Despite major advances in assisted reproductive techniques, the implantation rates remain relatively low. Some studies have demonstrated that intrauterine infusion of granulocyte colony stimulating factor (G-CSF) improves implantation in infertile women. To assess the G-CSF effects on IVF outcomes in women with normal endometrial thickness. In this randomized controlled clinical trial, 100 infertile women with normal endometrial thickness who were candidate for IVF were evaluated in two groups. Exclusion criteria were positive history of repeated implantation failure (RIF), endocrine disorders, severe endometriosis, congenital or acquired uterine anomaly and contraindication for G-CSF . In G-CSF group (n=50), 300 µg trans cervical intrauterine of G-CSF was administered at the oocyte retrieval day. Controls (n=50) were treated with standard protocol. Chemical, clinical and ongoing pregnancy rates, implantation rate, and miscarriage rate were compared between groups.Number of total and mature oocytes (MII), two pronuclei (2PN), total embryos, transferred embryos, quality of transferred embryos, and fertilization rate did not differ significantly between two groups. So there were no significant differences between groups in chemical, clinical and ongoing pregnancy rate, implantation rate, and miscarriage rateour result showed in normal IVF patients with normal endometrial thickness, the intrauterine infusion of G-CSF did not improve pregnancy outcomes. Despite major progression in assisted reproductive techniques, the implantation rates still remain relatively low. “Successful implantation needs good quality embryo, receptive endometrium, and good embryo transfer technique” . The recImmunological mechanisms in the endometrium are very important and crucial in implantation process . Granuloet al showed intrauterine G-CSF administration improved chemical and clinical pregnancy rate in infertile women with thin endometrium in frozen-thawed embryo transfer cycles but they found in their study endometrial thickness in their patients did not increased . Women with repeated implantation failure (RIF) , endocrine disorders, severe endometriosis, congenital or acquired uterine anomaly , contraindication for G-CSF  were excluded. After receiving informed written consent from all participants and their spouse, according to enveloped pocket method women were allocated randomly in two groups (G-CSF and control group). Standard agonist or antagonist protocol was used for ovarian stimulation in groups . When atIn G-CSF group at the day of oocyte retrieval, after oocytes collection, 300 mg G-CSF  was administered by slow transcervical intrauterine infusion with IUI catheter  . In contPregnancy outcomes were assessed based on positive serum βhCG test , 14 days after embryo transfer and observation of gestational sac on transvaginal ultrasound examination , three weeks after positive serum βhCG. Implantation rate was assessed by the number of gestational sacs divided by the number of transferred embryos in each group. The ongoing pregnancy rate was defined as the presence of fetal heart activity by ultrasonography after 12 wks of pregnancy. The miscarriage rate was assessed by the number of miscarriages before 20 wks gestation per number of women with positive βhCG test.Statistical analysis1=20%, p2=45% and the sample size=50 in each group was considered. Continuous data were presented as mean±SD and assessed by independent Student’s t-test. Qualitative data were compared by  2 or fisher exact test. P<0.05 was considered significant.Data were analyzed using Statistical Package for the Social Sciences 20.0 . With 95% confidence level, power of 80%, pTotally, 113 normal infertile women were participated in this study. 13 women were excluded and finally data of 100 women analyzed . DemograIn the present study, G-CSF effect on implantation and pregnancy rates in normal infertile women candidate for IVF treatment were evaluated. It was found that pregnancy outcomes did not improve significantly after intra uterine G-CSF infusion in women with normal endometrial proliferation. G-CSF is a factor that rising the synchronization between uterine environment and embryo development during endometrial remodeling , 16. Preet al represented a new option for thin endometrium treatment. They evaluated the G-CSF effect in four patients who underwent IVF that endometrial thickness had not increased with routine treatment. They reported successful endometrial thickness to at least 7 mm after G-CSF uterine infusion and all patients were conceived . We did not obtain significant differences between two groups in terms of chemical, clinical, ongoing pregnancy, implantation, and miscarriage rates. There are nor numerous studies on the effect of G-CSF in women with normal endometrial thickness. et al showed intrauterine G-CSF infusion in fresh embryo transfer cycles in IVF women with normal endometrial thickness do not affect endometrial thickness, implantation, and clinical pregnancy rates (Similar to our results, Barad cy rates . Therefocy rates .A systematic review and meta-analysis of 14 studies shown that there may be a relationship between endometrial thickness and pregnancy, but implantation is more complex than be determined by single ultrasound . Now, duIn summary, we showed that, in normal IVF women who had normal endometrium, the intrauterine infusion of G-CSF did not improve pregnancy outcomes. The available evidence does not support routine use of G-CSF in normal IVF women with normal endometrial thickness. More randomized controlled trials is needed for comparison of G-CSF effects on women with thin and normal endometrial thickness.


Closure of donor site of the flap has special problems. Reduction of this site will decrease the morbidity of operation. In this study, we present our experience in donor site size reduction. Between 2006 and 2008, 15 patients with skin and soft tissue defects underwent operation. In all patients, coverage of defect was performed with various flaps. Substratum horizontal mattress suture was used to reduce donor site dimensions. In all 15 patients, size of the flaps, the defect after the flap elevation and the scar size were measured.2, 69.4 cm2, and 32.2 cm2, respectively. There was 46.5% reduction in the donor site after using this suture.The mean size of the flap, the defect after flap elevation, and the scar after 3 months were 43.9 cmThe substratum horizontal mattress suture was shown to de- crease the donor site dimensions and also its scar size in flap surgery. This suture is highly recommend in order to reducedonor site dimensions. Eight pAfter elevation of the flap, the donor site reduction was carried out with 0 nylon suture . The nee2, 69.4 Cm2, and 32.2 Cm2, respectively. The overall reduction of donor area after using substratum horizontal mattress sutures was 46.5%.The donor site healed completely in all patients. The mean size of the flap, the mean size of the defect after flap elevation, and the mean scar dimensions after 3 months were 43.2 Cm2Flap surgery is an essential part of plastic and reconstructive surgery. Coverage of the donor site of the flaps has various complications.2These include: pain, prolonged healing, hypertrophic scar and aesthetic problems.3 The smaller the secondary donor site, the less morbidity and better aesthetic results ensues.4 Thin soft tissue layer over the suture facilitates complete taking of skin graft (4,5 According to our experience and results, we suggest using this simple and useful suture for reduction of the donor site size during flap surgery.Primary repair of donor site is possible when the flap is small. In larger flaps, skin graft is necessary for coverage of the defect.in graft . If sutuThe authors declare no conflict of interest.


Time-correlated Förster resonance energy transfer (FRET) probes molecular distances with greater accuracy than intensity-based calculation of FRET efficiency and provides a powerful tool to study biomolecular structure and dynamics. Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions.Here we develop a new approach based on Monte Carlo simulations of time-correlated FRET events to estimate the time-correlated single photon counts (TCSPC) histograms in complex systems. This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules. The simulations are consistent with the theoretical predictions of the dye behavior in FRET systems with defined dye distances and measurements of randomly distributed dye solutions. We validate the simulation results using a highly heterogeneous aggregation system and explore the conditions to use this tool in complex systems.This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems.The online version of this article (doi:10.1186/s13628-015-0020-z) contains supplementary material, which is available to authorized users. FRET is based on non-radiative energy transfer between two dyes dependent on their absolute distance ,2 which e labels . HoweverRecent developments include Monte Carlo analysis of FRET kinetic traces which are used to estimate FRET rates taking into account the full statistics of individual photon absorption, energy transfer, and photon emission events , or to gNeurodegenerative pathologies based in misfolding and aggregation, such as Alzheimer's and Huntington's diseases, are growing in medical prevalence . Aggregaκ2) is usually taken as 2/3, which is based on the assumption that the dyes have enough time to randomize their orientation during the time the donor stays excited [κ2 inaccurate. Thirdly, too many short range distances dramatically increase the simulation time by reducing the number of the collected photons because of the large competition rates for transfer deactivation of the donors. Furthermore, the fluorescence decay time of the donor should be sufficiently long during the measurements to provide enough time for the donors to randomize their orientation after excitation and limit the fraction of photons detected by a non-random distribution of photon emission angles at the onset of signal detection.We first selected a set of dyes for optimal consistency of simulated and experimental donor TCSPC histograms and to reflect the expected behavior in the simulations. Note that this approach allows also for calculating the acceptor TCSPC histograms; here, if not otherwise mentioned, we focus primarily on the donor TCSPC histograms. Typically, the Förster radius of the FRET pair should be chosen within a range of distances which are expected between the donor and its nearest neighboring acceptor for the following reasons. Firstly, FRET efficiency and measured donor TCSPC histograms respond sensitively to distances ranging from one half to two times the Förster radius, thus increasing the influence of small deviations between distances. For distances beyond twice the Förster radius, the FRET rates are extremely low with very little influence on time-dependent photon emission probability. Energy transfer for distances closer than half of the Förster radius is extremely efficient, but hardly any donor photons will be collected. Typically, those distances do not influence the donor photon distributions, since donors with very high quenching rates have negligible brightness compared to donors without acceptors at distances closer than half of the Förster radius. Additionally, since within the half of the Förster radius, the donor-to-acceptor transfer occurs almost instantaneously, no significant change in the donor TCSPC (or even in the acceptor TCSPC) is expected. Secondly, the factor describing the relative orientation of donor emission and acceptor absorption transition dipoles . The dyes were chosen because they implement the described requirements and criteria for suitable experimental setups, e.g. high photostability, extinction coefficient and quantum yield ,22.To read the information of the positions serving as an anchor point into the code, input files are created according to the following scheme. The files contain the three Cartesian coordinates that define the dimensions of the simulated volume, three binary values determining the periodicity of the system in each direction and the number and Cartesian coordinates of donors and acceptors in the system. All coordinates are given in nanometers.We randomly positioned our dyes according to the probabilities derived from (i) specific dye concentrations with randomly distributed dyes in the three dimensional space, or (ii) fixed labeled structures with known labeling sites according to the probabilities of labeling with donors or acceptors. Additionally, files with defined donor-acceptor distances in defined ratios were created to compare the simulations to theoretically calculated fluorescent decay time behavior.r, the FRET rates kT and the deactivation probabilities Pdeactivation are then calculated for every donor  and the emission was collected at λem = 525 nm using a polarizer set at magic angle position and a multichannel plate (Europhoton) as a detector. The repetition rate of the excitation light source was set to 10 MHz.Fluorescent decay times were recorded on a FL920 spectrometer (Edinburgh Instruments) operated in a TCSPC mode. In the measurements we used a time window of 50 ns and 1024 channels. Samples were excited at ΦD for the donor was determined to be 0.6. Based on that, the Förster radius was determined to be 5.4 nm (Eq. J (λ) was calculated from the recorded donor emission and acceptor absorption spectra determined with the QuantaMaster 40  with Felix32 software. The Förster radius is a function of κ2, describing the relative orientation of the donor and acceptor transition dipoles, the quantum yield of the donor ΦD, the refractive index n of the solution and the overlap integral J (λ) between the donor emission and the acceptor absorption spectra:Using a photoluminescence (PL) quantum yield measurement system , the quantum yield NA is the Avogadro number.where To verify the consistency of our code with the theory of Förster resonance energy transfer, we calculated the probability for photon emission. For multiple distances, the resulting distributions were set in relation to their weight according to the photons expected during the first time interval based on the probability to emit photons and on the fraction of excited donors in the particular surrounding.NP(t) is the amount of photons in dependence of time, fD,i is the fraction of donors with surrounding i, kemission is the emission rate of the donor and the sum of kj represents the combined deactivation rate constant for all deactivation processes. The derivative dDi,emission/dt represents the deactivations by emission per time unit at a given time point. It cannot be assumed to be constant due to photon decay events of donors with very close acceptors at earlier times than resolved by a typical experimental time resolution. Therefore in the first time interval it was determined for 1000 equal subintervals and subsequently averaged. [For additional information see Additional file where http://figshare.com/) and has the accession number: 1158992 (http://figshare.com/articles/FRET/1158992).The code is deposited in an open-access platform domain (Time-correlated photon count measurements offer a powerful tool to investigate dye surroundings. TCSPC histograms respond sensitively to environmental changes, such as solvent polarity, pH or presence of heavy atoms. Additionally, atomic distance information can be extracted using resonance energy transfer between donors and acceptors that are introduced at targeted labeling sites. However, when using a mathematical fitting function to gain many parameters it is often impossible to implement restrictions and the fitting will instead settle into different minima that represent meaningless dye arrangements. Connecting the knowledge of the possible distance distributions to fluorescent decay functions using Monte Carlo simulations provides a powerful way to bypass this limitation. It is also based on detailed mechanistic understanding of the underlying physical processes that can produce observed signals.To evaluate the predictions made with our code, we first performed simulations with simple dye systems whose fluorescent decay function can be calculated Eq. . We creaFor the two donor environments we simulated all possible combinations of pairs from the single donor environment to ensure that the code will weight them according to the expected stochasticity Figure B and C. A fraction of donors with acceptors at a distance of 2 nm or less had very little influence since the amount of photons contributed by such donors was small. Therefore, the calculated decay functions were mainly influenced by other donor environments. That led to a paradoxical situation: a slight increase of those donor-acceptor distances beyond 2 nm, which resulted in less quenching, seemed to increase the quenching effect by enhancing the relative contribution to the total signal (Figure Next, we created a system in which the dye distances follow a distribution instead of being fixed to discrete values. We therefore used a simple experimental system containing Alexa488- and Alexa594-maleimides with defined concentrations. High dye concentrations were used in order to observe significant quenching. As expected, increasing the acceptor concentration shortened the fluorescent decay times. An increase of the concentration of the donor, however, did not have any effect, since the donor excitation rates were chosen to be very low. The presence of additional donors did not change the donor environment as long as the excitation was sufficiently low to avoid competition for nearby acceptors Figure . The TCSTransfer processes between the dyes outside FRET are not considered in our code. The parameters and fluorescent dye pair should be chosen accordingly to minimize the influence of processes such as homo-FRET or Dexter energy transfer. When the expected dye distances are in the range of the Förster radius and the donor or acceptor properties are suitably chosen, ignoring these processes should not influence the simulation results.Subsequently, we assessed the relevance of predicting the correct change in distances for dyes attached to fixed positions Figure . Since tThe dye positions were reassigned to a sphere around the labeling position with different radii. Allowing for changes in the dye positions altered the shape of the fluorescent decay function Figure . For sinFor complex FRET systems, e.g. amyloid fibrils , where tAdditionally, FRET pairs labeled at positions closer than the half of the Förster radius had higher contribution to the signal. If some dyes change their position and influence the donor-acceptor distance growth, it will result in a relative increase in the number of photons detected shortly after excitation. For larger linker length, however, the average distance was dominated by another effect. The total volume accessible to the dyes increased in non-periodic systems in all three dimensions, thus reducing the effective dye concentration and increasing the average dye distances. At some point, the original distances between the dyes will become insignificant, since they are comparably small to the length of the flexible linker. This is a limitation that needs to be carefully considered when designing a system to be assessed with this approach.In sum, allowing for changes in the dyes positions can influence and potentially interfere with the success of the simulations, albeit within a limited distance scale. However, by linker size small compared to the Förster radius, it had minor influence on complex dye systems with high label densities. Therefore, more precise assumptions, such as effective ranges of linker-length rather than fixed length defined by theoretical models or molecular dynamics simulations, or excluded dye volumes based on angle restriction from the volume occupancy, are not necessary for most applications.To investigate how much signal needs to be obtained for stochastically reliable sample size, we performed a simulation using only Alexa 488 labeled protein within our polyglutamine amyloid fibrils  and inveP to obtain a photon within an excitation cycle is typically very low. This is necessary because in a given excitation cycle only one photon can be detected. If more photons would reach the detector within one excitation cycle, only the first photon would be counted, leading to a bias and thus to a misrepresentation of the time-dependent photon distribution. The probability to detect more than one photon should be negligibly small. In our Monte Carlo simulation we can simultaneously monitor several photons in the same excitation cycle. Given that the typical small fraction of excited donors in the experimental setup slows down the simulation, it is reasonable to increase the probability for donor excitation artificially, although the correct value can be easily calculated using:In time correlated single-photon count measurements, the probability I – irradiance, Δt – the time interval of the excitation light pulse, A – the surface area of the light pulse, EP – the energy of a single photon, ND – the number of donors in the simulated volume V, εD – the extinction coefficient of the donor, and l – the length of the hypothetically excited volume in direction of the light beam. Despite the capability of detecting multiple photons without any bias, the simulations will be affected by increasing fractions of excited donors. An increase of the amount of excited donors also results in an increased amount of electronically excited acceptors during an excitation cycle. In situations where two donors in close proximity competing for the same acceptor are too frequent, they could induce error in the prediction of the donor TCSPC histograms. Thus, we addressed at which excitation probability such competition would alter the time-dependent profile of the photon emission. For this purpose, we simulated the structure of polyglutamine fibrils composed of 20% of donor-labeled monomers and 20% of acceptor-labeled monomers. The simulated fluorescent decay function was dramatically influenced with increasing excitation probabilities (Figure with s Figure . ChanginIn conclusion, our approach is a step forward in exploiting the power of FRET to determine distances at the molecular level. The Monte Carlo-based simulation tool that we developed to predict the time-correlated single photon counts correctly assesses the donor behavior and allows for evaluation of precise structural models in complex FRET systems. Different key input parameters can change the predictions and reliability of the simulations. For a stochastically meaningful simulation at least 10,000 photons should be collected in the most frequently observed time interval. The simulation time can be shortened by artificially increasing the excitation probability of the donors in the simulated system. For bulky dye systems whose dimensions largely exceed the relevant distance for FRET, including a flexible linker flattens the distance distribution and alters the resulting photon decay. For isolated donor-acceptor pairs, increasing the linker length systematically reduces the quenching as previously observed . Taking This research does not involve human subjects, human material, or human data.


