Here, we provide a rationale for evaluation of DCreg therapy in clinical organ transplantation with the goal of promoting sustained, donor-specific hyporesponsiveness, while lowering the incidence and severity of rejection and reducing patients’ dependence on anti-rejection drugs. Generation of donor- or recipient-derived DCreg that suppress T cell responses and prolong transplant survival in rodents or non-human primates has been well-described. Recently, good manufacturing practice (GMP)-grade DCreg have been produced at our Institution for prospective use in human organ transplantation. We briefly review experience of regulatory immune therapy in organ transplantation and describe our experience generating and characterizing human monocyte-derived DCreg. We propose a phase I/II safety study in which the influence of donor-derived DCreg combined with conventional immunosuppression on subclinical and clinical rejection and host alloimmune responses will be examined in detail.Dendritic cells (DC) are rare, professional antigen-presenting cells with ability to induce or regulate alloimmune responses. Regulatory DC (DCreg) with potential to down-modulate acute and chronic inflammatory conditions that occur in organ transplantation can be generated  While rates of acute renal transplant rejection have improved dramatically since the advent of calcineurin inhibition (CNI) >30 years ago, similar improvement in long-term graft survival has not been achieved. This reflects the inability of conventional immunosuppressive agents to prevent late graft dysfunction leading to transplant failure , 2. Moreex vivo-expanded Treg, now entering phase I/II trials in organ transplantationex vivo to promote their inherent regulatory properties  approach that will promote sustained, donor-specific immune hyporesponsiveness, while lowering the incidence and severity of acute and chronic rejection and reducing patients’ dependence on anti-rejection drugs. There is recent evidence that, by exploiting inherent mechanisms of immune regulation, it may be possible to achieve this goal. Rare, naturally occurring regulatory immune cells, either innate [regulatory dendritic cells (DCreg)] or adaptive [regulatory T cells (Treg)], critically regulate immunity, can promote antigen (Ag)-specific T cell hyporesponsiveness, and prevent adverse immune reactions (self-tolerance) in the healthy steady-state , 9. Moreoperties , 22–24. operties . Equallyoperties , 26, an operties , 28.de novo, live-donor renal transplantation. To our knowledge, this promising donor-specific induction approach to regulatory immune cell therapy in clinical organ transplantation is unique. It is distinct from the testing of recipient blood monocyte-derived DCreg in live-donor renal transplantation currently being conducted at the University of Nantes, France, as part of The ONE Study (We have now generated good manufacturing practice (GMP) grade human DCreg from elutriated peripheral blood monocytes and demonstrated both their stable resistance to maturation under inflammatory conditions and their ability to negatively regulate alloreactive T cell responses. We have also established release criteria for clinical testing and plan to conduct a safety trial of donor-derived DCreg in adult, NE Study , 30.ex vivo in human transplantation is particularly compelling  of donor-derived DCreg, 1 week before transplant, safely prolongs renal allograft survival, with no evidence of host sensitization , 1 week before transplantation to patients receiving standard immunosuppression . Successful safety evaluation of our strategy and any evidence of inhibition of early, acute subclinical or clinical rejection, and/or attenuation of long-term anti-donor immunity would justify broader evaluation of DCreg efficacy in renal transplantation. This would potentially address unmet needs of CNI-free immunosuppression and/or realize the unmet goal of improving long-term allograft survival, without increasing the burden of immunosuppression.deceased donors. Indeed, rodent studies have shown that delaying DCreg infusion until 7 or 14 days post transplant is (still) effective in prolonging graft survival  to ascertain whether the incidence of rejection episodes encountered with Co-B  can be rsurvival , 47, thuFirst, we have generated a highly-purified GMP cell product  distinct from those  being investigated by other groups, which satisfies phenotypic and functional release criteria. The manufacturing process is relatively simple, comparatively short and highly reproducible. Second, while early pilot studies have begun to examine the safety of autologous DCreg in human autoimmune diseases  DCreg in human organ transplantation. Third, our proposed mechanistic studies will address our hypothesis that, in addition to inhibition of de novo T cell priming and memory reactivation against donor HLA Ags, DCreg infusion will selectively undermine early inflammation that fuels anti-donor effector/Tmem responses and promote specific T cell unresponsiveness to donor that we will monitor sequentially in blood and protocol biopsies. We will also generate novel insight into the persistence/longevity of donor-derived DCreg in graft recipients. Of particular relevance, based on our NHP transplant data, will be analyses of de novo-primed T cell and Tmem phenotype and function and the potential of establishing new biomarkers of donor-specific hyporesponsiveness based on the profile of donor-reactive T cells. Fourth, since protocol biopsies will be performed, we will gain preliminary insight into the influence of DCreg on the incidence of subclinical rejection, an important predictor of long-term graft outcomes by analyzing graft-infiltrating T lymphocytes. By contrast, traditional immunosuppression trials have focused on the incidence of clinically evident rejection as a principal endpoint.Several closely interrelated aspects of our proposed clinical trial of DCreg in live-donor renal transplantation are highly innovative. diseases –50 and odiseases , this wiin vitro-generated autologous DC) via local administration can control T cell responses to model Ags (flu matrix peptide and keyhole limpet hemacyamin) in human healthy volunteers  that we propose in a phase I clinical trial are, therefore, supported by experiments in rodents -oxine in one patient and whole-body single photon emission computed tomography imaging (SPECT) revealed that the Mreg located initially in the lungs, but after 2.5 h were evident in the circulation and had begun to accumulate in the liver and spleen. Twenty-four hours after Mreg infusion, signal from the lung had diminished substantially and the cells had accumulated in the liver, spleen, and bone marrow. Absence of signal from the patient’s urinary tract throughout the 30 h follow-up suggested that the majority of labeled infused cells remained alive. No unexpected adverse events were observed in either patient. At 3 and 2 year, respectively, post-transplant, the patients were taking once-daily or twice-weekly tacrolimus. Despite early minimization of immunosuppressive therapy, neither patient underwent an acute rejection episode during the 3-year follow-up period.In a further (2011) publication , the samin vivo mechanisms whereby infusion of donor (or recipient)-derived DCreg restrains alloimmunity and promotes long-term survival of experimental organ allografts are not well understood. In mice, there is evidence that donor-derived DCreg infused before transplantation are targeted by host NK cells and, thus, short-lived ] represents, on average, 25% of the total PBMC. The phenotype of the purified monocytes, determined by flow cytometry, is HLA-DR+ CD40lo CD80lo CD86+, programed death ligand (PD-L) 1lo, CD14+.To ensure sufficient DCreg yields, blood monocytes will be obtained and banked in high purity by elutriation from cryopreserved leukapheresis products of the prospective transplant donors approximately 28–15 days before scheduled transplantation  medium, supplemented with 5% certified human AB serum and recombinant human (rhu) GM-CSF (1000 units/ml) and rhu IL-4. These cytokines are added at the start of culture (day 0) and on day 4. VitD3 and rhu IL-10, which suppress DC maturation , 102, ar+T lymphocytes determined by flow cytometry. It is especially significant that the incidence of T cells is so low since these are the cells that are of concern regarding risk of graft-versus-host disease. The DCreg consistently exhibit an immature phenotype compared to control DC  and are HLA-DR+, CD11c+, CD14−, CD40lo, CD80lo, CD86lo, PD-L1hi, CCR7+, CD83lo. High expression of PD-L1(= B7-H1), a negative regulator of T cell responses and a consistently high PD-L1:CD86 ratio [determined as: PD-L1 mean fluorescence intensity (MFI) ÷ isotype control/CD86 (MFI) ÷ isotype control] conforms to that of DCreg with potential to subvert alloreactive T cell responses.The DCreg harvested at day 7 of culture are consistently >94% pure, with ≤0.1% contaminating CD3Investigation of the function of hu DCreg harvested at 7 days of culture, including their responses to the TLR4 ligand bacterial lipopolysaccharide (LPS), CD40 ligation, or a pro-inflammatory cytokine cocktail, will ensure their regulatory properties. The DCreg we have generated display robust resistance to phenotypic and functional maturation in response to factors that promote the maturation of control DC, confirming that they are refractory to stimulation under inflammatory conditions. In particular, the inability of DCreg exposed to LPS to release pro-inflammatory and immunostimulatory cytokines (tumor necrosis factor [TNF]α and IL-12p70) is profoundly inhibited, while production of anti-inflammatory IL-10 is preserved, resulting in marked reversal of the IL-12:IL-10 and TNFα:IL-10 ratios.