Furthermore, GM-CSF stimulation renders colon cancer cells more resistant to cytotoxic agents. Mechanistic investigation reveals that MAPK/ERK signaling and EMT-inducing transcription factor ZEB1 are critical to mediate these effects of GM-CSF. In specimen of CRC patients, high-level expression of GM-CSF positively correlates with local metastases in lymph nodes. Moreover, the co-expression of GM-CSF and its receptors as well as phosphorylated ERK1/2 are observed. Thus, our study for the first time identifies a progression-promoting function of GM-CSF in colon cancer by inducing EMT.Growing evidence shows that granulocyte macrophage colony-stimulating factor (GM-CSF) has progression-promoting potentials in certain solid tumors, which is largely attributed to the immunomodulatory function of this cytokine in tumor niches. However, little is known about the effect of GM-CSF on cancer cells. Herein, we show that chronic exposure of colon cancer cells to GM-CSF, which harbor its receptor, leads to occurrence of epithelial to mesenchymal transition (EMT), in time and dose-dependent manners. These GM-CSF-educated cancer cells exhibit enhanced ability of motility  Recently, clinical data show that GM-CSF levels in the circulatory system were frequently elevated in patients suffering from colorectal cancer compared with healthy controls6. Furthermore, high GM-CSF expression in neoplastic lesions significantly correlated with local and distant metastasis, more advanced histological grade, and poor prognosis in patients with breast cancer7 and pancreatic ductal carcinoma9. These disease-promoting effects of GM-CSF may be linked to modulation of immune reactions in tumor niche, involving macrophages10, myeloid-derived suppressor cells12, and plasmacytoid predendritic cells13.Granulocyte macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor produced by a variety of cell types, including macrophages, T lymphocytes, fibroblasts, endothelial cells, and keratinocytes following appropriate stimuli15, which suggests the possibility of responsiveness of cancer cells to GM-CSF challenge. Indeed, GM-CSF has been reported to support tumor growth and progression by autocrine stimulation of proliferation and migration in squamous cell carcinoma of skin or head and neck17, gliomas18 and osteosarcoma19. Furthermore, it also enhances the invasive capacity of human cancer cells in vitro by increasing production and activation of several matrix metalloproteinases22. The mechanisms underlying the metastasis-promoting function of GM-CSF are still incompletely known and need to be further addressed.It is worthy to note that the receptors for GM-CSF are constitutively expressed on the surface of certain nonhematopoietic cancer cells23. The EMT phenotype is characterized by the loss of cell-to-cell adhesion with the disintegration of tight and gap junctions and a phenotypic change from an “epithelial” morphology to a motile, fibroblast-like morphology25. The hallmark of EMT is the functional loss of E-cadherin, while additional cellular changes, such as reduced expression of epithelial markers cytokeratins and ZO-1, and the overexpression of mesenchymal markers N-cadherin, vimentin and fibronectin, are also often observed. Several transcription factors (TF) have been identified as master regulators of EMT, including the Snail, Zeb and Twist families, all of which interact with E-box elements located within the proximal region of the E-cadherin promoter24. Additionally, EMT-inducing TF expression is tightly regulated at different steps of transcription, translation and protein stability control by a variety of cell-intrinsic pathways as well as extracellular clues26.Epithelial to mesenchymal transition (EMT) is a developmental process that seems to play an important role in tumor progression and metastasis in diverse solid tumors, including colorectal cancerin vitro and in vivo. Chronic treatment of GM-CSF or ectopic expression of GM-CSF led to the occurrence of EMT in receptor-expressing colon cancer cells, but not in receptor-negative counterparts. These GM-CSF-educated cancer cells displayed enhanced ability of migration and invasion in vitro and more metastatic nodules in a mouse model of colorectal liver metastasis. Mechanistically, MAPK/ERK signals and ZEB1 were required for GM-CSF-induced EMT phenotype. Furthermore, the exposure to GM-CSF rendered colon cancer cells more resistant to drug-mediated cell death.In this study, we hypothesize that exogenous GM-CSF stimulation in colon cancer cells is competent to induce EMT and thereby enhance their motility 27, five of six cell lines virtually harbored two GM-CSF receptor subunits A and B, albeit different abundance . In contrast, the phosphorylation of MAPK/ERK and NF-κB was visible in SW480 cells upon stimulation with GM-CSF , and PI3KCSF Fig. , which wnce Fig. . Deletionce Fig. . Conversnce Fig. , includince Fig. . These fIntriguingly, ZEB1 expression was dramatically suppressed in ERK1 or 2-knockdown cancer cells with stimulation of GM-CSF Fig. , suggest30. To determine whether chronic exposure to GM-CSF led to resistance to cytotoxic compounds, SW480 cells with or without GM-CSF stimulation were treated with three first-line chemotherapeutic agents for colorectal cancer in the clinic. The results showed that, although GM-CSF stimulation did not affect cell viability, GM-CSF-educated cancer cells were resistant obviously to fluorouracil (5-FU), oxiliplatin or irinotecan-mediated cell death, by SRB assays  of specimens were positive for GM-CSF staining Fig. . The expin vitro27. Here, we have addressed mechanistic clues underlying these effects, by analyzing EMT-inducing potentials of GM-CSF. Of note, it has been reported that GM-CSF contributes to acquisition of the invasive phenotypes in breast cancer cells by educating infiltrating macrophages in microenvironment7. In this study, we provide evidence for the first time that GM-CSF has an effect on the induction of EMT program in receptor-expressing colon cancer cells through activating MAPK/ERK-ZEB1 signaling pathways. These data may provide mechanistic explanation for recent observations that high GM-CSF contents in serum of CRC patients significantly correlated with poor prognosis3.In a previous work we showed that GM-CSF stimulation in colon cancer cell lines could augment the migration and invasion et al. reported that GM-CSF production by tumor cells was associated with lower T stage and significantly longer survival as well as favorable prognosis in mismatch-repair proficient colorectal cancer31. This observation coincides with a previous report showing that 100% of the patients with human colon tumors that overexpressed GM-CSF and its receptors survived at least 5 years after diagnosis32. These divergences may be reconciled by the concept that the function of GM-CSF in tumor niche is context-dependent. GM-CSF stimulation alone is potent to drive M1 polarization of macrophage and maturation of dendritic cells, two populations mounting robust anti-tumor immune responses. On the other hand, cancer cell-derived GM-CSF can shape immunosuppressive microenvironment by promoting the generation and accumulation of myeloid suppressor cells and tumor-associated macrophages9. The latter indicates that synergistic effects of other soluble factor (s) from cancer cells or stroma may be indispensible for GM-SCF-induced tumor-promoting environment. Besides immunomodulation of tumor-surrounding stromal components, we showed a stimulatory effect of GM-CSF on cancer cells themselves. Clinical analysis also supports the metastasis-promoting role of this cytokine. These data are not in agreement with a previous study that colorectal tumors substantially overexpressed GM-CSF and this cytokine had an immune-independent antitumor effect on receptor-positive cancer cells32. It should to be noted that the expression of proinflammatory cytokines such as IL-17A, IL-6, IL-22 was dramatically increased in lesions of colorectal cancer and their high expression significantly correlated with cancer progression and poor prognosis35. As one member of proinflammatory mediators, it seems plausible that high expression of GM-CSF in tumor is related to more invasive phenotype and poor outcome. Considering a strongly positive correlation of high GM-CSF expression in tumor with distant dissemination and unfavorable prognosis in several types of solid cancers, further investigations of GM-CSF role in CRC development and progression in larger cohorts of CRC samples are warranted.However, the relevance of GM-CSF to CRC progression and prognosis remains controversial. Nebiker 27. Furthermore, MAPK/ERK signaling pathways are clearly linked to tumor invasion and metastasis in response to a variety of intracellular and extracellular stimuli in several types of solid cancers including colorectal cancer40. Of note, we showed that ERK1 and 2 are equivalently important for GM-CSF-induced EMT program, as knockdown of ERK1 or 2 had similar effects on the expression of EMT markers and cell motility. This is not consistent with a recent study showing that ERK2 but not ERK1 is critical for a oncogene placenta-specific 8-driven EMT phenotype in colorectal cancer41, suggesting that ERK1 and/or ERK2-mediated EMT lies on upstream clues. We also identified the activation of NF-κB in colon cancer cells upon GM-CSF challenge. The involvement of this key transcriptional factor in GM-CSF-induced EMT need to be addressed in the future through inactivation of NF-κB by ectopic overexpression of IκBα in cancer cells.Mechanistic investigations in this study reveal that MAPK/ERK signaling pathways are required for GM-CSF-induced EMT program and metastasis. Although several signaling pathways have been shown to be activated upon the engagement of GM-CSF with its receptor in hematopoietic cells, MAPK/ERK signals appear to dominantly mediate bioactivity of GM-CSF in epithelial cells, which is consistent with our previous results in a murine colon carcinoma cell line43 and lung cancer44, we showed that ZEB1 was also a critical mediator for GM-CSF-driven EMT. ZEB1 expression seems to be regulated by MAPK/ERK signals, because knockdown of ERK1 or 2 decreased ZEB1 expression in GM-CSF-stimulated cancer cells. As previously reported46, the activation of MAPK/ERK pathway induced Ets1 and Fra1 expression in cancer cells, and the latter activated ZEB1 promoter and induced its expression, which may provide some clues for the exact regulation of ZEB1 expression by MAPK/ERK signaling pathway in our model. In addition, one of metastasis-promoting function of ZEB1 is reported to enhance MMP secretion47, which was increased in GM-CSF-overexpressing HT-29 colon cancer cell lines as described recently22. Thus, ZEB1 could regulate colon cancer invasiveness by inducing EMT and MMP production.As described in previous studies that demonstrated a key role of ZEB1 in mediating EMT and tumor invasiveness in colorectal48. The role of GM-CSF, however, in this process is unknown. In this paper, we identified GM-CSF as a chemoresistance-inducing factor, at least in colorectal cancer. Given the bioactivity of GM-CSF in other types of tumors, it is plausible that GM-CSF induces chemoresistant phenotype in receptor-expressing cancer cells. Furthermore, we showed that chemoresistance induced by chronic stimulation with GM-CSF was tightly associated with EMT, which is consistent with the notion that EMT contributes to drug resistance, which is validated in many types of tumors. Accordingly, since knockdown of ERK1/2 or ZEB1 led to the failure of GM-CSF-driven EMT, it is reasonable that inhibition of MAPK/ERK-ZEB1 signals also abrogates acquisition of chemoresistance. Notably, the contribution of MAPK/ERK-ZEB1 signaling pathway to multidrug resistance has been reported in numerous types of cancer, including breast cancer49, hepatocellular carcinoma50, and glioblastoma51. Therefore, inhibition of MAPK/ERK signals using small-molecular compounds may be favorable to reverse chemoresistance in certain settings. The details in the regulation of chemoresistant traits in cancer cells by MAPK/ERK-ZEB1 pathways need to be further addressed in the future, although GM-CSF stimulation greatly suppresses chemo-induced active cleavage of caspase and PARP.It is well established that chronic exposure to a variety of growth factors or cytokines is linked to acquisition of chemoresistance in solid tumors27. Thus, further work is warranted to determine whether any one of them is responsible for mediating GM-CSF effect on EMT by adding neutralizing antibodies to some of these factors during the culture.In conclusion, in this study we demonstrate that GM-CSF is an EMT-inducing factor by binding its receptor on the surface of cancer cells through activating MAPK/ERK-ZEB1 signaling pathway Fig. . Of noteCurrently, functional contribution of GM-CSF to cancer progression and metastasis is controversial, which may be due to dual effects of GM-CSF on immune cells and non-immune cells. Considering that recombinant GM-CSF are routinely used to correct neutropenia subsequent to chemotherapy and radiation and that adjuvant GM-CSF treatment has been suggested to occasionally enable tumor growth, detection of GM-CSF receptor expression in cancer cells is warranted in the future prior to GM-CSF treatment, to better define eligile patient’s cohorts.Primary CRC tissue samples were obtained from 65 patients undergoing surgical resection at the Department of Surgery, Zhengzhou