We have recently shown that IgGs from serum and cerebrospinal fluid (CSF) of MS patients are active in hydrolysis of DNA and myelin basic protein. According to literature data, anti-DNA and anti-MBP abzymes may promote important neuropathologic mechanisms in this chronic inflammatory disorder and in MS pathogenesis development. At the same time, the involvement of antibodies with amylase activity in the pathogenesis of any autoimmune disease has not yet been identified. Electrophoretically and immunologically homogeneous IgGs were obtained by a sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We are able to present the first unpredictable evidence showing that IgGs from CSF possess amylase activity and efficiently hydrolyze maltoheptaose; their average specific Ab activity is ~30-fold higher than that of antibodies from sera of the same MS patients. Specific average RA (SAA) for IgGs from healthy volunteers was approximately ~1000 lower than that for MS patients. In addition, it was shown that a relative SAA of total proteins of CSF (including Abs) ~15-fold lower than that for purified IgGs, while the relative SAA of the total sera protein is higher than that of sera IgGs by a factor of 1033. This result speaks in favor of the fact that amylolytic activity of CSF proteins is mainly caused by the activity of amylase abzymes. One cannot exclude, that amylase abzymes of CSF can play a, as yet unknown, role in the pathogenesis of MS. Some possible reasons of these findings are discussed. Multiple sclerosis (MS) is a chronic demyelinating pathology of the central nervous system presenting a serious medical and social problem. Its etiology remains unclear, and the most valid theory of its pathogenesis assigns the main role in the destruction of the myelin-proteolipid shell of axons to inflammation, related to autoimmune reactions  and natural abzymes are novel biological catalysts that have attracted a lot of interest in recent years (reviewed in –8). ArtiNatural abzymes hydrolyzing DNA, RNA, oligopeptides and proteins are present in serum of patients with several autoimmune and viral diseases (reviewed in –17). HeaIt has recently been shown that myelin basic protein (MBP)-hydrolyzing –25 and DFor a long time it was not clear whether DNA- and MBP-hydrolyzing antibodies can exist only in the blood of patients with MS and SLE, or if they may also be found in the cerebrospinal fluid. We have recently shown, that the average content of IgGs in their sera is about 195-fold higher than that in their CSF , 33. We IgGs and IgMs from sera of patients with several autoimmune diseases –38 and sIn the present study we have compared a relative content of total protein and IgGs in sera and in CSFs of MS patients. Using different approaches, for the first time, we produce very strong direct evidence that amylase activity is intrinsic to IgGs from CSF of MS patients and we compare other parameters characterizing CSFs and sera of MS patients.Fifteen patients (11 women and 4 men) satisfying the criteria for clinically or laboratory-supported definite MS according to , 43 wereThe relative concentrations of total protein of CSFs (range 0.26–0.66 mg/ml) and sera (47–74 mg/ml) of fifteen MS patients varied in different ranges. The average concentration of total protein in the serum (62.0±6.7 mg/ml) was ~129-fold higher compared with CSF  as well health-IgGmix  was confirmed by SDS-PAGE with silver staining, which showed a single band under non-reducing conditions and two bands corresponding to the heavy and light chains after reduction  and sera (ser-IgGmix) efficiently hydrolyze maltoheptaose (MHO) (see below). Then, electrophoretically homogeneous MS csf-IgGmix preparation was used to prove that amylase activity of IgGs from the CSF of MS patients is an intrinsic property of these Abs , characterizing IgGs from serum and CSF at a fixed concentration of MHO  functions and sum of these characteristics) in the case of 49 patients with MS, was carried out . For theThe groups of primary progressing remitting course and secondary progressing course of MS patients were not “homogenous” with respect to the patients’ characteristics, and their further subdivision using cluster and factorial analysis revealed high statistically significant correlation coefficients . For exa-, and auto-Abs result in the formation of demyelinization nodi and the interruption of axon conductivity. The neurodegenerative phase of MS that ensues thereafter is directly connected to the neural tissue destruction in these patients [The absence of a definite dependence between titers of anti-DNA and anti-MBP Abs and these parameters with standard clinical indices may be caused by several reasons. MS is an extremely multifactorial disease, in which similar pathomorphological and clinical indices manifested as MS may result from very different underlying processes and conditions , 54. Forpatients –55. Therpatients –55. TherIn general, all data obtained demonstrate that the amylase activity is an intrinsic property of IgGs deriving from CSF and sera of MS patients. It is extremely unpredictable result showing that average amylolytic activity of IgGs from CSFs is 30-fold higher, than that for IgGs from sera. This result correlates with previously published data: average DNase and MBP-hydrolyzing activities of IgGs from CSFs are respectively 49- and ~54-fold higher than those for abzymes from sera , 33. It We have previously shown that the appearance of abzymes specifically hydrolyzing DNA, MBP, and significant increase in amylase activity is among the earliest and clearest signs of autoimmune reactions in a number of autoimmune diseases when titers of Abs to various auto-antigens have not yet increased significantly and correspond to their ranges for healthy donors , 49–51. Most chemicals, maltoheptaose, Protein G-Sepharose, and the Superdex 200 HR 10/30 column were from Sigma or GE Healthcare.Fifteen consecutive MS patients , satisfying the criteria for definite MS according to the classification of McDonald  and admiBlood and CSF sampling protocols were confirmed by the local committee for medical ethics in research (Comitato Etico della Provincia di Ferrara) that approved our study in accordance with the Helsinki ethics committee guidelines including a written consent of patients confined to present of their blood and CSF for diagnostics of a possible disease and scientific purposes. The protocol was approved on May 31 2007 and it was focused on the creation of a biological bank of CSF and serum samples, and related clinical data of patients with MS and other neurological diseases including: a) a study of potential markers  for diagnostic and prognostic significance in diseases of the nervous system; b) specific antibodies directed against antigens, potential exogenous and / or endogenous; c) presence of pathogens (mostly viruses or bacteria) for association studies and pathogenesis; d) neurotransmitters and their metabolites; e) a study of different properties of different markers.All participants provided their written consent before inclusion and the study design was approved by the Regional Committee for Medical Ethics in Research.CSF and serum samples were collected under sterile conditions and stored in aliquots at –80°C until assay. “Cell-free” CSF samples were obtained after centrifugation, at room temperature, of specimens obtained by atraumatic lumbar puncture performed for diagnosis purposes in the absence of contraindications. Serum samples derived from the centrifugation of blood specimens withdrawn by puncture from an anterocubital vein at the same time of a CSF extraction. Paired CSF and serum samples from MS patients were stored and measured under exactly the same conditions. Informed consent was given by all patients before inclusion and the study design was approved by the Regional Committee for Medical Ethics in Research. CSF and serum IgG levels were measured by immunochemical nephelometry with the Beckman Immage 800 Immunochemistry System  according to the procedure of Salden et al. .In all cases, protein concentration in the intact CSF, sera of MS patients and final solutions of Abs was measured using Bradford assay with a bovine serum albumin standard.Relative concentrations of total IgGs in the intact CSF and in sera of MS patients were analyzed using a special quantitative isoelectrofocusing and immunoblotting test system according to the standard manufacturer’s protocol and equipment . Concentration of IgGs (mg/ml) was estimated using calibration curves obtained according to the standard manufacturer’s protocol.Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF and serum proteins on protein A-Sepharose and FPLC gel filtration similarly to , 33. In IgGs from CSF were incubated in 50 mM glycine-HCl (Ph 2.6) containing 0.2 M NaCl for 20 min. at 25°C. Separation of the IgGs under “acid shock” conditions was carried out by FPLC gel filtration on a Superdex 200 HR 10/30 column equilibrated with 50 mM glycine-HCl (pH 2.6) containing 0.1 M NaCl as previously described , 33. AftIn some cases, electrophoretically homogeneous IgGs were chromatographed on Sepharose bearing immobilized polyclonal mouse IgGs against human IgGs. The protein was applied to the column (1 ml) equilibrated with 20 mM Tris-HCl (pH 7.5) containing 0.1M NaCl and the column was washed with the same buffer containing 0.3 M NaCl. Abs were eluted in 0.1 M glycine-HCl (pH 2.6), neutralized, dialyzed and sterilized as described above , 33. All3, 1.67 mM MHO and 0.001–0.2 mg/ml of IgGs was incubated for 1–6 h at 37°C. In the case of IgGs from sera of healthy donors we used a higher concentration of IgGs (0.25–0.5 mg/ml) and a longer time of incubation (12–36 h). Products of hydrolysis were identified by TLC on Kieselgel plates (Merck) using 1-butanol-acetic acid-H2O (12:4:4). The activities of IgGs were determined from the scanning data as a relative percentage of oligosaccharide in the spots of MHO and its hydrolyzed forms. All measurements were taken within the linear regions of the time courses and Ab concentration curves. If the Ab activity was low (< 5–10% of hydrolysis) the incubation time was increased to 5–36 h. If MHO hydrolysis exceeded 40%, the concentration of Abs was decreased 2-100-fold depending on the sample analyzed. Finally the catalytic RAs were normalized to 1 mg/ml IgGs and 1 h of incubation.Amylase activity was analysed as in , 50. TheSDS-PAGE analysis of Abs for homogeneity and for the polypeptide spectrum of sera and CSF was performed in a 5–16% gradient gel containing 0.1% SDS (Laemmli system) as described in , 33. The2O. Then 2-3-mm cross sections of longitudinal slices of the gel were cut up and incubated with 50 μl 50 mM Tris-HCl, pH 7.5, containing 50 mM NaCl for 6 days at 4°C to allow protein refolding and eluting from the gel. The solutions were removed from the gels by centrifugation and used for assay of MHO hydrolysis as above described. Parallel control longitudinal lanes were used for detecting the position of IgG on the gel by Coomassie R250 staining.Analysis of amylolytic activity of MS IgGs from CSF and sera after SDS-PAGE was performed similarly to the analysis of the amylolytic and proteolytic activities of different abzymes –21, 37. 2, 1 mM EDTA, 50 mM Tris-HCl, pH 7.5, 2 mM ATP and 0.2–0.5 mg/ml IgG. The reaction mixtures were incubated for 12–36 h at 37°C. The products of ATP hydrolysis were analyzed by thin-layer chromatography in 0.25 M KH2PO4 (pH 7.0) on PEI-cellulose plates (Merck) using the system dioxane—10% NH4OH- Н2O (6:1:4). After chromatography, the plates were dried and the positions of various products were identified. The relative amount of products was calculated using scanning data.ATP-hydrolyzing activity was analyzed as in , 51. Reat-test, p < 0.05 was considered statistically significant.The results are reported as mean ± S.E. of at least three independent experiments for each sample analyzed. Errors in the values were within 7–10%. The correlation coefficients (CC) between sets of different samples were analyzed using Statistica 10 (StatSoft Inc.). The differences between samples were analyzed by the Student’s


Ventriculoperitoneal shunts (VPS) are a critical feature of management of pediatric hydrocephalus. Unfortunately, shunt failure is often unrecognized before significant symptoms and focal signs present, requiring costly emergency room visits and neuroimaging. Ventricular CSF biomarkers of neuropathology and neurodevelopment are emerging as critical predictors of real-time physiology in pediatric hydrocephalus. Moreover, in the future, biomarkers of neuropathology could be pre-operatively analyzed from shunts in question, reducing unnecessary surgical exploration. In this pilot study, CSF samples taken during surgical exploration were compared, hypothesizing that that two critical CSF proteins, amyloid precursor protein (APP) and neural cell adhesion molecule (NCAM-1), known to be elevated in untreated hydrocephalus, may discriminate between functioning and malfunctioning shunts.Approval from the Washington University Human Research Protection Office was acquired, and a pediatric CSF repository was established. All patients prospectively enrolled were between 0-4 years of age and were undergoing surgical exploration of proximal, valve, and distal components of VPS for suspected CSF diversion failure. CSF was acquired in the operating room under sterile conditions, transported on ice, centrifuged, and supernatant was stored at -80°C until experimental analysis. Enzyme-linked immunosorbent assay (ELISA) was used to examine CSF concentration of APP and NCAM-1. Total protein was assessed using a standard Pierce Bicinchoninic Acid (BCA) protein assay kit. A microplate reader was used to assess biomarker concentration (ng/mL). Mann-Whitney U analysis was used for comparison of these non-parametric data, with statistical significance threshold set a priori at p<0.05.Twenty eight patients were found to have malfunctioned VPS, while 5 were found to have functioning VPS. There were no significant differences in age at the time of ventricle CSF sample (p=0.07), levels of total CSF protein (p=0.335), total APP (p=0.516), or total NCAM (p=0.816) concentrations. However, due to the fact that there is significant inter-individual variation in CSF total protein levels, we compared normalized APP (nAPP) and normalized NCAM-1 (nNCAM) between the two groups.Patients found to have nonfunctioning shunts had a significantly elevated nAPP (p=0.044) and tended to have elevated nNCAM (p=0.061) when compared to patients with functioning shunts.nAPP and nNCAM concentrations in ventricular CSF may be elevated in malfunctioning VPS, when compared to similarly aged patients with functioning shunts. While this is a small pilot study, active enrollment continues of new patients into this study given its potential implications.


