Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features. Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would provide a critical standard to allow meaningful comparisons with and between neurologic and psychiatric disorders. CSF contains both cellular and soluble components providing insights into processes occurring in the central nervous system (CNS). As much as 30 to 40% of CSF is formed by the extracellular fluid of the brain and spinal cord. CSF contains both normal and disease specific components, and provides an accessible liquid window into the brain. In fact, recent data suggest CSF may provide more relevant evidence for initial or propagating pathology than the brain parenchyma itself in certain neuropsychiatric diseasesUntil recently, technological limitations have prevented full characterization of the CSF proteome. Comprehensive analysis of CSF has been challenged by low protein levels (0.3 to 0.7 mg/ml) compared to plasma, protein concentration variability up to twelve orders of magnitude, potential masking of brain-specific proteins by highly abundant proteinsTowards our principal purpose of establishing a comprehensive list or proteome of normal CSF, we have prepared CSF samples from healthy normal people for analysis by using immunoaffinity depletion of abundant proteins  to enhance coverage and detection of low abundance proteins3 and protein levels ranged from 14 to 40 mg/dl with a median of 25 mg/dl). We also examined pairs of individual serial CSF samples, obtained at least 4 weeks apart, from 10 additional normal healthy volunteers to assess the potential variability of particular CSF protein levels in an individual from one time point to another.We examined pooled CSF from 11 normal healthy volunteers  who reported their health as excellent or good and were taking no medications. Standard clinical laboratory testing on their CSF was normal . Four sets of different types of normal CSF samples were analyzed. The first set, designated as true  normals was comprised of pooled CSF from 11 healthy normal individual volunteers from the United States  was used for the comprehensive analysis using immunoaffinity depletion and 2D-LC-MS/MS. A second set, also true normals included pairs of serial CSF aliquots taken at least 4 weeks apart from 10 healthy volunteers from the United States . A third set, designated as non-neurologic surrogate-normals, was a pool of 200 subjects from Sweden ). Ages ranged from 16 to 65 years with a median of 44 years; 50∶50 female:male. They were used in the direct LC-MS analysis using the AMT tag approach. These samples were collected on ice and cells removed by centrifugationAll specimens had normal clinical laboratory values with respect to microbiology, chemistry (including protein levels), and cell counts  using a 12.7×79.0 mm Seppro® IgY14 LC10 affinity LC column  on an Agilent 1100 series HPLC system , followed the protocols described previouslyA total of 18 mL of the pooled CSF sample  was subjected to the separations of 14 high-abundance proteins  at a trypsin/protein ratio of 1∶50. Sample cleanup was achieved using a 1-mL SPE C18 column  as described previouslyThe CSF proteins were incubated in 8 M urea and 10 mM dithiothreitol at 37°C for 60 min, followed by alkylation with 40 mM iodoacetamide in the dark for 30 min at room temperature. The samples were diluted 10-fold with 50 mM ammonium bicarbonate (pH 8) and 1 mM CaClFor tryptic digests of the IgY14 bound and flow-through fractions , 300 µg of tryptic peptides from CSF samples were resuspended in 300 µL 10 mM ammonium formate, 25% acetonitrile and fractionated by strong cation exchange chromatography as described previouslyThe SCX fractions were analyzed using a custom-built automated four-column high pressure nanocapillary LC system coupled on-line to either a linear ion trap mass spectrometer  or a linear quadrupole ion trap-orbitrap mass spectrometer , both modified in-house with an electrodynamic ion funnelwww.ebi.ac.uk/ipi). The search parameters used were: 3-Da tolerance for precursor ion masses and 1-Da tolerance for fragment ion masses with no enzyme restraint and a maximum of 2 missed tryptic cleavages. Static carboxyamidomethylation of cysteine and dynamic oxidation of methionine were used during the database search. LTQ-Orbitrap MS/MS data were first processed by an in-house software DeconMSnThe LTQ LC-MS/MS raw data were converted into .dta file by Extract_MSn (version 3.0) in Bioworks Cluster 3.2 (Thermo) and the SEQUEST algorithm (version 27 revision 12) was used to independently search all the MS/MS spectra against the human International Protein Index (IPI) database with a total of 69,731 total protein entries  from the CSF study by Zougman et al., reveals a 92% overlap see .In order to assess the CSF protein variability from serial sample collections, we next examined individual (non-pooled) samples from another group of 10 healthy volunteers  who had two CSF samples obtained at least 4 weeks apart using the AMT tag approach. Inter-subject differences were far greater than intra-subject differences . We perfAs an example of how CSF proteomic databases may be used to better understand disease states, we compared the proteomes of two similarly processed see  pooled sThis study provides the most comprehensive CSF protein coverage and list reported to date for healthy normal individuals including serial lumbar punctures. The protein set has immediate utility for investigators interested in using CSF to study neurological or psychiatric diseases. It will serve as a normative base to which disease states may be compared. Our study also suggests CSF protein variability over a short time is relatively limited in an individual. If this observation is supported by larger scale studies, it would further facilitate the utility of disease-state sample comparative analyses.The other major previous investigations of CSF from healthy individuals were published by Zhang et al Because of the challenge in obtaining CSF from healthy people, most previous studies may have used CSF from “surrogate-normals,” that is CSF collected from people with neurological complaints such as headache but with normal clinical CSF laboratory values.We compared proteomes of two different surrogate-normal groups. We found significant differences between the two groups. This study supports the potential usefulness of the normal human CSF proteome data library as an invaluable tool in investigating pathophysiological abnormalities in neurological and psychiatric disorders.Proteomic databases can be used in several ways. One of our own perspectives on using these proteomic databases for studying diseases involves a stepwise strategy. The first step would be a comparison of pooled samples representative of the disease to normal subjects or a comparator disease. The second step involves the selection of specific candidate proteins. The selection of candidate proteins is not likely to be predicted in advance and may require bioinformatic strategies and knowledge related to the disease under study. A third step would involve analysis of the individual samples contributing to the pool to ascertain how many of the samples actually contained one or more of the candidate proteins. This step provides a check in the event that a single individual in the pool disproportionately contributes a protein compared to other subjects. We would subject the results to statistical analyses. In the case of a search for biomarker proteins we strive to select those that meet clinically useful criteria, such as presence, absence or relative abundance in a large percentage of disease subjects and not so in most subjects without the disease under consideration. The fourth step would involve verification of the previous results using independent individual samples with the same disease. A final validation step may involve analyzing a larger number of subjects with the disease and controls using assays targeted to the candidate proteins. In contrast to the discovery phases, it would be advantageous, if feasible, to use assay platforms already having wide clinical use. Immunobased assays such as ELISA and Western blots may serve this purpose being relatively inexpensive. Steps 3 and 4 will likely employ a type of mass spectrometry which targets selected candidate proteins, such as Multiple Reaction Monitoring (MRM) using triple quadrupole instrumentation.The availability of the data presented here, detailing the normal human CSF proteome, should prove to be a critical base on which to compare proteins, both qualitatively and quantitatively, in studies of patients with a variety of neurological or psychiatric diseases.Figure S1Immunoaffinity depletion of plasma and CSF samples using the IgY14 LC10 column.(0.30 MB TIF)Click here for additional data file.Figure S2Comparison of the distributions of the gene ontology terms for all proteins identified from the healthy normal CSF sample and those for the 3654 plasma proteins reported by us previously (Text Reference 21). Biological process.(0.74 MB TIF)Click here for additional data file.Figure S3Comparison of the distributions of the gene ontology terms for all proteins identified from the healthy normal CSF sample and those for the 3654 plasma proteins reported by us previously (text reference 21). Cellular component.(0.85 MB TIF)Click here for additional data file.Figure S4Comparison of the distributions of the gene ontology terms for all proteins identified from the healthy normal CSF sample and those for the 3654 plasma proteins reported by us previously (text reference 21). Molecular function.(0.71 MB TIF)Click here for additional data file.Figure S5Unsupervised hierarchical clustering analysis of 88 proteins found to be present at significantly different levels  comparing serial CSF samples from 10 individuals . Log2 transformed protein abundances were used. M: male; F: female; numbers right after the hyphen indicate the two serial samples from the same individual.(0.31 MB TIF)Click here for additional data file.Figure S6Unsupervised hierarchical clustering analysis of 9 proteins found to be present at significantly different levels  comparing serial CSF samples from 10 individuals . Log2 transformed protein abundances were used. M: male; F: female; numbers right after the hyphen indicate the two serial samples from the same individual.(0.28 MB TIF)Click here for additional data file.Figure S7Unsupervised hierarchical clustering analysis of 2 proteins found to be present at significantly different levels  comparing serial CSF samples from 10 individuals . Log2 transformed protein abundances were used. M: male; F: female; numbers right after the hyphen indicate the two serial samples from the same individual.(0.26 MB TIF)Click here for additional data file.Table S1Peptides detected in CSF from healthy normal individuals using immunoaffinity depletion and 2D-LC-MS/MS.(2.36 MB PDF)Click here for additional data file.Table S2Analysis of overlap between proteins identified in normal CSF, plasma and previous CSF  proteomic study.(0.24 MB PDF)Click here for additional data file.Table S3Proteins identified and quantified from direct LC-MS analysis of CSF from non-neurologic and neurologic (headache) surrogate-normals.(0.02 MB PDF)Click here for additional data file.


