Laparoscopic right donor hepatectomy has been reported sporadically in several experienced centers for selected donors. This report introduced a case of a donor with an independent right posterior segmental portal branching from the main portal vein.A 47-year-old woman volunteered to donate her right liver to her 48-year-old husband.The recipient has been diagnosed as hepatocellular carcinoma meeting the Milan criteria and hepatitis B virus related cirrhosis.The parenchymal transection was performed by ultrasonic aspirator and Hem-o-Lok clips. The right hepatic artery, right hepatic duct, and the anterior and posterior branches of right portal vein were meticulously dissected, clamped, and transected. The right hepatic vein was transected by vascular stapler. A Y-graft of the recipient's own portal confluence was reconstructed with the donor's separate right anterior and posterior portal veins.The donor's operation time was 420 minutes and the warm ischemia time was about 9 minutes. Blood loss was less than 600 ml without transfusion. The donor was discharged at the 10th postoperative day without any complications.Laparoscopic right hepatectomy for donors with anomalous portal vein branching and subsequent inflow reconstruction for adult living donor liver transplantation is safe and feasible in highly experienced center. Concerning the donor's safety, the LDRH was principally recommended for selected donors with normal anatomy of vessels. For expanding the donor pool for laparoscopic donor hepatectomy, we reported a case of LDRH for a donor with a type III portal vein anatomy  which was reconstructed with a Y-graft of the recipient's own portal confluence.Laparoscopic donor hepatectomy for living donor liver transplantation (LDLT) has been widely accepted throughout the world in experienced centers since the first report in 2002.2A 47-year-old woman  volunteered to donate her right liver to her 48-year-old husband . The recipient was diagnosed as hepatocellular carcinoma meeting the Milan criteria and hepatitis B virus related cirrhosis, with a Child-Pugh score of 10, and a model for end-stage liver disease score of 13.The donor was comprehensively evaluated before operation. The preoperative computed tomography (CT) showed that the donor had a type III portal vein anatomy with an independent right posterior segmental portal branching from the main portal vein Fig. A. Howevehttp://www.cltr.org/). All the procedures performed in this case were in accordance with the ethical principles of the Helsinki Declaration and approved by the Ethics Committee of the West China Hospital of Sichuan University.Both the donor and recipient were informed about the risks of laparoscopic approaches in LDLT, and the written informed consents of LDRH were obtained. This donation was registered in China Liver Transplantation Register  was dissected and encircled with a silicone tube for hanging maneuver. Then, the right hepatic pedicle was dissected, and the RHA and right portal veins (RPV) were encircled  and an ultrasonic aspirator . The liver capsule transection line was along with the ischemic demarcation line by transiently clamping the RHA and RPVs. The laparoscopic ultrasound was used to identify MHV, which was reserved for the donor. The intrahepatic vessels were divided and sealed with a LigaSureTM dolphin tip 37 cm laparoscopic instrument  between Hem-o-Lok clips .The liver parenchyma was transected with a laparoscopic HarmonicAfter the parenchymal transection was completed, the RHD was clamped and divided at the appreciate point after rereading the MRCP images and performing intraoperative roentgenographic cholangiography. After a 10-cm caesarean incision was prepared and the heparin was injected, the RHA and RPVs were clamped and transected. The RHV was transected using a vascular stapler. The right liver was extracted from the suprapubic incision.The graft was perfused with 2000 ml of HTK solution immediately. The graft had separate openings of right anterior and right posterior portal veins. A Y-graft interposition technique using the recipient's own portal confluence Fig. E was appThe donor's operation time was 420 minutes. The warm ischemia time was about 9 minutes. The blood loss was less than 600 ml without transfusion and anotherintraoperative complications. The graft weighted 500 g with a GRWR of 0.83%. The donor was discharged at the 10th postoperative day uneventfully without postoperative complications. The recipient was discharged at the 8th postoperative day with normal graft function.3 However, the donors substantially suffered from medical, social, and psychological burden of heavy laparotomy trauma after donor hepatectomy. Application of laparoscopic procedures for donor hepatectomy with caesarean incision could alleviate the postoperative pain, increase the cosmetic satisfaction, and enhance the recovery to normal life. Laparoscopic donor left lateral hepatectomy has been recommended as a new standard practice for pediatric living donor liver transplantation in highly specialized centers. However, the laparoscopic donor major hepatectpomy remains a challenging innovative procedure and requires a high level of surgical skills. Concerning the donor's safety, LDRH was performed in selected donors without anomalies of bike duct or portal vein during a small number of highly experienced centers.,9,10LDLT has become an alternative to deceased donor liver transplantation for patients with end-stage liver disease. Laparoscopic hepatectomy was initially performed from 2009 in our center, and the experience of laparoscopic major hepatectomy for cirrhotic patients has been mature since 2015. Meanwhile, several minimally invasive laparoscopic techniques, including laparoscopic assisted technique, hybrid technique, and pure laparoscopy, have been performed for living donor liver grafts harvest in our center.,12,13 From October 2015 to December 2018, we have completed 19 laparoscopic living donor hepatectomies, including 6 left lateral lobes, 6 left lobes, and 7 right lobes (including this case).Furthermore, the surgical teams performing laparoscopic donor hepatectomy  are recommended for technical expertise of both the LDLT and laparoscopic hepatectomy. From January 2001 to December 2018, 401 LDLTs  have been performed in West China hospital. Fortunately, there was no donor death in our center. And the type III portal vein anatomy was discovered in 3.3% to 12.7% of right lobe donors.–16 The anomalous portal vein branching  may result in 2 portal venous openings in a right lobe graft. The candidates for right liver donation with the anomalous portal vein branching were often disqualified because of the technical difficulty in vascular dissection and reconstruction. One challenging issue for the type III portal vein anatomy is the dissection of anterior portal branch, which is located deeply in the hilum. And inappropriate transection of the anterior and posterior portal branch may injury the reserved left portal vein of donor.The portal vein variations are infrequent, but of immense clinical significance in right lobe LDLT. The variants of the portal vein were classified as type I (bifurcation), type II (trifurcation), and type III . Another challenging issue for the donor with anomalous portal vein branching is the reconstruction of separate portal branches. The separate branches can not be sutured together simply because the 2 graft portal openings are always far apart. And previous study has reported that direct double anastomoses of separate graft's portal branches with recipient's left and right portal veins might cause postoperative portal venous thrombosis. So we performed the double anastomoses with the recipient's portal vein Y-graft on the back table to avoid compromising the donor's reserved portal vein.With the accumulation of experience, we attempted to perform this LRDH for a donor with type III portal vein anatomy to extend the selection criteria of donors for laparoscopic surgery. For the donors with type III portal vein anatomy, the right anterior and posterior portal veins should be dived separately for the donor's safety.In conclusion, we present our initial experience in LRDH for the donor with anomalous portal vein branching. However, it could only be performed by experienced surgical team with expertise of laparoscopic liver surgery, living donor hepatectomy and phleboplasty.http://www.cltr.org/) for supporting the study data.We acknowledged the China Liver Transplant Registry (Conceptualization: Jiu-Lin Song.Formal analysis: Jiu-Lin Song.Investigation: Jiu-Lin Song.Methodology: Jiu-Lin Song, Hong Wu, Jia-Yin Yang.Resources: Jia-Yin Yang.Supervision: Hong Wu, Jia-Yin Yang.Writing – original draft: Jiu-Lin Song.Writing – review & editing: Hong Wu, Jia-Yin Yang.Jiu-Lin Song orcid: 0000-0003-3555-2641.


Diminishing cognitive and physical function, worsening psychological symptoms and increased mortality risk and morbidity typically accompany aging. Health needs of the aging population will continue to increase as the proportion of the population aged 50 years and older increases. Pet ownership (PO) has been linked to better health outcomes in older adults, particularly those with chronic health conditions. However, much of the evidence is weak with little known about the contribution of PO to successful aging in community living older adults. This study examines associations between PO and cognitive performance, physical functioning, and psychological status in community living older adults. Participants in the Baltimore Longitudinal Study of Aging  completed physical functioning, cognitive and psychological testing, and a ten-year PO history. Most participants (89%) had owned pets at some point and 24% currently have pets. Fourteen percent have 1-4 dogs, 12% have 1-4 cats, and 3% have others. PO was lower with older age (p<.001). In regression analysis controlling for age decade, pet ownership within the past 10 years (PO10) independently predicted cognitive function  but not physical function or psychological status. PO was lower in older age groups as was cognitive, physical functional, and psychological status, while PO within the past 10 years was associated with better verbal learning and memory independent of age. Longitudinal analysis is required to disentangle the sequential associations between PO and change in health status over time..


To the editor,L265P, using ddPCR in known MYD88L265P PCNSL.Primary central nervous system lymphoma (PCNSL) is a rare disease accounting for around 3% of primary CNS tumors. Its diagnosis is usually based on cranial MRI and brain biopsy (including immunophenotyping for faster diagnostic confirmation ). The idL265P PCNSL patients without ocular infiltration. The MYD88 mutation status was established either on brain biopsy (n = 7) or in cell DNA from CSF (n = 4) with an allele specific (AS) PCR technique. CSF samples at initial diagnosis (n = 9) or relapse (n = 5) were processed within 4 h after lumbar puncture. After CSF centrifugation  or AS PCR (#09D). The MYD88L265P detection rate in CSF combining both CSF fractions achieved 86% (12/14 cases). Two cases, at relapse, remained negative for MYD88L265P detection in CSF, most probably due to a low cfDNA input or possible clonal evolution. The median fractional abundance (FA) was 7%, varying from 2.6 to 92.9%. FA was higher than in previous studies using plasma [L265P was present in the absence of lymphoma cells using cytology and flow cytometry (FCM) in three cases at diagnosis , as it was described in recurrent/refractory CNS lymphoma [The presence of cfDNA was detected in PCNSL CSF with a median value of 3.1 cfDNA copies/μL ddPCR mix (Fig. AS PCR #0D. The MYThis is the first report comparing cell and cell-free tumor load in CSF from PCNSL, showing the contribution of cell-free tumor detection in CSF for diagnosis. This study shows that detection of tumor cell and cell-free DNA is feasible using a workflow combining FCM and molecular biology. Moreover, ddPCR could be used for the tumor characterization of actionable mutations and longitudinal monitoring of the disease. We anticipate that this technique might also be applicable to other brain tumors with known hotspot mutations.


