Kidney paired donation (KPD) remains an important strategy to facilitate transplantation in patients who have a healthy and willing donor, but are unable to proceed with directed donation due to either ABO incompatibility or a positive cross-match against their intended donor.Personal knowledge, The Canadian Blood Services Database for Living Donor Exchange, published reports and personal communications.The national Living Donor Paired Exchange Programme (LDPE) in Canada was established in 2009. 235 transplants were completed of which 190 were registered recipients and 45 were from the deceased donor (DD) wait list. At 1 year, patient survival was 100%, graft survival 98%, with a biopsy proven acute rejection rate of 8%. The mean serum creatinine (Cr) at the end of one year was 109 mmol/l. Donor survival is 100%. Key to success are national standards for antibody testing and cross-matching, and for evaluating donors and recipients, as well infrastructure (software and personnel) to run the program. The structure of the Canadian program is compared with that of other programs in the United Kingdom, Australia, the Netherlands, and the United States.This review does not include information on travel distances and difficulties, or patient satisfaction.National collaboration and acceptance of common standards is possible and leads to substantial benefits, especially for those patients who are hardest to match.What was known before: Kidney paired donation is considered ethically acceptable. National and regional programs have been created in a number of countries.What this paper adds: Key to the success of the Canadian national program are acceptance of standardized procedures and national and provincial support and oversight. The results of renal transplantation have improved substantially. As well, various strategies have been employed to increase organ availability. Despite this, the number of patients on the waiting list continues to grow and demand for organs is ever increasing , 2. The Living donor transplants provide a mortality benefit over deceased donor transplantation . HoweverSegev et al have shown that the percentage of registry matches is higher if more pairs are entered . As wellUntil several years ago, based partly on provincial funding and organization of health care, there was no national transplantation collaboration or oversight. Canadian Blood Services (CBS) was ultimately entrusted by provincial and territorial governments to establish national organ donation and transplant registries. The first registry, the Canadian LDPE programme was established in 2009 by a collaboration between Canadian Blood Services and the Canadian renal transplant community. Collaboration and discussion with key leaders in the field of KPD played an important role in setting out the principles and foundations of the programme. Table As of November 2013, 468 pairs were registered and a total of 58 non – directed donors (NDD) had been included. 235 transplants were completed of which 190 were registered recipients and 45 were from the deceased donor (DD) wait list. Of the 235 total transplants, 20 were paired exchanges or 2 way exchanges, 55 were from 16 multi way exchanges of 3, 4 or 5 chain lengths, and 160 resulted from domino exchanges; (46 dominos of 2-5 transplants each) , 129 days for multi way and 117 days for domino chains. 54% of matches have required travel. Sensitized patients with calculated panel reactive antibody (cPRA) <97% have had a 50% chance of a match within the program and sixty per cent of registered recipients with a cPRA between 80-96% have been transplanted. Transplant rates among the highly sensitized group of patients with cPRA >97% have been lower. 58% of proposed matches and 62% of proposed chains were completed. 55 chains collapsed due to 57 declined matches, of which, 6 were repaired and completed. 49 collapsed chains were not repaired (affecting 151 matched pairs). Of the 57 declined matches, 15(26%) were due to positive cross-matches, 24 were due to medical reasons, 14 due to non-medical reasons and finally 5 were as a result of surgical causes. In 2013, there were only 2 unexpected positive cross-matches.The outcomes of KPD transplants are comparable to that of directed living donor transplants. At 1 year, patient survival was 100%, graft survival 98%, with a biopsy proven acute rejection rate of 8%. The mean serum creatinine (Cr) at the end of one year was 109 mmol/l. Donor survival is 100%.The KPD programme in the UK has been operating since 2007. Donors and recipients are assessed according to guidelines set by the British Transplantation Society/Renal Association . MatchinChanges were made to the system in 2012, where no priority was given to local exchanges, blood group compatible transplants were allowed, as compared to previously, where blood group identical transplants only were permitted. An incremental prioritization for recipients who remained unmatched in the scheme after each matching run was adopted. Further, compatible donor–recipient pairs could be enrolled and an extended matching criteria form for registering patients with low titer HLA and ABO blood group antibodies was introduced .The first kidney paired donation (KPD) transplants were performed in Western Australia in 2007 and the national KPD programme was established in late 2010 as part of the Organ and Tissue Authority’s efforts to increase available organs from live donors . Match rAs of October 2012, 115 pairs had registered in the national programme and 51 transplants were completed under the auspices of the national programme .In the national programme, 63% of enrolled pairs in the first 2 years found a match with 46% of them receiving a transplant. 33% of registered candidates had a cPRA >95%. 42% of transplanted patients had a cPRA >75% and mean cPRA was 55%. Blood group O recipients are often considered to be disadvantaged in a KPD program as they can only receive an organ from a blood group compatible donor. In the Australian KPD program, 56% of transplant recipients were blood group O, this has been possible due to ABO desensitization being included as part of KPD . ABO-incEight year outcomes of the Dutch KPD programme were published in 2011 . All traIn the US, multiple independent registries exist in addition to a national registry administered by UNOS. KPD may be underutilized in the US and factors contributing to underutilization include insurance costs, reimbursement to donor when there are different insurance providers, and fragmented registries . The KPDThe national LDPE programme in Canada has been a rewarding success story. It is important to appreciate that the benefit of LDPE is not only that more patients get living donor transplants with the associated benefits, but also that all of those patients, who would previously have been on deceased donor lists, no longer are on them. This reduces waiting time for those without a living donor. The success of the programme is related to national collaboration, standardized antibody testing and standardization of both the workup and acceptance criteria for donors, centralized allocation software, operational oversight, dedicated central support staff, and government support of logistics for organization and travel.The Canadian national LDPE program was established later than similar programs in the UK and Netherlands, but has seen good success in a relatively short span of time. As of November 2013, 235 transplants were completed which is higher than the reported transplants in other national programmes worldwide. The relatively small geographic area in the Netherlands enables them to do cross matches in a central laboratory. Other national programs are yet to standardize HLA laboratory techniques. The publicly funded healthcare system in Canada has obviated the argument of financial responsibility unlike in the US where there is ongoing debate on establishing a standard acquisition charge for KPD by private insurers [There are important considerations for the future. Candidates with cPRA > 97% have a lower transplant rate than other highly sensitized patients. Strategies to improve this, including the national registry for highly sensitized patients, are under active consideration. For instance, consideration of desensitization in association with LDPE is suggested by some early encouraging results of ABO and low level DSA desensitization from programs in Australia and the US , 14. TheThe Canadian LDPE programme is an example of success achieved by national integration and collaboration. Hopefully, the success to date will emphasize the benefits of LDPE to both patients and centers, resulting in a further increase in pairs entered, matches and transplants.


Patients with serum concentrations of TT > 6.6 ng mL−1 showed a frequency of ultrasound criteria suggestive for bilateral form of prostatitis and prostate-vesiculo-epididymitis and significantly lower compared to the other examined groups. At multivariate logistic regression analysis adjusted for age and BMI, TT was an independent predictive factor of prostatovesiculitis  and prostate-vesiculo-epididymitis , which represent the main forms of complicated MAGI. The results of this study suggest that male hypogonadism could be associated with a different ultrasound characterization of these patients.Aim of the present study was to evaluate the different ultrasound characterization of fertile symptomatic patients with MAGI  according to different serum concentrations of total T (TT). We analyzed the ultrasound and hormonal data of 200 patients aged between 24.0 and 67.0 years. Patients were divided into six groups according to the sextile distribution of TT. Patients with serum concentrations of TT < 3.6 ng mL Male accessory gland infection (MAGI) represents a potential cause of male infertility , 2, withMAGI may affect sperm parameters through three possible mechanisms: (a) secretory dysfunction of the accessory glands  –6, (b) fThe ultrasound examination of the epididymis and prostate-vesicular tract represents an important diagnostic tool for the clinical evaluation , 15, altuncomplicated form (ultrasound criteria limited to the prostate: prostatitis (P)),complicated form , and the epididymis: prostatitis, vesiculitis, and epididymitis (PVE)) [s (PVE)) , 15,unilateral form  [-PV-PVE) , 15, 17,bilateral form  [PV-PVE ) , 15, 17:PV-PVE ) ,hypertrophic-congestive form (FSUF),fibrosclerotic form (HCUF).The ultrasound evaluation allows discriminating different diagnostic categories, in particular,There are several risk factors that may favor the onset of MAGI, for example, lifestyle, diet, cigarette smoking, gastrointestinal diseases, and sexual promiscuity , 20.The role of low levels of testosterone (T) in promoting the onset of P in an experimental model was recently reported . In partOn the basis of these premises, the aim of this study was to evaluate the different ultrasound characterization of fertile symptomatic patients with MAGI according to different serum concentrations of total T (TT).Out of a consecutive series of 2000 subjects, we selected 200 fertile (spontaneous pregnancy during the previous 12 months) symptomatic patients with MAGI evaluated during the period between January 2012 and January 2014 that were subjected to evaluation of serum concentrations of TT.The diagnosis of MAGI was carried out according to the criteria reported in All examined patients had ultrasound criteria suggestive of MAGI, as previously reported , 17, 18.Subjects with TT higher than the upper limit of the range were excluded. Other exclusion criteria were represented by hormone therapy with antiestrogens or gonadotropins or T.