ObjectiveTo assess efficacy and safety of dual therapy (DT) and triple therapy (TT) in patients with atrial fibrillation (AF) and acute coronary syndrome (ACS) with or without percutaneous coronary intervention (PCI) and evaluate the quality of evidence with respect to said outcomes based on contemporary randomized trials (RCTs). The efficacy outcome taken was major adverse cardiovascular events (MACE) while safety outcome was major bleeding events.IntroductionAppropriate anti-thrombotic therapy is still controversial in patients with AF and concomitant ACS or PCI. We conducted a conventional meta-analysis pooling data from major RCTs to assess the efficacy and safety of DT and TT. Additionally, we utilized advanced analytic properties of trial sequential analysis (TSA) to assess for quality of evidence in this realm.Methods and resultsA total of 8,732 patients from five major RCTs were enrolled in this study. There was a statistically significant reduction in major bleeding on the DT group compared to the TT group . The incidence of major adverse cardiovascular events (MACE) was similar in both groups . The trial sequential analysis showed strong evidence supporting reduction in bleeding from current major RCTs while being inconclusive based on MACE outcome.ConclusionSufficient quality evidence could be ascertained from contemporary RCTs on reduced incidence of bleeding in DT patients compared to TT patients. Further adequately powered RCTs are needed to ensure non-inferiority of DT over TT with respect to MACE outcome. Triple therapy (TT) with an oral anticoagulant and dual antiplatelet medications is currently endorsed as the therapy of choice by the European guidelines in this patient population . In contWe used the advanced meta-analytic properties of trial sequential analysis (TSA) to assess the quality of available evidence comparing TT vs. DT from current major randomized controlled trials (RCTs). For the purpose of our analysis, we used major adverse cardiovascular events (MACE) as an efficacy outcome while major bleeding was taken as a safety outcome.For the current study, data was pooled from five major RCTs that compared DT and TT in AF patients with associated ACS and/or PCI. The RCTs used to collect data for our current analysis included the recently published Open-label, 2x2 Factorial, Randomized Controlled, Clinical Trial to Evaluate the Safety of Apixaban vs. Vitamin K Antagonist and Aspirin vs. Aspirin Placebo in Patients with Atrial Fibrillation and Acute Coronary Syndrome or Percutaneous Coronary Intervention (AUGUSTUS) trial  and prevTSA can be applied to quantify the reliability of conclusions driven from meta-analysis by establishing monitoring boundaries to test the quality of evidence. By this method, if the cumulative Z curve crossed the TSA boundary, a sufficient level of evidence has been reached supporting the intervention. However, if the Z curve failed to cross the TSA boundary, evidence to reach a conclusion is insufficient and more studies are needed. We pooled the primary safety outcome of bleeding  and the primary efficacy outcome of major adverse cardiovascular events  using the random effect model from above RCTs comparing DT to TT at the maximum reported follow-up. We then performed TSA to maintain an overall two-sided type-I error rate at 5% and calculated the required sample size to achieve 80% power to detect a statistically significant difference. The analysis was conducted using RevMan 5.3  and Copenhagen Trial Unit, version 0.9.5.10 beta.LimitationsFew limitations of our study should be acknowledged. We do not have patient-level data for our current meta-analysis and utilized trial-level data so we cannot account for differences in baseline characteristics that may affect outcomes. Additionally, differential trial designs may have instituted heterogeneity although we tried to mitigate this by depicting statistical heterogeneity for each outcome studied.A total of five major RCTs with 8,732 patients were included in the current analysis. A statistically significant reduction in the rate of bleeding was seen in the DT group compared to the TT group  Figure . The corThe findings from our current analysis indicate firm evidence supporting lower rates of bleeding in patients treated with DT vs TT. Our conventional meta-analysis further showed that TT confers no additional benefit in preventing MACE outcomes when compared to DT. TSA analysis, however, concluded that sufficient evidence could not be obtained with respect to MACE outcomes among patients on DT and TT. The management of AF patients with concomitant ACS or PCI is fraught with uncertainty. While oral anticoagulants are shown to be superior to dual anti-platelet agents (DAPT) in reducing risk of ischemic stroke and systemic embolism in AF patients, they don’t confer any benefit in preventing complications such as stent thrombosis . On the Our conventional meta-analysis showed a significantly improved rate of bleeding in AF patients with concomitant ACS and/or PCI on DT compared to TT while at the same time showed similar incidence of MACE outcomes in both groups. TSA confirmed that sufficient quality evidence exists from current RCTs that proves low incidence of bleeding in the DT group compared to the TT group. However, more adequately powered RCTs are required to ensure non-inferiority of DT over TT in preventing MACE related outcomes.


Pythium insidiosum, a finding also confirmed by histopathology, serology, and DNA sequencing and thus, treated with itraconazole. Since no improvement was observed, Pythium-immunotherapy was initiated. The cat responded to the latter approach and in less than 10 days, the lesion had contracted around the edges and was crusting off to reveal healthy granulation tissue. Twenty-three days after immunotherapy was initiated the original wound had shrunken significantly to a small scabbed area. However, the cat acutely developed tachypnea, lung and intestinal complications and due to her rapid deterioration, humane euthanasia was elected. Unfortunately, necropsy was not conducted. The clinical presentation reported here suggests large ulcerative cutaneous lesions caused by P. insidiosum can also occur in domestic cats. Despite reports of unsuccessful treatment results of feline pythiosis using Pythium-immunotherapy, this report suggests this approach might be helpful in similar feline cases.Pythiosis is frequently reported in dogs and horses inhabiting tropical, subtropical and temperate areas of the USA, but the disease is rare in domestic cats. The clinical presentation of feline pythiosis includes subcutaneous masses without ulcerated tissue and involvement of the intestinal tract. Here in we report an eight-week-old female unvaccinated stray kitten with an unusual extensive circular ulcerated lesion on her left flank. The lesion did not respond favorably to administration of systemic antibiotics. Clinical specimens submitted for culture demonstrated submerged fungal-like flat colonies later identified as  Feline pergroup ,2. Bissopergroup  in 1991 pergroup  reportedPythium-immunotherapy without success. Subsequently the cat was treated with a combination of amphotericin B and itraconazole also without response. The authors reported the subcutaneous lesions in the latter case progressed very slowly. Based on these observations they concluded that the invasion of the subcutaneous tissues by P. insidiosum “may be slower in cats than in dogs” [7.8 suggesting clinicians are better recognizing the disease in this species. Herein, we report an unusual case of cutaneous feline pythiosis with an extensive ulcerative lesion on her left flank.In 1998 Thomas and Lewis  reportedin dogs” . More rein dogs” ,6 and th2An 8-week-old female kitten presented to South Orlando Animal Hospital with a 2-week history of a rapidly growing, round and pruritic lesion on her left flank. She was adopted after being found as a stray cat in the Central Florida area. At presentation, the lesion was circular and approximately 6.0 X 7.0 cm in diameter covered by a necrotic black crusted surface . SurrounP. insidiosum IgG detection . Histopathology showed the presence of eosinophilic granulomatous tissue with numerous hyaline slender sparsely septate hyphae (13) as P. insidiosum (GenBank accession number= MK772067) and thus, treatment with itraconazole (10 mg/kg q 24 h) was then initiated. Seven days later, the lesion showed no change and the kitten remained pyrexic. Since no improvement was observed with antifungal therapy, all previous treatments were discontinued and Pythium-immunotherapy, using 200 μg/ml of purified P. insidiosum immunogens, was initiated administering 1.0 mL of these antigens subcutaneously [P. insidiosum IgG antibodies (250%).Two weeks later, the wound showed no improvement so a biopsy was taken from the wound margin for histopathology and culture. On physical exam, the kitten had enlarged axillary lymph nodes bilaterally and remained pyrexic (39.6 °C). The wound was again debrided and a wet-to-dry bandage was applied and changed daily for 3 to 4 days to encourage granulation tissue formation. Amoxicillin clavulanate was continued (as above) and buprenorphine prescribed for pain management (0.4 mg/kg). A serum sample was also collected and sent to Pan American Veterinary Laboratories for an ELISA anti-e hyphae A–D. The Pythium-immunotherapy was administered at this time. The kitten improvement was monitored for the following ten days , another oomycete phylogenetically and taxonomically related to P. insidiosum, was also diagnosed causing protractive subcutaneous infections in cats with clinical and histopathological features similar to that in feline pythiosis [Lagenidium hyphae is broader than that displayed by P. insidiosum, the clinical and histopathological features of both infections are comparable [Feline pythiosis had also been diagnosed in captive big cats affecting intestinal and pulmonary tissues ,10. Furtythiosis . Althougmparable . In addimparable ,13.4Pythium-immunotherapy [Although the clinical features of previously reported feline pythiosis have displayed protractive cutaneous or subcutaneous lesions, the current case suggests rapidly progressive ulcerative cutaneous lesions can also be expected in this species. In addition, the management of feline pythiosis is difficult with only a few reportedly cured cases using surgery and antifungal drugs without otherapy ,8. The cThere are not.