The central nervous system was previously thought to draw oxygen and nutrition from the arteries and discharge carbon dioxide and other metabolic wastes into the venous system. At present, the functional role of cerebrospinal fluid in brain metabolism is not fully known.In this prospective observational study, we performed gas analysis on venous blood and cerebrospinal fluid simultaneously acquired from 16 consecutive preoperative patients without any known neurological disorders.2) (p < 0.0001) and lactic acid level (p < 0.001) in the cerebrospinal fluid were significantly higher than those in the peripheral venous blood, suggesting that a considerable proportion of metabolic carbon dioxide and lactic acid is discharged from the central nervous system into the cerebrospinal fluid. The oxygen partial pressure (pO2) was much higher in the cerebrospinal fluid than in the venous blood, corroborating the conventional theory of cerebrospinal fluid circulatory dynamics. The pCO2 of the cerebrospinal fluid showed a strong negative correlation with age , but the other studied variables did not show significant correlation with age.The carbon dioxide partial pressure (pCOCarbon dioxide and lactic acid are discharged into the circulating cerebrospinal fluid, as well as into the venules. The level of carbon dioxide in the cerebrospinal fluid significantly decreased with age. However, in addition to this conventional metabolic pathway directly from artery to vein, cerebrospinal fluid (CSF) is derived from the arterial system and drained into the venous system, with a turnover rate of three to five times per day [The central nervous system (CNS) is commonly believed to draw oxygen and nutrition from the arterial system and discharge carbon dioxide . Blood and CSF samples were collected at the Tohoku University Hospital . The study was approved by the Institutional Review Board of Tohoku University Hospital . Written informed consent was obtained from all enrolled subjects.All enrolled subjects were preoperative patients awaiting lumbar anesthesia, and all were aged ≥ 18 years. The following exclusion criteria were applied: (1) pregnancy, (2) severe spinal canal stenosis, (3) diagnosed neurological disorders, including dementia, (4) brain or spinal cord lesions.Based on these criteria, we selected 16 consecutive patients in December 2018. They were awaiting the following types of procedure: knee joint surgery, skin biopsy, hemorrhoid surgery, etc. All procedures were unrelated to the brain and spinal cord.3 blood collection syringe designed for use in blood gas analysis. From each patient, 1 cm3 of venous blood and CSF were extracted. Because the samples were aspirated directly from the needle, neither was exposed to room air. Thus, it is unlikely that the room air or the time period between collection and sample measurement affected the data. The extracted venous blood and CSF were subjected to gas analysis within 10 min of sample collection.Venous blood and CSF samples were extracted from each patient in the operating room of Tohoku University Hospital during the lumbar anesthesia procedure. Venous blood was extracted from each arm, while CSF was extracted at the lumbar level. Patients were conscious and received no supplemental oxygen during sample collection. None of the patients had any history of respiratory disease with ventilatory impairment. Both venous blood and CSF were extracted using a 5-cm2), CO2 partial pressure (pCO2), standard base excess (SBE), bicarbonate (HCO3−), sodium ion (Na+), potassium ion (K+), chloride ion (Cl−), anion gap (AG), glucose level, lactic acid level, and total bilirubin level.All analysis was performed using the same device  in the operating room of Tohoku University Hospital. The following variables were measured: pH, oxygen partial pressure  in one subject; a test of outliers confirmed that this result was an outlier (p < 0.0001). Thus, the lactic acid data from this subject were not included in the present study because the measured level suggested that the patient had lactic acidosis that could have biased the data interpretation.Gas analysis data were compared between the venous blood and CSF of each subject using either the paired Because multiple variables were compared simultaneously, p-value < 0.01 was regarded as statistically significant. Statistical analyses were conducted using either SPSS Statistics Base 22 software  or MATLAB R2015a .The 16 enrolled subjects  had a mean age of 59.5 years (range 25–87) and a mean body mass index (BMI) of 23.9 (range 16.5–38.9). Eight underwent lumbar anesthesia for hemorrhoid surgery, six for skin biopsy, and two for joint surgery.2 , pCO2 , and lactic acid level  were significantly higher in the CSF samples than in the venous blood samples.The gas analysis results of the venous blood and CSF samples are listed and compared in Table 2, pCO2, and lactic acid level were depicted . Meanwhile, the mean Na+ level and HCO3− level did not differ between CSF and venous blood . The mean Cl− level was significantly higher in CSF than in venous blood .The mean pH, K2, pO2, and HCO3− levels in CSF were evaluated for correlation with age. Figure 2 in CSF showed a significant negative correlation with age , while pO2 and HCO3− in CSF showed no significant correlation with age . None of the following factors showed any significant correlation with age: pCO2 in venous blood, lactic acid in venous blood, and lactic acid in CSF .The pCO2 and lactic acid were higher in CSF than in venous blood, suggesting that the CNS discharges CO2 and lactic acid into the CSF. Furthermore, the levels of pCO2 in the CSF significantly decreased by age, even though those in the venous blood did not change, suggesting that the efficiency of CO2 discharge from the brain parenchyma into the CSF decreases with age. However, this was an observational study, so the exact mechanism of CO2 and lactate discharge into CSF remains unclear. Nonetheless, our findings may offer new insight into the role of CSF in the drainage system of the CNS.In the present report, the levels of pCOThe movement of lactic acid across the cell membrane is mainly mediated by proton-linked mono-carboxylate transporters (MCTs) –15, whic2 in the brain parenchyma probably discharges into the circulating CSF via gradient-based diffusion. Previously, it was assumed that dissolved gases were allowed to diffuse across cell membranes almost without restriction [2 across the cell membrane is somewhat restricted, because the unstirred layer in the membrane vicinity functions as diffusional barrier [2 [2 would be surely restricted to some extent by the glial cells that delineate the ISF from the CSF, ependymal cells and perivascular astrocytic endfeet form a looser barrier than microvascular endothelial cells that form tight junctions. Because there are gaps of varying tightness in the ependymal cell lining and astrocytic endfeet, CO2 would be able to easily diffuse from the brain parenchymal ISF into the CSF [2 from the CNS drains into the CSF, possibly via para-vascular CSF flux or ependymal gap junctions, as well as into the venules via capillaries. Therefore, because the CSF turns over three to five times per day [2 and lactic acid from the CNS.The COtriction . Later s barrier , 30. Morrrier [2 . Althoug the CSF , 32. Con per day , 2, it i2 in the CSF significantly decreased with age, whereas that in the venous blood did not, suggesting that CO2 discharge from the CNS into the CSF gradually decreases with age, or that the turnover rate of circulating CSF increases with age. However, the CSF turnover rate is known to significantly decrease with age [2 cannot be explained in these terms. Therefore, it is likely that either CO2 production within the brain parenchyma or the efficiency of CO2 discharge into the CSF decreases with age. Future research is warranted to evaluate the efficiency of CO2 discharge into the CSF in patients with neurological diseases of unknown cause.Another notable finding of this study was that the pCOwith age , so the 2 using potentiometry, which is based on the Nernst equation; pO2 and lactic acid were measured using amperometry. One previous study validated and recommended the use of gas analysis to measure pH in pleural effusion samples [+ concentrations measured using potentiometry did not differ significantly between venous blood and CSF, nor did HCO3− levels calculated on the basis of pCO2 and pH using the Henderson–Hasselbalch equation .Finally, we shall consider whether the absence of blood cells in the CSF may have affected the measured data and derived conclusions. The gas analyzer used (ABL800 FLEX) measures pH and pCO samples , so gas 2 also remains unclear. To further characterize the association between CO2 discharge into the CSF and various neurological disorders, future researchers should compare controls with patients who have such disorders. Another limitation is that gas analysis of arterial blood was not performed in the present study, so it is not clear whether the enrolled subjects had normal blood gas levels in their arterial blood. Finally, peripheral venous blood was extracted from the arms in the present study, rather than from the venous sinuses around the arachnoid granulations. We did not confirm experimentally that the levels of solutes and dissolved gases in venous blood from the arms were comparable to those in the venous sinuses. To determine the concentration gradient between CSF and venous blood, samples must be acquired from adjacent sites.The principle limitation of the present study was the small sample size. In addition, the study only reported an observation of physiological phenomena, the exact molecular mechanism of which was not elucidated. The clinical significance of the age-dependent decrease in CSF pCO2 and lactic acid are discharged from the human CNS into the circulating CSF. The pCO2 of the CNS may decrease with age, even though that in venous blood does not. The clinical significance of such a CO2-drainage system in the CNS has not yet been elucidated and should be addressed in future research.The present results demonstrated that CO


Crossed testicular ectopia (CTE)/transverse testicular ectopia (TTE) is a rare condition occurring in only 1 in 4 million male patients, in which both testes migrate toward the same hemiscrotum.We report on two cases of TTE in first degree cousins (1 + 3 years of age).Both presented with right nonpalpable testis. On diffusion-weighted magnetic resonance imaging, the right testis was located above the left testis in both patients. Right orchiopexy was performed after passing the right testis through the median raphe of the scrotum followed by ipsilateral left scrotal orchiopexy. Transverse testicular ectopia (TTE) is a rare anomaly in which both testes descend or migrate through a single inguinal canal into the same hemiscrotum. Often, the diagnosis is made during surgical exploration (65%).Two first degree cousins presented with a right nonpalpable testis which could not be visualized on pelvic ultrasound (US). Therefore, they were referred for abdominopelvic conventional magnetic resonance imaging (MRI) plus diffusion-weighted imaging.General physical examination was unremarkable, no other congenital anomalies were found, and no family history for the same condition.On examination, the right testis was nonpalpable inguinally with an empty right hemiscrotum.The MRIs were performed with a 1.5-T MRI system  using a body coil under sedation.Physical examination showed the phenotype of a 3-year-old boy with a normal size penis. The testis was palpable in the left groin and a nonpalpable undescended testis was found on the right side.On MRI, the left testis was located in the left hemiscrotum, was 1.3 × 0.6 cm in size, and associated with mild hydrocele. Another testis measuring 1.2 × 0.6 cm was noted on the ipsilateral side within the left inguinal canal .Exploration of the left groin revealed the presence of both testes within the same side one above the other. They had separate vasa deferentia and testicular vessels for each testis and a common cremasteric muscle and tunical covering. After herniotomy, the ectopic testis was fixed transseptally to the right hemiscrotum .Physical examination showed a 1-year-old male with a normal size penis. The testis was felt in the left groin and a nonpalpable undescended testis was found on the right side. In comparison to the right side, the left scrotum was well developed.On MRI, the left testis was located in the left hemiscrotum, was 1.2 × 0.6 cm in size, and associated with mild hydrocele. Another testis measuring 1.3 × 0.6 cm was noted at the same side just above the first one near the neck of the scrotum andb. The suspected diagnosis of a right undescended testis was changed to a left-sided TTE.Left groin exploration showed a testis over the other near the scrotal neck. Both having common coverings. Both cords were released followed by bilateral orchiopexy using a subdartos pouch after passing one of them through the median raphe onto the right hemiscrotum .Ectopic testis may be located in different sites including the superficial inguinal pouch, suprapubic, femoral, perineal, and base of the penis. TTE is a rare anatomical abnormality where both gonads migrate toward the same hemiscrotum. It was first described by Von Lenhossek in 1886.Despite this variety of theories, the exact etiology of this condition is still not fully understood.The mean age of presentation is 4 yearsOur two cases of TTE in first degree cousins are unusual as there was no clinical evidence for an inguinal hernia or other anomalies indicating TTE.Patients with TTE are at increased risk of malignant transformation, Mullerian remnants, and subsequent malignancy. In one study, a yolk sack tumor developed in a patient who presented with transverse ectopic testes.Management of testicular ectopia includes either transseptal or extraperitoneal transposition orchiopexy. Our two cases were managed by transseptal orchiopexy with preservation of the vas deferens and testicular vessels.TTE associated with fused vas deferens is extremely rare.Our follow-up plan includes a physical examination after 1 month postorchidopexy and then after 6 and 12 months.TTE is a rare anomaly with unknown pathogenesis. Hereditary factors may play a role. It should be suspected in patients presenting with impalpable undescended testis on one side and cryptorchidism on the other side.Also, a thorough clinical examination and laparoscopy imaging such as US and MRI are useful for establishing the preoperative diagnosis of TTE and associated anomalies.