−1, FSH = 2.0–12.0 mIU mL−1, and total testosterone = 2.8–8.0 ng mL−1. Blood sampling was performed at 8:00 am, after at least 8 hours of sleep. The determination of serum LH was repeated after an interval of 30 minutes.Hormonal evaluations were performed by electrochemiluminescence with Hitachi-Roche equipment . The reference intervals were as follows: LH = 1.6–9.0 mIU mL2 .We analyzed the ultrasound and hormonal data (serum TT) of 200 patients with MAGI aged between 24.0 and 67.0 years (mean age = 36.0 ± 14.0 years) with BMI between 18.0 and 33.0 kg/mGroup 1: TT ≤ 2.7 ng mL−1,Group 2: TT > 2.7 and ≤3.6 ng mL−1,Group 3: TT > 3.6 and ≤4.4 ng mL−1,Group 4: TT > 4.4 and ≤5.3 ng mL−1,Group 5: TT > 5.3 and ≤6.6 ng mL−1,Group 6: TT > 6.6 ng mL−1.Patients were divided into six groups according to the sextile distribution of total testosterone: unilateral P, PV, and PVE, bilateral P, PV, and PVE, and HCUF or FSUF.The frequency of the following possible ultrasound conditions was evaluated for each group: P , PV (ultrasound criteria suggestive for prostatitis associated with criteria of vesiculitis), and PVE (criteria of prostatitis and vesiculitis associated with epididymitis),The study was approved by the Internal Institutional Board and all examined patients signed informed consent to the processing of personal data.t-test or Mann-Whitney U test on the basis of their normal or not-normal distribution, respectively. Pearson's or Spearman's correlation coefficients were used in order to test the associations between the different variables. Multivariate linear regression models adjusted for age and BMI were performed for factors significantly correlated at Spearman's analysis. Multivariate logistic regression analysis was performed to assess significant predictors of MAGI after adjusting for age and BMI.Normality of variables' distribution was tested by Kolmogorov-Smirnov test. Accordingly, variables are presented as means ± standard deviations or median (interquartile range) and differences between groups were tested by Student's independent P value was lower than 0.05.The software SPSS 9.0 for Windows was used for statistical evaluation . A statistically significant difference was accepted when the r = −0.929; P < 0.01).The linear regression analysis showed inverse association between TT and duration of symptoms after adjusting for age and BMI (P < 0.01), PV , PVE , UP , BP , BPV , BPVE , HCUF , and FSUF .At multivariate logistic regression analysis adjusted for age and BMI, TT was an independent predictive factor of P , ultrasonographic bilateral form of P, PV, and PVE, and ultrasonographic fibrosclerotic form. The increase in TT represents a predictive factor for ultrasonographic uncomplicated form (P), ultrasonographic unilateral form of P, and ultrasonographic hypertrophic-congestive form.−1). The same group showed a significantly higher frequency of the ultrasonographic hypertrophic-congestive form and a significantly lower frequency of the ultrasonographic fibrosclerotic form.The comparison between groups showed a significantly lower frequency of PVE (ultrasonographic form with greater anatomic extension) in group 6 . Furthermore, the hypogonadal patients showed a higher frequency of “bilateral” and “fibrosclerotic” ultrasound form of MAGI.Ultrasound evaluation of the epididymis, prostate, and seminal vesicles represents an important tool for the clinical characterization of MAGI; however, it is not a mandatory diagnostic step . In thisThe evaluation of serum levels of TT is often required during an initial andrological consultation for various clinical problems  and therefore we wanted to examine retrospectively the frequency of ultrasound criteria suggestive of MAGI according to the different serum concentrations of TT. From the methodological point of view, there are no similar studies in the literature; however, for the interpretation of the results, it is necessary to analyze some evidences that suggest a role of testosterone as a modulator of the inflammatory response in the prostatic tissue.Several mechanisms have been proposed to explain the potential anti-inflammatory effects of T on the prostatic tissue. In particular, it has been suggested that T could reduce the tissue expression of proinflammatory cytokines and T-lymphocytes and antagonize macrophage activation and neutrophil in the prostatic tissue and the progression of the fibrosis. Furthermore, T seems to positively modulate the expression of junctional proteins of the prostatic epithelium and negatively the activation of the immune response. Finally, the antiestrogenic effect and the negative modulation of the expression of toll-like receptor 4 represent other mechanisms , 28–30.β, and TNF (proinflammatory cytokines), T lymphocytes, macrophages, markers of neutrophil activation, and prostatic fibrosis. The hypogonadism is associated with a reduced expression of Claudina 4 and Claudina 8, the essential components in the formation of junctional structures of the prostatic epithelium, whose alteration induces inflammatory degeneration of the prostate tissue and the immune response [In detail, the study of Vignozzi and colleagues  showed tresponse ; insteadresponse . A reducresponse . Finallyresponse .Finally, the patients of group 1 have a condition of male hypogonadism probably related to the age, suggested by higher serum concentrations of LH and lower mean value of testicular volume. This aspect must be evaluated through further studies in order to understand a possible association between persistence of MAGI (these patients have an increased duration of symptoms) and the different serum concentrations of TT, as suggested in previous studies about a similar clinical model represented by LUTS (lower urinary tract symptoms) secondary to benign prostatic hyperplasia (BPH) , 32.An important difference between these two clinical models is represented by the increase in prostate volume for the patients with LUTS-BPH related that represent a potential contraindication to the hormonal treatment with T, while patients with MAGI have an inflammation of periurethral zone assessed by ultrasound evaluation associated with obstructive symptoms , 17, 18,In conclusion, the results of this study suggest evaluating the serum concentrations of total testosterone in patients with MAGI. In particular, male hypogonadism is associated with different clinical and ultrasound characterization of these patients.


Nannochloropsis sp. CCAP 211/78 cultivated in four different outdoor continuously operated pilot-scale photobioreactors under the same climatological conditions were compared. The optimal dilution rate was determined for each photobioreactor by operation of the different photobioreactors at different dilution rates.Microalgae are a potential source of sustainable commodities of fuels, chemicals and food and feed additives. The current high production costs, as a result of the low areal productivities, limit the application of microalgae in industry. A first step is determining how the different production system designs relate to each other under identical climate conditions. The productivity and photosynthetic efficiency of −2 day−1 and 2.4–4.2 %, respectively, were found in comparison to the horizontal systems; 12–15 g m−2 day−1 and 1.5–1.8 %. The higher ground areal productivity in the vertical systems could be explained by light dilution in combination with a higher light capture. In the raceway pond low productivities were obtained, due to the long optical path in this system. Areal productivities in all systems increased with increasing photon flux densities up to a photon flux density of 30 mol m−2 day−1. Photosynthetic efficiencies remained constant in all systems with increasing photon flux densities. The highest photosynthetic efficiencies obtained were; 4.2 % for the vertical tubular photobioreactor, 3.8 % for the flat panel reactor, 1.8 % for the horizontal tubular reactor, and 1.5 % for the open raceway pond.In vertical photobioreactors, higher areal productivities and photosynthetic efficiencies, 19–24 g mVertical photobioreactors resulted in higher areal productivities than horizontal photobioreactors because of the lower incident photon flux densities on the reactor surface. The flat panel photobioreactor resulted, among the vertical photobioreactors studied, in the highest average photosynthetic efficiency, areal and volumetric productivities due to the short optical path. Photobioreactor light interception should be further optimized to maximize ground areal productivity and photosynthetic efficiency. Microalgae are a promising feedstock for bulk commodities like chemicals, food, feed and fuels. High production costs hinder the current implementation of algal biomass as a feedstock for bulk commodities; production costs should decrease to less than 1 €/kg dry weight .−1 by a paddle wheel. Open raceway ponds are characterized by low cell densities up to 0.3 g L−1 ; EPAR: energetic content of the PAR fraction of sunlight (4.76 J mol−1), ASTM G173-03 [−1).The photosynthetic efficiency was calculated with Eq.  G173-03 ; and 0.4sunlightPEsunlight to biomass conversion efficiency (%)Px,ground−2 day−1)ground areal biomass productivity (g mVharvestharvested volume (L)Cx−1)biomass concentration (g LVrvolume of photobioreactor (L)Aground2)ground area occupied by photobioreactor volumetric biomass productivity daily ground areal photon flux density (mol mIground−2 s−1)ground areal photon flux density (µmol m−1)standard enthalpy of combustion (kJ gEPAR−1)conversion factor PAR photons to joule (J molD−1)dilution rate PFD−2 day−1)photon flux density (mol mgsv−1)superficial gas velocity (m sORPopen raceway pondHThorizontal tubularVTvertical tubularFPflat panel


During surgery, samples of CSF were collected from the lumbar and ventricular compartments and assayed for chloride, glucose and total protein. The lumbar/ventricular (L/V) ratio was calculated for each analyte. The ratio between measured and expected levels (Ibioch) was calculated for each analyte and compared with Idyn. Idyn varied from 0 to 100.103μl2.s2. In contrast to the L/V ratios for chloride and glucose, the L/V ratio for total protein varied markedly from one patient to another (mean ± standard deviation (SD): 2.63 ± 1.24). The Ibioch for total protein was strongly correlated with the corresponding Idyn .We observed correlated alterations in CSF flow and biochemical parameters in patients with CH. Our findings also highlight the value of dynamic flow analysis in the interpretation of data on CSF biochemistry.The equilibrium between the ventricular and lumbar cerebrospinal fluid (CSF) compartments may be disturbed (in terms of flow and biochemistry) in patients with chronic hydrocephalus (CH). Using flow magnetic resonance imaging (MRI) and CSF assays, we sought to determine whether changes in CSF were associated with biochemical alterations. Nine elderly patients with CH underwent phase-contrast MRI. An index of CSF dynamics (I In 1964, Dichiro .Chronic hydrocephalus (CH) is characterized by marked ventriculomegaly and alterations in CSF flow. The incidence of CH increases with age, and the diagnosis of this condition is based on both clinical and radiological data. Magnetic resonance imaging (MRI) can reveal severe ventricular enlargement  and may also evidence CSF “flow void” in the aqueduct . The total protein concentration is 2.5 times higher in the lumbar CSF than the ventricular CSF because of the gradual influx of proteins moving from the choroid plexus to the lumbar spinal canal  on the relatively high permeability to albumin of the blood-CSF barrier in the spinal subarachnoid space. However, this gradient also depends on several other factors, including: (i) mixing within the ventricles and the subarachnoid space; and (ii) removal mechanisms, which may operate in some compartments but not others . All the patients included in the present research project were informed by a medical doctor of the aim of this research. They were all free to reject their participation without any implication on the following of their care. All have signed the consent before to be included in the research