In this article, we present an approach to address how human CSF (hCSF) influences neuronal circuits in vitro. We validate our approach by comparing the morphology, viability, and electrophysiological function of single neurons and at the network level in rat organotypic slice and primary neuronal cultures cultivated either in hCSF or in defined standard culture media. Our results demonstrate that rodent hippocampal slices and primary neurons cultured in hCSF maintain neuronal morphology and preserve synaptic transmission. Importantly, we show that hCSF increases neuronal viability and the number of electrophysiologically active neurons in comparison to the culture media. In summary, our data indicate that hCSF represents a physiological environment for neurons in vitro and a superior culture condition compared to the defined standard media. Moreover, this experimental approach paves the way to assess the functional consequences of CSF on neuronal circuits as well as suggesting a novel strategy for central nervous system (CNS) disease modeling.For decades it has been hypothesized that molecules within the cerebrospinal fluid (CSF) diffuse into the brain parenchyma and influence the function of neurons. However, the functional consequences of CSF on neuronal circuits are largely unexplored and unknown. A major reason for this is the absence of appropriate neuronal  The cerebrospinal fluid (CSF), which fills the brain’s ventricles and subarachnoid space, communicates with the ISF via diffusion, since a barrier between the ventricular system and the brain parenchyma is absent in the adult brain . We also provided (first) evidence that these neuromodulatory effects were mediated by G-protein coupled receptors  of the brain parenchyma encompasses all brain cells, and thereby, represents the physiological environment of neurons in vitro exposure of hippocampal neurons to CSF influences the viability and function of neurons. Currently used in vitro models comprise the cultivation of isolated whole brains or specific brain regions as well as dissociated neuronal cells obtained from rodent brains or human pluripotent stem cells. In order to support survival and functionality of cultured neural cells, different types of cultivation media are used, such as DMEM or Neurobasal/B27-based culture media. Such chemically defined culture media represents, however, an artificial environment for neuronal cells, posing questions about the physiological relevance of the data collected. Moreover, recent works demonstrate that culture media such as Neurobasal or DMEM/F12 suppress neuronal activity  treated hippocampal neurons. In addition, we applied multielectrode array recordings to characterize the neuronal network properties of hCSF-treated mature hippocampal neurons. In a second set of experiments, we validated the impact of hCSF on neuronal viability and synaptic function in organotypic hippocampal slice cultures from the rat.In this article, we present an 2/95% air) at 37°C. Culture medium was replaced twice a week. Neuronal cultures cultivated for 26–28 days in vitro (div) were used for all experiments.Hippocampal neuronal cells were prepared from postnatal (P0–3) Fischer 344 rats  according to published protocols Brewer, . NeuronsBaseline activities of hippocampal neuronal cultures were recorded in cultivation media prior to application of either pure hCSF or fresh culture media. According to the number of spike detecting electrodes, the appearance or absence of population bursts (PBs), neuronal cultures were split in two groups. Each well of a six-well MEA contains nine planar electrodes, allowing it to record spontaneous activity of neurons which are in close vicinity (50–100 μm) of the recording electrode. High-active networks are characterized by 90–100% (8–9 out of 9) electrodes detecting spike activity showing partial or synchronous activity. Low-active neuronal networks are characterized by 10–70% (1–6 out of 9) electrodes detecting spike activity showing asynchronous, partial or synchronous activity. To investigate the influence of prolonged hCSF exposure on high- and low-active hippocampal neuronal cultures, cultivation media was removed and substituted with 100 μl pure hCSF/well. The six-well MEAs were then placed back into the incubator. For control experiments 100 μl Nb-A-based media was used. After 24, 48 and 72 h, MEA-recordings were performed to measure the electrophysiological activity of neuronal populations exposed either to hCSF or Nb-A-based media. Each well contained a square grid of nine planar Ti/TiN electrodes . Signals from all 54 (6*9) electrodes were simultaneously sampled at 25 kHz, visualized and stored using the standard software MC_Rack provided by Multi Channel Systems. Spike and burst detection was performed offline by custom-built Software . Detailed description of analysis procedures are described elsewhere ; n refers to the number of individual cultures treated either with hCSF or culture media. All statistical data analyses have been performed with GraphPad Prism, version 5.01. Stars indicates significant values .Data of spikes/2 min, number of PB, PB half width values and number of active electrodes were either analyzed by paired (before and after treatment) or unpaired (different treatments) ad libitum. All experiments were performed in accordance with the guidelines of the local ethical committee for animal research at the University of Gothenburg and were conducted in accordance with European Union directives on animal rights.Wistar rats 7–10 days old (P7–10) were used for this study. The animals were housed under a 12 h light/dark cycle with food and water 2PO4, 1.2 CaCl2, 7 MgCl2, 25 NaHCO3, and 11 Glucose. The solution was bubbled with carbogen gas (95% O2 and 5% CO2) in order to maintain the pH (7.4). Sagittal slices (300 μm) were cut using a microtome . Slices were maintained in a Hibernate-A media  at 4°C until transferred to the membrane. Slices were transferred to a membrane with hCSF and Nb-A-based culture media respectively. Slices were incubated under sterile conditions at 37°C with 5% CO2 and 90% humidity. Culture medium was changed at regular time intervals of 2–3 days up to 9 div.Rats were anesthetized with isofluorane and immediately decapitated. The brain hemispheres were isolated and transferred to ice cold solution (4°C) containing (in mM): 219 Glycerol, 2.5 KCl, 1.2 NaHOrganotypic hippocampal slices were prepared in accordance with previously described methods , supplemented with B27 and L-glutamine, according to the manufacturer protocol. Antifungal agent, Nystatin, and antibiotics, Penicillin and Streptavidin (P/S), were added to both Nb-A and hCSF. Slices from the same animal were alternately placed on a membrane into wells containing culture media or hCSF.hCSF samples  used for neuronal culture experiments were obtained via lumbar puncture at the Christian-Doppler-Klinik in Salzburg, Austria. Samples came from individuals admitted to the hospital and were collected for diagnostic purposes. hCSF samples were considered normal hCSF when routine laboratory CSF parameters , total protein, glucose, lactate, CSF cytology and CSF/serum albumin quotient) were in a normal range, and when inflammatory, neurodegenerative, ischemic, CNS neoplastic, or infectious processes were excluded by clinical examinations. The use of hCSF samples for research purposes was approved by the ethics board of the Land Salzburg (415-E/1160/4-2010).Pooled hCSF (consisting of 5–30 anonymized samples) obtained from patients which had been diagnosed with normal pressure hydrocephalus (NPH) was used for organotypic hippocampal slice cultures. The clinical procedure conformed to the declaration of Helsinki and was performed by means of lumbar puncture by neurologists at the Sahlgrenska University Hospital in Gothenburg, Sweden.All CSF-samples were handled according to an international accepted consensus guideline  and kept at room temperature (20–22°C). Whole-cell patch-clamp recordings were performed for at least 5 min in CA1 pyramidal cells, which were visually identified using infrared differential interference contrast video microscopy mounted on a Nikon Y-QT (Japan) microscope. The intracellular solution contained (in mM): 95 Cs-methanesulfonate, 2 NaCl, 10 HEPES, 5 QX-314, 4 Mg-ATP, 0.4 GTP, 15 phosphocreatine and 20 BAPTA . Patch pipette resistance ranged from 3–5.5 MΩ.After 9 div, hippocampal slices were placed in a submersion recording chamber and perfused at a constant flow (~2 ml/min) with aCSF containing (in mM): 129 NaCl, 3 KCl, 2 CaClMiniature excitatory postsynaptic currents (mEPSCs) were recorded at −70 mV in the presence of 0.5 μm tetrodotoxin (TTX) and 0.1 μm picrotoxin (PTX), using an EPC-9 amplifier . mEPSCs were sampled at 10 kHz, filtered at 2.9 kHz, and the first 100 mEPSCs were analyzed blinded using the Software Mini-Analysis .Cresyl violet staining was used for histological evaluation of organotypic slices after 9 div. Slices attached to a small rectangle of the supporting membrane were cut from the whole membrane insert, fixed with 4% paraformaldehyde at 4°C (24 h) and washed three times in phosphate-buffered solution (PBS) for 5 min. Slices were mounted on Superfrost Ultra Plus slides  and dried overnight. Slices were hydrated using decreasing concentrations of ethanol , rinsed twice with water for 2 min and stained with 1% Cresyl violet for 5 min at 60°C. Slices then were dehydrated using increasing concentrations of ethanol  and xylene and mounted with DPX mounting media .Morphological analysis was carried out using an Axio Observer microscope  objective NA 0.3 and camera NA 0.5. Area measurements of the organotypic hippocampal slices stained with Cresyl violet were performed using ImageJ Software (NIH) from images taken under 10× magnification. The width of the stratum pyramidale was measured as the average width over 500 μm from organotypic hippocampal slice cultures in which it was possible to clearly define the cell body layer. The analysis was performed blinded with regard to the experimental group.2, 5% CO2). Slices were then fixed by incubation in HistoFix  for 1 h. Preparations were mounted on glass slides using 40–60 μl ProLong® Gold Antifade supplemented with DAPI nuclear stain . Slides were stored at 4°C. An Olympus DP72 microscope equipped with an Olympus U-RFL-T mercury lamp was used for digital micro-imaging; the digital image analysis was captured with Olympus CellSens Dimension. Final analysis was performed in ImageJ . For every preparation three independent areas in the CA1 were selected and the intensity measured in gray scale. Intensities were averaged and normalized to the DAPI intensity to control for any difference in exposure. The CellEvent Caspase-3/7 Green ReadyProbes was evaluated prior to the experiments by comparing untreated controls to hippocampal slices exposed to 150 μM NMDA for 1 h.To measure the viability of the cultured hippocampal slices, 80 μl