Effective treatment for neuropathic pain is still lacking, because of poor understanding of pathological mechanisms at the molecular level. Chronic pain (inflammatory and neuropathic pain) is believed to be caused by aberrant neuronal responses along the pain transmission pathways. Both peripheral and central origins are likely to be involved in chronic pain, although their contribution may be different depending on the various forms of chronic pain. Glial cells have recently been implicated in neuropathic pain. These cells form close interactions with neurons and thus may modulate nociceptive transmission under pathological conditions. We will first examine the recent progress in the role of glia in neuropathic and inflammatory pain, with particular emphasis on microglia. Finally, we will discuss how the study of the interaction between neuronal and microglial mechanisms can open the door to new therapeutic opportunities, designed to act on the mechanisms underlying the disease (“disease-oriented”) using natural endogenous substances. I.To develop a better treatment for neuropathic pain, a comprehensive understanding of its pathogenesis is required. Chronic pain (such as inflammatory and neuropathic pain) is believed to be caused by aberrant neuronal responses along the pain transmission pathway from dorsal root ganglion (DRG) to spinal cord, thalamus and cortex. Both peripheral and central origins are likely to be involved in chronic pain, although their contribution may be different depending on the various forms of chronic pain. It has been recently reported that neurons are not the only cell type involved in chronic pain states. Glial cells, including astrocytes and microglia, are emerging as possible additional players in the initiation and maintenance of neuropathic and inflammatory pain. These glial cells have close interactions with neurons and thus modulate pain transmission particularly under pathological conditions –2.II.Current therapeutic strategies for neuropathic pain aim to reduce the excitability of neurons by modulating ion channel activity (such as gabapentin and lidocaine) or by enhancing endogenous inhibitory mechanisms (such as tricyclic antidepressants and opioids). As we know, NMDA receptors play a critical role in synaptic plasticity within pain transmission pathways and are thus likely to be important in neuropathic pain. NR2B subunit-containing NMDA receptors are localized in pain-relevant structures, such as in superficial layers of the dorsal spinal horn, thalamus, hippocampus and cortex. The restricted distribution of NR2Bmakes it promising as a candidate target of side effect free analgesic drugs. Indeed, NR2B antagonists, such as ifenprodil and related compounds, are effective in neuropathic pain in animals, and show better separation between efficacy and side effects in human patients than non-selective NMDA receptor blockers.Our understanding of pathological pain has evolved from solely neuronal mechanisms to neuronglial interactions. In particular, astrocytes and microglia act as possible modulators of neuropathic pain by releasing a number of cytokines and chemokines. Astrocytes and microglia play different roles in relation to neuronal activity; however, they do have some overlapping functions in mediating CNS innate immune response . Both asThese lead to an increase in the synthesis of inflammatory factors (interleukin 1β (IL-1 β), IL-6, tumour-necrosis factor-α (TNF α), prostaglandin E2 (PGE2) and nitric oxide(NO), which finally alter glial glutamate transporter function and gap-junction proteins, which are known to facilitate astrocyte–astrocyte activation through Ca2+ cascades. Although similar pathways are activated in microglia after nerve injury, the temporal patterns of enzyme activation and pro-inflammatory cytokine release are distinct for microglia and astrocytes.. Furthermore, chronic astrocyte activation after nerve injury has been shown to involve ERK activation and subsequent down-regulation of excitatory amino acid transporters (glutamate transporter 1 (GLT1) and glutamate–aspartate transporter (GLAST) leading to a decrease in glutamate uptake and an increase in excitatory synaptic transmission. During chronic neuropathic conditions, astrocytes remain activated in response to the initial microglia-derived inflammatory factors; this is likely to account for their ongoing responses during these conditions.Microglia are the resident macrophages and main immune-response cells in the CNS . They coResting ramified microglia rapidly transform into an activated state in most pathological conditions, including host defense against infectious organisms, autoimmune inflammation, ischemia, trauma, neurodegeneration and neuropathic pain ,7. ActivUsing transgenic mice in which microglia are selectively labeled with GFP, recent studies  performeIn the setting of neuropathic pain, peripheral neurons transmit signals to spinal dorsal horn neurons, releasing neurotransmitters such as calcitonin gene-related protein (CGRP), substance P, glutamate, and ATP. Locally in the dorsal horn, there are also other neurotransmitters involved, such as GABA, glycine, serotonin.3CR1, monocyte chemotactic protein (MCP-1) and CCR2. Similar to microglia in the brain, spinal microglia show fast chemotaxis in response to local application of ATP [Spinal microglia activation happens via several signaling pathways, which include ATP and its receptors (P2X and P2Y receptor), fractalkine and CXn of ATP . Microgln of ATP ,14. Actin of ATP , while an of ATP ,17. Inten of ATP ,19 while3CR1 are also involved in microglial activation associated with neuropathic pain. Fractalkine is a neuronal transmembrane glycoprotein that can be released after being cleaved by proteolysis; fractalkine activates p38 in spinal microglia and produces mechanical allodynia and thermal hyperalgesia [3CR1, and its expression is markedly upregulated in models of neuropathic pain.Fractalkine and its receptor CXralgesia . The cleralgesia . Microgl2+ elevation and phosphorylation of p38, which subsequently increase BDNF synthesis and release. BDNF released from microglia may produce a shift in neuronal anion gradient with potassium-chloride exporter KCC2 reduced in dorsal horn neurons. This prompts a disinhibition and thus facilitates mechanical allodynia after nerve injury.In addition, MCP-1 and its receptor CCR2 are involved in microglial activation and neuropathic pain. Intrathecal injection of MCP-1 produced tactile allodynia, Microglial cells in the dorsal horn express CCR2, which may mediate MCP-1's effect: microglial morphology transitions and p38 activation in microglia. How might activated microglia contribute to neuropathic pain? One intriguing pathway involving ATP and the P2X4 receptor has been proposed. Peripheral nerve injury leads to an upregulation of P2X4 receptors in activated microglia. ATP acts on the P2X4 receptor in microglia and induces intracellular CaIt has been shown that p38 activation turns on the transcription factor NF-κB, which leads to the expression of IL-1β, IL6, and COX-2 . These aAmong the receptors we know to be upregulated on immune response cells under neuropathic pain conditions, there are also the cannabinoids receptors (CB1 and CB2).Cannabinoids suppress behavioural responses to noxious stimulation and suppress nociceptive transmission through activation of CB1 and CB2 receptor subtypes. CB1 receptors are mainly expressed at high levels in the central nervous system (CNS), whereas CB2 receptors are found predominantly, but not exclusively, on immune cells outside the CNS.1 and CB2 receptors inhibits adenylyl cyclase [i/o protein.Activation of CB cyclase ,24 and a cyclase  through 1 receptors suppress calcium currents and activate inward-rectifying potassium channels: these effects are associated with depression of neuronal excitability and transmitter release. Thus, differences in receptor distribution and signal transduction mechanisms are likely to account for the relative absence of the CNS side effects induced by CB2 agonists.Only CBMicroglia or macrophages exspress CB2 receptors in a different way in vivo or in vitro: in vitro its levels depend on the local environment and the combination of inflammatory molecules ; in vivoIII.2 receptors may have considerable therapeutic potential for suppressing inflammatory and neuropathic pain states. In animal models of tissue and nerve injury-induced nociception, CB2-selective agonists suppress hyperalgesia and allodynia and normalize nociceptive thresholds without inducing analgesia [2 agonists is the failure of these compounds to induce adverse CNS side effects associated with activation of CB1 receptors.All these considerations together suggest that novel pharmacotherapies targeting CBnalgesia . CB2 sel2 agonists in suppressing persistent pain states following acute administration. However, the impact of long-term treatment with CB2 agonists on antihyperalgesic efficacy and immune system function remains largely unknown [2 receptors and to explore the therapeutic potential of multimodal analgesic strategies that combine CB2-mediated pharmacotherapies for pain with other agents directed at different analgesic targets. Such strategies offer the potential to produce synergistic antihyperalgesic effects with a more beneficial therapeutic ratio compared to conventional analgesics .The available literature supports the efficacy of CB unknown . More woTaking into account that the endocannabinoids are synthesized only “on demand” and that tissue accumulation of N-acylated glycerophospholipids, and free N-acylamides, such as anandamide (AEA) and palmitoylethanolamide(PEA), occurs in some pathological conditions known to be associated with inflammatory and pain reactions, several studies have been carried out to identify the beneficial effects associated with therapeutic use of PEA .PEA is an endogenous fatty acid and with properties comparable to anandaminde, as well as other endogenous cannabinoids. PEA may behave as local autacoids capable of negatively modulating mast cell activation (ALIA mechanism) . In keepLipids like N-palmitoylethanolamine can act as signaling molecules, activating intracellular and membrane-associated receptors to regulate physiological functions. The signaling lipid PEA is known to activate and membrane-associated, intracellular and nuclear receptors  and regulate many physiological functions related to the inflammatory cascade, and thus is of high interest in the treatment of neuropathic, or gliopathic pain.Meanwhile it is an established fact that anandamide (AEA) and PEA regulate directly or indirectly many of the same pathophysiological processes, including pain perception, inflammation, convulsions and neuroprotection. There are a number of biological effects of endocannabinoids which can be enhanced by related endogenous fatty acid derivatives which are devoid of some of these primary effects. The last mechanism of action is called the entourage effect .The fatty acids as PEA seem to be co-synthesized and co-released with the endocannabinoids such as anandaminde. Anandamide and PEA both are present in the spinal cord, but the concentration of PEA is most probably 10 times higher. The entourage effect of PEA most probably is due to its function of amplifier activity anti-inflammatory and antinociceptive of other endogenous compounds such as endocannabinoid anandamide. It has been suggested that PEA enhances the effects of AEA by acting as a competitive inhibitor of the enzymatic degradation of endocannabinoids, and increaseing the affinity of the AEA for the CB1 and vanilloid receptors (TRPV1), highly exspressed on neuronal and mast cells .Transient Receptor Potential Vanilloid 1 (TRPV1) is a Ca2+ permeant non-selective cation channel and they are exspressed highly on sensorial fiber nerve endings (FNE) and they can act as a transducers of noxious temperature and chemical stimuli .TRPV1 expression is increased in inflammation and neuropathic pain because of the retrograde transport of nerve growth factor (NGF) released at the site of peripheral tissue injury to the DRG soma.The possibility of using TRPV1 receptor agonists, such as capsaicin, to alleviate pain is an interesting concept. Capsaicin acts first exciting FNE, than leading to TRPV1 down-regulation. As a result of this FNE show a loss of pain sensibility known as analgesia. Moreover capsaicin inhibits the retrograde transport of NGF. Activation of TRPV1 by capsaicin can induce persistent depolarization of the nerve terminals causing a decrease in their ability to generate and propagate action potentials . On the