Yihe Hospital from February 2010 to June 2011. Sections were reviewed by two experienced pathologists to verify the histologic assessment. All the specimens were adenocarcinoma. Prior informed consent was obtained and the study protocol was approved by the Ethics Committee of Zhengzhou Yihe Hospital. All experiments were performed in accordance with relevant guidelines and regulations.6/ml/well) and stimulated with recombinant human GM-CSF (Peprotech) at 1–100 ng/ml for 1–3 weeks. To blocking GM-CSF activity, neutralizing anti-GM-CSF monoclonal antibody or isotype IgG  was added into the culture and maintained for one week. Then cells were collected and centrifuged at 1500 rpm for 5 minutes and resuspended in serum-free medium. In some settings, cells were treated with fluorouracil (5-FU), oxiliplatin and irinotecan at indicated concentration for 72 hours, which were purchased from Selleckchem. After incubation, cells were collected for analysis.Human colon adenocarcinoma cell lines  and human acute monocytic leukemia cell line THP-1 were purchased from American Type Culture Collection and maintained in appropriate medium according to product specification, supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin. Cells were seeded in 6-well culture plates  were anesthetized with ketamine/dexmedetomidine admixture and the spleen was exteriorized through a left lateral flank incision. Tumors were established by intrasplenic injection of 2 × 106 cells suspended in 50 μl of serum-free media using one 27-gauge needle. The injection site of the spleen was pressed with a cotton stick wet in iodine-polividone solution in order to remove extravasated cells and ensure homeostasis. The peritoneum and skin were closed in a single layer using surgical thread. Mice were sacrificed at four-to-six weeks post-injection and the burden of metastatic liver tumors was calculated. Primary (splenic) tumor mass was dissected and the protein was extracted conventionally for immunoblotting analysis. All animal experiments were performed in accordance with international guidelines for the care and use of laboratory animals and approved by the Animal Ethics Committee of the Institute of Basic Medical Sciences.The surgical procedure was performed according to splenic injection model as described previouslyIn vitro cell migration and invasion assays were performed using transwell chambers with polyethylene terephthalate membrane . For the migration assay, 2.5 × 104 cells were added to the top chambers. For the invasion assays, 5 × 104 cells were seeded to the top chambers coated with Matrigel (BD Biosciences). Complete medium was added to the bottom wells. After incubation for 36 hours, the noninvasive cells were removed with a cotton swab. The cells that had migrated through the membrane and adhered to the lower surface of the membrane were fixed with methanol for 10 minutes and stained with crystal violet solution (0.1%). For quantification, the cells were counted using a microscope from five randomized fields at ×200 magnification.in vitro scratch wound healing assay. Cells were starved overnight with serum-free RPMI1640 medium and a scratch was made using a sterile 1-mL pipette tip. The cell debris was removed with PBS and cells were cultured with GM-CSF (25 ng/ml) for 24 hours. An inverted microscope was used to observe cell migration.Cell migration was also assessed using an 3 cells per well in 100 μl medium) were seeded into 96-well plates in triplicate and exposed to recombinant human GMCSF (Peprotech) at titrated concentrations. After incubation for indicated time, 50 μl of 30% trichloroacetic acid was added and incubated for 60 min at 4 °C. After washing and drying the plate, 100 μl of 0.4% SRB was added for 30 min. The plates were rinsed with 0.1% acetic acid and air-dried, after which 100 μl of Tris base (10 mM/L) was added, and the plates were shaken for 5 min. The SRB value was measured at a wavelength of 590 nm.Cell growth was assessed by Sulforhodamine B (SRB) assay. Cells (5 × 10Samples were homogenized and sonicated in RIPA lysis buffer (Santa Cruz Biotech.), supplemented with protease inhibitors. After centrifugation at 20,000 g for 15 min, 30 μg of the supernatants were separated on 10% SDS-polyacrylamide gel and transferred onto an Immunobilon-P Transfer membrane (Millipore). After being blocked with 5% skim milk, the membrane was incubated with primary antibodies at1:1000 dilution. Rabbit anti-GAPDH antibody was used as an internal control (purchased from Tianjin Sungene Biotech.). ImmunoPure peroxidase-conjugated anti-rabbit IgG were used as secondary antibodies. The blotted membrane was then treated with the Super Signal West Dura Extended Duration Substrate (Pierce) and signals were detected by LAS-3000 mini CCD camera (Fuji Film). Primary antibodies used for immunoblotting were purchased from Cell Signaling.The spleen and liver was removed, fixed in 10% neutral-buffered formalin solution, embedded in paraffin, and cut into tissue sections, which were stained with hematoxylin and eosin. Paraffin-embedded sections were deparaffinized and immersed in 80 °C water bath in 10 mM sodium citrate buffer with 0.1% Tween 20 overnight for antigen unmasking. Slides were incubated with primary antibody against CSF2RA , CSF2RB , GM-CSF , P-ERK1/2 , E-cadherin  or Fibronectin  in PBS containing 1% BSA and 10% goat serum. Biotinylated secondary antibodies (Dako) were added and incubated at room temperature for 1 hour. Streptavidin-HRP (BD Pharmingen) was added, and after 40 min the sections were stained with DAB substrate and counterstained with hematoxylin.Cells were seeded onto glass coverslips in 24-well plates, washed with PBS, fixed in 4% formaldehyde solution and permeabilized with 0.2% Triton X-100/PBS. Cells were blocked with 2% BSA in PBS for 30 min. Coverslips were incubated with primary antibodies for 1 hour, followed by incubation with phycoerythrin (PE)-conjugated secondary antibodies for 1 hour, and then stained with DAPI. Finally, coverslips were observed under a fluorescence microscope.41. ShRNA plasmid targeting ZEB1 and human GM-CSF expression plasmid were purchased from Santa Cruz Biotech. SW480 or HCT116 colon cancer cell lines (1 × 107/ml/well) were seeded in 6-well culture plates and transfected with plasmids indicated above using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Stable cell lines were selected with 2 μg/ml puromycin.ShRNA plasmids targeting ERK1/2 and non-targeting shRNA control plasmid were kindly provided by Robert J. coffey Apoptosis was quantified using fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI), according to the manufacturer’s protocol . Cells were stained with 5 μl of FITC-Annexin V diluted 1:10 in buffer, and 2.5 μl of PI. After incubation for 15 minutes, cells were analyzed by flow cytometry with standard FACScan equipment (Becton Dickinson Co.).GM-CSF levels in the supernatants were measured using ELISA kits (eBiosciences) according to the manufacturer’s protocol.RNA was extracted and reverse-transcribed into cDNA using the SuperScript III First Strand cDNA synthesis system (Invitrogen). cDNA was synthesized from 0.5 μg RNA using random hexamer primers and SuperScript III (Invitrogen). Real-time RT-PCR was performed on a Bio-Rad iCycler to quantify mRNA levels. The primers for real-time were listed in Supplementary Table t test. One-way analysis of variance (ANOVA test) was used to define differences in cell viability in cultures treated with various concentrations of cytotoxic agents. A value of P < 0.05 was considered statistically significant.Data represent means of multiple determinations and were analyzed using the GraphPad Prism statistical PC program  by means of paired Supplementary Information


In order to investigate this hypothesis, we evaluated the association between systemic inflammatory markers  and BBB function  in 1273 consecutive lumbar punctures. In the absence of cerebrospinal fluid (CSF) abnormality, systemic inflammation did not affect the CSF/serum albumin ratio. When CSF abnormality was present, systemic inflammation significantly predicted the CSF/serum albumin ratio. Amongst the systemic inflammatory markers, C-reactive protein was the predominant driver of this effect. Temporal analysis in this association study suggested causality. In conclusion, the diseased BBB has an increased susceptibility to systemic inflammation.The blood-brain barrier (BBB) plays an important role in the clinical expression of neuropsychiatric symptoms during systemic illness in health and neurological disease. Evidence from  BBB perin vitro and in vivo preclinical studies have shown that inflammatory challenge results in an increase in BBB permeability. This effect appears to be a feature of the BBB, unrelated to the type of inflammatory trigger since it has been observed in a wide variety of experimental settings including lipopolysaccharide, poly I:C, bacteria, viruses, chemically-induced inflammation, anaphylaxis and cytokines  is a widely accepted indicator of blood–CSF barrier function , during a service evaluation, with institutional approval. The white cell count, neutrophil, lymphocyte, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) measurements within a five day period centred around the lumbar puncture were recorded  count >127 cells per microlitre. Since CSF total protein concentration rises by 1 mg/dl for every 100 red blood cells/μL that enter the CSF during traumatic lumbar puncture , the CSFCSF and blood were collected in sterile polypropylene tubes  and Vacutainers  respectively. CSF volume was not available. Samples were analysed on the same day, except for isoelectric focussing in which case samples were kept at 4 °C and batch analysed within one week. Blood counts were performed on a Sysmex XE-2100 automated hematology system. ESR was performed on a Vitech Starrsed system using the Westergren sedimentation method. CRP, albumin, protein and glucose were assayed on a Beckman Coulter AU5800 automated system. CSF cell counts were performed manually using a modified Fuchs-Rosenthal haemocytometer. Oligoclonal band assessment was performed manually using isoelectric focussing on CSF/serum pairs.2.2Blood) integrating all these indices was created to reflect systemic inflammation. To reflect central nervous system inflammation, the variable InfCSF was derived from the CSF white cell count. The derivation of InfBlood and InfCSF is detailed in Data preparation was performed in Excel v14. Cases were identified as having normal findings if the following conditions were met: CSF total protein <500 mg/L, CSF glucose >2/3 serum glucose, white blood cells ⩽5 cells/μL, polymorphs were absent, and there was no evidence of intrathecal synthesis of oligoclonal immunoglobulin G. Single systemic inflammatory variables included total leucocytes, neutrophils, lymphocytes, ESR and CRP; a composite variable , age, sex and Infvariable A,B,C. Ag3.3alb was significantly higher in cases with abnormal CSF versus those with normal CSF , and this effect was still significant in an analysis of covariance with age and sex (p < 10−8). This was expected since several neurological diseases affect BBB permeability . In cases with abnormal CSF only, an interaction between InfBlood and InfCSF was examined in the regression analysis as a second block after age, sex, InfBlood and InfCSF; a trend was observed but the interaction was not significant (p = 0.08).Infnificant , an inteCSF and Qalb, but CRP exerted the highest influence on BBB permeability . Hence CRP was the most sensitive systemic inflammatory marker impacting on BBB permeability.When individual systemic inflammatory markers were examined, high CRP and neutrophils exhibited the same relationship with Inf3.4alb was cause or effect, a temporal analysis was undertaken. The multiple linear regression was re-run in cases with abnormal systemic inflammatory markers  before the LP (n = 59), and in cases with normal systemic inflammatory markers before the LP but abnormal markers at or after the LP (n = 29). High systemic inflammatory markers  were only predictive of an increased Qalb when these systemic abnormalities were present before the LP . This result is in keeping with a causative relationship of systemic inflammation on BBB breakdown, in the presence of intrathecal inflammation.In order to investigate whether the relationship between systemic inflammation and Q4alb may underestimate, or fail to detect, changes in BBB permeability to molecules smaller than albumin. Despite this limitation, this study has clearly demonstrated that the BBB in the diseased brain is more susceptible to systemic inflammation compared to the healthy brain. The BBB alterations which occur as a consequence of neuropathology  or circulating biomarkers of BBB integrity will be useful. Although this study provided some evidence in support of causation, prospective and/or experimental human studies would provide concrete proof that systemic inflammation affects BBB function.Future work is needed to address limitations of this study. Here, we have not systematically determined the precise reason for systemic raised inflammatory markers or abnormal CSF. Although preclinical studies have shown that the effect of systemic inflammation on BBB permeability occurs across a broad range of systemic inflammatory stimuli and neuropathologies , future ulk flow , which culk flow . Finallys system , other mAll authors declare that there is no conflict of interest.