However, CSF collection is highly invasive and expensive. In contrast, blood collection is routinely performed, minimally invasive and cheap. In this work, we develop a blood-based signature that can provide a cheap and minimally invasive estimation of an individual’s CSF amyloid status using a machine learning approach. We show that a Random Forest model derived from plasma analytes can accurately predict subjects as having abnormal (low) CSF Aβ1−42 levels indicative of AD risk . Refinement of the modeling indicates that only APOEε4 carrier status and four plasma analytes  are required to achieve a high level of accuracy. Furthermore, we show across an independent validation cohort that individuals with predicted abnormal CSF Aβ1−42 levels transitioned to an AD diagnosis over 120 months significantly faster than those with predicted normal CSF Aβ1−42 levels and that the resulting model also validates reasonably across PET Aβ1−42 status (0.78 AUC). This is the first study to show that a machine learning approach, using plasma protein levels, age and APOEε4 carrier status, is able to predict CSF Aβ1−42 status, the earliest risk indicator for AD, with high accuracy.It is increasingly recognized that Alzheimer’s disease (AD) exists before dementia is present and that shifts in amyloid beta occur long before clinical symptoms can be detected. Early detection of these molecular changes is a key aspect for the success of interventions aimed at slowing down rates of cognitive decline. Recent evidence indicates that of the two established methods for measuring amyloid, a decrease in cerebrospinal fluid (CSF) amyloid  However, it is increasingly recognized that AD is a decades-long neurodegenerative process, with shifts in amyloid 7. It is hypothesized that the high failure rate of AD trials is in part due to the trials targeting AD patients with significant cognitive impairment, who are therefore in the late stages of the disease and likely have suffered a level of brain tissue loss that cannot be compensated for8. Compounding this is the discovery that many patients enrolled in clinical trials were retrospectively found to have normal levels of amyloid and hence did not have AD9, with this number as high as 20%10. Given these findings, there is a great interest in amyloid screening for clinical trial enrichment in order to recruit individuals at the earliest stages of AD, where intervention is thought to have the greatest chance of success8. It would also ensure that included individuals are amyloid positive , a necessary precondition for the development of AD. This sort of selective screening is an important precursor for the longer-term goal of population screening for AD11.There is currently no cure or disease-modifying therapy for this terminal illness despite hundreds of clinical trials being conducted since 2002in vivo in the form of reduced levels of Aβ1–42 in the cerebrospinal fluid (CSF) or increased uptake of radioactive tracers that bind selectively to the Aβ fibrillary aggregates by PET imaging. Unfortunately, existing methodologies for measuring an individual’s amyloid levels suffer drawbacks that limit their utility for screening. Lumbar punctures are highly invasive, with this factor alone limiting the applicability of CSF biomarkers for screening. While PET scans are less invasive, they are far more expensive and access to PET scanning facilities is limited in some regions. Despite this, many current trials that target amyloid now require positive amyloid imaging at baseline to ensure accurate diagnosis, a cost-intensive process7.There are currently two established methods to measure an individual’s amyloid burden: either Aβ1–42 may indicate AD risk long before these same changes are reflected in PET Aβ imaging14. Palmqvist et al.13 have shown compelling evidence that changes in CSF Aβ1–42 occur up to a decade before the same signal is found by PET Aβ imaging. These results indicate that CSF may be a more suitable measure for early detection, whereas Aβ PET contributes independent information that is more related to disease progression and downstream pathology.Despite their invasiveness, recent studies have found evidence that changes in CSF 24. Of particular interest is the recent study by Nakamura et al.25, whereby levels of Aβ1–40, Aβ1–42 and APP669−771 in plasma, measured using specialised immunoprecipitation (IP) coupled with Matrix Assisted Laser Desorption/Ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) (henceforth referred to as IP-MALDI-TOF-MS), were shown in combination to have strong performance (>0.94 area under the receiver operating characteristic curve (AUC)) in predicting PET Aβ1–42 status across two cohorts. This combination of biomarkers was also found to be predictive of CSF Aβ1–42 status, with an AUC of 0.88 on a smaller subset of patients (n = 46). The novel IP-MALDI-TOF-MS method employed by Nakamura et al.25 is still in its infancy and it is unclear how easily this will be translated into a clinical setting. Thus, there is still strong interest in finding blood markers for CSF Aβ1–42 using alternative approaches that rely on more established assays.To bypass the invasiveness of CSF collection, there is a strong interest in finding blood-based markers that yield the same information about amyloid status as would be obtained from CSF. There have been a number of studies which have shown that a blood protein signature can be found that reflects AD brain pathology as measured by PETβ1–42 using a Random Forest (RF) approach and explore which types of measurements lead to the strongest predictive performance. We then determine the minimal set of features required to achieve comparable predictive performance. Finally, we evaluate the robustness and utility of these predictive models across a held-out validation cohort of individuals with mild cognitive impairment (MCI), demonstrating that subjects with predicted abnormal CSF Aβ1–42 levels showed a faster rate of cognitive decline  than those with predicted normal CSF Aβ1–42 levels.Here, we evaluated the ability of proteomic and metabolomic data to predict the levels of CSF AThe Alzheimer’s Disease Neuroimaging Initiative (ADNI) is a large, multicenter, longitudinal neuroimaging study, launched in 2004 by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, the Food and Drug Administration, private pharmaceutical companies, and non-profit organizations.ADNI is a longitudinal study of older adults, designed to test whether serial magnetic resonance imaging (MRI), positron emission tomography (PET), other biological markers, and clinical and neuropsychological assessment can be combined to measure the progression of MCI and early AD.http://adni.loni.usc.edu/), downloaded 10th October 2017 as the R package, ADNIMERGE.The data used in this study were obtained from the ADNI database (http://www.adni-info.org/) and written informed consent was obtained from all participants or authorized representatives. All the analytical methods were performed on the de-identified data and were carried out in accordance with the approved guidelines. Study inclusion criteria and definitions for each diagnosis class have been previously reported in detail26. Briefly, individuals diagnosed as AD had to meet the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association criteria for probable AD27. These individuals had issues with global cognition and memory function and they, or their caretakers, reported significant concerns about their memory. In contrast, individuals with MCI exhibited subjective memory loss (CDR of 0.5 and were at least one standard deviation(SD) below the normal mean of the delayed recall of the Wechsler Memory Scale Logical Memory II) but showed preserved activities of daily living, the absence of dementia and scored 24–30 on the MMSE.The ADNI study protocols were approved by the institutional review boards of all participating sites  and a further 190 proteins measured on a Rules-Based Medicine (RBM) platform) and 186 LC-MS/MS metabolites and lipids. After applying previously documented quality control procedures (Supplementary Methods) and removing analytes with more than 15% missingness, 149 proteins and 138 metabolites remained. No samples were removed from the analysis as missingness levels were less than 5%. Any remaining missing data points were imputed using an unsupervised RF approach28, with the resulting 289 analytes listed in Supplementary Table Aβ1–42 status. After quality control, a total of 566 individuals, each with measures for 289 analytes, age, and APOEε4 carrier status, were present in the ADNI cohort , amnestic MCI (n = 396) or probable AD n = 112). A breakdown of the demographics of the 566 individuals by baseline diagnosis and CSF availability is shown in Table 12. A breAβ1–42, respectively. CSF Aβ1–42 was measured using the Luminex xMAP platform. The training set was used to build predictive models and evaluate their performance directly using the measured Aβ1–42 levels while the validation cohort was used to evaluate the generalizability and utility of the model’s predictions. For each cohort, we also considered a subset of individuals for whom Aβ1–42 status from PET was available at least one-time point (not just at baseline), either using 11C-Pittsburgh compound B (PiB) and 18F-AV-45  tracers, for further validation of our modeling. Further demographic information for these cohorts can be found in Supplementary Tables This cohort was split into training and validation cohorts with 356 and 210 individuals with and without measures of CSF Aβ1–42 levels are below the recognized clinical threshold of 192 pg/ml for the Luminex platform, indicating an abnormal CSF Aβ1–42 level, and hence increased AD risk. Given the continuous CSF Aβ1–42 measures in the ADNI cohort, two approaches were consideredAβ1–42 levels and thresholding these post-predictiona ‘regression’ task: learning the continuous CSF Aβ1–42 status based on clinical thresholds directly.a ‘binary’ task: learning the dichotomized CSF The primary aim of this work was to produce a model that predicts if an individual’s CSF While both tasks result in a binary classifier, they face different trade-offs. The regression task makes use of the full information in the CSF levels but needs to learn a suitable threshold to convert its continuous predictions into suitable binary labels whereas the binary task only learns from the dichotomized CSF levels. Given these trade-offs, we have investigated both modeling approaches throughout this work.