There is growing evidence that surgery can drive an inflammatory response in the brain. However, the mechanisms behind this response are incompletely understood. Here, we investigate the hypotheses that 1. Cerebrospinal fluid (CSF) cytokines increase after vascular surgery and 2. That these changes in CSF cytokines are interrelated. Patients undergoing either open or endovascular elective surgery of the thoracic aorta were invited to participate in this study. Cerebrospinal fluid samples were taken before surgery and on the first post-operative day. These were analysed for the presence of ten cytokines by immunoassay to examine for post-operative changes in cytokine levels. After surgery, there were significant increases in six out of the ten measured CSF cytokines . This included changes in both putative pro-inflammatory  and putative anti-inflammatory  cytokines. The greatest increases occurred in IL-6 and IL-8, which showed a 63-fold and a 31-fold increase respectively. There was strong intercorrelation between CSF cytokines after the operation. Following surgery on the thoracic aorta, there was a marked increase in CSF cytokines, consistent with a potential role in neuroinflammation. The ten measured cytokines showed intercorrelation after the operation, indicating that a balance between multiple pro- and anti-inflammatory cytokines may be present. It has been argued that the cytokine mediated neuroinflammatory process could drive peri-operative neurocognitive disorders (PND), such as post-operative delirium (POD), however this is a putative idea which has yet to be proven2. PND is the commonest post-operative complication in older patients undergoing surgery and is associated with worse clinical outcomes3; understanding its pathophysiology is essential for developing prevention and cure.Neuroinflammation is a complex and incompletely understood process which is partly mediated through cytokines4. An intact blood–brain-barrier (BBB) prevents these inflammatory mediators from affecting the closely regulated environment of the CNS. However if the BBB is weakened by aging, neurodegeneration5, or the response to a surgical insult6, inflammatory mediators can enter the CNS. The processes by which the BBB may become weakened are complex, but in Alzheimer’s disease (AD), this includes brain capillary leakage, cellular infiltration, degeneration of pericytes and loss of endothelial integrity5. Once inflammatory mediators enter the CNS, microglial activation occurs, which drives further inflammation, partly through cytokine release8. Cytokine release and the inflammatory response can then trigger neuronal injury and dysfunction 5, which theoretically could manifest as PND. There is currently no treatment for PND9. If the process of neuroinflammation can be better understood, this may open the door to cytokine manipulation, potentially reducing levels of consequent PND.One hypothesis for the aetiology of PND is that a physiological insult from surgery induces the release of local inflammatory mediators (such as cytokines) from macrophages at the surgical site12. This suggests a key role for CSF cytokines in post-operative neuroinflammation. However, the number of patients involved in these studies was small. There is a further suggestion that the CSF cytokines, IL-6 and IL-8, are increased in patients with worse neurocognitive outcomes, but again the sample sizes of these studies are inadequately powered to provide definitive evidence13.Previous investigations on peri-operative cytokine changes has shown greater increases in CSF cytokines than serum or plasma cytokines after an operation14. In this study of 23 patients, the cytokine IL-6 was increased in both serum and CSF after the operation. In patients with spinal cord injury, the CSF IL-6 increase was more marked. While this study examined a wide variety of proteins relevant to neurology, the number of cytokines studied was small.To date, only one study has investigated peri-operative changes in both CSF and serum following vascular surgery on the thoracic aorta15. Draining CSF intraoperatively with a spinal catheter has been shown to reduce the chances of this complication16, by increasing blood flow to the spinal cord17. As such patients already have a spinal catheter in place. This allows for easy access to CSF peri-operatively, making patients undergoing thoracic-vascular surgery a valuable cohort for studying neuroinflammation.Spinal cord injury leading to paraplegia is the most devastating consequence of morbidity following surgery on the thoracic aorta19 in a cohort of patients undergoing vascular surgery. The aim of this study was to evaluate the neuroinflammatory response to vascular surgery in order to:We investigated cytokines with both a pro-inflammatory role  and those with an anti-inflammatory role Determine changes in pro- and anti-inflammatory CSF cytokines before and after vascular surgery.Investigate potential intercorrelations between cytokines in the CSF, to establish whether multiple cytokines are involved in driving neuroinflammation.Establish if the changes in CSF cytokines are reflected in serum cytokine levels. If so, this would enable serum cytokines to be used as biomarkers of CSF changes.This was a prospective observational study looking at peri-operative changes in cytokine levels in the CSF and serum of patients undergoing elective vascular surgery for any pathology of the thoracic aorta.Patients were eligible for recruitment into the study if they were undergoing either open or endovascular repair for any thoracic aortic pathology in which a spinal catheter was to be used as part of standard peri-operative care. The decision about whether a spinal catheter would be used during an operation was a joint decision made pre-operatively in the vascular multidisciplinary team meeting involving the operating surgeon and anaesthetist. Patients did not have neuroimaging performed before or after the operation.Recruitment took place at St Mary’s Hospital, London, over a one-year period. Patients unable to give informed written consent were excluded from the study. The study received approval from the London Westminster Research Ethic Committee (13/LO/0210). The study conformed to the precepts set out in the Declaration of Helsinki of 1975. All patients gave informed, written consent prior to surgery.Study patients had a spinal catheter inserted prior to surgery by an anaesthetist, following administration of a general anaesthetic. The spinal catheter was used to maintain the CSF pressure at 10 mmHg. If the pressure rose above 10 mmHg, CSF was slowly drained until the target was met. CSF was collected by the same operator (author AP) prior to surgery and on the first post-operative day. Approximately 1-3 mL of CSF was collected each time. The spinal catheter was removed when it was no longer clinically indicated. Samples were centrifuged at 4000RPM for 5 min to remove any red blood cells present in the sample. Samples were then pipetted into cryo-tubes in 500 μL aliquots and stored in a −80 °C freezer.19. Analysis followed the manufacturer’s instructions . This method of cytokine measurement in peri-operative CSF and blood samples has been used previously10. It uses electrochemiluminescence to quantify the levels of ten cytokines in 25 μL of peri-operative CSF or blood samples.The V-PLEX Proinflammatory Panel 1 Human Kit  was chosen as it measures both pro-inflammatory  and anti-inflammatory  cytokines20. All cytokine analysis was completed in the Infectious Diseases Laboratory at Imperial College London, London.The range for the lowest level of detection (LLD) was between 0.00861 pg/mL for IL-4 and 0.605 pg/mL for IFN-γ. If samples were above the upper limit of detection (ULD) for the assay, they were diluted in Diluent 2 which was supplied with the kit assay and re-analysed, according to the manufacturer’s instructions. This was necessary for a small number of IL-8 measurements. For cytokine levels below the LLD or detected, but below the fit curve, the LLD value for the cytokine assay was used, as previously describedwww.python.org). A Wilcoxon Signed-Rank test with Bonferroni correction was used to examine changes in cytokines before and after surgery. Spearman’s rank correlation coefficient, again with a Bonferroni correction, assessed cytokine intercorrelation. An adjusted-p value of < 0.05 was considered statistically significant. Patient demographics (age and sex) were assessed for correlation with CSF cytokine changes using Spearman’s rank correlation coefficient with a Bonferroni correction and logistic regression respectively. A power calculation to determine the necessary sample size was not undertaken before starting the study, as the number of patients who would be able to consent and complete the study was anticipated to be small.Analyses were carried out using Python, version 3.7 . The study conformed to the precepts set out in the Declaration of Helsinki of 1975. All patients gave informed, written consent prior to surgery.During a 1-year period, ten patients were recruited into the study. Table All patients had pre-operative (timepoint 1: T1) and post-operative (timepoint 2: T2) CSF samples taken. Five patients also had simultaneous paired serum samples. Patient 5 had their post-operative samples taken on the day of surgery. Supplementary