of CellEvent Caspase-3/7 Green ReadyProbes  was added to 500 μl of culture media (Nb-A or hCSF) and the preparations were incubated for 30 min at 37°C .Statistical significance for immunohistochemical analysis was assessed using Mann-Whitney test. Significance level was set at invitro, we characterized their baseline electrical activity. Cultures consisted of spontaneously active (as determined by electrophysiology) mature neurons  showed a gradual increase in spiking activity in comparison to baseline activity  after 24 h, 7.0 ± 6.0-fold  after 48 h and 9.1 ± 8.1-fold  after 72 h of hCSF incubation , population bursting  and PB half width  in which nearly all electrodes detected spiking activity   Figure .2, N = 8. hCSF; 1.09 ± 0.18 mm2, N = 12. p = 0.0005, Mann-Whitney U test) in organotypic hippocampal slices cultured in Nb-A compared to slices cultured in hCSF . In contrast to the larger peripheral area of the organotypic hippocampal slices cultured in Nb-A, there was no significant difference in the width of the CA1 pyramidal cell body layer .To evaluate the feasibility of culturing organotypic hippocampal slices in hCSF we compared organotypic hippocampal slices cultured for 9 days in hCSF with those cultured for the same time in culture media. We first compared the gross morphology of the organotypic hippocampal slices using Nissl staining Figure . HippocaF Figure . The incN = 10; hCSF: 0.17 ± 0.04, N = 8, p < 0.0001, mean ± SD), indicating less apoptotic cell death.To compare the viability of organotypic hippocampal slices cultured in hCSF and Nb-A, we examined the expression of caspase-3/7, which is upregulated in association with neuronal cell death. We first tested the validity of the caspase-3/7 method by exposing acute hippocampal slices to 150 μM NMDA for 1 h, a treatment known to induce excitotoxic cell death , frequency , rise-time , or decay-time  of mEPSCs.As a functional comparison between organotypic hippocampal slices cultured in hCSF or Nb-A, we used whole-cell patch-clamp recordings of miniature AMPA receptor mediated EPSCs (mEPSCs) from CA1 pyramidal cells Figures . We founWe present a novel experimental approach to assess the influence of hCSF on neuronal circuits. This approach was validated by prolonged exposure of organotypic hippocampal slices and primary neuronal cultures to hCSF in combination with electrophysiology and analysis of cell survival. Our results indicate that hCSF is well tolerated by rodent neurons, providing new possibilities to examine the effects of healthy and pathological hCSF on neuronal function.in vitro cultivated neurons than currently used culture media. The development of chemically defined culture media that increase survival and preserve physiological properties of neurons is an important goal in neuroscience and stem cell research  as well as allowing analysis of several different hCSF samples simultaneously. Our results reveal that hCSF treatment resulted in a robust increase in spontaneous spiking activity and an increased number of electrodes detecting neuronal activity in hippocampal neuronal cultures in comparison to Nb-A media cultivated cultures. This is in line with a recent study demonstrating that Neurobasal media suppresses neuronal activity in cultivated rodent and human neurons  The cultivation of organotypic slices required several milliliters of CSF whereas the CSF volume in a rodent is in the microliter range. Thus, we used pooled hCSF for the cultivation of organotypic slices and individual hCSF samples for hippocampal neurons cultured in six-well MEAs. (ii) Since pathological CSF obtained from patients suffering different types of CNS disease could be used when creating relevant disease models, we were aiming to obtain proof-of-concept evidence that both rodent-based in vitro neuronal cultures and organotypic slices tolerate hCSF. We believe that the use of hCSF will be more relevant to predict human brain pathophysiological processes than the use of rodent CSF. However, the volume of CSF routinely collected during the diagnosing of neurodegenerative diseases, such as Alzheimer’s disease, is in a milliliter range. We illustrate that neuronal cells cultured on six-well MEAs can be used to analyze the impact of a limited volume of hCSF. (iii) Human pluripotent stem cell-derived neurons might represent a more appropriate pre-clinical in vitro model system to reveal the functional influence of hCSF. On the other hand, the rodent hippocampal neuronal circuit is well characterized and hippocampal slice cultures more reliably represent the in vivo hippocampal neuronal circuit than PSC-derived neuronal cultures.The use of CSF from human individuals in rodent-based in vivo  of hCSF increases neuronal activity both at a single cell and network level (Otto et al., in vitro. hCSF-based in vitro model systems may turn out to be the golden standard for evaluating artificial cultivation media. Moreover, our work may pave the way the chronic influence of normal and pathological hCSF on neuronal circuits.We demonstrate that the cultivation of hippocampal neuronal cultures and organotypic slices in hCSF constitutes a feasible and advantageous approach to studying neuronal activity MP-A, GC and FA performed the histological evaluations of organotypic slices. GC and AB performed the electrophysiological analysis of organotypic slices. TL and PW prepared organotypic slices cultures. KM and HS performed the capase-3/7 evaluations of organotypic slices. PW, AH and BI collected human CSF, clinical evaluation and CSF analysis for the identification of normal CSF samples. SI performed the MEA-recordings and analysis of hippocampal neuronal networks. LA did critical revision of the manuscript and interpretation of the data. EH and SI conceived the study and wrote 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. SI received financial support by EISAI to conduct research. However the study design, data collection, data interpretation and data presentation were not influenced by EISAI.


The nude phenotype results from null mutation of the forkhead transcription factor FOXN1, which has emerged as a pivotal regulator both of thymus development and homeostasis. FOXN1 has been shown to play critical roles in thymus development, function, maintenance, and even regeneration, which positions it as a master regulator of thymic epithelial cell (TEC) differentiation. In this review, we discuss current understanding of the regulation and functions of FOXN1 throughout thymus ontogeny, from the earliest stages of organogenesis through homeostasis to age‐related involution, contextualising its significance through reference to other members of the wider Forkhead family.Development of the primary T‐cell repertoire takes place in the thymus. The linked processes of T‐cell differentiation and T‐cell repertoire selection each depend on interactions between thymocytes and thymic stromal cells; in particular, with the epithelial cells of the cortical and medullary thymic compartments . The importance of the thymic epithelial cell lineage in these processes was revealed in part through analysis of  As discussed in detail below, cTECs are required for commitment of haematopoietic cells to the T‐cell lineage, and also mediate both the β selection and positive selection stages of T‐cell lineage development. Developing T cells  that successfully undergo positive selection can then enter the medulla, the site of central tolerance induction, with tolerance induction being mediated by both mTECs and thymic dendritic cells  by mTECs identifies these TEC sublineages and permits their isolation and subsequent analysis. In the adult thymus, Ly‐51+ cTECs and UEA‐1+ mTECs each constitute heterogeneous populations that can be subdivided into a number of different subsets based on expression of additional surface markers, including MHC Class II The thymus is a central organ of the adaptive immune system due to its obligatory role in T‐lymphocyte differentiation and repertoire selection lls Fig. . Not surThe epithelial component of the thymus arises from the endoderm of the pharyngeal pouches (PPs). These structures are bilateral outpocketings of the foregut endoderm. The number of PPs varies between species; in mouse and human it is the third PPs (3PPs) that generate the thymus, while other PPs also contribute in some species nude (nu/nu) mice, which carry an autosomal recessive mutation leading to congenital hairlessness and athymia Nu/nu mice are correspondingly immunocompromised as they lack normal T‐cell populations nu/nu mice results from a severe developmental block early in thymus organogenesis. The common thymus‐parathyroid primordium forms normally and thymus organogenesis proceeds until E11.5–E12.0. However, a maturational arrest in thymic epithelial progenitor cells occurs at around E12.0 nude thymic epithelium never becomes competent to support T‐cell development. Indeed, the nude thymic rudiment is never colonized by hematopoietic or vascular progenitors; instead, these remain in the perithymic mesenchyme nu/nu mice retain a small, cystic, alymphoid thymic rudiment, which does not support T‐cell development at any stage in ontogeny.Some of the earliest insights into the function of the thymus came from studies on Foxn1 was originally identified as the gene mutated in nu/nu mice using genetic approaches nu to chromosome 11 in mice and subsequent fine‐mapping, a member of the forkhead or winged helix superfamily, originally named winged helix nude , was identified as the nude gene by positional cloning Whn transcript in nu/nu mice carried a single base pair deletion in its third exon, resulting in the absence of Whn mRNA due to nonsense‐mediated decay Whn was expressed in the developing mouse embryo from E9.5, and was restricted to skin and thymus in adult tissues Whn was generated by inserting a lacZ M2‐neo cassette into exon 3 of Whn, close to the site of the spontaneous mutation in nu. Mice homozygous for this allele phenocopied nu/nu mice, confirming Whn as the nude gene FOXN1 was one of the first members of the forkhead (FOX) superfamily of transcription factors (TFs) to be implicated in a specific developmental defect in vertebrates Drosophila melanogaster gene fork head (fkh) which, when mutated, causes a spiked head phenotype in adult flies HNF3α) in 1990 revealed an approximately 100 amino acid region of high homology between the HNF3α and fkh proteins, that was suggested to be a DNA binding domain (DBD) The FOX family is evolutionarily ancient. Its canonical member is the FOXN1 is its paralog FOXN4. The evolutionary history of the FOXN subclass as currently understood is depicted in Table Foxn4 first appeared in cephalochordates (amphioxus) and is found in all higher organisms. Cephalochordates also contain a more ancient paralog Foxn4b, which is present in Echinodermata and Cnidaria but absent from urochordates and all vertebrates. Jawless fish possess a gene very similar to