matched testes. This suggests that Cyp7b1 is primarily expressed in ALCs.We determined that the timing of ficantly . When Le in PLCs . Cyp7b1 Cyp7b1) catalyzes DHEA into 7αOHD and that the epimerase activity of 11β-HSD1 converts 7αOHD into 7βOHD . 7αOHD increased 11β-HSD1 reductase activity with LOEL of 100 nM . 7αOHD is 0.7 μM . This su50 value of 7αOHD of inhibiting oxidase activity was 1.17 μM (11β-HSD1 oxidase activity was measured in intact ALCs in presence of 7αOHD (10 nM−10 μM). 7αOHD decreased 11β-HSD1 oxidase activity with LOEL of 100 nM . 7αOHD d 1.17 μM . We calc 1.17 μM . 7αOHD d50 value of 7αOHD of increasing 11β-HSD1 reductase activity was 200 nM (11β-HSD1 reductase activity was measured in ALC microsomes in presence of 7αOHD (10 nM-10 μM). 7αOHD increased 11β-HSD1 reductase activity with LOEL of 100 nM. However, at the highest concentration (100 nM), 7αOHD did not affect 11β-HSD1 reductase activity . The EC5s 200 nM .50 value of 7αOHD for inhibiting oxidase activity was 0.93 μM (11β-HSD1 oxidase activity was measured in ALC microsomes in presence of 7αOHD (10 nM−10 μM). 7αOHD decreased 11β-HSD1 oxidase activity with LOEL of 100 nM . 7αOHD d 0.93 μM . 7αOHD s 0.93 μM . The actWe measured the rates of reductase reactions in ALC microsomes in presence of different DHEA concentrations  in order to evaluate the mode of action. DEHA was a competitive inhibitor of 11β-HSD1 reductase .We measured the rates of oxidase reactions in ALC microsomes in presence of different 7αOHD concentrations  in order to evaluate the mode of action. 7αOHD was a competitive inhibitor of 11β-HSD1 oxidase .The present study demonstrated that DHEA stimulated 11β-HSD1 oxidase activity and inhibited 11β-HSD1 reductase activity in intact ALCs. The metabolite of DHEA by CYP7B1, 7αOHD, inhibited 11β-HSD1 oxidase activity and stimulated 11β-HSD1 reductase activity in intact ALCs and in ALC microsomes.Cyp7b1 mRNA and histochemical staining and Western blotting analysis of CYP7B1 indicate that ALCs are the major, if not exclusive, sites of Cyp7b1 expression. We showed that Cyp7b1 mRNA levels in purified ALCs were enriched by 33-folds when compared to age-matched testis samples  compared to 11β-HSD1 mRNA levels in ALCs   or DHEAS plus DHEA are much higher than those of cortisol –35, thes/pg RNA) . If the Cyp7b1 is about 1/1700 of Hsd11b1 and therefore the action of CYP7B1 may be limited.In summary, DHEA directly inhibits 11β-HSD1 reductase in the intact ALCs, thus default 11β-HSD1 is enhanced to drive 11β-HSD1 being more oxidative . CYP7B1 All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the Wenzhou Medical University. Written informed consent was obtained from the owners for the participation of their animals in this study.JS and R-SG conceptualized the study design and analyze the data. QZ, YD, XL, CN, and TH performed the experiments and collected the data. R-SG wrote the manuscript and has full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis, and all authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BMP2, RELA, TNF, and TRAF1, were found to be significantly suppressed by EMD and selected given their role in mediating hVIC calcification. In conclusion, our study shows that EMD inhibits TNF-α-induced calcification and phenotypical transformation of hVICs via the NF-κB signaling pathway, thereby preventing calcification events stimulated during acute inflammatory responses.Exploring effective therapies for delaying calcific heart valve disease (CHVD) is the focus of current cardiovascular clinical and scientific research. In this study, human aortic valve interstitial cells (hVICs) were isolated from patients with CHVD. After expansion, cultured hVICs were induced with the tumor necrosis factor-alpha (TNF-α) with or without anthraquinone emodin (EMD) treatments. Cytotoxicity and flow cytometric analysis were used to assess cell growth, while Alizarin Red S staining was used to detect hVICs calcification. Furthermore, RNA-sequencing analysis was utilized to investigate changes in mRNA profiles of cells cultured in TNF-α conditioned medium with or without EMD. Western blotting and qRT-PCR analyses were used for the verification of key selected genes. Our results indicated that EMD had limited influence on hVIC morphology, whereas in a dose-dependent fashion, EMD interfered with hVIC growth under TNF-α conditioned cell