It is usually very complicated to treat meningeal carcinomatosis, and it is important to treat it as soon as possible.The 19-Del mutation was found in the exon for the epidermal growth factor receptor gene in the pleural effusion of a patient on March 11th, 2015. He took 250 mg of oral gefitinib once a day for 11 months beginning in December of 2015. On the 3rd of November 2016, he arrived at the hospital and presented with dizziness, headache and transient blurred vision. At this time, he began to take 4 mg of oral zoledronic acid once a month to prevent bone metastases. The result of a cytology exam of the cerebrospinal fluid showed that the man had meningeal carcinomatosis. The 19-Del mutation and the 20-T790 M mutation in the exon of the epidermal growth factor receptor gene was found by the next generation sequencing of the CSF. Then, he discontinued taking gefitinib and began to take 90–100 mg of oral AZD9291 once a day in November 2016. After adjusting the medication dose based on the NGS, his headache was noticeably reduced, and his condition gradually stabilized.Cerebrospinal fluid ctDNA detection by next generation sequencing may become a suitable biomarker to monitor clinical treatment response in meningeal carcinomatosis. Meningeal carcinomatosis (MC) is a rare complication of extraneural solid tumors and consists of a focal or multiple malignant infiltration of neoplastic cells in the leptomeninges of the brain or spinal cord . It is wEGFR) mutations in plasma with high specificity and sensitivity.Circulating tumor DNA (ctDNA) consists of short, double stranded DNA fragments that are released by tumors. It has been shown that plasma ctDNA can be used to characterize and monitor tumors –6. ctDNAEGFR [CSF, which can have intimate contact with tumor cells, is an easily obtained body fluid that reflects the underlying pathological state of the central nervous system. Recently, ctDNA has been found in the CSF of patients with brain tumors , 10. AddEGFR .EGFR mutations in ctDNA with NGS technology. Here, we present a case of a MC patient, who was provided treatment based on analysis of EGFR mutations in ctDNA of their CSF.In this study, we used a highly sensitive and specific method to analyze According to the kit instructions, cell free DNA (cfDNA) and genomic DNA (gDNA) were extracted by the QIAamp Circulating Nucleic Acid kit (QIAGEN) Circulating Nucleic Acid Kit extraction kit and blood genome DNA extraction kit, respectively. The CSF samples were taken from a − 80 °C refrigerator and thawed at room temperature before extraction (8 ml CSF and 200 μl cell suspension). It was necessary to control the pressure of the vacuum pump  to ensure that the CSF samples were integrated with the adsorption column. Before the sample library was constructed, the concentration and size of cfDNA fragments were quantitatively detected by Qubit 3 and 2100-H. The KAPA Hyper Prep Kit was used to construct the precapture , connection and PCR amplification). The capture of the target area was based on chip hybridization and liquid hybridization capture products from the SureSelect QXT rapid hybridization kit (Agilent Company). A total of 120 cancer-associated genes were included in the gene panel, which was used for target region capture. The library was then tested by Agilent 2100 and Q-PCR . Lastly, the Illumina NextSeq 500 system was used for sequencing.https://sourceforge.net/projects/flexbar/), the clean fastq (the raw data file format of from sequencing) data were generated from the raw data from the NEXT SEQ 500 runs. Then, the clean fastq data was aligned to hg19 (GRCH37) and assembled through BWA-sampe . The polymerase chain reaction (PCR) duplicates were removed by MarkDuplicates in Picard Tools . Variant calling was performed in the CSF  by GATK . All variants were annotated by ANNOVAR . Lastly, variation frequency (> 0.5%) was used to eliminate erroneous base calling and generate the final mutations. Manual verification was performed by IGV .After trimming the adapter sequences, and filtering and removing poor quality reads using flexbar software  and a 20-T790 M mutation in the exon of the EGFR gene (mutation rate 0.85%) were found in the NGS. In November 2016, the patient discontinued gefitinib and began to take 90–100 mg of oral AZD9291 once a day. After adjusting the medication based on the results of the NGS, the headache of the patient was noticeably reduced, and his condition gradually stabilized. In February 2017, a pulmonary CT showed that the lesion in the superior lobe of the left lung was significantly reduced  in nonsmall cell lung cancer [EGFR-T790 M and the capability to penetrate the blood-brain barrier [EGFR gene is still present, which suggests that targeted medicines possess a certain sensitivity. After taking oral AZD9291, the patient’s clinical symptoms improved.It is an important potential application of CSF gene analysis to determine drug resistance mechanisms in patients. Through NGS, we detected ctDNA in the CSF of one MC patient with primary lung cancer, and conducted targeted therapy based on these results. Targeted gene therapy was effective for the primary tumor, however, tumor development of the central nervous system continued to progress. The patient appeared with symptoms in his head after taking oral gefitinib for 11 months. We took CSF from the patient diagnosed with MC to use for NGS. We found a 20-T790 M mutation in the exon of the g cancer . The mut barrier , 18. The, our study confirmed that ctDNA can be detected in the CSF from MC patients. In addition, we used NGS technology and found a 20-T790 M in the exon of the EGFR gene in ctDNA from CSF, which may be helpful in providing drug treatment guidance.In conclusion


Infection of donor sites in split-thickness skin grafts is one of thecomplications of skin transplantation. Nutrition status and associated diseasesplay important roles in healing of donor sites. There are different ways used totreat infected donor sites. Water-filtered infrared-A (wIRA), as a special formof heat radiation with a high tissue penetration and a low thermal load to theskin surface, can improve the healing of acute and chronic wounds both bythermal and thermic as well as by non-thermal and non-thermic effects.Water-filtered infrared-A (wIRA) increases tissue temperature, tissue oxygenpartial pressure and tissue perfusion. These three factors are decisive for asufficient supply of tissue with energy and oxygen and consequently also forwound healing and infection defense. This was confirmed in a case with a latesevere healing disturbance of the donor sites after skin transplantation.  The aim of this report is to highlight the benefit of using water-filteredinfrared-A (wIRA) in cases with infected donor sites.Skin transplantation is the treatment for second and third degree burn wounds.Infection of donor sites is a possible complication of skin transplantation. Weherein present a case of a 61-year-old male with infected donor sites that wasmanaged with a daily non-adherent antimicrobial alginate dressingbacteria. Patient was a heavy smoker and suffered from multiple sclerosis. Up to ourknowledge, neglect and inadequate wound care were responsible for the opening andsecondary infection of donor sites. One day after admission we performed a surgicaldebridement and removed the hypertrophic granulation tissue. After the operation westarted daily wound dressing with a non-adherent antimicrobial alginate dressing(Silvercell®) and irradiation with water-filtered infrared-A(wIRA). Water-filtered infrared-A (wIRA) protocols used at our department wereradiations all wound areas three times daily with a 60 cm distance for 30 minuteswith a “Hydrosun 505®” model . The patient tolerated this application well without anycomplaints. After 5 weeks the patient was discharged with healed donor sites leftthigh and left lower leg and nearly totally healed sites left forearm and left upperarm  and IIb-III degree, involvingthorax, abdomen, and right arm. Patient was treated with split-thickness skin graft.We discharged the patient after successful management with healed graft and nearlyhealed donor wounds. 8 months later, the patient was referred to us from his primarycare physician for management of infected open wounds in donor sites. Physicalexamination revealed infected area with hypergranulated tissue in donor sites, leftupper limb and left lower limb Figure 1 . Wound cFigure 2 . Infected donor sites often complicate split-thickness skin graft (STSG). Manyantimicrobials have been used in the management of infected donor sites, examplesinclude wound contact dressings containing silver, gentamycin cream, fusidic acidcream, or silversulphadiazine cream. In this case we combined local antimicrobialtreatment and irradiation with water-filtered infrared-A (wIRA) to improve theenvironment of the wound and wound healing. wIRA increases tissue temperature,tissue perfusion, and tissue oxygen partial pressure as basis of an increasedmetabolism and increased energy production. wIRA also has non-thermal andnon-thermic effects (on cells and cellular structures and substances). In generalwIRA decreases pain, inflammation, and hypersecretion and promotes infection defenseand regeneration in a wide range of indications including acute and chronic woundsand infected wounds , 2], , , 5]. wI. wI5]. wLonger irradiation times per day result in larger effects. Thus, more frequent andlonger lasting irradiations  with small irradiances arepreferred to shorter lasting irradiations with higher irradiances , 2]..2].Water-filtered infrared-A (wIRA) can optimize the environment of the wound and woundhealing in split-skin donor sites. These will reflect in decreased healing time andan optimized treatment result and in a better cosmetic result. Therefore wIRA isrecommended in addition to conventional treatment with antimicrobials. GH was working for the Dr. med. h.c. Erwin Braun Foundation, Basel, a charitable,non-profit Swiss scientific foundation approved by the Swiss FederalAdministration. The foundation supports clinical investigation of waterfilteredinfrared-A. The foundation was not involved in any content- or decision-relatedaspect of the case report. GH is not and was not employed by a commercialcompany and has not received fees or grants by a commercial company in the fieldof water-filtered infrared-A. Therefore, GH declares that no conflicts ofinterest exist according to the guidelines of the International Committee ofMedical Journal Editors. The three other authors declare to have no competinginterests.


To,The editorFirst of all please convey many thanks to author of “Letter to editor” for his interest in our paper . As per Whether the measured parameter should be called as “Patellar tendon–Trochlear Groove distance (PT–TG Distance)” or “Tibial tuberosity–Trochlear Groove Distance (TT–TG Distance)”;Methodology used in the study needs to be verified.It is a very well known fact that any parameter can have more than one method to measure it. Results obtained by one method may differ from another to an extent. But that does not undermine the importance of the actual parameter or does not necessitate to rename it every time when it is measured by a different method. If with each method we change the terminology of that parameter, then confusion is inevitable and importance of the parameter in discussion may fade away.According to literature, while measuring TT–TG Distance multiple methods are used to define tibial tuberosity and trochlear groove.To determine tibial tuberosity, the following landmarks are used in various studies:Anterior most point of tibial tuberosity (bony landmark) ;Center of tibial tubercle (bony landmark);Center of distal most part of patellar tendon attached over tibial tuberosity (cartilaginous/soft tissue landmark) , 4.On the trochlear side,The deepest portion of the bony trochlear groove (bony landmark) ;The deepest portion of the cartilaginous trochlear groove (cartilaginous/ soft tissue landmark).Also there are multiple studies with contradicting results regarding accuracy of bony vs cartilaginous landmarks and also regarding use of CT scan vs. MR imaging as imaging modalities , 6.Even though the landmarks varied in multiple studies, many of them have maintained a discipline to refer measured parameter as TT–TG Distance , 4.We preferred to refer this measurement as TTTG distance considering following points:It is a well-known fact that tibial tuberosity has its significance in discussion because it has patellar tendon attached over it. In other words, tibial tubercle without patellar tendon attachment has no clinical significance and should not be considered for measurement. So we took part of tibial tuberosity for measurement which has patellar tendon attached over it and referred it as “tibial tuberosity (TT)”. We did not refer it as “patellar tendon (PT)” because we are measuring the distance from clinically significant tibial tuberosity and not from the patellar tendon.Multiple studies have used the term “TTTG distance” for the same measurement methodology that we used. On the contrary, very few recent articles have come up with newly coined terminology as “PT–TG Distance” for this method. We decided to go by traditional and widely accepted terminology TT–TG distance and not PT–TG distance.patellar tip to trochlear groove distance (PTTG distance) ;Patellar tendon to trochlear groove distance (PTTG distance).In literature we found that two different measurements are being referred as PTTG distance:So we wanted to avoid confusion.The method used by us to measure the said parameter is unanimously accepted by orthopedic community as PTTG distance for future reference and research;The confusion about PT–TG distance terminology is resolved.But we will have no objection to refer this measurement as PTTG distance instead of TTTG distance ifNow coming to the second question to be answered, there are studies including ours in which the good inter-observer reliability for the measurement has been found. This should resolve the doubt in the methodology.Hope all the queries are answered to satisfaction.