Humans are exposed to the DNA damaging agent, ionizing radiation (IR), from background radiation, medical treatments, occupational and accidental exposures. IR causes changes in transcription, but little is known about alternative transcription in response to IR on a genome-wide basis. These investigations examine the response to IR at the exon level in human cells, using exon arrays to comprehensively characterize radiation-induced transcriptional expression products. Previously uncharacterized alternative transcripts that preferentially occur following IR exposure have been discovered. A large number of genes showed alternative transcription initiation as a response to IR. Dose-response and time course kinetics have also been characterized. Interestingly, most genes showing alternative transcript induction maintained these isoforms over the dose range and times tested. Finally, clusters of co-ordinately up- and down-regulated radiation response genes were identified at specific chromosomal loci. These data provide the first genome-wide view of the transcriptional response to ionizing radiation at the exon level. This study provides novel insights into alternative transcripts as a mechanism for response to DNA damage and cell stress responses in general. The transcriptional response to cellular stress is critical for cell survival. Ionizing radiation (IR) causes a broad spectrum of DNA damage for which the cells' commitment to repair, programmed death, cell division arrest or senescence, is required for an organism's survival. Humans are ubiquitously exposed to radiation, including during cancer treatment. About 1–5% of radiotherapy patients have severe side effects. The rate of these normal tissue reactions, for which there is nearly a normal distribution across the population, is determined by a therapeutic ratio . The outcome of this radiation response diversity is that the dosage all patients receive is limited by those few patients who are particularly radiosensitive in their normal tissue, and thereby ultimately preclude optimal treatment for the majority of radiotherapy patients. Identification of those patients who are radiosensitive given current dose regimes, is paramount to enable individualization of RT. These individuals can potentially be identified by interrogating the transcriptome. Furthermore, identifying the IR transcriptional response profile also benefits establishment of biological dosage predictors, understanding response to other radiological exposures and can contribute to the development of new radio-pharmaceuticals.DNA damage is a major cellular consequence upon exposure to radiation, where double-strand breaks (DSBs) are a critical type of damage that can lead to cell death and potentiate tumorigenesis. Two major pathways involved in DNA DSB repair are non-homologous end-joining and homologous recombination both involving many proteins The IR response at the transcriptional level has been characterized to some degree in different experimental settings for the human genome Post-transcriptional processing is a primary mechanism to generate protein diversity. In particular, the production of alternative transcripts leading to multiple isoforms is common for many genes. Different transcription isoforms can result in dramatically different cellular responses. Use of alternative transcripts is predicted for the majority of human genes Alternative transcripts can be grouped into different categories. These include alternative use of exons, different 5′ and 3′ splice sites, alternative transcription start sites, alternative termination sites, as well as intron retention and mutually exclusive alternative exons Alternative transcription is important in the regulation of genes involved in many cell processes and genetic diseases including cancer Drosophila melanogaster, the TAF1 gene (a subunit of TFIID involved in RNA polymerase II transcription), is alternatively spliced following IR RAD17, involved in cell cycle arrest, is another example of a gene that is alternatively spliced in response to radiation Inhibition of RNA polymerase II elongation has been shown to be a mechanism for genotoxic stress (ultraviolet radiation) induction of AS proposed to be due to allowance of weaker splicing sites to participate in AS MDM2PPM1DFBXW7Some specific genes have been found to use a secondary promoter following IR to produce a radiation-induced isoform. These genes include Human exon arrays were used to identify transcription changes, including the induction of AS, in cells exposed to IR. Unlike some other oligonucleotide array platforms that have probe sets only at the 3′ end of the transcripts, the exon array has an average of nearly four probe sets for every known exon. This allows the determination of relative levels of each exon for a given treatment, facilitating identification of exons that are differentially expressed after IR. Having a transcript profile for every exon has the advantage of being able to detect transcripts which classical 3′ assay platforms would miss. For example, exon arrays are able to detect transcripts missing the 3′ exon for a number of reasons such as degradation, splicing, or undefined 3′ ends. Transcripts with non-polyadenylated messages or alternative polyandenylation sites would also be commonly missed. Given present estimates that most genes can use AS Here we analyse the human transcriptome IR response for both dose and time in two human cell types, lymphoblastoid cell lines (LCLs) and primary fibroblasts at the level of individual exons. Alternative transcription and other genome-wide transcription features have been identified as a response to IR.CDKN1A, MDM2, PPM1D, GADD45A, SESN2, CCNG1 and XPCEDA2R, FAM72A and C1orf183) or in fibroblasts (ASAH3L (ACER1), EDA2R, PAG1, BCOR, CBL, FAM100B, FAM72A, SETD8 and TIGD1), although some of these genes are IR-responsive in other experimental settings.Whole genome transcript exon arrays were utilized to comprehensively characterize radiation-induced transcriptional expression products in two human cell types. The exon array is set up with 4 probes for most probe selection regions (PSRs), and represents an exon or potential exonic region of a gene. For these studies, the filtered ‘core’ set of PSRs  which are well-documented exon regions, were utilized. RNA processed from human lymphoblast and fibroblast cell lines exposed or sham-exposed to radiation was run on exon arrays to examine the transcriptional profile in response to radiation at the exon level. The extensive transcript coverage and relatively large number of samples allowed us to obtain robust whole gene expression levels inclusive of exon specific expression for LCLs and primary fibroblasts in response to 10 Gy at 4 hours post-IR. Therefore, the majority of the analyses were performed on data from this dose and time. Similar conditions have been used in previous IR response papers The differences in PSR transcripts four hours after exposure to IR for all genes were determined using an AS ANOVA (Partek Genomics Suite) and FIRMA and calculated splicing indexes for each exon. We have identified many genes that show different PSR expression changes within the gene in LCLs  and S6 aEDA2R showed a relatively consistent increase at each exon region across the entire gene  were run on polyacrylamide gels using cycle numbers determined to be in the linear amplification range  as indicated , 4, 5. Pon range . Primerson range  or down-on range . Likewison range . ExampleA PSR000 .VWCE gene exon array results suggested this gene was alternatively spliced. A region from PSR235 to PSR239 was amplified with PCR and the resulting amplification products run on an agarose gel. A 313 bp band full length product, as well as a 200 bp shorter product found only in the 10 Gy sample was observed  are shown in TNFRSF10B) to 3.79 (for PLK2) for LCLs (ZNF79) to 2.63 (CDKN1A) in fibroblast cells (INCENP) to −4.63 (KIF20A) for LCLs (SETD8) to −2.63 (AURKA) for fibroblasts  have not previously been reported to be modulated by IR  in four cell lines for both LCL and fibroblast samples. Selected genes, that show a modulation with time, are plotted . Exon ars tested . Unlike ll times . Whole gll times , S17. Maed by IR .HIST genes in the HIST cluster. Of the 61 genes down-regulated on chromosome 6 , we found 21 (38%) HIST genes, all of which were found at the HIST cluster on chromosome 6. We found down-regulation of HIST genes in both LCL and fibroblast cells although to a lesser extent in the fibroblasts. The gene olfactory receptor gene clusters on chromosome 11 showed many of these genes to be up-regulated (17/61 (28%) up-regulated genes ) ) . Comparichange)) . There wchange)) .CDKN1A, cyclins, TNF and PLK genes, was obtained  have low fold changes between −1.19 and −1.64 which may have made them difficult to identify as significantly modulated genes in previous studies. Similarly the five up-regulated genes in FB (ASAH3L (ACER1), EDA2R, PAG1, RNF19B and XPC) were among the genes with smaller expression differences on our list ranging from a fold increase of 1.39 to 1.57. However, the fold changes for the novel genes in LCLs are not as low. The differences in cellular responses, namely that the LCLs are more radiosensitive than fibroblasts, are at least in part due to the different gene expression response elicited by these two cell types.Gene transcription at the exon level in response to IR has been determined enabling identification of alternative transcription products across the whole genome in response to IR. Genes have been identified that show expression differences between exons in response