table 1 summarises the aetiology of the thoracic aorta pathology, the type of vascular surgery and the post-operative complications.18.Table The levels of most CSF cytokines tended to rise following surgery. However, levels of IFN-γ and IL-12p70 were generally below the LLD in both the pre-and post-operative CSF samples, so no change could be detected. For the cytokines IL-1β, IL-2 and IL-4, pre-operative levels were often below the LLD, but post-operative levels rose into the detection range.p < 0.001). At T2, there were a number of strong positive correlations between 20 pairs of different cytokines, as shown in Supplementary table 2.Figure p < 0.05). These included IL-2 and IL-1β, IL-4 and IL-12, IFN-γ and Il-2 and IL-1β and IL-1β. At T2 there were no significant correlations between the CSF and serum cytokine levels (data not shown).Only five of the ten patients had serum samples drawn at the same time as the CSF samples. The greatest increase was in IL-6 which showed a 71-fold increase in serum levels after the operation. There was limited intercorrelation between CSF and serum cytokines. At T1, four combinations of CSF and serum cytokines showed significant correlation 27, and dysregulated neurotransmission28.The results of this study need to be interpreted alongside the current postulated mechanisms of brain dysfunction. These include excessive neuroinflammation27 and cytokines29. Microglia are also key drivers of the kynurenine inflammatory pathway, which, in turn, can drive glutamatergic neurotransmission30. In the healthy brain, microglia are fundamental in maintaining tissue homeostasis by removing accumulated debris27. However, their overactivity may be harmful in disease states31.These mechanisms are all mediated through activation of microglia, which when stimulated can release ROS28. Indeed, pro-inflammatory cytokines have been shown to activate the kynurenine pathway, which in turn leads to the generation of ROS through quinolinic acid production32. The cytokine changes demonstrated in this study may therefore have implications for several mechanisms of brain dysfunction.The postulated mechanisms of brain dysfunction are not mutually exclusive, with multiple mechanisms likely to be acting together33. Currently, no effective treatment for PND exists34. The suggestion that CSF IL-6 and IL-8 hold a key role in the post-operative neuroinflammatory pathway, raises the question of whether direct cytokine inhibition could attenuate these effects. However, as we have demonstrated in this paper, multiple cytokines, both pro- and anti-inflammatory, increase after an operation and thus blocking the action of only one of these cytokines may not necessarily inhibit neuroinflammation. Furthermore, as has been suggested in Alzheimer’s disease, a degree of cytokine-driven neuroinflammation may be neuroprotective36.In contrast to neurodegenerative processes, peri-operative brain dysfunction occurs at a predictable time point, giving a potential opportunity for prevention12.Similar to many studies in this area, this study had a small patient cohort, which may have limited our ability to demonstrate the true magnitude of peri-operative cytokine changes. Future studies should involve larger sample sizes across multiple settings to address this problem. Samples were only taken at two time points, with one patient’s CSF sample taken after surgery on day 0 rather than day 1. This patient was not excluded due to an already small cohort. Within the cohort, the surgical approach was not homogenous, with some patients undergoing open surgery and others undergoing endovascular surgery, which could represent a further complicating factor. The small numbers of patients in different surgical groups limits meaningful comparisons. A further confounder was that seven out of ten patients had undergone previous vascular surgery, often with serious post-operative morbidity. Ideally, this study would have corrected for underlying co-morbidity and baseline inflammatory status. In future studies, neuroimaging, to look for radiological evidence of stroke, should be included to investigate how much of the neuroinflammatory burden may be driven by ischaemic strokes. Finally, we cannot exclude the possibility that the inflammatory response was driven by the insertion of the spinal catheter, rather than surgery or anaesthesia, but this is felt to be unlikely due to the magnitude of cytokine changes33. Measurement of the Q-albumin to determine the integrity of the BBB would also have been useful5. Future studies would ideally also examine the CSF cell count and immunoglobulin subtypes37, and other markers of neuronal injury6 to more fully understand pathophysiological processes. This was not possible within the scope of this study.This study looked solely at cytokine changes, which is only part of the neuroinflammatory process after surgery38 and neuropsychological testing, would allow for the more direct investigation of correlations between observed CSF cytokine changes and the magnitude of cognitive dysfunction in PND.A final key limitation of this study was that patients did not undergo peri-operative cognitive testing. Formal cognitive testing for delirium, using screening tools such as the 4ATAfter vascular surgery there is a large increase in cytokines in the CSF, particularly in the pro-inflammatory cytokines IL-6 and IL-8. This may be secondary to peripheral changes in the circulation crossing the BBB and driving neuroinflammation. A strong correlation was found between CSF cytokines on day one after the operation, suggesting that it may be the balance between multiple pro- and anti-inflammatory cytokines which drives neuroinflammation.Supplementary Information.


Neuropsychiatric symptoms are important treatment targets in the management of dementia and can be present at very early clinical stages of neurodegenerative diseases. Increased cortisol has been reported in Alzheimer’s disease (AD) and has been associated with faster cognitive decline. Elevated cortisol output has been observed in relation to perceived stress, depression, and anxiety. Dehydroepiandrosterone sulfate (DHEAS) has known anti-glucocorticoid effects and may counter the effects of cortisol.We aimed to examine whether CSF cortisol and DHEAS levels were associated with (1) neuropsychiatric symptoms at baseline, (2) changes in neuropsychiatric symptoms over 3 years, and (3) whether these associations were related to or independent of AD pathology.n = 78), i.e., mild cognitive impairment or mild dementia, and volunteers with normal cognition (n = 40), were included. Neuropsychiatric symptoms were assessed using the Neuropsychiatric Inventory Questionnaire (NPI-Q). CSF cortisol and DHEAS, as well as CSF AD biomarkers, were obtained at baseline. Neuropsychiatric symptoms were re-assessed at follow-up visits 18 and 36 months from baseline. We constructed linear regression models to examine the links between baseline neuropsychiatric symptoms, the presence of AD pathology as indicated by CSF biomarkers, and CSF cortisol and DHEAS. We used repeated-measures mixed ANCOVA models to examine the associations between the neuropsychiatric symptoms’ changes over time, baseline CSF cortisol and DHEAS, and AD pathology.One hundred and eighteen participants on a prospective study in a memory clinic setting, including patients with cognitive impairment . In particular, higher CSF cortisol was associated with higher baseline scores of depression/dysphoria, anxiety, and apathy/indifference. Elevated CSF cortisol was also associated with more marked increase in NPI-Q scores over time regardless of AD status , but this association was no longer significant after controlling for BMI and the use of psychotropic medications. CSF DHEAS was associated neither with NPI-Q scores at baseline nor with their change over time. Cortisol did not mediate the association between baseline NPI-Q and changes in clinical dementia rating sum of boxes over 36 months.Higher CSF cortisol was associated with higher NPI-Q severity scores at baseline after controlling for covariates including AD pathology status (Higher CSF cortisol may reflect or contribute to more severe neuropsychiatric symptoms at baseline, as well as more pronounced worsening over 3 years, independently of the presence of AD pathology. Our findings also suggest that interventions targeting the HPA axis may be helpful to treat neuropsychiatric symptoms in patients with dementia.The online version contains supplementary material available at 10.1186/s13195-022-01139-9. Neuropsychiatric symptoms (NPS) are increasingly recognized as core symptoms in dementia and Alzheimer’s disease (AD) , 2. WhilNPS have been linked to poorer quality of life and to more pronounced impairment in activities of daily living , 4. TheiPathophysiology of NPS in dementia and AD remains poorly understood: while there is evidence linking these symptoms to Aβ pathology, the relationships with tau pathology and neurodegeneration seem to be less clear . CortisoIt is possible that elevated cortisol levels and cortisol/DHEAS ratio can reflect and/or contribute to more pronounced NPS in older people not only through worsening AD pathology and neurodegeneration , but posIn the present study, we aimed to examine whether the CSF levels of cortisol and DHEAS and the cortisol/DHEAS ratio