β1–42 levels for both the binary and regression tasks. RFs are a widely-used machine-learning ensemble method that have a number of advantages for the small sample size and disparate types of features observed in the ADNI dataset. RFs are invariant to the scale of the observed features and make few assumptions about the distributions of observed data allowing them to be applied to multiple data modalities easily. It can also detect non-additive relationships between variables without needing them to be included explicitly29.We made use of Random Forests (RF) as the modeling approach to predict CSF Aranger30. Each forest contained 2000 individual trees, each making use of a random selection of p3/4 features, where p was the total number of variables used in a given model. These parameter choices were based on recommendations provided in Ishwaran et al.31. All other parameters in the ranger implementation were set to their default values. To get an estimate of the performance of our models, we have made use of a nested cross-validation (CV) framework, whereby an inner CV was used to determine model parameters, and the outer CV was used to gain an estimate of the model’s performance on unseen data32. In this study, we used 3 repetitions of 3 fold CV for the inner loop and 10 repetitions of 10 fold CV for the outer loop.All analysis in this work made use of the RF implementation in the R package OptimalCutoffs33 to evaluate six potential cutoff metrics (Supplementary Methods) and selecting the method which maximized the accuracy over all of the test folds from the inner cross validation loops. The best performing cutoff criterion was then used in the current iteration of the outer cross-validation loop and the accuracy, sensitivity, and specificity derived from this threshold was recorded for that fold. While this approach means that a different method could be used to derive the regression threshold for each fold in the outer CV loop, the resulting estimate of performance is unbiased and hence is likely to be more representative of performance on unseen data compared with selecting a threshold based on the entire set of training data.As the RF used pre-determined parameter values, only a single parameter had to be determined: the threshold on the continuous regression predictions necessary to generate binary labels. This threshold was selected based on performance in the inner CV loop, using the R package R2 values were also calculated for the regression task. Increases in AUC between models were tested for significance using a one-tailed Wilcoxon signed-rank test. Receiver operating curves were constructed by aggregating all of the test predictions from the outer cross-validation.Model performance was summarized by the mean and standard deviation of the area under the Receiver Operating Characteristic (ROC) curve (AUC), accuracy, sensitivity, and specificity from the testing performance across the different cross-validation runs. APOEε4 carrier status; Proteomics (P), which included the 146 analytes measured on the RBM panel as well as homocysteine and plasma Aβ1–40 and Aβ1–42; Metabolomics (M), including 138 metabolites and lipids. Four separate random forests were created using different subsets of these features to determine which were most useful for modeling CSF Aβ1–42. We denoted these models by the combination of features they included; for example ‘BPM’ refers to a model built using all three classes of features. The best performing model was selected for all subsequent analysis.The input variables were separated into three classes: a commonly used baseline model (B) including age and Variable Importance, the difference in the prediction error on the out-of-bag data when a given feature was permuted and unpermuted29 and removed the lowest ranking features in a stepwise fashion. The AUC of the resulting RF was recorded, and the procedure was repeated over increasingly smaller subsets of features until no features were left to be removed. After the inner CV loop finishes, we determine the number of features that achieved the optimal trade-off between model complexity and performance by selecting the smallest subset of features that achieved within 4% of the maximal observed AUC. A model using this subset of features was then trained on all training folds of the outer CV loop and evaluated on the test fold. Again, by determining the number of features to include within our nested cross-validation framework, we are able to determine an unbiased estimate of the model’s expected performance over unseen data.After evaluating the impact of the different input modalities, we determine the minimum set of individual analytes necessary to achieve high predictive performance. This was done by treating the number of included features as a parameter to be determined in our nested CV framework. Within each fold of the inner CV loop, we used a recursive feature elimination approach, ranking features according to their Aβ1–42 levels, enabling us to determine if our predictions lead to useful clinical outcomes in the validation cohort.Survival analysis was conducted to determine if the rate of conversion from MCI to AD was different between those with predicted low and normal CSF measured CSF status on the training set (n = 198)predicted CSF status from B model, the standard baseline, in the validation set (n = 198)predicted CSF status from BP model, the best performing model, in the validation set (n = 198)fs model, the most parsimonious model, in the validation set (n = 198),predicted CSF status from BPFour separate analyses were performed, using the:fs is the feature selected model with the smallest set of features. For each analysis, we have examined the hazard ratios using Cox regression and used log-rank tests to compare the survival distribution of low/normal CSF Aβ1–42 stratifications in the four analyses, as well to compare equivalence between the actual and predicted stratifications.where BPAβ1–42 abnormal and normal status. While it is known that Aβ1–42 status from PET can differ from that observed in CSF, measurements from the two modalities are correlated and should be very similar for individuals who are not close to the cutoff indicating pathology. This provides analysis provides further evidence of our model’s ability to determine Aβ1–42 status in individuals in the validation cohort, where CSF measurements are not available.In order to further validate our model, we have examined the ability of our model to differentiate PET Given that only a limited number of individuals had associated measures of PET imaging at baseline , we have made use of the earliest PET image available, leaving us with 108 and 68 individuals in the training and validation cohort to evaluate. The threshold for abnormality was defined as an SUVR of 1.5 and 1.11 for PET images using PiB and AV45 tracers respectively. The mean number of years past baseline that a scan was taken was 3.07 and 2.97 years for training and validation cohorts respectively.Aβ1–42 accumulation) and that few individuals are close to the defined threshold for abnormality. If these assumptions do not hold, it is likely to worsen predictive performance, making this analysis somewhat conservative.The use of imaging at non-baseline times assumes that differences between the baseline and the  time that the image was taken are relatively small , likely due to the noise inherent in both the analytes being used for prediction as well as in the CSF Aβ1–42 measurements.We observed strong overall predictive performance for both the regression and binary tasks within our cross-validation framework. All sets of features outperformed the base model of age and R2 of 0.29 for the regression task. The automatically derived threshold for this regression RF yielded a mean accuracy of 0.77, with a sensitivity of 0.78 and a specificity of 0.73. Across the 100 cross-validation runs, the chosen threshold ranged from 164 pg/ml to 194 pg/ml with a median of 185 pg/ml. Similar AUC and accuracy could be observed for learning the dichotomized CSF labels directly . For the binary task, a slight drop in both AUC, as well as an altered trade-off between sensitivity and specificity, was observed across all different feature sets compared to the regression task. Given this, we chose to focus on the regression model for much of the follow-up analysis.The BP models resulted in a mean APOEε4, models that made use of the protein level measurements consistently achieved the strongest predictive performance, whereas metabolites appeared to be of limited utility. In both the regression and binary tasks, models containing metabolites and proteomic data (BPM) achieved equivalent or worse AUCs than models containing only the proteomic data (BP). Furthermore, we observed that the use of the base features and metabolites alone (BM) lead to decreased performance compared to the baseline model, indicating that the set of measured metabolites may have contributed little predictive information or may have been too noisy to be useful for predicting CSF status. These findings are in contrast to the previously reported utility of metabolites in predicting PET Aβ positivity22.While all models making use of blood analytes outperformed the base model of age and β positive, it is worth considering only ‘pre-clinical’ individuals as this may be more relevant for selective screening in drug trials. Evaluating our model’s performance on CN and MCI individuals only, we find that similarly strong predictive performance can be obtained , obtaining similar AUCs of 0.81 for the regression and binary tasks  available features in this dataset. In practice, measuring hundreds of analytes is costly, negating a key advantage of using blood biomarkers for screening. Given this, we have applied feature selection to the BP regression model to identify the smallest number of features that still achieved high predictive performance. Within cross-validation, we find that the average performance of this feature selection approach, denoted BPβ1–42 (AB42), Eotaxin 3, and Apolipoprotein E (APOE). This combination of protein levels, together with APOEε4 is denoted as BP5. Figure β1–42 is with APOEε4 carrier status, where being a carrier (APOEε4 = 1) leads to a low predicted Aβ level. While the relationships between the proteins and CSF Aβ1–42 are non-linear (a common outcome given the nature of RFs), the overall correlation with CSF Aβ1–42 is positive for CGA, Plasma Aβ1–42, and APOE protein levels and negative for Eotaxin 3.When applying this feature selection procedure to the entire set of training data, we identified a subset of four plasma analytes as well as APOE4 genotype status critical for model performance: Chromogranin-A (CGA), AAβ1–42 status from either the B, BP or reduced BP5 model. These survival distributions could then be compared to those of the real Aβ status observed over the training cohort. Given the demographic similarity of the two cohorts, we would expect to see strong similarities in rates of conversion.To demonstrate the utility of our modeling on unseen data, we conducted a survival analysis over the validation cohort (n = 198), evaluating the probability of baseline MCI individuals transitioning to AD diagnosis over 120 months, stratified by predicted CSF β1–42 group transitioned to AD significantly faster than the Aβ normal group. Comparing the predictions from the BP, BP5 and B models on the validation cohort to the actual CSF Aβ status on the training cohort, we find that there is no significant difference between the survival distributions for either the normal  or abnormal  survival