CSF-1 and IL-34 share the CSF-1 receptor and no differences have been reported in the signaling pathways triggered by both ligands in human monocytes. IL-34 promotes the differentiation and survival of monocytes, macrophages and osteoclasts, as CSF-1 does. However, IL-34 binds other receptors, suggesting that differences exist in the effect of both cytokines. In the present study, we compared the differentiation and polarization abilities of human primary monocytes in response to CSF-1 or IL-34. CSF-1R engagement by one or the other ligands leads to AKT and caspase activation and autophagy induction through expression and activation of AMPK and ULK1. As no differences were detected on monocyte differentiation, we investigated the effect of CSF-1 and IL-34 on macrophage polarization into the M1 or M2 phenotype. We highlighted a striking increase in IL-10 and CCL17 secretion in M1 and M2 macrophages derived from IL-34 stimulated monocytes, respectively, compared to CSF-1 stimulated monocytes. Variations in the secretome induced by CSF-1 or IL-34 may account for their different ability to polarize naïve T cells into Th1 cells. In conclusion, our findings indicate that CSF-1 and IL-34 exhibit the same ability to induce human monocyte differentiation but may have a different ability to polarize macrophages. They have the unique property among peripheral blood cells to migrate into tissues where they are subjected to differentiation into morphological and functionally heterogeneous cells that include macrophages, myeloid dendritic cells, and osteoclasts, depending on the stimulus2. The differentiation of peripheral blood monocytes into resident tissue macrophages can be recapitulated ex vivo by incubation in the presence of colony-stimulating factor-1 (CSF-1)3. The biologic effects of CSF-1 are mediated by a unique receptor, the CSF-1R, which is encoded by the c-fms proto-oncogene4. Downstream signaling pathways activated by CSF-1R upon ligand binding include PI3K-AKT and AMPK pathways, which are implicated in the respective activation of caspases and autophagy, two key processes required for CSF-1-induced macrophage differentiation5. Our previous studies have established that physiological monocyte differentiation triggered by CSF-1R engagement is critically dependent on the oscillatory activation of the kinase AKT, which within 2–3 days leads to the formation of a multi-molecular platform that includes the adaptor Fas-associated death domain (FADD), the serine-threonine kinase RIP1, the long and short isoforms of FLIP, and procaspase-86. Caspase-8 activation in this platform provokes a limited activation of several downstream caspases that cleave specific intracellular proteins8. The contribution of these cleavages to the CSF-1–driven monocyte-to-macrophage differentiation remains poorly understood. More recently, we have also established that autophagy plays a crucial role during macrophage differentiation of monocytes9. We found that CSF-1 increases the expression of the purinergic receptor P2RY6 that in turn activates the CAMKK2-AMPK-ULK1 pathway leading to autophagy induction10. Notably, inhibition of this pathway abrogated both CSF-1-mediated induction of autophagy and differentiation.Monocytes are circulating blood leukocytes that play important role in tissue homeostasis and in the inflammatory response which is essential for the innate response to pathogens11. Although it lacks appreciable similarity with CSF-1 or any other proteins, IL-34 tightly binds to CSF-1R and promotes the differentiation, proliferation and survival of monocytes, macrophages and osteoclasts as CSF-1 does13. IL-34 actions have been rendered more complex by the discovery of two other distinct receptors: the receptor type protein-tyrosine phosphatase zeta (PTP-ζ)14 and CD138 (Syndecan 1)15, suggesting additional roles for IL-34, compared to CSF-1. Recently, IL-34 was found to be associated with the inflammation process seen in diseases such as rheumatoid arthritis (RA)16, inflammatory bowel disease17 and Sjogren’s syndrome18. Interestingly, emerging findings indicate that IL-34 levels are abnormally increased in serum and synovial fluid in patients with reactive rheumatoid arthritis20.IL-34 is a newly identified cytokine with only partially understood functions. IL-34 has been recently identified as the second ligand for CSF-1R through comprehensive proteomic analysesHere, we used human primary monocytes to characterize in details IL-34 and CSF-1-driven macrophage differentiation and polarization into the M1 or M2 phenotype. We demonstrate that IL-34, like CSF-1, induced caspase and autophagy activation and that both processes are required to induce differentiation of monocytes into macrophages in response to IL-34. Moreover, we report that IL-34 and CSF-1 macrophages display a different polarization potential when polarized by pro or anti-inflammatory stimulus, since we found some differences in the mRNA and protein expression profiles of some cytokines/chemokines in CSF-1 and IL-34 differentiated cells polarized into the M1 or M2 phenotype.When stimulated with 100 ng/mL CSF-1, human monocytes differentiate into macrophages as shown by an increase in cell adherence and the acquisition of specific markers such as TFRC/CD71 and CD163. At day 2, 60–70% of myeloid cells were found to be positive for both CD71 and CD163 expression Fig. . This hi10. This increased phosphorylation and activation of AMPK and ULK1 also correlated with an enhanced expression of both AMPK and ULK1 proteins  activation in both CSF-1 and lL-34 treated monocytes , in response to interferon gamma (lFNγ) and lipopolysaccharide (LPS), is characterized by up-regulation of interleukin lL-6 and enhancement of the Th1 immune response. M2 phenotypic activation (anti-inflammatory) is stimulated by lL-4 and characterized by increased expression of lL-1023.Globally, the results described above indicate that CSF-1 and lL-34-mediated intracellular signaling pathways, including auto-phosphorylation of the CSF-1R, activation of AKT, ERK 1/2, AMPK and ULK1 and induction of caspase activities and autophagy are similar in human primary monocytes induced to differentiate into macrophages, suggesting that the effects of lL-34 are mediated via the CSF1-R. As no obvious differences have been reported so far in the effects of CSF-1 and IL-34 on monocyte differentiation  or IL-4 (M2 inducer) was analyzed by flow cytometry in macrophages differentiated with CSF-1 or lL-34. ln CSF-1 differentiated macrophages, LPS + lNFγ induced M1 polarization with both an increase in CD80 and CD86 expression and a decrease in CD200R and CD206 expression, whereas lL-4 promoted M2 polarization with an inhibition in CD80 and CD86 expression and an increase in CD200R and CD206 expression Fig. . Interes26. Regarding M1 polarization, we found a significant increase in IL-10 and CXCL11 mRNA and protein levels when IL-34 macrophages were polarized in response to LPS + IFNγ compared to CSF-1 polarized macrophages  mice exhibit a less severe phenotype than their CSF-1R deficient counterpart suggesting that at least one part of the IL-34 effect could be triggered independently of the CSF-1R30. This has been recently confirmed following the identification of a second receptor for IL-34 in the brain. Indeed, IL-34 binds to and signals by an alternative receptor, namely PTP-ζ14. Moreover, it has also been reported minor differences in the production and secretion of some cytokines including eotaxin-2 in peripheral blood cells stimulated with CSF-1 or IL-3427. Although CSF-1 and IL-34 exert many redundant functions ex vivo, the profile of expression, the source and the kinetic of cytokine production are different in vivo. Indeed, in contrast to CSF-1, IL-34 shows specific expression by keratinocytes and neurons in mice and plays an important role in the development and maintenance of Langherans cells and microglia, respectively31. In human, IL-34 is more widely/differently expressed, albeit also highly expressed in neuronal tissues and skin (see FANTOM5 and GTEX databases). There is also increasing evidence that IL-34 expression is upregulated in pathological conditions and may play important functions in autoimmune disorders, infections, inflammation and cancer32. Regarding cancer, IL-34 promotes the recruitment of M2-polarized tumor-associated macrophages by a direct effect on CSF-1R in macrophages, promoting new vessel formation and extravasation of immune cells, as CSF-1 does33. However, clear evidence indicating different functions of both cytokines are still lacking.CSF-1 and IL-34 share the same receptorIn the present study, we scrutinized the effect of CSF-1 and IL-34 on human monocyte differentiation. We found that the early and late signaling pathways leading to the differentiation of monocytes into macrophages were strictly identical in both conditions. Moreover, we also described for the first time that IL-34 promotes caspase activation and the activation of the AMPK/ULK1 pathway in monocytes leading to LC3-I lipidation and cleavage into LC3-II, as CSF-1 does, confirming the similarity of action of both ligands on human monocyte differentiation and induction of autophagy.Whereas monocyte differentiation proceeded similarly in CSF-1 and IL-34 stimulated human primary monocytes, we identified however, differences in the production of some cytokines/chemokines when cells were polarized into the M1 or M2 phenotype using LPS + IFNγ or IL-4 following macrophage differentiation by either CSF-1 or IL-34. The most noticeable examples, were increased production of IL-10 and CXCL11 at the mRNA and protein level in M1 polarized macrophages. Regarding M2 polarization, we also observed a striking induction of CCL17 and CCL22 and a decrease in IL-10 mRNA and protein expression, which were found exclusively in M2-polarized IL-34 macrophages. Therefore, despite a strictly identical profile of monocyte differentiation, the cytokines/chemokines produced by CSF-1 or IL-34 macrophages are somehow different, suggesting that macrophages generated in both conditions may have different role in pro or anti-inflammatory pathophysiological conditions.26. It was also recently reported that GM-CSF drives CCL17 mRNA and protein expression in human and mice monocytes. Surprisingly, CCL17 seems to mediate at least some of the pro-inflammatory action of GM-CSF on arthritic pain and disease34. Several other cytokines including IL-435 and IL-1336 have been reported to be associated with upregulation of CCL17 mRNA and protein. The fact that CCL17 mRNA expression was selectively upregulated in macrophages differentiated in the presence of IL-34 and polarized into the M2 phenotype by IL-4 but not in their CSF-1 differentiated counterpart indicates that polarization rather than IL-4 itself is responsible for CCL17 mRNA induction in our experiments. Globally, these findings indicate that IL-34 could resemble more GM-CSF than CSF-1 in its ability to trigger M2 polarization.CCL17 (TARC) was originally implicated in the selective attraction of Th2 lymphocytes and thus considered as a M2 cytokine. Recent findings in the literature have reported that human monocyte-derived macrophages grown in CSF-1 do induce CCL17 in response to IL-4 as do mouse bone-marrow-derived macrophages grown in CSF-139. We showed here, that IL-10 protein expression is strikingly increased in M1-polarized IL-34 macrophages compared to M1-polarized CSF-1 macrophages. This may explain the impairment of Th1 polarization observed with the secretome of M1-polarized IL-34 macrophages.IL-10 is produced by Th2 lymphocytes and is known to suppress the differentiation and effector function of Th1s is currently unknown. However, a likely explanation could rely on the fact that IFNγR/TLR4 or IL-4 receptor expression is different in CSF-1 and IL-34 differentiated macrophages. We checked that it was not indeed the case . Human CSF-1 was purchased from Miltenyi (130-096-493). Bafilomycin A1 was from Tocris (1334). qVD was from Clinisciences (A1901-5mg). 