We aimed to assess the feasibility of determining Alzheimer's disease cerebrospinal fluid (CSF) cut points in small samples through comparison with amyloid positron emission tomography (PET).1–42 and total tau/Aβ1–42 ratio, and florbetapir PET. We compared CSF measures with visual and quantitative  PET measures of amyloid.Twenty-three individuals  had CSF measures of amyloid beta (Aβ)1–42 of 630 ng/L, and 20 of 20 on tau/Aβ1–42 ratio . Three discordant cases had Aβ1–42 levels between 403 and 729 ng/L and tau/Aβ1–42 ratios of 0.54–0.58.Seventeen of 23 were amyloid-positive on visual reads, and 14 of 23 at an SUVR of ≥1.1. There was concordance (positive/negative on both measures) in 20 of 23, of whom 19 of 20 were correctly classified at an Aβ1–42 ratio appears robust at predicting amyloid status, although there are gray zones where there remains diagnostic uncertainty.Comparing amyloid PET and CSF biomarkers provides a means of assessing CSF cut points in vivo, and can be applied to small sample sizes. CSF tau/Aβ Although not in routine clinical use at our center, we also measured Aβ1–40. We preferentially chose individuals with CSF Aβ1–42 levels in a potential border zone range of 400–700 pg/mL. Each patient's cognition was assessed using the Addenbrook's Cognitive Examination III, scored out of 100 We recruited 23 individuals: 19 patients with a range of dementia syndromes and four healthy controls. As part of their clinical evaluation, each had a diagnostic lumbar puncture, with CSF samples obtained using a 22G Quincke needle. Optimum CSF handling and transfer procedures were used Each patient had an F18 florbetapir PET scan on a Siemens 3-T PET/MR unit, with a 50-minute dynamic acquisition commencing immediately after intravenous injection of 370 MBq of florbetapir. A volumetric T1-weighted MRI scan was acquired concurrently. Attenuation correction was performed using synthetic computed tomographies (CTs) generated from the MR images The four age-matched healthy controls previously had a florbetapir PET/CT scan as part of another study, with a separate T1-weighted MRI acquisition. These images were processed in the same way as described previously, excluding the generation of synthetic CTs. Clinical studies were approved by the Queen Square Research Ethics Committee.PET images were analyzed in two ways. First, three trained nuclear medicine physicians blinded to the clinical diagnosis visually rated the images positive/negative according to clinical criteria 1–42, Aβ1–42/Aβ1–40, tau/Aβ1–42, and p-tau in subjects rated amyloid positive/negative on visual reads, and based on SUVR. Linear regression was used to assess the relationship between CSF and SUVR, covarying for the interval between lumbar puncture and PET. A secondary analysis compared amnestic and nonamnestic AD clinical syndromes for each of the CSF biomarkers.Statistical analyses were performed in STATA version 12.0 . Independent of clinical diagnosis, we compared CSF Aβ31–42 ranged from 343 to 1199 ng/L, tau/Aβ1–42 0.11–2.54, and p-tau 14–227 g/L.Patients and controls were well matched for age (63.7 ± 7.6 vs. 62.9 ± 7.0). Nine patients had amnestic and 10 nonamnestic  clinical syndromes . CSF exaSeventeen of 23 participants were rated as amyloid-positive on visual assessment. SUVRs ranged from 0.87 to 1.66. At an SUVR cutoff of 1.10, 14 of 23 were amyloid-positive. Comparing SUVR and clinical reads, 20 were concordant ; and three discordant , Table 21–42 , CSF Aβ1–42/Aβ1–40 , CSF tau/Aβ1–42 , and CSF p-tau , with no evidence for an influence of duration between CSF sampling and scanning.The SUVR correlated with CSF Aβ1–42 ratio cut point of 0.52 At a CSF tau/AβP = .092), which was higher in typical AD (91.6 ± 63.4 ng/L) compared with atypical AD , there was no significant age difference between the typical (amnestic) AD cases and the atypical (nonamnestic) AD cases (64.3 ± 5.1 vs. 63.8 ± 10.6). When comparing the two subgroups for each CSF biomarker, there were no significant differences, although trend significance was reached for p-tau , Aβ1–42/Aβ1–40 ratio , and p-tau . A CSF Aβ1–42 cutoff of ∼630 ng/L is very similar to those proposed by other recent studies 1–42 cut point Considering only individuals with concordant positive/negative PET clinical reads and SUVRs, there was almost complete separation (19 of 20 correctly classified) at a CSF Aβ1–42 (403–729 ng/L) and p-tau (26–49 ng/L). The tau/Aβ1-42 ratios for all three cases were remarkably similar, and in a “gray zone” between 0.54 and 0.58, very close to the previously proposed optimal cut off (0.52) 1–42 ratio of 0.52 had very good sensitivity/specificity for determining amyloid status, these results suggest that rather than a strict dichotomy, introducing a gray zone  might be more appropriate. However, the significantly narrower gray zone for tau/Aβ1–42 than for the other CSF measures assessed is consistent with previous findings that this is likely to be the most robust marker for underlying AD pathology 1–42/Aβ1–40 has until now been used less commonly in clinical practice than the other markers but does appear to potentially provide more precise separation of AD-positive and AD-negative cases compared with Aβ1–42 alone 1–42/Aβ1–40 values between the PET concordant and PET discordant groups would suggest, consistent with findings from other studies 1–42/Aβ1–40 may not be as reliable a marker as tau/Aβ1–42.Although the concordant cases produced relatively clear cut points, the three discordant cases showed considerable overlap between the positive/negative ranges for Aβ1–42 varied by over 400 ng/L, from 192 ng/L to 638 ng/L The gold standard for setting cut points to dichotomize any surrogate biomarker of a continuous biological variable would be to calibrate the cut points against direct measurements of the pathologic entity in question; in this case, brain amyloid and tau. However, the only available method of directly measuring these proteins in the brain is to perform an autopsy. Collecting CSF in a cohort of end-of-life patients to then validate postmortem is not straightforward, and it is unlikely to be feasible to collect sufficient numbers of samples in a relevant time frame in any individual center. In the absence of postmortem pathologic confirmation of diagnosis, we, like a number of other centers, have previously tried to determine individuals with AD or non-AD pathology based on clinical diagnosis, and defined cut points accordingly All amyloid-positive individuals in our study had dementia, thus fulfilling IWG-2 criteria for AD 1–42, suggesting that this is unlikely to have influenced results; and a previous large study has demonstrated excellent concordance between SUVR measurements made in different centers and pipelines This study has a number of limitations. The sample size is small, although in keeping with our aim to assess methods for determining cut points in samples appropriate for single centers. There were in some cases significant delays between the CSF and PET scan, although there was no evidence that this influenced the relationship between the two measures; and pragmatically, the fact that delays of some months between CSF and PET do not have a significant influence means that applying this approach in other clinical centers is more feasible. All the patients were scanned on the same PET/MR unit, whereas the controls were scanned on a PET/CT. However, all the controls were very clearly and consistently amyloid negative based on visual read, SUVR, and Aβ51–42 cut point of ∼630 ng/L and tau/Aβ1–42 ratio of ∼0.52 provide good group separation, these data provide evidence that incorporating biomarker gray zones  may be more biologically plausible.1.Systematic review: The authors used PubMed to review the literature pertaining to cerebrospinal fluid (CSF) cut points and, in particular, (1) what methods have been used to determine cut points and (2) what the optimum cut points are thought to be.2.1–42 ratio was found to be the most robust CSF measure. Although cut points have utility in clinical practice, these data show that in some cases there may be discordance, suggesting the need for biomarker gray zones to reflect diagnostic uncertainty.Interpretation: Our results demonstrate the value of comparing CSF and positron emission tomography biomarkers in relatively small cohorts to assess local CSF cut points. In keeping with previous studies, the tau:amyloid beta3.Future directions: Replication of our approach to determine CSF cut points in other centers will provide further validation. Other future studies should aim to further assess and quantify CSF biomarker gray zones to improve understanding of how best to incorporate these in to clinical practice.Comparing amyloid PET and CSF biomarkers provides a means of assessing CSF cut points in vivo, and can be applied to small sample sizes. Although in unequivocal cases, a CSF Aβ


Several immunomodulatory checkpoint inhibitors have been approved for the treatment of patients with advanced melanoma, including ipilimumab, nivolumab and pembrolizumab. Talimogene laherparepvec is the first oncolytic virus to gain regulatory approval in the USA; it is also approved in Europe. Talimogene laherparepvec expresses granulocyte–macrophage colony-stimulating factor (GM-CSF), and with other GM-CSF-expressing oncolytic viruses in development, understanding the clinical relevance of this cytokine in treating advanced melanoma is important. Results of trials of GM-CSF in melanoma have been mixed, and while GM-CSF has the potential to promote anti-tumor responses, some preclinical data suggest that GM-CSF may sometimes promote tumor growth. GM-CSF has not been approved as a melanoma treatment. We undertook a systematic literature review of studies of GM-CSF in patients with advanced melanoma (stage IIIB–IV). Of the 503 articles identified, 26 studies met the eligibility criteria. Most studies investigated the use of GM-CSF in combination with another treatment, such as peptide vaccines or chemotherapy, or as an adjuvant to surgery. Some clinical benefit was reported in patients who received GM-CSF as an adjuvant to surgery, or in combination with other treatments. In general, outcomes for patients receiving peptide vaccines were not improved with the addition of GM-CSF. GM-CSF may be a valuable therapeutic adjuvant; however, further studies are needed, particularly head-to-head comparisons, to confirm the optimal dosing regimen and clinical effectiveness in patients with advanced melanoma.The online version of this article (doi:10.1007/s00262-016-1860-3) contains supplementary material, which is available to authorized users. Malignant melanoma accounts for approximately 1 % of cancer deaths worldwide, equating to over 55,000 deaths from this tumor type in 2012 . The treBRAF V600 tumors [Several immunomodulatory agents work by overriding checkpoints in the cancer-immunity cycle, thus promoting the elimination of tumor cells by the immune system . Ipilimu0 tumors . Antibod0 tumors , aveluma0 tumors  and atez0 tumors ) are in More recently, oncolytic viruses expressing GM-CSF have been developed, and in 2015, talimogene laherparepvec, a modified herpes simplex virus type 1, became the first oncolytic virus to gain regulatory approval in the USA, where it is indicated for the local treatment of unresectable cutaneous, subcutaneous (s.c.) and nodal lesions in patients with melanoma recurrent after initial surgery. It has not been shown to improve overall survival (OS) or to have an effect on visceral metastases . TalimogGM-CSF Pivotal Trial in Melanoma (OPTiM), talimogene laherparepvec was compared with s.c. GM-CSF in patients with stage IIIB–IV unresected melanoma [In the randomized phase 3 Oncovexmelanoma . Studiesmelanoma  and a phmelanoma .GM-CSF is a hematopoietic growth factor that has pleiotropic effects on the