population. This study was approved by the local investigational review board . For obvious ethical reasons, it was impossible to obtain samples of ventricular and lumbar CSF from healthy volunteers; we therefore studied nine elderly, shunted patients  age: 73 ± 8; age range: 56–83) with a documented clinical history of CH and easy access to the CSF during the tap test and the surgical shunt procedure.2; pixel resolution: 0.6 × 0.6 mm2; slice thickness: 5 mm; views per segment: 2; velocity (encoding) sensitization: 5, 10 or 20 cm/s for the cervical aqueduct and 5 cm/s for the C2-C3 subarachnoid space. The acquisition time for each flow series was about 2 min, depending on the participant’s heart rate.All nine patients underwent morphological and CSF flow MRI of the brain Table . All acqData were analyzed using dedicated PCMRI image processing software  and the spinal canal (SVspine) over the CC  was derived as a guide to the CSF volume displaced in the ventricles and the spinal canal during the CC. It was defined as the product of the two CSF SVs and the two CC times (CCTaqu and CCTspine); Idyn = SVaqu × SVspine × CCTaqu × CCTspine.An overall index of CSF dynamics (“IThe CCT was measured using a plethysmographic sensor worn on the patient’s finger during the MRI. The sensor monitored the changes in blood volume in the finger during the CC. The PCMRI acquisition was synchronized with the systolic pulse.bioch”), in order to assess the difference between expected and measured protein levels in the ventricle:All patients underwent a CSF tap test during placement of their shunt. During surgery, CSF samples (at least one for the ventricular compartment and one for the lumbar compartment) were collected from each patient. The samples were collected between 9.00 and 10.30 am after overnight fasting, in accordance with our university medical center’s validated, aseptic protocols. The plastic vials were immediately sent to the university medical center’s central laboratory. Chloride, glucose and total protein levels were determined using conventional techniques . Briefly, chloride was assayed potentiometrically using an ion-selective electrode. The glucose level was determined in an enzymatic assay based on hexokinase and glucose-6-phosphate. The CSF total protein level was determined using pyrogallol red/acidic molybdate reagent. Levels of the resulting blue pyrogallol/molybdate/protein complex were quantified by reading the optical density at 596/694 nm. Ventricular and lumbar levels were measured for each CSF component. Under physiological conditions, the ventricular CSF protein level is around half the lumbar level  and 444 ± 188 (130–650) μL/CC, respectively. The mean values of CCTaqu and CCTspine were 0.8 ± 0.12 and 0.79 ± 0.12 s, respectively. The Idyn values ranged from 0 to 100.103 μl2.s2  when measured at the cerebral aqueduct or at the spinal level. The mean ± SD (range) values of SV−1, the mean ventricular and lumbar CSF glucose levels were respectively 3.8 ± 0.8 (2.9–5.3) mmol.l−1 and 3.4 ± 0.6 (3–4.9) mmol.l−1, and the mean ventricular and lumbar CSF protein levels were respectively 0.23 ± 0.08 (0.12–0.36) and 0.57 ± 0.3 (0.29–1.3) mmol.l−1 . The mean value was 2.63 ± 1.24 . Nevertheless, the respective values of Idyn and Ibioch were highly correlated , suggesting the presence of a strong, linear relationship between CSF dynamics and CSF total protein levels.There were no significant ventricular vs. lumbar differences in the chloride and glucose levels. The L/V ratios for chloride and glucose were respectively 0.99 ± 0.01 and 0.91 ± 0.10. The value of the L/V protein ratio varied greatly from one patient to another but was correlated with the CSF dynamic index  movement of the tracer between the subarachnoid space and the ventricles was proportional to the CSF pressure; and (ii) that CSF turnover may be proportional to the ICP , the L/V protein ratio and Ibioch strongly support this hypothesis.  were similar. Indeed, electrolytes diffuse passively from the CSF to the interstitial tissue. The glucose concentration appears to be much the same through the whole cerebrospinal system. In contrast, total protein levels differed markedly when comparing the ventricular and lumbar regions. In the lumbar compartment, most CSF proteins are derived from plasma proteins (as a result of close interaction with blood vessels; Laterre et al., et al.’s  study in. Figure .Lastly, the stable isotope labeling approach recently described by Lehmann et al.  could beIn conclusion, the results of our pilot study in a reliable human model suggest that the amplitude of the CSF flow during the CC influences CSF protein levels and the distribution of protein within the central nervous system’s cavities. This proof-of-concept study opens up new avenues in the investigation of the relationship between clinical biochemical parameters and dynamic MRI parameters, which should now be confirmed in a larger cohort. Although further research should seek to determine which proteins are the most informative, we have demonstrated the value of combining PCMRI with CSF biomarker assays. Recent studies have emphasized how difficult it is to diagnose Alzheimer’s disease—especially in patients with normal pressure hydrocephalus (Graff-Radford, VP: Data analysis, writing and editing the manuscript. OB: project design, acquisition and post-processing of MRI data, data analysis, writing and editing the manuscript. JZ-A: clinical investigation. CC: neurosurgical procedures. RB: statistical and data analyses. J-MS: ethical approval, project design. A-MB: biochemical assays. JZ-A: project design.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.


Many neurological diseases are accompanied by an increase in the cerebrospinal fluid (CSF) protein concentration, which indicates dysfunction of the blood-CSF/blood–brain barrier. However, the significance CSF protein concentration of patients with cryptococcal meningitis (CM) is not fully understood. The aim of the present was to determine whether CSF protein concentrations correlated with the responses of patients to treatment with antifungal drugs.We conducted a retrospective study of the analytical data of 623 lumbar punctures of 46 patients with CM who were treated at West China Hospital. We divided the patients into groups with good or poor responses to antifungal treatment. We used a generalized linear mixed model (GLMM) to evaluate the significance of the differences between the two groups.The baseline CSF protein concentrations of the good antifungal response group (GR-group) (median = 0.97 g/L) were higher compared with those of the poor antifungal response group (PR-group) (median = 0.72 g/L). Analysis using the GLMM indicated that the CSF protein concentration of the GR-group decreased at a rate of 1.8 mg/L per day after antifungal treatment started and was 2.1 mg/L higher compared with that of the PR-group.Compared with poor responders, we found that the baseline CSF protein concentrations of good responders were higher and decreased at faster rate after the initiation of antifungal treatment. Cryptococcus neoformans and Cryptococcus gattii [Cryptococcus gattii, which infects both immunosuppressed and immunocompetent individuals [C. neoformans [Cryptococcal meningitis (CM) caused by s gattii  is notors gattii –4. Thereividuals . In contoformans –8. This oformans .Lumbar puncture (LP) is advocated for the diagnosis and management of patients with CM , 10. AnaThe clinical interpretation of the levels of CSF protein concentrations is difficult, because the values change during treatment, and most studies report a single data point data rather than a time course. In the present study, we conducted a retrospective analysis of patients with CM who received antifungal treatment in our hospital and evaluated the clinical implication of the longitudinal data of CSF protein concentrations and the association with patients’ outcomes.We analyzed the data for 46 patients who were admitted to West China Hospital from 2009 to 2014. CM was diagnosed according to clinical symptoms and a positive culture of the CSF or the results of the India ink stain. Patients with CM were included in this study if they met all the criteria as follows: 1) monitored for more than 30 days, 2) treated with antifungals for no longer than 7 days before admission, 3) not suspected of intracranial infection with other pathogens, and 4) CSF data acquired on a regular basis. All patients were administered antifungal treatment according to ithe guidelines of the Infectious Diseases Society of America [LPs were routinely performed approximately once each week for each patient. The CSF samples were immediately sent to the laboratory and analyzed for cryptococcal antigen, cell counts, and biochemical parameters. India ink staining and fungal cultures were performed as well. The India ink stain, fungal cultures, biochemical tests, and cell counts were compulsory and the other tests were optional. The CSF protein concentration data for each patient were sorted in chronologically with the baseline data  listed first for each patient.We aimed to explore the association between patients’ responses to treatment with antifungal drugs and CSF protein concentrations. Patients were assigned to the poor antifungal response group (PR-group) according to the criteria as follows: persistent positive culture or positive India ink stain of the CSF 30 days after initiation of antifungal therapy. The other patients were assigned to the good antifungal response group (GR-group).t test and the Wilcoxon rank sum test were used to evaluate the significance of differences between the values of continuous variables. Spearman’s rank correlation test was used to test bivariate correlations. The Fisher’s exact test were used to evaluate the significance of differences between the values of categorical variables.A generalized linear mixed model (GLMM) was used for the statistical analysis. This model is suitable for analyzing repeated measures data . The datThere were no significant differences between age, sex, and underlying diseases of the GR and PR groups Table . The obsClinical treatment of patients with CM employs amphotericin B (AMB) and flucytosine to decrease the fungal burden in the central nervous system (CNS) . To achiThirty days after initiating antifungal treatment, the CSFs of 18 (39.1 %) of 46 good responders were sterile, and the India ink stains were positive for the other 28 (60.9 %) patients. However, the clinical responses of the groups did not correspond completely with the CSF data described below.P = 0.04)  Fig. . The medAnalysis using the GLMM revealed the relationship between CSF protein concentrations and time Fig. . FurtherWe show here that patients in the good antifungal response group had higher baseline CSF protein concentrations, which indicates an increased inflammatory response in the CNS . A plausP = 0.00). Because drug concentrations in the CSF may be higher during inflammation of the CNS compared with those of patients without inflammation [A prospective study of patients with CM indicates that the baseline number of CSF colony-forming units (CFUs) is a prognostic factor, because patients with a lower number of CSF CFUs had better clinical outcomes . Anotherammation , 24, an There are differences between the findings of the present study and those of other reports. For example, Lu et al., 1999) reported no significant difference in CSF protein concentrations between treatment failures and cured or improved groups . However999 reporThe present study supports the hypothesis that there is a correlation between antifungal responses and CSF protein concentrations. These findings must be confirmed by prospective studies of larger numbers of patients. However, we recommend that the treatment of patients with CM should include detailed analyses of CSF protein concentrations with focus on their variability.