To date, a specific marker to evaluate and predict the clinical course or complication of the liver-transplanted patient is not available in clinical practice. Increased procalcitonin (PCT) levels have been found in infectious inflammation; poor organ perfusion and high PCT levels in the cardiac donor appeared to predict early graft failure. We evaluated PCT as a predictor of early graft dysfunction and postoperative complications.PCT serum concentrations were measured in samples collected before organ retrieval from 67 consecutive brain-dead donors and in corresponding recipients from day 0, before liver transplantation, up to day 7 after liver transplantation. The following parameters were recorded in donors: amount of vasopressive drug doses, cardiac arrest history 24 hours before retrieval, number of days in the intensive care unit, age of donor, and infection in donor, and the following parameters were recorded in recipients: cold and warm ischemia time, veno-venous bypass, transfusion amount during orthotopic liver transplantation (OLT), and occurrence of postoperative complication or hepatic dysfunction.In the donor, the preoperative level of PCT was associated with cardiac arrest and high doses of catecholamines before organ retrieval. In the recipient, elevated PCT levels were observed early after OLT, with a peak at day 1 or 2 after OLT, then a decrease until day 7. A postoperative peak of PCT levels was associated neither with preoperative PCT levels in the donor or the recipients nor with hepatic post-OLT dysfunction or other postoperative complications, but with two donor parameters: infection and cardiac arrest.PCT level in the donor and early PCT peak in the recipient are not predictive of post-OLT hepatic dysfunction or other complications. Cardiac arrest and infection in the donor, but not PCT level in the donor, are associated with high post-OLT PCT levels in the recipient. Normall30 hours . The ori30 hours ,6 can beAfter approval by the local ethics committee, all patients admitted for liver transplantation at our institution, Pitié Salpétrière Hospital (Assistance Publique-Hôpitaux de Paris), between July 2003 and March 2005 were prospectively included in the study. The ethical committee waived the need for informed consent because alicots were taken from routine samples. For each recipient, the following were recorded: age, gender, presence of severe portal hypertension, need for veno-venous bypass, number of blood cell transfusions, and PCT serum concentration before anhepatic phase and then 12 hours after reperfusion and daily during the first week after OLT. Postoperative clinical course was analyzed from main clinical data: hepatic dysfunction, pulmonary and renal failure, and overall complications. For each donor, the following were collected: full cadaveric, age, occurrence of cardiac arrest (CA) 24 hours before the retrieval of the organs, occurrence of infection (I), possibility of retrieving the heart with the liver, amount of catecholamine (epinephrine or norepinephrin) administered before organ retrieval, and number of days in the intensive care unit before retrieval. All organs were retrieved and flushed using the same procedure: Wisconsin solution for preservation and 4% human albumin solution for hepatic flush before graft reperfusion.Blood samples were obtained for routine testing , and for each patient, serum aliquots were used for PCT determination. A blood sample from the donor was obtained after installation of the donor in the operating room. An investigator blinded to clinical data used a time-resolved amplified cryptate emission technology on a Kryptor analyser  to measure PCT in 100 μL of serum. The analytic sensitivity of the assay was 0.06 ng/mL and the detection threshold was 0.02 ng/mL; the normal range detected was from 0.1 to 0.5 ng/mL.9/L or less than 4,000 × 109/L or body temperature of greater than 38°C or less than 36°C. Severe portal hypertension in recipients was defined as hepatic venous portal gradient of greater than 20 mm Hg if the patient had a preoperative hepatic transjugular exploration or a decrease in portal output of less than 500 mL/minute, estimated with Doppler ultrasonography. Poor tolerance to vascular liver exclusion was defined as a macroscopic disturbance of bowel coloration or persistence of mean arterial pressure of less than 50 mm Hg and oxygen mixed venous saturation of less than 60% despite fluid loading.Graft dysfunction was defined as the occurrence of at least one of the following four criteria: the need for retransplantation , a rise in aminotransferases of above 2,000 UI/L , the neeAll patients were treated using our standard protocol for immunosuppression: cyclosporine A  or FK 507 (trough concentration of 10 to 15 ng/mL at day 7), prednisolone, starting at 10 mg/kg of body weight on the day of transplantation and reduced to half doses each day to 0.3 mg/kg at day 7, and mycophenolate mofetil (1 g per day) from the day of the liver transplantation. Acute rejection was diagnosed based on clinical and biochemical data and liver biopsy if required. All recipients received broad-spectrum antibiotic treatment with piperacilline-tazobactam for 7 days and ciprofloxacin for 3 days. Cytomegalovirus (CMV) infection was defined by the appearance of CMV antigen polymerase chain reaction in the blood. This measure was performed once a week.P value of less than 0.1 in univariate analysis were included in a multivariable linear regression model, with a stepwise variable selection method. Potential associations between graft dysfunction or overall complications and clinical or biological parameters were tested with univariate procedures, using Mann-Whitney tests for continuous variables and chi-square or Fisher exact tests for categorical variables. The evolution over time of recipient PCT concentration was studied with an analysis of variance for repeated measurements. The multiplicity associated with the comparisons between times was addressed using Scheffe adjustments. The alpha level was set at 0.05. All analyses were performed with the SAS software version 8.2 .Linear regression was used in univariate analysis to identify predictors of donor or recipient PCT concentrations. Predictors with a Sixty-seven patients (19 women and 48 men) were included. Thirty-eight were transplanted because of postviral hepatitis cirrhosis, 19 because of alcoholic cirrhosis, and 10 for other causes. The main characteristics of donors are reported in Table PCT concentration was normal, less than 0.5 ng/mL, in 61 recipients before OLT (D0). Cause of transplantation did not influence PCT level in recipients before total hepatectomy: median 0.1 ng/mL (range 0.1 to 0.8) versus 0.1 ng/mL (range 0.1 to 2) versus 0.2 ng/mL (range 0.1 to 1.1) in cirrhosis from viral, alcoholic, and other causes, respectively. Peak PCT values were observed at D1 or D2. Then, PCT mean concentrations decreased from D2 to D7. Mean values are reported in Figure -/CA-, 16.5 ng/mL (range 0.2 to 91) in 6 patients I-/CA+, 1.8 ng/mL (range 0.7 to 4.7) in 8 patients I+/CA-, and 1.1 ng/mL (range 0.1 to 10.1) in 4 patients I+/CA+. The multivariate analysis did not show any association between donor PCT concentration and OLT hepatic dysfunction or overall complications. The parameters studied for this analysis were donor age, donor PCT level, cold ischemia, warm ischemia, operative transfusion, veno-venous bypass, a high dose of catecholamines in donors, CA and infection in donors, days in intensive care of donor before organ retrieval, and age of recipient. Donor age was the only parameter associated with hepatic dysfunction in univariate analysis (P = 0.03). In this model (adjusted with age), the median levels of PCT were 6.7 ng/mL (range 0.56 to 85) in recipients without hepatic dysfunction and 8.7 ng/mL (range 1.13 to 45) in recipients with hepatic dysfunction. Donor parameters significantly associated in multivariate analysis with donor concentration of PCT were a dose of epinephrine or norepinephrin administered before liver retrieval and occurrence of CA in the 24 hours prior to retrieval  in 49 patients IThis study could not confirm the hypothesis that the donor PCT could be predictive of hepatic dysfunction or post-OLT complications in the recipient. Second, it showed that a peak in PCT in the recipient was associated with the clinical characteristics of donors but not with recipient characteristics and post-OLT complications. Third, our study confirmed the time course of PCT serum concentration in recipients after liver transplantation ,6. CondiOur results were not in agreement with those of Fazakas and colleagues , who fouper se but infection associated with a severe systemic response or poor organ perfusion is thought to induce PCT release [Our study did not find any association in donors between PCT concentration and infection, which is in contrast to the current concept . To asse release . In the  release  exposed  release ,19, makeAlthough some authors demonstrated toxicity of PCT , the phyPCT level in the donor could not be considered as a good predictive marker of hepatic dysfunction or postoperative complication. PCT in the donor was associated with CA but not with infection. Post-OLT PCT peak is associated with infection and CA in donor but not in recipient parameters.• Procalcitonin (PCT) level in the donor and early PCT peak in the recipient are not associated with postorthotopic liver transplantation hepatic dysfunction or other complications.• PCT in the donor is associated with cardiac arrest but not with infection.• High PCT peak levels in recipients are associated with infection and cardiac arrest in donors.CA = cardiac arrest; CI = confidence interval; CMV = cytomegalovirus; I = infection; OLT = orthotopic liver transplantation; PCT = procalcitonin; PNF = primary nonfunction.The authors declare that they have no competing interests.DE conceived, designed, and carried out the study. SBA and MB helped to conceive and carry out the study and helped to perform laboratory analyses. MLT helped to conceive and carry out the study and performed statistical analyses. JMS helped to conceive and carry out the study. CV and SF helped to perform the literature search. MM and J-CV helped to compile the data for the study and helped to perform analyses. PC and LH helped to conceive the study. All authors contributed to the writing of the manuscript and approved of its final version.