Foxn4, termed Foxn4‐like (Foxn4L). Foxn1 is thought to be an ortholog of Foxn4L, based on protein sequence and short‐range synteny relationships. The expression patterns of these genes further support this genealogy: Foxn4 (or Foxn4a) being expressed in the pharyngeal endoderm and other sites in amphioxus; FOXN4L, in the epithelium lining the gill basket in lamprey; and Foxn1 in the pharyngeal pouches giving rise to the thymus in cartilaginous fishes and all other jawed vertebrates Foxn4 in the pharyngeal endoderm in amphioxus suggests that this gene contributed to the evolutionary emergence of thymopoiesis Foxn1 and Foxn4 are expressed in the thymi of catshark, zebrafish, and medaka; however Foxn4 is either not expressed or expressed at very low levels in higher order organisms Foxn1 appears to have a unique role in the thymus in jawed vertebrates, which cannot be completely substituted by Foxn4The FOXN genes cluster separately from other FOX subclasses, and the gene most closely related to The classical ‘forkhead’ DBD consists of three N‐terminal α‐helices , three β‐sheets , and two C‐terminal ‘wing’ regions/loops , arranged in the order H1‐S1‐H2‐H3‐S2‐W1‐S3‐W2 Most FOX factors appear to bind to DNA as monomers Recently, much interest has focused on the capacity of some TFs to act at the level of chromatin organization, opening regions of previously compacted chromatin thus enabling their transcription. TFs with this activity are called ‘pioneers’. Pioneer factors are defined as being able to access their target sequence on nucleosomes and certain forms of compacted chromatin, to bind nucleosomes stably and before the binding of other TFs or initiation of the target gene expression, and to possess chromatin‐opening capabilities Foxn1 expression is evident in the pharyngeal endoderm as early as mouse E9.5—the time of the initial outpocketing of the 3PP Foxn1) can generate an intact and functional thymus containing both cortical and medullary thymic epithelial compartments, indicating that 3PP cells are already committed to the TEC lineage Foxg1) and interleukin 7 (Il7) specifically mark the thymus domain of the 3PP and for each, this expression occurs independently of FOXN1 Foxn1 have shown that TEPC that lack Foxn1 expression undergo a developmental arrest that can be reversed in neonatal and adult mice Foxn1 that confers TEPC identity and thus thymic epithelial lineage specification. Overall, these studies indicate that TEC lineage commitment does not depend solely on FOXN1, implicating an as yet unidentified genetic network in this process.As discussed above, low‐level Foxn1 allele, Foxn1SA2Foxn1 in a single cell in the thymic rudiment of Foxn1 null mice was shown to result in the generation of miniature thymi, each containing well‐defined cortical and medullary areas Foxn1−/− thymi contain cytokeratin 5hi, claudin 4hi (K5hiCldn4hi), and K5−Cldn4lo/− regions. Since mTEC‐restricted progenitors in the early fetal thymus are Cldn4hiFoxn1‐independent and thus that the divergence of the cTEC and mTEC sublineages may occur earlier than implied by this particular genetic analysis Foxn1‐independent development which results in generation of the undifferentiated thymic primordium containing specified TEPCs, and later Foxn1‐dependent development in which FOXN1 expression in TEPCs results in their differentiation and the concomitant orchestrated development of the fully patterned and functional thymus As discussed above, the absence of functional FOXN1 arrests fetal TEPC in a bipotent progenitor cell state, and normal functioning in these developmentally arrested fetal TEPCs can be restored by permitting normal FOXN1 expression. This was initially demonstrated using a revertible Vegf‐a and Pdgf‐b in TECs, thymic vasculature‐associated mesenchyme, and endothelium, was severely reduced under conditions of low Foxn1 expression, with the thymic rudiment showing fewer capillaries, leaky blood vessels, disrupted endothelium–perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization at later stages of organ development How does FOXN1 effect these later stages of development, and what are its subsequent roles in thymus maintenance and function? Studies on FOXN1 function in early thymus development have revealed a role in TEC proliferation Foxn1 in TEC differentiation has been provided by studies in which Foxn1 is either under‐ or overexpressed specifically in TECs. For example, mice homozygous for a hypomorphic Foxn1 allele, Foxn1Δ, whose transcript lacks the N‐terminal domain of FOXN1, develop severely hypoplastic and cystic thymi that lack distinct cortical and medullary regions Foxn1Δ/Δ thymus. However, the adult Foxn1Δ/Δ thymus supports only aberrant thymopoeisis, characterized by the absence of CD25+ thymocytes (DN3 cells), and reduced expression of TCR‐β at the DP stage. Analysis of Foxn1Δ/Δ mice is consistent with TEC differentiation being initiated, but then blocked at an intermediate stage of development, such that the Foxn1Δ/Δ thymus has impaired functionality compared with that of the wild‐type Foxn1 allele, RFoxn1, allowed investigation of FOXN1 function in TEC differentiation independently of its role in proliferation Foxn1R generates only around 15% of WT levels of normal Foxn1 transcripts, which results in development in R/RFoxn1 mice of a hypoplastic thymus which can only suboptimally support T‐cell development such that fewer thymocytes are generated during differentiation. Analysis of an allelic series based on the R, Foxn1−Foxn1, and wild‐type Foxn1 alleles revealed strong dose‐dependent effects of Foxn1, in brief showing that increasing levels of Foxn1 expression are required for progression through multiple intermediate stages of TEC development—from exit from the earliest progenitor cell state(s) through to terminal differentiation, in both cTEC and mTEC sub‐lineages in the fetal and adult thymus Further insight into the role of nude gene product, the molecular functions of FOXN1 have not yet been determined in full, and indeed no direct targets have yet been verified in TECs by chromatin immunoprecipitation. However, FOXN1 has been shown to be required for the expression in TECs of proteins with essential roles in promoting thymocyte development, including Chemokine (C‐C Motif) Ligand 25 (CCL25), C‐X‐C motif chemokine 12 , Delta‐like ligand 4 (DLL4), Stem cell factor , Cathepsin L (CTSL), the 20S proteasome subunit beta‐5t , and MHC Class II + and of CD8+ thymocytes, respectively + T cells. Notably, transgenic expression of Dll4, Ccl25, Cxcl12, and Scf conferred some capacity to support production of CD4+ and CD8+ SP T cells to the Foxn1−/− thymic rudiment, although the TEPCs within the rudiment remained in an undifferentiated state Foxn1−/− thymus expressing transgenic Dll4, Ccl25, Cxcl12, and Scf lacked functional TECs and normal thymus architecture, this study established that Foxn1 must regulate additional genes that are required for TEC differentiation and function. In keeping with this observation, in addition to the genes discussed above, the genes encoding transformation related protein 63 (Trp63), Paired Box 1 (Pax1), Fibroblast growth factor receptor 2 isoform IIIB (Fgfr2IIIb), Autoimmune regulator (Aire), Cluster of differentiation 40 (CD40), Cluster of differentiation 80 (CD80), and Cyclin D1 (Ccnd1), which have known roles in TEC differentiation, proliferation, or function, and of several genes involved in Wnt signaling, are all FOXN1 responsive in TECs Despite 20 years having elapsed since confirmation of its identity as the Foxn1hi cells expressing higher levels of Foxn1 than MHC Class IIlo TECs in each compartment Foxn1 expression in the thymic stroma is one of the earliest events in the age‐associated degeneration of the thymus Foxn1 allele (Foxn1lacZ), which expresses normal levels of Foxn1 in the fetal and newborn thymus, after which Foxn1 expression declines to 20–30% of wild‐type levels by 5 weeks after birth Foxn1lacZ/LacZ mice exhibit a premature loss of thymus homeostasis, correlating with Foxn1 downregulation, that phenocopies many of the hallmarks of age‐related involution Foxn1, indicating their continued FOXN1 dosage sensitivity Foxn1 or of FOXN1+ cells in postnatal mice, resulted in rapid thymic atrophy, further supporting a role for FOXN1 in thymus homeostasis TECs in the adult thymus continue to express Foxn1 expression in the involuting thymus has recently been investigated at the single cell level. Using a new antibody generated against the C‐terminus of FOXN1 protein and a tagged version of FOXN1, Rode and colleagues showed that in aging mice, Foxn1 expression progressively decreases and there is an age‐related accumulation of Foxn1−/low TECs Foxn1‐negative TECs has shown that these cells arise from Foxn1‐positive precursors Foxn1‐eGFP reporter mouse line (in which eGFP was knocked into the Foxn1 locus) to investigate transcriptional changes in Foxn1 expression with age, and similarly showed that the emergence of Foxn1− TECs correlates with the onset of age‐related thymic involution Foxn1 expression.Foxn1 cDNA, under control of the human K14 promoter, was introduced into the genome by random insertion, resulting in 20‐fold over‐expression of Foxn1 in TECs due to multiple copies of the transgene The emerging evidence, discussed above, suggesting that down‐regulation of FOXN1 might be a primary cause of age‐related thymic involution has recently been tested in two studies, which have respectively determined the outcome of maintaining high‐level FOXN1 expression throughout ontogeny Foxn1ERT2, which allows tissue‐specific expression of a tamoxifen‐regulatable form of FOXN1 (FOXN1ER) Dll4, Ccl25, Kitl, Cxcl12, Ctsl, Cd40, Cd80, Pax1, Trp63, Fgfr2IIIb, and Aire, to levels similar to those observed in the young thymus , is sufficient to reprogram the MEFs into functional TECs itl Fig. B, and toFoxn1 is surprisingly scarce. Concrete evidence supports positive autoregulation of Foxn1 in TECs Foxn1 promoter in vitro Foxn1 in TECs Foxn1 expression was revealed by analysis of compound transgenic mice that lack the Rb family genes Rb and p103, and carry only a single copy of the third Rb family member p107 . By 9 months old, these mice exhibit severe thymus hyperplasia, characterized by increased cellularity and increased Foxn1 expression levels in TECs. Further genetic analysis showed that reduced levels of Foxn1 expression  were sufficient to reverse this hyperplastic phenotype, implicating RB and hence E2Fs in Foxn1 regulation Foxn1 in TECs is the T box transcription factor TBX1, mutation of which is thought to cause DiGeorge Syndrome Foxn1 expressing cells of the E11.5 3PP resulted in down‐regulation of Foxn1 expression, indicating that TBX1 can repress Foxn1 transcription in TECs Foxn1 repression, the forced TBX1 expression in Foxn1Cre;R26‐iTbx1 thymi appeared to block TEC differentiation in an early progenitor state, evidenced by the accumulation of progenitor‐phenotype cells  and the