Macrophage colony-stimulating factor (M-CSF) is an important cytokine for monocyte/macrophage lineage. Secretory M-CSF (sM-CSF) and membrane-bound M-CSF (mM-CSF) are two major alternative splicing isoforms. The functional diversity of these isoforms in the activation of tumor-associated macrophages (TAMs), especially in lymphoma microenvironment, has not been documented. Here, we studied the effects of M-CSF isoforms on TAMs in xenograft mouse model. More infiltrating TAMs were detected in microenvironment with mM-CSF and sM-CSF. TAMs could be divided into three subpopulations based on their expression of CD206 and Ly6C. While sM-CSF had greater potential to recruit and induce differentiation of TAMs and TAM subpopulations, mM-CSF had greater potential to induce proliferation of TAMs and TAM subpopulations. Though both isoforms educated TAMs and TAM subpopulations to M2-like macrophages, mM-CSF and sM-CSF induced different spectrums of phenotype-associated genes in TAMs and TAM subpopulations. These results suggested the diverse effects of M-CSF isoforms on the activation of TAMs and TAM subpopulations in lymphoma microenvironments. They bientified . sM-CSF entified . Though entified . M-CSF ientified  and mM-Centified . mM-CSF entified .M-CSF takes part in the pathological process of tumors. High level of M-CSF was detected in breast cancer, ovarian cancer, endometrial carcinoma, and cervical cancer , 10. Furi.e. M1 and M2 macrophages. As important components of the tumor microenvironments, tumor-associated macrophages (TAMs) promote the progression of tumors in most cases by enhancing angiogenesis, stimulating proliferation, migration and invasion of tumor cells [Macrophages are essential cellular components of the host defense system and play important roles in both physiological and pathological processes . They haor cells . Though or cells . In factor cells  though Mor cells . Meanwhior cells –24.The significance of macrophages in hematopoietic malignancies is mainly discussed in lymphomas. Lymphoma-associated macrophages were proposed in follicular lymphoma, and suggested to be an independent predictor of overall survival , 26. HigIn this study, we studied the characteristics of TAMs in microenvironment with mM-CSF or sM-CSF in a xenograft mice model. We found that TAMs could be divided into three subpopulations based on the expression of CD206 and Ly6C. While mM-CSF was more potent to induce proliferation, sM-CSF was more potent to recruit and induce differentiation of TAMs and TAM subpopulations. Furthermore, mM-CSF and sM-CSF induced different spectrums of phenotype-associated genes though both isoforms educated TAMs to M2-like macrophages phenotypes.To investigate the effects of M-CSF isoforms in tumor microenvironment on macrophages, Namalwa cells, which lack endogenous M-CSF expression, were infected with blank MSCV-GFP retrovirus, retrovirus carrying sM-CSF or mM-CSF. After cell sorting, stably transfected cell lines were named Namalwa-V, Namalwa-S and Namalwa-M, respectively. M-CSF expression was verified by RT-PCR Figure , flow cy3. Flow cytometry analysis showed that the average of infiltrating macrophages was about 0.63% and 0.67%, respectively, in Namalwa-M and Namalwa-S formed tumor tissues , whereas it was about 0.12% in Namalwa-V formed tumor tissues (TAM-V)  Figure , 2B. Thi) Figure , 2D. The+ TAM-M were detected, whereas only 28.6±3.4% Ki67+ TAM-V and 28.0±7.5% Ki67+ TAM-S were detected, respectively  of gate I TAMs in TAM-V was designated 1.000 . Hence, sM-CSF is more potent to recruit TAMs while mM-CSF is more potent to stimulate the proliferation of TAMs. Interestingly, M-CSF isoforms had different effects on the proliferation of TAM subpopulations. Compared with TAM-V, little difference was detected in gate I TAM-M and TAM-S; slight decrease was detected in gate II TAM-M and TAM-S; whereas significant increase was detected in gate III TAM-M and TAM-S. Moreover, mM-CSF was more potent to stimulate the proliferation of gate III TAMs than sM-CSF. It was suggested that mM-CSF could provide stronger and more persistent signals, which accounts for stronger effects of mM-CSF on the proliferation of gate III TAMs, since the membrane integrated mM-CSF hinders the internalization and degradation process [High level sM-CSF in tumor tissues accounted for the accumulation of TAMs and blockage of CSF1/CSFR signal in tumors could significantly decreased TAMs infiltration , 40. We  process , 44.in vitro induced monocytes to M2 phenotype macrophages [in vivo studies. Blocking CSF1/CSF1R signaling in mouse pancreatic tumor model preferentially killed M2-like CD206hi TAMs, whereas M1-like CD206lo TAMs were much less affected [lo and M1-oriented MHC IIhi TAMs [lo or Tie+ TAMs [M-CSF affects activation phenotype of macrophages. But the mechanism(s) seem to be complicated. Administration of sM-CSF rophages , 32. Howaffected . HoweverIhi TAMs . In orthie+ TAMs . From an+ and Ly6C− monocytes are regarded as “inflammatory” monocytes and “patrolling or resident” monocytes, respectively [+ monocytes differentiate into Ly6C+ inflammatory macrophages while Ly6C− monocytes differentiate into Ly6C− macrophages [+ macrophages could also differentiate into Ly6C− macrophages in both tissue repairing model and tumor [+ and Ly6C− recruited peripheral blood monocytes [+ monocytes might be the origin of TAMs [+ subpopulations) were detected while neither high proliferation rates nor low apoptosis rate was detected in both TAM-M and TAM-S. Furthermore, more gate III TAMs (Ly6C− subpopulation) and high proliferation rate were detected in TAM-M. These results imply that both M-CSF isoforms are more potent to recruit Ly6C+ monocytes rather than Ly6C− monocytes.An interesting observation of our results is that TAMs in our model could further be divided into three sub-populations based on the expression of CD206, an M2 macrophage marker, and Ly6C, an important marker for monocytes. Ly6Cectively . Ly6C+ mrophages . Ly6C+ mnd tumor , 49. TAMonocytes , but rec of TAMs , 50, 51.M-CSF isoforms had different effects on phenotypes of TAM subpopulations. Three subpopulations in TAM-S showed more mature morphology with large cell size and abundant cytoplasm despite of differential expressions of F4/80, CD206, Ly6C and M-CSFR. Only gate I TAM-M showed more mature morphology though the majority of TAM-M was detected in gate II which expressed high level of F4/80, CD206 and M-CSFR. Furthermore, analysis of activation-related gene expression also demonstrated that M-CSF isoforms also had different effects on TAM subpopulations despite of similarities.Taken together, TAMs could be divided into three subpopulations based on the expression of CD206 and Ly6C in a lymphoma xenograft model. More TAMs could be detected in tumor microenvironment with M-CSF isoforms. While mM-CSF had greater potential to induce proliferation of TAMs and TAM subpopulations, sM-CSF had greater potential to recruit and induce differentiation of TAMs and TAM subpopulations. Though both M-CSF isoforms educated TAMs and TAM subpopulations to the states with more M2-like macrophage phenotypes, mM-CSF and sM-CSF induced different spectrums of activation-related genes in TAMs and TAM subpopulations.+ stable transfected cell lines, named Namalwa-V, Namalwa-M or Namalwa-S, were sorted by flow cytometry, respectively. All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum  and antibiotics  in a humidified atmosphere of 5% CO2 at 37°C. All culture supplies were screened and selected on the basis of being endotoxin free.Human lymphoma cell line Namalwa was obtained from American Type Culture Collection . Namalwa cells were infected with MSCV-PGK-GFP retrovirus or recombinant virus carrying mM-CSF or sM-CSF. The GFPTM 649-conjugated) were from Biolegend (San Diego). Antibodies against human M-CSF, mouse F4/80 were from Abcam ; antibodies against mouse STAT1 and pSTAT1 were from cell signaling technology ; antibodies against mouse STAT6 and pATAT6 were from Santa Cruz (CA).Fluorescence-conjugated antibodies against mouse F4/80, CD206 , Ly6C , Ki67  and IgG .4- to 5-week old female NOD/SCID mice were purchased from Center for Experimental Animals, the Academy of Military Medical Sciences, and housed in the sterile microisolators in the Animal Centre of the Institute of Hematology & Blood Diseases Hospital, CAMS & PUMC. Experimental procedures performed on the mice were approved by the Animal Care and Use Committee at the institutions involved in this study. After irradiated by + cells were enriched by anti-CD11b conjugated magnetic microbeads  following manufacturer's protocol. Then, enriched cells were resuspended in PBS containing 1%FBS and stained with fluorescence-conjugated antibodies. For sorting or analysis of TAMs, cells were stained with APC-conjugated anti mouse F4/80, whereas for sorting or analysis of TAM subpopulations, cells were stained with APC-conjugated anti mouse F4/80, PerCP-Cy5.5-conjugated CD206, PE-Cy7-conjugated Ly6C. PE-conjugated anti mouse F4/80 was used for BrdU incorporation assay.Mice were sacrificed by cervical dislocation, and tumor tissues were isolated by blunt dissection and grinded into the Petri dish with PBS buffer with EDTA. Then cell suspension was filtered through graded nylon filter and red cells were removed using erythrocyte lysis buffer . After washing, enrichment of macrophages and labeling of macrophage-associated antigens were performed. Briefly, CD11b+ population. Macrophages in bone marrow were gated as described previously [An LSR II cytometer  was used for FACS analysis and a FACS Aria III  was used for cell sorting. Flowjo software  was used for data analysis. Standard protocols were followed for all experiments. TAMs in tumor tissues and macrophage in peritoneal cavity were gated as F4/80eviously .i.p. with 100ul of 10 mg/ml BrdU 16hrs prior to preparation of tumor tissue samples. Then enriched and labeled cells were further stained with BD PharmingenTM APC BrdU Flow Kit  according to manufacturer's instructions. For Ki67 staining assay, enriched and labeled cells were fixed and permeabilized by Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit , then stained with PE-conjugated anti Ki67 according to the standard procedure. BrdU+ or Ki67+ cells in TAMs or TAM subpopulations were analyzed by flow cytometry.For BrdU incorporation assay, tumor-bearing mice were injected TM 649-conjugated anti-mouse IgG before performed FACS or dropped on slides. To study the distribution of TAMs in tumor tissues, immunofluorescence staining of tumor sections (4-μm thick) was performed as described previously [TM 649-conjugated anti-mouse IgG. Slides or sections were scanned analyzed under a confocal laser microscope .To identify Namalwa-V, Namalwa-M, and Namalwa-S, cells were collected and incubated with primary antibody against human M-CSF at a dilution of 1:100 followed by incubation with Dylighteviously . BrieflyTAM subpopulations were sorted and spun to slides and Wright-Giemsa staining was performed. The slides were examined under a light microscope .TAMs were sorted from different tumor microenvironments and cultured in 24-well plates for 24hrs with or without 100 ng/ml LPS  or 20ng/ml IL-4 .Total RNA was extracted with RNeasy mini Kit  according to the