were associated with (1) NPS at baseline, (2) changes in NPS over 3 years, and (3) whether these associations were related to or independent of AD pathology. We hypothesize that elevated CSF cortisol and CSF cortisol/DHEAS ratio would be associated with more severe NPS at baseline and with more pronounced worsening of NPS over time, even after controlling for the presence of AD pathology as indicated by CSF biomarkers. Considering that higher cortisol has been associated with faster cognitive decline, we additionally explored whether cortisol may mediate the association between NPS and cognitive decline at follow-up visits.We recruited 118 community-dwelling subjects aged between 49 and 88 years in an observational study on biomarkers of cognitive decline and AD conducted at the Department of Psychiatry and the Department of Clinical Neurosciences, University Hospital of Lausanne, Switzerland.n=78) were recruited among outpatients referred to the Memory Clinics, Department of Psychiatry and the Department of Clinical Neurosciences, University Hospital of Lausanne, and who were diagnosed with MCI [n=40) were recruited through journal announcements and word of mouth. They had the same clinical and neuropsychological examination as the cognitively impaired participants. They had no history or clinical signs of cognitive decline and a CDR=0. Subjects with any concomitant neurological, psychiatric, or somatic comorbidity or current medication that could affect cognition at baseline were not included. Patients on corticosteroids were excluded. However, patients on psychotropic medications were not excluded if the medication regimen was deemed stable and not interfering with cognition in the clinician’s opinion. The intake of antidepressants, antipsychotics, benzodiazepines, antiepileptics, and anti-dementia drugs has been systematically recorded.The study participants with cognitive impairment .We also used a battery of cognitive tests to assess the most important cognitive domains  and proxy questionnaires to evaluate basic and instrumental activities of daily living, as previously described . These tWe assessed NPS using the Neuropsychiatric Inventory Questionnaire (NPI-Q) in its validated French version . The NPIThe time between CSF sampling and NPI-Q questionnaire administration was required to be less than 6 weeks. The proxies were asked to state the situation at the time point of the CSF sampling.We collected 10–12 mL of CSF by lumbar puncture between 8 and 9 AM after overnight fasting. The CSF samples were spun down at 4°C, immediately aliquoted, snap frozen at −80 °C, and stored at −80 °C until assay, with no thawing before analysis . The fir1-42 (Aβ1-42), tau, and tau phosphorylated at threonine 181 (p-tau181) were measured using commercially available enzyme-linked immunosorbent assay kits .CSF amyloid-β1-42 ratio with a cut-off of 0.0779 to define the presence/absence of an AD CSF profile , previously determined as the center cutoff indicating the concomitant presence of amyloid and tau pathology [We used the CSF p-tau181/Aβathology .CSF cortisol and DHEAS concentrations were measured using commercially available chemiluminescence immunoassay with high sensitivity . We measured DHEAS rather than DHEA in the present study because DHEAS was shown to be a robust indicator of DHEA production . The intapolipoprotein E (APOE) genotype was determined as previously described [The escribed . We spliThe study was conducted in accordance with applicable laws and regulations, including the International Conference on Harmonization, Guideline for Good Clinical Practice, and the ethical principles that have their origins in the Declaration of Helsinki . The locStatistical analysis was performed using SPSS v26.0 .We determined absolute and relative frequencies for categorical variables. We calculated means and standard deviations for continuous variables  or the median and interquartile range (IQR) in case of non-normality.We also used Spearman’s correlations to examine the associations between baseline NPI-Q scores and changes in CDR-SB over 18 and 36 months, controlling for age, sex, and APOEε4 status.t-test to compare CSF cortisol, CSF DHEAS, and CSF cortisol/DHEAS ratio between participants with baseline NPS (NPI-Q>0) and those without (NPI-Q=0).We used the independent samples We constructed a multiple linear regression model to examine the links between baseline NPI-Q total severity scores, AD pathology, and CSF cortisol and DHEAS, controlling for APOEε4 status, sex, and age.To determine which neuropsychiatric subsyndromes are associated with CSF cortisol in particular, we used a multivariate analysis of covariance (MANCOVA) with the twelve NPI-Q severity subscores as dependent variables and with CSF cortisol, CSF DHEAS, CSF AD pathology, APOEε4 status, sex, and age as independent variables. It is worth mentioning that APOEε4 status was added to the model because APOEε4 significantly differed between participants with and those without NPS.η2).Preliminary assumptions for MANCOVA  were tested. Pillai’s trace test was used because the NPI-Q scores violated the normality assumption. The effect size was assessed using the partial eta squared  as within-subject variablesWith sex, AD pathology status, and APOEε4 status as between-subject factorsWith age, CSF cortisol, and CSF DHEAS as covariatesWe used repeated-measures mixed analysis of covariance (ANCOVA) models with:η2).Global analyses were followed by between-subject and within-subject analyses to highlight significant interactions. The Greenhouse-Geisser correction was applied whenever the sphericity assumption was not fulfilled. Effect sizes were determined using partial eta squares  and the regression between the changes in CDR-SB and CSF cortisol .For each of the repeated-measures ANCOVA and regression models above, we also constructed models adding psychotropic medications and body mass index (BMI) to covariates. Indeed, previous research indicated that psychotropic medications including antidepressants, antipsychotics, and stimulants can affect cortisol levels . Similarp values were adjusted according to Holm-Bonferroni’s method.For all statistical tests, the alpha value was set at 0.05. For multiple comparisons, n=100 subjects at baseline, n=75 at 18 months, and n=39 at 36 months. Baseline CSF cortisol and DHEAS as well as baseline cognitive assessment were available for all participants. CDR and MMSE were available in n=103 individuals at 18 months and in n=96 individuals at 36 months. The first follow-up actually took place at 17.8±2.7 months. The second follow-up was conducted at 35.5±3.5 months.Out of the 118 participants included, NPI-Q was available for Baseline sample characteristics of all subjects and subgroups defined according to baseline NPS are given in Table n=56) were older and more likely to be APOEε4 carriers than participants without (n=44). No differences were found between groups regarding sex or education level (Table Participants with NPS at baseline (el Table .n=37), apathy/indifference , depression/dysphoria , and irritability/lability   and 36 months , controlling for age, sex, and APOEε4 status.We also found positive correlations between baseline NPI-Q and changes in CDR-SB at 18 months .When comparing participants with baseline cognitive impairment (CDR≥0.5) to those without (CDR=0), we found a higher prevalence of baseline NPS in the cognitive impairment group  and higher CSF cortisol/DHEAS ratio  than participants without. CSF DHEAS did not differ significantly between groups . Even though the association between CSF cortisol and the NPI-Q severity score became “weaker,” it remained statistically significant , anxiety , and apathy/indifference   but not with CSF DHEAS. Higher CSF cortisol predicted more pronounced worsening in NPS over 3 years. However, when BMI and the use of psychotropic medications were added to the model, this association was no longer significant .Mixed design repeated-measures ANCOVAs found significant interactions between NPI-Q total severity score changes 36 months after baseline and CSF cortisol  levels, to reflect more accurately the levels in the brain , 55.A further strength is that CSF biomarkers of AD pathology were measured in all participants allowing to address the relationships of the observed associations with the presence or absence of AD.Our findings suggest that elevated CSF cortisol may be linked to both more severe NPS and more pronounced worsening of NPS over the years. These relationships are probably not merely explained by the previously described effects of cortisol on AD pathology and neurodegeneration . It is pCSF cortisol might be a useful biomarker to predict the course of NPS over time, while interventions targeting the HPA axis might be useful in reducing NPS severity and preventing their presence or worsening over time. Further studies are needed to confirm these hypotheses.Additional file 1: Supplementary Table 1. Frequency of neuropsychiatric syndromes at baseline.Additional file 2: Supplementary Table 2. Distribution of participants according to the AT(N) system classification of Alzheimer’s disease pathology.Additional file 3: Supplementary Table 3. Sobel’s test regressions coefficients between cortisol and baseline neuropsychiatric symptoms, and between changes in CDR-SB and CSF cortisol.