The neuroplastic mechanisms exerted by EPO combined with G-CSF included enhanced expression of the antiapoptotic protein of Bcl-2, augmented neurotrophic factors synthesis, and promoted neovascularization. Further, the combination therapy significantly increased homing and differentiation of bone marrow stem cells (BMSCs) and intrinsic neural progenitor cells (INPCs) into the ischemic area. In summary, EPO in combination with G-CSF synergistically enhanced angiogenesis and tissue plasticity in ischemic animal models, leading to greater functional recovery than either agent alone.Stroke and peripheral limb ischemia are serious clinical problems with poor prognosis and limited treatment. The cytokines erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF) have been used to induce endogenous cell repair and angiogenesis. Here, we demonstrated that the combination therapy of EPO and G-CSF exerted synergistic effects on cell survival and functional recovery from cerebral and peripheral limbs ischemia. We observed that even under normoxic conditions, G-CSF activates hypoxia-inducible factor-1α (HIF-1α), which then binds to the EPO promoter and enhances EPO expression. Serum EPO level was significantly increased by G-CSF injection, with the exception of Tg-HIF-1α Erythropoietin (EPO), a 30.4 kDa glycoprotein, acts mainly on late erythroid precursor cells to induce maturation. Exogenous administration of EPO and endogenous upregulation of EPO receptors have been demonstrated to promote neuronal survival in the injured brain Previous reports have described the beneficial effects from exogenous administration of multiple cytokines, such as EPO, G-CSF, stem cell factor (SCF) and IL-11 In this study, we intended to demonstrate that G-CSF upregulates EPO expression via the induction of HIF-1α activity, and that the co-treatment of G-CSF and EPO synergistically enhances neural survival and angiogenesis in both primary cortical cultures and animal models of ischemia.n = 24) were measured by ELISA at 1, 3, 7, and 14 days. We noted an increase in serum EPO level, which the peak level was seen in the 3th days after first G-CSF injection  were treated with G-CSF for different durations , and at different concentrations . HIF-1α and of EPO protein expression increased following G-CSF treatment . In addiThe subcellular location of HIF-1α in HUVECs with or without G-CSF treatment was determined by immunohistochemistry. Without G-CSF treatment, HIF-1α was primarily localized in the cytosol . G-CSF tAccording to results from an electrophoretic mobility shift assay (EMSA) using an 18-bp oligonucleotide probe containing the HRE with the HIF-1α-binding site of the EPO gene promoter , G-CSF aTo examine whether EPO combined with G-CSF exerts a neuroprotective effect through an antiapoptotic pathway, Bcl-2 protein expression, caspase-3 activity and synthesis of neurotrophic factors under OGD were measured in primary cultures of rat cortical cultures (PCCs) with or without co-treatment of EPO and G-CSF. In OGD, PCCs pretreated with EPO+G-CSF for 12 hours showed significantly less caspase-3 activity by fluorimetric methods than EPO alone, G-CSF alone or control groups . Levels n = 8) with different therapeutic recipes . EPO+G-CSF-treated rats showed much higher percentage of recovery in body swing tests than rats treated with EPO only, G-CSF only, or control rats  one week post-treatment (each group n = 8). The results indicate that FDG uptake in the right cortexes of the EPO + G-CSF group was significantly better than that in the EPO, G-CSF or control groups . The anti-apoptotic protein Bcl-2 was significantly upregulated in EPO+G-CSF, EPO, and G-CSF groups, compared with control group  (+f/+f mice (As measured by ELISA 3 days after G-CSF (250 µg/kg) or EPO (5000 U/kg)+G-CSF (250 µg/kg) injection, serum EPO levels in wild-type mice (C57BL/6 mice) increased significantly compared to control mice (each group n = 6) . In cont/+f mice .n = 8). The penumbral region surrounding the ischemic cores of EPO+G-CSF-treated rats contained significantly fewer TUNEL positive cells than the same regions in EPO alone, G-CSF alone, or ischemic control  (Bromodeoxyuridine (BrdU) labeling was used to trace the homing and engraftment of stem cells  to brain tissue at 7 days after cerebral ischemia. According to cumulative BrdU labeling immunohistochemistry, many BrdUboundary  and the boundary . BrdU+ cmisphere . Resultsp n = 8) .+c-Kit+ BMSCs were dispersed over the right striatum, hippocampus and the penumbral area in EPO+G-CSF-treated mice compared to EPO-, G-CSF-, or saline-treated mice (each group n = 8) , were significantly higher than those in the EPO-treated , G-CSF-treated  and control  groups  , G-CSF-treated  and control  groups  . Fractio) groups . In nestp n = 8) . Quantit) groups .+ cells from transgenic GFP-chimeric mice showed vascular phenotypes (vWF+ cells) around the perivascular and endothelial regions (n = 8) (n = 8)  . In addi (n = 8)  show bet (n = 8) .n = 8)  under anesthesia after cerebral ischemia. At one week after cerebral ischemia, CBF was significantly greater in the ischemic cortex of the EPO+G-CSF-treated rats than that in EPO, G-CSF or control (each group n = 8) .+ cells from transgenic GFP-chimeric mice co-expressed vascular phenotypes (vWF+ cells) around the perivascular and endothelial regions of the ischemic limb muscles of EPO+G-CSF-treated mice (n = 8). In evaluation of the blood perfusion of ischemic limbs by LDPI, EPO+G-CSF treatment led to significantly greater recovery of blood perfusion of ischemic limbs than G-CSF, EPO alone or control (each group n = 8)  analysis were performed on both limbs from each experimental mouse. Several GFPted mice . Accordited mice , ischemip n = 8) .Although many reports have noted the synergy between EPO and G-CSF in the treatment of anemia in myelodysplastic syndromes (MDS) HIF-1α is activated by G-CSF in HUVECs under normoxia condition, as evidenced by our observation that G-CSF stimulation leads to enhanced HIF-1α activity and increased DNA-binding activity driven by the HRE. G-CSF is known to enhance the expression of two HIF-1α target genes, VEGF and SDF-1, in human neural stem cell cultures and platelets Cells express many proteins in response to hypoxia and ischemia, which supports cellular adaptation in response to altered metabolic demands and the removal of toxic substances Recombinant EPO and G-CSF have been used clinically for over 20 years In the present study, the bone marrow stem cells (BMSCs) homing and differentiation were significantly enhanced by EPO in combination with G-CSF treatment. Previous investigators have also demonstrated the individual capability of EPO or G-CSF to stimulate proliferation and differentiation of neural stem/progenitor cells inside the animal brain We obtained serum samples from age-matched (Age  = 35–40 y), healthy donors  at sequential time points  after subcutaneous G-CSF administration  for 5 consecutive days according to the manufacture's instruction. All protocols and informed consents procedures were fully reviewed and approved by the Institutional Review Board of China Medical University Hospital. Informed consent was obtained from all participants. As a positive control, known level of EPO protein in serial dilution were measured using a human EPO ELISA kit (R&D Systems), with the results used to plot a standard curve. Each serum sample for EPO concentration measurement was run in triplicate and compared with the standard curve.2 humidified air incubator as previously described Human umbilical vein endothelial cells  were cultured in EGM-2 medium containing human epidermal growth factor, hydrocortisone, human fibroblast growth factor, vascular endothelial growth factor, ascorbic acid, gentamicin, amphotericin-B, human insulin-like growth factor, heparin and 2% FBS at 37°C in a 5% COIn order to evaluate the non-hypoxic activator effect of G-CSF, the levels of activated HIF-1α protein after G-CSF treatment were measured by ELISA. HUVECs were treated with different doses  of G-CSF (Kirin) for 6 hours. Nuclear extracts from these cells were incubated (50 µg/well) with biotinylated double-stranded oligonucleotide containing a consensus HIF-1α binding site from an active HIF-1α DuoSet IC ELISA kit (R&D Systems) according to the manufacturer's instructions. The level of activated HIF-1α was measured by absorbance and was expressed as optical density (OD) at 450 nm–540 nm as previously described HIF-1α and EPO expressions in HUVECs were measured by Western blot and immunohistochemical analyses. HUVECs were treated with 1 µg/mL G-CSF for different times , or treated with different concentrations of G-CSF ranging from 0.01 to 10 µg/mL for 6 hours. Western blot analyses of HIF-1α, and EPO expression from HUVECs were performed after each G-CSF treatment as previously described For HIF-1α and EPO immunostaining, HUVECs were washed with PBS and fixed for 30 minutes at room temperature in 4% paraformaldehyde as previously described 2 and 5% CO2 at 37°C to maintain pO2 of <1 mmHg  To prepare cellular extracts for analysis, HUVECs were harvested and washed twice with cold phosphate-buffered saline (PBS). Nuclear and cytoplasm extracts were prepared using a commercial kit (Pierce). For oxygen glucose deprivation (OGD) treatment, cells were placed in a hypoxic chamber  for 4 hours, continuously flushed with 95% N5′-agcttGCCCTAC GTGCTGTCTCAg-3′ and 5′-aattcTGAGACAGCACGTAGGG Ca-3′) corresponding to the hypoxia-response element (HRE) in the EPO gene promoter Detailed protocols to assess HIF-1α DNA binding activity using EMSA have been described previously Luciferase assays and transient transfection of pEpoEm1-luc and pEpoE-luc reporter plasmids  by Western blot as described previously th week) were used for this study. The rats were anesthetized with chloral hydrate  and subjected to right middle cerebral artery (MCA) ligation and bilateral common carotid artery clamping for 90 mins as previously described Adult male Sprague-Dawley rats  was performed on rats in order to measure the infarct size after treatment of each group as described previously Behavioral assessments were performed 3 days before and 3, 7, 14 and 28 days after cerebral ischemia. The tests measured: (a) body asymmetry (b) locomotor activity, and (c) grip strength as previously described 18F]fluoro-2-deoxyglucose (FDG) to measure relative metabolic activity by a protocol previously described Experimental rats were examined using microPET scanning of [For Western blot analysis of apoptosis-related protein expression, experimental rats were anesthetized with chloral hydrate  at 24 hours after initiation of each treatment. Expression of apoptosis-related proteins  in the right cortex and striatum region was also examined in each cytokine-treated and control rat +f/+f mice  were used to measure EPO levels in blood samples 1, 3, 7, and 14 d following treatment with G-CSF or G-CSF plus EPO in wild type (C57BL/6) and TgCAGGCreERTM-HIF-1αCellular apoptosis was assayed by histochemistry using a commercial TUNEL staining kit  