absence of differentiated TECs in the fetal thymus Foxn1 directly or indirectly, and in this light TBX binding sites have not yet been identified in the putative Foxn1 promoter regions Foxn1 in the hair follicle and skin may also provide some clues as to its regulation in TECs, and notably, a homeobox family member, Hoxc13, has been suggested to regulate Foxn1 in skin and hair follicle. However whether Hoxc13 is also involved in regulation of Foxn1 expression in TECs remains to be determined Foxn1 expression in TECs Given the importance of FOXN1 in thymus biology, there has been considerable interest in its upstream regulation. However, information regarding transcriptional regulators of Foxn1 expression in TEPCs or TECs are also still only poorly characterized. Several studies have investigated whether a minimal genomic region surrounding the Foxn1 gene on mouse chromosome 11 can reproduce the wild‐type Foxn1 expression pattern in skin and thymus. The largest region tested was 110kb, containing the entire Foxn1 locus with an additional 74kb of 5′‐flanking sequence and 12kb of 3′‐flanking sequence; this region rescued the nude phenotype in vivo, indicating that it contains all the regulatory elements required for normal expression of Foxn1Foxn1 plus 8.5kb of 5′‐flanking sequence and 3kb of 3′‐flanking sequence could rescue the hairless but not the athymic phenotype of nude mice, showing that it lacked at least some of the regulatory regions required for Foxn1 expression in TECs Foxn1 (exon‐2) and the upstream gene Slc13a2 can recapitulate Foxn1 expression pattern in the developing thymus Foxn1 expression in the postnatal and adult thymus has not been provided. Within these regions, the promoter and enhancers governing the expression of Foxn1 in TEPCs and TECs remain to be definitely identified and similarly the identity of the tissue‐restricted transcription factors important for its expression remains elusive.Surprisingly, the regulatory regions governing Foxn1 in different TEC subsets will also be crucial. This presents a major challenge, but should now become tractable in light of recent technological advances allowing interrogation of global gene expression and TF binding in single cells/small cell populations. In this regard, the recent identification of TBX1 and E2F as transcriptional regulators of Foxn1 should provide a tangible starting point for deciphering the molecular details of Foxn1 transcriptional regulation.FOXN1 plays a critical role in thymus biology, functioning as a master regulator of TEC differentiation, function, and maintenance in the fetal and adult thymus and displaying remarkable potency as a regeneration and reprogramming factor. Further investigation of FOXN1 function will thus illuminate TEC biology during development, homeostasis and aging, and contribute to a broader understanding of how master regulator TFs function to regulate and coordinate gene expression programs. Elucidation of the transcription factor networks responsible for regulating the initiation and maintenance of C.C.B. and A.B.L. declare no financial or commercial conflict of interest. H.J.V. is a consultant for a privately held company.cTECscortical thymic epithelial cellsDNdouble negativeDPdouble positiveETPsearly thymocyte progenitorsFOXforkheadmTECsmedullary thymic epithelial cellsMEFsmouse embryonic fibroblastsPPpharyngeal pouchSPsingle positiveTECsthymic epithelial cellsTEPCsthymic epithelial progenitor cells


To study the oxygen saturation profile in normal Asian Indian eyes.A cross sectional prospective study.Ninety eight consecutive patients presenting to our hospital with best corrected distance visual acuity (BCVA) of 20/20 and a normal ophthalmic examination were included in the study. Patients having any ocular or systemic disease were excluded from the study.Oximetry was performed on all subjects with the Oxymap T1 retinal oximeter .The images were analysed for oxygen saturation and diameter.2 = 0.264; p=0.001) and venous saturation  with age. There was no significant change in the arterio-venous saturation difference (AVSD). The inferotemporal quadrant had the lowest saturations. Age correlated positively with ocular perfusion pressure (OPP). OPP correlated positively with global arteriolar saturation .The mean age was 33 years . The average arteriolar saturation was 90.3 ± 6.6% and the venous saturation was 56.9% ± 6.3. The average A-V (arterio-venous) difference was 33.2% ± 5.2. There was an increase in arteriolar (RThis study provides the normative database for an Indian population and is comparable to previous studies. Age, vessel diameter and OPP were the significant factors that influenced the saturation. Arteriolar and venous saturations increased with age while the AVSD did not change significantly. Oxygen is important for the normal physiology and metabolism in the body. The retina has the highest metabolic demands of any tissue in the body with the photoreceptors having the highest demand in the retina. InadequaHemoglobin bound to oxygen has differential absorption of light at different wavelengths. This is the basic principle used in dual wavelength oximetry. Fundus photographs at 2 different wavelengths are taken and the relative oxygen concentration can be determined using photospectrometry. ,7 Jani ePrevious studies,9 have eIt has been shown that narrower retinal vessels show higher oxygen saturations. In contWe aim to study averages and variability of oxygen saturation in healthy individuals in an Indian population. We also aim to study the interocular variability, quadrant values, relationship with diameter and age.Healthy subjects presenting to our outpatient department at Narayana Nethralaya, Bangalore were enrolled in the study with their consent. The study follows the tenets of Helsinki. The Institutional Review Board and Ethics Committee approved the study.Enrolled subjects had a normal ophthalmic examination, BCVA 20/20–20/30, with spherical refractive error less than ±6.00 diopters and cylindrical error of less than ±2.00 diopters.Patients with BCVA vision less than 20/30, cataract greater than Lens Opacification Classification System—Nuclear sclerosis Grade 2/Posterior Subcapsular Cataracts (NS2/PSC) or other significant media opacities, history of ocular conditions such as diabetic or hypertensive retinopathy, macular degeneration, or retinal vascular occlusions or chronic systemic conditions like diabetes or hypertension that can confound retinal oximetry measurements were excluded. Patients regularly taking any systemic drugs or smokers were also excluded.All patients underwent dilation with 1% tropicamide and 10% phenylephrine and subsequent retinal imaging with the oximetry using the Oxymap T1 Retinal Oximeter . The procedure was explained to them in detail and a written informed consent was obtained. They were allowed to rest for 5 minutes before the images were captured. This was done to eliminate exercise induced fluctuations in readings. Resting blood pressure and pulse oximetry  readings were taken for all patients. None of the subjects had consumed caffeine within 2 hours of the examination.The aiming light was set at the lowest setting, flash intensity was 50W, small aperture and large pupil settings were applied to the TRC 50DX  Fundus camera.One experienced photographer obtained standardized images for all the subjects. We obtained 2 images per eye in all the subjects—50°—disk centered  and macuThe software used was version 2.4.2. The oxygen saturation was given by the equation: saturation = (a x ODR + b)100; a = -1.28 and b = 1.24. Segments of vessels  were theStatistical analysis was done using the IBM SPSS v22 software. All continuous variables were tested for normality using the Shapiro-Wilk test. The difference between means was tested using independent t-test for normally distributed data and the Mann-Whitney U test for non-parametric data. For normally distributed data the Pearson’s correlation was used, for non parametric data the Spearman’s correlation was used. The level of significance was p<0.05. Linear regression was performed after identifying significant correlations.There were a total of 98 eyes (98 subjects). The mean age was 33 years (18–63)(SD = 12.4) . There w2 = 0.263, p = 0.001) and venous saturation  with age. The A-V difference did not show a significant change with age. The trends have been summarized in 2 = 0.073; p = 0.007) and with the global arteriolar saturations There was an increase in arteriolar  and nasal  saturations correlated positively with the OPP. The inferotemporal quadrant had the lowest saturations with 86% and 51%, highest diameters with 136μm and 182μm, arteriolar and venous respectively.The average arteriolar saturation (%) was 87.1 (95% C.I 85.9–88.3) temporally and 93.5 (95% C.I 92.2–94.8) nasally (p = 0.001); whereas the venous saturation was 53.7 (95% C.I 52.7–54.7) temporally and 60.1 (95% C.I 59.0–61.3) nasally (p = 0.001). The average arterial and venous diameters (μm) were 135 and 177 temporally; whereas they were 111 and 142 nasally . Both the temporal . Globally average diameter and saturation showed a weak negative correlation which was not statistically significant.We found a negative correlation between diameter and saturation for individual vascular segments (arterioles: ROur results on normative Asian Indian eyes using the Oxymap T1 retinal oximeter yields an average saturation of 90.3% in the arterioles and 56.9% in the venules which is similar to previous studies ,9 which 2 is 7%, but no statistical correlation was found between the two parameters. The standard deviation of the pulse oximetry readings is only 2 as compared to 7 with oximetry.The difference between the average pulse oximetry readings and the average arterial SOGeirsdottir et al have in their study showed the ocular perfusion pressure increases with age. We foundTissue loss with aging should rThe vessel walls change with age owing to sclerosis, which may alter the amount of light that interacts with the blood column and thus prevents us from measuring the true saturation. Our patients were methodically screened and we ensured that none of them had systemic hypertension or diabetes. It would be interesting to see how saturations vary with varying grades of hypertensive retinopathy. On one hand increased perfusion pressure and blood flow velocity may alter saturation, and altered vessel wall characteristics may change saturation on the other.This study provides the normative database for an Indian population and is comparable to previous studies. Age was the most significant factor that influenced the saturation. We also see that diameter and saturation of individual vessel segments are inversely correlated. Arteriolar and venous saturations increased with age whereas A-V difference saturations remained unchanged in this study. It is essential to understand the normal variations in the saturation to be able to pick up subtle changes in pathology.