3-MethylAdenine was purchased from Sigma Aldrich (M9281). Phospho-CSF-1R (Tyr723), Phospho-AKT, AKT, Phospho-ERK1/2, ERK1/2, Phospho-AMPK (Thr172), AMPK, Phospho-ULK1 (Ser555), ULK1, Caspase-8, Caspase-3, Nucleophosmin, LC3-B, and Beclin antibodies were purchased from Cell Signaling Technology . CSF-1R, ACTIN, CD71, and CD163 antibodies were from Santa Cruz Biotechnology . Cathepsin B was from MERCK (IM27L). HRP-conjugated rabbit anti-goat or mouse was purchased from Dako (P0449 and P0260) and HRP-conjugated goat anti-rabbit was from Cell Signaling (5127).Human peripheral volunteers were obtained from healthy donors with informed consent following the Declaration of Helsinki according to the recommendations of an independent scientific review board. The project has been validated by The Etablissement Français du Sang, the French national agency for blood collection (protocol N°ALM/PLER/FO/001). Blood samples were collected using ethylene diamine tetra-acetic acid–containing tubes. Mononucleated cells were first isolated using Ficoll Hypaque . Then, we used the autoMACS® Pro Separator  to perform cell enrichment. An initial positive selection, which included antibody targeting CD14, was used for monocyte enrichment . Purified monocytes from human were grown in RPMI 1640 medium with glutamax-I  supplemented with 10% (vol/vol) foetal bovine serum (Life Technologies). Monocytes were seeded in tissue culture treated flasks or wells. Macrophage differentiation was induced by adding into the culture medium 100 ng/mL CSF-1 or 100 ng/mL IL-34 and was visualized using standard optics (20x/0.35 Ph1) equipped with an AxioCam ERc camera . Phase images of the cultures were recorded with the Zen 2 software (Zeiss).6 per mL in RPMI 1640 medium with glutamax-I supplemented with 10% (vol/vol) fetal bovine serum plus CSF-1 or IL-34 for 7 + 2 days to differentiate into M0 macrophages. 100 ng/mL lipopolysaccharide  and 20 ng/mL interferon gamma  were added after 7 days of differentiation for two days to polarize into M1-macrophages. M2-macrophages were obtained by addition of 20 ng/mL IL-4  for two days. To analyze the macrophage polarization, cells were detached using PBS/EDTA/BSA, washed with PBS, and incubated at 4 °C for 10 min in PBS/ bovine serum albumin with anti-CD80 , anti-CD86 , anti-CD200R , and anti-CD206  or isotype controls. Finally, cells were washed and fixed in 2% paraformaldehyde . Fluorescence was measured with a MACSQuant® Analyzer . To analyze the ability of macrophages to phagocyte bacteria, we used Vybrant® Phagocytosis Assay Kit according manufacture’s instruction . Briefly, macrophages were detached and incubated with fluorescein-labeled E. coli (K-12 strain) for 30 min. Next, cells were washed twice with PBS and resuspended in PBS. Fluorescence, that indicate the internalization of particles, was measured with a MACSQuant® Analyzers . Trypan blue solution was used to quench the fluorescence from particles that were not internalized.To analyze the macrophagic differentiation of monocytes, cells were washed with ice-cold phosphate buffered saline , incubated at 4 °C for 10 min in PBS/bovine serum albumin  with anti-CD71 and anti-CD163 or an isotype control . Finally, cells were washed and fixed in 2% paraformaldehyde . To detect caspase activity, we used FITC-DEVD-FMK according to the manufacturer’s instruction . To perform macrophage polarization, purified monocytes were plated at 0.3 × 10Cells were lysed for 30 min at 4 °C in lysis buffer . Lysates were centrifuged at 20,000 g  and supernatants were supplemented with concentrated loading buffer (4X laemmli buffer). Fifty micrograms of proteins were separated and transferred following standard protocols before analysis with chemiluminescent detection kit .6 monocytes in 100 μL of nucleofector solution with 15 nmol of siRNA. Cells were incubated for 24 h with 5 mL of prewarmed complete medium, and CSF-1 or IL-34 was subsequently added. We used siRNAs (Life Technologies) targeting Caspase-8 (HSS141461), BECLIN1 (HSS112741) and LUCIFERASE as a negative control (Sense: 5′-CUUACGCUGAGUACUUCGAtt-3′).Small interfering (si) RNAs were introduced into monocytes by nucleoporation  of 5 × 106 cells using the RNeasy Mini Kit according to manufacturer’s protocol . Each cDNA sample was prepared using AMV RT and random primers . Real-time polymerase chain reaction (PCR) was performed using the SyBR Green detection protocol . Briefly, 5 ng of total cDNA, 500 nM (each) primers, and 5 µL SyBR Green mixture were used in a total volume of 10 µL. Detection of multiple endogenous controls  were used to normalize the results. Specific forward and reverse primers are accessible upon request.RNA was prepared from 5 × 106 per mL in RPMI 1640 medium with glutamax-I supplemented with 10% (vol/vol) fetal bovine serum plus CSF-1 or IL-34 for 7 + 1 days to differentiate into M0 macrophages. 100 ng/mL lipopolysaccharide  and 20 ng/mL interferon gamma  were added after 7 days of differentiation for one day to polarize into M1-macrophages. M2-macrophages were obtained by addition of 20 ng/mL IL-4  for one day. Next, cells were washed and seeded in RPMI 1640 medium with glutamax-I supplemented with 10% (vol/vol) fetal bovine serum during one day. The cytokines productions were finally evaluated using MultiAnalyte ELISArray kit .To perform macrophage polarization, purified monocytes were plated at 0.3 × 10t test and significance was considered when P values were lower than 0.05. The results are expressed as the mean ± SEM.Statistical analysis was performed using a paired Student Supplementary information


Since TT has not yet been scientifically proven as per Western standards, leaders of the health care community are likely wary of lending support to TT at this time. If TT can be found to be a scientifically sound therapeutic technique, then it will be more readily accepted in the health care community. This paper reviews TT.The growing popularity and use of therapeutic touch (TT) is an issue that has generated controversy and concern within the medical community. While anecdotal and traditional scientific evidence suggest that TT would be an advantageous addition for clinics and hospitals to include in their armamentarium of complementary interventions within the realm of traditional medicine, TT has not become widely available in the U.S. One reason for the lack of availability may be the dearth of conclusive scientific support for TT's efficacy and, therefore, its inclusion in clinic and hospital treatment planning would give it the appearance of legitimate practice, which it may not yet deserve. Whether or not deserved, if TT were added to hospital and clinic treatment protocols without substantial scientific support, it would be thought to have the implicit support of the scientific community, at which point the question of its efficacy would be moot in the minds of many people; thus patients would utilize it, because they


In this paper we calculate these energy levels from first principles using density functional theory. The wavefunction of the donor electron’s ground state is found to have a form that is similar to an atomic s orbital, with an effective Bohr radius of 1.8 nm. The corresponding binding energy of this state is found to be 41 meV, which is in good agreement with the currently accepted value of 45.59 meV. We also calculate the energies of the excited 1s(T2) and 1s(E) states, finding them to be 32 and 31 meV respectively.The  The phosphorus donor electron has been shown to have long spin coherence times in the laboratory, which make these donors excellent candidates for spin qubits4. Moreover, during the last decade a technique of phosphorus δ doping, based on scanning tunneling microscope (STM) lithography, has led to a variety of new electronic devices in silicon6. This δ doping technique has been used to make a quantum dot of seven donors7 and a transistor with a gate island that consists of only one phosphorus donor3. Another novel electronic device is the quantized electron pump of Tettamanzi et al.8, which demonstrates charge pumping of single electrons through a phosphorus donor. Finally, the wavefunction of the donor electron has even recently been imaged using an STM9. These images have been analysed using tight binding10 and effective mass theory11; whilst the latter provides a qualitative description, the tight binding method is precise enough to pinpoint the atomic position of a single phosphorus donor in the silicon lattice. Although semi-empirical approaches have successfully been used to model the properties of these donor devices, a full ab initio treatment of the electronic structure of these donors has to-date not been possible. Here we present such a treatment.Phosphorus donors in silicon have long been important for electronic devices but are now seen as central to the development of silicon based quantum information processing13, resulting in three nondegenerate states. These states are, in order of increasing energy, a singlet [1s(A1)], a triplet [1s(T2)], and a doublet [1s(E)]14. Only the ground state [1s(A1)] is populated14 at liquid helium temperatures (~4K), whereas at higher temperatures (≥30 K) the populations of the excited 1s(T2) and 1s(E) states become observable due to thermal broadening15.At low doping densities it is well known that the phosphorus donor electrons occupy the lowest energy conduction band of silicon. In bulk silicon this band is sixfold degenerate but the degeneracy is lifted by a valley splitting when silicon is doped16. Traditionally, shallow defects in silicon like phosphorus donors could not be treated by ab initio methods because the wavefunctions of such defects are partially delocalized. Today, however, this statement does not hold true, as in the last ten years innovations in modern computing technologies have made much larger computational resources available to scientific research. Recently it has been shown that shallow defects are now within the reach of ab initio methods such as density functional theory (DFT)17.Over a decade ago, theoretical methods for describing point defects in semiconductors were separable into two categories: “methods for deep defects and methods for shallow defects: the former defect class is treated by ab initio methods, … while for the latter class approximate one-electron theories … are used”s manifold states [s manifold states. This is a known problem and, as will be discussed, we correct these energies using the ground state wavefunction, via the method described in ref. s manifold energy levels for a phosphorus donor in silicon.In this paper we calculate the energies of the et al. in 196518. These measurements do not give the binding energy of the donor electrons but rather the energy splitting between the ground and excited states; namely, the energy splitting between the p± states. The binding energy of the phosphorus donor electron that is reported in ref. p± state12 to the energy of the transition 19.The Lyman spectrum for Group V donors