The role of colony stimulating factors (CSFs) in cystic fibrosis (CF) circulating neutrophils has not been thoroughly evaluated, considering that the neutrophil burden of lung inflammation in these subjects is very high. The aim of this study was to assess granulocyte-CSF (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) levels in CF patients in various clinical conditions and how these cytokines impact on activation and priming of neutrophils. G-CSF and GM-CSF levels were measured in sputum and serum samples of stable CF patients (n = 21) and in CF patients with acute exacerbation before and after a course of antibiotic therapy (n = 19). CSFs were tested on non CF neutrophils to investigate their effects on reactive oxygen species (ROS) production, degranulation , and chemotaxis. At very low concentrations found in CF patients (0.005–0.1 ng/ml), both cytokines inhibited ROS production, while higher concentrations (1–5 ng/ml) exerted a stimulatory effect. While either CSF induced elastase and MMP-9 secretion, lactoferrin levels were increased only by G-CSF. Chemotaxis was inhibited by GM-CSF, but was increased by G-CSF. However, when present together at low concentrations, CSFs increased basal and fMLP-stimulated ROS production and chemotaxis. These results suggest the CSF levels that circulating neutrophils face before extravasating into the lungs of CF patients may enhance their function contributing to the airway damage. The CFTR protein is a chloride and bicarbonate channel and also a regulator of other channels, such as the epithelial sodium channel (ENaC). In the CF airways, its absence/dysfunction leads to the lack of chloride and bicarbonate secretion coupled with an exaggerated ENaC activity, increased sodium flux and water absorption from the lumen, thickening and abnormal viscosity of mucus overlaying the epithelium, and eventually abrogation of the mucociliary clearance2. The acidification of the airway secretions determines the functional loss of antimicrobial peptides3. Bacterial species, such as Pseudomonas aeruginosa, took advantage of such alteration of the airway microenvironment by penetrating in the mucus and creating macrocolonies which defend themselves from the attack of the immune system5. Neutrophils are thereby continuously attracted into the CF airways and activated. The continuous secretion of reactive oxygen species (ROS) is implicated in the damage to the airway epithelial cells, whilst the secretion of granule proteases, such as elastase, is involved in the degradation of extracellular matrix protein of bronchioles, determining bronchiectasis, and thus further worsening the mucociliary clearance. Metalloproteinases (MMPs), in particular MMP-9, also produced by neutrophils, may contribute to matrix breakdown and repair, thus contributing to the damage and remodeling of CF airways6. Moreover, neutrophil cytotoxic peptides, granule enzymes, extracellular DNA, and neutrophil extracellular traps (NETs) are associated with increased mucus clogging and lung injury in CF7. During NETosis, neutrophils release decondensed chromatin coated with elastase, myeloperoxidase, and other cytotoxic granular proteases, thus perpetuating severe inflammation that characterizes CF lung disease9. Pro-inflammatory cytokines (IL-1β and TNF-α) and chemoattractants (such as IL-8 and LTB4), are elevated in the CF airway milieu, especially during acute exacerbation episodes, and are involved in continuous neutrophils’ extravasation and activation12. The attraction of neutrophils in the CF airways has been more recently attributed to high burden of the IL-23/IL-17 axis which occurs in patients infected by P. aeruginosa and undergoing a pulmonary exacerbation15. IL-17A and IL-17F up-regulate granulopoietic factors and CXC chemokine in airway epithelial cells, thus contributing to the massive neutrophil influx in the CF airways13. In the more advanced stages of lung disease, a skewed immune response with a TH2-dominated alveolar inflammation and tolerance towards Gram-negative infections, disfavoring a protective TH1 response, is associated with poor prognosis16.Cystic fibrosis (CF) is a life-threatening condition and the most lethal among the autosomal recessive diseases. It is caused by the absence/dysfunction of CFTR (CF Transmembrane Conductance Regulator) protein, expressed on the plasma membrane of epithelial cells. Although CF is characterized by the dysfunction of many organs, lung disease represents the main cause of morbidity and mortality of these individuals. A neutrophil-dominated inflammatory response is the hallmark of CF lung disease, along with pulmonary infections by opportunistic bacterial pathogens19. Mechanistically, GM-CSF is involved in the accumulation of neutrophils in the airways caused by IL-17 and TNF-α, probably via effects on both recruitment and survival of neutrophils20. Moreover, GM-CSF has been found to prime neutrophils for NET release by other stimuli, such as LPS or C5a21.The role of colony stimulating factors (CSFs) in the survival and function of neutrophils is well recognized. In CF, granulocyte-CSF (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) have been found to be elevated in serum and sputum and to correlate negatively with lung disease23. In this paper, we have aimed to quantify serum and sputum CSF levels in a cohort of stable CF patients and compare them with those of CF subjects in acute exacerbation and to elucidate whether they are modified by a course of antibiotic therapy. Moreover, based on these results, we have studied the effect of GM-CSF and G-CSF on various neutrophil functions, including oxidative burst, degranulation, and chemotaxis. We chose to carry out this experimental part on non CF neutrophils to avoid biases linked to pre-exposure of CF neutrophils to a systemic inflammatory milieu as has been demonstrated in these patients24.However, notwithstanding these findings, the role of CSFs in modifying circulating neutrophils in CF has not been ascertained yet. In the past, we have undertaken a thoughtful study of blood neutrophils’ functions in CF before and after an antibiotic course for a pulmonary exacerbation, finding that oxidative burst and expression of HVCN1, a protein channel involved in the acidification of endosomal vesicles, were modified by the antibiotic therapy1, as a parameter of respiratory function, was reduced in patients in acute exacerbations as compared with stable patients and showed a non-significant increase upon antibiotic therapy.Clinical parameters of stable and acute patients, before and after a course of antibiotic therapy, are shown in Table Patients in stable state and in acute exacerbation before and after antibiotic therapy were analyzed for G-CSF and GM-CSF levels in sputum and serum specimens and their concentration values are depicted in Supplementary Fig. 1 in stable patients .There was a weak but significant positive correlation observed between sputum GM-CSF levels and WBC prior to therapy in acute patients  and between sputum G-CSF levels and FEVi.e. neutrophils incubated firstly with either CSF and then with fMLP, a similar bimodal behavior was found although with some differences  on the ROS production by circulating neutrophils obtained from non-CF individuals. Neutrophils were incubated with different concentrations of either CSF followed, in some experiments, by fMLP stimulation. When neutrophils were incubated with either G-CSF or GM-CSF only Fig. , a bimodces Fig. . G-CSF dces Fig. . OverallTo investigate degranulation, we analyzed elastase as a marker of primary granules Fig. . fMLP in25. Although fMLP mobilized CD66 on neutrophils plasmamembrane (P < 0.01 as compared with untreated cells), both G-CSF and GM-CSF did not modify the CD66b expression significantly, either in the absence or in the presence of fMLP  granules, we studied CD66b and lactoferrin. CD66b is a receptor whose levels are increased during exocytosis of specific granulesMLP Fig. . LactofeMLP Fig. .Figure 3To study tertiary granules, we measured the levels of MMP-9 in the supernatants of neutrophils Fig. . fMLP inCSFs were tested for induction of chemotaxis in a Transwell system. As shown in Fig. Chemotaxis was evaluated when neutrophils were incubated with both cytokines at the same time and at the lowest concentrations, either when used as direct agonists or primers of fMLP effect Fig. . InteresP. aeruginosa infection versus those with no chronic infection18. Although a direct comparison of results obtained in these studies with ours cannot be made, since almost all patients were chronically infected with S. aureus, P. aeruginosa or B. cepacia, CSFs levels in CF patients reported in this study are quite in good agreement with those previously found. In sputum, G-CSF concentrations were higher than GM-CSF ones  and locally in inflammatory fluids in both mice30 and in humans33. On the other hand, GM-CSF is normally produced locally by the alveolar macrophages34, being the first line of defense when clearing bacteria35. It appears that its main functions are to regulate surfactant homeostasis and primarily macrophage activity and differentiation. Our data suggest that chronic infection of CF lungs stimulates the production of G-CSF rather than of GM-CSF.G-CSF production is primarily confined to monocytes/macrophages, fibroblasts, and endothelial cells and is enhanced in response to bacterial products and inflammatory mediators36. G-CSF and GM-CSF bind to their cognate cell surface receptors on neutrophils, i.e. G-CSFR (encoded by CSF3R) and GM-CSFR (encoded by CSF2R). While G-CSFR is a homodimeric receptor, GM-CSFR is a heterodimer comprising the cytokine-specific α subunit and the βc subunit common also to IL-3 and IL-5 receptors. Both G-CSF and GM-CSF are type I receptors that lack intrinsic catalytic activity but trigger the activation of the JAK-STAT pathway, SRC family kinases, and the PI3K and MAPK pathways38. The β-chain-containing GM-CSF and the homodimeric G-CSF receptors utilize STAT2 and STAT5 or STAT3, but it is unclear how the specificity of the receptor is carried over through different JAK and STAT family members and how a limited number of JAK and STAT components are able to trigger specific signals39.G-CSF and GM-CSF are chemoattractants for neutrophils and could prime these cells for an enhanced respiratory burst activity after stimulation with several agents. Moreover, both cytokines enhanced the neutrophil  antibody-dependent cellular cytotoxicity (ADCC), adherence, phagocytosis and killing of microorganismsThis complexity is reflected by the results obtained in this paper. Indeed, we show that both cytokines, tested as single stimulus, had similar effects on ROS production but exerted different modifications of degranulation and chemotaxis. To make this outcome even more complex, when the two CSFs were present at the same time as direct agonists or priming agents for fMLP, the results showed that they increased ROS production by an effect that has to be determined yet, possibly given by an overlapping of the signaling pathways.42, others showing that they were negative factors for chemotaxis44. The timing of exposure and the chemotactic agent to which CSFs prime neutrophils are likely responsible for these different results. It is worth noting that all these studies were carried out by using single CSFs as chemotactic agents, and indeed we obtained similar differing results under these conditions. However, studying chemotaxis with both cytokines present contemporarily, which has not been done before to the best of our knowledge, showed that they could exert a positive effect on neutrophils chemotaxis, either as direct agonists or when used as priming agents.In the case of chemotaxis, previous studies had reported divergent results, some demonstrating G-CSF and GM-CSF as chemotactic for neutrophils46, and inducing superoxide production where it is required most. Specifically, at the lowest concentrations found in CF patients (0.005–0.1 ng/ml), CSFs exerted a negative effect on the respiratory burst, whereas higher concentrations increased ROS levels. Interestingly, a signaling switch for GM-CSFR has been observed. At concentration as low as 1 pM (0.014 ng/ml), GM-CSF mediates βc Ser585 phosphorylation, leading to 14–3–3 binding, PI3K activation, and hematopoietic cell survival, whereas at concentrations of 10 pM (0.14 ng/ml) or more, GM-CSF mediates βc Tyr577 phosphorylation, Shc recruitment, and PI3K activation, thereby promoting both survival and proliferation47. However, under our experimental conditions, the incubation of neutrophils with both cytokines at low concentrations enhanced ROS production, which may simulate higher concentrations. Whatever the experimental set-up, the two cytokines exerted their effect directly, and the magnitude of ROS levels was similar to that