to radiation. These results are consistent with previously reported alternative transcript gene products in response to IR for single gene examples VWCE and GADD45G produce increased amounts of alternative transcripts lacking internal exons in response to radiation. These proteins are involved in signalling pathways, GADD45G being involved in the p38/JNK pathway. Our array data analysis suggests an IR specific induction of alternative transcripts for many genes. The genes identified include involvement in cell cycle regulation, chromatin dynamics, p53 regulation and cell growth. We have identified other genes that show complex transcript isoforms prior to and in response to IR. Exon arrays have the limitation that one cannot know the isoforms when multiple isoforms are present without thorough investigation using other methodologies such as PCR. The array data has given important clues for which exons are involved in AS. For genes, such as CDKN1A, SUN2, LRDD and SAT2, we were able to identify alternatively spliced products, and we showed an induction of both products following radiation, but failed to show a difference in the ratio of isoforms after IR. This may be limited by the sensitivity of our PCR assay (not quantitative PCR) or these differences may be teased out at different times or doses. Nevertheless, an induction of an alternative isoform can also be an important contributor to the cellular response to radiation.We have identified a large number of genes that show transcript expression modulation characteristic of alternative transcripts in response to IR. Some genes that show AS following IR have important functional roles in cell fate decisions in response to IR-induced cell and DNA damage. Our array data indicate that genes such as VWCE, FBXW7, CCNG1 and MDM2 . Some of these response differences may contribute to the higher cellular radiosensitivity that LCLs have compared to fibroblasts. Consistent with this, genes involved with apoptosis were induced more in LCLs compared with fibroblasts. Alternatively, these two cell types may have different thresholds for advancing certain fate decisions such as cell death. For instance, the more robust induction of apoptosis genes in LCLs compared to the fibroblasts, may be the key to higher LCL radiosensitivity.LCLs and fibroblasts respond differently to the same radiation treatment, and therefore, have a cell type specific response to IR. We found that many transcripts had an exaggerated response in LCLs compared to fibroblasts and some transcript responses differed dramatically between the two cell types . Many genes that had relatively large expression change tended to peak at around 8 hours, for example, TP53I3. Finally, groups of related genes were co-ordinately down-regulated as is the case for HIST genes. Therefore, there are many patterns of co-ordinately regulated genes which direct the cellular response to IR.A variety of expression kinetic profiles were observed with dose and time course studies. For example, a number of genes  was added, mixed and the aqueous layer was mixed with and equal volume of 70 percent ethanol and added onto a RNeasy column . The RNA extraction was continued by using the RNeasy method as per manufactures recommendation except starting with the addition of the sample of Buffer RW1. RNA concentration and integrity was determined by analysing on a bioanalyzer . RNA was determined to be high enough quality if a minimum RIN of 8.5 was obtained.GeneChip Human Exon 1.0 ST Array analysis was performed as per the ‘GeneChip Whole Transcript (WT) Sense Target labelling assay Manual’ . The rRNA from 1 ug of total RNA was reduced using a RiboMinus Human/Mouse Transcriptome Isolation Kit . The experimental designs for each experimental group and which and how many patient samples were used can be found in For this investigation we have analysed the ‘core set’ that is defined by over 228,000 probe set regions (Affymetrix.com). Assessment of array quality was determined using Expression Console (Affymetrix.com). For differential gene expression, all exon arrays were normalized with RMA background correction and quantile normalization, and then overall transcript expression estimated using Exon RMA linear model The first method to detect AS was determined using AS ANOVA from Partek Genomics Suite statistical analysis package . Secondary methods used to detect alternatively spliced exons, in order to compare all 0 Gy and 10 Gy samples for LCLs and fibroblasts independently, utilized Splicing Index (ie. log NI scores) Two cohorts of samples were designed specifically for the interrogation of time and dose dependent genes. The time-dependent cohort consists of samples at 0, 2, 4, 8 and 24 hours post irradiation (10 Gy). The dose-response cohort consists of samples irradiated with 0, 1, 2, 5, 10 and 20 Gy, taken 4 hours post-irradiation. Time and dose response genes were identified using EDGE, a significance analysis method designed for time course experiments Primers were designed to candidate exons or genes using ‘Primer3’ on-line software PCR amplicons were cut out of poly acrylamide gels and the DNA was eluted in elution buffer overnight at room temperature. Direct sequencing from PCR amplicons was completed by cutting out the appropriately sized band and purifying the amplicon using a Qiagen gel purification column or by stabbing the band of interest using a pipet tip followed by re-amplification and clean-up using a Qiagen PCR product spin column. In other cases the isolated PCR amplicon was ligated to the pGEMeasy-T linearized vector as per manufacturer's recommendations (Promega). For sequencing from clonal inserts, we utilized the amplicon primers. Big Dye terminator sequencing was performed using T7 and SP6 primers and the splice sites at the nucleotide level were determined by sequence comparison.5′-RLM-RACE was performed using FirstChoice RLM-RACE kit  recommended by the manufacturer except the CIP digested RNA was purified using RNAeasy kit . The MDM2 transcript was amplified using nested PCR (as recommended by Ambion) with forward (inner and outer) primers to the adaptor (provided with the FirstChoice RLM-RACE kit) and reverse (inner and outer) MDM2 specific primers .Figure S1Gene ontologies that are enriched after IR. The top 10 gene ontology functional  and pathway  categories using the top 100 up- and down-regulated genes 4 hours after 10 Gy in LCLs  and fibroblasts . The threshold for significance is indicated . The ratio of total number of genes in a gene ontology pathway category divided into the number of genes from the 100 gene input is indicated by the squares.(TIF)Click here for additional data file.Figure S2Gene networks after IR. (A) Gene network of IR-modulated genes in LCLs. Genes such as CCNB1 and NFkB complex are central in this network. Solid lines represent direct protein interactions and dashed lines represent indirect interactions. Shaded genes are genes that are in the top 100 up-regulated (red) and top 100 down-regulated (green) genes shown to be modulated at 4 hours following 10 Gy IR in LCLs. (B) Gene network of IR-modulated genes in fibroblast cells. Genes such as CDKN1A and MDM2 are central in this network. Solid lines represent direct protein interactions and dashed lines represent indirect interactions. Different shapes correspond to different gene ontological groupings . Shaded genes are genes that are in the top 100 up-regulated (red) and top 100 down-regulated (green) genes shown to be modulated at 4 hours following 10 Gy IR in fibroblast cells.(TIF)Click here for additional data file.Table S1PCR primer sequences.(XLSX)Click here for additional data file.Table S2Genes modulated 4 hours post 10 Gy IR in LCLs.(XLSX)Click here for additional data file.Table S3Genes modulated 4 hours post 10 Gy IR in fibroblast cell lines.(XLSX)Click here for additional data file.Table S4Novel IR-modulated genes in LCLs.(XLSX)Click here for additional data file.Table S5Novel IR-modulated genes in fibroblasts.(XLSX)Click here for additional data file.Table S6Genes predicted to be alternatively spliced in LCLs at 4 hours post 10 Gy IR based on Partek.(XLSX)Click here for additional data file.Table S7Genes predicted to be alternatively spliced in LCLs at 4 hours post 10 Gy IR based on FIRMA.(XLSX)Click here for additional data file.Table S8Genes predicted to be alternatively spliced in LCLs at 4 hours post 10 Gy IR based on SI.(XLSX)Click here for additional data file.Table S9Genes predicted to be alternatively spliced in fibroblasts at 4 hours post 10 Gy IR based on Partek.(XLSX)Click here for additional data file.Table S10Genes predicted to be alternatively spliced in fibroblasts at 4 hours post 10 Gy IR based on FIRMA.(XLSX)Click here for additional data file.Table S11Genes predicted to be alternatively spliced in fibroblasts at 4 hours post 10 Gy IR based on SI.(XLSX)Click here for additional data file.Table S12Dose response in LCLs.(XLSX)Click here for additional data file.Table S13Dose response in fibroblasts.(XLSX)Click here for additional data file.Table S14Time course in LCLs to 24 hours.(XLSX)Click here for additional data file.Table S15Time course in LCLs to 48 hours.(XLSX)Click here for additional data file.Table S16Time course in fibroblasts to 24 hours.(XLSX)Click here for additional data file.Table S17Time course in fibroblasts to 48 hours.(XLSX)Click here for additional data file.Table S18The percentage of genes modulated on individual chromosomes.(XLSX)Click here for additional data file.Table S19Identification of the patient derived cell lines used for each experiment.(XLSX)Click here for additional data file.


Familial hemophagocytic lymphohistiocyosis is a severe inflammatory condition due to genetic defect in cytotoxic activity (e.a. :perforin). This disease is similar to the macrophage activated syndrome (MAS).In this disorder cytotoxic CD8 lymphocytes are highly proliferating due in part to a cytokine storm condition (IFNγ mediated). In this inflammatory climate macrophages gets activated and are forming fused cells called histiocytes which are pathognomonic for the disease.Using murine model, we are interested in studying the immune suppressive mechanisms of this disease to understand the reason why they fail to prevent the inflammation onset.As previously shown, perforin deficient mice do not have significant immunophenotype difference compared to wild type (wt) controls .In the regulatory T cell compartment (Treg) which are specifically natural immunosuppressive cells, the percentage of Treg between perforin deficient mice and wt are similar from the thymic formation to the periphery  +/- 0,8333 (SD), perforin deficient: 2,228% +/- 0,6372). Moreover their homing phenotype does not differ from wt for molecules such as CCR6, CD103, CTLA-4, CCR4, CCR7, CD62L.In vitro supressive assay based with CFSE labeling showed a normal suppressive function for perforin deficient Treg to suppress wt effector T cells.For the forthcoming meeting, comparison of the rescue ability of the wt and perforin deficient Treg in an autoinflammatory model in vivo (Treg depleted mice) will be presented.


Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP) sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR) for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs). Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by  The structure and physiology of the intestinal tract mature during postnatal growth in order to meet changing absorptive and digestive requirements. While the mechanisms and timing of certain critical events differ between species, the maturation of bowel structure and function is universally important. In rodents, the postnatal growth of the epithelium has been studied in considerable detail Prior work has characterized crypt fission in various mammalian species during the postnatal time period Recent developments in the characterization of ISCs have led to the identification of multiple putative ISC markers. To date however, there is no consensus regarding the ideal marker and no single one appears to universally identify these important pluripotent cells Lgr5, Ascl2 and Olfm4 are markers of the crypt base columnar cell population Bmi1 and mTert identify the supra-Paneth “quiescent” ISCs Various lines of evidence suggest that the intra-Paneth cell ISC (crypt base cells or CBCs) and the supra-Paneth cell ISC (often referred to as “plus 4”) represent distinct sub-populations that may be active and quiescent, respectively Lgr5-EGFP-IRES-creERT2 (Lgr5-EGFP–enhanced green fluorescent protein) mice were obtained from The Jackson Laboratory  and paired in order to obtain timed pregnancies. Lgr5-EGFP genotyping was performed on tail clippings using Extract-N-Amp Tissue PCR kits  with common (CTGCTCTCTGCTCCCAGTCT), WT reverse (ATACCCCATCCCTTTTGAGC) and mutant reverse primers (GAACTTCAGGGTCAGCTTGC) to perform the PCR as described by The Jackson Laboratory (http://jaxmice.jax.org/strain/008875.html). Offspring were euthanized on postnatal days (p) 0, 4, 7, 14, 21, 28 and adulthood (8–12 weeks) after induction of 2% isoflurane general anesthesia. Each data point is represented by 3 or more animals from both sexes with actual numbers given in the respective figure legends. All animals were weaned to standard chow  on p21 and kept under a 12 hour light-dark cycle.All animal experiments were carried out after approval by the University of North Carolina School of Medicine Institutional Animal Care and Use Committee (protocol number 07-202.0). Adult male and female wild-type mice  and breeder pair heterozygote Lgr5-EGFP mice, an adjacent 3rd cm was harvested and sequentially placed in 4% paraformaldehyde, 30% sucrose, and then OCT compound  and stored at −80°C until slide preparation.The entire small bowel was eviscerated and its length recorded. The proximal 1 cm was discarded as duodenum and the remaining small bowel was divided in half with the proximal portion being the jejunum. The middle 2 cm of jejunum was isolated and flushed with phosphate-buffered saline. The first cm was placed in 10% buffered zinc formalin  for histology and the second 1 cm was cut in several smaller pieces, and then placed in FastPrep-24 Lysing Matrix D tubes  filled with 600 µL of RLT lysis buffer, a component of the Qiagen RNeasy Mini Kit . These samples were snap frozen in liquid nitrogen and kept at −80°C until RNA isolation. For For studies with WT mice, slides were stained with hematoxylin and eosin (H&E) and analyzed for intestinal morphometry in a blinded manner. Images were obtained using an AxioImager microscope . Crypt depth and villus height were obtained from at least 10 well-oriented crypt-villus axes per animal. Circumference measurements were obtained from the submucosa using AxioVision 4.6 as described previously Lgr5-EGFP mice, we utilized a laser scanning spectral confocal microscopy system and software  to visualize Lgr5-EGFP sections by fluorescence and take representative photographs. To quantitate the number of EGFP positive cells per crypt: a) only complete, well oriented crypts were chosen; b) only cells exhibiting bright fluorescence were counted; and c) within the focal range of the section all such cells (i.e. from both sides of the crypt) were counted. For the same crypts the total number of cells were quantitated by scoring the number of nuclei that stained positive for 4′,6-diamidino-2-phenylindole (DAPI). Preliminary analyses did not show gross differences in the absolute number of EGFP positive cells per crypt or the% EGFP positive cells/crypt from p7–p28, therefore animals were grouped to provide an n of 3–5 during early (p7–p14) and late (p21–p28) development for comparison with adulthood. For quantification of the percentage of EGFP positive crypts, at least 2 transverse sections were examined per animal.For studies with Lgr5 (Mm00438890_m1), Ascl2 (Mm01268891_g1), Bmi1 (Mm03053308_g1), Olfm4 (Mm01320260_m1), and mTert (Mm01352136_m1) from Applied Biosystems were used and data analyzed using a relative standard curve method normalized to β-actin (Mm00607939_s1). We evaluated 18S, GAPDH and β-actin and found that the latter changed the least across our developmental time points and thus was used for the housekeeping gene.Total RNA was isolated using the Qiagen RNeasy Mini Kit per the manufacturer's instructions after addition of 6 µL β-mercaptoethanol  to the lysing tubes. Complimentary DNA was then created using a High Capacity cDNA Reverse Transcription Kit . Quantitative RT-PCR was performed on cDNA samples in triplicate using an Applied Biosystems StepOne Plus system. Individual probes for In situ hybridization was performed on WT animals as previously described 35S-labeled antisense or sense cRNA probes. These were prepared using nucleotides 2538 to 3268 (NCBI reference sequence NM_010195.2) of Lgr5 cDNA cloned into a pBluescript vector. RNase A-resistant hybrids were detected by autoradiography. Sections were post-stained with H&E. Sections hybridized with the sense probes did not exhibit any positive signals and served as negative controls as shown in − SP cells was quantitated using flow cytometry.Jejunal mucosal cell suspensions were prepared from p21 and adult mice as previously described using a collagenase/dispase method Data are presented as means ± standard error. All timepoints and ranges are represented by an n ≥3 animals. Statistics were performed using SAS version 9.2 . To analyze the qRT-PCR results, a regression model was fit with each stem cell marker as the response and dummy variables for the timepoints as predictors. Levene's test for homeogeneity of variance was performed and if it showed heterogeneity then a weighted-least-squares model was fit with weights inversely proportional to the variance of the response at that time. For each marker, a test of any overall difference between times was done and if statistically significant, pairwise tests were performed. P-values less that 0.05 were considered significant. A full table of p-values for the analysis of qRT-PCR results and crypt fission is shown in 2 values of 0.91, 0.99, 0.94 and 0.92, respectively , we utilized the commercially available Lgr5-EGFP mouse in order to visualize the Lgr5-expressing cells in the crypts. Representative confocal photomicrographs of Lgr5-EGFP animals are shown in To assess whether there are increased numbers of Mature bowel possesses an ability to respond to challenges by augmenting several characteristics after a loss of length or functional capacity Two parameters included in our study but not reported in the previous study were bowel circumference and the number of crypts per circumference. Bowel circumference has particular clinical relevance as dilation is one of the properties of small intestinal adaptation after massive bowel resection both in mice and in human patients The process of crypt fission has been described in rodents and humans Cheng and Bjerknes have previously quantified the overall proliferative activity of murine intestinal crypts during postnatal development by flow cytometry and found proliferative indices between 7–14% mTert, at p4. This finding was followed by surges in Lgr5 and Bmi1, which peaked at p14, corresponding with the peak of crypt fission. Interestingly, the early increase in mTert mRNA correlates with recent data indicating that putative ISCs marked by mTert may give rise to Lgr5+ cells Olfm4 mRNA levels were significantly lower at all timepoints in development compared with adult while Ascl2 was statistically similar to adulthood at p7–14 during the time of elevated crypt fission and lower than adult levels at the other timepoints  have been reported to precede the increase in crypt fission Lgr5, Bmi1, and mTert are elevated during and/or preceding the surge in crypt fission while Olfm4 and Ascl2 are not. Crypt fission is a unifying theme in 3 distinct scenarios that involve expansion and/or repair of the intestinal epithelium. Neonatal development is strictly a growth phenomenon while the adaptive process after ileocecal resection is a response of normal bowel to a loss of length elsewhere in the gastrointestinal tract. The response to damage arises from crypts that have sustained an insult whether from radiation or chemotherapeutic agents.Clearly, techniques to directly evaluate actual numbers of ISCs are lacking. With this limitation, surrogates are used to indirectly study ISC expansion. In development, elevated levels of crypt fission correspond with rapid increases in small bowel length, circumference, and the number of crypts per circumference over a discrete time period. We describe patterns of mRNA expression in this time frame for several putative ISC markers and show that Lgr5 mRNA expression, Lgr5-EGFP, and Lgr5 in situ hybridization) and quiescent , may play a role in neonatal intestinal growth. While the surge of crypt fission seen at the end of the second postnatal week suggests increased numbers of ISC at this time, we were not able to demonstrate a clear correlation between this surge and either of these populations. This raises the question that both active and quiescent ISC subpopulations may be interacting in a complex manner during neonatal intestinal growth. Further characterization is difficult within the current limitations of the field, namely the lack of any specific, commercially available antibodies for putative ISC markers. All of the measures currently available are surrogates. A better understanding of the regulatory mechanisms controlling crypt fission and the expansion of ISCs has clear translational application to patients suffering from short bowel syndrome and other intestinal pathology. Enhanced techniques to more directly identify, quantify, and manipulate putative ISCs have significant potential. Further characterization of ISC biology may reveal components that are subject to beneficial clinical intervention.We provide evidence that during development, a heterogeneous population of ISCs, both active  and high power (panel B).(TIF)Click here for additional data file.Figure S2Lgr5 in situ hybridization.Anti-sense and sense controls for (TIF)Click here for additional data file.Table S1Statistical analysis of mRNA expression and crypt fission. P-values indicate comparison with adult values for each mRNA and for crypt fission.(TIF)Click here for additional data file.