Esophageal cancer was the fifth most commonly diagnosed cancer and the fourth leading cause of cancer-related death in China in 2009. Genetic factors might play an important role in esophageal squamous cell carcinoma (ESCC) carcinogenesis.p21, p53, TP53BP1 and p73 single nucleotide polymorphisms (SNPs) on the risk of ESCC, we conducted a hospital based case–control study. A total of 629 ESCC cases and 686 controls were recruited. Their genotypes were determined using ligation detection reaction (LDR) method.To evaluate the effect p21 rs3176352 GG homozygote genotype was used as the reference group, the CC genotype was associated with a significantly increased risk of ESCC. When the p73 rs1801173 CC homozygote genotype was used as the reference group, the CT genotype was associated with a significantly increased risk of ESCC. After Bonferroni correction, for p21 rs3176352 G>C, the pcorrect was still significant. For the other six SNPs, in all comparison models, no association between the polymorphisms and ESCC risk was observed.When the p21 rs3176352 G>C and p73 rs1801173 C>T SNPs are associated with increased risk of ESCC. To confirm the current findings, additional, larger studies and tissue-specific biological characterization are required. As the fourth leading cause of cancer deaths and the fifth most common diagnosed cancer in China in 2009 p53 gene is on chromosome 17p13.1. A well-studied p53 polymorphism, Arg72Pro  has been reported to have functional significance p53 rs1042522 G>C has been associated with risk of numerous kinds of cancers Tumor suppressor protein p53 is frequently mutated in diverse types of cancers and is implicated in cell proliferation and tumor progression p21 encodes a 21-kDa protein, is located on chromosome 6p21.2 and consists of three exons and two introns P21 (Waf1/Cip1/CDKN1A), a non-specific cdk inhibitor and a key mediator of G0-G1 cell cycle arrest, is upregulated by wild-type p53. p21 functions during gene repair and angiogenesis p53 and participates in both DNA repair and cell cycle control. By cooperating with damage sensors and signal transducers, TP53BP1 helps mediate the DNA damage checkpoint Tumor protein 53-binding protein 1 (TP53BP1) interacts specifically with P73 shares structural and functional similarities to p53. p73 is located at 1p36.33, mapping to a region that is often deleted in cancers p73 gene has some significant differences from p53. In contrast to p53-deficient mice, those lacking p73 show no increased susceptibility to spontaneous tumorigenesis p21, p53, TP53BP1 and p73, may contribute to the development of ESCC. In a hospital-based case-control study, we performed genotyping analyses of eight functional p21, p53, TP53BP1 and p73 SNPs in 629 ESCC cases and 686 controls in a Chinese population.Genetic variations in the p53 pathway genes, such as The data has been deposited in Supporting Information files. Regarding ethical conduct of research involving human subjects and/or animals, we complied with the World Medical Association Declaration of Helsinki. The review Board of Jiangsu University  approved this hospital-based case-control study. Written informed consent was provided by all subjects in the study.Between October 2008 and December 2010, from the Affiliated People's Hospital of Jiangsu University and Affiliated Hospital of Jiangsu University , 629 subjects with esophageal cancer were recruited consecutively. By pathological means, all cases of esophageal cancer were diagnosed as ESCC. Exclusion criteria were: patients who previously had cancer; any metastasized cancer and radiotherapy or chemotherapy. 686 patients without cancer were matched to the cases with regard to age (±5 years) and sex, as controls. The controls were recruited from the above-mentioned two hospitals at the same time period. Most of the controls were being treated for trauma .Using a pre-tested questionnaire, trained interviewers questioned each subject personally and obtained demographic data information  and related risk factors . Venous blood samples (2-mL) were collected after the interview from each subject. The definition of “smokers” was smoking one cigarette per day for >1 year. The definition of “alcohol drinkers” was consumption ≥3 alcoholic drinks a week for >6 months.From whole blood, genomic DNA was isolated χ2 test, between the cases and controls, the distributions of demographic characteristics, selected variables, and genotypes of the p21, p53, TP53BP1 and p73 variants differences were evaluated. Using logistic regression analyses, the associations between the eight SNPs and risk of ESCC were estimated for crude ORs and adjusted ORs when adjusting for age, sex, smoking and drinking status. Because of the number of comparisons, the Bonferroni correction procedure was applied. By a goodness-of-fit χ2 test, the Hardy-Weinberg equilibrium (HWE) was tested to compare the observed genotype frequencies to the expected ones among the control subjects. With SAS 9.1.3 , all statistical analyses were performed.Using the χ2 tests, the cases and controls are adequately matched on age and sex. Between the cases and the controls, significant difference was detected on smoking and drinking status, which is shown in p21 rs3176352 G>C . For all SNPs, minor allele frequency (MAF) in our controls was similar to MAF for Chinese in database. In the controls, for these eight polymorphisms, the observed genotype frequencies were all in HWE . In the dominant model, the p21 rs3176352 GC/CC variants were not associated with the risk of ESCC, compared with the p21 rs3176352 GG genotype. In the recessive model, when the p21 rs3176352 GG/GC genotypes were used as the reference group, the CC homozygote genotype was associated with a 63% increased risk of ESCC  (When the  0.0006) .p73 rs1801173 CC homozygote genotype was used as the reference group, the CT genotype was associated with a significantly increased risk for ESCC ; the TT genotype was not associated with the risk of ESCC. In the dominant model, the p73 rs1801173 CT/TT variants were associated with a significantly increased risk for ESCC , compared with the p73 rs1801173 CC genotype. In the recessive model, when the p73 rs1801173 CC/CT genotypes were used as the reference group, the TT homozygote genotype was not associated with the risk of ESCC  and both risk variant genotypes carrier group  were associated with a significantly increased risk for ESCC.When the p21 rs3176352 G>C genotypes on ESCC risk according to different age, sex, smoking and alcohol drinking status; we performed the stratification analyses. A significantly increased risk of ESCC associated with the p21 rs3176352 G>C polymorphism was evident among all subgroups except in female patients after stratification  is located in intron 2 of p21, 16 bp downstream from the splicing site. This C-to-G transition is predicted to affect the p21 messenger RNA splicing et al. demonstrated that p21 rs3176352 G/C polymorphism appeared to be in linkage disequilibrium with Ser31Arg in a Korean population. Analysis of this haplotype for lung cancer susceptibility demonstrated a protective effect that was dependent on the number of variant alleles. In a previous study involving 80 esophageal cancer patients and 200 cancer-free controls from Ningxia Region of China, the p21 rs3176352 G/C polymorphism was not associated with esophageal cancer risk p21 polymorphisms (rs1801270 and rs1059234) with ESCC risk p21 polymorphisms, both alone and in combination, are not ESCC genetic susceptibility biomarkers, which agrees with our results.p73 polymorphisms were not associated with ESCC susceptibility p73 rs1801173 C/T polymorphism merits further functional study to elucidate the etiology of this SNP and ESCC.P73, a p53 homolog, has some p53-like activities and plays an important role in modulating the cell cycle, apoptosis and DNA repair. In a high incidence region of China, p21 rs3176352 C was 0.410 in 686 control subjects, which is consistent with the values reported in the SNP database for the Chinese Han (0.422) and Japanese populations (0.455), higher than that of the Sub-Saharan African (0.233) population and African American population (0.250), and but lower than that of the European population (0.758). The allele frequency of p73 rs1801173 T was 0.230 in 686 control subjects, which is consistent with the values reported in the SNP database for the CHB+JPT (Chinese Han+Japanese) populations (0.267), higher than that of the Sub-Saharan African (0.102) population and European population (0.150).The frequencies of genetic polymorphisms often vary between ethnic groups. In the present Chinese study, the allele frequency of p21, p53, TP53BP1 and p73 genetic variability. Fine-mapping studies are required. Third, because of the moderate sample size and absence of a validation cohort, the statistical power was limited. Finally, the viral infections and immune parameters information was not available, thus the power of our analyses was restricted.This case-control study had several limitations. First, because the patients and controls were enrolled from hospitals, inherent bias may have resulted in spurious findings. Second, the polymorphisms we studied may not provide a comprehensive view of p21 rs3176352 G/C and p73 rs1801173 C/T polymorphisms may contribute to ESCC risk. Tissue-specific biological characterization and replication studies with larger populations are required to confirm our findings.In conclusion, our study provides strong evidence that Table S1p21 rs3176352 G>C polymorphism and ESCC risk by sex, age, smoking status and alcohol consumption.Stratified analyses between (DOCX)Click here for additional data file.Table S2p73 rs1801173 C>T polymorphism and ESCC risk by sex, age, smoking status and alcohol consumption.Stratified analyses between (DOC)Click here for additional data file.Data S1Data of p21, p53, TP53BP1 and p73 polymorphisms.(SAV)Click here for additional data file.