distributions, respectively, reflecting the overlapping confidence intervals of the hazard ratios. However, it can be observed that due to differences in the thresholding of the Aβ levels, fewer individuals are deemed as CSF Aβ1–42 ‘normal’ in the actual data (n = 53), compared with any of the three models applied to the validation datasets , highlighting the well-recognized issues of defining standardized cutoff values across studies34. The significant differences in conversion rates between the predicted normal/low strata, especially from the more parsimonious BP5 model, together with their similarity to the survival distributions of the actual CSF measures, provide strong evidence that our blood-based model can help stratify individuals based on their risk of developing clinical AD .The resulting ROC curves in Fig. ata Fig. , with a 8. Some AD clinical trials are enriching pre-symptomatic AD individuals with PET screening. However, recent findings that shifts in CSF amyloid can be observed up to a decade before those from PET may indicate that CSF positive individuals are even more suitable for clinical trial enrichment35. Direct measurement of CSF biomarkers is too invasive to be used in such a screening test36 motivating the development of a minimally-invasive, low-cost solution that provides the same type of information to overcome these limitations.The most positive results from AD trials to date have been found in patients with early forms of the disease, leading to an increasing awareness that treatments are likely to be most successful if applied at the earliest stages of ADβ1–42 status using a Random Forest approach. We demonstrated that CSF Aβ1–42 normal/abnormal status using age, APOEε4 carrier status, and protein levels can be predicted with a high AUC, sensitivity and specificity of 0.84, 0.78 and 0.73 respectively. Compared to the baseline model (age and APOEε4 genotype) the inclusion of the plasma analytes improved the performance (AUC) by 6%. To make the model more suitable for clinical application, we identified four plasma analytes which, together with APOEε4 carrier status, still achieved a high AUC, sensitivity, and specificity of 0.81, 0.81 and 0.64 respectively. These predictive models were then validated on a separate cohort of individuals to demonstrate that MCI subjects with predicted abnormal CSF Aβ1–42 (low) levels transitioned to an AD diagnosis at a significantly higher rate than those predicted with normal CSF Aβ1–42 levels. Furthermore, these rates were similar to those observed in a demographically similar cohort of MCIs using actual CSF Aβ1–42 levels. This is a strong validation of our modeling as the blood-based biomarkers for CSF Aβ1–42 status is only useful if they can replicate the behavior of the actual Aβ1–42 status for clinically relevant endpoints for individuals that were not used to build the predictive model. Strong predictive power of PET Aβ1–42 status on the validation cohort provides further evidence for the generalizability and robustness of our modeling.This current study evaluates the utility of a blood-based signature of CSF A23. Some of these studies showed similar performance metrics to those reported in this work (>0.80 AUCs25 or >0.78 accuracy17), indicating that prediction of PET Aβ and CSF Aβ1–42 status are of similar difficulty. PET Aβ is directly related to brain fibrillar amyloid, whereas CSF amyloid is a marker of soluble Aβ1–42 and they may, therefore, give different insights into AD progression and mechanisms. For example, CSF Aβ1–42 has been shown to be associated with APOEε4 whereas PET Aβ has been shown to have a greater association with tau37. Thus, the development of a blood-based screening test for CSF Aβ1–42 levels is a complementary approach to existing blood-based biomarkers of PET amyloid status.A number of studies have previously investigated the use of blood analytes to predict the burden of amyloid in the neocortex, as measured by PETet al.25 showed a very high AUC in discovery and validation datasets for PET Aβ1–42 status (AUC 0.94 and 0.96 respectively) as well as a strong performance for predicting abnormalities in CSF Aβ1–42 levels (AUC 0.88%), in a small cohort (n = 46) of their validation set. While these results are promising, the automation of the novel technique used (IP-MALDI-TOF-MS), and hence transfer to a clinical setting, is non-trivial, motivating the search for complementary approaches. The protein signature presented in this study, based on a multiplex immunoassay, is likely to require a far shorter timeframe for clinical translation given the high level of automation that already exists for multiplex immunoassays, and that biomarkers from such platform have already been used in commercially available diagnostic tests that have been approved by the FDA.Of the above studies, the study by Nakamura β1–42. In both the regression and binary tasks, models containing metabolites achieved equivalent or worse AUCs than models without. These findings can be contrast with the utility of metabolites in predicting PET Aβ positivity22 and their association with AD more broadly38. Alternative methods for integrating this source of data39 may be required in order to find robust associations with CSF Aβ1–42 status.The use of metabolites appeared to be of limited utility for predicting CSF AAPOEε4 genotype and plasma levels of Chromogranin-A (CGA), Eotaxin 3, Aβ1–42 (AB42), and Apolipoprotein E (APOE). Several of these identified proteins have known associations with Alzheimer’s disease. Unsurprisingly, the levels of plasma APOE are associated with CSF amyloid levels. APOEε4 is the strongest genetic risk factor for AD. APOE is involved in the clearance of Aβ1–4242 and there is a strong relationship between APOEε4 genotype and APOE plasma levels, where APOEε4 carriers have lower plasma levels44. Plasma Aβ1–42 showed a positive relationship in our model for CSF Aβ1–42, in line with a prior observation45. This is interesting as the link between alterations of Aβ1–42 levels in the blood and the progression of the disease is still controversial and studies assessing the Aβ1–42 concentration in blood of AD patients have produced conflicting results51. Chromogranin A (CGA) is associated with synaptic function and has traditionally been used as an indicator of neuroendocrine tumors52. More recent work has shown that CGA has a degree of co-localisation with amyloid plaques in the brain54. However, levels of CGA in the CSF and blood serum do not appear to be correlated55 and serum CGA has not previously been linked to AD. Eotaxin 3, also known as C-C chemokine ligand 26 (CCL26), plays an important role in the innate immune system and has been found to be dysregulated in AD patients56. CSF Eotaxin 3 has been shown to be significantly elevated in patients with prodromal AD, however, Eotaxin 3 levels in plasma or the CSF has not been shown to correlate with rates of disease progression57.The subset of features used in our BP5 model included 58. Furthermore, the current cohort is neuropathology biased, i.e. 84% of the cohort have MCI or AD, and thus likely to have neuronal damage, potentially confounding the analysis of CSF Aβ1–42 status. Finally, it needs to be noted that there are other medical conditions that are known to affect CSF Aβ1–42 levels and it is unclear whether these affect any of the patients in our cohort.This study has several limitations. The training and validation cohorts are both composed of individuals in the ADNI study and thus all measures were conducted on the same platforms. Hence further cross-cohort and cross-platform replication is required. This remains an ongoing issue within the development of all AD biomarkers relating to early screening and requires significant future investmentβ1–42 and PET Aβ in preclinical stages of AD are likely to have implications for clinical trial enrichment. Blood-based biomarkers of amyloid can serve as the first step in a multistage screening procedure, similar to those that have been clinically-implemented in cancer, cardiovascular disease, and infectious diseases58. Inconjunction with biomarkers for neocortical amyloid burden, the CSF Aβ1–42 biomarkers presented in this work may help yield a cheap, non-invasive tool for both improving clinical trials targeting amyloid and for  population screening.The early identification of AD disease is paramount and a major global focus as the success of disease-modifying or preventative therapies in AD may depend on detecting the earliest signs of abnormal amyloid-beta load. The differences between CSF ASupplementary Information


GM-CSF promotes myeloid differentiation of cultured bone marrow cells into cells of the granulocytic and monocytic lineage; the latter can further differentiate into monocytes/macrophages and dendritic cells. How GM-CSF selects for these different myeloid fates is unresolved. GM-CSF levels can change either iatrogenically  or naturally  resulting in different immunological outcomes. Therefore, we asked whether the dose of GM-CSF may regulate the development of three types of myeloid cells. Here, we showed that GM-CSF acted as a molecular rheostat where the quantity determined which cell type was favored; moreover, the cellular process by which this was achieved was different for each cell type. Thus, low quantities of GM-CSF promoted the granulocytic lineage, mainly through survival. High quantities promoted the monocytic lineage, mainly through proliferation, whereas moderate quantities promoted moDCs, mainly through differentiation. Finally, we demonstrated that monocytes/macrophages generated with different doses of GM-CSF differed in function. We contend that this selective effect of GM-CSF dose on myeloid differentiation and function should be taken into consideration during pathophysiological states that may alter GM-CSF levels and during GM-CSF agonistic or antagonistic therapy. BM cellin vitro , 3; such . How GM-in vitro differentiation of moDCs, it is thought to be not essential for in vivo moDCs differentiation  ascribed to GM-CSF.