in silicon was first measured by Aggarwal 20. For the phosphorus donor electron the binding energies of the 3p± and 20. The theoretically calculated binding energy of the excited 3p± state is in good agreement with experiment, whereas the binding energy of the 20. Later, in 1981, using the theoretical correction of Faulkner20 and a new experimental technique that produced narrower linewidths in the excitation spectra, Jagannath et al.19 reported a binding energy of 45.59 meV for the phosphorus donor electron. Finally, the charging energy of a single phosphorus donor in the presence of two densely-doped, phosphorus leads was recently found to be 47 ± 3 meV in experiment. This value agrees, within experimental uncertainty, to the earlier optical measurements19.In 1969, Faulkner used effective mass theory (EMT) to calculate the energy levels of the ground and excited states of a donor electron for Group V donor atoms in silicons manifold states21. In addition, a model for a phosphorus donor in silicon that goes “beyond effective mass theory” has been introduced22. In ref. s manifold of states. This study shows that the binding energy is also an important quantity for theoretical modelling. The same fact is highlighted by ref. 23. The truncation of the Coulomb potential was found by adjusting a free parameter “to obtain the experimental ground state energy of 45.6 meV”23.More recently it has been demonstrated that EMT, with effective potentials calculated from ab initio methods, is capable of reproducing the accepted values for the binding energies of the 24. EMT has been shown to be capable of reproducing the wavefunction of a phosphorus donor electron that is predicted by tight binding theory25. The results in ref. 25.The binding energies of Group V donors in silicon have also been used as input parameters to modelling of the hyperfine Stark effect with EMT26. However, as we will show, these latter system sizes are not large enough to isolate the phosphorus donor electron from its periodic images. The confinement of the donor electron is thereby increased, which artificially raises the binding energy of the electron. The binding energy of the phosphorus donor electron was therefore unable to be reported in ref. 16. However, this study was also not able to report meaningful binding energies for the donor electrons because the long-range tail of the impurity potential was ignored16.The first large-scale atomic simulations performed on a Group V donor in silicon using DFT were those presented in ref. 33. For more information on this method and its benchmarking see Appendices A and B. We have employed the SIESTA package35 to carry out calculations on cubic supercells that range in size up to 10,648 atoms.In this paper we present the results of electronic structure calculations performed on a single phosphorus donor in silicon with DFT. This approach has previously been benchmarked in a number of other studiess orbital. The corresponding probability density can be seen to decay to approximately 2% of its maximum value at a distance of ±1.5 nm from the donor site in the We have calculated the wavefunction, mentclass2pt{minimmentclass2pt{minimA is a normalisation constant and F(r) is radially symmetric and The apparent hydrogenic character of the donor electron’s wavefunction is compatible with an effective Bohr model of the electron. Phosphorus is a shallow defect in silicon so it is reasonable to treat the wavefunction of the Kohn-Sham eigenvalue, calculated within DFT, as an independent single particle state that can be modelled by a simple exponential function. The wavefunction of the donor electron can therefore be described by the envelope function ψ(r)|2), plotted against radial distance from the donor site, r. The domain in this figure includes the core region of the phosphorus atom, which in our model is described by a Troullier-Martins pseudopotential36. A pseudopotential will deviate from a Coulombic potential in the core region. The envelope function is not applicable within the core region because a hydrogenic wavefunction is not a valid solution here. We have therefore fitted the wavefunction of the donor electron on the domain R is termed the model radius. The model radius must be chosen such that the effects of the core region on the wavefunction do not influence the accuracy of the exponential fit. Nor can the model radius be so large that the whole of the wavefunction’s exponential decay is not captured by the fit. We have set the model radius equal to the atomic nearest neighbour distance, which has a value of 0.235 nm in silicon37. As can be seen from Fig. Figure In EMT, it is possible to derive two Bohr radii for the donor electron: one corresponds to the longitudinal effective mass, mentclass2pt{minims manifold energy levels for the phosphorus donor electron are shown in Fig. The 39. It is therefore incorrect to calculate the binding energy of the donor electron by taking the difference of this energy and the CBM of bulk silicon. We now need another way to calculate the binding energies of the s manifold energy levels. This is provided by the method described in ref. The binding energies of the 17;V′(q) is the Fourier transform of the unscreened impurity potential. The dielectric screening is described by a nonlinear function41;A = 1.175, α = 0.7572, β = 0.3123, γ = 2.044, and the relative permittivity of silicon A, α, β, and γ were found by fitting the above function to the q dependent dielectric screening in silicon, which was calculated from the random phase approximation41. The calculation of the binding energy therefore relies on the assumption that the screening of the impurity potential is well-described in the random phase approximation, within which dynamical effects on this screening are ignored. The kinetic and potential energies of the donor electron can then be computed;The method of ref. s manifold states calculated using experiment, EMT, and DFT. The binding energy of the 19 and a theoretical correction20. In addition, we find the binding energies of the excited Table mentclass2pt{minims manifold energy levels for a single phosphorus donor in silicon.In summary, we have calculated the wavefunction of a phosphorus donor electron in silicon with DFT. This wavefunction is then used to compute the effective Bohr radius of the donor electron. We employ a hydrogenic model of this electron and thereby find its Bohr radius to be 1.8 nm. In addition, we compute the binding energy of the donor electron’s ground state, which is found to be in good agreement with the currently accepted value. The energies of the excited 10. The effect of a surface on the donor’s properties cannot be studied using a one-electron theory, like EMT, because these theories implicitly assume the defect is surrounded by a homogeneous bulk. In addition our ab initio model could easily be extended to investigate many donor systems, which are also of current interest42.In the future this atomistic model could be expanded beyond the bulk case, to investigate the effect of a nearby surface on the electronic and structural properties of the phosphorus donor. Recent experiments on single donor atoms are commonly performed in the presence of such an interface35. Table 43. Application of the GGA to phosphorus-doped silicon systems in the past has produced results that are in good agreement with experiment44. The total energies of each of the supercells were converged to within 0.1 meV using a planewave energy cutoff of 300 Ry and a Fermi-Dirac occupation function at a temperature of 0 K. Atomic potentials were described by norm-conserving Troullier-Martins pseudopotentials36.The electronic structure calculations were performed with density functional theory (DFT) using the siesta package28. The basis set was double-δ doped monolayer in silicon. Despite the higher precision of the planewave basis, the double-30.We have variationally solved the Kohn-Sham equations using a basis set of localised atomic orbitals that was optimised for phosphorus-doped silicon using the simplex method45. The overestimation of the lattice constant by approximately 0.5% is lower than the usual systematic deviation of the lattice constant that is expected from the PBE XC functional, which is a 0.7% deviation46.We relaxed the crystallographic structure of bulk silicon using this basis set and found the lattice constant to be 5.4575 Å. This value is in good agreement with the experimental value of 5.431 Åk point grid that contains only a single k point: the k = . For the supercell of 10,648 atoms, an increase in the size of the k point grid would result in these calculations being computationally impractical. When the number of k points is increased up to 8 × 8 × 8 for the supercell of 512 atoms, we find the eigenvalue of the 17.To reduce the computational expense of performing these electronic structure calculations, we have used a We geometrically optimised the ionic positions of supercells that ranged in size from 64 to 4096 atoms. We found the maximum displacement of a silicon atom was largest for the supercell of 64 atoms. When the size of the supercells is increased up to 4096 atoms the maximum displacement decreased to less than 0.02 Å. This displacement is equivalent to less than 0.5% of the lattice constant of bulk silicon. We therefore conclude it is unnecessary to relax the ionic positions of silicon atoms beyond their bulk values for supercells larger than 4096 atoms.k| = 0, not The conduction band minimum (CBM) of silicon is located at 30. Consequently, the amount by which the energies of the The size of the Brillouin zone is decreased when the size of the supercell is increased. Decreasing the size of the Brillouin zone causes the bands, and therefore CBM, to be folded towards the centre of the zone, i.e. the kx axis, in Fig. k point grid of 6 × 6 × 6. For the sake of clarity, we do not show the part of the bands that are reflected back into the Brillouin zone at the zone boundary. Neither do we show the conduction valleys of supercells with more than 4096 atoms in Fig. 30.The value of The energy of the lowest conduction valley of bulk silicon at the k point grids that were used to calculate the quantity We find the CBM for each of the supercells do not agree when the energies are corrected for band folding only. We also need to account for the differences in the valence band maximum (VBM) of each supercell. The conduction band minima are shifted by the difference between the VBM of each supercell and the VBM of the supercell containing 10,648 atoms. Once this is done, the CBM in Fig. In this section, we give the mathematical details of the calculation of the binding energy for the donor electron in full. This method was first proposed in ref. T is the kinetic energy and U is the potential energy of the donor electron. In , thenl = 0 component of the norm-conserving pseudopotentials for phosphorus and silicon when evaluating Eq. V′(q) is the Fourier transform of the unscreened impurity potential. The dielectric screening is described by a nonlinear function41A = 1.175, α = 0.7572, β = 0.3123, γ = 2.044, and A, α, β, and γ were found by fitting the above function to the q dependent dielectric screening in silicon, which was calculated from the random phase approximation41. We can then use (The binding energy of the donor electron is given bymentclasspt{minimamentclass2pt{minimThe binding energies of the donor electron’s ground state, calculated using supercells of 216 to 10,648 atoms, are shown in Fig.