as previously described BrdU (Sigma), a thymidine analog that is incorporated into the DNA of dividing cells during S-phase, was used to label cells undergoing mitotic division. Labeling protocol and BrdU immunostaining procedure have been described previously In order to verify the enhancement of bone marrow stem cell (BMSC) and intrinsic neural progenitor cell (INPC) mobilization and homing into the infarcted brain following one of four treatments , transgenic GFP-chimeric mice and nestin-GFP mice were built as previously reported + cells was examined by double immunofluorescence using laser scanning confocal microscopy as previously described The expression of cell type-specific markers in GFPCerebral microcirculation was analyzed by intravenous administration of the fluorescent plasma marker FITC-dextran (Sigma), and CD31 immunohistochemical analysis as previously described Rats anesthetized with chloral hydrate were positioned in a stereotaxic frame. Baseline local cortical blood flow (bCBF) was monitored with a laser doppler flowmeter  as previously described Transgenic GFP-chimeric mice were anesthetized with chloral hydrate . The femoral artery ligation, limb blood flow measured by laser Doppler perfusion imaging  and immunohistochemical studies were performed as previously described P<0.05 was considered as significant.Observers were blind to the experimental conditions of each measurement. Results are expressed as mean ± SEM. The behavioral scores were evaluated for normality. We used one-way or two-way ANOVA with appropriate post hoc Newman-Keuls testing to evaluate mean differences between different groups with different treatments. A value of


Groove pancreatitis, a form of chronic pancreatitis affecting the head of the pancreas, is localized within the groove between the pancreas head, duodenum, and common bile duct. We report a case of a male patient with groove pancreatitis who initially underwent a duodenal preserving gastrenteranastomosis. Unfortunately, the patient's symptoms were only partially controlled, necessitating a pancreaticoduodenectomy in due course as the definite surgical restoration procedure. The surgical approach selected proved inadequate since the patient's symptoms did not resolve over time. This reflects that by-pass operations like these are not indicated for the management of patients with groove pancreatitis. The term “groove pancreatitis”, pertaining to a form of segmental pancreatitis affecting the head of the pancreas, localized within the groove between the head of the organ, the duodenum, and the common bile duct, although coined after Stolte et al. in 1982, was first described by Becker and Bauchspeichel, almost ten years earlier . A 34-year-old patient diagnosed with groove pancreatitis was admitted to our department because of recurrent abdominal pain, vomiting, and weight loss. The patient had been initially evaluated (6 months ago) in another hospital for similar episodes of pain and vomiting for which he had received treatment with omeprazole and pancreatic enzymes. The patient had a history of alcohol abuse for 5 years, but abstinence for the last year. Eventually groove pancreatitis was diagnosed, and duodenal stenosis by-pass surgery was performed. Unfortunately, no other information regarding imaging studies, histopathology at that time, and the selection of the aforementioned surgical procedure as the treatment of choice were available. Furthermore, the patient reported poor only symptom remission, so he sought medical advice elsewhere. γ-GT: 118 U/L, normal range: 9–40 U/L) with normal bilirubin. Tumor marker levels  were within normal limits.Upon admission in our hospital, physical examination was notable for malnutrition. There was a mild decrease of bowel sounds and a moderate tenderness periumbilically. Laboratory exams showed mild leucocytosis, increased serum amylase  and urine amylase , and mild elevation of liver function tests , a Computed Tomography (CT) scan, and a Magnetic Resonance Imaging (MRI) examination of the upper abdomen. A thickening of the second part of the duodenum, causing concentric obstruction of the lumen, was noted. Cystic formation on the duodenal wall was also prominent. The presence of tissue between the duodenum and the pancreas was shown. There was longitudinal narrowing of the pancreatic duct and a mild dilatation of the common bile duct. Several cystic lesions were noted on the head of the pancreas. The above findings were confirmed on the Magnetic Resonance cholangiopancreatography (MRCP) that followed Figures . The upper gastrointestinal endoscopy showed the gastrenteranastomosis with well functioning proximal and distal loops revealing severe edema and stenosis of the second part of the duodenum with erosive inflammation. Histological examination of the biopsy specimens from the duodenal mucosa showed mild nonspecific gastritis and hyperplastic Brunner's glands. The patient was discharged with his symptoms partially controlled, and pancreatoduodenectomy has been planned in due course as the definite surgical restoration procedure. He was put on a strict diet, proton pump inhibitors, and pancreatic enzyme substitutes, and he had an uneventful period of two months .Although, some authors have tried to unify the concept of “groove pancreatitis”, “cystic dystrophy of heterotopic pancreas”, and “paraduodenal wall cyst” as the same clinical entity, based on distinct clinicopathological findings, under the term “paraduodenal pancreatitis”, the pathogenesis of groove pancreatitis remains controversial as several factors are implicated. While heterotopic pancreas is only occasionally found in groove pancreatitis, the presence of this feature is an inherent precondition for cystic dystrophy of the duodenal wall in the heterotopic pancreas and is characterized by the presence of cysts surrounded by inflammation and fibrosis in the duodenal wall, along with pancreatic ducts and lobules. Peptic ulcers, gastric resections, true duodenal wall cysts, and pancreatic head cysts as well as previous diseases of the biliary system are also believed to be triggering factors , 3–5. OtMacroscopic examination of the surgical specimen usually reveals an abundant whitish firm mass of the groove area that produces a stenosis in the terminal common bile duct, with cystic lesions, either true cysts or pseudocysts, being frequently encountered in the groove or the duodenal wall. Major histopathological findings include the presence of scar tissue with fibrosis in the pancreaticoduodenal groove (pure form) or in the groove and the superior portion of the pancreatic head . On microscopic examination, extensive fibrosis of the duodenal wall with concomitant Brunner gland hyperplasia in the submucosa can be seen. Extensive fibrosis, acinar involution, and intimal fibrosis of the pancreatic arterioles are frequently observed in the pancreatic biopsy , 3, 4.The disease mainly affects middle-aged men with a preceding history of alcohol abuse. Clinical presentation resembles that of chronic pancreatitis, with postprandial abdominal pain of varying degrees. Duodenal stenosis often leads to early satiety, vomiting and weight loss. These symptoms last from weeks to months, commonly remitting upon resuming of enteric feeding. Jaundice is rare. The course of the disease is often chronic and debilitating. Blood tests often show a slight elevation of serum pancreatic enzymes and occasionally of liver function tests. Tumor markers are rarely elevated , 3.Upper gastrointestinal endoscopy often reveals an inflamed and polypoid duodenal mucosa with stenosis of the duodenal lumen . EndoscoMRI findings are also demonstrative of the pathologic features characteristic of this entity: the fibrous tissue in the pancreaticoduodenal groove, the duodenal wall inflammation, and the groove and/or duodenal wall cyst formation. The most characteristic finding on MRI is a sheet-like mass between the head of pancreas and the C-loop of duodenum. A T1-weighted image reveals a mass that is hypointense when compared to the pancreatic parenchyma, while a T2-weighted image reveals a hypo-, iso-, or slightly hyperintense mass. Cystic lesions are well shown in the groove or the duodenal wall, especially on T2-weighted images. Since some degree of common bile duct stenosis is almost always found, the sign of common bile duct tapering is characteristically seen in contrast to the abrupt and “shouldered” aspect of stenosis in pancreatic cancer. A progressive pattern of narrowing of the main pancreatic duct in the head of the gland can also be depicted, especially in the segmental form of the disease .Endoscopic retrograde cholangiopancreatography (ERCP) or MRCP also depicts the relationship between the ductal system and the cystic changes. Duodenal evaluation is important in differentiating groove pancreatitis from pancreatic cancer, because marked inflammatory duodenal parietal thickening is not a common feature associated with tumors in the pancreatic head. ERCP can demonstrate dilatation of the Santorini's duct and its branches, depicting intraductal stones. MRCP reveals a widening of the space between the distal pancreatic and common bile ducts and duodenal lumen. Finally, while groove pancreatitis is not usually associated with a significant degree of biliary dilatation, distention of the gallbladder is usual  .Regarding the differential diagnosis, in the pure form, the physician should rule out duodenal cancer, common bile duct cancer, or, even, acute pancreatitis. In the segmental form, pancreatic adenocarcinoma should be excluded . Some auAs far as treatment is concerned, there are two therapeutic options: (1) conservative medical measures and (2) surgery. The second is the most common due to the severity of the symptoms and in order to rule out malignancy. The surgical treatment of choice is a pancreatoduodenectomy using the Whipple procedure . HoweverIn our case, the surgical approach selected proved inadequate since the patient's symptoms did not resolve over time. This reflects that by-pass operations like these are not indicated for the management of patients with groove pancreatitis. It is our belief that such patients should be medically managed more aggressively. Radical operations, such as the Whipple procedure, should be performed whenever possible and feasible.New imaging techniques have markedly improved the accurate diagnosis of pancreatic disease; however, it is still difficult to differentiate groove pancreatitis from pancreatic carcinoma. Groove pancreatitis presents various clinical features and, often, mimics pancreatic head carcinoma. This condition should be kept in mind in all cases of focal pancreatitis involving the region between the head of the pancreas, the duodenum, and the common bile duct. Awareness of this disease may lead to more reliable preoperative diagnosis avoiding unnecessary radical surgery. Patients with groove pancreatitis having undergone conservative treatment or pancreatic preserving operations should be carefully followed up due to the risk of coexistent carcinoma.