culture. Additionally, hVICs treated with TNF-α plus EMD, presented a gradual decrease of positive Alizarin Red S staining, when compared with cells treated with TNF-α only. Notably, cells treated with TNF-α plus EMD showed 1874 differential expression genes (DEGs), among them, 1131 were upregulated and 743 were downregulated. These DEGs displayed an enrichment of biological functions and signaling pathways, among them,  Calcific heart valve disease (CHVD) is a common and frequently occurring valvular disease that severely harms human health and is rapidly increasing with the growing aging population . CurrentBMP-2, and RUNX2, among others  containing 10% fetal bovine serum  for primary cultures. After expansion, cells from the fourth or fifth passage were used in all the experiments. The treatments used included, 30 ng/ml of TNF-α alone (TNF-α treated group) or TNF-α plus EMD at different concentrations ranging from 1 to 20 μM (TNF-α + EMD treated group). For experiments investigating signaling mechanisms, 10 μM EMD was chosen as the concentration used for RNA-sequencing, qRT-PCR, and western blotting assays.Human aortic valves were obtained from patients presenting CHVD  , who und4 cells/well in 24-well plates. The cells were then treated with 30 ng/ml of TNF-α with or without the different concentrations of EMD (see section above), for 1, 2, and 3 days. After that, at each time point, the cells were washed with phosphate-buffered saline (PBS) and incubated with serum-free medium containing 10% of the CCK-8 reagent. After 2 h of incubation, aliquots were pipetted into 96-well plates and measured at a wavelength of 490 nm using an enzyme labeling instrument .Cell viability and cytotoxicity analyses were performed using the Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich). In brief, cells were seeded at a density of 1 × 10Cells were seeded into 24-well plates and grown for 3 days until confluency and further cultured in a calcification-inducation culture medium containing 30 ng/ml of TNF-α plus different final concentrations of EMD ranging from 1 to 20 μM for 12 and 18 days, respectively. The degree of cell calcification was measured using Alizarin Red S staining. In brief, cells were incubated in a 100 mM aqueous solution of cetyl-pyridinium chloride and the amount of Alizarin Res S dye released from the extracellular matrix was quantified by spectrophotometry at a wavelength of 550 nm.TM flow cytometer .Cells were harvested and washed, followed by fixation using 70% alcohol at 4°C overnight. After washing the cells with PBS, the cells were treated with ribonuclease RNase A, then incubated with propidium iodide (PI) for cell cycle analysis on a FACSVerseTM SYBRTM Green Master Mix (Applied Biosystems) following the manufacturer’s guide. All the primers were designed via NCBI primer blast and synthesized by Invitrogen , followed by RNA isolation. Each sample was reverse transcribed to cDNA according to the protocol . Then, ten Table . The finAfter 3 days of treatment with TNF-α (30 ng/ml) with or without EMD (10 μM), the cells were then lysed with standard lysis buffer plus 1% of phenylmethylsulfonyl fluoride (PMSF). Proteins were then separated in 8–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to 0.2 μm polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Then the membranes were incubated overnight at 4°C with primary TRAF1 (26845-1-AP) and RELA (10745-1-AP) antibodies . After washes with TBST, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody  for 1 h at 37°C. The targeted proteins were detected in Pierce ECL Western Blotting Substrates .RNA-sequencing (RNA-seq) technology was utilized to investigate changes in cell mRNA profiles among the different treatments performed. Isolated RNA was sent to BGI Co., LTD.  for RNA-seq performed on BGISEQ-500, and all the samples were replicated three times for confirmation purposes. Sequencing results were further analyzed using the “R Project” in order to identify DEGs, and perform GO and KEGG pathway enrichment analysis.p-value < 0.05 was accepted as statistically significant.Sequencing results were further analyzed using the R Project, and all other data were analyzed using IBM’s Statistical Package for Social Sciences (SPSS), expressed as the mean ± standard deviation (SD). Statistical comparisons were made by analysis of variance (ANOVA) to