Stillbirth affects 1 in 200 pregnancies and commonly arises due to a lack of oxygen supply to the fetus. Current tests to detect fetal hypoxia in-utero lack the sensitivity to identify many babies at risk. Emerging evidence suggests that microRNAs derived from the placenta circulate in the maternal blood during pregnancy and may serve as non-invasive biomarkers for pregnancy complications. In this study, we examined the expression of miRs known to be regulated by hypoxia in two clinical settings of significant fetal hypoxia: 1) labour and 2) fetal growth restriction. Six miRs  were differentially expressed in pregnancies complicated by fetal hypoxia. In healthy term pregnancies there was a 4.2 fold increase in miR 210 (p<0.01), 2.7 fold increase in miR 424 (p<0.05), 2.6 fold increase in miR 199a (p<0.01) and 2.3 fold increase in miR 20b (p<0.05) from prior to labour to delivery of the fetus. Furthermore, the combined expression of miR 21 and miR 20b correlated with the degree of fetal hypoxia at birth determined by umbilical cord lactate delivery . In pregnancies complicated by severe preterm fetal growth restriction there was upregulation of the hypoxia-regulated miRs compared to gestation-matched controls: 3.6 fold in miR 210 (p<0.01), 3.6 fold in miR 424 (p<0.05), 5.9 fold in miR 21 (p<0.01), 3.8 fold in miR 199a (p<0.01) and 3.7 fold in miR 20b (p<0.01). Interestingly, the expression of miR 373 in gestation matched controls was very low, but was very highly expressed in FGR (p<0.0001). Furthermore, the expression increased in keeping with the degree of in-utero hypoxia estimated by fetal Doppler velocimetry. We conclude quantifying hypoxia-regulated miRs in the maternal blood may identify pregnancies at risk of fetal hypoxia, enabling early intervention to improve perinatal outcomes. Fetal hypoxia ultimately leads to fetal death and disability. Hypoxia may occur acutely during labour and birth, or develop gradually across pregnancy in cases of fetal growth restriction (FGR) due to placental dysfunction.Fetal growth restriction accounts for 50% of unexplained stillbirths and is strongly associated with permanent neurological disability in survivors Fetal hypoxia may also occur acutely during labour in an otherwise healthy baby, as a result of cord occlusion or restricted placental perfusion. 1 in 200 newborns are affected by perinatal asphyxia while 2 per 1000 newborns develop hypoxic-ischaemic encephalopathy (HIE), which accounts for 1 million neonatal deaths globally per year Therefore, clinicians utilize a combination of tests to assess fetal well-being to determine whether the fetus is at risk of succumbing to hypoxia. These include: clinical judgment; ultrasound assessment of fetal growth, fetal movement and fetal vessel velocimetry; and recording fetal heart rate patterns The exciting discovery that microRNAs circulate in the blood and may reflect gene expression in a distant tumour, has led to enthusiasm for the role of circulating miRs as biomarkers for cancer detection and prognosis All subjects were recruited from two tertiary hospitals in Melbourne, Australia  between 2008–2012. Written informed consent was obtained prior to inclusion and the study protocol was approved by both institutions’ Human Research and Ethics Committees. Two separate cohorts were recruited: acute hypoxia in labour and chronic hypoxia in FGR.Maternal whole blood samples were prospectively collected from consecutive women undergoing induction of labour at term (n = 8). A second intravenous cannula was inserted for serial blood collection for the study: prior to the onset of uterine contractions and at the moment of delivery . Fetal hypoxic status was determined by measuring umbilical artery blood lactate levels collected at delivery.th centile  requiring iatrogenic delivery before 34 weeks with antenatal evidence of uteroplacental insufficiency . FGR due to infection, chromosomal or congenital abnormalities, and multiple pregnancies were excluded. Preterm control blood samples were collected from women with an appropriately grown fetus, at a gestation matched to the FGR cases, but who subsequently delivered appropriately grown (birthweight 20–80th centile) neonates at term without obstetric complications.Maternal whole blood samples were collected from women carrying a preterm severely growth restricted fetus (n = 12) and gestation matched controls (n = 12). Severe preterm FGR was defined as customized birthweight <10Peripheral whole blood samples (2.5 ml) were collected in PAXgene blood RNA tubes . As per manufacturers instructions, they were stored at room temperature for 24 h, transferred to −20°C for 24 h, and then stored at −80°C until processing.miRNA was extracted from peripheral whole maternal blood using the PAXgene Blood miRNA kit  according to manufacturer’s instructions, as previously described miRNA Taqman assays (Applied Biosystems) were used for the quantification of the miRNAs according to manufacturer’s instructions. 50 ng of total RNA was reverse transcribed for each target miRNA with the pool of housekeeping miRNAs: RNU-48 and RNU-6b. qPCR was performed in triplicate, with multiple negative controls, using TaqMan Universal PCR Master Mix on a CFX 384  with the following cycling conditions: 50C for 2 mins, 95C for 10 mins, and 40 cycles of 95C for 15 sec, 60C for 1 min. Fold changes in expression were determined by the comparative CT method normalized against the mean expression of RNU-48 and RNU-6b as reference genes.http://www.r-project.org) or Graphpad Prism v 5. Differences in RT-PCR gene expression was assessed using Mann–Whitney U or Kruskal –Wallis test, or Wilcoxon signed rank test as appropriate. Patient characteristics were compared using χ2 where appropriate. Data was presented as mean +/− SD. Significance was defined as p<0.05.All data was statistically analyzed using R version 2.12.0 . Interestingly, this significant increase observed in FGR, a situation of chronic hypoxia, was opposite to the trends seen in our labour ward study, where there was a reduction in expression with acute hypoxia. There was also a 3.6 fold increase in miR 210 , 3.6 fold increase in expression of miR 424 , 5.9 fold increase in miR 21 , 3.8 fold increase in miR 199a (p<0.01) and 3.7 fold increase in miR 20b  .Ultrasound velocimetry of the umbilical artery is used clinically to determine the severity of preterm FGR and the likelihood of fetal hypoxia. Doppler assessment of the umbilical artery evaluates blood flow from the fetus to the placenta and therefore reflects placental vessel resistance and end organ function of the fetus We therefore divided the FGR cohort into those fetuses who had a raised pulsatility index (associated with less severe hypoxia) and compared miRNA expression to those with AREDF (associated with more severe hypoxia). There was a trend to increased expression with all hypoxia induced miRs with AREDF but a significant increase was only observed in miR 424  and miR 21  in those FGR with AREDF .Placental transfer of oxygen to the fetus is essential for growth and survival. Birth asphyxia and placental insufficiency lead to placental and fetal hypoxia, and are the commonest causes of stillbirth, neonatal morbidity and long-term neurological sequelae In this study, we have identified a novel approach to non-invasively diagnose fetal hypoxia by measuring circulating miRNAs in the maternal blood. We demonstrate that a panel of hypoxia-induced miRNAs are detectable in the maternal blood, and that their expression changes when the fetus is subjected to both acute and chronic hypoxia. Furthermore, quantification of hypoxia-induced miRNAs correlated with the degree of in-utero hypoxia.Electronic monitoring of fetal heart rate patterns is currently the mainstay of assessment of fetal well-being during labour, but has a low predictive value for fetal hypoxia. As a result, research into adjunctive methods of assessing the degree of fetal hypoxia has been recommended In addition, we have demonstrated that hypoxia-induced miRNAs differentiated severe FGR pregnancies from healthy gestation matched controls. This is in contrast to current antenatal surveillance, which fails to detect 75–85% of FGR cases, pregnancies at considerable risk of antepartum stillbirth The discovery of miRNAs in the maternal circulation has yielded much potential for biomarker discovery Several studies have demonstrated altered expression of miRNAs in placental tissue but although these studies provide biological insight, their clinical utility is limited by the necessity of awaiting delivery to obtain placental tissue In this study we choose to screen hypoxia–induced miRNAs as potential biomarkers of fetal hypoxia. The cellular response to hypoxia results in induction of a complex set of gene regulatory mechanisms, largely controlled by the hypoxia inducible factors (HIFs). More than 90 microRNAs have been identified that are induced by hypoxia and modulate the cellular response to hypoxia via HIFs Although our results are promising several limitations are recognized. The small sample size requires further validation in a larger cohort to determine the diagnostic potential of such an approach. The speed at which analysis can be performed currently prohibits the potential of such a test as a biomarker for acute hypoxia but may still be useful for the detection of chronic hypoxia in cases of severe FGR.In conclusion, we have demonstrated an elevation in hypoxia-induced miRNAs in both acute and chronic fetal hypoxia that may be a promising approach to clinically assess fetal hypoxic status in-utero. This may help clinicians to identify hypoxic fetuses allowing timely intervention to improve perinatal outcomes and decrease the rates stillbirth.