obtained with priming experiments, suggesting that priming for fMLP stimulation is not important with low CSF concentrations on blood neutrophils. Whether this phenomenon occurs also in sputum neutrophils has to be ascertained, but we can anticipate that since the concentration of bacterial stimuli should be higher in the local lung environment, the already stimulated neutrophils should react more to these agonists. Together with the combined effect of local CSFs, neutrophils should be more active and secrete more ROS than in the blood. While this would increase the bactericidal activity of neutrophils during non-CF bacterial infections, however, this would create more tissue damage than benefits in the CF lung, since opportunistic CF bacteria, such as P. aeruginosa, are protected by alginate exopolysaccharides. Moreover, GM-CSF primes neutrophils for NET formation by other stimuli, such as LPS or C5a, and ROS are implied in their formation21. One caveat to these considerations is that sputum cytokines levels, including CSFs, reflect heterogeneity of airway inflammation across lung regions48 and also bacterial-derived products are present in sputum as a consequence of the complex CF lung environment49. Thus, the sputum CSF levels are likely to be an average of higher and lower local G-CSF/GM-CSF concentrations in CF lungs, implying that our results should be confirmed in vivo by exploring different lung regions (e.g. upper and lower lobes). Finally, since NETs have been correlated with CF lung disease progression, it would be interesting to study NETs in blood neutrophils upon CSF stimulation. Since we could appreciate the secretion of granule enzymes in our assays with differences between GM-CSF and G-CSF stimulation, and NETs or partially cleaved NET fragments can sequester active proteases27, the evaluation of NETs, with both lower and higher concentrations of both CSFs present contemporarily, will allow to obtain crucial information on neutrophils’ activation already in the blood and avoid the issue of local heterogeneity. This brings the attention to blood neutrophils as possible non-invasive markers of CF lung disease.Once attracted into the airways, CSFs would assist in maintaining the neutrophils at the site of inflammation, promoting their survivalAs for the degranulation, we found that G-CSF and GM-CSF were able to induce elastase and MMP-9 release by neutrophils, at concentrations as low as 0.005–0.1 ng/ml, while lactoferrin secretion was enhanced significantly only by G-CSF. As pointed out above, differences in signal transduction pathways may generate different responses, however this issue has been studied more deeply in hematopoietic progenitors and less in mature neutrophils, and should be thoroughly investigated by means of pathway inhibitors or in neutrophils bearing mutations inactivating these pathways.50. Neutrophil elastase has been recognized as a key risk factor for the onset and early progression of CF lung disease52. The role of elastase in the pathogenesis of CF lung disease is highlighted by the fact that this protease has been implicated in several key features of CF lung disease including airway inflammation, goblet cell metaplasia and mucus hypersecretion, and proteolytic damage of airway walls59. Airway and serum/plasma MMP-9 protein and activity were shown to be elevated in CF and negatively correlated with the respiratory function and with its decline rate65. Moreover, plasma MMP-9a is associated with bacterial infection65. Although this is not the only MMP found to be elevated in CF airway secretions, these findings highlight its possible role in neutrophil-mediated progression of CF lung diseases.The direct and priming effects on elastase and MMP-9 shown by neutrophils upon stimulation with both CSFs might be important for the pathogenesis of matrix breakdown and remodeling occurring in infected CF bronchioles leading to bronchiectasis66. Lactoferrin exerts several functions as inhibition of microbial growth, biofilm development, and bacterial invasion of host cells68. Also due to its demonstrated anti-inflammatory activities, it has been suggested that lactoferrin is an important molecule for interrupting the vicious circle involving iron homeostasis dysregulation, bacterial infection, and inflammation in CF69. Thus, lactoferrin increase might be part of a negative feedback mechanism operating when hyperinflammation occurs, as in CF lungs, however other studies are mandatory to validate this hypothesis.G-CSF had an enhancing effect on unstimulated and fMLP-induced release of lactoferrin, which is an iron-chelating glycoprotein of innate immunity and represents a major endogenous antimicrobial constituent of airway secretionsAlthough the number of replicates in degranulation studies witnesses the robustness of data, conclusive remarks should be made only when the levels of granule enzymes as well as NETosis, which is strictly interrelated, will be analysed following neutrophil stimulation with both cytokines present at the same time, which is the focus of ongoing studies.All-in-all, our data strongly suggest that circulating CSFs stimulate neutrophils to display a heightened chemotactic phenotype, and to produce more ROS and granule enzymes. Thus, neutrophils once entered into the CF lungs might be more damaging than protecting the airways from the infection. Our data show that G-CSF is highly produced than GM-CSF in the infected lung of CF patients and it might be the factor promoting further neutrophils’ activation and then more damage. Therefore, it could be a target of inhibitory therapy.In conclusion, CSF levels found in serum of CF patients modify the behavior of circulating neutrophils by modulating ROS production, degranulation of proteases, and chemotaxis. On the basis of these results it might be reasonable to assume that, influenced by these levels, circulating CF neutrophils are already activated before entering CF lungs.1) compared with previous best, weight loss and fever70. Patients in acute exacerbation were studied before and after a 10-day course of antibiotic therapy. The ratios between males and females were 11:10 and 6:13 in stable and acute patients, respectively. Patients age was 30.9 ± 2.2 for stable and 30.0 ± 2.9 for acute (mean ± SEM). Upon admission, all clinical and laboratory parameters were collected: white blood cell count, neutrophil percentage, CRP (mg/dl), FEV1 (% predicted for sex and height), and pathogen colonization. The stable patients were colonized, as single or coupled, by P. aeruginosa (n = 10), S. aureus (n = 8), B. cepacia (n = 3), nontuberculous mycobacteria (n = 1) and Enterobacter (n = 1), while acute patients presented P. aeruginosa (n = 10), S. aureus (n = 4), nontuberculous mycobacteria (n = 5), E. coli (n = 3), and B. cepacia (n = 2). Most patients, i.e. 20 in stable condition (95%) and 17 in acute exacerbation (89%), were chronically infected, as established by international guidelines72. The study was approved by the ethics committee of the Azienda Ospedaliera Universitaria “Policlinico” of Bari (no. 1373/CE/2012) and performed in accordance with the 2013 amended Declaration of Helsinki. Written informed consent from the adult study subjects or informed consent from a parent and/or legal guardian for study participation of subjects under the age of 18 was obtained.In this study, 21 patients in stable condition and 19 patients in acute exacerbation were considered. Exacerbation was defined as a deterioration in symptoms perceived by the patient and included an increase in coughing, sputum production, dyspnea, decline in forced expiratory volume in 1 sec  were diluted 1:1–1:6, with phosphate-buffered saline (PBS), immediately vortexed and then centrifuged for 5 min at 7000 × g. Each supernatant was immediately removed and stored in aliquots at −80 °C before assaying.G-CSF and GM-CSF levels in sputum and serum specimens were measured by an ELISA kit . The sensitivity of the Hu GM-CSF kit (cat. #KHC2011) and of the Hu G-CSF kit (cat. #KHC2031) is <3 pg/ml and <20 pg/ml, respectively. Obtained values were normalized according to the initial dilution of the sample.Some of the samples were below the detection limit of the assay: serum G-CSF ; sputum G-CSF ; serum GM-CSF (post-therapy = 1); sputum GM-CSF .6 cells25. An irrelevant mouse FITC-conjugated monoclonal antibody was used as negative control. In total, 10,000 events were recorded per staining. Neutrophil viability was assessed by Trypan blue exclusion assay, with viable neutrophils >98%.Neutrophils were obtained from whole blood of healthy human donors. Briefly, neutrophils were isolated in LPS–free conditions by Dextran sedimentation followed by Ficoll-Hypaque density gradient centrifugation. The lower phase containing neutrophils was resuspended in RPMI  medium, washed and contaminating erythrocytes were removed by hypotonic lysis. The resulting cell population contained greater than 95% mature neutrophils as assessed by flow cytometric analysis with BD FACScalibur  using a FITC-conjugated mouse monoclonal antibody against CD16b , used at 1 μg/1 × 106/mL) were incubated either with G-CSF or GM-CSF or primed with CSFs for 50 min and then stimulated with 1 μM fMLP. For ROS measurement experiments, DMSO was used as a negative control for fMLP and TNF-α (50 ng/ml) was used as positive control for priming. In some experiments, both CSFs were present together at low concentrations (0.005–0.1 ng/ml) both as direct stimulators or priming agents for fMLP.GM-CSF, G-CSF, TNF-α, and N-formylmethionine-leucyl-phenylalanine (fMLP) were purchased from Sigma-Aldrich . Neutrophils  for 10 min, washed twice in PBS and exposed to 1 μM fMLP. After 10 min, ROS-mediated oxidation of H2DCFH-DA into the highly fluorescent 2′,7′-dichlorofluorescein (DCF) was measured by flow cytometry  within 30 min after termination of stimulation. In total, 10,000 events were recorded per staining. Control neutrophils (i.e. incubated with DMSO) and neutrophils stimulated with fMLP gave an intracellular fluorescence (MFI = mean fluorescence intensity) of 160.5 ± 14.4 and 207.0 ± 17.1 , respectively. MFI obtained with study samples was calculated as % of controls mean .Neutrophils were either unstimulated or stimulated with CSFs, incubated with 2 μM H6 cells/ml) were incubated with G-CSF or GM-CSF  for 50 min and evaluated either unstimulated or stimulated with 1 μM fMLP for 5 min. Supernatants were snap-frozen at −80 °C. The release of granule proteins  were assessed by enzyme-linked immunosorbent assay (R&D System). As an alternative approach to degranulation of secondary granules, the membrane expression of CD66b was investigated. Neutrophils were fixed in 4% (w/v) paraformaldehyde following stimulation and analyzed by flow cytometry  using a fluorescein isothiocyanate (FITC)-labeled mouse anti-human CD66b antibody  used according to manufacturer’s instructions. An irrelevant FITC-conjugated mouse IgG1 was used as negative control. In total, 10,000 events were recorded per staining. Data were expressed as MFI.Neutrophils (7 × 106 per filter) were added in the upper compartment of a Transwell insert , while in the lower compartment, 1 μM fMLP, GM-CSF or G-CSF was added. After  2.5 h, neutrophils that crossed the insert were recovered in the lower compartment and quantified by flow cytometry , and data were expressed as percentage of total neutrophils added in the upper compartment. In some experiments, neutrophils were pre-incubated with either CSF for 50 min, added to the upper compartment and allowed to migrate along a fMLP gradient for 2.5 h.Neutrophils , the differences were computed by parametric and non parametric tests, respectively. Clinical parameters, CSFs levels in serum and sputum, and ROS levels showed a non normal distribution, thus Kruskall-Wallis analysis of variance (with Dunn’s test as post hoc test) and Mann-Whitney U-test for pairwise comparisons were used. Degranulation and chemotaxis data presented a normal distribution, hence we used ANOVA with Tukey’s post hoc test and Student t-test for pairwise comparisons. Correlation analysis was carried out by computing Spearman’s rank correlation coefficients. The difference was considered significant when P < 0.05. The analysis was carried out using the software Prism 4 .Supplementary Material