check differences among groups. A ∗p < 0.05), and the addition of 10 or 20 μM of EMD further inhibited cell proliferation, showing a statistically significant difference when compared to the TNF-α group (#p < 0.05). Similarly, at day three this inhibition appears more obvious, as seen in Figure The viability and cell cycle of hVICs with treatments at the concentration of EMD ranging from 1 to 20 μM. Cell morphologies were monitored with the different treatments for 3 days, and there were no significant changes observed in the basic morphology of the cells, when comparisons between the groups were made Figure . Furtherp < 0.05)  Figure . In summ∗p < 0.05)    Figure . Similar) Figure . TherefoGlobal gene expression profiles exhibited under TNF-α conditioned cultures, demonstrated that with the addition of EMD, great differences of gene expression regulation were observed Figure , when coRELA gene, is a full-contained consensus DEGs involved in each of the above signaling pathways. In addition, some important functional genes were selected, such as TNF, TNFRSF1A, PTGS2, TRAF1, VCAM1, and CXCL2, among others.Based on the DEGs analysis and GO functional annotations described above, KEGG signal pathway enrichment analysis was performed on the identified DEGs described above. Our results showed that these DEGs were highly enriched in functions related to cytokine-cytokine receptor interaction, chemokine and calcium signaling pathways, among others Figure . Among tBMP2, RELA, TNF, and TRAF1 genes were highly inhibited by EMD. Then, qRT-PCR results showed that TNF-α significantly upregulated the expression of these genes, when compared with the blank control group (∗p < 0.05), and promoted expression genes: BMP2, RELA, TNF, and TRAF1 were downregulated with presence of EMD . Furthermore, the protein synthesis of TRAF1 and the key regulatory factor RELA were investigated. These results were consistent with the trend observed with the gene expression profiles described above. As predicted, EMD was able to effectively inhibit the upregulated expression of the TRAF1 and RELA proteins in the presence of TNF-α -2 pro-calcification gene. This is consistent with results reported in the literature (BMP2, RUNX2, and other genes mediating osteoblastic differentiation. Our results show that EMD has a significant inhibitory effect on the transformation to a calcification phenotype by hVICs. In order to further understand the mechanism underlying EMD mediated inhibition on calcification, we used RNA-sequencing to analyze the global gene expression of hVICs treated with EMD in the presence of TNF-α induction. In this study, we identified 1874 DEGs with 1131 upregulated genes and 743 downregulated genes. Among them, TNF, TRAF1, and RELA were confirmed to have important roles in the process of calcification inhibition via EMD mediated mechanisms.hVICs grown in TNF-α-induced conditions have a tendency to transform into an osteogenic phenotype, which leads to a significant increase of calcification deposits, accompanied by an upregulation of the terature . For insTRAF1 is a receptor for TNF, responsible for transducing the signaling pathway across the cell membrane . MoreoveA more important finding is that the expression of RELA at both the gene and protein levels, was significantly inhibited by EMD. RELA, also known as p65, is a REL-associated protein involved in the heterodimer formation, nuclear translocation, and activation of the NF-κB protein . In our BMP2, TNF, TRAF1, and RELA.EMD was found to interfere with cell proliferation pathways, while suppressing the calcific transformation of hIVCs under the induction of TNF-α, possibly via the NF-κB signaling pathway by inhibiting the gene expression of KX, PZ, and QC designed the project, collected the data, and wrote the manuscript. KX, TZ, YH, and JS analyzed the data, wrote and revised the manuscript. ND designed the project, gave financial support, wrote and revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is increasing evidence pointing to the important role of tumor necrosis factor-alpha (TNF-α), a key inflammatory and apoptotic mediator in urticarial inflammation. However, the role of the TNF-α system and Fas/Fas ligand (FasL) in the apoptosis-inducing pathways in chronic spontaneous urticaria (CSU), remain unclear.To determine circulating concentrations of TNF-α, soluble TNF-α