manufacture's instructions. Reverse transcription was achieved using Super Script First-Strand Synthesis System . Real-time PCR was performed using a StepOnePlus Real-Time PCR System . The sequences for all primers are listed in 4 cells/μl. Cell lysis was mixed with 2.7M urea/53mM DTT (1:3) and treated for 5 min at RT. Then samples were mixed with Premix G2 5-8 , including pI standard  at a ratio of 1:3, and loaded into 384-well microplate. For each experiment, samples were analyzed in duplicate. Data analysis was carried out using Compass Software .Nanopro immunoassay (NIA) was performed using Nanopro 1000  following manufacturer's protocol. TAMs were sorted and lysed with Bicine/CHAPS Lysis buffer plus 1×DMSO inhibitor mix and 1×aqueous inhibitor mix  at 104 cells were sorted directly into lysis buffer RLT . RNA was extracted and reverse transcribed into cDNA for further specific target amplication (STA). STA consists of 2 min at 95°C to inactivate reverse transcriptase and activate Taq enzyme, followed by 14 cycles of 15 s at 95°C and 4 min at 60°C. Preamplified cDNA was diluted with TE buffer (1:5) before used for real-time PCR. Gene expression was analyzed using BioMark™ 96·96 Dynamic Array . The PCR consists of 10 min hot-start at 95°C to activate the Taq polymerase, followed by a 40 cycles two-step program (15 s at 95°C and 60 s at 60°C). All The TaqMan primers and probes  used were listed on table S2. Data were analyzed using BioMark™ Real-Time PCR Analysis Software .Dynamic array analysis of gene expression in TAM subpopulations was performed following instructions of BioMark™ real time PCR system. Briefly, 10P < 0.05 was considered statistically significant.The results were represented as means ± SD. Analysis was done using GraphPad Prism 6.0 software. Significance was determined by one-way or two-way analysis of AVONA.


Meningitis is a serious concern after traumatic brain injury (TBI) or neurosurgery. This study tested the level of reactive oxygen species (ROS) in cerebrospinal fluid (CSF) to diagnose meningitis in febrile patients several days after trauma or surgery.Febrile patients (temperature > 38°C) after TBI or neurosurgery were included prospectively. ROS were measured in CSF within 4 hours after sampling using luminescence in the basal state and after cell stimulation with phorbol 12-myristate 13-acetate (PMA). The study was conducted in a single-center cohort 1  and then in a multicenter cohort 2  in the Intensive Care and Neurosurgery departments of two teaching hospitals. The performance of the ROS test was compared with classical CSF criteria, and a diagnostic decision for meningitis was made by two blinded experts.The production of ROS was higher in the CSF of meningitis patients than in non-infected CSF, both in the basal state and after PMA stimulation. In cohort 1, ROS production was associated with a diagnosis of meningitis with an AUC of 0.814  [0.684–0.820]) for steady-state and 0.818 (95% CI [0.655–0.821]) for PMA-activated conditions. The best threshold value obtained in cohort 1 was tested in cohort 2 and showed high negative predictive values and low negative likelihood ratios of 0.94 and 0.36 in the basal state, respectively, and 0.96 and 0.24 after PMA stimulation, respectively.The ROS test in CSF appeared suitable for eliminating a diagnosis of bacterial meningitis. Traumatic brain injuries and neurosurgical procedures ) confer a high risk of nosocomial meningitis. The incidence varies between 1.4%  and 8%  with a mPMNs are the first line of defense of the innate immune response to bacterial infection and are activated when infection is present. Such activation is essential for many PMN functions, especially crossing the blood brain barrier and releasing radical oxygen species (ROS) . In a reThis study reports the results of a prospective multicenter study testing ROS measurement by luminescence to diagnose nosocomial meningitis. The test was initially applied to a single-center cohort (cohort 1) as a training cohort and prospectively applied to multicenter cohort 2 for validation.This study was approved by the ethical committee of the Société de Réanimation de Langue Française  and conducted according to instructions; patients or their next of kin received written information. Signed informed consent was not required because measurements were conducted with routine CSF samples without further biological collection. Patients from two teaching hospitals were enrolled in the intensive care unit (ICU) or in the neurosurgery department when they had a fever over 38°C in a context of TBI, after neurosurgery or CSF drainage by catheterization (EDV). The daily enrolment time was between 7 AM to 7 PM because ROS measurements should be performed on fresh samples with a delay after sampling of less than 4 hours. The first single-center cohort (cohort 1) was prospectively studied from Feb 2009 to Oct 2010 . The multicenter cohort (cohort 2) was prospectively enrolled for validation of the test from Oct 2010 to Mar 2012 . The physicians in charge of the patients were not aware of the investigation and ignored the results of ROS measurements when managing their patients.For each patient, the following data were collected: demographic characteristics, type of surgery if any, presence of external CSF drainage, method for CSF sampling (lumbar puncture or catheter drainage), symptoms of inflammation; routine CSF laboratory analysis , medical treatments, especially antimicrobial therapy at the time of sampling, and outcome. If present, other concomitant sites of infection were considered.In the absence of a consensus for the definition of variables and thresholds for bacterial meningitis in the context of secondary infection after trauma or neurosurgery , we askePlus LB 953; Berthold Technologies, Bad Wildbad, Germany) with luminol  measured under basal conditions and after cell stimulation with phorbol 12-myristate 13-acetate  as previously described [2O2 . These tests showed a good reproducibility of the signal.ROS production in CSF was evaluated by luminescence intensity . Statistical analyses were performed using R-statistical software (http://www.r-project.org/). A two-sided P value <0.05 was considered statistically significant.Values were expressed as median and interquartile range or count and percentage. Groups of patients were compared using the chi-square test for categorical variables and the Student’s t test or Wilcoxon test for continuous variables as appropriate. A diagnosis of meningitis was based on the consensus of two experts. To determine the diagnostic accuracy of the luminescence level measured either under basal conditions  or associated with cell function (AUC ROS production under PMA conditions), operating characteristics of both tests were evaluated using receiver operating characteristics curves (ROC) with calculation of the area under the curve (AUC). Confidence intervals 95%) of AUCs were estimated using bootstrap operations [5% of AUC3 and positive microbiology culture [In the sensitivity analysis, the performance of the ROS test was compared to formerly published meningitis criteria including CSF cell count > 100/mm culture .th percentiles)) or major neurosurgery with a leak or in an emergency ), or external ventricular drainage (delay of 9 days (4.5–13)). Six patients were taking hydrocortisone, 5 were taking prednisone and 2 had recently received non-steroidal anti-inflammatory drugs.The characteristics of the two cohorts are presented in Pseudomonas, 2 for Staphylococcus, 2 for enterobacteria, Enterococcus, Acinetobacter and Gemella). A discrepancy between the diagnosis and microbiology findings was observed for 7 patients (13.0%)  in two patients because of low CSF cell counts, low protein and high glucose levels in a context of extraventricular drainage. The experts thought that this pattern was more likely due to material colonization than infection. Differences in clinical and bacteriological criteria according to the diagnosis of meningitis are detailed in Fifty-four patients were included in cohort 1. Meningitis was diagnosed by the experts in 14 patients from this cohort when CSF was positive for microbes in 11 patients  . The peBecause the routes of sampling, lumbar puncture (LP) or external ventricular drainage (EVD), have different risk factors for meningitis or ventriculitis, we tested ROS production for both types of sampling. The performance of the ROS test was similar to that of expert diagnosis if samples were obtained by LP or by EVD in the basal state . HoweverBecause the concomitant administration of antibiotics for other infections could alter the course of meningitis, we analyzed the results of the ROS test in patient subgroups who were or were not taking antibiotics . The datAdditionally, ROS results were not influenced by age or gender.3 in the total population of 190 patients. The ROS test in the basal state and after PMA stimulation and high CSF cell counts (>100/mm3) had sensitivities of 0.74, 0.88 and 0.89, specificities of 0.73, 0.53 and 0.75, NPVs of 0.93, 0.95 and 0.97 and PPVs 0.38, 0.30 and 0.44, respectively.Because early suspicion of meningitis is limited to biological criteria, we compared the performance of the ROS test with patients who had CSF cell counts >100/mmThis study aimed to validate the production of ROS in CSF as a marker of meningitis in one-center cohort first, then with a second multicenter cohort of febrile patients (over 38°C) in a context of TBI or neurosurgery. The proportion of meningitis diagnosed by experts was 25.9% in cohort 1 and 15.4% in cohort 2. In both cohorts, the production of ROS in CSF was higher in diagnosed meningitis compared with negative CSF under both measurements condition . The test demonstrated high sensitivity and negative predictive value for prediction of CSF infection. This test may constitute a rapid and reliable tool for eliminating meningitis and particularly when it is suspected in a context where biological diagnosis is difficult, for example soon after intracranial hemorrhage or surgical procedures.In neurocritical care, suspicion of meningitis is a daily concern when fever occurs after brain injury or neurosurgery. In this context, the presence of PMN in CSF may result from sterile inflammation or from a proven infection . Even thLooking at ROS production is a reasonable approach because the main source of ROS is activated PMNs, mainly via the NADPH oxidase pathway ,10. In aA suspicion of meningitis is very frequent in neurocritical practice and is a daily concern, with a heterogeneous incidence ranging from 1 to 20% ,14. Such3 and positive microbiology [i.e., global CSF infection and ventriculitis. Finally, antibiotics were administered to 44% of our population for other infections, which could alter a diagnosis of central nervous system infection, but the ROS test still performed well and especially in comparison with the expert diagnoses. The clinical context and comorbidities like diabetes might have an impact on ROS release but were not tested in this study. Further data collection will help to clarify these important questions.We described the sensitivity analysis of our parameters because the requirement to diagnose/exclude a diagnosis of meningitis occurs in different contexts and also because the interpretations of CSF parameters by physicians may vary in the absence of recommended definitions and thresholds for variables . Our intobiology . ROS res3. ROS measurements would be superior to cell counts for diagnosing infection if, for the same number of cells, ROS production was higher in infected than in non-infected CSF. This hypothesis was supported by in vitro studies which have demonstrated increased NADPH oxidase subunit expression after stimulation of cells by LPS or interferon gamma [Other types of biomarkers, such as serum procalcitonin ,16, a coon gamma . As illuThe limitations of the current study include the lack of a consensus for postoperative or post-trauma meningitis diagnostic criteria . This haThis data obtained in this prospective multicenter study confirmed the interest studying ROS measurements in the diagnosis of meningitis. The performance of the test was excellent for its sensitivity and negative predictive value in comparison with diagnoses made by two independent experts. In order to validate the use of ROS measurements in the detection of CSF infection, the test requires further standardization and development for rapid bedside use. Such a test would help decision-making by clinicians and limit numerous specialized CSF lab tests and the administration of broad-spectrum antibiotics.S1 FigROC curves associated with a diagnosis of meningitis in cohort 2 after PMA stimulation (solid line) and in the basal state (dotted line).