Orexin-A (OXA) and -B (OXB) are involved in the regulationof multiplephysiological functions including the sleep–wake states; therefore,it is critical to monitor their levels under various conditions. Unfortunately,the widely used radioimmunoassay has insufficient specificity forOXA. Although liquid chromatography–tandem mass spectrometry(LC–MS/MS) has higher specificity for OXA, previously reportedOXA levels in human cerebrospinal fluid (CSF) measured using thistechnique are still inconsistent. Moreover, to the best of our knowledge,OXB has not been detected in the CSF. In this study, we establisheda novel method for OXA and OXB measurement. We noticed that OXA andOXB in the CSF was sticky; thus, citric acid and Tween 80 were usedto prevent their nonspecific binding. Then, highly specific and sensitivenanoflow liquid chromatography–high-resolution mass spectrometry(nanoLC-HRMS) was used to measure OXA and OXB levels. Evaluation ofthe diurnal fluctuations of OXA and OXB in cisternal and lumbar CSFsamples from cynomolgus monkeys revealed a sharp increase in the earlylight period, followed by a gradual increase to the maximum levelsat the end of the light period, and then a sharp drop to the minimumlevels during the early dark period. OXB levels were lower than OXAlevels in cisternal CSF. Although basal OXA levels in individual monkeysshowed substantial variations, the ratios between the maximum andminimum OXA levels of each monkey were similar. Our method for accurateOXA and OXB measurement should help improve our knowledge of orexinbiology. OXB is a 28-amino acid peptide with a C-terminal sequencesimilar to that of OXA including C-terminal amidation, while its N-terminushas no modifications. OXA and OXB are ligands of two G-protein-coupledreceptors, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R).1 Loss of orexin neurons and the resultant orexinneuropeptide reduction are associated with narcolepsy type 1 (NT1),which is a severe neurological disorder characterized by multiplesymptoms including hypersomnia, cataplexy, disturbed nighttime sleep,hypnagogic/hypnopompic hallucinations and sleep paralysis.7 Because OX2R knockout (KO) mice show clear narcolepsy-like phenotypesand OX1R KO mice do not, OX2R-selective agonists are a promising optionfor treating NT1.8 Recently, two compounds (YNT-185and TAK-925) have been discovered as novel OX2R-selective agonists.10 As NT1 is related to selective loss of orexin neurons, the OXA levelin the cerebrospinal fluid (CSF) has been used as a diagnostic biomarkerfor NT1.12 The OXA level in the CSF has been measuredby competitive radioimmunoassay (RIA).12 However, arecent report suggested that more than 90% of the signal detectedby RIA reflects biologically inactive OXA-related metabolites.13 Detailed analysis has shown that antibodies(Abs) employed in the RIA methods, including Phoenix Ab used for diagnostictesting, could capture not only OXA but also its metabolites, possiblyC-terminal-truncated OXA.13 Therefore,improved methods to precisely measure OXA should be established. Sandwichimmunoassay using two Abs that bind to different sites on the ligandmay have higher specificity toward authentic bioactive OXA comparedwith RIA using a single Ab. However, no sandwich immunoassays forOXA, including electrochemiluminescence (ECL), have fully demonstratedspecificity for authentic OXA.14 Liquidchromatography–tandem mass spectrometry (LC–MS/MS) isalso a promising method for measuring OXA with high specificity becauseits detection is based on differences in the mass-to-charge ratio(m/z). Theoretically, LC–MS/MScould distinguish OXA from its metabolites like truncated OXA unlessthe metabolites give remarkably similar m/z to OXA; for example, deamidation at the C-terminal couldshow quite similar m/z to that ofOXA (Δm/z = −0.984).Thus far, three LC–MS/MS methods have been reported for measuringOXA in human CSF.17 However, the median OXA concentrations from thosestudies differed from each other by factors as high as 10 for healthyhuman subjects. It is well known that some peptides adsorb to thesurface of materials, such as syringes, needles, and sample tubes.18 Therefore, the nonspecific binding of OXA couldbe a possible reason for this discrepancy. Moreover, reported methodsusing conventional LC–MS/MS may not be able to discriminateOXA from its metabolites with slight modification such as deamidation.Furthermore, no LC–MS/MS methods have detected OXB in the CSF,although OXA and OXB are produced in the same amount by the cleavageof prepro-orexin.17 Thus, little is knownabout the distribution and physiological functions of OXB.1 A combination of nanoflow LC and high-resolutionmass spectrometry (HRMS) is a promising approach to address this problembecause of their higher specificity, sensitivity, and separation efficiencycompared to conventional LC–MS/MS.Orexin-A (OXA) and orexin-B (OXB)  are released from orexin neurons in thehypothalamus. They are involved in various physiological functionsincluding regulation of the sleep–wake states.20 The results showed a similarpattern among these animals: a higher level of OXA during the activeperiod and a lower level during the resting period that dropped below50% of the peak levels. In comparison, human OXA levels in lumbarCSF display smaller fluctuations, within ±10% from the 24 h average.21 This difference could be due to (1) limitedspecificity of the RIA and ECL methods for OXA, (2) species difference,and (3) the site of CSF correction . More specificmethods for OXA measurement would help to clarify these questions.Characterizing thediurnal fluctuation of orexins is critical forunderstanding orexin signaling. Immunoassays  havebeen used to measure diurnal fluctuations of OXA in cisternal CSFsamples collected from not only nocturnal animals like mice and ratsbut also diurnal animals like dogs, rhesus monkeys, and squirrel monkeys.In this paper, we report a novel method that uses additives toprevent the nonspecific binding of OXA and OXB, and nanoflow LC coupledwith HRMS (nanoLC-HRMS) for highly specific and sensitive detectionof these two peptides. Using this method, we examined the diurnalfluctuations of both OXA and OXB in cisternal and lumbar CSF samplescollected from cynomolgus monkeys.22 Nonspecific binding of peptides to syringesand sample tubes is a serious issue in bioanalytical assays, especiallywhen using CSF which lacks proteins.23 We assessedthe magnitude of nonspecific binding of orexins and evaluated antiadsorptiveagents to overcome the nonspecific binding.We took advantage of the nanoLC-HRMS approach to precisely measureOXA and OXB. The nanoflow LC system provides higher sensitivity andbetter separation than conventional high-performance liquid chromatography.Survivor-selected ion monitoring (SIM) analysis using HRMS enableshighly sensitive and specific detection of the target analytes.Figure S1). Carry-over was assessed by injectingblank samples immediately after a calibration standard at the upperlimit of quantification. The carry-over was lower than 20% of LLOQfor orexins and 5% for the ISs. The stability of OXA and OXB in thereal matrix was evaluated using unspiked and spiked QC samples intriplicate. OXA and OXB were stable during three freeze–thawcycles at −80 °C, at room temperature for 2 h, and at−80 °C for 60 days. The percent difference in QC samplesfor stability evaluation was within ±15% of the initial sample.The processed sample stability was evaluated using three replicatesof real matrix (endogenous levels ranging from 93.7 to 179.5 pg/mLfor OXA and from 14.9 to 26.6 pg/mL for OXB). The percent remainingafter 48 h in an autosampler at 10 °C was within ±15% ofthe initial analysis. The result indicated that OXA and OXB were stablefor at least 48 h in an autosampler at 10 °C. The preparationand storage of all CSF samples were conducted under the stable conditionsdescribed above. Compared to the reported LC–MS/MS method forOXA (LLOQ > 3.6 pg/mL), our method can measure OXA using only approximately1/10 of the CSF sample volume and achieved lower LLOQ (2.5 pg/mL).17Theestablished nanoLC-HRMS method seemed to have sufficient specificityand sensitivity for measuring OXA and OXB in monkey CSF 1A,B. TheSelective detection of OXA and OXB in monkey CSF wasfurther assessed based on retention time and characteristic productions obtained by parallel reaction monitoring (PRM). The concentrationsof OXA and OXB in the CSF sample used in this analysis were 179.5and 26.6 pg/mL, respectively. The same product ions with referencestandard OXA at 500 pg/mL were detected in monkey CSF 1G,H. Sim13 Thus, those methods cannot discriminate OXAfrom its metabolites that have an intact N-terminal region and a modifiedC-terminal region such as truncations. In contrast, the LC–MS/MSmethods can theoretically separate OXA from truncated metabolitesaccording to their detection principle. However, even these methodscould not easily discriminate OXA from its metabolites with similar m/z, such as deamidation with Δm/z < 1 due to multiple charges. A putativemetabolite is an OXA-related molecule modified with deamidation atthe C-terminal leucine, OXA-Leu33-OH (m/z 1187.24237) was completely separated from that ofOXA-Leu33-OH (m/z 1187.57230) usingHRMS . A tube containing neat CSF spiked with the standardsfor OXA and OXB was prepared. Then, the spiked CSF was collected fromthe tube using a needle and a syringe. To the tube, 6 μL of1M citric acid and 3 μL of 10% (w/v) Tween 80 were added, andthe 300 μL of spiked CSF was returned from the syringe to thetube. The tube, the needle, and the syringe were rinsed by pipettingwith CSF containing 20 mM citric acid and 0.1% (w/v) Tween 80 . The results demonstrated satisfactory recovery . Similar recovery was observed with the additionof 0.01% (v/w) Tween 80 alone . Thus, 0.01%(w/v) Tween 80 would be a sufficient