receptor type 1 and type 2  as well as soluble Fas (sFas) and FasL (sFasL) in CSU subjects.Serum TNF-α, sTNF-R1, sTNF-R2, sFas, sFasL concentrations were measured using enzyme-linked immunosorbent assay in CSU subjects and in the healthy subjects.TNF-α concentrations were significantly higher in CSU subjects and moderate-to-severe CSU than in the controls, while there were no significant differences in TNF-α concentrations between subjects with mild CSU and the controls. sTNF-R1 and sTNF-R2 concentrations were significantly higher in all CSU and moderate-severe CSU subjects vs. the controls. Serum concentrations were also significantly higher in mild CSU vs. the controls, but not in moderate-severe CSU vs. mild CSU. No significant differences were observed in sFas and sFasL concentrations between CSU subjects and the healthy controls. Significant correlations were found between concentrations of TNF-α and its receptors, as well as sTNF-R1 and sTNF-R2, but not with the urticaria activity score (UAS). There was no relationship between TNF-α/sTNF-R1/sTNF-R2 and sFas/sFasL pathways in CSU.CSU is associated with the activation of the TNF-α/receptors signaling pathway, marked by increased circulating concentrations of TNF-α, sTNF-R1 and sTNF-R2, which are related to each other in this disease. In contrast, the circulating sFas/FasL system is not up-regulated in CSU, and sFas/sFasL may not be a useful marker of the activity/severity of urticarial processes. Considering the lack of significant changes in sFas/sFasL (mainly reflecting systemic apoptosis) in CSU patients, it appears that elevated serum TNF-α concentrations are related to its pro-inflammatory function rather than an enhanced systemic apoptotic response in CSU. Chronic spontaneous urticaria (CSU) is associated with autoimmune processes and a systemic inflammatory response –3.Tumor necrosis factor alpha (TNF-α) plays a central role in inflammatory processes, immune regulation and apoptosis. TNF-α exerts its action via cell-surface receptors. They exist in two cell-bound forms (TNF-Rs) type 1  and 2 TNF-R1, p75), as well as soluble forms (sTNF-Rs), sTNF-R1 and sTNF-R2, respectively. Binding to TNF-Rs, TNF-α activates the constitutive shedding processes of soluble receptors, that play an important role in regulating its in vivo activity –7. High 5, as welThe study involved 17 men and 41 women with active CSU [median age 39 (range 21–45 years)]. The control group consisted of 22 healthy people who were matched for age and sex with CSU subjects; recruited from the hospital staff   and 19.01 (17.34–20.24) vs 16.89 (16.45–18.40) pg/ml; p < 0.00001].sTNF-R1 and sTNF-R2 concentrations [median and IQR 1.28 (1.03–1.56) and 2.79 (2.09–3.66) ng/ml] were significantly higher in CSU subjects as compared with the healthy subjects , but not vs. mild CSU subjects   and 2.38 (1.87–3.45) vs. 0.73 (0.67–0.86) and 1.45 (1.26–1.77) ng/ml, respectively; p < 0.0001]  and 87.59 (54.64–106.6), 17.09 (14.33–24.15) and 76.78 (45.23–97.03), 13.79 (10.20–21.50) and 102.03 (67.76–114.88) vs. 12.78 (10.3–19.26) and 75.71 (53.76–91.58) ng/ml and pg/ml, respectively; p > 0.05).CRP concentrations were significantly higher in all CSU subjects, mild, and moderate-severe CSU as compared to the controls as well as between the groups  .Significant correlations were observed between: (i) TNF-α and sTNF-R1  involved in the activation of the acute phase response. We have previously found a significant correlation between increased IL-6, IL-8 and CRP concentrations in CSU subjects , 21. In However, circulatory TNF-α is unstable, therefore to get a more precise insight into TNF-α/receptors system activity, both TNF-α and its soluble receptors, which maintain a more stable form should be measured. The soluble TNF-α receptors are more useful for monitoring the activity of the TNF-α system activity , 22. TheIn our study, the concentrations of sTNF-R1 and sTNF-R2 were significantly higher in all CSU subjects, moderate-to-severe CSU, and mild CSU as compared with the healthy group.However, no significant differences in these values were observed between subjects with moderate-to-severe and mild CSU. There was no correlation between TNF-α sTNF-R1, sTNF-R2 and UAS in CSU.It has been suggested that circulating concentrations of sTNF-Rs may reflect the state of activation of the TNF-α/receptors system , 