Delirium is a marker of brain vulnerability, associated with increasing age, pre-existing cognitive impairment and, recently, cerebrospinal fluid (CSF) biomarkers of Alzheimer’s disease. This nested case-control study used a targeted quantitative metabolomic methodology to profile the preoperative CSF of patients (n = 54) who developed delirium following arthroplasty (n = 28) and those who did not (n = 26). The aim was to identify novel preoperative markers of delirium, and to assess potential correlations with clinical data. Participants without a diagnosis of dementia (≥65 years) undergoing elective primary hip or knee arthroplasty were postoperatively assessed for delirium once-daily for three days. Groups were compared using multivariate, univariate and receiving operator characteristic (ROC) methods. Multivariate modelling using Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) of metabolomic data readily distinguished between delirium and control groups . Three metabolites  significantly differed between groups , and performed well as CSF biomarkers (ROC > 0.75). The biomarker performance of the two polyamines (spermidine/putrescine) was enhanced by ratio with CSF Aβ42 (ROC > 0.8), and spermidine significantly correlated with Aβ42 . These findings suggest that spermidine and putrescine levels could be useful markers of postoperative delirium risk, particularly when combined with Aβ42, and this requires further investigation. It is associated with significant negative outcomes including dementia, institutionalisation and death3. Postoperative delirium is common, developing after 17% of planned joint replacement surgeries4. Delirium is a marker of brain vulnerability, associated with increasing age, pre-existing cognitive impairment6 and, recently, cerebrospinal fluid (CSF) biomarkers of Alzheimer’s disease (AD)7. Whilst the pathophysiological basis for delirium remains unclear, it is thought to represent, at least partly, an aberrant stress response8.Delirium is an acute confusional state10. As metabolomics directly measures chemical processes involving metabolites, it holds huge potential as a discovery platform for identifying novel diagnostic biomarkers and elucidating disease pathogenesis. The metabolomic analysis of brain tissue, CSF and plasma has advanced our understanding of the processes leading ultimately to dementia13.Metabolomics is a relatively new technique which can comprehensively and simultaneously detect disturbances in the metabolome in a high-throughput manner14 and one utilising both blood and CSF15. Guo et al. reported multiple preoperative metabolic pathways disturbance in postoperative delirium groups including deficiency of ω3 and ω6 fatty acids, energy metabolism and oxidative stress with interactions between hypoxia and mitochondrial dysfunction, as well as disorders in the Glu–Gln cycle14. Watne et al. applied high performance liquid chromatography (HPLC) to measure amino acids and monoamine metabolites in CSF and serum and found increased levels of methionine, phenylalanine, tryptophan and tyrosine in fracture patients who went on to develop postoperative delirium15. Fracture patients are older and sicker at baseline17, have by definition already experienced trauma and often have preoperative delirium18. Elective orthopaedic populations, by contrast, permit comprehensive preoperative and premorbid cognitive assessment and CSF sampling at a clinically homogenous point, whilst still seeing a significant incidence of delirium.Two previous studies have investigated the alteration of metabolites to samples from delirium and no delirium groups in a fracture population, one studying plasma samplesThe aim of the current nested case-control study was to employ a targeted quantitative metabolomic methodology to profile the CSF metabolome of patients who developed delirium following arthroplasty compared to those who did not. The aim was to test the hypothesis that CSF metabolomic profiles differ in those who developed delirium following arthroplasty from those who did not.Participants aged 65 years or older admitted for primary elective hip or knee arthroplasty to a single surgical centre and planned to undergo spinal anaesthesia with intrathecal diamorphine analgesia were eligible for inclusion. Exclusion criteria included a pre-existing diagnosis of dementia or other neurodegenerative condition. Between March 2012 and October 2014, 315 participants completed the study. Average age was 74.4 years and 57% were female. American Society of Anesthesiologists (ASA) physical status classification was available for 53 of the 54 participants. Of these, one participant was ASA 1, 43 were ASA 2 and nine were ASA 3. The incidence of postoperative delirium in our cohort was 44/315 (14%).19, level of education, estimated IQ20 and the Vertical Visual Analogue Pain Scores21 were collected preoperatively and included in analyses. Baseline neuropsychological tests included Colour Trails 222 and Mini Mental State Examination23.Study methods are summarised in Fig. 24 and daily assessments were supplemented by post-discharge reviews of the nursing and medical notes. In all cases where there were reports of possible delirium but CAM criteria were not fulfilled, cases were then discussed between ELC and APP and a consensus reached as to whether they reflected delirium according to the Diagnostic and Statistical Manual of Mental Disorders (DSM) IV criteria25.Postoperatively participants were assessed for delirium once daily by a single researcher for the first three days. Delirium was diagnosed using the Confusion Assessment Method (CAM)Venous blood was sampled preoperatively into PAXgene Blood DNA tubes . All samples were transported to the laboratory within 12 hours of collection.DNA samples were processed according to the manufacturer’s instructions .APOE status was inferred from the genotype at each of the two alleles, rs7412 and rs42935826.DNA was analysed using the TaqMan Single Nucleotide Polymorphism Genotyping Assay  as per the manufacturer’s instructions. Spinal anaesthesia was carried out, fasting, in the sitting or lateral positions using 25 gauge  Whitacre type spinal needles with graduated metal introducers (Vygon). Once CSF was obtained, a 5 ml syringe  was attached to the spinal needle and up to 5 mls of CSF withdrawn by the anaesthetist. The CSF was immediately transferred to a 30 ml sterile polypropylene universal container (Unisurge) which was placed on wet ice in an insulated container.g for 5 minutes with no brake and 500 mcl aliquots of the supernatant pipetted  into 1.5 ml polypropylene tubes  and stored at −80 °C.All samples were transported to the laboratory within 12 hours of collection. CSF samples were centrifuged at 4 °C at 3000 rpm/1811 27.CSF samples were transported to the Leonard Wolfson Biomarker Laboratory, University College London on dry ice. There Aβ42, T-tau and P-tau were analysed by a single, trained, technician, blinded to the clinical data, using the INNOTEST β-amyloid (1–42), hTau Ag and Phospho-tau (181 P) ELISA kits respectively  run on a FLUOstar Omega BMG LABTECH instrument (chemiluminescent plate reader) according to the manufacturer’s protocols. Samples were thawed at 21 °C in an air-conditioned lab and vortexed prior to use. CSF biomarker values were determined using Omega software (version 5.10 R2) and a 4-parameter curve fit. Intra- and inter-assay CVs, respectively, were 2.0% and 3.9% for Aβ42, 3.3% and 8.1% for T-tau and 1.6% and 5.1% for P-tau. Longitudinal stability in the measurements is further monitored in the laboratory by participation in the Alzheimer’s Association Quality Control program29 and analysed on a triple-quadrupole mass spectrometer . The data were recorded in a 96-well format and seven calibration standards were integrated in the kit. Metabolites (amino acids and biogenic amines) were derivatised using phenylisothiocyanate (PITC) in the presence of isotopically labelled internal standards, separated using a UPLC  system with a reverse phase column  and quantified using a triple-quadrupole mass spectrometer  operating in the multiple reaction monitoring (MRM) mode. All the remaining metabolites  were quantified using the same mass spectrometer without column separation by the flow injection analysis (FIA) operating in MRM mode. Metabolite concentrations were calculated and expressed as micromolar (µM). The mean of the coefficient of variation (CV) for the 180 metabolites in repeated quality controls was 0.12, and 85% of the metabolites had a CV of <0.15.Quantitative mass spectrometry-based metabolomic profiling was performed using the Biocrates AbsoluteIDQ p180 . All sample preparatory steps were carried out according to the manufacturer’s instructionsAPOE ε4 positive and negative groups based on an estimated APOE ε4 carrier rate of 25.5%30 and a rate of delirium in the APOE ε4-positive and APOE ε4-negative groups of 28% and 11%, respectively, in an elective non-cardiac surgery population31. It was estimated that 316 participants would be required to enable detection of significant difference, at the 5% significance level, with 90% power.The observational cohort study was powered to detect a difference in the risk of postoperative delirium between the 7. Delirium and control groups were matched for age and gender. Initially two groups of n = 27 each were identified but when additional medical notes became available and were reviewed one participant was reclassified as having had delirium using the methods outlined above, leaving n = 28 cases and n = 26 controls.Participants, with and without postoperative delirium, with sufficient CSF available to facilitate metabolomic analysis were identified. Samples for this study were selected primarily based on CSF availability. Availability of CSF was not associated with delirium in this cohortet al. 2017, Cunningham et al. 2018) were included in the analyses. Data for perioperative intravenous opioid use was insufficient and this was therefore excluded.Preoperative clinical variables previously shown to be associated with delirium in our cohort  plots was created to Identify the top metabolites responsible for discriminating between groups. The normality of data were tested using SPSS (version 24). Student’s t-test for metabolites exhibiting a normal distribution or the Wilcoxon Mann-Whitney test for metabolites exhibiting non-normal distributions, and false discovery rates  were performed to determine if metabolites were statistically different between two groups  . The FDR adjustment of P-values for multiple comparisons within Metaboanalyst applies the Benjamini & Hochberg correction method33. In order to assess and compare the biomarker performance of metabolites and/or Aβ42 receiver operating characteristic (ROC) curves were constructed . This initially examined individual concentration data and subsequently assessed metabolite/Aβ42 ratios as a method of enhancing overall biomarker performance.Concentration data for 187 metabolites were appropriately reformatted and exported to Simca 15  for multivariate analysis. Data were grouped into delirium and control prior to analysis by principal component analysis (PCA) identifying any potential outliers, followed by partial least squares-discriminant analysis (PLS-DA). The validity of the model was evaluated based on the residuals  and the model predictive ability parameter (Q2) determined through the default leave-1/7th-out cross validation. The number of components for the PLS-DA model was optimized using y-table permutation testing (n = 500) and an ANOVA based on the cross-validated predictive residuals (CV-ANOVA)The baseline characteristics of the delirium and control groups, matched for age and gender, are shown in Table −3. The PLS-DA loading plots showed the most influential variables that led to the separation between two groups. Combining metabolomic and clinical data improved the predictive ability  for discrimination between two groups. The model was also validated using a permutation analysis  predicts postoperative delirium. Amyloid pathology causes impaired synaptic function and ultimately damages neurons, resulting in cognitive impairment and finally dementia43; and likely therefore increases vulnerability to the effects of stressors, including opiates, pain, and inflammatory changes seen in surgery44, which may result in delirium.The elevation of polyamine pathway metabolites has previously been reported in brain tissue, plasma and CSF from AD patients17, have by definition already experienced trauma and often have preoperative delirium18. The incidence of delirium is sufficiently high to study this condition4, but low enough to likely indicate an underlying vulnerability. Although the findings of this nested case-control study are based on a relatively small sample and are not immediately translational, the key strengths include the use of fasting samples , the detailed clinical information, and the annotated and quantitative metabolomic data.Understanding the biochemical basis for the development of postoperative delirium is a necessary first step if we are to intervene to reduce the incidence of this common, unpleasant and potentially dangerous syndrome. Identifying the pathways responsible may also enhance our understanding of neurodegeneration which predisposes to delirium. The study of elective orthopaedic populations permits comprehensive preoperative and premorbid cognitive assessment in contrast to research undertaken in fracture populations when participants are older and sicker at baselineper se. Replication of these findings, in both delirium and AD cohorts, is now necessary.This study indicates the benefits of applying metabolomic methodologies to conditions such as delirium to determine susceptibility risk: our findings suggest that CSF levels of spermidine, putrescine, and their metabolite precursor glutamine, could be useful markers of postoperative delirium risk, particularly when combined with Aβ42. The development of rapid and inexpensive methods of measuring spermidine and putrescine is needed to evaluate their utility in larger prospective patient cohorts, and to study whether CSF amines/polyamines are markers of underlying neurodegeneration , or for delirium Cerebrospinal Fluid Spermidine, Glutamine and Putrescine Predict Postoperative Delirium Following Elective Orthopaedic Surgery