+ T cells over 4 days in culture. This analysis provides further assurance of the inability of the DCreg, despite exposure to a potent pro-inflammatory stimulus (LPS), to stimulate allogeneic effector T cell responses. Similarly, marked attenuation of alloreactive CD8+ T cell responses is observed.The ability of the DCreg exposed to LPS to induce hyporesponsiveness of normal allogeneic T cells is of paramount importance. Therefore, DCreg (ratio 1 DCreg:40 T cells) should induce minimal CD4 and CD8 T cell proliferation, IFNγ and IL-17 release, or granzyme B production in the responder CD4in vitro will not induce sensitization of prospective recipients following their adoptive in vivo cell transfer and rather, will induce donor-specific T cell hyporesponsiveness. Our plan to closely monitor study patients for evidence of development of donor-specific alloAb production and anti-donor T cell reactivity will allow detection of any increase in anti-donor immune effector activity in the unlikely event it should occur.Thus, we contend that infusion of DCreg that are (1) phenotypically immature, (2) resistant to maturation under inflammatory conditions, and (3) able to induce allogeneic T cell hyporesponsiveness The DCreg generated for infusion will undergo rigorous testing at specified time points during their manufacture from blood monocytes. The following release criteria will be considered as crucially important: DCreg yield , percent purity , and viability (>70%); sterility; DCreg phenotype: phenotypic characterization will be performed by flow cytometry to monitor CD86 and PD-L1 expression before and after LPS stimulation, compared to conventional DC cultured in GM-CSF + IL-4 and not VitD3 and IL-10. High PD-L1 and low CD86 expression, before and after LPS stimulation, with a PD-L1:CD86 ratio >3.5  will be used as a release criterion. The 3.5 ratio is based on many analyses in which a ratio of 3.5 or above was associated with a cytokine profile and T cell stimulatory profile consistent with the induction of alloreactive T cell hyporesponsiveness. DCreg function: supernatants from cultures (DCreg exposed or not to LPS) will be tested by ELISA to assess the lack of IL-12p70/TNF-α and the presence of IL-10 production, consistent with their regulatory properties and their resistance to maturation. We consider this, in addition to the tests above, a simple, reproducible, release criterion that can be applied before release of the DCreg product for infusion.n = 5/group, with 4 receiving DCreg and 1 “control” subject receiving concomitant pre- and post-transplant immunosuppression without DCreg): dose 1: 0.5 × 106 cells/kg body weight; dose 2: 2.5 × 106 cells/kg body weight; dose 3: 5 × 106 cells/kg body weight.We plan to test three dose levels of DCreg in three separate groups of recipients  will be initiated 7 days before transplant, at the time of donor DCreg infusion, instead of on the day of transplantation. This is in order to minimize any risk of sensitizing the patient. Historically, pre-treatment of kidney transplant recipients with unmodified donor-specific transfusions and low-dose azathioprine (that acts similarly to MPA) significantly reduced the risk of sensitization –107. Fursafety trial, we have chosen to adhere to a safe and proven immunosuppression regimen that does not interfere with DCreg action.The rationale for not using ATG, alemtuzumab anti-CD52 mAb) or basiliximab (anti-IL-2Rα mAb) as induction therapy at transplant, is to avoid potential targeting of DCreg infused 7 days before transplant or dampening of immunoregulatory pathways triggered in host T cells by DCreg. In our NHP study, we established that such an approach (pre-transplant immunosuppression at the time of DCreg infusion and avoidance of lymphocyte-depleting induction agents) is both safe and effective. We have opted, however, not to use a Co-B and/or mTOR inhibition-based immunosuppressive regimen, such as that employed in our NHP study, because of the high incidence of acute rejection episodes, including higher grade rejection, in patients receiving Co-B (belatacept), MPA, and steroid therapy and increased side effects in clinical trials of rapamycin-based regimens, either with CNI or MPA , 5. Sinc2 mAb or −, HLA-DR+, BDCA1(CD1c)+, CD209 (DC SIGN)+, CD11c+ DC, in conjunction with staining for a miss-matched donor MHC allele. This approach will allow us distinguish between recipient and donor-derived DCreg. Others  for short-term tracking by SPECT imaging following their infusion in renal transplant patients and we will consider using this as a complementary approach.Monitoring DCreg persistence in the circulation and their tissue homing is essential for understanding their survival and distribution. Flow cytometry techniques to detect donor T cells in peripheral blood of transplant recipients with a threshold sensitivity of one donor cell in 1000 recipient cells (0.1%) are readily available . We plan. Others  have usede novo-primed naive T cells . While it is believed that DCreg are effective in blunting Tmem responses , 38–40 a T cells , 34, it  T cells  will indTo address these questions, we will collect blood samples pre-transplant on day −7 (pre DCreg infusion), on day 0, and at 3 months, 1 year, and 2 year post-transplant. We will (1) characterize the phenotype, memory differentiation, and function of different T cell subsets, (2) assess donor-reactive T cell clonality and function, (3) identify effector and regulatory cells and molecules in for-cause and protocol biopsy samples. While no single immunologic test can identify peripheral hyporesponsiveness after organ transplantation, we will attempt to assess multiple essential T cell immune parameters methodically at the same time, an approach expected to provide a possible signature and mechanism of peripheral anti-donor hyporesponsiveness after DCreg infusion.+, CD4+ TFH, and CD4+ Tconv effectors with Treg, DSA titer, plasma cytokines and effector and regulatory cell, IgG, and complement (C4d) deposition in the allograft. Age-matched healthy controls and renal transplant patients who did not receive DCreg infusion will serve as controls.We will assess T cell expression of co-stimulatory receptors , which are critical for cross-talk with DC, as well as co-inhibitory receptors  that are up-regulated on recently activated/exhausted T cells in conjunction with expression of Annexin V/7-AAD to track apoptosis. We will also track EBV-specific and anti-TT T cells as controls for recall responses. We will correlate the levels of memory CD8There is extensive evidence that DCreg of donor origin can regulate alloimmune responses and promote long-term organ transplant survival in rodents. The recent observation that DCreg can safely prolong renal transplant survival in a robust, pre-clinical NHP model, in which the graft recipients received a minimal immunosuppressive regimen, provides further justification for a clinical trial. Appropriate culture conditions, leading to the manufacture of GMP grade DCreg, which are resistant to maturation and have potential to regulate host alloimmunity, have been developed for clinical testing.AT, AZ, ME, LB, FL, and DM each contributed to the conception and writing of the work, revised it critically, finally approved the submitted version of the manuscript, and agreed to be accountable for all aspects of the accuracy and integrity of the work.Angus W. Thomson is co-inventor of a US patent for generation of dendritic cells to enhance transplant tolerance. The remaining co authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many attempts have been made to effectively improve a prosthetic system controlled by the classification of surface electromyographic (SEMG) signals. Recently, the development of methodologies to extract the effective features still remains a primary challenge. Previous studies have demonstrated that the SEMG signals have nonlinear characteristics. In this study, by combining the nonlinear time series analysis and the time-frequency domain methods, we proposed the wavelet-based correlation dimension method to extract the effective features of SEMG signals. The SEMG signals were firstly analyzed by the wavelet transform and the correlation dimension was calculated to obtain the features of the SEMG signals. Then, these features were used as the input vectors of a Gustafson-Kessel clustering classifier to discriminate four types of forearm movements. Our results showed that there are four separate clusters corresponding to different forearm movements at the third resolution level and the resulting classification accuracy was 100%, when two channels of SEMG signals were used. This indicates that the proposed approach can provide important insight into the nonlinear characteristics and the time-frequency domain features of SEMG signals and is suitable for classifying different types of forearm movements. By comparing with other existing methods, the proposed method exhibited more robustness and higher classification accuracy. The surface electromyographic (SEMG) signals can be collected noninvasively at the surface of the skin by the appropriate electrodes. Because the SEMG signals are approximately the accumulation of all motor unit action potentials (MUAP) around the pick-up area of the electrodes, they reflect the neuromuscular activities of the examined muscle . In addiMany feature parameters have been successfully applied to the classification of SEMG signals. Hudgins et al. extracted some simple time-domain statistics such as integral of absolute value (IVA), zero crossing (ZC), variance (VAR), Willison amplitude (WAMP), and histogram of EMG (HEMG) from upper limb SEMG signals . Due to Two channels of SEMG signals were collected by two pairs of bipolar Ag/AgCl electrodes with conductive paste. One pair was located over the flexor carpi radialis (FCR) and the other over the extensor carpi radialis longus (ECRL). All disc electrodes were put on the skin surface of the right forearm of each subject. Electrodes in each bipolar pair had a center-to-center spacing of 20 mm. The reference electrode was an Ag/AgCl disc electrode and was placed on the top side of the wrist. To help minimize the cross interference between the two channels, electrodes of 5 mm diameter were used. Differential amplifiers with bandpass filters between 10 Hz and 500 Hz were used to reduce the effects of high-frequency noises and low-frequency movement artifacts. The sampling frequency was 1000 Hz. The sampling was triggered when the energy value of the SEMG signals based on the myoelectric activity exceeded a set threshold value.Five normally limbed subjects  took part in this experiment. The mean age of all subjects was 23.2 (±1.64) years. Each subject was instructed to perform four different types of movements: forearm pronation (FP), forearm supination (FS), hand close (HC), and hand open (HO). The subjects were first asked to initiate a movement while the energy value of the SEMG signals was examined with a moving window. After a threshold was exceeded and the sampling was triggered, continuous SEMG samples were recorded for 800 ms with the intention of signals analysis. For each subject, all forearm movements were performed in four trails. One category of forearm movements was repeated for 8 times in each trial. So there were 32 groups of two channels of SEMG signals for each subject and 160 groups in total. In order to avoid mental and muscle fatigue, the subjects were asked to relax for 5 s between each movement and for 1 min between each trial. For comparison, one hand amputee was also recruited in the study. The procedures of data acquisition for hand amputee were the same as the normally limbed subjects.The wavelet transform is a local time-frequency analysis method which has the fixed analysis window and the variable resolution both in time domain and frequency domain. Due to the properties of the flexible and self-adaptive multiresolution of the wavelet function, the wavelet analysis has been widely applied in very diverse problems –24. In tx(n)  is a finite energy signal and is decomposed to level  J. Firstly, the original signal  x(n)  was filtered through a discrete low-pass filter  h0  and a high-pass filter  h1, which resulted in an approximation signal and a detail signal. The approximation signal describes the high-scale and low-frequency components of the signal and the detail signal describes the low-scale and high-frequency components of the signal. Secondly, the approximation signal was further decomposed into a second-level approximation signal and detail signal. Finally, this process was repeated until level  J  was achieved. The original SEMG signal  x(n)  can be denoted by the approximation signal  A0  at level 0. Then, the approximation signal  Aj  and the detail signal  Dj  of the DWT at level  j  can be expressed byk is the translation parameter in time domain and  j  is the scale parameter which determines the inflation or deflation of the wavelet function. The low-pass filter  h0  is related