Particle swarm optimization (PSO) is an evolutionary computing method based on intelligent collective behavior of some animals. It is easy to implement and there are few parameters to adjust. The performance of PSO algorithm depends greatly on the appropriate parameter selection strategies for fine tuning its parameters. Inertia weight (IW) is one of PSO’s parameters used to bring about a balance between the exploration and exploitation characteristics of PSO. This paper proposes a new nonlinear strategy for selecting inertia weight which is named Flexible Exponential Inertia Weight (FEIW) strategy because according to each problem we can construct an increasing or decreasing inertia weight strategy with suitable parameters selection. The efficacy and efficiency of PSO algorithm with FEIW strategy (FEPSO) is validated on a suite of benchmark problems with different dimensions. Also FEIW is compared with best time-varying, adaptive, constant and random inertia weights. Experimental results and statistical analysis prove that FEIW improves the search performance in terms of solution quality as well as convergence rate. Swarm intelligence is an exciting new research field still in its infancy compared to other paradigms in artificial intelligence . One of The rest of this paper is organized as follows: Section 2 presents the principles of particle swarm optimization algorithm. A review on inertia weight strategies is stated in section 3. Proposed inertia weight and its properties will be discussed in section 4. In Section 5, parameter settings and performance evaluation criteria is introduced. The numerical analysis, statistical tests and discussion of results is performed under section 6 and the conclusions are given in section 7.M and the search space is D − dimensional. The position of the ith particle is presented as xi =  where xid ∈ , d ∈ , and ld and ud are the lower and upper bounds of the dth dimension of the search space. The velocity of each particle is represented with a vector. The ith particle velocity is presented as vi = . At each time step, the position and velocity of the particles are updated according to the following equations D.The basic idea of the PSO algorithm is to search out the optimum value by collaborating and sharing information between the individuals, and the particle’s quality could be measured according to the fitness value of particles. First, the positions and velocities of a group of particles are initialized randomly, and then the optimal solution can be searched out by updating generations in the search space. Suppose that the size of the swarm is = xi1, xi, …, xiD = xi1, xi, …, xiD onstants  and t isω) in which the velocity of each particle is updated according to the following equation  is an integer number. In this strategy, t and Imax are the current iterative time and the maximum iterative time, respectively. The parameters ω1 and ω2 are inertia weight at the start and inertia weight at the end of a given run, respectively. In the other wordω(t), the Flexible Exponential Inertia Weight (FEIW) strategy because it can adapt with each problem, i.e., with suitable parameters selection, we can construct many increasing or decreasing inertia weights, or even a lot of strategies with one global minimum in , thus FEIW encompasses a wide range of IW strategies. There is a trade-off between accuracy and efficiency of the PSO algorithm and one of the most important of applications of FEIW is that according to each problem, one can easily change the parameters ω1, ω2 and ψ, to achieve better accuracy or better efficiency or both of them. We call ω(t) In this subsection, for a mathematical analysis of FEIW, suppose that t ∈  be a real number instead of integer number. We define a new function byBefore using FEIW, we should have some information about its behavior. In particular, to select its parameters, we need a careful analysis of the function Lemma 1. The check function has the following properties:Proof. According to definition of FEIW, ψ > 0 thus 1 –eψ2 < 0, therefore based on Similarly one can prove the other term.Lemma 2. The equation ω(t) = 0 has at most one root. This equation has a root if and only ifImax] if and only ifIn addition, this only root, if it exists, is at Proof. By using ψ > 0 and Imax > 0, thusFrom Lemma1 and Using Eqs Corollary 1. For all t ∈ , ω(t) ≥ 0.Proof. Suppose ∃t0 ∈ : ω(t) < 0. First note that based on relations ω(t) have positive values. Since ω(t) is a continuous function, thus it has at least two roots, a contradiction, because according to Lemma 2, the equation ω(t) = 0 has at most one root.Corollary 2. If sgn * Tψ) = 1 then Tψ < 0 and Tψ < 0.Proof. Let Tψ > 0 and Tψ > 0. Thus from Lemma 1, it follows that α1 < 0 and α2 < 0, Hence from t, ω(t) < 0, a contradiction, because according to Corollary 1, ∀t ∈ , ω(t) ≥ 0.Theorem 1. The function ω(t) has an extremum if and only ifImax] if and only ifIn addition, this only extremum, if it exists, is a global minimum at Proof. We first calculate ω′(t) and ω″(t) as follows:ω(t), we set its derivative equal to 0. The equation ω′(t) = 0 implies α1α2 > 0. Using Lemma 1 and Corollary 2, it is equivalent to sgn * Tψ) = 1. To use the second derivative test, we evaluate ω″(t) at this critical number:To find the critical numbers of differentiable function ω″(t) > 0, ω(t) has a local minimum at t**, but α1 > 0 and α2 > 0 thust** is a global minimum of differentiable function ω(t). The proof of the second part of this Theorem is similar to that of Lemma 2.Because of Theorem 2. Ifω(t) is increasing on Proof. From Lemma 1 and α1 < 0 and α2 > 0, soThus ω(t) is increasing on Therefore Lemma 3. If Tψ = 0 and ω1 < ω2 then ω(t) is increasing. Also If Tψ = 0 and ω1 > ω2 then ω(t) is decreasing.Proof. If Tψ = 0 then α1 = 0 and ω2 − ω1eψ = 0. This implies ψ > 0 because of ω1 < ω2. In this case, we can conclude from α2 = ω1, thus using ω(t) is increasing. Now suppose Tψ = 0 thus α2 = 0 and ω1 − ω2eψ = 0. This implies ψ > 0 because of ω1 > ω2. Also α1 = ω1 and from Therefore ω(t) is decreasing.Therefore Corollary 3. For all t ∈ , ω(t) > 0.Proof. By Corollary 1, ∀ t ∈ , ω(t) ≥ 0. Suppose that ∃t* ∈ , ω(t*) = 0. Using Lemma 2, we have sgn * Tψ) = −1. By Theorem 2, ω(t) is increasing or decreasing. Thus according to relations Imax], ω(t) ≠ 0, a contradiction. Therefore ∀ t ∈ , ω(t) > 0.Corollary 4. If ω1 = ω2 then ω(t) takes its global minimum in  at Proof. Suppose that ω1 = ω2 = Ω. From Eqs α1 = eψα2, thus using In this special case, the check functions are as follows:By Theorem 1, has a minimum at t** ∈  and Thus Lemma 4. As ψ approaches 0 from the right, FEIW function approaches linear inertia weight function. If ω1 > ω2, then this linear function is decreasing, while if ω1 < ω2, the function is increasing.Proof. Differentiating ω(t) with respect to t, from Eqs m is the slope of line through  and . Thus the limit of FEIW function as ψ approaches 0 from the right equals Imax > 0, ω1 > ω2, and is increasing if ω1 < ω2.Since All of above results are summarized in ω1 ≈ 0, ω2 ≈ 1, ψ ≈ 2.6 for increasing FEIW and ω1 ≈ 1, ω2 ≈ 0, ψ ≈ 2.6 for decreasing FEIW and ψ ≈ 5 for cases ω1 ≈ ω2. In this paper, the parameters of different variations of FEIW strategy are selected such that include all the different situations such as increasing (decreasing) functions and functions with a global minimum. Let ψ as follows:The massive experiments indicate the proposed algorithm performance can enhance greatly for most problems when ω1,ω2). These variations of FEIW strategies in Also six pairs of positive numbers are selected for Maximum velocity: ➢Imax = 500, 1000.Maximum number of iterations allowed: outf − fmin| < ε, where outf is the best solution found when the algorithm terminates and fmin is the known global minimum of the problem, is considered to be successful. In this case, ε is error of the algorithm. In order to evaluate the performance of different IW strategies, we need to define different terms for termination of the PSO algorithm, so the termination criterion for all considered PSO variants is one of the following conditions:According to the “no free lunch theorem” , one opt➢Imax.Condition 1: achieving to ➢Imax or when the known optimum is within 1 –ε of accuracy, whichever occurs earlier.Condition 2: achieving to For each method and problem the following are recorded:SR) is number of successful runs (Srun) per total number of runs (Trun)Success rate (ANS).Average number of iterations of successful runs (MNS).Minimum number of iterations of successful runs (AE),Average error (ME) over 100 runs.Minimum error (STD) of error over 100 runs.Standard deviation  over 100 runs. The numerical results are shown in Tables According to the numerical results obtained from this study Tables –14, we cf23) in terms of ANS, are GLBIW and AIW, also in f22) in terms of AE, are FEIW-3, FEIW-5 and NEIW. Thus the notations S-IW and S-FEIW are used in the f23 and f22 columns of Tables T (15 ≤ T ≤ 22) which achieve the best result with variations of FEIW strategy in terms of PEC. Also let T. If we define T. Using this definition, we can summarize Tables S-IW indicates several inertia weights except variations of FEIW. Also S-FEIW indicates several inertia weights including some variations of FEIW. For example in  table T  ≤ T ≤ 22In this section, the numerical results obtained using FEIW strategy and other strategies are statistically analyzed based on non-parametric tests as: Wilcoxon test; Friedman test and Bonferroni-Dunn test –37. The R+ and R−, which represent the sum of positive and negative ranks of an algorithm in comparison to other algorithms in the column. During statistical analysis on Imax = 1000 and D = 10. Imax = 1000 and D = 10. Imax = 500 and D = 50 can be evaluated. Imax = 1000 and D = 50 can be evaluated. Wilcoxon sign rank test is nonparametric statistically hypothesis test which can be used as an alternative to the paired t-test when the results cannot be assumed to be normally distributed. The results for Wilcoxon’s test are summarized as α = 0.05 and α = 0.01 thus there are significant differences among the observed results. The speed in obtaining the global optimum is a salient yardstick for measuring the algorithm performance. From Imax = 1000 and D = 10, Friedman test proves the advantage of FEIW-1 and FEIW-5 over other IW strategies in terms of convergence speed and solution precision. From Imax = 1000 and D = 10, Friedman test proves that FEIW-6 and FEIW-1 are the best strategies for better accuracy. Under condition 1, Imax = 500 and D = 50, from Imax = 1000 and D = 50, from The Friedman test is a non-parametric statistical test developed by the Friedman , 39. TheNi and Nf are number of IW strategies and benchmark functions, respectively. Also critical values qα at the probability level α is given in [Here we have employed Bonferroni-Dunn test  to detecgiven in  as folloUsing Eqs  and 42)42) critiThe difference among mean ranking of PSO variants is illustrated by Bonferroni-Dunn’s graph in Figs For average error criteria, CIW, RIW, LDIW, CHIW, GLBIW, AIW, FEIW-4 and FEIW-5 emerge as worst performers and FEIW-1 and FEIW-6 as best performers; For minimum error criteria, CIW, RIW, LDIW, CHIW, GLBIW, AIW, FEIW-3 and FEIW-4 come as worst performers and FEIW-1 and FEIW-6 as best performers. For standard deviation criteria, CIW, RIW, LDIW, GLBIW, AIW, FEIW-3, FEIW-4 and FEIW-5 emerge as worst performers and FEIW-1 and FEIW-2 as best performers. Therefore, in general manner, Bonferroni-Dunn bar charts show that FEIW-1 strategy has the best performance among all considered strategies.In addition to using statistical tests to observe the performance of considered PSO variants, boxplot analysis is also performed for benchmark functions and shown in Figs D = 10 and Imax = 30000. From convergence graph, we can discover that the convergence rate of the mentioned IW strategies is clearly faster than the other strategies on the benchmark functions. At the same time, the best solution get by FEPSO is more optimum than by CIWPSO, RIWPSO, LDIWPSO, CHIWPSO, GLBIWPSO, AIWPSO, NEIWPSO and EDIWPSO.The convergence graph for FEIW-1, FEIW-3, FEIW-5 and FEIW-6 is demonstrated in There are many modifications have been done to the standard PSO algorithm. Some of modifications to the basic PSO are directed towards introducing new strategies of inertia weight which tuned based on trial and error. Suitable selection of the inertia weight provides a balance between global and local searching. This paper proposed a new flexible exponential time-varying inertia weight (FEIW) strategy to improve the performance of PSO. The algorithm named as FEPSO is proposed based on FEIW strategy. We confirmed the FEPSO’s validity in terms of convergence speed and solution precision by testing it with a suit of well-known standard benchmark unimodal and multimodal functions and by comparing obtained results with eight inertia weight strategies of the best time-varying, adaptive and primitive inertia weight strategies. The comparisons are made in terms of convergence speed and solution accuracy and the results are tabulated and graphs are plotted for dimensions 10 and 50 separately. Statistical tests show that this novel strategy converges faster than others during the early stage of the search process and provide better results for problems. Thus experimental results clearly prove the superiority of the proposed model over other inertia weight models. The future work includes the implementation of the FEPSO to solve a real world problem with lots of complexity such as brain MR image segmentation to compare the efficiency of the FEPSO with other recent optimization techniques.