additive to measure orexin levelsin monkey CSF, and addition of citric acid had no negative impacton OXA and OXB measurement. These results supported that the improvedrecovery was not derived from the inactivation of degrading enzymesby acidification but rather from inhibition of nonspecific binding.Given the low recovery of orexins in neat CSF, a portion was consideredto be adsorbed to the tubes, needles, and syringes before the additionof the antiadsorptive additives. The observed adequate recovery indicatedthat the antiadsorptive additives could not only prevent nonspecificbinding of orexins during sample transfer but also recover orexinsthat were once adsorbed to syringes and tubes.The recovery of spiked OXA and OXB at thefinal concentration of 100 pg/mL in neat monkey CSF was 41.6 and 40.2%,respectively, and these low values could be due to nonspecific bindingand/or enzymatic degradation 2. DetergS3). Theconcentrations of OXB in four of eight cisternal samples from animal-1and 28 of 40 lumbar samples were below the LLOQ, and these were givena value of 0 pg/mL. The mean molar ratio of OXA to OXB was 5.3 ±3.9 (mean ± SD) in cisternal CSF. A higher OXA level can be causedby stabilization of OXA by PTM against enzymatic degradation. Theconcentrations of OXB were below the LLOQ in lumbar CSF at severaltime points, especially during the dark period . Further improvement in sensitivity is needed tofully understand OXB fluctuation in lumbar CSF.The concentrations of OXA and OXB were determined by nanoLC-HRMSin cisternal and lumbar CSF treated with Tween 80 during collectionat 3:00, 7:00, 8:00, 12:00, 17:00, 19:00, 21:00, and 23:00. Samplesat different time points were collected on different days. The meanconcentrations of OXA in cisternal and lumbar CSF ranged from 37.9to 129.1 pg/mL and 16.9 to 97.8 pg/mL, respectively 2A. The OS3). However, the individualmonkeys were ranked in similar orders in terms of both OXA and OXBconcentrations in cisternal and lumbar CSF. Interestingly, althoughthe individual monkeys showed substantial variation in the absoluteOXA concentrations, the percent changes in OXA in cisternal and lumbarCSF at the same time point were similar .Interday variation of OXA and OXB levels was assessed using cisternalCSF collected from the same five individual animals on three differentdays 4. ThesaN = 5) for OXA andfrom 18.0 to 24.0%  for OXB. Inthe sample collected from animal-1 without additives, the OXB levelwas below the LLOQ and therefore excluded from the ratio calculation.The large variability in the ratio of OXA made it difficult to correctthe concentration using a fixed coefficient. These results show thatCSF samples collected without antiadsorptive additives were not appropriatefor OXA and OXB determination. We hypothesize that severe nonspecificbinding of OXA and OXB could cause the variability of OXA concentrationsand undetectable OXB reported previously for human CSF. Further investigationsshould be conducted using newly collected CSF samples with appropriatereagents added for each sample.To assess the effectiveness of antiadsorptiveadditives, we compared the concentrations of OXA and OXB in cisternalCSF collected with and without 20 mM citric acid and 0.1% (w/v) Tween80  5. The saWe established a novel method to preciselymeasure OXA and OXBlevels in monkey CSF. Antiadsorptive additives—citric acidand Tween 80—were added during sample collection and preparationsteps to prevent the nonspecific binding of orexins. Then, OXA andOXB levels in the CSF were measured using the nanoLC-HRMS approach.Employing this method, we revealed diurnal fluctuations of OXA andOXB in cisternal and lumbar CSF samples from monkeys. Better characterizationof orexin levels, including diurnal fluctuation under various conditions,would improve our understanding of orexin physiology, as well as helpto establish the ideal pharmacokinetic pattern of OX2R agonists fortreating related diseases. Further studies of both OXA and OXB levelsusing this method should provide valuable information on the rolethat orexins play in human health and diseases.13C6, 15N]Leu  and OXB with a mass shift of 17 Daby one Leu and one Arg . OXA metabolite with amide hydrolysis at the C-terminalleucine (OXA-Leu33-OH) was obtained from Scrum Inc. Antiadsorptivereagents used were 20 mM citric acid and 0.1% (w/v) Tween 80 . All other chemicals, reagents, and solvents wereanalytical grade.Synthetic standards for OXA and OXB were purchasedfrom Peptide Institute . Stable isotope-labeled OXAand OXB were used as ISs for nanoLC-HRMS: OXA with a mass shift of42 Da by six +.NanoLC-HRMS was performed on an EASY-nLC1200 system  and a QExactive HF-X Orbitrap mass spectrometer (Thermo Fisher Scientific)equipped with an electrospray ionization interface. The reconstitutedsamples were kept in an autosampler at 10 °C, and then, 5 μLof each sample was loaded onto an Acclaim PepMap 100 C18 trap columncoupled with an NTCC-360/75–3–15 analytical capillarycolumn  at room temperature. TFA/water  and TFA/water/acetonitrile were used as mobile phases A and B, respectively.The total flow rate was 300 nL/min, and the following gradient elutionwas used: 0–30 min, 5–30% B; 30–38 min, 30–50%B; 38–45 min, 50–95% B; and 45–55 min, 95% B.The mass spectra were obtained in the positive ion mode under thefollowing conditions: resolving power of 60,000 , automatic gain control (AGC) target of 200,000, spray voltageof 2.00 kV, capillary temperature of 275 °C, and funnel RF levelof 45.0. The precursor ions selected with an isolation width of The experiments were approved by the InstitutionalAnimal Care and Use Committee of Shonan Research Center, Takeda PharmaceuticalCompany Limited. Five male cynomolgus monkeys  were used in this study. Each cagehoused a single animal, and two cages were connected to allow theanimals to interact with each other. Starting from 1 week before aseries of CSF collections, the animals were single-housed for 1 month.Then, two cages were connected again for at least 1 week before anotherseries of CSF collections. Throughout the experiments, one animal was housed singly because the total number of five is odd.The monkeys were kept under a 12-h light/dark cycle with light between7:00 and 19:00. Food was provided daily in the morning.The animals were anesthetized in thehome cage by intramuscular injection of 10 mg/kg ketamine . CSF was collected from anesthetizedanimals only once a day, and there was an interval of more than 48h before the next collection. CSF samples were collected via eithercisterna magna puncture or lumbar puncture.Figure S2).For cisterna magnapuncture, CSF was sampled with an injection needle  and a syringe .Injection needle consists of a needle and a needle hub. The needleis made of stainless steel and the needle hub is made of polypropylene.For syringe, the parts that contact CSF samples are the rubber stopperon the plunger and the barrel. The rubber stopper is made of elastomerand the barrel is made of polypropylene. The volume of collected CSFsample was approximately 250 μL for diurnal fluctuation experiment.Orexin levels in CSF samples collected at 7:00 and 17:00 were alsoused as orexin levels in the CSF of first collection in interday variationexperiment. For other collections, the volume of CSF sample was approximately500 μL. Tween 80  in PBS was generally added to thecollected CSF  to reach a final concentrationof 0.01%. However, for the second and third collection in the interdayvariation experiment, 10% Tween 80 (v/v) in PBS and 1 M citric acidwere added to the collected CSF  to reach a final concentration of 0.1% and 20 mM, respectively,instead. Pipetting was conducted in the collection tube (EppendorfProtein LoBind) with the syringe and injection needle used for CSFcollection . The supernatant was transferred to a new collectiontube and stored in a freezer at −80 °C until use. Thetime interval between sample collection and placement in the freezerwas within 1 h.For lumbarpuncture, an injection needle  was inserted between L3 and L5, and CSF leaked throughthe injection needle was sampled into a collection tube. The volumeof collected CSF sample was approximately 250 μL. Tween 80  was added to the CSF  to reach a finalconcentration of 0.01%, and pipetting was conducted in the collectiontube with the used injection needle and a brand new syringe. All collectedCSF samples were kept on ice until centrifugation , the animals were anesthetizedin the dark, blindfolded, and then transferred to a bright room forsample collection to mitigate the potential effect of light on CSForexin levels. It took 7 weeks to collect all samples used to analyzediurnal fluctuations.CSF was collectedvia cisterna magna puncture. Orexin levels in CSF samples collectedat 7:00 and 17:00 in the diurnal fluctuation experiment were alsoused as orexin levels in the CSF of first collection in this experiment.For samples collected at 7:00, the interval was 10 months betweenthe first and second collection and 11 days between the second andthird collection. For CSF collected at 17:00, the interval was 10months between the first and second collection and 5 days betweensecond and third collection.CSF was collected via cisterna magna puncture at 17:00. Orexinlevels in CSF samples collected at the third collection in the interdayvariation experiment were also used as orexin levels in CSF with 20mM citric acid and 0.1% (w/v) Tween 80  in thisexperiment. After the third collection in the interday variation experiment,CSF samples without antiadsorptive additives were collected with a3-month interval.Xcalibur 4.1 and TraceFinder4.1 (Thermo) were used for data acquisition, peak detection, integration,calibration, and quantification. Results of CSF analysis are expressedas the mean ± standard error of the mean (SEM). Graphs were createdusing GraphPad (v6) software.