to the scaling function  ϕ  and the high-pass filter  h1  is related to the wavelet function  ψ. These functions can be defined asdj = {dj(k) | k = 1,2,…, Kj}. If the depth of wavelet analysis is too small, the classification information would not be extracted efficiently. If the depth is too large, the computation complexity would be increased remarkably. Therefore, in this study, the SEMG signals were decomposed to the fourth level using the DWT. The Coiflet wavelet of order 5 was selected as the mother wavelet, because the waveform of Coiflet wavelet can fit the SEMG signals appropriately.Given that a SEMG signal  =∑k∈Zh0k−nAj−1(k),=∑k∈Zh0k−nAj−1(k),The physiological signals generated by complex self-regulating systems may exhibit complex temporal fractal patterns in which the large scale structure of the signal resembles the small scale structures, as in the branchings of a tree. The fractals can exhibit this self-similar property and the fractal dimension can be used to characterize the fractal properties of a structure. Hence, the fractal dimension has been widely applied in the analysis of the physiological signals which has self-similar property , 25, 26.D = {dk, k = 1,…, K}  denote the wavelet coefficients corresponding to a fixed resolution level of the multiresolution analysis. Then, a new phase space can be reconstructed by embedding the time series  D  into an m-dimensional Euclidean space and the elements of this new phase space can be given bym  is the embedding dimension,  τ  is the delay time, and  N  is the number of points in the reconstructed phase space (N = K − (m − 1)τ). For a reference point  Di  selected from  N  points of the reconstructed phase space, the distance between the reference point and the other  N − 1  points can be defined asThe correlation dimension can be directly calculated from the time series by using the GP algorithm proposed by Grassberger and Procaccia , 28. ThiDi  , the correlation integral function can be obtained byr  in the m-dimensional phase space and the Heaviside function  θ  is defined asBy repeating the calculation procedure of the distance described above, for all  N  is usually far greater than 1,   versus  lnr  [K  was equal to 800 in the multiresolution analysis. In addition, it is very important to choose appropriate values of the embedding dimension  m  and the delay time  τ  in the reconstructed phase space as their precision is associated with the accuracy degree of the invariants characterizing the strange attractors. A large number of investigations demonstrated that the embedding dimension and the delay time were correlated with each other since the time series could not have infinite length and could be inevitably contaminated by various noises in the real environment. The C-C algorithm, which was developed by Kim et al. [τ  can be calculated by using the correlation integral and the BDS statistic. Secondly, a suitable value can be achieved for the delay time window  τw  which is the entire time spanned by the components of each embedded point. Finally, the embedding dimension  m  can be obtained by the formula  τw = (m − 1)τ. By using the C-C method in this study, the embedding dimension  m  and the delay time  τ  were determined as 16 and 10, respectively. After identifying  m  and  τ, the number of points in the reconstructed phase space could be calculated as 650.In practice, the correlation dimension  s  lnr  . In thism et al. , was a cThe SEMG signals were firstly segmented from the onset of a contraction by the length of 800 samples. The amplitudes of segmented SEMG signals were normalized to avoid the effect of different amplitude scales on the following calculation. Specifically, the segmented SEMG signals were normalized to center it at zero mean and scale it to unit standard deviation. For each subject, the SEMG signals were then analyzed by the wavelet transform. The SEMG signals are a result of the summation of the similar MUAPs originated from different locations of the muscles. Because the patterns of SEMG signals obtained at one sampling rate are statistically similar to patterns obtained at other sampling rates, the SEMG signals have the property of self-similarity . TherefoIn this study, the classification of SEMG signals was performed for five normally limbed subjects and one hand amputee, respectively. At the first and the second resolution level, the classification results of the wavelet-based correlation dimension method for two channels of SEMG signals recorded from the flexor carpi radialis (FCR) and the extensor carpi radialis longus (ECRL) in five normally limbed subjects are summarized in For the third and the fourth resolution level, the scatterplots of the wavelet-based correlation dimension of two channels of SEMG signals in five normally limbed subjects are illustrated in In this study, we have examined the ability of classifying four different types of forearm movements by using the SEMG signals from five normally limbed subjects and one hand amputee. All subjects were asked to perform four different types of movements: forearm pronation (FP), forearm supination (FS), hand close (HC), and hand open (HO). The SEMG signals of these subjects were acquired and were decomposed by the wavelet analysis. Then, the wavelet-based correlation dimension of the SEMG signals was calculated to extract the features of different movements by using the GP algorithm , 28. BecIn order to evaluate the performance of the wavelet-based correlation dimension method, the comparison with other algorithms was made. We used the correlation dimension without multiresolution analysis in this comparison task. To facilitate the effectiveness of the comparison, this method was applied to the same database of the SEMG signals. The length of the analysis window of SEMG signals was also selected as 800 ms. The calculation of the correlation dimension was directly performed on the SEMG signals without the multiresolution analysis. The scatterplots of the correlation dimension without multiresolution analysis for five normally limbed subjects are illustrated in In addition to the correlation dimension method, we compared the wavelet-based correlation dimension method with the wavelet packet transform (WPT) method which was demonstrated to offer the best performance for the classification of SEMG signals . A coif5For further investigating the wavelet-based correlation dimension method, the SEMG signals were decomposed to the fifth level and the sixth level by using wavelet transform. The correlation dimension was then calculated for the decomposed wavelet coefficients. The classification results of the wavelet-based correlation dimension method for the two-channel SEMG signals recorded from the FCR and the ECRL in five normally limbed subjects are illustrated in For the classification of SEMG signals in a prosthetic control system, previous studies focused on the time-domain analysis, the frequency domain analysis, and the time-frequency domain analysis in order to extract the features of the SEMG signals for distinguishing the different forearm movements. These methods had certain limitations because they did not use the nonlinear intrinsic characteristic of the SEMG signals. In this study, according to the nonlinear of the SEMG signals, we combined the time-frequency domain analysis approach and nonlinear dynamics analysis to explore the features of the SEMG signals. Comparing with the traditional SEMG analysis methods, the proposed method in this study can obtain more useful information and can better identify different forearm movements. The experimental results demonstrated that the proposed method can exhibit good performance for the classification of the SEMG signals.One limitation of this study was that the SEMG database for classification performance evaluation of the SEMG signals was the same as the one used for developing the algorithm and the classification criterion. Therefore, a larger database would be required for assessing the classification performance of the SEMG signals during different forearm movements for this proposed method.In summary, the wavelet-based correlation dimension method can provide a novel description of the SEMG signals during the different forearm movements and the SEMG signals can be characterized efficiently by this method. The fractal analysis was performed by the GP algorithm after the SEMG signals were decomposed by the wavelet transform in order to obtain the features of the SEMG signals. Our results reveal that the wavelet-based correlation dimensions of SEMG signals are determined by the different types of forearm movements. Thus, the wavelet-based correlation dimension extracted from two channels of SEMG signals can be employed to distinguish four different types of upper arm movements. By comparing with the correlation dimension method without multiresolution analysis, the wavelet-based correlation dimension method proposed in this study represented more robustness and higher classification accuracy. Although the data analysis approach in this study was applied to the classification of SEMG signals in a prosthetic control system, the wavelet-based correlation dimension method presented here provides a new idea for analyzing other nonstationary physiological signals and can be easily generalized to other applications.


Since 2003, the established paradigm of T cell immunology has defined interleukin (IL)-17 as a dominant Th17 cell derived cytokine driving autoimmune disease. Recent murine studies have challenged this, identifying GM-CSF as a Th17 related cytokine necessary and sufficient for the induction of autoimmunity. The origin of GM-CSF+ T cells and their relationship with IL-17 secreting cells is unclear in human autoimmune disease. Trials of biologic agents targeting the GM-CSF pathway show promise in rheumatoid arthritis, so it is important to establish if GM-CSF contributes to the inflammatory environment of the arthritic joint in Juvenile Idiopathic Arthritis (JIA).in vitro.To analyse T cell GM-CSF production in the JIA joint and investigate the origin of GM-CSF+ T cells by testing for co-expression of the Th17 marker CD161 and modelling the plasticity of Th17 cells towards a GM-CSF phenotype Peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from 17 patients with JIA were stimulated with PMA and ionomycin in the presence of Brefeldin A and analysed for IL-17, interferon gamma (ifnγ), GM-CSF and CD161 expression by flow cytometry. In some experiments Th17 cells were purified from healthy donor PBMC using a cytokine capture assay and upregulation of GM-CSF was examined after culture, in the presence of IL-12.SFMC from patients with JIA were enriched for GM-CSF-secreting CD4 T cells, compared to matched PBMC . The enrichment was most marked within the synovial CD161+ Th1 cell compartment. Following culture in the presence of IL-12, purified Th17 cells preferentially upregulated GM-CSF compared to IL-17- CD4 T cells (62% vs 35% of CD4 T cells).GM-CSF secreting T cells are enriched within the JIA joint. Our data shows for the first time that synovial GM-CSF+ T cells demonstrate a phenotype previously associated with ex-Th17 cells, namely IL-17-ifnγ+CD161+. We propose that synovial Th17 cells may drive ongoing local inflammation by undergoing plasticity towards a GM-CSF expressing phenotype in response to elevated synovial IL-12.None declared.