−/− mice , CCR2.CFP.DTR, GM-CSF transgenic (GMtg) mice, and CCR2.CFP.DTR/GMtg , 25, A1−−/− mice , and Fuc−/− mice  were houCells from spleen and pooled subcutaneous lymph nodes  unless specified were prepared by digestion in collagenase/DNase I as described . Single 6/ml containing titrated doses of GM-CSF. After 3–4 days, the cultures were added fresh media with cytokines. Cell cultures were harvested on different day over 7 days.BM cells from mice were isolated by flushing femurs and tibias with 5 ml PBS supplemented with 2% heat-inactivated fetal bovine serum (FBS) . The BM cells were centrifuged once and then re-suspended in tris-ammonium chloride at 37°C for 30 s to lyse red blood cell. The cells were centrifuged again and then strained through a 70-μm filter before being re-suspended in RPMI-1640 supplemented with 10% FBS. For GM-CSF stimulated culture, BM cells were re-suspended at 0.5 × 10in vivo source and from 7-day BM cultures with GM-CSF as in vitro source were purified by flow sorting. Purified cells were cultured at 1–5 × 104 in 200 μL RPMI-1640 supplemented with 10% FBS in U-bottom 96-well plates in the absence or the presence of graded doses of GM-CSF. Upon harvesting, cells were stained for cell surface markers. Cell survival was measured by flow cytometry with FITC or APC-conjugated FACS calibration beads (BD Biosciences) and PI to determine the number of viable cells.moDCs were enriched from spleen cells by using a Nycodenz density gradient as previously described  and furt5 in 200 μL RPMI1640 supplemented with 10% FBS in flat-bottom 96-well plates in the absence or the presence of graded doses of GM-CSF. Cell cultures were harvested every day and cells were stained for cell surface markers.BM or Blood cells from Fucci mice were cultured with or without CTV  labeling (as per manufacturer's protocol). Labeled cells were cultured at 1 × 104 in 200 μL RPMI1640 supplemented with 10% FBS in U-bottom 96-well plates in the absence or the presence of LPS (1 μg/mL) or CpG (1 μM) for 20 h. For cytokine detection from the supernatants of in vitro assays, the indicated cytokines were detected using Cytometric Bead Array (CBA) technology (BD Biosciences) according to the manufacturer's instructions using a FACS Verse (BD Biosciences) cytometer.Myeloid cells -derived from 7-day BM cultures were purified by flow sorting. Then cells were cultured at 5 × 10t-test, and data presented as dose response curves or time courses were made using ANOVA. The analysis was performed with Prism v.5.0 software . P < 0.05 were considered statistically significant.Statistical comparisons of mean difference between two groups from independent experiments were made using a student's hiCD11b+, whereas monocytic myeloid cells as Ly6GloCD11b+ . Granulocytes were identified as Ly6G+ Figure . When cu+ Figure . On the + Figure . Proport+ Figure . Thus, t+ Figure .in vivo, we compared GM-CSF overexpressing mice (GMtg) with WT mice (with lower levels of endogenous GM-CSF). Previous studies showed that GM-CSF overexpression resulted in an increase in both Ly6G+ granulocytes and monocytic myeloid cells  of moDCs  and Ly6C−CCR2− cells, increase in CCR2+ cells in GMtg mice was more prominent  Figures . The yieAs phenotypic analysis and transcriptome analysis concluded that mo/mΦ and moDCs derived from mouse BM cultured under GM-CSF belong as distinct entities , howeverin vivo influence of GM-CSF on moDCs is complicated by two factors: that GM-CSF is not essential for development of moDCs . An additional moDC population expressing lower levels of CD11c also existed (termed as CD11clo moDCs)  Figure . Althoug1 Figure . TogetheOne of the determinants of population size is cell survival. To examine the effect of GM-CSF on survival of generated granulocytes, mo/mΦ, and moDCs from BM cultures, we checked viability of the three myeloid cells in the presence or absence of GM-CSF at the end of culture period. We found that granulocytes had poor spontaneous survival in contrast to higher and similar survival rate of mo/mΦ and moDCs. In response to GM-CSF stimulation, however, the survival of both granulocytes and moDCs increased significantly, although the fold of increase were less in moDC than that of granulocytes. On the contrary, additional GM-CSF did not alter the survival of mo/mΦ Figure .−/− mice. In vitro, survival of three types of cells under different doses of GM-CSF was evaluated. We found that A1 deficiency significantly reduced survival by granulocytes and moDCs with or without GM-CSF. On the other hand, survival of mo/mΦ was not grossly affected by A1 deficiency  with different doses of GM-CSF. Unsurprisingly, we did not find cells that were actively proliferating i.e., there was a lack of FucciGreens Figure . When mos Figure , suggestloCD11bhi) vs. granulocytes (Ly6Ghi). We found that the proportion of cycling cells was much higher in monocytic myeloid cells than granulocytes over 7 days in culture; for example, on day 3 after culture with 5 ng/mL GM-CSF, 30% of monocytic myeloid cells were cycling, compared with 1.3% of granulocytes. Moreover, proliferation by monocytic myeloid cells , whereas proliferation by granulocytes was similar regardless of GM-CSF dose at least for the range between 0.5 and 10 ng/mL . By then the rate of proliferation of monocytic myeloid cells had already reduced. Over 5–7 days in culture, the proportion of moDCs was highest when intermediated doses of GM-CSF (2.5–5 ng/mL) were used Figures . We alsoOverall, high doses of GM-CSF favored the proliferation of mo/mΦ. Most moDCs were differentiated after active proliferation stopped. Thus, appearance of moDCs between day 5 and 7 most likely represented differentiation. The rate of moDCs differentiation was highest with intermediate doses of GM-CSF.In a previous report, cells generated in GM-CSF stimulated cultures may differ in function, including the production of certain cytokines by mo/mΦ and dendritic cells . Here weMo/mΦ can be generated with low or high dose of GM-CSF, albeit with different yield. When isolated mo/mΦ were stimulated with various doses of LPS  for 20 h, mo/mΦ generated with high dose GM-CSF in most cases produced higher levels of cytokines/chemokines than those generated with low dose GM-CSF (not shown). As survival of mo/mΦ can be impacted by LPS, we found out that survival or death of mo/mΦ generated with low or high dose of GM-CSF behaved similarly in response to TLR stimulation (not shown). However, we found that overall spontaneous survival of mo/mΦ generated with low GM-CSF was poor than those generated with high dose of GM-CSF (not shown), implying that reduced survival by mo/mΦ generated with low GM-CSF could contribute to low production of cytokines.+ cells that was induced with lower doses of GM-CSF. Granulocytes had very poor survival that were greatly improved with low concentration of GM-CSF while mo/mΦ had better spontaneous survival and addition of GM-CSF only moderately improved survival. We also revealed that differentiation of moDCs from monocytic cell population occurred at intermediate levels of GM-CSF. This differentiation occurred relatively late (from day 4 in the culture).GM-CSF plays an important role in inflammation and immunity to pathogens, cancer and inflammation. It is released during emergency leukopoiesis and its main therapeutic use is to hasten leukopoiesis after BM suppression. Its influence in the development of multiple types of myeloid cells from BM cells has been extensively documented. Yet, how GM-CSF differentially affects each myeloid cell type has not been defined. Here we showed that GM-CSF quantity had a selective effect on which cell type was favored; moreover, the cellular process by which this was achieved was different. High doses of GM-CSF favor mo/mΦ production, largely through enhancement of cell proliferation while high doses of GM-CSF did not further increase proliferation of Ly6Gin vitro data indicate that GM-CSF quantity had a selective effect on differentiation of granulocyte and macrophages. High dose of GM-CSF mainly increases the production of monocytic myeloid cells. The finding is also largely vindicated in vivo. Two previous reports had showed that high doses GM-CSF lead to an increase in the monocytic:granulocytic ratio, even though they did not look at monocytic:granulocytic ratio directly. One study showed that their GMtg mice had >25-fold higher levels of circulating GM-CSF (−CD11b+), particularly CCR2+ cohort in blood, spleen, lung, and liver was larger in GMtg mice. We also observed previously that GM-CSF deficiency had differential effect on macrophages over neutrophils during listerial infection  in monocytic myeloid (Ly6GloCD11bhigh) cells was positively correlated to GM-CSF doses. On the other hand, proportion of cells actively cycling (FucciGreen+) in Ly6Ghi cells reached a plateau with much lower doses. The proliferation data provides an explanation of how mo/mΦ generation is favored.We further examined the impact of GM-CSF doses on proliferation of two lineages using mice with cell cycle reporters. In general, most cell proliferation occurred in the first 4 days of culture. Even with replenishment of fresh media, cell proliferation was not remarkably increased after 4 days. This probably reflects depletion of proliferating progenitors and limited proliferating capacity of fully differentiated myeloid cells in culture. During the first 4 days, proportion of cells actively cycling ; an intermediate dose of GM-CSF favored moDCs differentiation. In support, there was a recent study showing that high concentration of GM-CSF (10–100 ng/mL) favored macrophage differentiation (GM-CSF has been used for a long time as a cytokine critical for moDCs differentiation in BM cultures , 3, 5. Wntiation . Our datntiation  are somentiation  showing in vivo isolated Flt3+CD11c− MHCII+ PU.1hi monocyte subset can act as precursors of GM-CSF dependent moDCs (in vitro GM-CSF stimulated BM cultures might also contain “DC-precursors” that expressed intermediate levels of MHC class II although there was no clear “subpopulation” based on expression of MHC class II in mo/mΦ population (In our study, we isolated mo/mΦ and moDCs from GM-CSF stimulated cultures for 7 days and cultured further with GM-CSF and fresh media. In the secondary cultures, moDCs maintained their phenotype while proportion of mo/mΦ could turn into moDCs. This would suggest that moDCs were terminally differentiated whereas at least a substantial proportion of mo/mΦ might not be. It has been reported that nt moDCs . It is cpulation . These cpulation . Thus, iin vivo GM-CSF overexpression also resulted in highly activated mo/mΦ causing severe inflammation (GM-CSF dose likely has different impact on cell function in several ways. Firstly, GM-CSF dose generates different myeloid cells with different function. mo/mΦ and moDCs generated from the same cultures have been reported to greatly differ in cytokine production and T cell priming . We exteammation .The precise mechanism affecting cell differentiation and function by GM-CSF is not well-understood. Given that GM-CSF signaling can lead to the activation of multiple intracellular signaling modules, including JAK/STAT, MAPK, PI3K, it may be that different GM-CSF dose has different impact on the balance of these signaling events. Transcriptome analysis also revealed that GM-CSF induces multiple cellular pathways required for function of inflammatory monocytes of mouse and human . How GM-Together, we demonstrated that GM-CSF dose acts as a rheostat regulating survival, proliferation, differentiation and function of three myeloid cell types generated under GM-CSF stimulation. These findings could provide some answers why different GM-CSF doses could be either immunostimulatory or immunosuppressive ; why GM-YZ, YX, AL, and LS designed research. LS, JR, RD, FS-F-G, and EC performed research. RS and MH contributed vital new reagents or analytical tools. LS, JR, RD, FS-F-G, EC, and RS collected, analyzed, and interpreted data. LS, YZ, YX, AL, and NH wrote the paper.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.