immune system Fig. . It playOne of the mechanisms of cancer immunotherapy is the protection of anti-tumor immune cells from the suppressive effects of certain cell types, such as regulatory T cells (T-regs) and myeloid-derived suppressor cells (MDSCs). MDSCs are a heterogeneous population of cells that mediate tumor-induced immune suppression. The expansion and recruitment of MDSCs is mediated by several soluble factors, including GM-CSF . TherefoThere is some evidence to suggest that the dose of GM-CSF is important in determining immune activation, but other regulatory mechanisms may underlie this process . In normThere are few studies reporting data from direct comparisons between GM-CSF and other treatments in patients with advanced melanoma. Therefore, we conducted a systematic review of the available evidence on efficacy, immunological effects and safety of GM-CSF in adult patients with stage IIIB–IV melanoma.We systematically reviewed published English language studies according to a pre-specified protocol. We searched Embase  and PubMed . Abstracts from the annual congresses of the following organizations were searched for the period January 1, 2010–April 24, 2015: American Society of Clinical Oncology, European Association of Dermato-Oncology and Society for Melanoma Research. Complete search strings are listed in electronic supplementary material.Studies were included if they enrolled adults (≥18 years old) with advanced melanoma (defined as stage IIIB–IV) who received treatment with GM-CSF as monotherapy, as part of combination therapy or in an adjuvant setting. Studies that used modified viruses, cell lines, plasmid DNA or complementary DNA (cDNA) to deliver GM-CSF were excluded. Studies of prevention or detection of melanoma, or those that included patients with non-cutaneous melanoma , were also excluded.The systematic review process described here is compliant with the 2009 preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines . The titFor all studies that were deemed eligible for inclusion, and for those for which eligibility remained uncertain following the title/abstract screen, the full texts of the study manuscripts were reviewed by two independent reviewers to confirm or refute their eligibility. Data were extracted from full-text publications when available. Data from congress abstracts were included unless the same data had also been published in a peer-reviewed manuscript, in which case only the latter was included in the analysis. A protocol amendment was made at this stage and studies in which fewer than 10 patients received GM-CSF were excluded to focus the analysis on larger studies.All data were presented as reported by the authors of the publications analyzed. Mean or median values reported in the studies were used as a basis for summarizing the efficacy, immunological effects and safety of GM-CSF.n = 16), and there were six studies that actively compared GM-CSF with other treatments and three that made comparisons with historical controls.We identified 589 full-text articles and 44 congress abstracts Fig. . FollowiThe study designs and dosing schedules are described in Table Overall, six of the included studies made direct comparisons between GM-CSF treatment and non-GM-CSF treatments –51, and P = 0.01) [P = 0.01), but median progression-free survival (PFS) was not significantly different between treatment arms  [P = 0.04); however, there were high numbers of deaths in both treatment groups. The improvements in OS observed in the GM-CSF group may not necessarily be due to increased efficacy when GM-CSF is added to ipilimumab. Reduced toxicity and the resulting lower treatment dropout rate in the GM-CSF arm may have contributed to the observed OS improvement, but this remains to be determined. Furthermore, it is unclear whether treatment discontinuation in the ipilimumab group was due to adverse events or other reasons, such as disease progression [Significant improvements in OS in patients who received GM-CSF treatment as an adjuvant to systemic therapy were reported in two studies. A phase 2 randomized study reported significantly prolonged median OS when GM-CSF was added to ipilimumab compared with ipilimumab alone  . The 1-y = 0.37) . The res = 0.37) . In thisgression .P = 0.0004) [P = 0.0015), and multivariate analyses showed that maintenance biotherapy was predictive of PFS and OS [In another phase 2 study, patients who received a novel GM-CSF-containing maintenance biochemotherapy regimen after induction biochemotherapy had significantly longer median OS than historical controls who had received an identical biochemotherapy regimen without GM-CSF  . The medS and OS .P = 0.012) [Eroglu et al.  investig= 0.012) .GM-CSF used in combination with peptide vaccines had no significant effect on survival in two randomized phase 2 studies. In one study, median OS values for patients receiving no GM-CSF, GM-CSF 75 μg or GM-CSF 100 μg were 10, 6 and 9 months, respectively. This peptide vaccine was deemed clinically ineffective . The othP = 0.02). The low patient numbers and lack of important information on patient characteristics, such as mutation status or subsequent therapy, make the interpretation of this data set difficult.In studies that included patients who received adjuvant GM-CSF following complete surgical resection, most of the participants had stage IIIB or higher disease , 54, 55.P = 0.001). The 1-year OS and 2-year OS were also significantly higher in the GM-CSF group than in the control group  [Spitler et al.  evaluatectively) .In another study, prolonged GM-CSF treatment was given to patients for at least 3 years following surgical resection. The 5-year MSS rates were 67 and 40 % for patients with stage III and IV disease, respectively . The majOverall, 16 studies in which GM-CSF was not compared with another treatment arm were included. These are summarized in Table Several single-arm studies evaluated the use of GM-CSF in combination with chemotherapy , 56–62. P = 0.027) [Two studies assessed biochemotherapy followed by maintenance biochemotherapy , 62. Boa= 0.027) . O’Day eScheibenbogen et al.  administIn a phase 2 trial of GM-CSF in combination with an autologous DC vaccine, 1-year OS and 5-year predicted OS were 85 and 54 %, respectively . DespiteIn a phase 2 trial, patients with stage IV disease who had undergone surgical resection were given a peptide vaccine and adjuvant GM-CSF followed by IFN-α. After one treatment cycle, 55 % of patients had stable disease and 5 % were disease-free. The median OS was 583 days . Two stuP = 0.0045) [Most studies reported evidence of immune activation, and these data are summarized in Table  0.0045) . When im 0.0045) . Markovi 0.0045)  reported 0.0045) .Table 3EAdamina et al.  used GM-Although signs of peptide-specific immune activation were present in most studies, it appears likely to be non-functional due to the lack of correlation with anti-tumor responses . Celis  suggest P = 0.01) [P < 0.001) and CD8+ (P = 0.007) cells and a minor increase in the number of natural killer (NK) cells when GM-CSF was used as an adjuvant to chemotherapy. In another study, in which GM-CSF was given as an adjuvant to chemotherapy, induction of melanoma-specific T cells was observed in one of four evaluable patients. GM-CSF was used to support granulocyte recovery in this study, and IL-2 was also given to patients at the same time [GM-CSF enhanced the immune response to ipilimumab, as measured by activation of inducible costimulator (ICOS) T cells. In a phase 2 study evaluating ipilimumab with or without GM-CSF, the median change in CD8+ ICOS T cells was significantly greater in patients who received ipilimumab and GM-CSF than in those who received ipilimumab alone  . Groenew = 0.01)  reportedame time .When GM-CSF was combined with tumor-derived autologous heat-shock protein gp96, increased T-cell reactions against autologous and allogeneic melanoma cells were observed relative to baseline in 53.8 % (7/13) and 26.7 % (4/15) of patients, respectively. Increases in the number of NK cells were also observed in 61.1 % (11/18) of patients . In anot2.This is the first systematic review of the efficacy of GM-CSF in patients with advanced melanoma. Most studies were phase 1 or 2, but study phase was not reported in some studies; no phase 3 data were available. The study groups varied in size, but most enrolled fewer than 50 patients, and those that had multiple comparator arms often had fewer than 10 patients per treatment group. Furthermore, several studies used historical controls or calculated survival rates as comparators, which makes conclusions from these studies problematic. A range of dosing schedules and routes of administration was used for GM-CSF, but GM-CSF was most often delivered by s.c. injection at a dose of 125–250 mg/mThere was some evidence of clinical benefit in patients who received GM-CSF in combination with ipilimumab  or as paThe role of chemotherapy in immune activation is less clear, although there is accumulating evidence to suggest that off-target effects of chemotherapy may modulate the innate and adaptive arms of the immune system . ChemothActivation of the immune response may correlate with anti-tumor activity. Boasberg et al. assessed a novel biochemotherapy regimen containing GM-CSF and IL-2 and reported that median OS was significantly improved in patients exhibiting immune activation, as indicated by the development of vitiligo , compareGM-CSF in combination with peptide vaccines showed limited activity. Although T-cell activation was observed in several studies, this was not accompanied by a clinical anti-tumor response , 50, 63.Generation of anti-GM-CSF antibodies could limit the activity of GM-CSF in vaccines and other treatments. Mice treated with adenoviruses and plasmids engineered to express GM-CSF produced neutralizing antibodies against GM-CSF that were associated with suppressed CD8+ T-cell responses . When GMThe use of GM-CSF as an adjuvant to surgery has shown some clinical benefit, including improvements in OS , 55. AltOverall, GM-CSF in combination with peptide vaccines appears insufficient to induce an immune response that correlates with anti-tumor activity, whereas GM-CSF in combination with chemotherapy , 58, 59,This systematic review has several limitations. Few studies made direct comparisons between GM-CSF and other treatments, and most involved the use of GM-CSF as an adjuvant to other therapies. Therefore, it was often difficult to distinguish between the effect of the treatment and the effect of GM-CSF on clinical and immunological outcomes. Wide ranges of GM-CSF doses, treatment regimens and comparators were evaluated in the included studies. Establishing the optimal dose of GM-CSF for adjuvant treatment is important to achieve the maximum response , and theIn conclusion, it is clear that GM-CSF has been used as an adjuvant in many different clinical trial settings; however, evidence for clinical efficacy is controversial. Some clinical benefit has been observed in patients who received GM-CSF in combination with surgery, chemotherapy or immunomodulatory agents. In general, however, outcomes for patients receiving peptide vaccines were not improved when GM-CSF was used as an adjuvant. GM-CSF is a promising therapeutic adjuvant, but there is a lack of controlled phase 3 trials investigating the direct effects of GM-CSF in patients with advanced melanoma. Small, single-center studies conducted mostly in academic institutions have not led to the design and implementation of well-controlled phase 3 clinical studies. The results presented here indicate that further studies are needed to identify the optimal treatment regimen and effectiveness of GM-CSF in patients with advanced melanoma.Supplementary material 1 (PDF 31 kb)Below is the link to the electronic supplementary material.