(TIFF)Click here for additional data file.S2 FigCorrelation between CSF cell counts and ROS production (AUC) in the total population according to the experimental condition  and a diagnosis of meningitis (presence (red) or absence (blue) of infection).(TIFF)Click here for additional data file.S3 Fig3 and positive microbiological results [The performance of ROS measurements (PMA-stimulated) in the total cohort of 190 patients according to sampling by LP or EVD and the presence or absence of previous antibiotic administration for a diagnosis of meningitis by the experts or biological criteria .(TIFF)Click here for additional data file.S1 TableIndividual information and values in patients with discrepancies between adjudicated diagnosis of meningitis and microbiology. Clinical context and biological data are reported.(DOC)Click here for additional data file.


TCR) repertoire in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) can reveal antigen‐specific immune responses potentially implicated in the disease process. We applied a new unbiased deep‐sequencing method for TCR repertoire analysis to accurately measure and compare receptor diversity and clonal expansions within the peripheral and CSF‐trafficking T‐cell populations of patients with MS and control individuals with idiopathic intracranial hypertension (IIH).Analysis of the T‐cell receptor (CSF TCR β‐chain libraries from five MS patients and five IIH controls were sequenced, yielding a total of 80 million reads.Paired blood and TCR repertoire diversity was greater in the blood and CSF compartments of MS patients when compared with IIH controls, it is notable that the frequency of clonal expansions was also significantly higher in both compartments of MS patients. Highly expanded T‐cell clones were enriched in the CSF compartment of MS patients compared to peripheral blood, very few of them were detected in both compartments.Although MS patients.Collectively, our data provide a proof of principle that private compartmentalized T‐cell expansion exists in the intrathecal space of  Multiple sclerosis (MS) is a neuroinflammatory disorder that affects over 2.5 million people worldwide. The occurrence of clinical relapses and inflammatory lesions in MS patients is associated with the infiltration of T cells in the central nervous system (CNS), which potentially react with myelin proteins and drive the inflammatory and demyelinating process.+ and CD8+ T cells infiltrating actively demyelinating MS lesions reveals that the majority of CD4+ T‐cell population was more heterogeneous compared to CD8+ T cells, which were represented in 35% of total clones in one MS case.T‐cell receptor (TCR) complementary‐determining region 3 (CDR3) length spectratyping has been used as a tool to identify skewing of the TCR repertoire in the peripheral blood (PB) of patients with MS,β‐chain rearrangements present in unsorted and nonstimulated cells from these compartments. As controls, samples were also processed from individuals with idiopathic intracranial hypertension (IIH), a disorder characterized by increased intracranial pressure of unknown cause.To interrogate the peripheral and CSF T‐cell repertoire in MS patients, we applied an unbiased method to amplify and deep sequence TCR TMPlus; Amersham Bioscience, Piscataway, NJ, USA) and a total of 106 cells was stored in RNAlaterTM solution  at −80°C until RNA extraction. All CSF samples were obtained via nontraumatic collection assessed at the time of lumbar puncture by visual inspection and microscopic examination. CSF cells were also cryopreserved in RNAlaterTM solution.Paired PB and CSF samples were collected from five relapsing–remitting MS patients and five IIH controls. All patients were assessed under the care of the Neurology Unit at Charing Cross Hospital, Imperial College Healthcare NHS Trust, London, UK. None of the MS patients included in this study was receiving any form of immunotherapy at the time of sample collection. Peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation (Ficoll‐HyPaqueβ chain was amplified from unsorted PBMCs (1 × 106) and CSF cells (3.7 × 104 to 2.2 × 105) isolated from MS patients and IIH controls. The TCRβ library generated for high‐throughput DNA sequencing was prepared according to a previously published protocol with minor modifications.β chain, a nested PCR was performed using a pair of primers, one binding the SMARTer oligonucleotide region (5’‐AAGCAGTGGTATCAACGCAGAGTAC‐3’), and the other binding the TCRβ constant region (5’‐TGCTTCTGATGGCTCAAACACAGCGACCT‐3’). The qPCR assays were run using the Kapa HiFi system  as follows: one cycle at 95°C for 5 min; five cycles at 98°C for 20 sec and 72°C for 1 min; and 35 cycles at 98°C for 20 sec and 68°C for 1 min. TCRβ DNA library from each sample was purified using the e‐Gel 2% agarose system  and a unique band of ≅650 bp size was collected into a well filled with RNase/DNase‐free water. A second PCR was performed to add an Illumina adaptor and a unique index barcode to each individual sample. Library size and quality were measured using an Agilent Bioanalyzer High Sensitivity DNA Chip  and library concentration was calculated using Kapa Illumina Library Quantification Assay .The V‐CDR3‐J region of the TCRn = 20) was pooled for high‐throughput sequencing on the HiSeq 2500 platform . A total of 80‐million paired TCRβ sequences consisting of 150 bases at 5’ forward and 3’ reverse orientation were generated. This sequencing protocol was developed and validated as described previously.β J region at a final concentration of 100 μM each . TCRβ annotation was performed by combining a custom Java program written in‐house and the BLAST program developed by the National Center for Biotechnology Information. Briefly, the V and J germline genes of a TCRβ read were identified first, and the CDR3 was determined by finding the conserved cysteine at 5’ end and the conserved phenylalanine at the 3’ end. Unique and productive TCRβ combinations (V‐D‐J) were collapsed to determine the count (TCR in‐frame reads). For each combination, the number of nucleotides contributed by the germline V, D, and J genes was determined together with the number of nucleotide additions. Shannon entropy (diversity), species richness, and evenness were calculated for each TCRβ repertoire by using the R package, Vegan. Entropy and richness were normalized by calculating the maximum Shannon entropy and maximum combination richness for each repertoire based on the cell count used for library generation. The calculated Shannon entropy and combination richness were then divided by the respective maxima to return a number between 0.0 and 1.0. For each repertoire, the average and standard deviation were calculated for germline index, CDR3 length, and number of nucleotide additions. Custom Perl scripts were used to calculate the distribution of CDR3 length, V‐J pairing percentage, and amino acid compositions of each CDR3 position, as well as whole CDR3s from all of the annotated TCRβ sequence reads in each subject and group.High‐throughput sequencing was performed by the Genomic Core Facility of the Medical Research Center (MRC) at Imperial College London. A total of 2 nmol of each sample (http://www.graphpad.com/scientific-software/prism/) or R (http://www.r-project.org/). Clone frequencies observed in each subject (http://data.ninds.nih.gov/Jacobson/Alessandra/index.html) were first converted to a percent total frequency value by dividing each clone frequency by the cumulative frequency observed across all clones for the same sample. Degree of expansion per clone was then calculated by taking the Log2 transform of the percent total frequency value. To compare richness, diversity, and clone expansion between sample classes , the unpaired nonparametric Wilcoxon signed rank T‐test was used (α = 0.05). To compare expansion for clones observed across compartments , the paired nonparametric Wilcoxon signed rank sum test was used (α = 0.05). In addition, we also calculated the p‐value by applying the Leave‐One‐Out (LOO) test on the Figures All statistical analyses were performed in Prism .Clinical, demographic, and biological sample information for the five relapsing–remitting MS patients and five IIH controls included in this study is shown in Table β in‐frame reads between the PB and CSF  enabled us to compare repertoires across the two compartments with confidence. Our initial analysis evaluated the number of unique productive TCRβ combinations (defined here as richness) between PB and CSF across all subjects. As shown in Figure P = 0.0019). Likewise, we also compared the level of T‐cell diversity, estimated as the number of unique TCRβ clonotypes per sample normalized by cell count, between PB and CSF across all subjects. As with richness, diversity in the PB compartment was significantly higher compared with CSF  in the total repertoire, was significantly higher in the PB and CSF of MS patients compared with IIH controls  in a repertoire with the greatest frequency would be represented by <5% of the total repertoire  with the shared clonotypes in PB across IIH controls (n = 252), we found that a subset (n = 94) of these clonotypes were common  those with <100 in‐frame reads ; and (2) those with >10,000 in‐frame reads . Significantly lower numbers of clonotypes in the small expansion group were observed in the CSF of MS patients (P = 0.007) and IIH controls (P = 0.003) relative to PB, whereas no significant difference was observed between disease cohorts with respect to CSF , whereas this trend was not observed in IIH controls . These observations are consistent with a disease‐specific enrichment of highly expanded clonotypes in the CSF of MS patients.As the overall frequency of expanded T cells was greater in the PB and CSF of MS patients compared with IIH controls Fig. A and B, CSF Fig. A. Higherols Fig. B. HoweveTo determine whether hyperexpanded clonotypes in the CSF of MS patients were also present in the PB Fig. B, we exaβ chain repertoire profiles in the CSF of patients with MS,β rearrangements present in unsorted T cells isolated from the PB and CSF of relapsing–remitting MS patients and IIH controls. It is notable that IIH is widely regarded as a disorder resulting from abnormalities in CSF volume homeostasis. Chronic inflammation has been implicated in this condition,Immune cell entry into the CNS is tightly controlled under physiological conditions and immune surveillance is localized to the perivascular and subarachnoid spaces (part of the CSF compartment).