Ventriculoperitoneal (VP) shunting is a permanent form of cerebrospinal fluid (CSF) diversion that can be performed for hydrocephalus. Sterility of the CSF is an important prerequisite for permanent shunt placement. It has been hypothesised that in early stage of meningitis, ventricular CSF remains sterile. A study is conducted on the first CSF sample taken from patients suspected to have meningitic hydrocephalus.A retrospective review case records of patients who had undergone external ventricular drainage (EVD) for suspected meningitic hydropcephalus in Hospital Sultanah Aminah Johor Bahru (HSAJB), Johor, Malaysia.P = 0.001), CSF protein , CSF gram stain  and CSF appearance . Multivariate analysis had proven that gram stain was the main parameter in the CSF analysis . No significant differences in CSF results were observed from EVD and lumbar puncture.Fifty-one cases were analysed. Mean age of patients was 37.27 years old, with 64.7% of them was male. Univariate analysis revealed that the main parameters to determine CSF sterility were CSF glucose (95% CI, 0.852, 10.290, The most significant parameter in CSF to determine infection was gram stain. Meningitis refers to the inflammation of the membranes (meninges) surrounding the brain and spinal cord . The incClinically, meningitis is diagnosed using the classic triad of neck stiffness, fever, and altered consciousness. However, any two of these three symptoms are more common and are exhibited by up to 95% of meningitis patients . In addiThe pathogenesis of bacterial meningitis is not yet completely understood. However, there are four main pathogenic processes: colonisation, invasion into the bloodstream, survival in the bloodstream, and entry into the subarachnoid space. The subsequent damage occurs due to interaction between bacterial and host factors. Invasion of the bloodstream refers to both passing through or between the cells. Survival in the bloodstream requires evasion of the immune system. Meningitis, in the majority of patients, occurs following bacteraemia; however, the diagnosis of sinusitis and otitis media suggest direct spread to the central nervous system.3, ratio of CSF glucose levels to blood glucose levels < 0.23, protein level > 80 mg/dL, presence of Gram-positive bacteria, and other clinical manifestations that are compatible with meningitis  is the gold standard among the diagnostic techniques employed for meningitis. High proportion of white blood cells (WBCs) indicates inflammation of the meninges; however, some meningitis patients exhibit normal level of WBCs. Such patients exhibit poor prognosis . Upon CSningitis . The conThere are several complications related to meningitis such as hydrocephalus, subdural collection, infarction cerebritis, ventriculitis, and brain abscess .Hydrocephalus is most likely associated with an increased fatality. Radiological assessment for hydrocephalus includes ventricular enlargement with CSF seepage, effacement of sulci and gyri, ballooning of third ventricle, temporal horn dilatation > 3 mm, and bicaudate index (BCI). BCI is the width of the frontal horn at the level of caudate nuclei and foramen of Monroe divided by the corresponding diameter of the brain. Hydrocephalus is diagnosed when relative BCI index is more than 95% percentile of age.Ventriculitis refers to the inflammation of the ventricles in the brain, and it is one of the complications of meningitis. The ventricles are responsible for the circulation of CSF throughout the brain. Ventriculitis is caused by infection in the ventricles, leading to swelling and inflammation .When a patient is diagnosed with both meningitis and hydrocephalus, CSF diversion needs to be performed to maintain normal pressure in the brain. The best approach for CSF diversion is the external ventricular drainage (EVD) . EVD is In this study, the first CSF sample taken from EVD was analysed, from patients who diagnosed with meningitis along with hydrocephalus. The aim of this study was to determine the CSF status of first EVD sample after the diagnosis of the disease. To prove the hypothesis that the CSF sample from ventricle was sterile, it was also noteworthy to know how many patients eventually ended up with internalisation.A retrospective study was conducted on 51 patients who had undergone EVD insertions for treatment of obstructive hydrocephalus due to meningitis in Hospital Sultanah Aminah Johor Bahru (HSAJB), Johor, Malaysia during the period of January 2012 to December 2016. Convenient sampling method was employed in this study. The inclusion criteria included all meningitis patients who underwent EVD insertion and from whom the CSF sample was obtained from the surgery site. Meanwhile, the exclusion criteria included patients with ventriculitis, intracranial abscess, and other space occupying lesions, and from whom the CSF samples were obtained from lumbar puncture. Meningitis was diagnosed clinically based on clinical triad: headache, neck stiffness, and reduced consciousness. The radiological criteria were signified by the roundedness of the anterior horn, the ballooning of the third ventricle, the effacement of sulci and gyri, and lucent periventricular without meningeal enhancement . The priP < 0.05. Comparison between CSF status and number of shunted patients is presented in Sex, CSF appearance, CSF protein, CSF glucose, CSF Gram stain status, WBC proportion and status of patient (either shunted or not) were collected to describe the variability of the study population. Data entry and analysis were done using SPSS programme for Windows version 22.0. The demography is presented in tabular form using the mean and standard deviation (SD) for numerical variables and numbers and percentages for categorical variables. Outcome predictors were analysed using simple and multiple logistic regression analyses so as to provide an odd ratio. The statistical significance was defined at The data was obtained from the medical reports of a centre and we faced some difficulties in its assessment, especially when the patient was discharged from tertiary centre and subsequently underwent medical treatment in other centres.Data was obtained from 51 meningitis patients that underwent EVD surgery at HSAJB, Johor, Malaysia during 2012 to 2017. The mean age of the patients was 37.27 years. Ten patients suffered from hydrocephalus as indicated by tumour and abscess and five patients exhibited ventriculitis as seen by CT contrast. Four patients were of young age (paediatric age group) and were, thus, were excluded.The outcome cross tabulation is depicted in P < 0.005). Thus, CSF Gram staining status was the most important parameter associated with CSF culture.In Meningitis is characterised by fever, headache and meningismus with inflammation in the subarachnoid space as revealed by CSF pleocytosis. The time to presentation  and degree of illness differ based on the aetiology and appropriate initial management and treatment. Acute meningitis manifests within a few hours to several days. It is often infectious with a bacterial or viral causal factor, along with non-infectious aetiologies as revealed in the differential diagnoses .In this study, only the first CSF samples obtained during EVD were analysed. Meningitis was diagnosed by evaluating the presence of clinical symptoms of meningitis, namely headache, fever and reduced consciousness level. CT scan of the brain was done, and hydrocephalus that needed intervention was noted. The EVD was performed to release the intracranial pressure due to hydrocephalus, as well as for CSF sampling. The first CSF sample taken during EVD was then analysed. Males exhibited higher tendency of developing meningitis along with hydrocephalus, as they were sexually active, busy working, and travelling, which can make them more susceptible to infection . Male pa3 or > 400 RBC per mm3) and/or bacteria, and therefore, might suggest bacterial meningitis  had normal levels CSF protein. High CSF protein level (>80 mg/dL) is one of the predictors of bacterial infection . HoweverStreptococcus pneumonia and Hemophilus influenza . Some patients came after few days of infection, from district hospitals, after completion of a course of antibiotics. Further investigation showed that their CSF was sterile. The most common pathogen were nfluenza . This renfluenza .Normal CSF glucose is 60%–70% of the patient’s serum glucose concentration. CSF glucose levels are used to discriminate bacterial meningitis from viral meningitis . PatientThe level of WBCs in the blood is another parameter that needs to be considered for meningitis diagnosis. High levels of WBCs indicated an inflammatory process in the blood, which could be due to infection. From this study, the proportions of patients having normal and abnormal levels of WBCs were comparable. If WBC level is high, further investigation must be carried out before shunting procedure is carried out . It shouGram staining status of CSF was the most useful factor to determine whether the cause of meningitis was bacterial; 89% of bacterial meningitis patients exhibited a positive initial Gram stain. In this study, 54.9% patients had sterile CSF exhibiting negative Gram staining. Only 3.97% patients had sterile CSF with positive Gram staining and 29.4% patients had infected CSF with negative Gram staining. Gram staining differentiates bacteria by the chemical and physical properties. Only the bacteria having peptidoglycan in their cell walls are presented as Gram-positive bacteria. In early acute meningitis, it is important to know which microorganism has infected CSF so as to devise a management approach accordingly  in indicating CSF infection. Hyponatremia, combined with a negative Gram stain, was highly suggestive of tuberculous meningitis. However, in this study, the natrium level was not analysed. The inflammation and the protein level obtained from pathogen was believed to be the cause of increased protein levels in the CSF and the inflammatory processes. The results of CSF culture were positive in 70%–85% patients who had not received any prior antimicrobial therapy. Therefore, since most of the patients had a negative Gram staining result and a clear CSF, the majority of patients with meningitis had sterile CSF.As shown in In this study, the CSF culture status was also analysed based on the first CSF sample, and its association with the VP shunt was obtained. Twenty-three out of 51 (43.5 %) patients with sterile CSF ended up with shunts. Ten patients with infected CSF were shunted, mainly after completion of the antibiotics course. The rest of the patients were not shunted. Seven patients with clear CSF were not shunted because of multiple comorbidities, such as fragility and sepsis, and patients getting better with good medical management. Most of the remaining patients with infected CSF and those that were not shunted died due to medical problems. From this table, no conclusion could be derived.P-value of 0.039, showing significant association between CSF culture and VP shunt. Shunting only can be done on patient with sterile CSF.The association between CSF culture and VP shunt was also analysed. From the univariate logistic regression study, the outcome was positive, showing a Analysis of the first CSF sample could help in devising of better approach for management of meningitis patients. Based on this study, high protein levels (> 0.8) showed that the CSF was not sterile. The most important parameter to determine CSF status was the CSF gram staining status. Most patients exhibited normal parameters, and more than 60% of the patients had sterile CSF. Half of the patients ended up with shunting, which showed that most of them were diagnosed with meningitis, requiring a permanent diversion of CSF. However, it was not possible to confirm that the first sample of CSF obtained from a meningitis patient was sterile.