All pts had visceral metastatic sites. 9 had only one metastatic site. The median number of cycles given was 2 in arm A and 3 in arm B. 100% and 89.4% of the planned dose of GM-CSF was given in arm A and arm B respectively. No objective response was obtained. 19 pts experienced at least WHO grade 3 toxicity. All pts had fever, 29 had a decrease in performance status and 23 had pain. Grade 3 toxicity were fever (38.7%), decrease in performance status (32.3%), pain (19.4%) and dyspnoea (12.5%). In this study, GM-CSF alone or in association with DTIC did not induce any antitumoural activity with subsequent toxicity. © 2001 Cancer Research Campaign   http://www.bjcancer.comThe potential antitumoural effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) led us to evaluate GM-CSF alone or with dacarbazine (DTIC) in metastatic melanoma in first line randomized phase II. Treatment was arm A: GM-CSF: 5 μg kg


In addition to their antimicrobial activity, antibiotics modulate cellular host defence. Granulocyte-colony stimulating factor (G-CSF) is also a well known immunomodulator; however little is known about the interactions of G-CSF with antibiotics. We investigated in septic rats the effects of two antibiotic combinations with G-CSF.In two clinic modelling randomised trials (CMRTs), male Wistar rats were anesthetized, given antibiotic prophylaxis, had a laparotomy with peritoneal contamination and infection (PCI), and were randomly assigned (n = 18 rats/group) to: 1) PCI only; 2) PCI+antibiotic; and, 3) PCI+antibiotic+G-CSF prophylaxis . This sequence was conducted first with 10 mg/kg coamoxiclav, and then with ceftriaxone/metronidazole . In additional animals, the blood cell count, migration and superoxide production of PMNs, systemic TNF-α and liver cytokine mRNA expression levels were determined.Only the combination coamoxiclav plus G-CSF improved the survival rate . Improved survival with this combination was accompanied by normalised antimicrobial PMN migratory activity and superoxide production, along with normalised systemic TNF-α levels and a reduced expression of TNF-α and IL-1 in the liver.There are substantial differences in the interaction of antibiotics with G-CSF. Therefore, the selection of the antibiotic for combination with G-CSF in sepsis treatment should be guided not only by the bacteria to be eliminated, but also by the effects on antimicrobial functions of PMNs and the cytokine response. Source control and antibiotic treatment are the mainstays in the treatment of infectious disease. Interestingly, antibiotics not only directly reduce bacterial growth; they also modulate cellular host defence, especially the antimicrobial functions of monocytes and granulocytes . Some anGranulocyte colony-stimulating factor (G-CSF) is a glycopeptide which stimulates the granulocyte lineage and stem cells. G-CSF also suppresses release of pro-inflammatory cytokines such as TNF-α by macrophages . G-CSF sTo evaluate the interaction of different antibiotics with G-CSF, we used clinic modelling randomised trials (CMRTs). The inclusion of an i.v. antibiotic prophylaxis is one important feature of CMRTs for modeling clinical complexity. Others include appropriate use of anaesthesia, volume loading, laparotomy, peritoneal contamination and infection with human stool bacteria (PCI), suitable postoperative analgesia, and complicating risk factors such as hypothermia and blood loss which we have assessed previously ,16. FurtUsing this model, we have evaluated cephalosporins, aminoglycosides, fluoro-quinolones, and carbapenemes in combination with G-CSF. All antibiotics are routinely used for clinical peritonitis treatment at our institution. Interestingly, 7 antibiotic/G-CSF combinations improved survival, but 4 others did not ,17. For The study was performed with permission of the animal welfare committee in Giessen, Hessen, Germany. 179 male Wistar rats, 220–280 g  were used. They were given standard diet  and water ad libitum.Two independent trials were performed to analyse survival. Separate animals were used for cytokine and PMN function analyses. At arrival, animals were assigned to study groups by simple random permutation using ear marks to: 1) peritoneal contamination and infection (PCI) only; 2) PCI + antibiotic prophylaxis, and 3) PCI + antibiotic + G-CSF prophylaxis (n = 18 rats/group). In the first trial, 10 mg/kg coamoxiclav was given; and in the second trial, ceftriaxone/metronidazole  was given. Liver samples for mRNA analysis were taken from additional animals (3 rats/group) from all six treatment groups. Blood cell parameters and PMN functions were assessed in main group animals and in addition in the groups G-CSF only and PCI only (n = 9 rats/group).The rats were deprived of food 12 hours before surgery. Appropriate animals received 20 μg/kg G-CSF  or placebo (Ringer's solution) as a subcutaneous injection at three times: 12 hours before surgery and 12 and 36 hours after surgery (PCI). This dose regimen was most effective as determined in dose response curves with G-CSF in the rat PCI model . The othOne hour before surgery, the animals were anaesthetised with 0.08 mg/kg fentanyl and 4 mg/kg droperidol, given intraperitoneally. In the analgo-sedated, spontaneously breathing animals a tail vein was cannulated. Animals received intravenous antibiotic prophylaxis (one hour before and one hour after surgery) with coamoxiclav , or with ceftriaxone  combined with metronidazole .Using an antiseptic technique, a 2-cm-long midline incision was performed and 1.5 ml/kg standardized human stool inoculum (diluted 1:2.5 in Ringer's solution) was injected into the pelvic region. The wound was closed in two layers using an interrupted vicryl 3-0 suture.ad libitum food and water was provided. The animals were checked carefully at least twice daily for mortality and clinical signs. When an animal appeared to be weak, it was kept separately from the others to prevent cannibalism. Animals surviving throughout the 120-hour observation period were sacrificed by inhalation of CO2.Postoperative analgesia consisted of 20 mg/kg tramadol  injected subcutaneously once daily. Postoperatively, Under fentanyl 0.08 mg/kg and droperidol 4 mg/kg narcosis, 2000 I.E. heparin  was given intravenously. Blood was withdrawn by puncture of the retro-orbital venous plexus. The white blood cell count (WBC) was determined in an automated counter optimized for rat blood . Granulocytes were purified on a discontinuous histopaque 1119/1077 gradient for 30 minutes at 700 × g at room temperature. The cells were washed with PBS buffer on ice and contaminating erythrocytes were lysed with distilled water for 30 seconds. Viability, purity, and the cell number were determined by trypan-blue staining and counting in a Neubauer chamber.Migration of PMNs was analyzed in Boyden chambers as described by Cates at al. . Zymosan2- production was determined by adding 10 μM superoxide dismutase.The production of superoxide anions was analyzed in a cytochrome-C microplate assay using a modified method from Mayo et al. . Each teEDTA anti-coagulated blood was immediately centrifuged and plasma was stored at -70°C until assayed. Cytokine TNF-α concentration was quantified with rat-specific ELISA kits .® . For semi-quantitative, competitive RT-PCR, a multi-specific competitor fragment for rat cytokines with the conditions published by Siegling et al. [Rats were sacrificed with a lethal dose of fentanyl and droperidol 24 hours after PCI. Pieces of excised liver weighing about 100 mg were frozen in liquid nitrogen and then stored at -70°C. RNA was extracted with RNA-Clean kits . Briefly, extracted RNA was stabilized with 40 U/μl RNasing et al.  was usedBefore amplification of the cytokine TNF-α, the housekeeping gene β-actin was amplified to check the efficiency of the reverse transcriptase reaction. When needed a correction was included in order to start with the same amount of c-DNA in each tube . The competitor fragment was diluted 1:4 in 3 dilution steps starting with 2 pg; if needed, a further dilution step was included. After PCR the samples were separated in a 1% agarose gel, stained with ethidium bromide, digitalized and analyzed with the Gelscan Software . For quantification, only bands with similar intensity on the grey scale between the competitor fragment and the cytokines were used.post hoc tests were performed with a Bonferroni correction. All tests were performed using the SPSS statistic software package [Survival rates were analysed with the Chi-square test and survival curves with the log rank tests. Cellular and cytokine data were assessed by ANOVA analysis of variance. If significant results were obtained in the global test,  package . CytokinOne rat died during anaesthesia, but before PCI, in the first trial in the G-CSF group; all other rats were included into the analysis. There were no further complications related to the perioperative injections or surgery. No rat survived without antibiotic prophylaxis. Coamoxiclav