Lymphatic filariasis (LF)-related disability affects 40 million people globally, making LF the leading cause of physical disability in the world. Despite this, there is limited research into how the impacts of LF-related disability are best measured. This article identifies the tools currently being used to measure LF-related disability and reviews their applicability against the known impacts of LF. The findings from the review show that the generic disability tools currently used by LF programs fail to measure the majority of known impacts of LF-related disability. The findings from the review support the development of an LF-specific disability measurement tool and raise doubt about the suitability of generic disability tools to assess disability related to neglected tropical diseases (NTDs) globally. Brugia malayi, Brugia timori, and most commonly, Wucheria bancroftiLymphatic filariasis (LF) is caused by three filarial nematodes: It is estimated that 40 million people are chronically disabled by LF, making LF the leading cause of physical disability in the world The Global Program to Eliminate Lymphatic Filariasis (GPELF) recently released their progress report for 2000–2009 Globally, the approach to disability measurement has shifted over the past two decades. Previously, disability and the impact of disease were conceptualized using a medical model. Measures of mortality, years living with impairment Within the LF community, a number of generic disability tools have been recommended and trialled. These include The International Classification of Functioning, Disability and Health (ICF) However, others have advocated for the development of an LF-specific disability measurement tool, arguing that an LF-specific tool would be more sensitive to the clinical features of LF and for detecting small changes in function that occur with disease progression In order to determine if current generic tools are suitable for disability measurement within LF programs, this article aims to summarize the reported issues of LF-related disability and review generic tools being used for LF measurement for relevance to these known issues of LF disability.A database search of MEDLINE, CINAHL, Scopus, and ProQuest databases was conducted to identify disability measurement tools that had been used to measure LF-related disability. Keywords used in the search (both separately and in Boolean combination) included but was not limited to: “lymphatic filariasis,” “elephantiasis,” “lymphoedema,” “lymphocele,” “hydrocele,” “disability,” “rehabilitation,” “morbidity,” “evaluation,” “measurement,” “assessment,” “monitoring,” “severity of illness,” “examination,” and “health screening.” Grey literature was also reviewed. Inclusion criteria were as follows: (a) the tools measured the experience of disability from a patient perspective and (b) the tools measured the lived experience or impacts of LF-related disability rather than solely the physical or medical attributes of the disease. Exclusion criteria were as follows: (a) tools that measured the clinicians' perspective of disability rather than the patients', (b) tools that measured solely medical issues of LF-related disability rather than the impact/experience of living with LF-related disability, and (c) survey instruments were specific to study rather than psychometrically tested generic disability tools for generic disability measurement. From the search, 12 studies were identified that had used generic disability measures, of which five were excluded as they were non-standardized, self-developed research questionnaires rather than psychometrically tested generic disability instruments.The questions included in each tool used for LF measurement were reviewed against the key issues of LF-related disability as reported in the literature see . These kThe most commonly reported issues relevant to LF-related disability were impact of LF on work (nine studies), stigma surrounding LF within local communities (nine studies), impact on personal relationships (nine studies), depression (seven studies), impact of LF on social events (seven studies), feelings of shame/humiliation experienced by LF patients (six studies), fear (six studies), and reduced social status (six studies) see . Least cThree tools have been previously used to measure LF-related disability: The seven domains five levels (7D5L) instrument, which is a seven-item, extended form of the European Quality of Life Instrument (EuroQol 5D3L) Another three tools have either been advocated for use in LF measurements or have been used for LF disability surveys but have not been formally published The 34 issues reported by people living with LF-related disability were compared against the items from each of the six disability measurement tools see . Some itContent analysis revealed five broad domains that the issues encompassed. These were psychological impacts, impact on daily activities, impact on participation, and the influence of environmental factors and personal factors. In total, the domains that would most comprehensively be assessed by the tools items were found to be daily activities (tools covered 37.5%–75% of issues), participation (tools covered 33%–100% of issues), and personal factors (tools covered 33%–100% of issues). More poorly measured by the tools were psychological issues (tools covered 0%–20% of issues) and environmental factors (tools covered 0%–60% of issues) relevant to LF-related disability.The ICF checklist was found to have items that captured the most issues (50%), followed by the WHODAS 36 (47%). However, neither of these tools included any items that would identify the 10 specific issues included within the psychological domain. The other tools had very few items that would identify issues related to LF disability: 7D5L (29%), WHO-QOL 100 (28%), DLQI (24%), and WHOQOL Bref (22%).The research that informs our knowledge of the impact of LF-related disability is limited and still emerging This review identified that the tools currently being used to measure LF-related disability are inadequate. This review revealed 34 issues across five domains that are consistently reported by people living with LF-related disability. Of the six measurement tools  that have been used to measure LF-related disability, only one measurement tool  Health Related Quality of Life, an appropriate indicator to assess the impact of morbidity management and disability prevention activities towards elimination of lymphatic filariasis. Filaria J 6: 8.Babu B, Nayak A, Rath K, Kerketta A (2006) Use of the dermatology quality of life index in filarial lymphoedema patients. Trans Roy Soc Trop Med Hyg 100: 258–263.Chandrasena T, Premaratna R, Muthugala M, Pathmeswaran A, de Silva N (2007) Modified Dermatology Life Quality Index as a measure of quality of life in patients with filarial lymphoedema. Trans Roy Soc Trop Med Hyg 101: 245–249.Harichandrakumar K, Krishnamoorthy K, Kumari A, Das L (2006) Health status of lymphatic filariasis assessed from patients using seven domains five levels (7D5L) instrument. Acta Trop 99: 137–143.McPherson T (2003) Impact on the quality of life of lymphoedema patients following introduction of a hygiene and skin care regimen in a Guyanese community endemic for lymphatic filariasis: a preliminary clinical intervention study. Filaria J 2: 1.


This core set exhibits evolutionarily conserved binding regions and is enriched in functional responses to hypoxia. Computational prediction of enriched transcription factor binding sites identified sequence motifs corresponding to several stress-responsive transcription factors, such as activator protein 1 (AP1), cAMP response element-binding (CREB), or CCAAT-enhancer binding protein (CEBP). Experimental validations on HIF-regulated promoters suggest a functional role of the identified motifs in modulating HIF-mediated transcription. Accordingly, transcriptional targets of these factors are over-represented in a sorted list of hypoxia-regulated genes. Altogether, our results implicate cooperativity among stress-responsive transcription factors in fine-tuning the HIF transcriptional response.The transcriptional response driven by Hypoxia-inducible factor (HIF) is central to the adaptation to oxygen restriction. Despite recent characterization of genome-wide HIF DNA binding locations and hypoxia-regulated transcripts in different cell types, the molecular bases of HIF target selection remain unresolved. Herein, we combined multi-level experimental data and computational predictions to identify sequence motifs that may contribute to HIF target selectivity. We obtained a core set of  Oxygen is essential for the survival of all eukaryotic cells, and metazoans are heavily dependent on this element to meet their large metabolic demands. At the cellular level, 90% of oxygen is consumed in oxidative phosphorylation. Consistent with a central role of oxygen in aerobic metabolism, all metazoan cells respond to an imbalance between demand and supply of oxygen (hypoxia) by activating a gene expression program aimed at restoring oxygen supply and reducing its consumption. The cellular response to hypoxia is mainly controlled by the evolutionarily conserved Hypoxia Inducible Factor (HIF) family of basic helix-loop-helix transcription factors. HIFs are heterodimers of a beta subunit , and an alpha subunit (HIFα) A large number of studies focusing on single genes have identified individual HIF targets that, collectively, account for the functional responses to hypoxia, mainly metabolic adaptation and induction of angiogenesis ET-1 under hypoxia PAI-1 induction by hypoxia has been linked to cooperative promoter activation by CEBPα, HIF1α and EGR-1 LDHA promoter) or identical sites (BNIP3) Understanding the mechanisms that explain HIFs transactivation selectivity is of paramount importance to expand our knowledge on transcriptional regulation and to improve the sensitivity and specificity of genome-wide efforts to characterize the HIF transcriptional response. DNA accessibility of transcription factor binding sites (TFBSs) can clearly contribute to binding selectivity Gene expression profiling indicates that the expression of thousands of genes changes with hypoxia, with vast cell-type differences in the specific genes being regulated http://www.ncbi.nlm.nih.gov/geo/) Gene profiling experiments of hypoxic cell cultures were downloaded from the Gene Expression Omnibus database .HeLa cells were maintained in Dulbecco’s modified Eagle medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin and 5% (v/v) fetal bovine serum. Cells were grown at 37°C and 5% CO7 HeLa cells were subjected to hypoxia (1% oxygen) for six hours or left untreated . Following treatments, cells were crosslinked with 1% formaldehyde for 12 min at 4°C. Cross-linking was stopped by the addition of glycine . Cell lysis was achieved by scraping in 1 ml of lysis buffer . Cell lysates were incubated on ice for 10 min and then sonicated to shear DNA to fragments between 200 and 1500 bp. Only experiments that showed homogeneous sonication across all samples (from normoxia and hypoxia treatments) were continued. 