Pancreatic groove cancer is very rare and can be indistinguishable from groove pancreatitis. This study is to clarify the characteristics, clinical features, managements, and survival outcomes of this rare tumor.Brief descriptions were made for each case of pancreatic groove cancer encountered at our institute. Individualized data of pancreatic groove cancer cases described in the literature were extracted and added to our database to expand the study sample size for a more complete analysis.A total of 33 patients with pancreatic groove cancer were included for analysis, including 4 cases from our institute. The median tumor size was 2.7 cm. The most common symptom was nausea or vomiting (89%), followed by jaundice (67%). Duodenal stenosis was noted by endoscopy in 96% of patients. The histopathological examination revealed well differentiated tumor in 43%. Perineural invasion was noted in 90%, and lymphovascular invasion and lymph node involvement in 83%. Overall 1-year survival rate was 93.3%, and 3- or 5-year survival rate was 62.2%, with a median survival of 11.0 months. Survival outcome for the well-differentiated tumors was better than those of the moderate/poorly differentiated ones.Early involvement of duodenum causing vomiting is often the initial presentation, but obstructive jaundice does not always happen until the disease progresses. Tumor differentiation is a prognostic factor for survival outcome. The possibility of pancreatic groove cancer should be carefully excluded before making the diagnosis of groove pancreatitis for any questionable case. Because of its rarity and limited clinical experiences, differentiation of pancreatic cancer from groove pancreatitis might be difficult in clinical diagnosis, and, moreover, groove pancreatitis can sometimes coexist with pancreatic cancer or may even mask the underlying problem.,5,7–9The definitive diagnosis is frequently only obtained by histopathological examination after surgical intervention.Pancreatic groove cancer refers to the pancreatic cancer located in an anatomic area between the head of the pancreas, the duodenum and the common bile duct.The purposes of this article are to present our clinical experience with groove pancreatic cancer and to analyze an expanded sample size by adding cases from the literature to our pool of study cases. Thus, an attempt is made to clarify the characteristics, clinical features, managements, and survival outcomes of this rare tumor.2–5,7,9–12 Cases without individualized data and duplicate cases reported in literature were excluded from the analysis. The data pool from the related literature and our patients’ cases were analyzed to determine the characteristics of pancreatic groove cancer including demographics, tumor size, clinical presentations, histopathology, treatment, and survival outcomes.Brief descriptions were made for each case of pancreatic groove cancer encountered at our institute between 2012 and 2016. This study was approved by our institutional review board, and appropriate informed consent was obtained for each patient. To clarify the characteristics of pancreatic groove cancer, individualized data of pancreatic groove cancer cases described in the literature were extracted and added to our database to expand the study sample size for a more complete analysis. Two methods were utilized to search for relevant cases in the literature. First, to identify the relevant articles dealing with pancreatic groove cancer in the literature, a computerized search was performed through the PubMed electronic database, covering data from 2003 to 2015. The following keywords were used for the PubMed search: pancreas groove cancer, groove cancer, and pancreas groove adenocarcinoma. Second, the reference lists of PubMed-selected pancreas groove cancer articles were screened systematically for additional studies of interest. A total of 10 related articles were selected for the study.P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 21.0 software . All continuous data were calculated using median and mean ± standard deviation (SD), and frequency, as appropriate to the type of data. Actuarial survival was estimated via the Kaplan–Meier method, and a log rank test was used to determine difference in the subgroups. 3There were 4 cases of pancreatic groove cancer in our institute, and 3 of them presented nausea/vomiting due to duodenal obstruction Table . One patA total of 33 patients with pancreatic groove cancer were included for analysis after adding cases from the literature to our pool of study cases Table . It coulMalignancy was proved by endoscopic biopsy for pancreatic groove cancer in 58% and by intraoperative biopsy in 50%. Resections of the pancreatic groove cancer with pancreaticoduodenectomy were achieved in 76%, and 24% underwent bypass surgery. The histopathological examination revealed well differentiation of the tumor cells in 43% and moderate/poor in 58%. Perineural invasion was noted in 90%, and lymphovascular invasion and lymph node involvement in 83% , instead of jaundice (67%). In contrast, duodenal obstruction in periampullary cancer is often a late presentation and sometimes means a preterminal event at the advanced stage that causes nausea, vomiting, and ultimately cachexia symptoms.Like the usual pancreatic ductal adenocarcinoma, pancreatic groove cancer also appears in the sixth or seventh decade with less incidence of alcohol abuse.,9Radiologically, T1-weighted and fat-suppressed T1-weighted MRI could show hypointensity, and T2-weighted images could be hyper- or isointensity. Dynamic computerized tomography (CT) and MRI could reveal hypovascularity in the early phase and delayed enhancement in the late phase of the lesion. However, these imaging findings can also be revealed in groove pancreatitis. The distinction between pancreatic groove cancer and fibrous scar in groove pancreatitis is often difficult on CT and MRI studies. claimed that pancreatic groove cancer could infiltrate the duodenal submucosal layer histopathologically with circumferential pattern. Cancer cells could be found only in the mucosal layer at the early stage, and common bile duct could only be involved at the advanced stage. The feature of invasion of pancreatic groove cancer into the submucosal layer around the wall might explain that although duodenal stenosis was noted in 96% of patients, 38% of the duodenal mucosa could still look normal. Malignancy could be preoperatively confirmed by endoscopic biopsy in 58%. Our findings seemed to be in agreement with Aimoto report. Therefore, pathological findings of endoscopic biopsy specimens might show no evidence of malignancy at the early stage.Aimoto et al,3 Nevertheless, some features might tell the difference. Groove pancreatitis appears as a sheet-like mass, whereas pancreatic cancer manifests as an irregular mass. Stenosis of the distal common bile duct is smooth and long in groove pancreatitis, but abrupt and short in pancreatic cancer. Cystic dystrophy of the duodenal wall is more commonly seen in the duodenal wall in groove pancreatitis than in that of pancreatic cancer. However, arteries in pancreatic head lesions are often encased in pancreatic cancer but rarely in groove pancreatitis.,14 Nevertheless, it is vital to include the differential diagnosis of pancreatic groove cancer in any questionable case, before making the diagnosis of groove pancreatitis. The possibility of pancreatic groove cancer should be carefully excluded.It is crucial but challenging to differentiate pancreatic groove cancer from groove pancreatitis. Usually groove pancreatitis can be treated by conservative measures, and surgical resection should be reserved for patients with severe symptoms or in order to rule out malignancy. Similar to pancreatic groove cancer, patients with groove pancreatitis also frequently present with vomiting due to duodenal stenosis and obstructive jaundice due to common bile duct stenosis.Although a very high positive rate of perineural invasion (90%), lymphovascular invasion (83%), and lymph node involvement (83%) for pancreatic groove cancer is noted in this study, the overall 1-year survival is 93.3% and 5-year survival 62.2%. However, with limited case number and short follow-up period, it is still hard to conclude that pancreatic groove cancer is better than that of pancreatic ductal adenocarcinoma. Tumor differentiation is a prognostic factor, and the well-differentiated pancreatic groove cancer can be associated with a favorable survival outcome, with 100% 1- and 5-year survival rates.In conclusion, nausea/vomiting resulting from early involvement of duodenum is often an initial presenting symptom for most cases, but obstructive jaundice is not always inevitable until the disease progresses. Well-differentiated pancreatic groove cancer is associated with favorable survival outcome. Nevertheless, more cases and longer follow-up period are needed to confirm this before solid conclusion is made. Since it is often difficult to differentiate the pancreatic groove cancer from groove pancreatitis on either clinical features or imaging studies, the possibility of pancreatic groove cancer should be carefully excluded before making the diagnosis of groove pancreatitis for any questionable case.