β repertoire diversity was higher in both the PB and CSF of MS patients compared with IIH controls, expanded clonotypes were significantly more frequent across both compartments in MS patients. In the frequency ranges best differentiating MS and IIH, few clonotypes were shared among all subjects in PB and none was shared in CSF. Phylogenetic tree analysis of the shared clonotypes from PB revealed potentially distinctive CDR3 motifs in MS patients.Although TCRβ clonotypes,β repertoire was much lower in CSF compared with PB, likely explained by the higher number of T cells obtained from unsorted PB samples compared with CSF (approx. 5 × 105 T cells in PB vs. 1 × 105 T cells in CSF). However, the number of input cells was used to normalize TCRβ repertoire diversity by applying Shannon's entropy algorithm, and the number of total in‐frame reads were not significantly different between groups. We therefore felt confident comparing repertoire diversity by compartment and subject. In addition, we applied the Leave‐One‐Out statistical test to reinforce the significant p‐value between our groups of five subjects. Diversity of the TCRβ repertoire was lower in CSF compared with PB in all subjects. A recent study using a different molecular approach (multiplex PCR for library preparation) reported a highly diverse TCRβ repertoire in the PB and CSF of MS patients and controls with other neurological disorders.β diversity in the PB and CSF of MS patients compared with IIH controls. Different methodologies and patient cohorts may underlie the diverging results. Using deep sequencing of the TCRβ repertoire across four different groups of healthy donors, Britanova et al. demonstrated that repertoire diversity decreased significantly with age.Our analyses of the dataset first examined richness, defined as the absolute number of unique TCRn = 204) as well as in all five controls (n = 158), very few shared clonotypes were observed in the CSF across IIH controls, and no clonotypes were common in the CSF across all MS patients. These results suggest that the CSF compartment may have a more private or “nonshared” TCR repertoire compared with PB. Accordingly, pathogenic T‐cell responses may be driven by individual clonotypes specific for a variety of antigen(s) present solely in the CNS. Recently, sharing of predominant T‐cell clones between CNS lesions, CSF, and blood CD8+ T cells was found in three MS patients when the TCR β‐chain repertoire was analyzed using a multiplex amplification approach for V‐ J genes.+ and CD4+ T‐cell in demyelinating MS lesions pattern II were also identified in the CSF compartment of MS patients after ex vivo culture expansion.The analysis of clonotype frequency revealed significantly higher mean clonal frequencies in MS patients compared with IIH controls across both compartments, albeit with a more prominent difference in CSF. The greater diversity of PB and CSF repertoires in MS patients, discussed above, reassures us against the possibility that these higher clonal frequencies may be due to sampling error . By analyzing the cumulative frequency curves, we were able to delineate the range of frequencies that contributed most to the observed differences. Through a density plot representation, we were then able to define a specific range of clonal expansion that revealed differences in the number of clonotypes between MS patients and IIH controls in both PB and CSF. This analysis allowed us to more selectively target a search for the presence of shared T‐cell clonotypes β repertoire.The phylogenetic tree analysis of “public” T‐cell clonotypes found exclusively in the PB of MS patients revealed a group of 26 clusters, which showed amino acid sequence relatedness across the CDR3 loop. Although the antigen specificity of these TCRs remains unknown, we found a consensus motif with a conserved glycine (G) residue at CDR3 positions eight and nine in MS patients that was not conserved in IIH controls. Further studies are needed to determine whether this motif constitutes an MS‐specific signature and potential biomarker of disease in the peripheral TCRAfter investigating the clones present at different density in the intermediate‐ to high‐frequency ranges in MS and controls and their relatedness, we focused our attention on “hyperexpanded” versus “small” expanded clonotypes. While the number of small clonotypes was significantly greater in PB than in CSF, both in MS and in IIH, and did not differ in the CSF between MS and IIH Fig. A, there β sequences for such downstream studies.In conclusion, we used an unbiased method for TCR repertoire analysis to demonstrate clonal T‐cell expansions in the CSF of MS patients. The detection of highly expanded private clonotypes in the intrathecal compartment is consistent with the possibility of disease‐inducing T‐cell activation by CNS‐resident stimuli and signals. Although the antigen specificity of these clonotypes remains unknown, our analysis will inform the selection of relevant TCRAlessandra de Paula A. Sousa performed research, analyzed data, and wrote the manuscript; Kory R. Johnson analyzed data and wrote the manuscript; Richard Nicholas designed research; Sam Darko contributed for the TCR bioinformatics data analysis; David A. Price discussed the data; Daniel C. Douek contributed for the TCR molecular technique; Steven Jacobson designed research and wrote the manuscript; and Paolo Muraro designed research and wrote the manuscript.Dr. De Paula Alves Sousa reports grants from the National Multiple Sclerosis Society. Dr. Nicholas reports grants, personal fees, and non‐financial support from Biogen, personal fees from Genzyme, personal fees and non‐financial support from Novartis, personal fees from Roche, outside the submitted work. Dr. Price reports grants from WELLCOME TRUST, NHMRC, outside the submitted work. Dr. Muraro reports grants from UK MS Society, during the conduct of the study; personal fees and non‐financial support from Merck Serono, personal fees from Biogen, personal fees and non‐financial support from Buyer, personal fees from Novartis, outside the submitted work.Table S1. High‐throughput sequencing dataset of TCRβ repertoire in the PB and CSF compartment of MS patients and IIH controls.Table S2. CDR3 amino acid sequences (clonotypes) shared among the five IIH controls in the CSF compartment.Table S3. CDR3 amino acid sequences (clonotypes) shared among the five MS patients in the PB compartment. By phylogenetic analysis, the clonotypes sequence‐relatedness was investigated.Table S4. CDR3 amino acid sequences (clonotypes) shared among the five IIH controls in the PB compartment.Click here for additional data file.


Parkinson’s disease (PD) is a devastating age related neurodegenerative disease that is believed to have a lengthy prodromal state. It is critical to find methods to harness compensatory recovery processes in order to slow or prevent the eventual progression of clinical symptoms. The current perspective paper argues that immune system signaling molecules represent such a promising therapeutic approach. Two cytokines of interest are granulocyte macrophage-colony stimulating factor (GM-CSF) and erythropoietin (EPO). These hematopoietic cytokines have been protective in models of stroke, neuronal injury, and more recently PD. It is our belief that these trophic cytokines can be used not only for cell protection but also regeneration. However, success is likely dependent on early intervention. This paper will outline our perspective on the development of novel trophic recovery treatments for PD. In particular, we present new data from our lab suggesting that EPO and GM-CSF can foster neural re-innervation in a “mild” or partial lesion PD model that could be envisioned as reflecting the early stages of the disease. Parkinson’s disease (PD) is characterized by a loss of dopamine (DA) neurons within the nigrostriatal pathway and the presence of Lewy body pathological protein aggregates , is routinely used to induce PD-like pathology, inducing a loss of substantia nigra pars compacta (SNc) DA neurons and downstream striatal terminals  within the SNc of PD patients  and granulocyte macrophage-colony stimulating factor (GM-CSF). Indeed, GM-CSF had protective effects in models of Alzheimer’s disease : Saline, EPO, and GM-CSF. On post-6-OHDA infusion Days 13 and 28, the animals received an intraperitoneal injection of either saline, recombinant human EPO solution  or recombinant rat granulocyte-macrophage colony-stimulating factor solution . Animals were sacrificed on Day 30.It is thought that a slow progressive “wave” of neurodegeneration occurs over many years before a critical threshold of neuronal loss is reached and clinical PD pathology is manifested. Accordingly, it is of interest to study the effects of potential treatments while the disease is still in an early or possibly even a prodromal stage. In the present paper, it was of interest to assess whether EPO or GM-CSF treatment could influence striatal innervation following the establishment of a partial or “mild” lesion that might be analogous to the early onset of the disease. To this end, male Sprague Dawley rats received a single intra-striatum  infusion of 6-OHDA (20 μg). Animals were then randomly divided into three groups  provoked a significant recovery (presumed re-innervation) of striatal terminals. The statistical analysis revealed a significant treatment effect, F = 10.7, P < 0.01, with regards to striatal lesion size (Figure Importantly, the two EPO and GM-CSF injections that were given The present findings are consistent with our own previous data and those of others showing beneficial effects of GM-CSF (Mangano et al., It is important to underscore that the 6-OHDA paradigm presently used provoked a very modest loss of striatal terminals and future studies are required to ascertain whether GM-CSF and EPO might also have reparative properties in PD models with more significantly sized lesions. Nonetheless, GM-CSF and EPO may be ideal trophic treatment candidates based on their biological profiles, preclinical data and track record of clinical applicability. Mechanistically, these cytokines are potent inducers of BDNF and GDNF (Bouhy et al., in vivo procedures. KF performed the immunohistochemical stains and data analysis. NP performed the stereological SNc counts. KF and SH prepared the manuscript. All authors have read and approved the final manuscript.CR and SH conceived the study. KF, CR, and SH designed the study. KF and CR performed the 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.