P = .029) and overall survival (OS) , while none of the other models predicted for survival in these patients. Moreover, using exploratory classification and regression tree analysis, we found that donor age <58 years combined with cytomegalovirus-nonreactive recipient was associated with the best OS, whereas donor age >58 years was associated with the worst OS. In the rest of our cohort (80%), cytomegalovirus-reactive recipients with a donor <58 years old, a higher CF-iKIR was associated with superior OS. The 3-year OS rates were 73.9%, 54.1% , 44.5% , and 18.5%  in the best, better, poor, and worse donor groups, respectively. Our results suggest that KIR alloreactivity assessed by CF-iKIR score can help optimize donor selection in haplo-HSCT.With the continuous increase in the use of haploidentical donors for transplantation, the selection of donors becomes increasingly important. Haploidentical donors have been selected primarily based on clinical characteristics, while the effects of killer cell immunoglobulin-like receptors (KIRs) on outcomes of haploidentical-hematopoietic stem cell transplantation (haplo-HSCT) with post-transplant cyclophosphamide (PTCy) remain inconclusive. The present study aimed to thoroughly evaluate the effect of KIRs and binding ligands assessed by various models, in addition to other patient/donor variables, on clinical outcomes in haplo-HSCT. In a cohort of 354 patients undergoing their first haplo-HSCT, we found that a higher Count Functional inhibitory KIR score (CF-iKIR) was associated with improved progression-free survival (adjusted hazard ratio [HR], 0.71;  Haplo-HSCT donor with NK cell alloreactivity predicted by count functional inhibitory KIR score is associated with improved survivalA predictive algorithm incorporating NK cell alloreactivity and donor characteristics can aid haplo-HSCT donor selectionAllogeneic hematopoietic stem cell transplantation (HSCT) is the curative therapy for patients with advanced hematologic malignancies. T cell-mediated alloimmunity in allogeneic HSCT is associated with a beneficial graft-versus-leukemia (GVL) effect accompanied by an increased risk of graft-versus-host disease (GVHD). With the successful control of GVHD using prophylactic cyclophosphamide and graft engineering –3, in thPost-transplant cyclophosphamide (PTCy) has been widely used in haplo-HSCT to prevent GVHD mostly arising from the mismatched HLA haplotype. Moreover, robust immune reconstitution and preferential recovery of regulatory T cells  allow HSNatural killer (NK) cells have been hypothesized to contribute to the GVL effect without increasing GVHD, and the process is thought to be regulated by recognition between polymorphic killer cell immunoglobulin-like receptors (KIRs) and class-I HLA ligands expressed on the target cells . Pioneer+ T cell survival and response against viral infections   and patifections . In a cofections . WhetherPTCy administration transiently reduces mature NK cells  and may In the present study, we aimed to comprehensively evaluate the effect of KIRs and their binding ligands along with other donor variables, on clinical outcomes in patients undergoing haplo-HSCT with PTCy-based GVHD prophylaxis.Our cohort included 354 consecutively treated patients aged 18 years or older with hematologic malignancies who underwent their first unmanipulated haplo-HSCT at The University of Texas MD Anderson Cancer Center (MDACC) between May 2009 and September 2019. All patients received PTCy-based GVHD prophylaxis with tacrolimus and mycophenolate, as previously described by our group . PatientPatients eligible for the study had donor and recipient HLA typing performed at the HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 loci using sequence-based typing methods at high resolution . KIR genWe examined and compared the following models to assess NK cell alloreactivity: donor NK cell benefit, KIR2DS1/C1C2 epitope combination, donor centromeric motif, donor telomeric motif, KIR B-content score, inhibitory KIR score, and CF-iKIR score.https://www.ebi.ac.uk/ipd/kir/ligand.html. NK cell alloreactivity in the graft-versus-host direction was assigned when the recipient lacked at least one of the HLA ligands that were present in the donor. For the KIR2DS1/C1C2 epitope combination model, binding between the KIR2DS1 and C1C2 ligands was classified as described by Venstrom et al. , and secondary outcomes included progression-free survival (PFS), relapse, non-relapse mortality (NRM), acute GVHD (aGVHD), chronic GVHD (cGVHD), and viral reactivation. All outcomes were measured from the time of stem cell infusion. OS was defined as the time from stem cell infusion to death from any cause. PFS events included death or relapse. NRM was defined as death without a previous relapse. Patients without the event were censored at the time of the last contact. The Kaplan-Meier method was used to calculate unadjusted PFS and OS, and the cumulative incidence with competing risks method was used to calculate aGVHD, cGVHD, NRM, relapse, and viral reactivation. For NRM, relapse was the competing risk, and for relapse, the competing risk was NRM. For aGVHD and cGVHD, death without the event and relapse were the competing risks. The effects of NK cell alloreactivity as assessed by various models and the effects of baseline clinical factors on survival outcomes were analyzed using univariate and multivariable Cox proportional hazards regression models, and the effects of these factors on relapse, NRM, aGVHD, cGVHD and viral reactivation were determined by univariate and multivariable proportional subdistribution hazards regression models. The median cutoff of the inhibitory KIR score and CF-iKIR score was used in the analyses due to it provided the lowest Akaike, Bayesian information criterion, as well as maximum log-likelihood in a univariate regression model for each outcome of interest.Baseline clinical factors included in the univariate models were recipient age (continuous), sex, recipient-donor sex combination , hematopoietic cell transplant comorbidity index (HCT-CI), transplant protocol (on protocol vs. standard of care), donor age (continuous), ABO matching (match vs. mismatches), recipient-donor CMV serostatus (nonreactive-nonreactive vs. nonreactive-reactive vs. reactive-nonreactive vs. reactive-reactive), stem cell type , conditioning regimen intensity (myeloablative vs. reduced intensity/non-myeloablative), donor-specific anti-HLA antibodies (presence vs. absence).Variables with a P-value <0.10 in the univariate analysis were included in the multivariable analysis. All variables of interest were tested for the proportional hazards assumption and interaction terms. All tests were two-sided. The type 1 error rate was fixed at 0.05. No adjustments for multiple testing were made.Classification and regression tree analysis  of OS waStata statistical software  was used for statistical analyses.The analysis included 354 patients with a median age of 49 years (range 18-72 years), of which 84 (23.7%) were ≥60 years. Patient and HSCT-related characteristics, as well as NK cell alloreactivity predicted by various models, are listed in Based on HLA typing from donor and recipient pairs, the NK cell benefit occurred in 28.5% of our cohort. For the KIR B-content score derived from the donor KIR genotype, donors were categorized as neutral (74.3%), better (15.3%), or best (10.5%) based on the number of centromeric and telomeric B motifs. The inhibitory KIR score and CF-iKIR score, calculated based on the presence of donor inhibitory KIRs and cognate HLA ligands from the recipient, had mean values of 2.29 for the inhibitory KIR score and 2.12 for the CF-iKIR score. Not surprisingly, the neutral/better/best classification for B-content score partially correlated with donor centromeric motif and donor telomeric motif. Inhibitory KIR scores largely overlapped with CF-iKIR scores but with a higher median cutoff (P = .029) for PFS (P = .016) for OS . The risk of viral reactivation significantly increased in patients who received a stem cell graft from a donor with NK cell alloreactivity predicted by the NK cell benefit model . None of the NK cell alloreactivity models predicted relapse, NRM, or cGVHD after HSCT. Results of multivariable analyses of the effect of NK cell alloreactivity according to various models on HSCT outcomes are shown in While the other models failed to predict survival they did show correlations with other transplant outcomes. A high