An 8-month-oldboy diagnosed with T-B+NK- SCID underwent peripheral blood hematopoietic stem cell transplantation (HSCT) from MSD without conditioning. However, he developed pancytopenia and became transfusion dependent by post-transplant 2nd month. Bone marrow aspiration/biopsy revealed an aplastic marrow with 98% donor chimerism. With a diagnosis of T-cell engraftment of the donor but no engraftment of the other lineages, a 2nd HSCT with conditioning (BU/FLU/ATG) was performed at post-transplant +23rd month from the same donor. Due to hyperferritinemia pre-transplant desferoxamine was given. On post-transplant day +2, he developed hyperpigmented patches . PlateleGeneralized hyperpigmentation, after conditioning, is a common finding after HSCT . However


Volunteers were free to manually increase or decrease the work rate on the cycle ergometer. Heart rate (HR), arterial oxygen saturation (SaO2), and rating of perceived exertion (RPE) were collected every 5 min during the TT, and the mean was calculated.Two groups of healthy fit men were matched on physical performance and demographic characteristics and completed a 720-kJ time trial on a cycle ergometer at sea level (SL) and following approximately 2 h of resting exposure to either HH (P < 0.05) in HH (121.0 ± 12.1) compared to NH (99.5 ± 18.1). The percent decrement in TT performance from SL to HH (65.1 ± 23.6%) was greater (P < 0.05) than that from SL to NH (35.5 ± 13.7%). The mean exercise SaO2, HR, and RPE during the TT were not different in HH compared to NH.Both groups exhibited similar TT performance (min) at SL (73.9 ± 7.6 vs. 73.2 ± 8.2), but TT performance was longer (2 equivalent to 4,300 m despite similar cardiorespiratory responses.Cycling time trial performance is impaired to a greater degree in HH versus NH at the same ambient PO Due to increasing interest in using hypoxic training to induce improvements in endurance performance in athletes, mountaineers, and military personnel, the use of intermittent exposures to either hypobaric hypoxia (HH) or normobaric hypoxia (NH) as an adjunct or in addition to regular sea-level training has generated a plethora or research -7. Whereacute exposures to HH and NH. If NH does not induce the same degree of hypoxic stimulus as HH, its usefulness as a training aid for endurance performance improvements at sea level or altitude may be limited. One meta-analysis suggested that live-high, train-low protocols conducted in HH were more effective for enhancing sea-level endurance performance than live-high, train-low protocols conducted in NH  and serum ferritin levels. All volunteers performed regular sea-level aerobic training (1–2 h · week-1) before and during the study. Each gave written and verbal acknowledgment of their informed consent and was made aware of their right to withdraw without prejudice at any time. The study was approved by the Institutional Review Board of the US Army Research Institute of Environmental Medicine in Natick, MA, USA. Investigators adhered to the policies for protection of human subjects as prescribed in Army Regulation 70-25, and the research was conducted in adherence with the provisions of 45 CFR Part 46.Two groups of healthy fit men were exposed to either HH  a cycle endurance test, (3) five 45-min cycle training sessions conducted at 55% VO2peak, and (4) a second cycle endurance test. These preliminary measurements were made to stabilize performance improvements due to familiarization with the cycle ergometer and/or training effects. The VO2peak was not measured in HYP but estimated by decrementing the SL VO2peak by 26% which is an established group decrement at 4,300 m  and Hct. The samples were analyzed immediately in duplicate using the i-STAT portable clinical analyzer . Percent change in plasma volume (PV) from SL to HYP and from pre- to post-exercise in both SL and HYP was calculated according to the Dill equation .2, and RPE were collected as previously described and respiratory gas measurements  were made using open-circuit spirometry . The metabolic cart was calibrated with certified gases and volume standard and used in the mixing chamber mode for exercise testing. The respiratory exchange ratio (RER) was calculated from VO2 and VCO2 measurements. The ventilatory equivalents for O2 (VE · VO2-1) and CO2 (VE · VCO2-1) were calculated from each subject's VE, VO2, and VCO2 data. Cardiac output (CO) was also measured at rest and during the two steady-state work rates using a validated non-invasive continuous finger blood pressure measurement system  [Cycle endurance tests were performed on an electromagnetically braked cycle ergometer . At rest and during the two steady-state work bouts, HR, SaOerlands) -30.2, and RPE by methods previously described were collected every 5 min during the TT, and the mean value was calculated.After the steady-state exercise, each volunteer completed a 720-kJ TT on a cycle ergometer. Respiratory gas measurements were not made during the TT in order to eliminate the impact of breathing through a mouthpiece on an all-out endurance performance effort. A shorter duration to complete the fixed 720-kJ time trial from one testing condition to another was considered an improvement in performance and vice versa. Due to the extensive familiarization period, volunteers were well acquainted with their capabilities on the cycle ergometer and were free to choose the initial wattage at the start of the TT. During the TT, volunteers were visually aware of the current wattage on the cycle ergometer at all times and free to manually increase or decrease the work rate by 5-W increments. There were no restrictions on the number or direction of 5-W changes. Volunteers were continually informed of the distance completed but not the time elapsed during the TT. This type of TT performance test has been shown to have a high repeatability and low coefficient of variation . The HR,2 of 93 mmHg by reducing the fractional inspired concentration of O2, while the hypobaric chamber was maintained at an ambient PO2 of 93 mmHg by decompression to 446 mmHg over a 15-min period. The inspired PO2 in the hypobaric chamber was 83.4 mmHg, while the inspired PO2 in the hypoxia room was 87.4 mmHg. The ambient PO2 was maintained in the normobaric hypoxia room with a combination of automatic feedback loops using the True Max gas analyzers as well as manual adjustments in flow to add room air if ambient PO2 levels dropped from desired levels. The ambient PO2 fluctuated very little (±0.1 mmHg) due to constant monitoring of the room. The SL testing was performed at ambient barometric pressure (approximately 760 mmHg), and all HYP exposures were conducted at an altitude equivalent of 4,300 m. The mean ambient PCO2 values in the hypoxia room (0.9 ± 0.2 mmHg) and hypobaric chamber (0.7 ± 0.2 mmHg) were similar.All testing was performed at SL in Natick, MA, USA in a hypobaric chamber or hypoxia room  maintained at a temperature and relative humidity of 21 ± 2°C and 45 ± 5%, respectively. The hypoxia room was maintained at an ambient POP < 0.05. All data are presented as means ± SD.A two-way repeated measures ANOVA was used to analyze differences between the repeated measures test condition factor (SL and HYP) and independent group factor (HH and NH) for all resting ventilatory and cardiovascular assessments as well as cycle TT performance. Three-way ANOVAs, with repeated measures on the additional factor of exercise duration, were used for physiological measurements during the two steady-state work rates. Pearson product-moment correlations were utilized to determine significant associations between variables of interest. Significant main effects and interactions were analyzed using Tukey's least significant difference test. Effect size was calculated as a 15 ± 8 min difference in cycle TT performance between groups. This effect size was based on the standard deviation from previous TT data collected in our laboratory  as well The cycle TT improved  from the first to second preliminary test at SL in both the HH group (96.7 ± 5.0 min vs. 79.4 ± 4.9 min) and NH group (93.7 ± 12.6 min vs. 79.3 ± 9.8 min). However, the cycle TT did not change from the second preliminary test to the first cycle TT at SL for either the HH (73.9 ± 7.6 min) or NH (73.2 ± 8.2 min) group. Thus, any learning or training effects due to unfamiliarity with the cycle ergometer were minimized prior to definitive data collection.2 and PETO2 were lower in HYP compared to SL values, but other physiologic measures did not change from SL. The percent change in resting PV from SL to HH (4.8 ± 7.5%) or SL to NH (6.1 ± 8.9%) was also not different between groups or from zero.Resting ventilatory and cardiovascular measurements are presented in Table 2peak from SL to HYP, the work rates (W) at 45% and 65% of altitude-specific VO2peak were decreased (P < 0.05) in HYP compared to SL in both groups. Between groups, there were no differences in the 45% or 65% altitude-specific VO2peak work rates at SL or HYP. These results attest to adequate matching of volunteers and were thus ‘by design’.Ventilatory, cardiovascular, and perceptual responses measured during the two steady-state exercise bouts on the cycle ergometer are presented in Table 2, VCO2, VE · VO2-1, VE · VCO2-1, or SaO2) between groups at 45% and 65% of altitude-specific VO2peak at SL or in HYP. As expected, the exercise VO2, VCO2, and SaO2 were lower (P < 0.05), and VE was similar in HYP compared to SL within groups at the same relative work rates. The VE · VO2-1 and VE · VCO2-1, therefore, were both increased (P < 0.05) in HYP compared to SL at 45% and 65% of altitude-specific VO2peak.There were no differences in ventilatory responses  and 65%  altitude-specific VO2peak in HYP. The percent change in PV from pre- to post-exercise was not different in the HH and NH groups at SL (-9.0 ± 3.4 vs. -11.9 ± 6.3) or HYP (-9.1 ± 6.0 vs. -8.1 ± 4.8), respectively.There were no differences in any of the cardiovascular or perceptual responses  between groups at 45% and 65% of altitude-specific VOP < 0.05) in HH compared to NH  than the decrement from SL to NH (35.5 ± 13.7%). Moreover, for each pair of men matched on physical performance and demographic characteristics, the individual exposed to HH had a greater decrement in TT performance than the one exposed to NH  to be approximately 20 W lower in the HH group compared to the NH group.Both the HH and NH groups exhibited similar TT performance at SL. Not only was the mean TT performance similar between groups, but also individuals in the HH and NH groups were closely matched on their TT performance at SL Figure a. The TTH Figure a. The peH Figure b. The me2 was similar in the HH and NH groups at SL (97 ± 2% vs. 97 ± 1%) and in HYP , respectively. The HR during the TT was also similar in the HH and NH groups at SL (168 ± 13 bpm vs. 165 ± 12 bpm) and in HYP (150 ± 9 bpm vs.158 ± 13 bpm), respectively. The RPE during the TT was similar in the HH and NH groups at SL (14 ± 3 vs. 15 ± 2) and in HYP (14 ± 2 vs. 15 ± 1), respectively.During the TT, the mean SaO2 delivery in NH compared to HH were responsible for the differences in TT performance. In fact, nothing we measured was able to identify the reasons for the TT performance differences between HH and NH. This result suggests the need for additional physiologic and metabolic measures  to determine the reason for such large differences in TT performance [The major finding from this study was that cycle TT performance was impaired to a greater degree in HH compared to NH which supports the first part of our hypothesis. As such, exposure to NH may not induce the same hypoxic stimulus and training benefit as exposure to HH. The two groups were closely matched at sea level on physical performance and demographic characteristics which makes the observed TT performance differences between HH and NH more convincing. Moreover, the fact that each individual exposed to HH demonstrated a larger decrement in TT performance compared to their fitness and age-matched pair exposed to NH demonstrates that this response was consistent across a wide range of individuals. Our findings do not support the second part of our hypothesis that increased ventilation and improved Oformance ,34.2peak, and the cycle TT do not indicate clear