According to the traditional understanding of cerebrospinal fluid (CSF) physiology, the majority of CSF is produced by the choroid plexus, circulates through the ventricles, the cisterns, and the subarachnoid space to be absorbed into the blood by the arachnoid villi. This review surveys key developments leading to the traditional concept. Challenging this concept are novel insights utilizing molecular and cellular biology as well as neuroimaging, which indicate that CSF physiology may be much more complex than previously believed. The CSF circulation comprises not only a directed flow of CSF, but in addition a pulsatile to and fro movement throughout the entire brain with local fluid exchange between blood, interstitial fluid, and CSF. Astrocytes, aquaporins, and other membrane transporters are key elements in brain water and CSF homeostasis. A continuous bidirectional fluid exchange at the blood brain barrier produces flow rates, which exceed the choroidal CSF production rate by far. The CSF circulation around blood vessels penetrating from the subarachnoid space into the Virchow Robin spaces provides both a drainage pathway for the clearance of waste molecules from the brain and a site for the interaction of the systemic immune system with that of the brain. Important physiological functions, for example the regeneration of the brain during sleep, may depend on CSF circulation. It is also believed that CSF circulates through the ventricles, the cisterns, and the subarachnoid space ultimately to be absorbed into the venous blood at the level of the arachnoid villi. Minor portions of CSF may be drained into the cervical lymphatics that run via the perineural spaces of the cranial nerves. Traditionally, the circulatory character of CSF flow is accepted and coined as the "third circulation"  system includes the cerebral ventricles as well as the spinal and brain subarachnoid spaces, cisterns and sulci. The traditional understanding of CSF physiology assumes that 80% of CSF is secreted by the choroid plexus into the ventricular cavities. Other structures, e.g. the brain parenchyma, add the remaining 20%. The rate of CSF formation in humans is 0.3 – 0.4 ml minThe traditional understanding of CSF physiology is mainly based on animal experimentation, which was criticized as early as 1947. It was argued that neuropathological observations fail to support the experimentally-based hypothesis that the origin of the cerebrospinal fluid is largely from the choroid plexus. Recent Most CSF is formed in the cerebral ventricles. Possible sites of origin include the choroid plexus, the ependyma, and the parenchyma. AnatomiDue to its glandular appearance and ventricular location, the choroid plexus has been suggested to be the major site of CSF secretion. This view was mainly based upon the historical canine experiments of Dandy. In these experiments the foramen of Monro was occluded and a choroid plexectomy of one lateral ventricle was performed. The authors reported collapse of the ventricle without choroid plexus and dilatation of the other ventricle. They coThere were two other sets of experiments that were thought to be "crucial" in support of Dandy’s thesis: First, Historically, the absorption of CSF into the circulating blood is most notable across the arachnoid villi,31,32. I-1, while no movement was observed during the expiration phase[Considerable portions of CSF may be absorbed into the cervical lymphatics. The peron phase. It is ion phase).3H2O from the bloodstream enters osmotically loaded cerebrospinal fluid significantly faster[Extracranial organs feature fluid exchange across the capillary bed that is driven by hydrodynamic and osmotic pressure gradients. However, absorption of CSF into cerebral capillaries has been disputed because it was thought that the absorption of CSF is not dependent on osmotic forces. This notion was based on experiments in which dextran solutions of different osmolality were infused into the ventricles of cats at a constant pressure of 27 mmHg. The measured infusion rate, which should equal the CSF absorption rate, decreased by the same extent. The decrease of the absorption rate was explained by the increased CSF viscosity. Interesy faster. Since, y faster. These s-1. The validity of results obtained in this way was criticized because the Masserman technique assumes that neither formation nor absorption rates are changed by alterations in pressure. However, the absorption of CSF varies greatly with changes in intracranial pressure[In 1931, Masserman calculated the rate of CSF formation in patients by measuring the time needed for the CSF pressure to return to its initial level following drainage of a standard volume of CSF by lumbar puncture. After dpressure,50. Modipressure). Despit-1, the maximum absorption capacity was 1.3 ml.min-1[-1[The ventriculo-cisternal perfusion ("Pappenheimer") technique represents a more quantitative approach for the assessment of CSF formation rate. Inulin or other macromolecules, which pass through the ventricular space without being absorbed, are infused at a constant rate into the cerebral ventricles. CSF formation is calculated from the measurement of the extraventricular  CSF concentration of inulin. It is assumed that any dilution of inulin between the inflow cannula and outflow cannula results from the admixture of freshly formed CSF. In addition, the test procedure allows for the calculation of the CSF absorption rate from the clearance of inulin at the extraventricular site . An impo ml.min-1. These r ml.min-1. Further ml.min-1. Though  ml.min-1. Finding.min-1[-1,56. Henc.min-1[-1.The concept of the "third circulation" suggesting that CSF flows through the ventricles, cisterns and subarachnoid space (SAS) and is reabsorbed into the blood at the arachnoid villi, was introduced by Cushing in 1926,58. ThisAnatomically the VRS refers to a histologically-defined space, which surrounds blood vessels (arterioles and venules) when penetrating from the subarachnoid space into the brain tissue. Originally, it was thought that the VRS is connected to the subarachnoid space, allowing for a free fluid communication. It was suggested that interstitial fluid may be outwardly drained along these pathways into the SAS and eventually towards the arachnoid villi. Later tThe current understanding of the microscopic anatomy of the VRS is more complex Figure . ActuallUltrastructural electron microscopic studies agree that pial membranes separate the VRS from the cortical subarachnoid space,68,70. Set al. found no spread of HRP from the subarachnoid space into the VRS[Although pial cell layers obviously separate the VRS from the cortical subarachnoid space, physiologically there is strong evidence indicating that fluid circulates along the VRS Figure . Followi the VRS. Also, a the VRS. Further the VRS.Since there is obviously at least some circulation of CSF into and out of the VRS, it raises the question how fluid and tracers could cross the pial membranes separating the VRS from the subarachnoid space. Ultrastructure studies have depicted the pial barrier as a delicate, sometimes single-cell layered structure. There aIt has also been shown both experimentally and clinically that the PVS and possibly more importantly intramural pathways between the basement membranes of the wall of arterioles and arteries provide drainage for the ISF and waste molecules of the brain. There is experimental evidence that the para-arterial drainage pathways are connected to the lymphatics of the exterior skull base,82. Actu-1 min-1[Traditionally, movement of fluids through the brain interstitial space has been attributed to diffusional processes-91, whic-1 min-1,61, revi-1 min-1. Very re-1 min-1.-1 and a CSF turnover rate of more than 140 times a day were calculated. This is far greater than the traditional views of CSF physiology[The discovery of water transporters (‘water channels’) located at the end-feet processes of astrocytes has decisively improved our understanding of the physiology of the blood brain barrier and has led to the concept that large water fluxes take place continuously between the different compartments of the brain, i.e. the blood, CSF and ISF (reviewed in-98). Intysiology. Of noteysiology and AQP4ysiology is simil+/Cl- cotransporter is co-localized with the Na+/K+ -ATPase. Together, these transporters expel water from the cell into the CSF space. Little is known about the water transport at the basolateral membrane. There is a K+/Cl- cotransporter, but its role is not yet well understood[CSF formation at the choroid plexus occurs in two stages: passive filtration of fluid across the highly permeable capillary endothelium and a regulated secretion across the single-layered choroidal epithelium. The choroidal epithelium forms a fluid barrier since tight junctions are expressed at the apical, CSF facing, cell membrane. The ratderstood. The molderstood,105,106.+/K+ - ATPase, on the brain side of the barrier. Accordingly, the BBB was proposed as a secretory endothelium, which produces ISF[Traditionally the properties of the blood–brain barrier (BBB) are considered to be those of the capillary endothelium in brain. This endothelium contrasts with that elsewhere in the body by being sealed with tight junctions, having a high electrical resistance and a low permeability to polar solutes. Early ruces ISF. Recent uces ISF-110). Thuces ISF. The roluces ISF. The astuces ISF. The roluces ISF,114, revuces ISF. It is auces ISF. Insteaduces ISF).The physiology of aquaporins (AQPs) and transporters in the brain has been comprehensively reviewed,116-118.+ released during neuronal activity; AQP1 plays a role in cerebrospinal fluid (CSF) formation, and AQP9 may play a role in energy metabolism[Currently, at least 14 different aquaporins have been identified,117. At tabolism. Positrotabolism. Structutabolism,124). Astabolism, AQP5 istabolism.-1). Accordingly it was discussed that AQP1-facilitated transcellular water transport accounts for only part of the total choroidal CSF production. As a more controversial possibility, it was suggested that the choroid plexus may not be the principal site of CSF production and that extrachoroidal CSF production by the brain parenchyma may be more important[217O is significantly reduced in ventricular CSF in AQP4 but not in AQP1 knockout mice. The authors concluded that AQP4 is more important for CSF production than AQP1[Notably, AQP1 is expressed in vascular endothelial cells throughout the body but is absent in the cerebrovascular endothelium, except in the circumventricular organs. As alremportant,127. Thehan AQP1,128.+ channel Kir4.1[AQP4 is strongly expressed in astrocyte foot processes at the BBB, glia limitans of brain surface and VRS, as well as ventricular ependymal cells and subependymal astrocytes. Actually, it is expressed at all borders between brain parenchyma and major fluid compartments,113,114.l Kir4.1. Since Kl Kir4.1.AQP4 is involved in water movements under pathological conditions + homeostasis during neuronal activity has been postulated . AQP4 diewed by and a teiewed by. Howeveriewed by). In AQPiewed by. Furtheriewed by. Howeveriewed by. Considenditions. Howevernditions.+/K+/2Cl- cotransporter, NKCC1, may contribute significantly to transendothelial water transport . In addiewed by). The idiewed by. As a pr sinuses. Figures-1[-1 in the craniocaudal direction[-1 was reported in children younger than two years[Phase-contrast magnetic resonance imaging (MRI) can provide quantitative blood flow velocity information in humans. It was -1-155. Basirection. Hence, wo years. Furtherwo years. In NPH wo years. Technicwo years. Unfortuwo years.There are numerous limitations of the early experiments that form our classical understanding of CSF physiology. Recent progress in neuroanatomy, molecular and cellular biology, and neuroimaging challenge the traditional model. The pillars of the classical model, i.e. CSF production at the choroid plexus, directed bulk flow and absorption across the arachnoid villi are currently being questioned. More recent experimental and clinical data have caused a growing number of researchers to reach the consensus that ISF and CSF are mainly formed and reabsorbed across the walls of CNS blood capillaries, which implies that there is no need for a directed CSF circulation from CP to the arachnoid villi. Eventually, a number of "unequivocal" findings, often more than 100 years old and still governing the customary understanding of CSF physiology, must be revised,158,159.et al. failed to reproduce the historical experiments of Dandy[et al. that CSF production and absorption just depend on hydrodynamic and osmotic gradients is not substantiated by current cellular and molecular biology findings. In fact, the proposed model does not consider the complex regulation of water movement between the brain compartments as discussed above. Finally, as in the original experiments of Dandy, the experiments of Klarica et al. may be criticized since they are surgically invasive and therefore results should be interpreted cautiously.However, the novel concepts are also challenged mainly by the lack of validated supporting data. For example, Klarica of Dandy, since nof Dandy. Subsequof Dandy. Since tof Dandy, they coof Dandy. Consideof Dandy. FurtherThere are similar concerns with the most recent publications of Nedergaard and her group. In a series of experiments, fluorescent tracers of different molecular weight were injected into the cisterna magna of mice. Applyinet al.