inhibitory KIR score predicted a reduced risk of clinically significant aGVHD (grade 2-4), with a cumulative incidence at 100 days of 37.4% for low inhibitory KIR score and 28.9% for high inhibitory KIR score , higher HCT-CI , and high/very high DRI  were clinical factors independently associated with poor PFS , higher HCT-CI , high or very high DRI , recipient-donor CMV serostatus  for reactive recipient–nonreactive donor and  for reactive recipient–reactive donor when compared with nonreactive recipient–nonreactive donor), and older donor  were predictors of poor OS , higher HCT-CI , and older donor age . We also found a significant interaction between donor age and donor sex on the risk of NRM. Using a stem cell graft from a female donor aged 40 years or older was associated with a significantly increased risk of NRM compared with a male donor younger than 40 years, with an adjusted HR of 2.00 . The increased NRM associated with an older female donor likely resulted from the development of severe aGVHD as we also found a significantly increased risk of grade 3-4 aGVHD associated with using a stem cell graft from a female donor aged 40 years or older compared with a male donor younger than 40 years . Results of multivariable analyses of the effect of clinical factors on post-HSCT outcomes are detailed in Independent predictors of an increased risk of NRM were older recipient age , 2.70 , and 5.44 , respectively. Internal validation using a dataset created from the bootstrapping method showed satisfactory predictive performance of this algorithm for OS, with a concordance index of 0.72.We developed an algorithm for donor selection using classification and regression tree analysis of OS, incorporating NK cell alloreactivity predicted by CF-iKIR score and other donor characteristics. Classification and regression tree analysis identified four groups of donors by OS outcome , as shown in In haplo-HSCT, unlike other forms of transplantation, a significant immune disparity exists in both HLA and KIR systems between donor and recipient, which may elicit the corresponding innate and adaptive alloreactivities. Recent studies have provided compelling evidence that the dynamics of NK cell recovery are remarkably affected by PTCy prophylaxis  and the Contradictory results have been previously reported among studies of NK cell alloreactivity in TCR haplo-HSCT. It was initially believed that T cells contained in the graft may inhibit NK cell function and eliminate their GVL effect . SolomonRusso et al. extensively investigated the dynamics of NK cell recovery and concluded that most mature donor NK cells are eliminated by PTCy and therefore NK cell alloreactivity does not affect outcomes . A later+ T cell survival as well as a response against viral infections  or relapse protection  yet the combined effects on both could explain this finding. A registry study showed that higher donor age is associated with inferior mortality in haplo-HSCT .The authors would like to thank Dr. Jill Hollenbach from the University of California San Francisco for the helpful discussion on KIR analysis. We thank Erica Goodoff, Senior Scientific Editor in the Research Medical Library at The University of Texas MD Anderson Cancer Center, for editing this article.Authors JS, FH, HB are/were employed by DKMS gemeinnützige GmbH, Tübingen, Germany.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aim was to evaluate the performance of the latest quantitative marker for intrathecal IgG synthesis and to compare it with other established markers used for the same purpose. We retrospectively applied Auer’s and Reiber’s intrathecal IgG synthesis formulae in a cohort of 372 patients under investigation for central nervous system demyelination who had undergone lumbar puncture and oligoclonal bands (OCBs) detection for demonstrating intrathecal IgG synthesis. A ROC analysis revealed Auer’s formula had lower sensitivity (68%) compared to Reiber’s formula (83%) and IgG index (89%), in our cohort of patients that exhibited normal to mildly elevated albumin quotients (4.48 ± 3.93). By excluding possible sources of errors, we assume that Auer’s formula is less sensitive than other established tools for the “prediction” of the detection of OCBs in routine cerebrospinal fluid (CSF) analyses due to the mathematical model used. Given the ability of Reiber’s hyperbolic formula to describe the blood–CSF IgG distribution across a wide range of blood–brain barrier functionality, its use and the use of similar formulae are recommended for the discrimination between CNS-derived and blood-derived molecules in clinical laboratories. The demonstration of intrathecal immunoglobulin synthesis (ITIS) as a part of the immune process within the central nervous system (CNS) has always been of great diagnostic significance and a major diagnostic challenge for all methodological developments in cerebrospinal fluid (CSF) analysis. For the qualitative demonstration of ITIS, isoelectric focusing (IEF) coupled with immunodetection (immunoblotting or immunofixation) has become the leading technique for the detection and study of oligoclonal bands (OCBs) in CSF . In addiFrom this perspective, we conducted this study to evaluate the new formula by Auer et al. for IgG in a clinical cohort considering OCBs as the reference method for the first time and to compare the performances of different kinds of quantitative ITIS markers.To perform this retrospective study, we collected data from a laboratory computerized database for patients who have been hospitalized between 2015 and 2017 in the Demyelinating Diseases Unit of the Neurology Department of Eginition University Hospital. All patients enrolled in the study had imaging features suggestive of demyelination with or without clinical manifestations and underwent diagnostic lumbar puncture. Most of them were in stable condition during sample collection and drug naïve. Diagnosis of MS was in accordance with McDonald2017 criteria .From theg for 10 min at room temperature and part of the supernatants was used for the nephelometric determination of IgG and albumin  levels . Serum and CSF aliquots from the rest of the supernatant were kept frozen in −20 °C for further analyses.After overnight fasting, a CSF and a blood sample were collected between 8.00 and 10.00 in the morning by lumbar and venous puncture, respectively. The maximum time interval between them was one hour and during that no major therapeutic interventions occurred that could affect measurements of both biological fluids . CSF red- and white-blood-cell concentrations were estimated using a Fuchs–Rosenthal chamber and this procedure was completed within two hours after lumbar puncture. Afterwards, CSF and blood samples were centrifuged at 2000× OCB detection by IEF on agarose gel was performed on the semi-automatic HYDRASYS system within a maximum of one week after lumbar puncture. Initially, CSF and serum IgG concentrations were adjusted to each other. Then, isofocusing with the use of Hydragel 9 CSF isofocusing kit followed by immunofixation with the use of antiserum anti-IgG conjugated to peroxidase  were performed. Recognized positive and negative controls ran with each set of samples. CSF findings were directly compared with serum samples’ findings, running simultaneously in the same assay in an adjacent track. Finally, the IgG immunodetection patterns of CSF and serum from the same patient were visually compared and categorized into five OCB constellation types (I–V), according to the European consensus paper of 1994 . Type II2 + 6 × 10−6) −1.7 × 10−3) , and the negative predictive value (NPV) as [true-negative/], with reference positive values in the presence of OCB.Classification accuracy (ACC) was calculated as follows: True Positive + True Negative/True Positive + True Negative + False Positive + False Negative. Using a 2 × 2 traditional confusion matrix for binary classifications, Cohen’s kappa formula was calculated as follows: κ = 2 × (TP × TN−FN × FP)/[(TP + FP) × (FP + TN) + (TP + FN) × (FN + TN)], where TP is true-positive, FP is false-positive, TN is true-negative, and FN is false-negative. Cohen’s kappa minimum value is −1 (wrong prediction) and the maximum value is +1 (perfect classification). Furthermore, if κ ≈ 0, the prediction made was similar to random prediction .A total of 372 neurological inpatients were included in the analysis. The clinical and demographic characteristics of the patients are presented in We performed an ROC