Seven days of actigraphy were collected to assess habitual total sleep time.One-night in-lab nocturnal polysomnography (NPSG) and morning to early afternoon CSF collection were performed to measure CSF Aβ42, P-tau and T-tau. From the three, CSF T-tau was the most significantly associated with spindle density, after adjusting for age, sex and ApoE4. Spindle duration, count and fast spindle density were also negatively correlated with T-tau levels. Sleep duration and other measures of sleep quality were not correlated with spindle characteristics and did not modify the associations between sleep spindle characteristics and the CSF biomarkers of AD.Spindle density during NREM stage 2 (N2) sleep was negatively correlated with CSF AβReduced spindles during N2 sleep may represent an early dysfunction related to tau, possibly reflecting axonal damage or altered neuronal tau secretion, rendering it a potentially novel biomarker for early neuronal dysfunction. Given their putative role in memory consolidation and neuroplasticity, sleep spindles may represent a mechanism by which tau impairs memory consolidation, as well as a possible target for therapeutic interventions in cognitive decline.The online version of this article (10.1186/s13024-019-0309-5) contains supplementary material, which is available to authorized users. With 10% of adults over the age of 65 suffering from dementia  and thisCortical sleep spindles are 11–16 Hz bursts of activity generated within the thalamo-cortical network that occur during N2–3 and have been defined as slow or fast based on their spectral frequency . A decreFifty subjects were recruited from a pool of healthy elderly participating in NIH-supported longitudinal studies on normal aging and biomarkers of AD at NYU. All subjects had ≥12 years of education, received the standardized Uniform Data Set II diagnostic assessment , and wer42) were measured using enzyme-linked immunosorbent assays  conducted at the Sahlgrenska University Hospital (Sweden) blind to both clinical and sleep data.Procedures for the lumbar punctures (LP) performed at NYU have been previously published , 42. BriSleep recordings were performed following American Academy of Sleep Medicine (AASM) guidelines . BrieflyWrist-based actigraph  data was collected to measure at home objective measures of TST. Subjects wore the actigraph for seven consecutive days on the non-dominant hand and maintained sleep logs to help confirm actigraphy measures.p < 0.001; WASO and SE: r = 0.931, p < 0.001; AHI4% and AHI-all: r = 0.709, p < 0.001; AHI-all and arousal index: r = 0.754, p < 0.001) were not included in the same regression models. Three predefined models of adjustment were used in which spindle density in N2 was the response variable. Model 1 included age, sex and ApoE4 status as predictors. Model 2 added each CSF biomarker individually as a predictor variable to age, sex, and ApoE4 status. Model 3 included combinations of CSF biomarkers (i.e. CSF Aβ42 and T-tau or Aβ42 and P-tau) along with age, sex, and ApoE4 status. After establishing T-tau as the strongest CSF predictor of spindle density, on a final analysis we assessed the extent to which spindle density best predicted variance in CSF T-tau (Models 4 and 5) or the CSF T-tau/Aβ42 ratio (Models 6 and 7) among several alternative sleep physiology variables by performing hierarchical regression with CSF T-tau or the CSF T-tau/Aβ42 ratio as the response variable. Models 4 and 6 included age, sex and ApoE4 status as predictors. Models 5 and 7 added individual sleep measures as predictors including N2 spindle density, frontal SWA, WASO, SE, AHI4%, AHI-all, in-lab TST, and habitual TST. All statistical analyses were performed with SPSS version 23.0 . Statistical significance was set at p < 0.05 using two-tailed tests.CSF and spindle properties were not normally distributed (Shapiro-Wilk test), therefore Spearman correlations were performed for bivariate analysis. Although correlations do not imply directionality, in order to better understand how individual CSF AD biomarkers could be influencing spindle density, we performed linear regression analyses where spindle density was the response variable. To restore normality for regression analyses, a square root transformation was applied to spindle density in N2, spindle count in N2 and fast spindle density in N2, while natural log transformations were applied to WASO, AHI4%, AHI-all, arousal index and CSF variables. Box-Cox transformation was applied to spindle duration and SE. Before conducting the regression analyses, we checked for inter-correlations between all the above variables. A correlation matrix was computed on transformed variables using Pearson’s correlations. Variables that inter-correlated with a value higher than 0.6  of 0, mean Mini-Mental State Examination (MMSE) of 29.1 ± 1.1, 16.7 ± 2.1 years of education, and were generally non-obese (BMI = 25.4 ± 3.5). Thirty-four percent (17 subjects) were ApoE4+. Overall, the cohort consisted of cognitively normal elderly in good health with 4 of 50 that were both amyloid-positive and tau positive , 14 of 50 that were amyloid-negative and tau-positive, 8 of 50 that were amyloid-positive and tau-negative, and 24 of 50 that were both amyloid-negative and tau-negative .A total of 50 subjects  with mean age 67.2 ± 7.3 (range 53–83) participated in this study. Table p < 0.001). N2 spindle density was negatively correlated with all CSF measures, and despite the strong correlation between both CSF tau measures, spindle density was more strongly associated with T-tau than with P-tau. The pairwise correlation coefficients adjusted for age, sex and ApoE4 status, given above the main diagonal, show a similar pattern and indicate that the conditional associations were even stronger than the unadjusted ones. In addition to individual CSF protein levels, the ratios between T-tau/Aβ42 and P-tau/Aβ42 have been used as AD biomarkers. Spindle density during N2 sleep was also found to be negatively correlated with the ratio of T-tau/Aβ42  and P-tau/Aβ42 .Pairwise unadjusted and conditional  correlations between the CSF biomarkers of AD and N2 sleep spindles density are presented in Table 42 by 8.8%, P-tau by 18.1%, T-tau by 25.9%, and the T-tau/Aβ42 ratio by 12.9% . We did not observe any correlations between T-tau or P-tau and any of these measures at baseline =3.77, p = 0.010), 45% of the variance in spindle duration =9.23, p < 0.001), and 41% of the variance in fast spindle density =7.90, p < 0.001)  [42 was inversely correlated with frontal SWA . In contrast, while we observed an unadjusted correlation between frontal SWA and T-tau, frontal SWA was not correlated with either T-tau or P-tau  . ConsistBoth levels of tau in the brain and sleep spindle density have been associated with measures of cognition. In particular, sleep spindle density has shown to be correlated with the Bells Test, Connors Continuous Performance test, and Auditory Verbal Learning Test when completed the morning following sleep testing . In the 42 in a population of older cognitively normal adults. Spindle density in N2 sleep was significantly correlated with CSF T-tau, P-tau and Aβ42 both unadjusted and after adjustment for age, sex, and ApoE4. Nonetheless, because of the inter-correlations between CSF T-tau, P-tau and Aβ42, we posit that the association between Aβ42 and spindle density might be largely driven by the primary association of CSF tau with spindle density. This was supported by the loss of significant associations between CSF Aβ42 and spindle density when CSF T-tau or P-tau was included as a covariate.Growing evidence suggests that disruptions in the sleep-wake cycle may increase AD risk prior to clinical symptoms. While attenuated SWS has been shown to be associated with elevated CSF Aβ , 13 as w42 is also reflective of increased AD risk, given that CSF Aβ42 is lower in bonafide AD dementia subjects when compared to age matched controls [42 changes over time, particularly in cognitively normal individuals who may be remote from developing symptoms remains a matter of some debate. For example, individuals with dominantly inherited AD have higher concentrations of CSF Aβ42 on average than controls 20 or more years from estimated symptom onset that then decline more rapidly [42 which were largely observed in younger age quartiles (between 45 and 70 years of age), indicating that elevated CSF Aβ42 might also be associated with very early preclinical AD risk [42 in isolation, we cannot rule out the possibility that low spindle density being associated with elevated Aβ42 might be protective. However, it is worth highlighting that spindle density was also significantly inversely correlated with CSF T-tau/Aβ42 and P-tau/Aβ42 which is meaningful because these CSF ratios have predicted decline from normal cognition in cognitively normal older adults over a 1–8 year follow-up period [We interpret the association of low spindle density with elevated CSF T-tau and P-tau as representative of increased AD risk. It is less clear that the association of low spindle density with elevated CSF Aβcontrols . That sa rapidly , 54. Add AD risk . When vip period .42, however, SWA, SE, WASO and number of arousals during sleep were not correlated with CSF tau proteins in this sample.In addition to total spindle density, we also found evidence that spindle duration and fast spindle density during N2 sleep are significantly associated with CSF T-tau. Of note, a decrement in total spindle density and fast spindle density has also been observed in AD patients, who presumably have high brain tau load . When ev42 and P-tau/Aβ42 ratios [This seeming specificity of CSF T-tau for spindles stands in potential contrast to recent findings suggesting that measures of subjective sleep quality by questionnaire were negatively correlated with both CSF T-tau/Aβ2 ratios . This ma2 ratios , OSA [602 ratios  and arou2 ratios  increaseWhile any correlation can represent an epiphenomenon, the observed associations between sleep spindles and CSF T-tau warrant thinking about possible mechanistic underpinnings that link the two. One possibility to consider is that accumulating tau negatively impacts the generation of spindles. Spindles are thought to largely evolve from a thalamo-cortical feedback loop involving thalamic reticular neuron inhibition of thalamic relay neurons. Tau is not thought to accumulate in thalamic reticular neurons, but tau in cortical neurons could affect this circuit. Tau in brainstem neurons projecting to thalamic reticular neurons could be involved –65, but The converse possibility is that sleep spindles reduce the production or increase the metabolism of tau. Animal studies have shown that neuronal activity can promote tau production , 67, howFinally, spindle density and CSF tau levels could be associated by being common downstream consequences of an upstream process. For example, tau is released in response to axonal injuries (often from traumatic brain injury) at the time of injury and before frank evidence of neurodegeneration is present. Because we observed associations of sleep spindles with both CSF T-tau and P-tau, we cannot completely infer whether any association is more strongly tied to axonal injury versus formation of neurofibrillary tangles. In subjects with moderate to severe diffuse axonal injury, sleep spindle amplitude and peak frequency were significantly reduced acutely with subsequent return to baseline levels commensurate with functional improvement over 1 year . Tauopat42 [42 or tau to 8% that have presence of both CSF Aβ42 and tau above biomarker cutoffs. Therefore caution is warranted