differences between groups. The resting PETCO2 was the same in HH and NH in the present study, which supports the findings of some [2 was not different between HH and NH in any of the reported studies, the functional significance of a slightly lower resting PETCO2 in NH is not conclusive. During steady-state exercise in the present study, PETCO2 was not measured but the ventilatory equivalents for O2 and CO2 were not different between groups at either of the two work rates. In addition, the SaO2 was not different between groups during steady-state exercise at either of the work rates or during the cycle TT. Our results confirm and extend the previously reported lack of differences in resting SaO2 in the two environments [2peak or during a cycle TT. It seems clear that if differences do exist in resting or exercise ventilation, they are subtle and do not affect the end product of pulmonary gas exchange.Our ventilatory data during rest, steady-state exercise at 45% and 65% of altitude-specific VO of some ,20 but n of some ,19,21. D of some ,21. Giveronments -19 to no2peak, and the cycle TT also do not indicate clear differences between groups. Others have also reported no differences in either resting HR, CO, or MAP between the two environments [Our cardiac and perceptual response data during rest, steady-state exercise at 45% and 65% altitude-specific VOronments -22, whilronments . No studronments  and exteP = 0.08) to select an approximately 20-W lower power output compared to the NH group right from the beginning of the TT. Even though the difference was not statistically significant, it suggests that perceptual clues were influencing their self-selected power output prior to even starting TT exercise. The HH group also perceived a lower power output as being equally hard despite a tendency for a lower HR compared to the NH group. This finding suggests a subjective feeling that exercise is more difficult in HH compared to NH.Given that volunteers were able to self-select the power output at the beginning and during the cycle TT, we analyzed pacing strategies between groups during different quarters of the TT. The volunteers in the HH group tended  should not be disregarded [2 was kept the same, but PIO2 can differ between groups based on the differences in water vapor calculations [2 was slightly lower in HH (83.4 mmHg) compared to NH (87.4 mmHg). However, the resting SaO2 was similar in both groups, indicating that the same level of hypoxemia, regardless of PIO2, was induced in both environments. Fourth, a 26% decrement in VO2peak was assumed in both HH and NH. There is individual variability in this decrement at altitude [2peak was decremented to a larger degree in HH than NH, the volunteers may have started their TT in a more fatigued state in HH. The RPE, however, did not differ between groups during the submaximal work rates or during the time trial. Last, independent groups of volunteers were utilized. A cross-over design was not utilized due to potential carry-over effect of a previous exposure to hypoxia on performance. The volunteers, however, were closely matched on fitness levels and demographic characteristics, which make the results more convincing.Limitations of this study should be acknowledged. First, although our sample size was adequate to detect differences in cycle TT performance, borderline differences in SaOregarded . Second,ulations . In thisaltitude , and if The practical implications of this study are important for athletes, mountaineers, military personnel, and others employing NH to induce health and performance benefits at terrestrial altitude. Although the physiologic responses appear to be similar between the two environments, it is clear that TT performance is impacted to a greater degree in HH. If the objective of training programs is to improve performance at terrestrial altitude, HH appears to be a more stressful stimulus and would likely induce greater performance adaptations. The specificity of training principle as well as a recent review article citing no improvements in endurance performance, measured in hypobaric hypoxia, following intermittent exposures to NH but great improvements with intermittent exposures to HH supports this argument .In conclusion, this study found a greater decrement in cycle TT performance in HH compared to NH. Clearly, HH appears to be a more stressful stimulus, and NH should not be utilized as a substitute for HH when endurance performance is the main outcome variable of interest. Ventilatory, cardiovascular, and hematologic responses did not differ during rest, steady-state exercise at the same relative or absolute work rates, or a cycle TT performance test between environments. Further research is needed to elucidate potential physiologic mechanisms responsible for the observed differences in TT performance between HH and NH.2; PO2: Ambient PO2; PV: Plasma volume; RPE: Rating of perceived exertion; SaO2: Arterial oxygen saturation; SBP: Systolic blood pressure; SL: Sea level; TT: Time trial; VE: Minute ventilation; VE · VCO2-1: Ventilatory equivalent for carbon dioxide; VE · VO2-1: Ventilatory equivalent for oxygen; VCO2: Carbon dioxide production; VO2: Oxygen consumption; VO2peak: Peak oxygen consumption.CO: Cardiac output; DBP: Diastolic blood pressure; HH: Hypobaric hypoxia; HR: Heart rate; HYP: Hypoxia; MAP: Mean arterial pressure; NH: Normobaric hypoxia; PETCO2: Partial pressure of end-tidal carbon dioxide; PETO2: Partial pressure of end tidal oxygen; PIO2: Inspired POThe authors declare that they have no competing interests.BAB, CSF, and SRM conceived and designed the study, collected and analyzed the data, and wrote the manuscript. JES and SPA collected and analyzed the data. All authors read and approved the final manuscript.


M-CSF receptor mRNA was expressed in naive rats and increased in spinal microglia following SNI. Intrathecal injection of M-CSF receptor inhibitor partially but significantly reversed the proliferation of spinal microglia and in early phase of neuropathic pain induced by SNI. Furthermore, intrathecal injection of recombinant M-CSF induced microglial proliferation and mechanical allodynia. Here, we demonstrate that M-CSF is a candidate molecule derived from primary afferents that induces proliferation of microglia in the spinal cord and leads to induction of neuropathic pain after peripheral nerve injury in rats.Peripheral nerve injury induces proliferation of microglia in the spinal cord, which can contribute to neuropathic pain conditions. However, candidate molecules for proliferation of spinal microglia after injury in rats remain unclear. We focused on the colony-stimulating factors (CSFs) and interleukin-34 (IL-34) that are involved in the proliferation of the mononuclear phagocyte lineage. We examined the expression of mRNAs for macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF) and IL-34 in the dorsal root ganglion (DRG) and spinal cord after spared nerve injury (SNI) in rats. RT-PCR and  In vitro, microglia showed chemotaxis against ATP, and migrated towards sites of higher concentrations of ATP [in vitro [via the P2Y12 receptor, an ATP receptor, leading to neuropathic pain [Microglia colonizes the central nervous system during development, originating from hematopoietic stem cells found in yolk sac and accous of ATP . Phosphoin vitro . Moreovehic pain . PreviouColony stimulating factors (CSFs) are cytokines that are involved in the proliferation and differentiation from hematopoietic stem cells to a specific kind of white blood cells, such as macrophages and granulocytes . Three tin vitro [in vitro [Elmore et al. demonstrated that continuous administration of a M-CSFr inhibitor depleted microglia in the brain, and then repopulation of microglia occurred after discontinuation of the administration of the M-CSFr inhibitor . Facial in vitro , 17. Thein vitro .in vivo and that M-CSF/M-CSFr signaling leads to generation of neuropathic pain after peripheral nerve injury using different methods and species that were used by Guan et al [In the very recent past, Guan et al. demonstrated that M-CSF produced by injured primary afferent is involved in microglial proliferation in the spinal cord and in neuropathic pain after peripheral nerve injury in mice . Herein,an et al .ad libitum.Male Sprague Dawley rats 200–250 g) were anesthetized with sodium pentobarbital  and the tibial and common peroneal nerves were transected, while the sural nerve was left intact  [0–250 g wAll animal experimental procedures were approved by the Hyogo College of Medicine Committee on Animal Research (Permit Number: 13–048) and were performed in accordance with the National Institutes of Health guidelines on animal care.35S UTP-labeled antisense and sense cRNA probes were synthesized. The 35S-labeled probes in hybridization buffer were placed on the section, and then incubated at 55°C overnight (>18 hours). Sections were then washed and treated with 1 μg/ml RNase A. Subsequently, sections were dehydrated and air-dried. After the hybridization reaction, the slides were coated with NTB emulsion  and exposed for 4–8 weeks. Once developed in D-19 (Kodak), the sections were stained with hematoxylin-eosin and coverslipped.The rats were killed by decapitation under deep ether anesthesia. The ipsilateral side of spinal cords (L4-L5) and DRGs (L4-5) were removed and rapidly frozen with powdered dry ice and stored at -80°C until use. Extraction of total RNA was done using a single step extraction method with ISOGEN  as described in a previous paper . PCR pri5 μm2 was placed onto areas of the medial 2/3 of the dorsal horn (lamina II-III) under x20 magnification using a microscope, and the number of profiles that were positive for each maker was counted in this area. These measurement protocols were performed on 8–10 spinal sections from each animal.Rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with 250 ml of 1% formaldehyde (FA) in 0.1 M phosphate buffer (PB), pH 7.4, followed by 500 ml of 4% FA in 0.1 M PB. The spinal cords and DRGs were dissected out and postfixed in the same fixative at 4°C overnight, followed by immersion in 20% sucrose in 0.1 M PB at 4°C for 2 days. The tissue was frozen in powdered dry ice and cut on a cryostat at a 16 μm thickness for the spinal cord. The sections were processed for IHC using the ABC method . The folAll DAB stained and emulsion coated slides were digitized with a Nikon Eclipse 80i microscope connected to a Nikon DXM-1200F digital camera . Immunofluorescence images were taken by the Olympus FLUOVIEW FV1200 confocal microscope . A maximum of 21 images of confocal z stacks were sectioned at 8 μm. Z projection of stacks series was averaged using ImageJ (1.46r). Adobe Photoshop CS4  was used to optimize the images and compose all figures.At the same time as SNI surgery, the L5 vertebra was laminectomized under adequate anesthesia with sodium pentobarbital, and a 7 cm soft tube  filled with saline was inserted into the subarachnoid space for an ~0.5 cm length. After the incision was closed, the tube was fixed, and the cut end was ligated. Then, the tube was laid under the skin and the incision was closed. M-CSF receptor inhibitor GW2580 (5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-pyrimidine-2,4-diamine) and rat recombinant M-CSF  were carefully injected in a volume of 10 μL followed by 5 μL saline. The concentrations of GW2580 was 10 nmol diluted in dimethyl sulfoxide (DMSO), M-CSF was 10 μg diluted in saline. An adequate concentration of DMSO and BSA were used as a vehicle control. The M-CSF inhibitor was given 3 times at the same time as SNI surgery, 25 and 40 hours after SNI. Rat recombinant M-CSF was given twice at a 24 h interval. Rats were anesthetized and drugs were injected using a Hamilton syringe.All rats were tested for mechanical allodynia on the plantar surface of the hindpaw in pretreatment (0), 1 and 2 days in each experiment. Mechanical allodynia was assessed with a dynamic plantar anesthesiometer  . DetaileMeasurements of the density of silver grains over randomly selected tissue profiles were performed using a computerized image analysis system , where only neuronal profiles that contained nuclei were used for quantification. At a magnification of 200× and with bright-field illumination, upper