50 µl of each sample was stored (input), while 100 µl were diluted in 1 ml of immunoprecipitation buffer . Lysates were precleared with 200 µg of a Salmon Sperm DNA/Protein A agarose 50% slurry  for 1 h at 4°C; and then immunoprecipitated twice, initially with whole rabbit serum for 6 h (IgG control) and then overnight at 4°C with a polyclonal anti-HIF1 alpha antiserum . Immunocomplexes were recovered by addition of 400 µg of Salmon Sperm DNA/Protein A agarose 50% slurry, and then sequentially washed in Low Salt Wash Buffer , High Salt Wash Buffer , LiCl buffer , and twice in TE buffer . Elution of protein-bound DNA was performed twice with 1% SDS 0.1 M NaHCO3. Eluates were pooled, and crosslinking was reversed by the addition of 200 mM NaCl  and overnight incubation at 65°C. Protein and RNA were removed by the addition of proteinase K (30 µg/sample) and RNAse (200 mg/µl) for 2 hours at 42°C, and immunoprecipitated DNA was purified by phenol-chloroform extraction and ethanol precipitation. Successful ChIP was assayed by standard PCR using two primer pairs, targeting the functional EGLN3 HRE and a control region in the same locusChromatin immunoprecipitation was performed as previously described The ChIP-chip method was carried out as previously described Amplified DNA from normoxic and hypoxic chromatin immunoprecipitation samples was labelled with Cy3 and Cy5 fluorescent dyes, respectively, and hybridized to microarrays following guidelines from the microarray manufacturer . Hybridized microarray slides were scanned in an Agilent DNA microarray scanner  at 5 µm resolution, and acquired microarray images were quantified with GenePix 6.0 software . A total of six hybridizations were conducted, corresponding to four biological replicates. The two technical replicates were dye-swap experiments, where normoxic samples were labelled with Cy5 and hypoxic samples with Cy3.A custom alternative promoter microarray was used for ChIP-chip hybridizations The R/Bioconductor packages “Ringo” and “limma” were used to analyze ChIP-chip readouts The microarray experiments described above have been deposited in ArrayExpress under accession number E-MEXP-3499.http://frodo.wi.mit.edu/primer3/). All measurements were carried out in triplicate. Threshold cycle (Ct) values for each sample were interpolated in a standard curve of input DNA dilutions to obtain % of input absolute values. Enrichment of HIF1 alpha binding to target loci was calculated as the ratio of the amounts of target sequence (measured as % of input) detected in hypoxic vs noxmoxic ChIP samples (% of input hypoxia/% of input normoxia). For validation of ChIP-chip candidates, three negative control regions  following the manufacturer’s recommendations. Primers targeting candidate ChIP-enriched regions were designed with Primer Express software v2.0 (Applied Biosystems) and Primer3 (Custom scripts written in Perl were used to indentify evolutionarily conserved HIF binding sites (HBSs) within ChIP-chip regions and to select HBSs that showed evolutionary conservation. Conservation of the HIF binding consensus RCGTG in four mammalian species, including mouse, was required for the evolutionary conservation filter. HBSs were further selected to map to genes robustly induced by hypoxia, as indicated by the results of a meta-analysis of gene expression profiling experiments in hypoxic cell cultures loci that contained conserved RCGTG motifs in their non-coding sequences were first selected. For these genes, each of their probes was examined, and only genes for which all of their associated probes exhibited a mean fold value within 0.25 standard deviations of the global mean in each of the 19 datasets employed in the meta-analysis were considered as not induced by hypoxia. The selected coordinates of conserved RCGTG motifs mapping to these loci were extended as previously described for the set of core HIF binding sites. Genomic regions from this collection were further selected to match the frequency of genomic locations (relative to the TSS) found in the core HBR set. Briefly, Perl scripts were used to annotate core HBRs as promoter, 5′UTR, intronic or 3′UTR genomic locations and to choose, from the whole collection, a random sample according to the proportions of genomic locations found in the core HBR set. Similarly as with the set of core HIF binding regions, multiple sequence alignments corresponding to the selected control regions were retrieved.To obtain a set of background (control) genomic regions, custom perl scripts were used to exploit the microarray meta-analysis results for the identification of genes harbouring conserved RCGTG motifs but that are unlikely to be modulated by hypoxia. To this end, gene In silico transcription factor binding site predictions were carried out employing custom scripts written in Perl. Position-weight matrices (PWMs) collections were downloaded from the public databases JASPAR (2010 release) and Transfac (7.0 version) PWM conversion:The weight of base b in position i is calculated as:The corrected probability is obtained from the raw matrix by adding a pseudocount:Information content calculation:Subsequently, log-odd matrices were used to screen core HIF binding regions and genomic-matched background regions for the presence of putative TFBSs or other sequence motifs. Perl scripts were employed to split each sequence into overlapping fragments of length equal to that of the PWM under analysis. For each fragment, a score value was calculated by summing up the log-odd frequencies obtained by substitution of nucleotides found in the fragment in the corresponding position of the PWM. The contribution of each base to the score was weighted by the information content of its position in the matrix, as detailed below.i is the information content of position i and Wb,i the log-odd weight of base b in position i.Score calculation:Finally, the resulting score was normalized by subtraction of the minimum score and division by the score range, and compared with a threshold value. Fragments showing a score above the threshold were considered as putative TFBSs, and the evolutionary conservation of nucleotides in each motif was evaluated for matrix positions with information content over 60%. Putative TFBSs showing evolutionary conservation in four mammalian species (including mouse) and whose score was over the threshold value were recorded as present (respresented as 1). Otherwise, they were considered absent from the analyzed sequence (represented as 0).For each PWM, the three strategies proposed in MATCH versus the background collection. In particular, we considered significant PWMs with a p value lower than 0.05. No correction for multiple comparisons was applied to these p values.Fisher’s exact test was used to identify PWMs showing significant enrichment in the set of core HIF binding regions Additionally, the Weka machine learning workbench CA9 promoter region (hg19 coordinates chr9∶35673508–35673956), which was subcloned into the pGL3-Basic plasmid (Invitrogen). The Human GYS1 reporter construct (hg19 chr19∶49496421–49496978) has been previously described LDHA promoter construct (mm9 chr7∶54101027–54101258) and the derived HRE and CREB binding site mutations were a kind gift from Peter Ratcliffe Human genomic DNA extracted from HeLa cells was used as template for PCR amplification of the Reporter assays were performed using the human cervical-carcinoma cell line HeLa. Cells were seeded on six-well plates (2.5·105 cells/well) 6 h prior to transfection. Per well, a 4.5 µg DNA mixture containing 1.5 µg of the indicated reporter construct or empty plasmid and 0.25 µg of a plasmid encoding for Renilla (sea pansy) luciferase under the control of a null promoter  was used for transfection using the calcium phosphate method. 16 h after transfection, cells where washed, replated in 24-well plates, and incubated in normoxia, in the presence of DMOG  or in hypoxia for an additional 16 hours. After treatments, cells were lysed and the firefly and Renilla luciferase activities of the lysate were determined using a dual-luciferase system . The firefly luciferase activity was normalized to that of Renilla luciferase. Each experimental condition was assayed in duplicate. Hypoxia or DMOG fold induction values for each experiment were analyzed by repeated measures ANOVA with a Dunnet posthoc correction, comparing values of the wild-type promoter construct to each of the introduced mutations.http://www.broadinstitute.org/gsea/index.jsp). We employed a ranked list of core hypoxia-regulated genes derived from a meta-analysis of 16 hypoxia gene expression experiments Gene-set enrichment analysis (GSEA) was carried out as previously described  for six hours. Amplified samples from normoxic and hypoxic cells were competitively hybridized to a proximal promoter microarray that tiles a subset of human 7000 genes Next, we compared HIF1α ChIP-chip predictions in the four cell lines by analyzing the overlap of reported binding locations . The majbona fide HBSs to computationally identify enriched TFBSs that could be indicative of transcription factor cooperation. To focus on binding locations for which there is evidence of transcriptional modulation of nearby genes in hypoxia, we sought to combine the core set of HIF1 binding locations with HIF transactivation data. To this end, we employed our previous microarray meta-analysis study in silico sequence analyses, we focused on HIF binding sites whose sequence showed evolutionary conservation in mammalian species  databases and custom scripts based on the MATCH algorithm As an independent assessment of enriched motifs that is less dependent on the composition of the core set, we compared the results of the previous analysis with a variable selection approach implemented in the Weka machine learning software bona fide HIF binding regions.We next asked whether the TFs associated to the enriched TFBSs may share any common characteristics. Gene annotation enrichment analysis  of thesein silico, we next set out to validate some of these predictions experimentally. To this end, we selected hits for enriched TFBSs focusing on: 1) HREs located close to the TSSs of genes, to be able to study these promoter regions in in cellulo reporter assays, and 2) TFBS predictions located close to the Hypoxia Response Element (HRE). According to these criteria, we evaluated a CREB binding site prediction in the LDHA promoter  and upon treatment with the prolyl hydroxylase inhibitor DMOG (500 µM). As expected, mutation of the HRE in all the studied promoters completely abrogated induction by either hypoxia or DMOG treatment . ImportaHRE core . This reHRE core . Finally or DMOG , reachinbona fide HIF binding regions play a functional role in the transactivation by hypoxia or DMOG treatment of HIF-responsive promoters. Moreover, our data also suggests that diverse stress-responsive transcription factors, probably through modulation of basal transcription or recruitment of common cofactors, contribute to the specification of HIF target selectivity.Collectively, these results indicate that at least some of the TFBSs computationally predicted as enriched in a core set of http://www.broadinstitute.org/gsea/index.jsp). This collection comprises over 3000 gene sets from various sources including experimentally derived lists of targets for specific transcription factors. Thus, this analysis identifies sets of functionally related genes, such as those co-regulated in response to specific genetic and chemical perturbations, that are significantly enriched in the top positions of a list of genes induced by hypoxia.The results in the