To determine whether clinical information influences endoscopic scoring by central readers using the Ulcerative Colitis Endoscopic Index of Severity .with  or without  clinical information. A total of 21 virtual Central Reader Groups [CRGs], of three blinded readers, were created. Agreement criteria were pre-specified. Kappa [κ] statistics quantified intra- and inter-reader variability.Forty central readers performed 28 evaluations, including 2 repeats, from a library of 44 video sigmoidoscopies stratified by Mayo Clinic Score. Following training, readers were randomised to scoring p = 0.97]. Correlation between UCEIS and visual analogue scale [VAS] assessment of overall severity was high . Scores for control videos were similar . Intra- [κ 0.47–0.74] and inter-reader [κ 0.40–0.53] variability for items and full UCEIS was ‘moderate’-to-‘substantial’, with no significant differences except for intra-reader variability for erosions and ulcers . The SD of CRGs was lower than for individual central readers . Correlation between blinded UCEIS and patient-reported symptoms was high .Mean UCEIS scores did not differ between blinded and unblinded readers for any of the 40 main videos. UCEIS standard deviations [SD] were similar [median blinded 0.94, unblinded 0.93; The UCEIS is minimally affected by knowledge of clinical details, strongly correlates with patient-reported symptoms, and is a suitable instrument for trials. CRGs performed better than individuals. The final model consisted of three items: vascular pattern [3 levels], bleeding [4 levels], and erosions and ulcers [4 levels]  to 8 .8 The UCEIS accounted for 88% of the variance between observers in the overall assessment of endoscopic activity in the test cohort5 and 86% in the validation cohort.8The UCEIS was developed using a predefined protocol. Initially, the level of disagreement for 10 endoscopic items, each with 3–5 levels of severity, was determined among 10 investigators. levels] .5,8 In p9 and to compare scores with other indices and patient-reported symptoms.The aim of the present study was to understand the UCEIS in a clinical context. The primary objective was to determine whether the UCEIS is affected by clinical information, using an independent cohort of central reader investigators. Henceforth, the term ‘readers’ rather than ‘investigators’ or ‘observers’ will be used for consistency, since ‘investigators’ implies clinicians who recruit patients to trials and often have knowledge of symptoms, whereas ‘readers’ implies independence from the patient, and ‘observers’ is a generic term for either. Secondary objectives were to investigate potential benefits of a group of central readers for reducing variability,8 In short, the resource was a library of 670 videosigmoidoscopies from patients in three clinical trials, supplemented by individuals without UC and hospitalised patients with acute severe UC.5 In phase 1, the authors determined agreement in overall endoscopic assessment and defined descriptive terms [‘items’]. Phase 2, conducted in a separate cohort of 30 investigators, rated items in 25 of 60 different videos and assessed overall severity on a 100-point VAS [0 completely normal and 100 = worst ever seen].8 The UCEIS developed from this study included three items  and unblinded [n = 20] assessment groups as they completed the study enrolment requirements [but prior to training or receipt of any study videos]. All underwent training on the UCEIS, which excluded clinical information and involved scoring four standard videos; to qualify [details in 3. Results], assessments on each video had to align with the level of ‘erosions and ulcers’ assigned by the authors , and within one level for vascular pattern and bleeding. For readers initially failing to qualify, a retest was permitted; they had correctly to score two of three different videos for the item[s] that they had previously scored incorrectly. Readers who failed the second qualifier were excluded.A new cohort of 40 investigators [‘readers’] was enrolled from Europe, North America and Australasia, all of whom were experienced in evaluating endoscopies in patients with UC. Three had previously participated in phase 2 or 3 of UCEIS development and all were blinded to the objectives of the study. To investigate the effect of clinical information on UCEIS scoring, investigators were alternately assigned to the blinded  assigned on the date that they were derived. 11 No video had been used in the earlier phases of UCEIS development. Three further videos were taken from patients with severe UC [recorded before colectomy] and three from subjects without UC [colorectal cancer screening]. Four videos were repeated as common controls between readers, drawn from the 34 videos of patients with active disease [two videos with MCS 1–2 and two with MCS 10–11].A total of 34 of the videos were selected [by PK and BRY] from sigmoidoscopies conducted to a standard procedure as part of clinical trials.11Each reader performed 28 evaluations from the 44 videos, which included two repeats of non-control videos to evaluate intra-reader variation and the four common controls . Readers1 Readers in the blinded group were not provided with any clinical information for the common control videos, as with their other videos.To ensure some disparity between clinical information and endoscopic assessment, the two common control videos with MCS 1–2 in the unblinded group were assigned information more severe than reported [originally: rectal bleeding [RB] = 1 / stool frequency [SF] = 0 / Physician’s Global Assessment [PGA] = 0; and RB = 0/SF = 1/PGA = 0; both changed to: RB = 1/SF = 2/PGA = 1]. The two control videos with MCS 10–11 were given symptom information less severe .n = 28, including the two duplicates] were created with 10 unblinded and 10 blinded readers randomly allocated to each pool.To ensure that sufficient numbers of readers viewed the same videos to power the analyses, two ‘pools’ of 26 videos [8 still photographs from the training were provided for reference during the evaluations and the overall assessment of endoscopic severity recorded on a 100-point VAS [0 = completely normal and 100 = worst ever seen]. Data were captured using a programme developed by one of the authors [PS] that ran simultaneously and saved responses after scoring each video.The 40 readers who passed training evaluated the UCEIS  in the w9The effect of central reading to improve consistency of scoring the UCEIS was examined through ‘virtual’ central reading groups [CRGs] of three readers. Some of the readers [6/40] were recruited specifically for their experience as central readers in other trials [see acknowledgements]; three were randomly allocated to the blinded group and three to the unblinded group . In the The UCEIS represents the simple sum of ‘vascular pattern’ [scored 0 to 2], ‘bleeding’ [0 to 3] and ‘erosions and ulcers’ [0 to 3]. Thus, UCEIS scores range from 0 [normal] to 8 [most severe].p-value with Holm’s multiplicity adjustment12]. Standard deviations [SD] of the assessments on a per video basis were compared using the Wilcoxon signed rank test. The correlation between the UCEIS and overall endoscopic assessment of severity by VAS, as quantified by Pearson correlation coefficients, was compared between the blinded and unblinded groups. Comparison of these correlations allowed the accuracy of UCEIS assessments to be checked, in addition to quantifying the similarity of accuracy estimates between the blinded and unblinded groups. Pearson correlation coefficients were calculated from reader’s scores of the UCEIS for their set of videos and the mean VAS for the appropriate videos, derived from the responses of all other readers in the blinded or unblinded group. This addressed any lack of independence between UCEIS and evaluation of overall severity. Correlations were summarised by median, minimum, and maximum within each group, and compared using the Wilcoxon rank sum test. The UCEIS and overall [VAS] severity scores for the common control videos, which were presented with misleading symptom information to the unblinded group, were also compared using the Wilcoxon rank sum test.UCEIS and overall severity by VAS [0–100] for each of the 40 videos in the main analysis dataset were compared between the blinded and unblinded groups using the Wilcoxon rank sum test  ordinal scale, a weighted kappa was also calculated to take account of close agreement, assigning a weight of 1 for precise agreement, 0.5 for scores that differed by 1 level, and 0 in all other cases. For intra-reader variability analyses, only data from duplicate videos were used. Inter-reader κ values were calculated by stratifying by reader pairs and using the common videos that they scored, but excluding second scoring of duplicate videos. An average of reader-pair κ values [‘overall κ’] was calculated, where the weighting was the inverse of their variance.Intra- and inter-reader variability in blinded and unblinded groups was evaluated through kappa [κ] statistics, as qualitatively interpreted by Landis and Koch .To evaluate the effect of blinded central reader groups, for the 18 non-control videos read by all three experienced central readers, SDs were calculated across the seven blinded ‘standard readers’ and compared with those across the 21 virtual CRGs. Differences in SDs between groups were assessed by nonparametric methods.14 partial MCS [excluding endoscopic subscore],15; and the modified Baron score,14 as recorded in the original clinical trials. To compare the UCEIS with patient-reported symptoms and variables recorded in practice, stool frequency [SF], rectal bleeding [RB], both SF and RB, and patient functional assessment were derived from subscores of the MCS contemporaneously recorded for each video. These variables were compared with UCEIS scores for the blinded readers, excluding those for ‘normal’ and ‘most severe’ videos [for which there was no MCS] and common controls [which were repeats], by Spearman rank correlation. SF, RB, PFA, and partial MCS were also correlated with the modified Baron score, to determine how results compared with those calculated for the UCEIS. The relationship between the UCEIS bleeding item [scored 0–3] and patient-reported RB on a 3-point scale [0 = none to 2 = visible blood with stool > half the time or passing blood alone] was also evaluated. Finally, the distribution of RB, SF, and RB and SF items across UCEIS scores 0–8 was assessed for blinded video evaluations, excluding ‘normal’, ‘most severe’, and common control videos.The comparative indices selected were the Mayo Clinic Score [MCS],p < 0.05], using the Statistical Analysis System  software, version 9.2.Except as noted for comparison of mean scores by video, statistical significance was assumed at the 5% level .A total of 47 readers underwent UCEIS training to reach the target of 40 for the study. Of those that qualified, 20 succeeded on their initial assessment and the remainder after scoring the additional set of three videos. The remaining seven physicians failed to qualify. Variance between qualifiers was not evaluated, because of disparity between reader numbers and variables.A total of 1120 evaluations were performed across the 44 videos [40 readers x 28 evaluations]. The main analysis dataset consisted of 880 evaluations [440 each for blinded and unblinded groups] across 