Efforts have been made by the transplant community to expand the deceased donor pool in paediatric liver transplantation (LT). The growing experience on donation after circulatory death (DCD) for adult LT has encouraged its use also in children, albeit in selective cases, opening new perspectives for paediatric patients. Even though there has recently been a slight increase in the number of DCD livers transplanted in children, with satisfactory graft and patient outcomes, the use of DCD grafts in paediatric recipients is still controversial due to morbid outcomes associated with DCD grafts. In this context, recent advances in the optimization of donor support by extracorporeal membrane oxygenation and in the graft preservation by liver machine perfusion could find application in order to expand the donor pool in paediatric LT. In the present study we review the current literature on DCD liver grafts transplanted in children and on the use of extracorporeal donor support and liver perfusion machines in paediatrics, with the aim of defining the current status and future perspectives of paediatric LT.  Organ shortage is the main challenge in liver transplantation (LT) and the discrepancy between the number of waiting list patients and potential donor offers is greater in the paediatric field due to the scarcity of size-matched donors/grafts. The paediatric waiting list mortality rate nowadays is about 10%; however mortality appears to be highest in children younger than 6 years . FurtherIn the last few decades, there has been a great interest in donation after circulatory death (DCD), the use of which has rapidly increased . DCD donAlthough the use of DCD livers in adult recipients is increasing, paediatric experience is limited. The use of DCD grafts in paediatric candidates for LT remains controversial and mainly single-center experiences have been reported. Nevertheless, paediatric DCD donors could constitute an important portion of the paediatric donor pool, since withdrawal of life-sustaining therapy accounts for 30–65% of deaths of patients in paediatric intensive therapy units (ITU) . MoreoveRecent advances in the optimization of donor stability with extracorporeal membrane oxygenation (ECMO) and the improvement of organ preservation with perfusion machine might also start a new era in graft availability for children, as these technological advances may help to minimize the morbid complications associated with conventional DCD grafts as well as to expand the safe utilization of extended-criteria DBD. Here we have reviewed the current literature on DCD liver grafts transplanted in children and the use of extracorporeal donor support and liver perfusion machine in paediatric donors. Our aim was to define the current status and future perspectives of paediatric LT. paediatric transplantation, liver transplantation in children, donation after cardiac death, donation after circulatory death, non–heart-beating donors, liver splitting, partial liver graft, reduced liver graft, extracorporeal donor support, extracorporeal membrane oxygenation, organ preservation, normothermic regional perfusion, normothermic machine perfusion, hypothermic machine perfusion, and sub-normothermic machine perfusion. All papers published in English between January 1995 and October 2017 and focusing on the following topics were retrieved: (1) donation after circulatory death used in paediatric LT (recipient age defined <18 years); (2) extracorporeal donor support used for paediatric LT; (3) liver perfusion machine used in paediatric LT; (4) splitting liver during perfusion machine. Studies of nonliver organs were excluded, as were studies where ECMO was used as a support for recipients awaiting LT or after transplantation. The selected papers included randomized controlled trials, conference consensus reports, meta-analyses, review articles, and pilot studies and case reports. Given the heterogeneity of the published papers and data, the present paper is in the form of a conventional review where important aspects of the aforementioned studies are highlighted.Two authors of this manuscript  independently searched the PUBMED/MEDLINE database with the following key words:We identified 48 relevant records on the use of DCD grafts in children, 41 of which were excluded as ineligible for the following reasons: 19 studies included non-LT from DCD donor to paediatric recipients, 18 reports focused on the ethical aspects and the implementation policy of paediatric DCD donation, and 4 studies included LT from paediatric DCD donor to adult recipients; the remaining 7 records, focusing on DCD grafts used in paediatric LT recipients, were included in the review –24.Regarding the use of ECMO in paediatric LT, 19 records were identified, 17 of which were excluded because ECMO was used as bridge therapy while awaiting LT or as a support for complications after LT . Therefore, only 2 records on the use of ECMO in liver donors for paediatric recipients were eligible , 26.Out of 10 papers selected on the use of liver machine perfusion in human LT, none included paediatric recipients; therefore all of them were excluded. Finally, since split liver represents the most common type of graft used in paediatric LT, we considered 2 reports on the split liver procedure during machine perfusion, even if the grafts were not transplanted, in order to open new prospective on the machine perfusion applicability in children , 28.Donation after circulatory death (DCD) involves donors with permanent absence of respiration and circulation, which have been defined by the Maastricht classification of uncontrolled DCD donors  and controlled DCD donors  . Due to Despite the growing experience with DCD grafts in adult LT in recent years, the limitation of the use of DCD livers in paediatric LT recipients might be related to the inferior graft outcomes and higher complication rates reported in adults as well as the scarcity of DCD donors with adequate size-match. In the United Network of Organ Sharing (UNOS) database, the use of paediatric DCD donors represented 0.4% of the whole paediatric donor pool in 1995–2005, this figure increasing to 8–10% in 2006–2016 , 1817, 1p = 0.3). The authors concluded that paediatric recipients of DCD organs appeared to have results similar to those of patients transplanted with DBD organs, but the small size of the population was insufficient to draw solid conclusions.In 2006, a UNOS report on DCD paediatric transplantation described the largest experience till then of DCD liver grafts in children . Out of Later, the Birmingham group described in different series 7 children transplanted with DCD liver grafts from paediatric and young adult donors –22. In 2 in vivo into the biliary tree during organ procurement and tissue plasminogen activator was infused into the donor hepatic artery at the back table. These data suggest that the good outcomes were also possibly related to the large experience of the center in adult DCD transplantation, leading to technical refinements of DCD organ retrieval and optimization of logistic planning to minimize the cold ischemia time.In 2010, Hong et al. from the University of California Los Angeles (UCLA) compared the long-term outcome of 7 children transplanted with DCD paediatric livers to that of 21 children transplanted with DBD paediatric grafts . At 10-yFinally, a recent report from van Rijn et al. described the long-term outcome of 20 paediatric DCD liver grafts, 3 of which (15%) were transplanted in children . In thisAs recently Schlegel et al.  reportedIn summary, 50 DCD liver grafts transplanted in paediatric recipients have been reported so far. The DCD donors were all classified as Maastricht category III with a median donor age of 10 years (0.3 months–64 years) and a median donor WIT of 15 10–25) minutes. Notably, the donor WIT was defined in 5 reports –23, 25 a minutes.n = 1; 2%), ischemic cholangiopathy , anastomotic biliary stricture , hepatic artery thrombosis , and portal vein thrombosis . Despite the small numbers, altogether these findings suggest that children transplanted with DCD livers present satisfactory graft and patient outcomes at medium-to-long-term follow-up, with different features when compared to adult DCD LT. In particular, the incidence of ischemic cholangiopathy seems to be significantly lower in children than in adult DCD recipients, which may reflect the superselective nature of the grafts [Except for one (2%) child who died, all recipients were reported to be alive with a median follow-up of 4.7 years (1.7 months–10 years). The retransplantation rate was 6%. A total of 8 (16%) recipients presented postoperative complications including PNF  is used to provide cardiac and pulmonary support when heart perfusion and lung oxygenation are inadequate to sustain life .The use of ECMO in organ donation has been implemented in the last decades worldwide, and it is currently used in hemodynamically unstable DBD donors or in DCD donors to improve organ preservation for transplantation and to expand the donor pool , 38. AftIn paediatrics, the use of grafts from donors supported by ECMO prior to organ procurement has been reported primarily in kidney transplantation , 47, whiRecently, Assalino et al. reported two cases of in situ liver splitting in DBD young donors  in whom refractory cardiac arrest and hemodynamic instability were supported by venoarterial ECMO; after the in situ liver splitting, two children were transplanted with a left lateral segment graft and two adults with an extended-right lobe graft with uneventful postoperative course . AlthougIn recent years, there has been increasing interest in the possible use of liver machine perfusion to minimize the effects of organ preservation and to resuscitate suboptimal donor grafts. Recent trials have shown that normothermic or hypothermic machine perfusion of liver grafts is associated with short-term superiority over cold storage in terms of early biochemical liver function (lower peak serum aspartate aminotransferase), decrease of early graft dysfunction, and reduced rate of discarded organ , 48, 49.Recently, normothermic machine perfusion (NMP) showed a useful role in reducing ischemia/reperfusion liver damage and in “resuscitating” organs from extended-criteria donors such as DCD . NMP in The application of extracorporeal liver perfusion in children has not yet been explored probably because paediatric LT recipients are usually transplanted with “optimal grafts” deriving from (a) split livers, which are normally obtained from “ideal donors”  or (b) whole grafts harvested from young size-matched donors. Therefore, the use of machine perfusion to improve the “graft quality” might not be considered necessary in the paediatric transplantation setting.So far, the largest application of NMP is in DCD grafts, but no cases of DCD livers perfused on NMP have been reported in children. Thus, what is not documented in the literature is the potential paediatric donor offers as DCD grafts, even from very young and small donors, whose use could be increased and safer preservation of these organs could be optimized by machine perfusion.Recently, there have been some reports on the technical feasibility of splitting during liver machine perfusion , 28. BroLater, four pig livers were split during NMP in order to verify the effectiveness and stability of NMP system . During These two reports proved the technical feasibility of splitting during liver machine perfusion and could lead to new perspectives in the application of NMP in paediatric LT. Though not proven by clinical outcomes, these options open up new possibilities for paediatric patients where even marginal liver grafts that are well preserved could be used for small children. How far could we go? Potentially, splitting the liver during NMP could combine the advantages of in situ and ex situ splitting techniques by reducing prolonged CIT and minimizing the ischemia-reperfusion injury to the partial grafts, influencing split liver outcomes . FurtherGreat efforts have been made by the transplant community to expand the donor pool for paediatric LT. The recent advances in transplantation of extended-donor criteria grafts in adult recipients have led to the selective use of these grafts also in children, opening new perspectives for paediatric LT. In the last few years, an increasing number of DCD grafts have been transplanted in children, with satisfactory graft and patient outcomes at medium and long terms and a low incidence of morbidity. A careful donor selection and short donor WIT and CIT are all mandatory for good outcomes of DCD grafts in paediatric candidates, and any additional donor risk factor should be avoided. The use of DCD liver grafts in children could be increased by the donor support with ECMO to optimize the graft quality.In paediatric LT like for adults, graft preservation on machine perfusion could permit assessing the viability of the organ and reducing the ischemia-reperfusion injury. Moreover, the technical feasibility of splitting liver during NMP could lead to new applications: splitting livers during machine perfusion could increase the number of partial grafts available for paediatric recipients and change the logistics of splitting. The use of new technologies for donor optimization and graft preservation could find selected applications in paediatric LT, but much work still needs to be done.