and lower thresholds of gray level density were set such that only silver grains were accurately discriminated from the background in the outlined cell or tissue profile and read by the computer pixel-by-pixel. Subsequently, the area of discriminated pixels was measured and divided by the area of the outlined profile, giving a grain density for each cell or tissue profile. To reduce the risk of biased sampling of the data because of varying emulsion thickness, we used a signal-to-noise (S/N) ratio for each cell in each tissue. The S/N ratio of an individual neuron and its cross-sectioned area, which was computed from the outlined profile, was plotted. Based on this scatter gram, neurons with a grain density of five-fold the background level or higher (5 < S/N ratio) were considered positively labeled for M-CSF mRNA. Because a stereological approach was not used in this study, quantification of the data may represent a biased estimate of the actual numbers of neurons. At least 500 neurons from the L4/5 DRG of each rat were measured.p < 0.05.Data are expressed as mean ± SEM. Differences in changes of values over time of each group were tested using one-way ANOVA, followed by individual post hoc comparisons (Fisher's exact test) or pairwise comparisons (t test) to assess differences of values between naive versus each time point of the SNI groups. A difference was accepted as significant if We first examined the gene expression levels of CSF receptor ligands, M-CSF, IL-34, GM-CSF and G-CSF in the rat dorsal root ganglion (DRG) after peripheral nerve injury using a conventional RT-PCR method . We usedin situ hybridization histochemistry (ISHH) using radioisotope (RI)-labeled cRNA probes (2), 4.4 ± 0.8% in medium-size (600–1200 μm2) and 2.4 ± 0.7% in large-sized (>1200 μm2) neurons. In SNI model (2 days), M-CSF mRNA was expressed in 40.2 ± 3.5% in small-, 43.0 ± 2.2% in medium- and 60.5 ± 4.1% in large-sized neurons of DRGs, respectively. The definition of neuronal size was described previously [To clarify the expression pattern of M-CSF and IL-34 mRNAs in the DRG, we performed A probes . As showA probes  indicatiA probes . BrightfA probes . To inveA probes . NeuronaA probes . M-CSF mA probes . M-CSF meviously , 28.To clarify upregulation of M-CSF mRNA in the DRG was injury-dependent, we combined ISHH for M-CSF mRNA with immunohistochemistry (IHC) with a widely used antibody, activating transcription factor 3 (ATF3), an injured-neuronal maker . Two dayThe low-magnification darkfield images of ISHH revealed that IL-34 mRNA seemed to be expressed in non-neuronal cells . The higWe next asked whether expression of mRNAs for M-CSF, IL-34, GM-, and G-CSF in the spinal cord were affected by peripheral nerve injury using same techniques . RT-PCR We narrowed the candidate down to one receptor, a M-CSFr, for detection in the spinal cord after nerve injury, since M-CSF and IL-34 mRNAs, M-CSFr ligands, were selectively detected in the DRG and spinal cord but not GM- and G-CSF mRNAs . RT-PCR Given that M-CSF increased in injured-DRG neurons and its receptor was induced mainly in spinal microglia after peripheral nerve injury, we performed behavioral testing to determine the possible role of M-CSF in the neuropathic pain mechanisms. We examined whether the M-CSFr inhibitor, GW2580, could ameliorate mechanical allodynia in both early and late phase of neuropathic pain . Since Gin vivo and neuropathic pain-like behaviors.We asked whether M-CSF itself leads to mechanical allodynia, we intrathecally injected recombinant M-CSF . The recCSFs are well known as hematopoietic cytokines that regulate survival, differentiation and proliferation of hematopoietic linages. G- and GM-CSF have often been applied to patients with acute radiation syndrome after irradiation due to carcinoma . Recentlvia induction of proliferation cell nuclear antigen in vitro [A previous study showed that M-CSF increased in the brain of patients with Alzheimer’s disease (AD), brain tumor and in several brain injury models –34. In tin vitro .in vivo.Previous report suggested that the induction of migration or proliferation of macrophage in injured fibers of primary afferent occurred from 3 days after nerve injury . It is wvia p-p38 MAPK in activated microglia and are involved in pain hypersensitivity [Activated microglia are also known to release many proinflammatory molecules and neurotrophic factors, such as IL-1ß, IL-6, TNF-alpha and brain-derived neurotrophic factor (BDNF) , 46. Thesitivity . In the sitivity , 49. We sitivity  recentlysitivity , we convsitivity , but thesitivity  and the sitivity . The presitivity , IL-34 isitivity . Our curIn contrast to expression of M-CSF and IL-34 mRNA, GM- and G-CSF could not be detected in the DRG and spinal cord of both naive mice and in the SNI model as shown in Figs in vivo, and proliferation of microglia induced by M-CSF leads to the generation of neuropathic pain in rats. Our data is strongly consistent with the data in mice that was recently reported by Guan et al [M-CSF, derived from injured-primary afferents, induced a microglial proliferation in the spinal cord after peripheral nerve injury an et al . Accumul


Background: Stroke is the third leading cause of death. Hypothermia has been recognized as an effective method in reducing brain injury. In this study, we assessed the effects of granulocyte colony-stimulating factor (G-CSF) as a neuroprotective agent and mild hypothermia on mortality, behavioral function, infarct volume, and brain edema in Wistar rats. Methods: Forty male rats were used in five groups (eight rats in each group): control, hypothermy, G-CSF, combination hypothermy + CSF, and sham. Rats were anesthetized by injection of chloral hydrate (400 mg/kg) intraperitoneally. Transient cerebral ischemia was induced by 60-min intraluminal occlusion of left middle cerebral artery. Hypothermia, initiated at the time of reperfusion and G-CSF was started one hour after reperfusion at a dose of 15 mg/kg subcutaneously. The motor behavior was measured using Garcia’s index and animals were assigned for the assessments of infarction, brain swelling, and mortality rate. Results: The mortality was 38.46% (control group) and reduced in other groups. Neurological deficit score of control group (40.31 ± 1.56) was significantly lower than in treatment groups. The total cerebral infarct volume of treatment group was significantly lower than control group (43.96 ± 44.05 mm3). Treatment with hypothermy plus G-CSF (2.69 ± 0.24%) could significantly reduce brain swelling volume than other treatment groups. Conclusion: Our major finding is that mild hypothermic treatment plus G-CSF significantly reduced mortality rate and edema and improved neurological function. The results suggest that the combination of hypothermia and G-CSF is more effectively than other treatment groups being used alone.  Stroke is a leading cause of human death and disability in worldwide , and infHypothermia has been recognized as an effective method in reducing brain injury caused by a variety of neurological insults . The neuSo far, there are no reports in the literature about the effect of post-ischemic hypothermia and G-CSF simultaneously.In the present study, we assessed the treatment effects of G-CSF and mild hypothermia on mortality, behavioral function, infarct volume, and brain edema in Wistar rats.Animals. A total of 56 male Wistar rats (280-330 g) supplied by Animal Lab of Iran University (Tehran) were used. The rats were kept in standard cages in a temperature (22°C), humidity (40–60%), and light period (07.00–19.00 hour) controlled environment with free access to food and water. Of 56 rats, 16 were withdrawn from the study for various reasons . The numbers reported in the results refer to the number of animals that survived after the surgery. The other 40 rats were randomly divided into five groups:Control ischemic group (n = 8) that submitted to 60 minutes of ischemia , 14 and Hypothermia group (n = 8) that submitted to 60 minutes of ischemia and 24 hours of reperfusion. Post-ischemic hypothermia 33.5-35°C) [-35°C [5]G-CSF group (n = 8) that submitted to 60 minutes of ischemia and 24 hours of reperfusion, and then G-CSF  treatment was started one hour after reperfusion at a dose of 15 mg/kg subcutaneously and continued daily for seven days [Hypothermia and G-CSF group (n = 8) that submitted to 60 minutes of ischemia and 24 hours of reperfusion. Then, procedures in groups 2 and 3 were carried out together. Next, the rats were kept for seven days.Sham group (n = 8) simulation of the surgical procedure, with the introduction of the obstructer, but without occlusion of the MCA.Induction of transient focal cerebral ischemia.  Rats were anesthetized by injection of chloral hydrate (400 mg/kg) intraperitoneally. Body temperature was intermittently recorded using a rectal thermometer  and kept between 37 and 38°C using a 220 V lamp next to the animal. Post-ischemic hypothermia was obtained by placing the animals in a container containing ice on neck and temporal region immediately after surgery [ surgery . The lef surgery , 17. Thi surgery .Assessment of cerebral infarction and brain swelling. For defining the size of cerebral infarction, the animals were euthanized under ketamine  and xylazine  anesthesia. The brain was then rapidly removed, cut into 2-mm thick coronal sections by using brain matrix, stained with 2,3,5-triphenyltetrazolium chloride at room temperature for 30 minutes and then fixed in 10% buffered formalin [2) were calculated to obtain the infarct volumes per brain (mm3) by Image J 1.46r software   . InfarctStatistical analyses. All data were analyzed by SPSS 16.0. ANOVA was used as appropriate for comparison among different groups followed by post hoc test (Tukey) for multiple comparisons. All data are expressed as mean ± SEM, and P≤0.05 was considered statistically significant.Mortalities. The mortality of groups after ischemia was 38.46% (ischemic group), 11.11% (G-CSF group) and zero (other groups). Mortality was decreased in G-CSF group and was not observed in other treatment groups.Neurological deficit score. To determine the neurological deficit score of ischemic rat, as described in somatosensory and motor test (Garcia's index), the area under a curve was used . However, all of the treatment groups could reduce the size of infarction especially when the rats were treated with hypothermy plus G-CSF (335 ± 18.23mm3).mic rats  indicateBrain swelling volumes . BSV was caused by ischemic edema as shown in In this study, we used mild hypothermia because fewer side effect  and reduBrain edema is life-threatening complication of cerebral ischemia, and one of the major determinants of patient’s survival after the first few hours of stroke . Our resA previous study reported that G-CSF treatment in aged rats led to a reduced mortality, an improved functional recovery of motor function . In addiHypothermia has been considered the most effective resource to reduce brain injury caused by ischemia in experimental studies , and ourMale animals were chosen in this study, because there is evidence that females have neuroprotection, due to sex specific hormones. Adult animals were used, because younger rats, usually weighing less than 280 g, have more developed collateral vessels, providing greater resistance to infarction.Morphometric analysis of area and volume of focal cerebral ischemia represents an objective way to estimate the extent of ischemic injury, and it is commonly used for measuring the efficacy of neuroprotective agents .Our study demonstrated that hypothermy and G-CSF could reduce mortality rate. We observed that treatment with hypothermy plus G-CSF reduced edema more effectively than other treatments .Our major finding is that short hypothermic treatment plus G-CSF significantly reduces mortality rate, and edema as well as improves neurological function between this group and control ischemia group. The results suggest that the combination of hypothermia and G-CSF is more effectively than other treatment groups in our study, and this combination is important in improving motor function. Thus, hypothermy may provide the beneficial effect for the recovery of stroke patients by decreasing edema, and G-CSF may protect neurons from apoptosis.