For the purpose of successfully developing a prosthetic control system, many attempts have been made to improve the classification accuracy of surface electromyographic (SEMG) signals. Nevertheless, the effective feature extraction is still a paramount challenge for the classification of SEMG signals. The relative frequency band energy (RFBE) method based on wavelet packet decomposition was proposed for the prosthetic pattern recognition of multichannel SEMG signals. Firstly, the wavelet packet energy of SEMG signals in each subspace was calculated by using wavelet packet decomposition and the RFBE of each frequency band was obtained by the wavelet packet energy. Then, the principal component analysis (PCA) and the Davies-Bouldin (DB) index were used to perform the feature selection. Lastly, the support vector machine (SVM) was applied for the classification of SEMG signals. Our results demonstrated that the RFBE approach was suitable for identifying different types of forearm movements. By comparing with other classification methods, the proposed method achieved higher classification accuracy in terms of the classification of SEMG signals. Millions of people in the world were amputated every year due to diseases, industrial injuries, traffic accidents, and accidental injuries. They hoped to possess a voluntarily controlled prosthetic limb for retrieving the basic human movement capabilities. At the present time, the key point of the prosthetic system is the selection of the information source of movement control. Surface electromyographic (SEMG) signals are one-dimensional and nonstationary time series which can be noninvasively recorded by using electrodes on the skin surface. These signals are the summation of all motor unit action potential (MUAP) within the pick-up area of the electrodes. Hence they are deeply related with the activities and the functional statuses of muscle and can reflect the activities of the neuromuscular system . MeanwhiIn order to control the myoelectric artificial limb, it is very important to extract the effective features to characterize the SEMG signals , 3. So fIn recent years, the feature extraction methods of multichannel SEMG signals mostly focused on the combination of the two or more methods mentioned above , 13, 17.A telemetric EMG system  was used for SEMG data acquisition. The sampling rate of the signals was 1500 Hz for each channel. Differential amplifiers with bandpass filters between 10 and 500 Hz were used to reduce the effects of high frequency noises and low frequency movement artifacts. Four-channel SEMG signals were collected from the extensor digitorum, extensor carpi radialis, palmaris longus, and flexor carpi ulnaris of the right forearm by using four pairs of differential silver-silver chloride disc electrodes (5 mm in diameter) with a center-to-center spacing of 20 mm. The reference electrode was attached to the elbow of a subject. In addition, because the SEMG signals were relatively weak and can be easily submerged in the noise, the exfoliating cream was used to clean the surface skin of forearm of each subject before experiments to reduce the interference generated by the skin impedance.For the purpose of data acquisition, seven healthy adults voluntarily accepted to participate in this experiment. The mean age of all subjects was 22 (±2) years. As shown in The Muscle Research Center at Boston University has discovered that the spectra of SEMG signals were distributed within 20–500 Hz, mostly concentrated in 50–150 Hz, when the bipolar model was used to collect the SEMG signals . In addiFor the pattern recognition of prosthetic limbs based on the SEMG signals, it is very critical to effectively extract the features of SEMG signals. In this study, the RFBE method based on wavelet packet decomposition was proposed for the feature extraction of different movements of multichannel SEMG signals. The computation method of the RFBE is then presented as described below.J using wavelet packet transform, the Symlet wavelet of order 5 was selected as the mother wavelet. 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, every SEMG signal was decomposed to the third level using wavelet packet transform. Let i denote the channel index of SEMG signals and Wi,jm   denote the decomposed subspace corresponding to node ; j is the decomposition level and m is the index of the subspace occurring at the jth level. Hence, the wavelet packet coefficients of the subspace Wi,jm can be described asKjm is the size of the subspace corresponding to node  in the wavelet packet decomposition. Because wavelet packet basis functions are orthogonal to each other, the energy in each subspace is defined asAssuming that the original SEMG signal is a finite energy signal and is decomposed to the level Then the total energy of four-channel SEMG signals in the same subspace or frequency band can be calculated byIn the end, the RFBE of four-channel SEMG signals in each frequency band is given byk principal components were used to obtain a k-dimensional feature space and regarded as the inputs to a classifier for the classification of the SEMG signals. At the same time, the DB index of this feature space should have the minimum value and the accumulative variance contribution of this feature space should be larger than 90%.In this study, the SEMG signals were decomposed to the third level using wavelet packet transform. For each forearm movement, a 32-dimensional feature vector can be obtained. In order to improve the generalization capability of the classifier and facilitate its design , 20, thek SVM models where k is the number of classes to achieve the multiclass classification. In addition, in order to estimate the performance of the classifier, the classification results were calculated using five-fold cross-validation method [As for the classifier, the support vector machine (SVM) method was used to evaluate the classification effectiveness of the proposed method. Because the SVM classifier is originally designed to solve the problem of binary classification, it cannot be directly applied in multiclass classification which is usually encountered in practice . At presn method . For eacThe extraction of the initial phase of forearm movement is a key issue for the control of prosthetic limbs based on the SEMG signals. In this study, the sliding time window method  was applk principal components which had the minimum value of DB index were chosen to consist of the new feature space of SEMG signals. The DB index values varying with different number of features for subject 1 are illustrated in According to the feature selection approach mentioned above, the first Every SEMG signal was decomposed to the third level by wavelet packet transform and the RFBE of SEMG signals in each frequency band was calculated. Because there were four channels of SEMG signals, this resulted in a feature vector including 32 elements. The feature selection was performed by means of the PCA and the DB index criterion. Then, the new feature space was used as the inputs to a nonlinear SVM classifier for the classification of the SEMG signals. The classification accuracy of prosthetic pattern recognition was obtained by the fivefold cross-validation method. The classification results of the different lengths of SEMG signals for 7 subjects are shown in In order to further investigate the performance of the RFBE method, we compared this one with other existing algorithms. The relative wavelet packet energy (RWPE) method was used in this comparison task, which was thought to present the highest classification accuracy for the prosthetic pattern recognition . To compWhen the RFBE method and the RWPE method are performed for the SEMG signals with different data lengths, the averaged values of DB index across 7 subjects are summarized in P < 0.05. The proposed method achieved significant improvement of classification accuracy as compared with the RWPE method for each data length. Thus, the RFBE method was more effective than the RWPE method when the features of SEMG signals were extracted. For the RWPE approach, it can be supposed that the magnitude of signal energy values is distinctly different in various frequency bands. The classification accuracy of the RWPE method is primarily dependent on the frequency band with large signal energy, whereas the RFBE method normalized the energy values of four-channel SEMG signals in every frequency band. Then, the RFBE method made use of all frequency bands to classify the SEMG signals. So the RFBE method can provide more useful information for the control system of powered prostheses than the RWPE method.The classification results of the RFBE and the RWPE for the SEMG signals with different data lengths are illustrated in When the PCA and the DB index were used to determine the feature space, the DB index was not decreased monotonically while the feature dimensionality increased, as shown in When using the RWPE method, the highest mean classification accuracy of surface electromyographic (SEMG) signals was achieved by the Symlet wavelet as compared with other wavelets . Hence, P > 0.05). The classification accuracy of 200 ms SEMG signals was also significantly lower than those of other data lengths (P > 0.05) except for the SEMG signals of 300 ms. When the data length ranged from 300 to 600 ms, there was no significant difference of classification accuracy among different data lengths (P < 0.05). These demonstrated that the data length of 300 ms can achieve good classification results of SEMG signals and the classification accuracy was not significantly improved after the data length exceeded 300 ms. It is generally considered that a 200 to 300 ms interval is the maximum delay time which can be acceptable for prosthetic users in clinical applications [In the control system for powered prostheses based on SEMG signals, the classification accuracy and the delay time were regarded as two important criteria on evaluating the classification results of SEMG signals. Therefore, in terms of the SEMG signals with different data lengths, the classification results of the proposed method were compared statistically by the one-way ANOVA. The analysis results are summarized in ications , becauseIn this study, the RFBE method based on wavelet packet decomposition was presented to extract the features of SEMG signal for the prosthetic pattern recognition. Compared with other existing methods, the proposed method provided significantly better classification results for each data length of SEMG signals, which demonstrated that the RFBE method was suitable for identifying different types of forearm movements. Additionally, when the data length was 300 ms, the RFBE method obtained good classification results. This satisfied the requirements of the high classification accuracy and the low delay time for the prosthetic control system based on the SEMG signals. The approach proposed in this study will also provide a new idea for analyzing other physiological signals and be easily extended to other applications. The future work will consider recruiting more subjects including the hand amputees into our database. At the same time, more motion classes and more repetitions for each movement class will be also extended for integrating a multifunctional system of prosthetic limbs.