[It should be noted that the glymphatic concept assumes transport from the SAS INTO the parenchyma along periarterial pathways. This notion is supported by previous findings of Rennels et al.. Howeveret al. and Wellet al.,70 suppoet al.. CurrentRegarding the glymphatic concept, one may criticize that it is based on two-photon laser scanning microscopy applying a 1 min scan time, which was optimized to acquire a 240 μm stack at 20 μm intervals. This appears to be a limitation of the scanning technique in terms of temporal resolution. It seems important to provide data with higher frequency imaging to clarify the direction of perivascular flow. Furtherin vivo two-photon laser scanning microscopy, fluorescent microscopy and measurements of the ISF volume comparing awake, asleep and anaesthesized animals. However, again, this is a very complex study design possibly prone to experimental errors: investigating animals with multiple brain catheters and fixated in a stereotactic or microscopic holder, one may assume that awake animals are under massive stress and may fell asleep just because of exhaustion. Although microdialysis was used to measure norepinephrine levels as a gauge for stress levels and norepinephrine did not increase in the experiments, important stress parameters may differ between the experimental groups, i.e. arterial blood pressure, venous blood pressure, stress hormone blood levels, heart and respiratory rate and blood gases. The fact that none of these parameters was recorded during the experiments is a major drawback, since each of the parameters may alter cerebral blood flow, cerebral blood volume, intracranial pressure and even the perivascular pump[A subsequent publication of Nedergaard’s group reports the exciting possibility of an increased clearance rate of brain waste along the glymphatic pathway during sleep. This colar pump. Each ofet al.[In spite of this criticism, the observation that astrocytes are involved in the clearance of interstitial waste molecules including soluble amyloid is exciting. In this regard the experiments of Iliff et al. revive tet al., who shoet al..The new findings do not render all of the previous, sometimes historical, work invalid. However, CSF researchers and clinicians have to recognize that much of the earlier findings need to be re-interpreted.For example, the discovery of aquaporins and other water transporters, all highly selective just for water molecules, implies that the extent of water exchange across the barriers may be heavily underestimated by the classical flow studies,49. The The authors declare no financial or non-financial competing interests.TB drafted and wrote the manuscript. ES, JM and PK commented and revised the manuscript. All authors have read and approved the final version of the manuscript.


The analysis of cerebrospinal fluid (CSF) is usually done under steady-state conditions, when proteins  reach diffusion equilibrium between blood and CSF. However, little data has been published on CSF analysis under non-steady-state conditions after therapeutic apheresis. By reducing serum proteins , while leaving CSF unchanged, therapeutic apheresis might cause spuriously altered intrathecal immunoglobulin fractions.Based on the incidental finding of plasma exchange-induced increased intrathecal immunoglobulin fractions in a cohort of 12 unsystematically selected patients with various neurological disorders, we retrospectively investigated CSF results that had been raised during routine diagnostic work-up from 41 consecutive neurological patients  treated with plasmapheresis or immunoadsorption in a tertiary care university hospital in whom lumbar puncture (LP) was performed after a varying number of treatments of therapeutic apheresis.Only when LP was performed 1 day after therapeutic apheresis, spurious quantitative intrathecal immunoglobulin (Ig) synthesis of at least one subclass  was found in 68.4 % of the patients, irrespective of the number of treatments, in all age groups and independent of other previous immunotherapies . This phenomenon occurred only transiently and was almost always accompanied by an elevation of the IgG index. In one patient, an elevated IgG index was noticed even 2 days after plasmapheresis. Neither quantitative Ig synthesis, nor elevated IgG index was observed when the LP was performed three or more days after therapeutic apheresis.Spurious quantitative intrathecal Ig synthesis and increased IgG index are common findings shortly after plasmapheresis or immunoadsorption due to altered serum immunoglobulin levels. Knowledge of this phenomenon is needed for clinicians to prevent false interpretations leading to unnecessary diagnostic and therapeutic procedures. Misdiagnoses can be avoided by considering the characteristic CSF constellation including absence of oligoclonal bands and the close temporal relation to therapeutic apheresis. IgG). In some inflammatory neurological disorders of infectious or autoimmune origin , the disturbance of immunoglobulin (Ig) and albumin quotients leads to an increase of the quantitatively calculated intrathecal Ig fractions indicative of an intrathecal antibody production by local B-cells. In this regard, intrathecal Ig synthesis strongly suggests a neuroinflammatory process  × 100 [%] established by Reiber et al. [In some patients LP was performed within, in others after a complete apheresis cycle consisting of up to six treatments. The methodology of CSF and serum analysis was equivalent in both centers and according to the German Society for Cerebrospinal Fluid Diagnostics and Clinical Neurochemistry. The following routine laboratory CSF data were collected from medical records: time interval between the last treatment of PE or IADS and LP; total protein concentration; albumin concentration in CSF and serum; albumin quotient; immunoglobulin  concentrations in CSF and in serum; immunoglobulin  quotients, as well as a quantitative analysis of intrathecal immunoglobulin  fractions  according to the formula Igr et al. ; IgG indr et al. ; and finU test was used for statistical analyses comparing CSF results before and after therapeutic apheresis within the Freiburg study group. A p-value of < 0.05 was regarded as statistically significant.Because of the unsystematic patient selection of the Berlin study cohort we did not pool data with the Freiburg study group. The two-tailed Mann–Whitney Of the 12 patients treated between 2013 and 2014, ten were females, and two were males. The average age was 46 years (range 22–77 years). Eight patients were diagnosed with autoimmune encephalitis, two with neuromyelitis optica, one with multiple sclerosis, and one with myelitis. In these 12 patients, between five and ten treatments of PE were conducted. Six of the 12 patients had an LP 1 day after PE that showed increased quantitative intrathecal immunoglobulin fractions , supporting the hypothesis of spurious intrathecal Ig synthesis. By contrast, the remaining six patients who had an LP more than 1 day after PE with a median time interval to CSF analysis of 18.0 days (range 3–26 days) did not demonstrate increased intrathecal immunoglobulin fractions. Of the six patients with spurious intrathecal Ig synthesis, one patient with autoimmune encephalitis and IgG as well as IgA synthesis had persistence of an increased IgA fraction in the further course, whereas the other five  had a CSF analysis before PE without increased intrathecal immunoglobulin fractions. In these six patients LP showing increased intrathecal Ig fractions was performed after a median number of three apheresis treatments (range 1–6 treatments).At University Medical Center Freiburg, 346 neurological patients who were treated with either PE or IADS between 2005 and 2014 were identified. The vast majority (234 cases) had to be excluded since no CSF analysis had been performed within two months after therapeutic apheresis; an additional 50 had incomplete CSF data . In addition, eight had neurological disorders with intrinsic intrathecal Ig synthesis  and had no other CSF analysis showing the absence of quantitative intrathecal Ig synthesis. In 13 patients, data on therapeutic apheresis were not available. Finally, 41 patients fulfilled all study criteria.The mean age at the time of therapeutic apheresis was 58 years (range 24–80 years); gender distribution was balanced , and in a variety of neurological diagnoses (Table data not shown). According to quantitative IF analysis (data not shown), none of the 22 patients who underwent LP more than 1 day after therapeutic apheresis showed elevated intrathecal Ig fractions. In one patient, an elevated IgG index was noticed 2 days after PE, but in none of the patients with a CSF analysis three or more days after therapeutic apheresis (data not shown). Strikingly, positive or predominant OCBs in CSF were detected in none of the 19 cases with elevated intrathecal Ig fractions or an elevated IgG index up to 2 days after therapeutic apheresis.Elevated intrathecal immunoglobulin fractions were seen after one or more treatments, in patients from all age groups, independent of other previous immunotherapies es Table . Elevatees Table . Five ofBerlin study cohort), as well as in 13 of 19 patients (68.4 %) from a well-defined, independent, confirmatory cohort of patients with various neurological disorders (the Freiburg study group). Additionally, 18 of these 19 patients (94.7 %) had spuriously elevated IgG indexes. While being well-acknowledged among CSF experts, to our knowledge this is the first systematic study on these spurious phenomena.In this study, we describe spurious intrathecal immunoglobulin (Ig) synthesis of all three subclasses  occurring 1 day after therapeutic apheresis in 6 of 12 selected patients  with respect to molecular size [Freiburg study group mainly consisted of patients with neurological diagnoses typically not associated with intrinsic intrathecal Ig synthesis or elevated IgG index  [. (3) Elevated intrathecal Ig fractions and elevated IgG index occurred only transiently, i.e., no more than 1 day after PE or IADS for intrathecal Ig fractions and 2 days for IgG index, respectively, and disappeared in the further course, indicating a “steady-state equilibrium” of CSF and serum proteins over time (data not shown) [The assumption that this phenomenon is spurious is supported by several facts: 1) The yndrome) . In case The yndrt shown) . As statTo ensure correct interpretation, CSF and serum samples should be analyzed under steady-state conditions allowing proteins  to reach a diffusion equilibrium, since the Reibergrams commonly used for CSF analysis were developed under steady-state serum/CSF conditions , 2. HencFreiburg study group. Additionally, we included both patient groups receiving plasmapheresis or immunoadsorption, which are different therapeutic measures [A limitation of this study is its retrospective character and the small sample size of only 41 systematically selected patients in the measures . BrieflyTransiently elevated intrathecal Ig fractions and increased IgG index , both due to dropped serum Ig levels, are frequent phenomena occurring in the first 2 days following therapeutic apheresis. To avoid unnecessary diagnostic and therapeutic procedures, clinicians should be aware of this spurious phenomenon. Recognition of “spurious intrathecal synthesis” is possible when taking into account the absolute concentrations of CSF and serum Ig. In addition, OCBs should be considered to distinguish between “real” and “spurious” Ig synthesis, as they remain negative in the CSF despite spuriously elevated intrathecal Ig fractions and/or IgG indexes. If CSF analysis of intrathecal Ig synthesis is indicated after therapeutic apheresis, we recommend a minimum time interval of 3 days between apheresis and LP.