previous section were restricted to a limited set of validated promoters. However, if the involved transcription factors are of general relevance to HIF mediated transcription, some of their targets would be expected to be common with HIFs. To judge the potential generality of the involvement of CEBPs, CREB and AP1 in modulation induction of HIF transcriptional targets, we employed a gene-set enrichment analysis (GSEA) As expected, GSEA analysis revealed a statistically significant enrichment of well-characterized HIF targets in this sorted list . These gThe complete elucidation of the molecular principles governing the translation of genomic information to gene regulation remains a central question in biology. In particular, understanding the mechanisms dictating target selection by HIF transcription factors is of fundamental importance to truly dissect the genes directly modulated by HIFs, and therefore to completely characterize the transcriptional response to hypoxia that these factors orchestrate, and its interactions with other transcriptional pathways. Several mechanisms have been proposed to contribute to selective DNA binding and gene regulation by transcription factors with largely generic DNA binding domains Chromatin accessibility has been shown to play an important role in dictating transcription factor binding BNIP3HIF1ADNA methylation of a HIF binding site was originally shown to block HIF1α binding to the 3′ erythropoietin enhancer bona fide HIF binding regions  of transcription factor targets in a sorted list of genes regulated by hypoxia In conclusion, the data presented herein demonstrates that integration of high-throughput chromatin immunoprecipitation and gene expression data is a successful approach to select high-quality core HIF binding regions, and provides experimental proof of principle for the biological relevance of enriched transcription factor binding sites other than the HIF binding consensus in HIF-mediated transcription. Specifically, our results suggest that diverse stress-responsive transcription factors, in particular CEBPs, contribute to fine-tuning of the HIF-mediated transcriptional response.Figure S1Construction of a background set of control sequences resembling core HIF binding regions.(PDF)Click here for additional data file.Table S1HRE, AP1, CREB, CEBPB binding sites and control mutations introduced in LDHA, CA9 and GYS1 promoter constructs.(XLSX)Click here for additional data file.Table S2Correlation between normoxic basal expression and hypoxia inducibility (known HIF targets).(XLSX)Click here for additional data file.Table S3Candidate HIF1alpha-binding regions identified by ChIP-chip analysis in HeLa cells.(XLSX)Click here for additional data file.Table S4Distribution of conserved HBSs in ChIP-chip results across different cell types.(XLSX)Click here for additional data file.Table S5Integrated set of core HIF binding regions.(XLSX)Click here for additional data file.Table S6Enriched gene annotation clusters in core HIF binding regions.(XLSX)Click here for additional data file.Table S7Enriched gene annotation terms in transcription factors associated to sequence motifs overrepresented in core HIF binding regions.(XLSX)Click here for additional data file.


To determine whether there is a significant association between maternal haemoglobin measured before delivery and short-term neonatal outcome in very preterm neonates.st 2009 and December 31st 2011. Outborn infants and infants presenting with lethal malformations were excluded. Three hundred and thirty-nine mothers and 409 infants met the inclusion criteria. For each mother-infant pair a prospective record of epidemiologic data was performed and maternal haemoglobin concentration recorded within 24 hours before delivery was retrospectively researched. Maternal haemoglobin was divided into quartiles with the second and the third one regarded as reference as they were composed of normal haemoglobin values. Short-term outcome was defined as poor in case of death during hospital stay and/or grades III/IV intraventricular haemorrhage and/or periventricular leukomalacia and/or necessity of ventriculoperitoneal shunt.We included prospectively all live births occurring from 25 to 32+6 weeks of gestation in a tertiary care centre between January 1The global rate of poor short-term neonatal outcome was 11.4% and was significantly associated with low maternal haemoglobin values. This association remained significant after adjustment for antenatal corticosteroids therapy, gestational age, parity, mechanism of preterm birth, mode of delivery and birth weight (aOR = 2.97 CI 95% [1.36–6.47]). There was no relation between short-term neonatal outcome and high maternal haemoglobin concentration values.We show that low maternal haemoglobin concentration at delivery is an independent risk factor for poor short-term neonatal outcome in very preterm neonates. This study is one of the first to show such an association within the preterm population. Preterm delivery accounts for 75% of perinatal mortality and more than half of long-term morbidity.Several authors have reported a potential association between maternal haemoglobin concentration and various neonatal outcomes in the whole neonatal population. Described associations usually follow a U-shaped distribution with adverse outcomes at both ends of the haemoglobin range. The main underlying mechanisms involved are preterm birth and low birth weight.We decided to check if this association remained in very preterm neonates born before 33 weeks of gestation. We tested this hypothesis in a hospital based observational study with prospective data collection.This study was approved by our institutional review board . After deliberation, the board decided that no written consent was needed.st 2009 and December 31st 2011. All infants were hospitalized in our neonatal intensive care unit after primary care in the delivery room.We included prospectively all live births from 25 to 32weeks + 6 days of gestation that occurred in the university-based tertiary care centre in Dijon  between January 1Outborn infants were excluded as maternal haemoglobin concentration values were usually unavailable in those cases. Inborn infants with missing data for maternal haemoglobin and infants presenting with lethal malformations  were also excluded.For each mother-infant pair, a prospective and continuous record of epidemiologic data is routinely performed within our perinatal network since 1998.In addition, for each birth, we retrospectively reported both the maternal haemoglobin concentration value and the maternal mean cell volume (MCV) recorded within the 24 hours before delivery. The recorded data were divided into quartiles (Q) with Q2 and Q3 clustered together and regarded as reference.Our main outcome was poor short term neonatal outcome defined as: death during hospital stay and/or grades III–IV intraventricular haemorrhage (defined as per the Papile classification)2) and maternal smoking (defined as one cigarette or more per day during pregnancy). Pregnancy characteristics: gestational age, foetal gender, number of foetus and mode of delivery. Pregnancy characteristics also included mechanism of preterm birth , premature rupture of membranes (PROM), gestational hypertension and others) and antenatal corticosteroids therapy (defined as at least one dose of betamethasone before delivery). Neonatal characteristics: intubation in the delivery room, surfactant use, blood cells transfusion, patent ductus arteriosus, hemodynamic instability, necroticizing enterocolitis (as defined per the Bell classification)In order to examine poor short-term neonatal outcome in relation to maternal haemoglobin concentration, we defined potential confounding variables classified as follows. Maternal characteristics: maternal age at delivery , parity (nulliparous or multiparous), maternal pre-pregnancy body mass index .All analyses were performed using chi2 test, Fisher test, variance analysis and logistic regression when needed. When variance analysis could not be used , Kruskal-Wallis test had to be used. P values <0.05 were considered statistically significant. All analyses were performed with STATA 9® software , from 10.8 g/dl to 12.4 g/dl within Q2 and Q3 (n = 199) and from12.5 g/dl to 15.7 g/dl within Q4 (n = 107).Associations between short-term neonatal outcome, maternal characteristics and pregnancy characteristics are shown in Associations between quartiles of maternal haemoglobin concentration values, maternal MCV, maternal characteristics and pregnancy characteristics are shown in Infants whose mothers had high haemoglobin concentration values (Q4) had a significantly lower mean birth weight than the ones whose mothers had haemoglobin concentration values within Q1 or Q2–Q3 .There was no statistically significant association between maternal haemoglobin concentration values and rates of intubation in the delivery room, surfactant use, need for blood transfusion, patent ductus arteriosus, hemodynamic instability, necrotizing enterocolitis and chronic lung disease.The global rate of poor short-term neonatal outcome was 11.4%. There was a statistically significant association between maternal haemoglobin concentration values and short-term neonatal outcome with an increased rate of poor outcome in infants whose mothers had low haemoglobin concentration values (Q1). This association remained significant after adjustment for antenatal corticosteroids therapy, gestational age, parity, mechanism of preterm birth, mode of delivery and birth weight . There wThe association between poor short-term neonatal outcome, gestational age and maternal haemoglobin concentration value is shown in This observational study showed that neonates born before 33 weeks of gestation had a poorer short-term neonatal outcome when prenatal maternal haemoglobin concentration was low. This association remained strong and significant after adjustment for gestational age, birth weight, mechanism of preterm birth, antenatal corticosteroids therapy, maternal age, parity and mode of delivery. To our knowledge, our study is the first to focus on the potential impact of maternal haemoglobin concentration on very preterm neonates. It is also to be noted that the upper limit of Q1 concurs with the definition of anaemia in pregnancy by World Health Organization.The association between maternal haemoglobin and neonatal outcomes has already been studied in the general population of neonates and seems to follow a U-shaped distribution with adverse outcomes at both ends of the haemoglobin range largely mediated through preterm birth and low birth weight.A deeper analysis of our results could lead to question a potential association between maternal haemoglobin concentration and mechanisms of preterm birth. At the higher end of haemoglobin values range (Q4), we find both a significantly higher proportion of women presenting with preeclampsia and a significantly higher proportion of neonates presenting with a lower birth weight. It is known that the ability to produce a large increase in plasma volume between the first and the second pregnancy trimester is one of the hallmarks of successful pregnancy as it is probably linked with correct uterine perfusion. Failure to produce this expansion may lead to insufficient uterine perfusion and thus to preeclampsia and intra uterine growth restriction.