T. b. gambiense-infected patients used other staging criteria than those recommended by the WHO. We compared WHO criteria (cell count and parasite identification in the CSF) with two biomarkers (neopterin and CXCL-13) which have proven potential to diagnose disease stage or relapse. Biological, clinical, and neurological data were analysed from a cohort of 83 patients. A neopterin concentration below 15.5 nmol/L in the CSF denoted patients with stage 1 disease, and a concentration above 60.31 nmol/L characterized patients with advanced stage 2 (trypanosomes in CSF and/or cytorachia higher than 20 cells) disease. CXCL-13 levels below 91.208 pg/mL denoted patients with stage 1 disease, and levels of CXCL-13 above 395.45 pg/mL denoted patients with advanced stage 2 disease. Values between these cut-offs may represent patients with intermediate stage disease. Our work supports the existence of an intermediate stage in HAT, and CXCL-13 and neopterin levels may help to characterize it.Human African Trypanosomiasis may become manageable in the next decade with fexinidazole. However, currently stage diagnosis remains difficult to implement in the field and requires a lumbar puncture. Our study of an Angolan cohort of  It is hoped that sleeping sickness will become manageable within the next decade, as suggested by the WHO , 2. In 2gambiense-HAT) remains the molecule indicated for the treatment of stage 1 of the disease. Thus, stage diagnosis is still necessary. Furthermore, fexinidazole has so far only been used in the context of field trials. What will happen when it is used routinely? Moreover, the infection mechanism remains incompletely understood; the disease may reappear at cyclic intervals as chronically infected individuals without clinical signs of disease or animal reservoirs can lead to reemergence [Pentamidine , the parasite VSG coat and DNA, by the immune system . This tr disease , 16. Alt disease . Neoptertivation . In facttivation  and virativation , 21 incltivation , 23, justivation , 25. CXCtivation . Perturbtivation , and CXCtivation –31. CSF tivation , neurosytivation , and multivation . Previoutivation –37; rapiIn this study, classical staging criteria, clinical signs, and new potential biomarkers  were reevaluated in patients from a cohort study in Angola , 37. TheEthical clearance was obtained from the Angolan Direccao Nacional de Saude Publica, Ministerio da Saude. Written informed consent was received from these individuals prior to enrolment and/or from their parents or guardians for participants below 18 years of age. Any individual who declined to participate was followed up according to the standard procedures of the national control programme of Angola.bertrand.courtioux@unilim.fr) for researchers who meet the criteria for access to confidential data.The clinical data used to support the findings of this study are restricted by the Angolan Direccao Nacional de Saude Publica, Ministerio da Saude in order to protect patient privacy. Data are available from the authors (Trypanosoma brucei gambiense-infected patients (n = 228) from diagnosis to the end of follow-up  and noninfected controls (n = 19).Samples were obtained retrospectively from a cohort study conducted in Angola between 2008 and 2011 . The stuIt is not acceptable to perform a lumbar puncture on a patient for whom no neurological pathology is suspected. Seronegative Angolan controls for HAT could therefore not be included in this study. Thus, the 19 controls are derived from the 601 patients with a positive CATT test. Initially, these individuals were suspected of HAT and finally considered healthy following further negative tests.μL, whereas those with trypanosomes in the CSF and/or a CSF WBC count above 20 cells/μL were classified as advanced stage 2. Patients who were deemed to be in the intermediate stage were those between stage 1 and advanced stage 2 with a CSF WBC count between 5 and 20 cells/μL and/or with trypanosomes in CSF without an increase in CSF WBC numbers. The Angolan national control programme differentiates the intermediate stage, as they treat those patients with first-stage drugs unless some clinical signs are suggestive of CNS involvement, in which case second-stage drugs are used. All participants were examined clinically, and a questionnaire was used to note all clinical and neurological characteristics , including sleep and psychiatric disturbances using the Mini-International neuropsychiatric interview [Subjects were enrolled during both active and passive screening activities by teams of the national sleeping sickness control programme. The CATT (Card Agglutination Trypanosomiasis Test) screen was used  and follnterview  and the nterview , 42.For our analysis of neopterin and CXCL-13, we selected 83 patients for whom we had maximum of neopterin and CXCL-13 CSF concentrations data. When the concentration data were missing and we had CSF samples available in the biobank, we determined the neopterin and CXCL-13 concentration in the CSF via ELISA. The ELISA kits for the dosage of neopterin and CXCL-13 used are the same throughout the study .μL blood was used to perform capillary tube centrifugation test (4 capillary tubes of 75 μL), and 300 μL blood was used for the miniature anion exchange centrifugation technique [A lymph node aspirate was taken from any subject who presented with swollen lymph nodes and examined for trypanosomes by microscopy. 10 mL venous blood with heparin as anticoagulant was collected from CATT-positive individuals as well as from patients with swollen lymph nodes. 600 echnique . For indechnique , 43.All plasma, buffy coat, and CSF samples that remained after the diagnostic procedures were aliquoted and stored in liquid nitrogen before being transported to Limoges on dry ice and then stored at −80°C. Patient medical data were anonymized. Samples were further selected for neopterin and CXCL-13 determination at diagnosis .Commercially available ELISA assays were used to measure CSF levels of neopterin  and CXCL-13  as performed previously by Tiberti et al. , 37. Allp < 0.05). Tests were performed using R Core Team version 3.3.2 [Descriptive analysis was carried out on Microsoft Excel. Receiver operating characteristic (ROC) curves were drawn to determine cut-off points as the best sensitivity/specificity ratio for each marker . ROC curves were further used to determine the disease stage  on diagnostic samples. The significance was assessed using the Kruskal–Wallis test and Fisher's exact test for testing the null of independence  patients in stage 1 and four (21%) patients in the intermediate stage showed neurological signs, and there was an increase in neurological signs in advanced stage 2 patients (32 out of the 34 (94%)) compared with previous stages , whereas a concentration above 60.31 nmol/L characterizes advanced stage 2 disease . Values between these two cut-off points could be used to characterize an intermediate stage .Our findings also suggest that CSF CXCL-13 levels below 91.21 pg/mL define stage 1 disease , and CXCL-13 levels above 395.45 pg/mL define advanced stage 2 disease . Values between these two cut-off points could be used to characterize an intermediate stage .μL of CSF (AUC = 0.80), and the invasion of the CNS by trypanosomes was associated with the presence of more than 50 cells/μL of CSF (AUC = 0.93). The sensitivity and specificity of each of these cut-off points are shown in ROC curves show that neurological signs and sleep disorders appeared when CSF neopterin concentrations were above 21.20 nmol/L  and/or CXCL-13 concentrations were above 330.26 pg/mL . Trypanosomes were found in the CSF when neopterin concentrations were higher than 31.40 nmol/L (AUC = 0.94) and/or CXCL-13 concentrations were higher than 688.85 pg/mL (AUC = 0.94). Sleep and neurological disorders were associated with the presence of a minimum of 16 cells/in vitro [in vitro and/or the beginning of the BBB crossing with the presence of some parasites in the CSF which have not been detected yet on CSF after lumbar puncture. However, the relevance and the importance of the intermediate stage are still not completely understood [μL [Our study addressed and characterized a specific group of patients with intermediate stage sleeping sickness, or early stage of CNS involvement. There have been reports of patients cured with first-line drugs at this disease stage, which suggests that either pentamidine , 47, 48 in vitro , 53. In derstood . In thretood [μL . In the tood [μL –37. In oOne of the difficulties encountered was to define cut-off points for markers of stage 1 disease in the absence of seronegative Angolan controls for HAT in our cohort. This is why we have grouped all patients in the intermediate stage and in stage 2 of the disease and therefore defined cut-offs to discriminate patients who are not in stage 1 (non-stage 1). Values below these thresholds are therefore characteristic of stage 1 patients.Previously, CSF neopterin levels below 14.3 nmol/L were indicative of stage 1 disease, and neopterin levels greater than this were indicative of stage 2 disease (determined from 412 samples) . SimilarIn our study, the cut-off point of CSF CXCL-13 to define stage 1 disease was found to be a concentration of less than 91.21 pg/mL; a CSF CXCL-13 concentration greater than 395.45 pg/mL was indicative of advanced stage 2 disease. Concentrations of CXCL-13 between these two values were indicative of the intermediate stage. The concentrations of CXCL-13 that define stage 1 and stage 2 diseases were originally found in the study by Tiberti et al. in 2012 ; their dμL), but rather to an intermediate stage between stage 1 (a WBC count below 5 cells/μL) and an advanced stage 2 (a WBC count greater than 20 cells/μL).This observed difference between the CXCL-13 and neopterin cut-off points is due to the fact that in the studies by Tiberti et al. , 37, patThe differences in levels of neopterin and CXCL-13 in patients with sleeping sickness may be explained by their production mechanisms. Neopterin is produced by microglial cells in response to the parasite , 55, wheThe main criticism of this work is the absence of a HAT-seronegative Angolan controls population, which is justified because it is not ethical to perform a lumbar puncture on a healthy person. It is for this reason that we had to define cut-off points for neopterin and CXCL-13 above which patients are considered non-stage 1; all patients with values below these thresholds are thus stage 1. According to the literature, the normal concentration of neopterin is less than 5.1 nmol/L of CSF , and theμL of CSF (91% sensitivity and 96% specificity) and confirms the lack of sensitivity of parasite detection. The most sensitive and current test to define CNS involvement is cytorachia but this test lacks specificity [μL; this has been reported previously [rhodesiense infection where patients are cured with first-stage drugs [μL. We established cut-off points for neopterin and CXCL-13 related to the onset of clinical signs of the disease instead of using cytorachia but the neopterin and CXCL-13 cutsoff points were not discriminative enough to be used as diagnostic markers.The difficulty in all studies of neurological stage discrimination during sleeping sickness is the definition of the gold standard as recently reviewed . The curcificity , 62. Theeviously , 64, conge drugs . Thus, tOur work suggests that the levels of the two biomarkers, neopterin and CXCL-13, are good markers of disease staging, in the patient. Using the two biomarkers in conjunction, rather than individually, could be a stronger predictor of disease stage after its diagnosis.Biomarker identification should not be limited to the CSF and should be continued on other biological fluids to avoid lumbar puncture. Even if no correlations were observed in serum, neopterin and CXCL-13 must be in urine and lacrimal fluid. With the development of new methods to identify specific markers, as proteomics, diagnosis algorithms should be simplified in the near future.