in interpreting the prognostic value of sleep spindle density, and studies tracking how sleep spindles change longitudinally along the progression of AD should be insightful.Limitations of this work include the fact that the NPSG can itself alter sleep quality  in ways 42 . Finally42 , our coh42. Further, sleep quality measures were not correlated with these CSF measures. These results indicate that poor sleep quality alone does not reduce spindle occurrence and is unlikely to be an explanatory factor in the observed associations between spindle density and levels of CSF tau. While reductions in NREM sleep oscillations, including SWS and spindles, may reflect a preclinical AD state, it remains unclear just how these attenuated oscillations are mechanistically linked to the cellular processes regulating Aβ and tau. Candidate mechanisms would include patterned activity-driven synthesis or degradation of such molecules or the coupling of such oscillations to neuro-immune function. Longitudinal and interventional studies to assess changes in sleep oscillations as well as AD biomarkers are needed.In conclusion, we report that spindles during N2 sleep are negatively correlated with CSF T-tau levels and to a lesser degree to CSF P-tau and CSF AβAdditional file 1:Figure S1.Distribution of CSF biomarkers in this cohort. Histograms of CSF Aβ42 (A), T-tau (B), P-tau181 (C) in our cohort of 50 cognitively normal elderly. Figure S2.Sleep quality measures are not associated with either CSF T-tau at cross section. Scatter plots of WASO (min.) (A), Sleep efficiency (%) (B), Arousal Index (#/min. of sleep) (C) or total sleep time  (D) with CSF T-tau show no relationship at cross-section. (PDF 1529 kb)Additional file 2:Table S1. Hierarchical linear regression examining CSF T-tau/Aβ42 ratio as a function of sleep measures (DOCX 15 kb)Additional file 3:Table S2. Hierarchical linear regression examining CSF T-tau as a function of N2 spindle density with comorbidities (DOCX 15 kb)Additional file 4:Table S3. Linear regression examining spindle properties as a function of CSF T-tau (DOCX 15 kb)Additional file 5:Table S4. Correlations between neurocognitive tasks and spindle densities (DOCX 15 kb)


To investigate the relationship between cerebrospinal fluid (CSF) β-amyloid peptide (Aβ42) and CSF Tau in a large population of patients referred to memory clinics for investigation of cognitive dysfunction.We analyzed Alzheimer’s disease (AD) biomarkers in CSF taken from 3565 patients referred to 18 French memory clinics. Patients were classified into four profiles according to levels of CSF biomarkers . The association between CSF Tau and CSF Aβ42 were analyzed using general linear regression models, in the overall population and stratified by biomarkers profiles. We compared linear and quadratic models using Akaike information criterion. We also assessed change in biomarker profiles in a subset of patients who had 2 assessments of biomarkers.CSF Tau was negatively associated with CSF Aβ42 in the overall population, following a non-linear quadratic model. However, the nature of this association was different in the 4 profiles: positive association in A-N- profile, negative association in A-N+ and A+N+ profiles, lack of association in A+N- patients. When considering patients with longitudinal data on profiles, 36% of those initially classified as A-N+ evolved to an A+N+ profile.The nature of the association between CSF Aβ42 and CFS Tau depends on the A/N profiles of patients. These results suggest an increase in CSF Aβ42 early in the disease before its decline while tau pathology progresses, this pattern is particularly observed in non-APOE4 subjects. This phenomenon may explain why some patients with neurodegeneration only markers convert to an AD profile (A+N+) over time. Alzheimer’s disease (AD), the most common cause of dementia, is neuropathologically characterized by extracellular β-amyloid peptide Aβ42) deposits, neurofibrillary tangles composed of intraneuronal abnormally phosphorylated Tau, and neuronal and synaptic loss . Accordi depositsThe development of AD biomarkers during the past decade allows new opportunities for studying the in-vivo kinetics of amyloid and tau lesions , 7. CereThe concept of SNAP (A-N+) remains the subject of debate . SeveralCSF allows assessment of both Aβ42 and Tau, the hallmarks of AD, but their relationship remains poorly understood. A recent study based on data on 766 subjects from 3 independent cohorts found a non-linear quadratic association between CSF Tau and Aβ42 . In the st 2008 and January 1st 2018 for whom AD biomarkers from CSF were available were included in the analysis; methods and characteristics of the population have been previously reported elsewhere [Following national recommendations, CSF analyses are routinely undertaken in patients with cognitive signs referred to hospital-based memory centers in France , 24. Forlsewhere , 26. TheLumbar puncture was performed on patients in a fasting state, typically between 9 and 12 am. All centers used the same model of 10 mL polypropylene tube to collect CSF . CSF samples were centrifuged at 1,000 g for 10 minutes at 4°C within 4 hours of collection, and then aliquoted in 0.5 mL polypropylene tubes and stored at -80°C for further analysis. CSF levels of Aβ42, total Tau, and pTau-181 were measured with the commercially available sandwich ELISA INNOTEST, using the manufacturer’s procedures . The Alzheimer’s Association quality control program for CSF biomarkers validated the quality of CSF evaluations of the hospital biochemistry department .The CSF biomarker profiles were used to determine the “profile” of every subject using the NIA-AA staging framework . The amyIn this study we chose not to use the recently proposed A/T/N classification, 28 as wst 2016, requiring adjustment of thresholds at these 2 dates. The cut-offs for CSF Aβ42, Tau and p-Tau 181 moved respectively from 500/300/65 pg/mL [Abnormal values of CSF biomarkers were defined using thresholds used routinely in clinical practice based on higher Youden index in order to discriminate AD patients from non-AD patients (i.e. best compromise for sensitivity and specificity). The type of the polypropylene tube changed in November 2012, and the ELISA INNOTEST sandwich changed on May 165 pg/mL  to 730/365 pg/mL  after thThese included age, sex, and performance on the 30-item Mini Mental State Examination (MMSE) for an assessment of general cognitive status. APOEe4 status was known for a sub-sample of 1034 individuals.2 test or Student t-test as appropriate.Participant characteristics were presented overall and by profiles defined using NIA-AA CSF biomarkers . Proportions were calculated for categorical variables, while means and standard deviations were computed for continuous variables. Differences as a function of profiles were assessed using a χThe association between CSF Tau and Aβ42 was examined using general linear regression models with CSF Aβ42 as the dependent variable (PROC GLM of SAS). The analysis was initially adjusted only for the type of collection tube (unadjusted model), and then further adjusted for age, sex, and APOEe4 status. Analysis were first undertaken in the total sample and then stratified by the 4 profiles . The difference in the association between CSF Tau and Aβ42 in the 4 profiles was tested formally using interaction terms in the linear regression models.2) following the Akaike information criterion (AIC), with lower AIC values designating better model fit [We compared the linear model (Aβ42 = α + β xTau) to the quadratic model (Aβ42 = α + β xTau + γ x Tauodel fit . Linear In the sub-sample with APOEe4 data (N = 1034) we examined the association between CSF Aβ42 and CSF Tau by first plotting the mean values of CSF Aβ42 as a function of deciles of CSF Tau in those with an APOEe4 allele versus none. These associations were also examined in linear regression analysis, adjusted for collection tube, age and sex, in two groups based on median CSF Tau.Finally, in persons with 2 lumbar punctures with CSF AD biomarkers, we examined the evolution of their biomarkers profiles to assess conversion in stage of AD pathology.The CSF biomarkers were not normally distributed and we followed the approach used by other teams by log-transforming CSF Tau and using CSF Aβ42 without any transformation in the statistical models . CSF TauAll p-values were two-tailed and p≤0.05 was considered statistically significant. Statistical analyses were performed using SAS version 9.3 .CSF biomarker data were available on 3565 persons, assessed between January 1st, 2008 and January 1st, 2018 in one of the 18 memory clinics included in the study. Their characteristics are summarized in There was an inverse association between CSF Tau and CSF Aβ42 when the study population was considered together , and then decreased for subjects with higher CSF Tau levels . For APOE4 positive subjects, there was no association between CSF Aβ42 and CSF Tau stable until the 5th decile , and then decreased for those with higher CSF Tau values .d assessment, and 13% of them evolved to A+N+ profile. For the 14 patients initially classified as A-N+ profile, 50% had the same profile and 36% evolved to an A+N+ profile.Finally, We report the following findings on the relationship between CSF Tau and CSF Aβ42 in a large, multicentric cohort of 3565 patients assessed for cognitive disorders:We confirmed the previously reported quadratic relationship  between Further analysis revealed that the association between CSF Tau and CSF Aβ42 is strongly dependant on biomarkers profiles. This association was positive in those with a normal profile, no association was found in amyloidosis only subjects, and an inverse association in those with SNAP and AD profiles. The association between CSF Tau and CSF Aβ42 was linear and not quadratic when examined separately in each profile group.Analysis of change in biomarkers showed that 36% of those who were initially classified as A-N+ evolved to an A+N+ profile, compared to 13% for those who were initially classified as A+N-.We propose a theoretical framework to explain these results which suggest two distinct patterns, illustrated by The kinetics of CSF Aβ42 in very early stages of sporadic AD remains largely unknown. Data from studies on carriers of autosomal-dominant AD mutations,, 33 as wIn the recently proposed biological diagnosis of AD based on the A/T/N classification, A-T+ profiles are considered to be non-AD pathologic change . Our finThis study has several strengths, including its large size which allowed us to cover a wide spectrum of AD pathology, the inclusion of data from 18 memory clinics, and the fact that the analyses of CSF were performed in a single biochemistry department which prevents intersite variability . One of In conclusion, this report describes the relationship between CSF Aβ42 and CSF Tau in a large and multicentric population of patients who were referred to memory clinics for further assessment of cognitive disorders. We found that the nature of the association between these biomarkers was dependent of the biomarker based profile of the patients: positive association in normal profile, negative association in SNAP and AD profiles, lack of association in amyloidosis only subjects. These results plead for an increase of CSF Aβ42 early in the disease, before its decline, especially among non-APOE4 subjects. This pattern may explain why some patients with neurodegeneration only markers convert to AD profiles only over time.S1 TableProfiles are based on CSF Aβ42, CSF Tau, and CSF p-Tau (NIA-AA classification).(DOCX)Click here for additional data file.