Implications of Cardiac troponin (cTnI) release after cardiac transplantation are still unclear. This study disclosed risk factors and prognostic implication of cTnI early levels in a single centre cohort operated on between January 1999 and December 2010.Data on 362 consecutive recipients  were analyzed using multivariable logistic regression modeling. Target outcomes were determinants of troponin release, early graft failure (EGF), acute kidney injury (AKI) and operative death.Mean cTnI release measured 24 hours after transplant was 10.9±11.6 μg/L. Overall hospital mortality was 10.8%, EGF 10.5%, and AKI was 12.2%. cTnI release>10 μg/L proved an independent predictor of EGF  and AKI . EGF, in turn, proved a determinant of hospital mortality. Risk factors for cTnI>10 μg/L release were: status 2B , duration of the ischemic period , previous cardiac operation , and left ventricular hypertrophy .Myocardial enzyme leakage clearly emerged as an epiphenomenon of more complicated clinical course. The complex interplay between surgical procedure features, graft characteristics and recipient end-organ function highlights cTnI release as a risk marker of graft failure and acute kidney injury. The search for optimal myocardial preservation is still an issue. Troponin release after cardiac surgery, first described in 1991, is detectable virtually in every patient. Given the inherent high sensitivity and specificity for myocardial injury, the release of such biomarkers is a valuable tool to estimate both the burden of the surgical procedure and the underlying vulnerability of the myocardium. The evidence from a recent authoritative meta-analysis and several publications is that, despite a variety of confounding factors prevents definitive conclusion about effect size and cut-off value, such a release is a predictor of both early and midterm survival . Unlike Study setting was the Department of Cardiothoracic and Respiratory Sciences of the Second University of Naples located in an affiliated teaching hospital . As described earlier by these authors, at this institution, patients’ information is collected on a daily basis using standardized case report forms for relevant clinical peri-operative variables. Data are entered into a computerized database including 100 variables, which was programmed to accept only data falling within pre-specified ranges. All queries are resolved by referring to the patients’ original records . These tThis retrospective single center cohort study aimed to determine predictors and clinical implications of troponin I release early after orthotopic heart transplantation (HTx).Donor-recipient matching was mainly based on ABO blood type compatibility and size. Size matching generally did not exceed 20% of body weight as per center policy, favoring moderate oversizing in those recipients displaying severe pulmonary artery hypertension. Prospective human leukocyte antigen (HLA) matching was deemed mandatory for recipients with high levels (>20%) of panel reactive anti- HLA antibodies. A retrospective HLA matching was obtained in almost all the other cases. Graft harvesting was allowed in younger donors  when there was no evidence of preexisting heart disease and impaired myocardial dysfunction at echocardiography. When considering acceptance of older heart donors, beside thorough evaluation of past medical history and risk factors, every effort was made to obtain a coronary artery angiography to rule out atherosclerotic lesions. Particular care was taken in evaluating echocardiographic data on left ventricular hypertrophy (LVH) of the donor organ. A case by case thorough evaluation implied, on the one hand, the overall technical quality of the ultrasound examination and the eventual execution of a second scan and, on the other hand, the congruity of these evidences with hemodynamic parameters, ECG findings (Sokolow-Lyon and Cornell criteria), inotropic support and donor history. As described earlier by these authors, high inotropic support of donor heart, defined as dopamine or dobutamine at a dose of 10 μg/kg/min or as the need for norepinephrine, did not prevent graft usage when there was echocardiographic evidence of LVEF >40% after haemodynamic optimization. Need for excessive inotropic support (dopamine or dobutamine at a dose of 20 μg/kg/min or similar doses of other adrenergic agents despite optimization of preload and after load) was instead considered a contraindication to organ harvesting as per current guidelines –10. OtheLVH of donor organ was graded according to the recommendations by the American Society of Echocardiography: the greater of two measurements recorded in the Database  was used to classify the allograft as having no LVH (< 1.1cm), mild LVH (1.2–1.3 cm) and moderate-severe LVH (>1.4cm) .2, higher filling pressures  in the first 24 h with the need of high inotropic support , systemic  and/or pulmonary vasodilators , intra-aortic balloon pump (IABP), prolonged intubation with high inhaled oxygen concentrations [Early graft failure (EGF), as reported elsewhere, was defined as a mono- or biventricular low output syndrome with a cardiac index < 2 l/min/mtrations , 9. Patitrations . The clatrations . Δ GFR >t test, Mann-Whitney test) were appropriately executed in order to address clinical and statistical relevance of variables on the primary end points. Variables that were not linearly related were also mathematically transformed, categorized along appropriate cut points or converted into multiple dichotomous variables. The logistical fit of each continuous variable was examined and dichotomization was performed by choosing, as a cutoff, the value that indicated an endpoint risk greater than the median risk of the overall population. Factors with values of P ≤ 0.10 on univariate tests were included in the multivariable models. Multiple logistic regression was conducted using the forward stepwise method. Since the series included patients operated on over a decade, changes in the pattern of perioperative care may have occurred. To account for such changes date of operation was forced into the multivariable models. All statistical analyses were performed with SPSS 10.0 .Data were summarized using standard statistical descriptors such as means, standard deviations, frequencies and percentages. Univariate parametric or non-parametric tests  length, troponin I release > 10μg/l, and transfusion of > 4 blood units. Univariate predictors for hospital mortality were: non-idiopathic cardiomyopathy, previous cardiac operation, hospitalization at time of transplant, donor age, early graft failure, AKI, troponin I release > 10μg/L and blood transfusions. Multivariable logistic regression analysis for such outcomes is summarized in Univariate predictors of cTnI > 10μg/L release were: status at transplantation, donor gender, PVRI, length of ischemic time, previous cardiac operation, donor/recipient (D/R) size matching < 0.8 and left ventricular hypertrophy. Independent predictors for such a myocardial enzyme leakage are reported in This study depicts the largest group of adult cardiac transplantation patients who have had cTnI levels correlated with perioperative morbidity and mortality reported so far. Rates of hospital mortality, incidence of graft failure as well as that of acute kidney injury, the two most dreaded early post-transplant complications, are consistent with contemporary reports. The pattern of troponin release observed in this series is similar to that reported by Fiocchi and Minami , 16. TnIA recent report from the United Kingdom Cardiothoracic Transplant Audit has newly brought attention on the importance of both cold and warm ischemia for survival after heart transplant . In the Several study limitations should be considered for a thorough data interpretation. First the single centre setting, though guarantee of a uniform process of care, closely reflects the influence of specific standards of clinical practice and a unique patient population that may have led to results not readily transferable to other patient populations. On the other side, correctness of statistical tools in balancing for confounders make results of this analysis, in an unselected consecutive series of patients experiencing almost all clinically relevant events, transferable. Second, the observational design intrinsically prevents definition of causality, allowing just the disclosure of association between clinical factors. Third, all the biases inherent to a retrospective design apply. Fourth, the series includes patients operated on during nearly a decade and patterns of referral and perioperative care may have changed. Nevertheless timing of surgery never showed up as an outcome predictor (see paragraphs on Present study significantly adds to current knowledge on this topic, and, to the best of our knowledge, this is the largest series so far that evaluated the prognostic implications of cTnI release after heart transplant –33. More