Cognitive dysfunction is a frequent complication to open-heart surgery. Cerebral inflammation caused by blood-brain barrier (BBB) dysfunction due to a systemic inflammatory response is considered a possible etiology. The effects of the glucocorticoid, methylprednisolone, on cerebrospinal fluid (CSF) markers of BBB function, neuroinflammation, and brain injury in patients undergoing cardiac surgery with cardiopulmonary bypass were studied.n = 15) or placebo (n = 15) as a bolus dose administered after induction of anesthesia. CSF and blood samples were obtained the day before and 24 h after surgery for assessment of systemic and brain inflammation , axonal injury , neuronal injury (neuron-specific enolase), astroglial injury , and the BBB integrity .In this prospective, randomized, blinded study, 30 patients scheduled for elective surgical aortic valve replacement were randomized to methylprednisolone 15 mg/kg (p < 0.001). The increase of the CSF levels of the astroglial markers was not affected. A postoperative BBB dysfunction was seen in both groups as the CSF/serum albumin ratio increased from 6.4 ± 8.0 to 8.0 in the placebo group (p < 0.01) and from 5.6 ± 2.3 to 7.2 in the methylprednisolone group (p < 0.01) with no difference between groups (p = 0.98). In the CSF, methylprednisolone attenuated the interleukin-6 release (p < 0.001), which could be explained by the fall in systemic interleukin-6, and the serum to CSF gradient of IL-6 seen both at baseline and after surgery. In the CSF, methylprednisolone enhanced the interleukin-8 release (p < 0.001) but did not affect postoperative changes in CSF levels of tumor necrosis factor alpha. Serum levels of S-100B and neuron-specific enolase increased in both groups with no difference between groups. CSF levels of total tau, neurofilament light chain protein, and neuron-specific enolase were not affected in any of the groups.In the control group, there was a 54-fold and 17-fold increase in serum interleukin-6 and interleukin-8, respectively. This systemic activation of the inflammatory cytokines was clearly attenuated by methylprednisolone  contains supplementary material, which is available to authorized users. Neurological complications to open-heart surgery remain a common cause of postoperative morbidity , 2. TheyExperimental studies have suggested that surgical trauma induces a systemic inflammatory response causing a disruption of the blood-brain, neuroinflammation, and cognitive dysfunction , 5. CardIn a previous study, we studied the release of cerebrospinal fluid markers of inflammation, neuronal and glial cell injuries, and BBB function after cardiac surgery with CPB . A pronoHigh doses of corticosteroids can be used to suppress the postoperative systemic inflammatory response syndrome in cardiac surgery . MethylpIn this randomized trial, we aimed to assess the effects of an intraoperative high dose of methylprednisolone on CSF markers of neuroinflammation, brain injury, and blood-brain barrier function in patients undergoing cardiac surgery with CPB. Our hypothesis was that the previously described neuroinflammatory response to cardiac surgery is attenuated by a high dose of methylprednisolone.www.epn.se/goeteborg/) and the Swedish Medical Product Agency, and all patients signed an informed, written consent preoperatively. The study was registered at www.clinicaltrials.gov; identifier: NCT01755338, first posted 24 December 2012 by Mattias Danielson. This study adheres to the applicable CONSORT guidelines  elective open aortic valve replacement surgery (SAVR) with a biological valve prosthesis due to aortic stenosis with or without concurrent coronary artery bypass grafting (CABG), (b) normal preoperative coagulation tests , (c) absence of recent (< 1 week) treatment with thrombolytic or potent anti-platelet drugs, and (d) preoperative left ventricular ejection fraction ≥ 50%. Exclusion criteria were (a) stroke with sequelae and (b) abnormal coagulation tests (see above) or thromboelastograms in the morning on the day after surgery.Ⓡ, Pfizer) 15 mg/kg or placebo (0.9% sodium-chloride) administered after induction of anesthesia. A nurse, not involved in the study or care of the patients, randomized the patients to the study drugs in a 1:1 ratio, using concealed, sequentially numbered opaque envelopes. This nurse prepared the drugs and performed the blinding.A randomized block design was used based on gender and type of surgery (SAVR or SAVR plus CABG). Patients were randomized to treatment with either methylprednisolone  was used during CPB. MAP was maintained within the range 70–80 in the pre- and post-CPB period, and a MAP within the range of 60–80 mmHg during CPB using phenylephrine and/or norepinephrine when necessary.Premedication consisted of oxazepam 10 mg, oxycodone 10 mg, and half the dose of any ongoing beta-adrenergic blocker treatment orally. A catheter was placed in the radial artery before the induction of anesthesia for blood sampling and continuous monitoring of mean arterial blood pressure (MAP). Anesthesia was induced with fentanyl (5–10 μg/kg), propofol (1–1.5 mg/kg), and rocuronium (0.6 mg/kg). Before and after cardiopulmonary bypass (CPB), anesthesia was maintained with sevoflurane 0.5–2.5% in a 50% O2 and the target hematocrit of 25–35%. PaCO2 was maintained at 40–48 mmHg and was uncorrected for body temperature. PaO2 was maintained at 180–260 mmHg, and SvO2 was maintained at > 65%. During CPB, the body temperature was maintained at 36 °C in all patients. During the peri- and postoperative period, the target serum glucose level was 4.2–6.3 mmol/l using a standardized insulin infusion protocol when appropriate.The CPB perfusion system consisted of a hollow fiber membrane oxygenator and a Stöckert S-5 (Sorin Group). The pump was primed with acetated Ringer’s solution and mannitol, with the target CPB flow rate of 2.4 l/min/mA lumbar puncture for quantitative analysis of CSF biomarkers was performed the day before surgery and approximately 24 h after surgery. We used a 27-G Whitacre spinal needle  for median approach at L3–4 level in the sitting position. A CSF sample volume of 2.5 ml and a 4 ml blood sample were collected at both time points. Detailed information on the measurements of CSF and serological markers of inflammation, brain injury, and blood-brain barrier function are shown in Additional file t test or Wilcoxon signed-rank test when appropriate. The comparison of the effects of surgery on changes of the various variables between the placebo and intervention groups were tested using an unpaired Student’s t test or Mann-Whitney U test. All data are presented as mean ± standard deviation (SD) or median and interquartile range (IQR) when appropriate. A probability level  of less than 0.05 was considered to indicate statistical significance. Statistical analysis was performed using SPSS for Macintosh, version 24.The primary end-point was the intergroup difference in postoperative IL-8. A power analysis was performed, based on our previous study . To deteThe clinical trial profile is summarized in Fig. p < 0.001). Serum levels of TNF-α were not changed in the control group but decreased in the methylprednisolone group after cardiac surgery. Serum S-100B increased in both groups, but showed no difference between groups. Serum albumin decreased in both groups with no difference between groups.In the control group, there was a 54-fold and 17-fold increase in serum IL-6 and IL-8, respectively. This systemic cytokine activation by cardiac surgery was clearly attenuated by methylprednisolone treatment  decreased (− 19%) after cardiac surgery in the control but not in the methylprednisolone group.The CSF to serum albumin ratio increased postoperatively in both the control and methylprednisolone group by 20–25%, with no difference between groups.p = 0.001). Cardiac surgery induced a 3.4-fold increase in CSF-IL-8 in the control group. In the methylprednisolone group, cardiac surgery caused a 7.5-fold increase in CSF-IL-8 (p < 0.001).CSF levels of TNF-α increased slightly in both the control and the methylprednisolone group after cardiac surgery with no difference between groups. There was a marked (8-fold) increase in CSF IL-6 in the control group, which was attenuated by methylprednisolone  behind the increased permeability of the BBB are not provided in the present study. Irrespective of the cause of the dysfunction of the BBB, it could not be prevented or attenuated by methylprednisolone.IL-8 is a pro-inflammatory cytokine which attracts neutrophils as mediators of inflammation . In the One limitation was that CSF levels of biomarkers were obtained only once after the surgical procedure. Thus, a more detailed evolution of the CSF biomarkers over time could not be assessed. However, it was too risky to perform more than one postoperative lumbar puncture for quantitative analysis of CSF, as anticoagulant and anti-platelet therapy was commenced on the first postoperative day. Furthermore, we did not measure anti-inflammatory cytokines, which could have provided information on the effects of cardiac surgery on the balance between pro- and anti-inflammatory mediators in the CSF and the potential effect of preemptive methylprednisolone on this balance.In conclusion, we showed that preventive treatment with high-dose methylprednisolone attenuated the systemic inflammatory response to cardiac surgery with CPB, but this treatment did not prevent or attenuate the increase in BBB permeability or the neuroinflammatory response seen after this procedure.Additional file 1:CONSORT 2010 Checklist for reporting of a randomized trial. (DOC 218 kb)Additional file 2:Measurements of cerebrospinal fluid (CSF) and serological markers of inflammation, brain injury and blood-brain barrier function. (DOCX 14 kb)