Background: We often see patients with a thin endometrium in ART cycles, in spite of standard and adjuvant treatments. Improving endometrial growth in patients with a thin endometrium is very difficult. Without adequate endometrial thickness these patients, likely, would not have reached embryo transfer.Objective: We planned this study to investigate the efficacy of intrauterine granulocyte colony-stimulating factor (G-CSF) perfusion in improving endometrium, and possibly pregnancy rates in frozen-thawed embryo transfer cycles.Materials and Methods: This is a non-randomized intervention clinical trial. Among 68 infertile patients with thin endometrium (-7 mm) at the 12th-13th cycle day, 34 patients received G-CSF. G-CSF (300 microgram/1mL) to improve endometrial thickness was direct administered by slow intrauterine infusion using IUI catheter. If the endometrium had not reached at least a 7-mm within 48-72 h, a second infusion was given. Endometrial thickness was assessed by serial vaginal ultrasound at the most expanded area of the endometrial stripe.Results: The cycle was cancelled in the patients with thin endometrium  until 19th cycle day ultimately The cycle cancelation rate owing to thin endometrium was similar in G-CSF group (15.20%), followed by (15.20%) in the control group (p=1.00). The endometrial growth was not different within 2 groups, an improvement was shown between controlled and G-CSF cotreated groups, with chemical (39.30% vs. 14.30%) and clinical pregnancy rates (32.10% vs. 12.00%) although were not significant. Conclusion: Our study fails to demonstrate that G-CSF has the potential to improve endometrial thickness but has the potential to improve chemical and clinical pregnancy rate of the infertile women with thin endometrium in frozen-thawed embryo transfer cycle. The endometrium has received much less attention in reproductive research compared with the ovary, fertilization, or embryo development .Casper . EndometFor frozen embryo transfer (FET), the endometrium is frequently artificially prepared with estrogen and progesterone supplementation in order the match the endometrial stage during the critical implantation window. Women preparing for FET will often require additional estrogen supplementation, or other medication, if their endometrium is thin . Conventet al described the presences of G-CSF and its receptor in various human fetal tissues to  is a cytokine that produced primarily by hematopoietic cells. Recently G-CSF and G-CSF receptors have been also shown to be produced by endometrium, decidua and placenta, that suggesting that it may play a role in decidual and placental function Calhoun ssues to , 12.et al first used intrauterine G-CSF in four patients undergoing IVF with thin endometrium after standard endometrial preparation. All the patients successfully underwent ET and conceived. This demonstrates G-CSF as an novel remedy in patients with unresponsive, inadequate, and thin endometrium  implantation failure, repeated (≥3) abortion, congenital or acquired uterine anomaly, were excluded from the study.This interventional, non-randomized clinical trial was performed at the Yazd Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences between September 2013 and March 2014. The study was approved by the Ethics Committee of the university. Informed written consent was obtained from all couples. 68 women with endometrial thickness below 7mm at 12Also third party reproduction cycles were excluded. The patients were allocated into two groups: G-CSF cotreated group and control group. Considering that G-CSF administration was an experimental treatment, based on the antecedent reports, the choice of the patients affects their following treatment. Both the patients and the physicians were aware of the allocated arm. The artificial endometrial preparation program was similar in two groups. th-13th day of cycle. Endometrial thickness was measured at its thickest part in the longitudinal axis of the uterus. When the endometrial thickness was below 7 mm, the same ultra-sonographers measured it repeatedly for 2 times to confirm thin endometrium, and the average value of the 2 different measurements was recorded. All women received estradiol valerate tablet  6 mg daily and low-dose aspirin  from the second day of the menstrual cycle. Ultrasonography was done from the 12The estradiol was increased to 8 mg in both groups. 34 patients received uterine infusion of 300 microgram recombinant human G-CSF  by the use of IUI catheter, while the standard treatment was continued in the 34 patients who did not accept G-CSF treatment. After 2-3 days, the thickness of endometrium was measured again and if it was at least 7mm, the patient received 100mg intramuscular progesterone in oil  for 3 days. Then 2 or 3, day-3 embryos were transferred. If not, the second injection was done in GCSF group and continued estradiol with 8 mg per day in control group. th cycle day ultimately. Estradiol, progesterone and aspirin were continued after the transfer and till the tenth week of gestational age if pregnancy occurred. Vitrification and warming was performed according to vitrolife instruction. Warmed embryos were transferred to a culture media and evaluated 1 day later. The criteria for embryo quality was consulted from the embryo morphology assessment according to Hill et al criteria, and cleavage-stage embryos scored as grade A, B,C and D. The grade D embryos were not transferred. The cycle was cancelled in the patients with thin endometrium  until 19th-13th day of transfer cycle till 19th day ultimately. The secondary outcomes were chemical pregnancy that was determined by serum βhCG titer, 14 days after embryo transfer and clinical pregnancy that was determined by presence of gestational sac in vaginal sonography 28-30 days after embryo transfer.The primary outcome was the expansion of endometrial thickness that was measured by serial vaginal sonography from the 12Statistical analysisStatistical analysis was performed using the Statistical Package for the Social Sciences . Continuous data were presented as mean±SD and assessed by independent samples t-test between the groups. Enumeration data were compared by chi-square or fisher exact test. P<0.05 was considered significant.A total of 68patients were included in this study. Among 68participants, 34women were treated with G-CSF and defined as G-CSF cotreated group, and 34 without G-CSF were defined control group. The baseline characteristics of the patients were summarized in . Basic cth-13th cycle day) was similar in both groups (p=0.88.) (The proportion of thin endometrium in previous attempt for frozen-thawed embryo transfer was greater in G-CSF cotreated group . Mean ofp=0.88.) . The end1 patient in G-CSF group developed ectopic pregnancy. Higher chemical (39.30% vs. 14.30%) and clinical pregnancy rates (32.10% vs. 12.00%) were observed in patients with adequate endometrial thickness after G-CSF treatment in comparison with the control group although were not statistical significant. It might be a result of the mechanism of GCSF effect in increasing LIF and decreasing CD16, CD 56 which is one of the most important factors in implantation. We were worried about the safety and side effects of G-CSF. We did not find any side effect at all.et al a rose our great interests  and clinical pregnancy rates (32.10% vs. 12.00%) but due to small sample size these were not statistical significant. So endometrial Growth appeared to be unaffected by G-CSF treatment, but the outcome of pregnancy improved. Gleicher et al study can be due to synergistic effects between G-CSF and sildenafil citrate, which all the patients in that study received before and during G-CSF administration. Li et al failed to demonstrate that G-CSF has the potential to improve embryo implantation and clinical pregnancy rate of the infertile women with thin endometrium in frozen-thawed embryo transfer cycles. They also found no marked difference in endometrial thickness before and after G-CSF infusion. This study was nonrandomized, retrospective and small sample size, and the women with G-CSF treatment seemed to have poorer endometrium than those without G-CSF treatment. Their administration of G-CSF was a relatively low dose (100 microgram) and only once  enhanced endometrial development inpatients without synechea. Ongoing pregnancy and implantation rates were significantly higher. In patients with intrauterine synechea did not achieve an endometrial expansion (Kim xpansion . In clinxpansion -18. In tG-CSF, GM-CSF and M-CSF are, however, distinct: G-CSF facilitates stem cell and progenitor proliferation in neutrophilic granulocytes, while GM-CSF facilitates proliferation and differentiation in macrophages, granulocytes and eosinophils. Because of proliferative effects on fibroblasts, one, therefore, can hypothesize that GM-CSF may expand endometrial thickness more than G-CSF and M-CSF or GM-CSF may have even better effectiveness on endometrial thickness . Local GFinally, we would like to point out the limitation of our study. This study was nonrandomized, and small sample size. Our study demonstrates that G-CSF has the potential to improve embryo implantation and clinical pregnancy rate of the infertile women with thin endometrium. This observation, however, has to be confirmed by larger prospective RCT. We would like to conduct further studies in terms of different dose and times for G-CSF administration to reveal the efficiency. Our study fails to demonstrate that G-CSF has the potential to improve endometrial thickness but has the potential to improve chemical and clinical pregnancy rate of the infertile women with thin endometrium in frozen-thawed embryo transfer cycle. It might be a result of the mechanism of GCSF effect in increasing LIF and decreasing CD16, CD56 which is one of the most important factors in implantation.