40 videos, obtained by removing evaluations of the four common control videos and the second evaluation in each of the two repeated videos. The dataset for the four common control videos consisted of 160 evaluations [40 readers x 4 videos]. There were also 160 evaluations in the dataset for assessing intra-reader variability [40 readers x 2 repeat pairs].Mean assessments of overall endoscopic severity by VAS [0–100] ranged from 1.65 for videos in the ‘normal’ stratum to 92.75 for videos in the ‘most severe ever seen’ stratum according to the 40 readers. Using the same videos, this corresponded to mean UCEIS scores of 0.15/8 for ‘normal’ subjects to 7.90/8 for ‘most severe’, indicating that the videos comprehensively covered the range of endoscopic severity of UC .p-values ≥ 0.05] [p = 0.045] [p = 0.97], although as expected, the SD was lowest at the severe end of the video spectrum [p = 0.02]. For the four common control videos, there were no statistically significant differences in UCEIS [p ≥ 0.55] or overall severity scores [p ≥ 0.07] between reader groups.Mean UCEIS scores did not differ between blinded and unblinded readers for any of the 40 videos in the main analysis set  . There w= 0.045] . There wspectrum . Median p = 0.047] just reached statistical significance. Clinical information tended to increase variability [i.e. reduced the κ] for the bleeding item and improved consistency between readers for erosions and ulcers. Weighted intra-reader kappas for the full UCEIS [sums of the three descriptors] were 0.51  and 0.56  for the blinded and unblinded readers, respectively, and were not significantly different [p = 0.66].Overall, intra-reader variability for the three items ranged from κ 0.47 to 0.74, indicating ‘moderate’ to ‘substantial’ agreement . Intra-rInter-reader agreement for the items was also ‘moderate’ to ‘substantial’ . This was most apparent in videos representing UCEIS scores between 3 and 5, most likely to represent the spectrum mild or moderate endoscopic disease severity  or patient functional assessment , through 0.81 for both stool frequency and rectal bleeding  to 0.82 for RB [95% CI = 0.78–0.85] and 0.86 for the full MCS . The UCEIS correlated well with patient-reported symptoms of stool frequency and rectal bleeding [or both], such that when endoscopic bleeding was scored 0 on the UCEIS [none], this corresponded to contemporaneously patient-reported RB of 0 or 1 in 95% of the time. The UCEIS had least variance for defining remission or severe endoscopic activity, but where variance was greatest , central reading by a group of three readers had most impact. Central reading matters for clinical trial design, whereas the lack of impact of symptoms on endoscopic assessment matters in clinical practice.8 Agreement in scoring of individual items is modest but it is the overall score that matters most, assessed in the most severely affected area at flexible sigmoidoscopy. In this study, the intra- [κ 0.47 to 0.74] and inter-reader [κ 0.40 to 0.50] agreement for the three components of the UCEIS  was consistent with that reported in phase 3 .8 Although the impact of training was not quantified in this study, it was considered appropriate as with any descriptive process such as endoscopy reporting. Consistency highlights a strength of the UCEIS in providing a simple, standardised reporting system for the endoscopic appearances of UC and confirms that it is a reliable instrument for assessing endoscopic disease severity.The collective performance of the three items accounts for 86% of the variance in the overall assessment of endoscopic severity.p = 0.047; The influence of clinical information had minimal effect on the variability and reproducibility of UCEIS scoring. No significant differences between readers were demonstrated for any of the items, whether readers were provided with clinical information or not . This is internally consistent with the finding that providing unblinded readers with clinical information apparently too severe or too mild for the common control videos did not influence their scoring of the items compared with blinded readers . Although the items of ‘erosions and ulcers’ registered statistical significance  and patient functional assessment [rank correlations 0.76 to 0.82]. There were strong correlations between the scores for individual items and overall UCEIS , encourap = 0.069].7 After blinded central reading of endoscopic videos had excluded 31% of patients for being ineligible for the criterion of a MCS subscore of > 2, the difference between the treatment groups readily achieved significance . This has implications for a charter on central reading, since the optimal configuration of central reading for scoring is unknown.9 Options include an index reader, polling multiple central reader results, adjudicating reads on ‘outliers’, or random selection of videos to be read centrally. The effect of training and revalidation on central readers needs to be determined, since all factors will affect conclusions on drug efficacy and registration.This study has shown that variation is both least and most similar for blinded/unblinded groups at the extremes of the UCEIS range [≤ 2 and ≥ 6]. It means that the UCEIS has least variance for defining either remission or severe endoscopic activity. This is an asset when determining responsiveness [the ability to detect change], which was not evaluated in this or previous studies. Relevant to clinical trials, however, is the variance in the mid range [UCEIS 3–5], which was significantly reduced by central reading groups. This was assessed through random groups of three readers, one of whom was ‘experienced’ in central reading, to act as an adjudicator should there be disagreement between ‘standard’ readers, representing normal investigators. Central reading of endoscopy can affect the outcome of clinical trials in mild-moderately active UC. A study of a well-established mesalazine product for mild-moderately active UC did not reach significance compared with placebo [> 6 represents a threshold for severe disease with prognostic implications, although thresholds and their implications need to be defined by prospective study in clinical trials, currently in progress.18 A score of 0 is likely to become the aspirational goal for both regulatory trials and clinical care;18 which should be index independent, since remission is remission. On the other hand, the UCEIS also seems likely to become the favoured instrument for early drug development, when a binary endpoint of remission / no remission is not efficient and there is enormous value in assessing relative changes in mean scores.18 Further limitations of the current study are that the readers in this study may not reflect endoscopists in clinical practice. Readers were selected, trained, and had images available during the rating, so the question of whether clinical information has an impact in real life may be questioned. The answer is training. Although the UCEIS is simple in concept and easy to apply, that 7/47 proposed readers failed to agree with defined interpretations indicates that training is a necessary component of its application, no less for evaluating colitis than for endoscopic polyp detection.What still needs to be defined are the levels of the UCEIS for remission, mild, moderate, and severe disease. Taking account of the variance and performance characteristics, the authors speculate that a UCEIS of 0–1 indicates remission and 20 On the other hand, caution should be exercised to prevent the UCEIS becoming more complex than necessary. Of greater interest is correlation with histological disease activity or biomarkers, especially for the prognostic value in remission. Central readers can markedly decrease the variability in UCEIS scoring, particularly in the mild-moderate disease spectrum, which is most relevant to clinical trials. The UCEIS is simple to use, derived from the sum of just three items, and accurately accounts for a widest range of disease severity associated with UC, is affected minimally if at all by clinical information, and is ready for practice after appropriate training.Next steps in the development of the UCEIS include establishing thresholds for remission and severity, and responsiveness to change. It would be valuable to examine how the UCEIS is affected by evaluation of colonic segments at full colonoscopy.None declared.ST, PK, DS, CB, BY, and WS contributed to the concept and design of the study. DS performed the statistical analysis with further evaluation by JYM and input from DA. PS created the data capture program for video evaluation. PK, BY, and CB coordinated the planning and implementation of the study. ST, MA, JFC, BF, SH, GL, PM, WR, BS, BY, JYM, and WS contributed to the clinical interpretation of the data. All authors contributed to the manuscript.


Immunizations have made an enormous impact on the health of children by decreasing childhood morbidity and mortality from a variety of vaccine-preventable diseases worldwide. The eradication of polio from Nigeria and India is one of the most recent victories for one of the greatest technological advances in human history. Despite these international successes, the United States has experienced the re-emergence of measles, driven largely by increasing parental refusal of vaccines. Pediatricians should be trained to be very knowledgeable about vaccines and should continue to advocate for parents to immunize their children. Immunizations have made an enormous impact on the health of children by decreasing childhood morbidity and mortality from a variety of vaccine-preventable diseases worldwide. The eradication of polio from Nigeria and India is one of the most recent victories for one of the greatest technological advances in human history. Despite these international successes, the United States has experienced the re-emergence of measles, driven largely by increasing parental refusal of vaccines ,2. WhileTo be successful in discussing vaccine benefits and risks pediatricians and other health care providers must be well-informed and confident about the overwhelming benefits and safety of immunizations. However, some of these providers and pediatric resident trainees do not feel prepared or informed enough to discuss vaccines and vaccine safety with families ,14. A reet al., supports this more paternalistic approach [Knowledge alone is not sufficient. In order to be effective immunization advocates, health care providers must confidently convey that parents should adhere to the immunization schedule approved by ACIP. Rather than asking parents if they plan to accept vaccines, pediatricians should take a directive role when discussing vaccines. We do not ask parents if they plan to use a car seat or place their infant on his or her back to sleep. A study by Opel, approach . Their sapproach . Familieapproach , howeverapproach . InsteadResidency training programs should continue to emphasize immunizations and immunization safety within their curricula. In addition, residents should learn how to talk to parents about immunizations in an effective manner. In order to communicate effectively and build trust with parents, pediatricians and other health care providers, as a whole, must be better informed about the safety and efficacy of vaccines and the rigorous mechanisms that safeguard the vaccine development process.