prophylaxis increased survival to 44% (8/18) and survivial further increased to 82% (14/17) when prophylactic G-CSF was included  which was nearly identical to the 39% (7/18) survival when Cef/met was combined with G-CSF Figure .6 cells/ml and increased the PMN fraction to 31 ± 3.5% .Systemic protein levels of TNF-α were significantly increased 24 hours after PCI in all infected groups Figure . TNF-α pIn liver biopsies, mRNA expression of TNF-α, IL-1, MIP-2 and IL-10 were elevated in the antibiotic prophylaxis groups compared to uninfected controls with the exception MIP-2 after antibiotic prophylaxis Figure . TNF-α wThe cytokine G-CSF is a glycopeptide that antagonizes the neutrophil depression associated with bone marrow transplantation or sepsis ,23. Cef/No differences were observed in the white blood cell count or PMN ratio after G-CSF prophylaxis in combination with the two antibiotics. The absolute number of the white blood cells thus does not appear to be a crucial factor explaining the efficacy of various antibiotics in combination with G-CSF.Antimicrobial PMN functions, namely the migratory activity and superoxide production are differently regulated. In obstetric patients without antibiotic treatment reduced PMN migration was predictive for infections in cases of preterm premature rupture of membranes . From thUnfortunately, only very few results are available assessing the antimicrobial activity and interaction of G-CSF with different antibiotics. Beneficial effects of G-CSF in combination with fluoro-quinolones such as ofloxacin on the antimicrobial functions were reported . With thThere is a close interaction of the antimicrobial PMN functions and the cytokine cascade activated during infections. In our trial, only the combination of G-CSF and coamoxiclav decreased systemic TNF-α. In the liver, the cytokine mRNA expression showed a heterogeneous picture. TNF-α was also significantly reduced by the combination coamoxiclav plus G-CSF compared to antibiotic only. This was not the case with the Cef/met, but in the group without G-CSF prophylaxis, a lower expression of TNF-α was observed after Cef/met prophylaxis. IL-1 mRNA levels were not different from control after prophylaxis with coamoxiclav plus G-CSF, but after prophylaxis with Cef/met plus G-CSF they remained still elevated. The combinations showed no difference between the two antibiotics regarding the cytokines MIP-2 (macrophage inflammatory protein-2) and IL-10. MIP-2 is a chemokine predominantly produced by mononuclear leucocytes which attracts PMNs and IL-10 is a well known anti-inflammatory cytokine. Surprisingly these two factors seem to be less important for the different antibiotic effect in combination with G-CSF, at least as measured in the liver. We can not exclude that they may play an important role in other organs or systemically.in vitro study demonstrated that coamoxiclav reduces mRNA expression of pro-inflammatory cytokines such as TNF-α, IL-1, IL-6 from PMNs stimulated with Klebsiella pneumoniae; in contrast, expression of the anti-inflammatory cytokine IL-10 did not increase [In vitro work also demonstrates that TNF-α release is increased by ceftriaxone, whereas other cytokines including IL-1, IL-6, and IL-8 were unchanged [Antibiotics have different effects on Cytokines. A recent nchanged ,34. TNF-nchanged . Grutosknchanged . Elevatenchanged  and the nchanged . The antnchanged . In thisThere are substantial differences in the interaction of antibiotics with G-CSF. G-CSF was only able to improve survival in combination with coamoxiclav. Increased survival may be explained by an improvement of antimicrobial PMN functions and by an altered cytokine expression which regulate these functions. Therefore, the selection of the antibiotic for combination with G-CSF in sepsis treatment should be guided not only by the bacteria to be eliminated, but also by the effects on antimicrobial functions of PMNs and the cytokine response. The antimicrobial activity will be optimised by a positive combination of direct antibiotic effects and indirect effects of the innate immune system like the PMNs.AB: antibiotic; Cef/met: ceftriaxone/metrodinazole; CMRT: clinic modelling randomized trial; FMLP: formyl-methionyl-leucyl-phenylalanine; G-CSF: granulocyte-colony stimulating factor; IL: interleukin; MIP: macrophage inflammatory protein 2; PCI: peritoneal contamination and infection; PMN: polymorph nuclear granulocyte; TNF-α: tumour necrosis factor alpha; WBC: white blood cell count.The authors declare that they have no competing interest. The work was in part supported by a grant of the Deutsche Forschungsgemeinschaft (DFG).AB designed the study, supervised the experiments and wrote the manuscript in most parts. MH performed the experiments in most parts. DS helped to improve the manuscript. WL had the study idea and gave input to the study design and experimental procedure. AT was involved in data analysis, experimentation and writing of the manuscriptThe pre-publication history for this paper can be accessed here:Characteristics of clinic modelling randomized trials (CMRTs)Click here for file


This investigation describes features of patients undergoing in vitro fertilisation (IVF) and embryo transfer (ET) where both gametes were obtained from anonymous donors.Gamete unsuitability or loss was confirmed in both members of seven otherwise healthy couples presenting for reproductive endocrinology consultation over a 12-month interval in Ireland. IVF was undertaken with fresh oocytes provided by anonymous donors in Ukraine; frozen sperm (anonymous donor) was obtained from a licensed tissue establishment. For recipients, saline-enhanced sonography was used to assess intrauterine contour with endometrial preparation via transdermal estrogen.p < 0.001). Infertility duration was ≥3 yrs for couples enrolling in dual gamete donation, and each had ≥2 prior failed fertility treatments using native oocytes. All seven recipient couples proceeded to embryo transfer, although one patient had two transfers. Clinical pregnancy was achieved for 5/7 (71.4%) patients. Non-transferred cryopreserved embryos were available for all seven couples.Among commissioning couples, mean±SD female and male age was 41.9 ± 3.7 and 44.6 ± 3.5 yrs, respectively. During this period, female age for non dual anonymous gamete donation IVF patients was 37.9 ± 3 yrs (Mean age of females undergoing dual anonymous donor gamete donation with IVF is significantly higher than the background IVF patient population. Even when neither partner is able to contribute any gametes for IVF, the clinical pregnancy rate per transfer can be satisfactory if both anonymous egg and sperm donation are used concurrently. Our report emphasises the role of pre-treatment counselling in dual anonymous gamete donation, and presents a coordinated screening and treatment approach in IVF where this option may be contemplated. For some patients, parenthood hinges on the successful completion of a medical odyssey comprising a fretful maze of tests and procedures. Among the advanced reproduction techniques currently available, one of the more complicated is anonymous donor oocyte IVF. The complexity and cost of this treatment relates to the interlock of screening and clinical management of the oocyte donor, the embryo recipient, and the sperm source . Although anonymous donor oocyte IVF generally requires eggs to be collected fresh, since cryopreserved spermatozoa have long been available from tissue establishments ("sperm banks") this has made anonymous acquisition of male gametes comparatively simple. But while the frequency of IVF incorporating either donor oocyte  or donorMedical records were reviewed for all in vitro fertilisation (IVF) cases at Sims International Fertility Clinic  to identify cases where both oocyte and sperm from anonymous gamete donors was used during treatment. We studied de-identified IVF records of all couples who completed embryo transfer during 2009. Given the emerging regulatory environment for assisted fertility services in Ireland, cycle commencement and monitoring occurred in Dublin, while embryo transfer was performed at our affiliate clinic site in Ukraine .IVF patients were considered for dual anonymous gamete donation if prior donor oocyte IVF treatment using partner sperm had failed. However, in cases where prior ovarian response to gonadotropin ovulation induction was poor and where the male partner evaluation also identified severe abnormality with semen parameters and/or sperm DNA fragmentation (and no donor gametes were used), couples could request to move directly to dual anonymous gamete donation without first having to complete one cycle of anonymous donor sperm or egg IVF. Anonymous oocyte donors underwent comprehensive medical and psychological evaluation in Ukraine, as described previously . RecipieAfter undergoing further counselling specifically on dual anonymous gamete donation, couples were offered this treatment as an alternative to