The differentiation between multiple system atrophy (MSA) and Parkinson’s disease (PD) is difficult, particularly in early disease stages. Therefore, we aimed to evaluate the diagnostic value of neurofilament light chain (NFL), fms-like tyrosine kinase ligand (FLT3L), and total tau protein (t-tau) in cerebrospinal fluid (CSF) as biomarkers to discriminate MSA from PD. Using commercially available enzyme-linked immunosorbent assays, we measured CSF levels of NFL, FLT3L, and t-tau in a discovery cohort of 36 PD patients, 27 MSA patients, and 57 non-neurological controls and in a validation cohort of 32 PD patients, 25 MSA patients, 15 PSP patients, 5 CBS patients, and 56 non-neurological controls. Cut-offs obtained from individual assays and binary logistic regression models developed from combinations of biomarkers were assessed. CSF levels of NFL were substantially increased in MSA and discriminated between MSA and PD with a sensitivity of 74% and specificity of 92% (AUC = 0.85) in the discovery cohort and with 80% sensitivity and 97% specificity (AUC = 0.94) in the validation cohort. FLT3L levels in CSF were significantly lower in both PD and MSA compared to controls in the discovery cohort, but not in the validation cohort. t-tau levels were significantly higher in MSA than PD and controls. Addition of either FLT3L or t-tau to NFL did not improve discrimination of PD from MSA above NFL alone. Our findings show that increased levels of NFL in CSF offer clinically relevant, high accuracy discrimination between PD and MSA. Parkinson’s disease (PD) is the most common movement disorder with typical age of onset around 60 years although some patients ~3–5%) develop PD before the age of 40 . PD is c–5% develMultiple system atrophy (MSA) is a relatively rare and sporadic adult-onset disease characterized by a variable combination of parkinsonism, cerebellar ataxia, autonomic dysfunction (particularly orthostatic hypotension), and pyramidal signs . MSA is Two recent studies have investigated the utility of fms-like tyrosine kinase ligand (FLT3L) as a potential cerebrospinal fluid (CSF) biomarker to differentiate MSA from PD but had conflicting results. While one paper reported that CSF levels of FLT3L could differentiate between MSA and PD with high accuracy  another Neurofilament proteins are highly phosphorylated neuronal cytoskeleleton proteins composed of three subunits of which the smallest, the 68 kDa neurofilament light chain (NFL), forms the backbone and is essential for neurofilament assembly . ElevateThe present study was performed at the Radboud University Medical Centre . We studied patients initially referred to our tertiary movement disorder center between December 2000 and November 2008 Figure , with a Disease severity was established using the (modified) Hoehn and Yahr (H&Y)  stages an = 15) and corticobasal syndrome  with previously unreported, retrospective CSF NFL levels were included to show differences in NFL levels between PD patients and other atypical parkinsonisms , written informed consent was obtained from a next of kin of the patient. The local institutional review board (“Commissie Mensgebonden Onderzoek region Arnhem-Nijmegen”) approved of this study. For the remaining patients , CSF samples were obtained as part of the clinical diagnostic work-up of a patient. Patients were informed that their data, including CSF, could be used for further scientific purposes and were given the option to object against this use, in which case their data were not used. This procedure has been approved as well.g at room temperature), and stored at −80°C. Patient information was decoded to maintain confidentiality. Undiluted CSF samples were measured in duplicate using commercially available enzyme-linked immunosorbent assays (ELISAs) for Human FLT3L , NFL , and t-tau . ELISAs were performed according to manufacturer’s instructions except the capture antibody for the FLT3L ELISA was used at 1 μg/mL.Cerebrospinal fluid samples obtained by lumbar puncture were collected in polypropylene tubes, centrifuged . Spearman rank correlation was used to determine correlations. We performed analysis of covariance (ANCOVA) to control for possible confounding variables . Binary logistic regression was used to identify variables contributing to discrimination of MSA from PD and receiver–operator curves (ROCs) were used to determine the diagnostic accuracy of CSF parameters and models developed from the binary logistic regression. Statistical analyses were performed using GraphPad PRISM 5 software  and SPSS software version 20.0 . Comparison of the ROC curves was performed using MedCalc® software version 12.5.0.0. Bootstrapping analyses using data from both cohorts were also performed for additional validation of the measures using Medcalc 12.7.0 9 Trial version.Cerebrospinal fluid parameters with non-Gaussian distribution were log transformed and between-group differences were tested using one-way analysis of variance (ANOVA) followed by Tukey’s We analyzed 233 CSF samples: 52 MSA patients, 68 PD patients, and 113 non-neurological controls. Of these, 61% (32/52) of the MSA and 87% (59/68) of the PD patients had been studied prospectively for 3 years. The discovery group consisted of 36 PD, 27 MSA, and 57 controls. Patient characteristics and CSF parameters are reported in Table p < 0.01) and MSA  compared with controls (47.8 ± 14.3 ng/L) but similar in MSA and PD  and controls  but not between PD and controls  but not MSA  or controls . Details of the correlation data are provided in Table S1 in Supplementary Material. Four of the PD patients exhibited levels of t-tau that were markedly different from the remainder of the group  yielded similar sensitivity (75.0%) and specificity  for discriminating MSA from PD whereas the combination of FLT3L and NFL (Model 2: y = −1.646 + 0.001*NFL-0.0308*FLT3L) yielded a sensitivity of 81.8% and specificity of 94.8% (AUC = 0.89). The combination of NFL, FLT3L, and t-tau (Model 3: y = −3.054–0.001*NFL + 0.003*t-tau − 0.028*FLT3L) yielded a higher AUC (0.92) with increased sensitivity (94.7%) but reduced specificity (83.3%). Comparison of the ROC analyses showed that this improvement was not significantly better at discriminating between MSA from PD than NFL alone (p > 0.05).Neurofilament light chain alone provided high discrimination (AUC 0.85) between MSA and PD with 74.1% sensitivity and 91.7% specificity. Logistic regression models of combination biomarkers were then analyzed  in MSA. Details of these correlations are provided in Table S3 in Supplementary Material. When we repeated our analyses controlling for age, gender, UPDRS, and disease duration using ANCOVA, significance levels were maintained for NFL but not FLT3L or t-tau, suggesting that NFL levels are robust but FLT3L and t-tau levels may be influenced by other factors that give rise to heterogeneous values.Gender was not correlated with CSF parameters for any group. Age was correlated with, or tended to be correlated with, all CSF parameters in PD and controls but not in MSA . Disease duration and severity (ICARS and H&Y) were not correlated with CSF parameters for either PD or MSA in the discovery cohort. UPDRS was not correlated with CSF parameters in the PD group but, intriguingly, showed a significant negative correlation with NFL (p < 0.001) and controls . CSF NFL levels were also significantly higher in other atypical parkinsonsisms  than in PD and controls . This significance was maintained after controlling for age, gender, and disease duration with AUC ≥ 0.9 . FLT3L levels were non-significantly lower in both PD and MSA compared with the controls although a small significant difference between MSA and controls was found after controlling for age, gender, disease duration, and disease severity (p < 0.05). As with the discovery group, we also observed higher levels of t-tau in MSA than PD but this failed to reach significance (p = 0.06). We noted that t-tau levels in both PD and MSA in the validation groups were overall lower than in the discovery group and for MSA the difference in t-tau levels in discovery (335 ± 164 ng/L) versus validation (244 ± 93 ng/L) was significant (p < 0.05). The methodology used to measure t-tau (Innotest ELISAs) was the same for all patients but CSF samples collected prior to 2004 were analyzed retrospectively, which may have influenced our results.In the validation cohort, we confirmed higher levels of CSF NFL in MSA (5938 ± 4267 ng/L) compared with PD  than MSA  in the validation group, but controlling for this variable using ANCOVA did not alter the significance level for the CSF parameters.The models developed using the CSF parameters from the discovery group were applied to the validation group and diagnostic values were calculated using cut-offs obtained from the discovery group. We could correctly identify the majority of MSA patients (sensitivity = 80% and specificity = 97%) using NFL alone (AUC = 0.94). Again, ROC curve comparison showed that none of the models significantly improved the discrimination of MSA from PD.Bootstrapping analysis of the combined data to further validate our result, produced an ROC curve for NFL (PD versus MSA) that was highly comparable with ROC curves from the individual cohorts . Bootstrapping of the combined FLT3L data revealed significantly lower levels of FLT3L in both the PD and MSA groups compared with controls as was observed in the discovery group but not the validation group. We found no significant differences in CSF FLT3L levels between PD and MSA patients in the individual cohorts nor when using bootstrapping of the combined data.In the current study, we showed that CSF levels of NFL can be used for clinically relevant discrimination of MSA from PD. These results confirm our previous findings using a different method of detection for NFL  and the We found significantly decreased CSF FLT3L levels in PD and MSA compared to controls in our discovery cohort but not in the validation cohort. Bootstrapping of the combined data was consistent with the discovery group, revealing significantly lower levels of FLT3L in both PD and MSA compared with controls but we and others  found noUnlike the first study , we did Neurofilament proteins are essential for maintaining the neuronal cytoskeleton and increased levels of NFL in the CSF of MSA patients likely reflects extensive axonal degeneration. In keeping with earlier findings , 14, CSFFLT3L is a hematopoietic growth factor expressed in various tissues including the brain  and has A major strength of our study is that diagnosis was made prospectively for the majority of PD and MSA patients using detailed neurological examination in combination with imaging studies, and final diagnosis was confirmed after long follow-up by case review. Our findings emphasize a consistency with other studies –14, 31 sFollowing the successful identification of the CSF biomarkers t-tau, p-tau, and Aβ42 that support the clinical diagnosis of Alzheimer’s disease, there has been a growing interest in the discovery of similarly specific CSF biomarkers for PD and AP. Many studies have been reported on the quantification of α-synuclein in CSF. Although there is a general consistency that decreased concentrations of this protein are observed in PD and AP with α-synuclein pathology, large overlap between these patient groups and neurological controls has hindered the introduction of its quantification into clinical practice , 35. SimNiklas Norgren is the CEO of UmanDiagnostics. UmanDiagnostics did not play any role in the study design and did not restrict or affect the data analysis in any way. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://journal.frontiersin.org/article/10.3389/fneur.2015.00091The Supplementary Material for this article can be found online at Click here for additional data file.


We wish to comment on the correspondence regarding the question of efficacy and mechanism of action of acetazolamide in the treatment of idiopathic intracranial hypertension (IIH), published in JAMA September 10, 2014  hold thaIn the 1950s, a similar discussion concerned the mechanism of acetazolamide in the treatment of epileptic seizures. Initially, it was thought that the effect was indirect via the kidneys and was related to an induced metabolic acidosis. Subsequently, acetazolamide was shown to prevent seizures in nephrectomized animals; the anticonvulsant effect was independent of the effect on the kidneys and was caused by a direct inhibition of carbonic anhydrase in the brain .In patients with IIH treated with acetazolamide, the inhibition of the enzyme in the choroid plexus results in a reduction of CSF production and flow. The acid–base status of the patient may also alter the distribution of acetazolamide in the CSF and brain, but its effect on the CSF flow is secondary to that mediated by the choroid plexus. Based on the pharmacology and distribution of acetazolamide and carbonic anhydrase in the brain, the theory that emphasizes the effect of acetazolamide on CSF production in IIH is most likely primary and direct, and weight loss, when recognized as a factor, is secondary and indirect, and frequently the result of toxic doses in excess of the amount needed for complete enzyme inhibition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.