To evaluate the relationships of brain iron and heme with the inflammatoryresponse of the systemic and central nervous systems and to investigate therole of defensive systems against the toxicity of iron and heme in thecentral nervous system.We assessed a prospective cohort of patients presenting with intracerebraland subarachnoid hemorrhage. We assayed plasma and cerebrospinal fluidsamples for the presence of iron, heme, hemopexin, haptoglobin, enolase,S100-β and cytokines for the first three days following hemorrhagicstroke. We also analyzed the dynamic changes in these components within bothfluids and their relationship with early mortality rates.Hemopexin and haptoglobin concentrations were nearly negligible in the brainafter intracerebral and subarachnoid hemorrhage. Cerebrospinal fluid ironand heme concentrations correlated with a pro-inflammatory response in thecentral nervous system, and plasmatic and cerebrospinal fluid inflammatoryprofiles on the third day after hemorrhagic stroke were related to earlymortality rates. Interleukin 4 levels within the cerebrospinal fluid duringthe first 24 hours after hemorrhagic stroke were found to be higher insurvivors than in non-survivors.Iron and heme are associated with a pro-inflammatory response in the centralnervous system following hemorrhagic stroke, and protections againsthemoglobin and heme are lacking within the human brain. Patient inflammatoryprofiles were associated with a poorer prognosis, and localanti-inflammatory responses appeared to have a protective role. In experimentalstudies, it is well known that iron promotes redox imbalance by mediating freeradical generation and reducing antioxidantdefenses. Iron has also been found to prevent DNArepair, augment glutamate release, and amplify inflammatoryresponses within the brain. In human studies, ferritin levels were correlatedwith poorer prognoses in intracerebral hemorrhage (ICH)patients.Inflammatory response is a well-documented consequence of brainhemorrhage, to proteins such as albumin andhemopexin (Hx). Free heme has been related to an increased mortalityin sepsis and to the severe systemic manifestations ofmalaria. Free heme can also stimulate a pro-oxidantreaction,13 and can augment inflammatory reactions by directlystimulating toll-like receptor 4 (TLR4). Neurons were found to be moresensitive to the toxic effects of heme than astrocytes, which contributes toperpetuating brain injury.Once released from hemoglobin polypeptide chains, heme binds to many extracellularcomponents, from phospholipid membranes,17Haptoglobin (Hp) and Hx are plasma proteins synthesized by the liver that function bybinding free hemoglobin and heme that have been released during intravascularhemolysis, thereby removing them from circulation. Some evidence suggests that Hpand Hx are involved in protecting the brain against injury following ICH. FollowingICH, hypohaptoglobinemic and Hx-knockout mice present with higher neurologicaldeficits and reduced striatal cell viability, respectively.centralnervous system (CNS)-based mechanisms of protection against iron- andheme-induced damage and the relationships among inflammatory and blood metabolismparameters with regard to early mortality rates.Despite ample experimental inquiry, the roles of iron and heme in the pathophysiologyof human brain injury after hemorrhagic stroke, as well as the potential protectivemechanisms provided by Hp and Hx, are not fully understood. In this study, we aimedto evaluate the role of blood-derived products in the pathophysiology of braininjury following brain hemorrhage. We also strove to clarify the Hospital Copa D'Or, Hospital Quinta D'Or and ComplexoHospitalar de Niterói. In all cases, written informed consentwas obtained from the patient or a surrogate.Approval for the study was obtained from the local Ethics Committees ofHospital Copa D'Or, Hospital QuintaD'Or or Complexo Hospitalar de Niterói  were included. The eligibility criterion was spontaneous ICH or SAHwith both intraventricular hemorrhage and external ventricular device insertionwithin 24 hours following the onset of symptoms. Exclusion criteria were as follows:aged less than 18 years, diagnosed with pregnancy, or expected to survive less than48 hours after admission.Fifteen patients with CT-documented ICH or subarachnoid hemorrhage (SAH) admitted tothe neurocritical care units at Demographic information of each patient was recorded upon admission. The severity ofillness in the SAH patients was assessed by calculating the Simplified AcutePhysiology Score (SAPS) II, as well as the Glasgow Coma, Hunt-Hess and Fisherscales. In the ICH patients, hematoma volume was used to assess illness severity.Clinical information  and laboratory results  were recorded sequentially. The primaryoutcome was 7-day mortality.Blood and cerebrospinal fluid (CSF) were collected at 24, 48 and 72 hours followingintensive care unit (ICU) admission. Blood was collected from either an arterialline or a peripheral vein, and CSF was collected from the external ventriculardevice. Blood and CSF samples were assayed for cytokines, iron, heme, Hx, Hp,S100-β and enolase concentrations. We also determined the levels of CRP-t,d-dimer, fibrinogen, prothrombin and prothrombin total time in the blood, as well asthe cell count, glucose and protein concentration in the CSF.Plasma and CSF samples were centrifuged at 800g for 15 minutes at 4ºC, and thesupernatant was aliquoted and stored at -70ºC until analysis. A multiplex cytokinekit  was used according to the manufacturer'sinstructions . Only the cytokines that were recoverablefrom more than 70% of the samples were analyzed. Briefly, iron is simultaneously released fromprotein and reduced by hydrochloric-thioglycolic acid. The ferrous dialysate reactswith buffered ferrozene, a monosodium salt of3-(2-pyridyl)-5,6-bis-(4-phenylsulfonic acid), at a controlled pH and is thenmeasured colorimetrically at 560nm. Total heme levels were measured using achromogenic assay , which utilizes peroxidaseactivity in the presence of heme to convert a colorless probe to a strongly colored(λ = 570nm) compound. Trace amounts of heme can be quantified in the 5 -160pg (10 - 250fmol) range.Iron was measured using a colorimetric assay previously described byCarter.Hemopexin, Hp, enolase and S100-β concentrations were measured using acommercial enzyme-linked immunosorbent assay (ELISA) kit according to themanufacturer's instructions . In this assay, any Hx, Hp,enolase or S100-β present in the samples reacts with their respectiveantibodies, which have been adsorbed to the surface of the polystyrene microtiterwells. Then, these antibodies conjugated with horseradish peroxidase are added. Theenzyme bound to the immunosorbent is then assayed with the addition of a chromogenicsubstrate, 3,3',5,5'-tetramethylbenzidine, and measured at 450 nm.2 and Fisher's exact test . Spearman analysis was employed to detect correlationsamong continuous variables.Statistical analyses were performed using Statistical Package for Social Science(SPSS) for Windows 17.0  and GraphPad Prism version6.0 for Mac . Numeric variables areexpressed as the median values (interquartile range) and were assessed using theMann-Whitney U-test and the Kruskal-Wallis test. Dichotomous variables wereanalyzed using χOf the 15 patients included in this study, 6 (40%) died within the first 7 days afterICU admission. All patients were on mechanical ventilation upon admission to theICU, and 11 (73.3%) were using vasoactive amines .We measured the concentrations of iron, heme, Hx and Hp in the plasmatic and CSFcompartments throughout the first 3 days following hemorrhagic stroke.Interestingly, Hx and Hp levels were almost undetectable within the CSF andsignificantly lower than in the plasma during the first three days following theevent. Furthermore, their concentrations did not increase during the early phaseof hemorrhagic stroke. These findings suggest that these protection mechanismsagainst hemoglobin and heme are deficient within the brain parenchyma .versus 74.85 versus 94.4mg/dL; p = 0.02).Concentrations of heme, Hx and Hp during the first three days after the eventremained stable .Interestingly, there was a strong correlation between the CSF concentrations ofiron at 48 hours after the ictus and the CSF IP-10 levels at 72 hours after theevent .There was a strong correlation between the CSF levels of heme during the first 24hours after the ictus and the MIP-1b concentration at 48 hours after hemorrhagicstroke . The CSF heme concentration in the first 48 hoursafter the event was also negatively correlated to the CSF MCP-1 levels at 72hours after the ictus . Combined, these data suggest thatiron and heme are associated with an inflammatory response within the humanbrain after a hemorrhagic event. We found no correlation between iron or hemeconcentrations and other cytokines throughout the study period.versus 4.85versus 38.06mg/dL; p = 0.02). In parallel, CSF enolaseconcentrations progressively decreased in the first 72 hours after the ictus.These results suggest a preferential death of neurons versusastrocytes, with subsequent antigen spillover from the CSF into the blood.Surprisingly, we found no change in the S100-β concentration kinetics ineither the plasmatic compartment or the CSF.In evaluating the kinetics of brain injury biomarkers, we measured a steadyincrease in plasmatic enolase concentrations during the first three daysfollowing hemorrhagic stroke . This finding suggests that ironoverload may contribute to brain injury and early mortality. There was noobserved difference between survivors and non-survivors regarding Hx or Hpconcentrations throughout the first 3 days after hemorrhagic stroke, in eitherplasma or CSF.In assessing patients over the first 7 days after hemorrhagic stroke, we foundthat plasmatic iron and heme concentrations were higher during the first 48hours following the ictus in non-survivors than in survivors (iron: 496.04Systemic (IL-6 and IL-8) and CNS inflammatory profiles  measured at 72 hours after the event exhibited aconsistent relationship with 7-day mortality rates .versus 26.15pg/mL, p = 0.04; IL-8: 134.8versus 3.83pg/mL; p = 0.). Conversely, localanti-inflammatory responses seem to exert a protective role. In CSF, IL-4 wasfound to be higher in survivors than in non-survivors during the first 24 hoursfollowing hemorrhagic stroke . There were no measurable differences between survivors and non-survivorsamong the remaining cytokines in either plasma or CSF. Surprisingly, there werealso no differences between survivors and non-survivors with respect to enolaseand S100-β concentrations in either plasma or CSF.We analyzed the concentrations of IL-1b, IL-2, IL-6, IL-8, GM-CSF, IP-10, MIP-1a,MIP-1b, IP-10 and RANTES in plasma. For CSF, in addition to these cytokines, weevaluated IL-4 and FGF levels. Three days after the ictus, plasmatic IL-6 andIL-8 were found to be significantly higher in non-survivors than in survivors. Iron andheme serve only as markers of the extension of bleeding. Inflammatory response components, suchas fever and leukocytosis, are markers of increased mortality,,24 and the frequency ofinflammatory response parallels the severity of cerebral insult; it is more commonand with a greater degree of higher-grade radiographic and clinical SAH. Both thesurge in ICP and sympathetic nervous systemactivation may be contributing factors to this strongrelationship between the severity of SAH and the degree of systemic inflammatoryresponse syndrome. In our study, we found that increased concentrations of IL-6 andIL-8 in plasma were related to mortality. However, a local anti-inflammatoryresponse, as shown by increased CSF IL-4 levels, served as a protective factor. Thefinding that CSF IL-4 is related to survival after hemorrhagic stroke agrees withother studies that show a neuroprotective role of anti-inflammatory activityfollowing brain injury.,28The burden of systemic inflammatory response as a factor governing poor prognosis iswell known in hemorrhagic stroke patients. Systemic inflammatory response syndromeis observed in up to one-third of patients with SAH and is related to extracerebralorgan dysfunction and poorer patient outcomes.The limitations of our study include the small sample size and the mixed profile ofpatients with ICH and SAH. Despite this heterogeneity in the root causes of ICH,iron and heme still play a key role in the pathophysiology of both diseases.This study provides preliminary evidence that iron and heme play a role in triggeringinflammatory response in the CNS and that the human brain lacks protectionmechanisms against hemoglobin by-products. Moreover, our study reinforces theconcept that systemic inflammatory response syndrome. is an important determinant ofthe outcome of hemorrhagic stroke patients. More extensive clinical studies of thesebiomarkers will be required to define their mechanistic and prognostic rolesfollowing hemorrhagic stroke.