adoption. They were apprised of the procedure's complexity and the international nature of the treatment was acknowledged. Each recipient selected an anonymous oocyte donor via secure internet portal with an electronic lock-out mechanism to prohibit multiple recipients from accessing the aggregate donor pool at the same time. Following registration of each provisional donor-recipient match, the corresponding anonymous oocyte donor entry was deleted from the donor library , thus crAfter match verification and institutional receipt of anonymous (frozen) donor sperm, the anonymous oocyte donor commenced controlled ovarian hyperstimulation. Programmed transvaginal ultrasound-guided oocyte collection followed 36 h after s.c. hCG administration. Anonymous donor sperm was used to fertilise all freshly retrieved eggs obtained from the anonymous oocyte donor; intracytoplasmic sperm injection (ICSI) was performed in all cases due to high variation in post-thaw sperm motility. The recipient underwent endometrial preparation by transdermal estrogen. Fresh embryo transfer occurred at the blastocyst stage under abdominal ultrasound guidance, and any non-transferred embryos were cryopreserved ; a karyotype was obtained on each donor in addition to statutory requirements [pn stage. Mean number ±SD of embryos transferred in this series was 2.1 ± 0.3, and commissioning couples had an average of 7.6 non-transferred embryos cryopreserved for storage (range = 1-17). Embryo transfer was completed in all seven cases, but one couple underwent two transfers. Clinical pregnancy was achieved for five (5/7 = 71.4%) patients. Transvaginal ultrasound at 7-10 weeks' gestation confirmed an intrauterine pregnancy for five patients, and two of these had twins . After fertilisation with anonymous donor sperm, 17.6 ± 4.5 advanced to the 2Donor gamete IVF typically joins the path to pregnancy as a late entry. As a fertility treatment generally reserved for the most refractory cases, anonymous donor oocyte IVF is usually preceded by multiple failed treatment cycles. For such patients, substantial emotional stress may coalesce around donor gamete IVF because of feelings of reproductive inadequacy or disappointment. Indeed, some fertility patients never accept the donor gamete alternative and steadfastly resist any reproductive option involving gamete donation . These pWhile previous investigations have focused on either anonymous donor oocyte or sperm with IVF, this work is the first to collect data on an IVF patient series where both gametes derived from anonymous donors. Irrespective of the clinical indication(s) leading to use of donor gametes with IVF, this treatment modality brings unique and sensitive issues not encountered with other assisted fertility pathways. For example, a successful anonymous donor oocyte IVF cycle may introduce family concerns regarding a "separated biological and social parenthood" , and feede novo. In contrast, donor embryo IVF is conditional on the availability of surplus embryos derived from someone else's prior IVF treatment. Studies on family dynamics after donor embryo IVF have suggested higher emotional overinvolvement and defensive responding are more often observed along with greater secrecy about the child's origins [It should be noted that IVF with dual anonymous gamete donation shares some features with embryo donation, in that neither provides a genetic linkage between parents and offspring. Key differences must be acknowledged between the two approaches, however. For example, in IVF with dual anonymous gamete donation, the commissioning couple initiates the entire treatment sequence  origins . Moreove origins . Notably origins . Some ofPerhaps not surprisingly, our research on IVF with dual anonymous gamete donation revealed that each couple undergoing this treatment develops their own narrative describing how this key decision is made. A theme of "engagement with technology" as a means to attain parenthood was commonly identified in recipient counselling sessions here. While the notion of dual gamete donation IVF was never portrayed as an ideal route to pregnancy, the alternative of childlessness was clearly regarded as unacceptable. Moreover, all seven recipient couples were conversant with traditional adoption and none objected to having a family in this way . But in Ireland, a government-regulated state adoption scheme does not favour continuation of fertility treatments in parallel to the adoption process. Curiously, while this agency "encourages" prospective adopters "to complete fertility investigations prior to pursuing adoption" , in pracA novel feature of this dual anonymous donor gamete IVF programme is that neither gamete donor was of Irish origin. As previously reported, the paucity of domestic anonymous gamete donors in Ireland together with the regulatory vacuum with respect to all assisted fertility treatments frustrates the provision of this service here . For theShould anonymous gamete donation be restricted by statute in Ireland, then Irish patients who specifically seek this treatment may travel elsewhere for this reproductive option. Evidence exists supporting such a consequence in the E.U., as in Sweden sperm donors are required by law to be identifiable, while anonymous sperm donation is permitted in Denmark. The shortage of Swedish sperm donors has contributed to cross-border "reproductive tourism" between Sweden and Denmark . How regThe authors declare that they have no competing interests.ESS was principal investigator and lead author; LOM and USD were chief clinicians at the Ukrainian site; DJW was senior reproductive endocrinologist; US, ABO and GDC were programme associates in Dublin; IAD and KMB were nurse coordinators in Dublin; OMT was donor welfare coordinator; APHW was chief of service and supervised overall research design. All authors read and approved the final manuscript.


These findings suggest that excessive GM-CSF production in the NOD bone marrow may interfere with the temporal sequence of GM-CSF and M-CSF signaling needed to mediate normal STAT5 function in myeloid cell differentiation gene regulation.Defects in macrophage colony-stimulating factor (M-CSF) signaling disrupt myeloid cell differentiation in nonobese diabetic (NOD) mice, blocking myeloid maturation into tolerogenic antigen-presenting cells (APCs). In the absence of M-CSF signaling, NOD myeloid cells have abnormally high granulocyte macrophage colony-stimulating factor (GM-CSF) expression, and as a result, persistent activation of signal transducer/activator of transcription 5 (STAT5). Persistent STAT5 phosphorylation found in NOD macrophages is not affected by inhibiting GM-CSF. However, STAT5 phosphorylation in NOD bone marrow cells is diminished if GM-CSF signaling is blocked. Moreover, if M-CSF signaling is inhibited, GM-CSF stimulation  Myeloid cell differentiation gives riseto 3 populations of professional antigen-presenting cells (APC) in the immune system: monocytes, macrophages, and dendritic cells (DC). Sequential signaling from interleukin-3 (IL-3), granulocyte-macrophage-, macrophage-, and granulocyte-colonystimulating factors  sets the microenvironmentnecessary for bone marrow myeloid precursor cells to differentiate and function. The timThe cytokine GM-CSF has a unique dual influence in myeloid cells, acting first as a differentiation factor in myeloid hematopoiesis, and then later as an activation stimulus in mature monocytes and macrophages. Both overexpression and knockout deletion of GM-CSF in mice can lead to dysregulation of myeloid differentiation andautoimmune disease , suggestWork by Piazza et al.  suggests Incomplete myeloid differentiation is emerging as a common component of multipleautoimmune pathologies, both in mice and man. Serreze et al. 1993)  foundth993  founin vitro. We find that both GM-CSFoverproduction and persistent STAT5 phosphorylation phenotypes are amplified inautoimmune NOD bone marrow cells, but can be overridden by in vitro inhibition of endogenousGM-CSF signaling. Furthermore, the persistent STAT5 phosphorylation phenotypeseen in NOD myeloid cells can be recreated in non-autoimmune C57BL/6 bonemarrow cultures by blocking M-CSF while stimulating with NOD level GM-CSF. Thesefindings in vitro suggest thatthe defect in NOD M-CSF signaling in vivo may occurduring the switch of influence between GM-CSF to M-CSF during myeloiddifferentiation, and indicate that prolonged expression of GM-CSF promotes thedysregulation of STAT5, which may interfere with M-CSF signaling during NOD myeloidcell differentiation.In our studies of APC dysfunction inautoimmunity, we have found that both NOD and autoimmune human myeloid cellsexpress abnormally high levels of GM-CSF and have prolonged STAT5 activationafter even brief exposure to GM-CSF –20. In tEight to twelve-week old NOD and C57BL/6female mice were used for all studies. These strains were maintained as breeding stock inour Pathology Department SPF colony, in microisolator cages with food and waterad libium. All procedures were conduced to maintain humane treatment of allanimals according to IACUC approved protocols B083 and D754, and humanelyeuthanized by cervical dislocation while over-anesthetized. All tissue collections weredone postmortem. A minimum of 3 animals per strain was used per treatment for each run of eachexperiment.