22. TheBinding of TNF-α to TNF-Rs activates the constitutive shedding process of soluble receptors, which plays an important role in regulating its in vivo activity , 22. sTNIn the negative feedback mechanism, high concentrations of TNF-R act as inhibitors/antagonists of TNF-α activity, preventing its binding to membrane-bound TNF-R, maintaining homeostasis and protecting against overwhelming immunological-inflammatory activation , 22, 25.On the other hand, it has been shown that sTNF-Rs at low/intermediate concentrations may preserve and enhance some of the effects of TNF-α by stabilizing its structure in complexes and prolonging its half-life , 25.We observed significant correlations between concentrations of TNF-α and its receptors as well as, sTNF-R1 and sTNF-R2 in CSU.The open question is whether the increased release of sTNF-Rs in CSU is sufficient to neutralize TNF-α activity to prevent over-response of the TNF/receptors system, systemically and locally at sites of inflammation. One the one hand, we speculate that the higher release of TNF-α in CSU stimulates shedding of sTNF-Rs into the circulation, probably leading to neutralization of TNF-α activity and attenuation/limitation of the inflammatory response, similarly as suggested in other immune-inflammatory diseases , 26. HowHowever, the source of circulating forms in CSU is still unknown. In contrast to widely expressed TNF-R1, TNF-R2 is expressed typically by immune cells , 27, thuThe balance between TNF- activity and sTNF-Rs determines the course of immune activation and inflammatory response, and its dysregulation may play a role in susceptibility and resistance to various diseases , includiIn addition, persistent expression of the nonsheddable form of TNF-R1 has been shown to increase susceptibility to hyperreactivity and inflammatory response in mice .The TNF-R1 mutation may result in a decrease in receptor cleavage and a reduction in the shedding of potentially antagonistic sTNR-1 and is associated with certain autoinflammatory syndromes called TNF-R1-associated periodic syndromes .It has been suggested that the Fas/FasL system may have pro-inflammatory activity , 30. TheIn contrast to other immune-inflammatory diseases, CSU is not associated with up-regulation of circulating soluble Fas/FasL system. No significant differences were observed in sFas and sFasL concentrations between CSU subjects and the control group.We only measured circulating concentrations of sFas and sFasL, which may be less important than their local levels at the site of inflammation .Our results do not exclude the importance of the Fas/FasL system and its soluble forms in urticarial inflammation. It seems, however, that concentrations of soluble apoptotic factors in serum/plasma would not be useful indicators of the activity/severity of urticarial inflammation.TNF-α/receptors and Fas/FasL systems are two key cell death-inducing systems in one apoptotic pathway . ConsideThe use of TNF-alpha inhibitors beyond therapeutic indications may be effective in a significant proportion of patients with refractory chronic urticaria, also previously unsuccessfully treated with omalizumab (anti-IgE) , 35.Sand et al. observed complete or almost complete response in 60% and a partial response in a further 15% of subjects with refractory chronic urticaria during therapy with TNF‐alpha inhibitors  .Taking into consideration our and previous results, inhibiting the TNF-α system activity as a therapy for refractory chronic urticaria unresponsive to omalizumab and cyclosporine or associated with unacceptable drug side effects seems to be interesting for further investigations.Nevertheless, our study has some limitations, therefore we can not prove the existence of any causal relationship. We have not studied the correlation between TNFα/sTNF-Rs and the effectiveness of anti-inflammatory or anti-IgE medications.CSU is associated with the activation of the TNF-α signaling pathway, marked by increased circulating concentrations of sTNF-R1 and sTNF-R2, probably as part of the systemic inflammatory response in the disease. TNF-α and its soluble receptors are related to each other in CSU. In contrast, the circulating soluble Fas/FasL system that mainly reflects systemic apoptosis is not upregulated in CSU patients. There is no relationship between TNF-α/sTNF-R1/sTNF-R2 and sFas/sFasL pathways in the disease.