μg/mLanti-M-CSF blocking antibodies , or medium containing 500 U/mL M-CSF (Invitrogen) + anti-GM-CSF blocking antibodies (Pierce-Endogen). Cultures weremaintained for 24 hours at 37°C/5%CO2, then washed, and resupplemented for an additional 24 hours in culture at 37°C/5%CO2. Cells were then analyzedfor myeloid phenotypic identification and STAT5 phosphorylation by flowcytometry and deconvolution microscopy.Long bones from the legs were collected postmortem into cold RPMI + 10%FCS medium and the marrow flushed into a steriletube by cold medium injected into the bone through a 30-gauge needle. Themarrow samples were washed with cold media by centrifugation and red bloodcells in samples lysed by incubation in nonisotonic 0.84% ammonium chloride buffer.The remaining bone marrow cells are then plated at 5–10 millioncells/well on tissue culture dishes and fed with fresh sterile medium alone or withmedium containing 1000 U/mL of GM-CSF  + 2 ex vivo and after in vitro stimulation. Cells werefirst labeled with anti-CD11b antibody FITC or PE conjugates  to identifymonocytes and macrophages in the sample, then fixed with 2% (v/v Cf)formaldehyde  and permeabilized using 0.5% saponin(Sigma-Aldrich) in a high protein, isotonic FACS buffer [t-test or Mann-Whitney Uanalyses and multiple group analyses done using one-way ANOVA.Flow cytometric analysis of fluorescently conjugated antibodysurface and intracellular binding was used to phenotypically identify myeloid cells and quantifythe level of STAT5 tyrosine phosphorylation (STAT5Ptyr) in cells S buffer . IntraceS buffer , 20 for μM Na vanadate  for 30 minutes at 378°C/5%CO2. Half of the cultures +/− vanadate were then supplemented with 1000 U/mLGM-CSF, and all were incubated for an additional 90 minutes at 378°C/5%CO2. The cells were then fixed in situ, with 1% formaldehyde in 1xPBS ,for 10 minutes at 37°C prior to lysis with high SDS lysis buffer (Millipore-Upstate). Thefixed lysates were sonicated to disrupt membranes and shear chromatin toapproximately 1000 bp fragments then frozen at −80°C until analyzed. The samplewas split into five1 × 106 cell aliquots for use in anti-STAT5Ptyr/anti-Histone H3mediated double ChIP (dbChIP) protein isolations for Western blot analysis ofSTAT5 associated with histone/chromatin complexes. Aliquots used for dbChIP wereprecleared with salmon sperm DNA Protein A or G agarose beads (Millipore-Upstate), thenincubated overnight at 4°C with antityrosine phosphorylated STAT5 (STAT5Ptyr)antibodies (Millipore-Upstate). After incubation, the antibody-bound chromatincomplexes were precipitated using salmon sperm DNA Protein A agarose beads, andwashed extensively with a series of increasing stringencybuffers .A nonspecific antibody control ChIP  and a sham IP containing noextract were used as negative controls.Five million bone marrow cellswere cultured with or without 100 ChIP extract aliquots dissociated from the beads in 1%SDS, 0.1 M bicarbonatebuffer (Fisher Scientific).To both unprecipitated  extractsand ChIP samples, NaCl was added to a final concentration of 500 mM and thesamples wereincubated for 4 hoursat 65°C to reverse the formaldehyde cross-links. The released anti-STAT5Ptyr-selectedcomplexes were neutralized and reprecipitated  overnightat 4°C using precoupled anti-Histone H3-Protein G Salmon sperm DNA-coated beads(Millipore-Upstate).The dbChIP complexes were then washed with the same sequence of buffers as usedin the first ChIP, and then boiled in 1x Leammili buffer  for Westernblot analysis. Western blots were probed with anti-STAT5 Ptyr (Millipore-Upstate)antibodies and cross-linked and uncoupled proteins were visualized usingAmersham ECL plus chemilumenescence reagents .Densitometry analysis using BioRadImager and Quantity One Software was used to estimate molecular weight ofcross-linked dimer complexes and freed monomeric isoforms in STAT5Ptyr positivebands.in vitro myeloid cell differentiationexperiments, NOD and C57BL/6 mouse bone marrow cells were stimulated with onecytokine (1000 U/mL GM-CSF or 500 U/mL M-CSF) in the presence of blockingantibodies for the other cytokine  for 48 hours at 37°C/5%CO2.Mouse bone marrowcells can differentiate to mature different macrophage phenotypes within 4 daysculture in M-CSF or 4–7 days in GM-CSF, 21, 22.P = .0240, ANOVA, In earlierstudies, we found that blocking GM-CSF signaling by anti-GM-CSF antibodies orwith the Jak2/3 inhibitor AG490 in mature monocytes and macrophages did notaffect the persistence of phosphorylated STAT5 . In contP = .0167, Mann-Whitney U test, in vitro in the presence of blockingantibodies to GM-CSF, STAT5 phosphorylation was also stimulated in C57BL/6cultures, and remained statistically unchanged in NOD bone marrow cells (*P = .0336and .5556, respectively, Mann-Whitney U test, Deconvolutionmicroscopic image analysis see  suggesteTaken together, thesefindings suggest that the interplay M-CSF and GM-CSF signaling is required toproperly regulate STAT5 function, and support the role of temporally sequencedGM-CSF and M-CSF signaling on controlling STAT5 activation during myeloid celldifferentiation. Furthermore, this interplay is modulated by the timing andconcentration of GM-CSF stimulation early in differentiation, which appears toset the stage for STAT5 activation in later stages, regardless of furtherstimulation by GM-CSF or M-CSF. Because the NOD cells have higherendogenous GM-CSF production , this tein situ assembly ofchromatin-associated protein complexes which contain both STAT5 and Histone H3 (To evaluate the possibility thatincreased STAT5 phosphorylation could lead to increased chromatin binding inresponse to GM-CSF, we performed double ChIP analyses on NOD and C57BL/6 bonemarrow cells that had been cultured with GM-CSF for 2 hours, and looked for stone H3 . We contstone H3 . The preOur studies indicate thatoverexpression of GM-CSF in both bone marrow myeloid precursor cells in NOD,but not non-autoimmune mice, can prolong the activation of STAT5 and itsbinding to chromatin. Furthermore,treatment of non-autoimmune mouse bone marrow cells with anti-M-CSF antibodieswhile stimulating them with GM-CSF at levels endogenously produced by the NODcan recreate the activation of STAT5 but not its chromatin binding. The levelof STAT5 phosphorylation and function in NOD cells is independent of concurrentphosphatase or kinase  activityBlocking GM-CSF or M-CSFsignaling alone reversed the high STAT5 phosphorylation seen in the NOD, but did notalter it in non-autoimmune bone marrow cells. In addition, M-CSF treatment onlysignificantly increased STAT5 activation in non-autoimmune cells, and only whenGM-CSF signaling was concurrently blocked. These findings suggest that the regulationof STAT5 activation is affected by the interplay of GM-CSF and M-CSF signalingin differentiation, and that this interplay is disrupted in NOD myeloid cellsby the persistence of GM-CSF influence on these cells.β,which induceepigenetic control modifications on its promoter [Csf2 gene promoter, which STAT proteins can mediate through their function as anadaptor-DNA-binding proteins for these enzymes [ Csf2 gene promoter [Csf2 gene expression, persistenceof its binding at the Csf2 promotercould promote the aberrant GM-CSF expression seen in the NOD. Furthermore, ourdata indicate that M-CSF signaling may be able to override NOD defects in theactivation/deactivation of STAT5 related to the overproduction of GM-CSF inthese cells, but its subsequent aberrant binding may be dependent on specific,unique binding site polymorphisms found within the NOD genome, especially newsites that allow STAT5 binding in the promoter of the gene for GM-CSF itself ([These data leave open thequestion of how GM-CSF is aberrantly activated in NOD bone marrow myeloid cellsin the first place. GM-CSF autocrine stimulation of myeloid cells is a keyregulatory component in myeloid differentiation and activation responsivenessin mature monocytes and macrophages and is under strict temporal regulation , 3. GM-Cpromoter , 23, 24.enzymes  –25. STATenzymes  , 7, 25. promoter . If such itself (and GarrThe autoimmune STAT5 dysfunction seen in the NOD may be best described as a “broken switch” in epigenetic control of cytokine signaling during myeloid differentiation. In autoimmune cells, this switching mechanism is stuck in the “on” position; thereby, “shorting out” the activationof cells by the normal sequence of cytokines and blockingtheir proper maturation. The incompletion ofmyeloid cell maturation blocks the eventual functionality of this importantclass of APC in the initiation and maintenance of self-tolerance, which hasbeen implicated as a major defect involved in immunopathogenesis.