analysis at the cut-off levels described in the Materials and Method section. The sensitivity, specificity, and positive and negative predictive values of all the investigated parameters are given in To distinguish OCB-positive  patients from OCB-negative patients, the IgG index showed the highest AUC with a value of 0.9515 , as compared with the other quantitative formulae. Reiber and Auer’s formulae exhibited AUC levels of 0.9404  and 0.9507 , respectively .Based on past experience, we applied cut-off levels of 0.65 for the IgG index and we found it had the highest sensitivity (89%) in verifying positive OCB results, albeit with lower specificity (90%) compared to the other two formulae . For the detection of OCBs, cut-off levels of >0.93 for Reiber’s formula and >0.882 for Auer’s formula were used, resulting in sensitivities that showed the superiority of Reiber’s formula (83%) compared to Auer’s formula (68%) . Auer’s As the ROC curves among the different formulae  were similar, we applied further statistical tests and metrics to better measure each method’s classification performance. To assess the accuracy rate, we used the proposed cut-off levels by Auer (>0.88) and Reiber (>0.93) and the standard cut-off level for the IgG index that had already been validated by our lab (>0.65) . We calcIn 2016, Auer et al.  introducAlthough ITIS is found in various, mainly inflammatory CNS diseases , it reacNowadays, agarose IEF methods combined with immunodetection (immuno-fixation or immune-blotting) to increase detection sensitivity and provide a high specificity have been proposed for detecting IgG OCBs in the human CSF in diagnostic laboratories ,21. HoweThe IgG index, exhibiting a 89% sensitivity rate and 90% specificity rate, performed quite well in this particular cohort of patients with normal to mildly elevated QAlb (4.38 ± 3.93). However, it is well known that, due to its linearity, it can be proved unreliable in patients with severe blood–brain barrier (BBB) dysfunction and high QAlb in contrast to Reiber’s formula. Similarly, our results showing 68% sensitivity for Auer’s formula cannot be extrapolated to patients with other diseases than demyelinating diseases, particularly to patients with moderate-to-severe BBB dysfunction.To our knowledge, this is the first and only evaluation of this kind for Auer’s et al. formula, besides the initial one . A diffeSince the calculation of the new formula was based on nephelometric determination in CSF and serum from OCB-negative patients, a possible explanation of the new formula’s failure in “predicting” OCB positivity could be a methodological error in the techniques used. Nephelometric determination used in both studies is standardized and the use of CSF/serum quotients eliminates any possible inaccuracy, almost excluding them from being the source of error. Regarding the detection of OCBs, Auer et al. used IEF and subsequent immunoblotting according to Keir et al. with the modification of using polyacrylamide instead of agarose gel. The same technique has been repeatedly proposed as the “gold standard” for detecting the presence of OCBs in CSF ,28,29,30A final possible source of error is the mathematical model that was used for describing a biophysical model. The IgG index describes the correlation between QIgG and Qalb as a linear function and ignores the nonlinear relationship between two blood-derived molecules of different sizes . Auer etUsing IEF followed by immune fixation as our reference method, Auer’s et al. simple power function for IgG, although scoring high in specificity (95%), lacked sensitivity (68%) in patients with demyelinating diseases. Our data revealed the superiority of Reiber’s hyperbolic function over it, exhibiting a 83% sensitivity. The IgG index performed quite well in our cohort of patients with normal to slightly impaired BBB, and it can be used as an additional linear formula in these cases. Efforts to introduce an ITIS estimate of the new promising KFLC molecule are in progress and close to a final consensus. Our results favor the use of a hyperbolic function for the optimal discrimination between blood-derived and CNS-derived CSF molecules, such as KFLC, within a broad range of CSF functionality.


Fecal microbiota transplantation (FMT) targeting gut microbiota has recently been applied to the treatment of ulcerative colitis (UC). However, preliminary trials showed that only a subset of patients responded to FMT, and the heterogeneity in donor gut microbiota probably played important roles in patients’ responses, implying the significance of matching an appropriate donor to a specified patient. We developed a strategy to build a donor-recipient matching model to guide rational donor selection for UC in FMT. We collected and uniformly reanalyzed 656 fecal 16S rRNA gene sequencing samples  from 9 studies. Significantly lower α-diversity indexes were observed in UC patients by random effects model. Thirty-four bacterial genera and 34 predicted pathways were identified with significant odds ratios and classification potentials for UC patients. Based on six bacterial indicators, including richness, overall distance, genera, and pathways , the analytic hierarchy process-based donor-recipient matching model was set to rank and select appropriate donors for patients with UC. Finally, the model showed favorable classification powers (>70%) for FMT effectiveness in two previous clinical trials. This study revealed the dysbiosis of fecal bacterial diversity, composition, and predicted pathways of patients with UC by meta-analysis and hereby developed a donor-recipient matching strategy to guide donor selection for UC in FMT. This strategy can also be applied to other diseases associated with gut microbiota.IMPORTANCE Modulation of gut microbiota by FMT from donors has been applied to the treatment of UC and yielded variable effectiveness in clinical trials. One possibility is that this variable effectiveness was related to donor selection, as a patient’s response to FMT may rely on the capability of the used donor’s microbiota to restore the specific gut disturbances of the patient. However, the biggest issues on the practical level are what should be considered in the selection process and how to set up such a donor-recipient matching model. In this study, we presented a bacterial profile-based donor-recipient matching strategy to guide donor selection for UC in FMT by first meta-analysis of 656 fecal 16S rRNA gene sequencing samples from 9 studies to identify significant indicators and then setting up the model by an analytic hierarchy process. The applicability and accuracy of this model were verified in the data sets from two previous FMT clinical studies. Our data indicate that the donor-recipient matching model built in this study enables researchers to rationally select donors for UC patients in FMT clinical practice, although it needs more samples and prospective trials for validation. The strategy adopted in this study to leverage existing data sets to build donor-recipient matching models for precision FMT is feasible for other diseases associated with gut microbiota. Ulcerative colitis (UC), one subtype of inflammatory bowel disease (IBD), is a remitting and relapsing inflammatory disease comprising symptoms of abdominal pain, diarrhea, fecal urgency, gastrointestinal bleeding, and weight loss. UC has become a global disease in 21st century, with a high coalescing incidence range of 6 to 15 per 100,000 in the Western world and accelerating incidence in newly industrialized countries , many paGut microbiota, as an important environmental factor, play important roles in UC progress –8. Gut m–8–Lachnospiraceae family and Ruminococcus genus in donors . When all samples were combined, the overall gut microbial communities were significantly different between UC patients and healthy individuals  and were significantly associated with the top abundant genera, including Bacteroides, and Prevotella_9  were first compared between UC and controls. We found significantly higher Shannon and evenness (J) indexes in healthy individuals than in those with UC in 7 of 9 studies and significantly higher microbial richness in controls in 8 of 9 studies , indicatAkkermansia, Alistipes, and Odoribacter. Ten genera had significant ORs lower than 1.0 (enriched in UC), four of which were generally thought to be harmful for human health, including Enterococcus, Escherichia-Shigella, Peptostreptococcus, and Streptococcus (n = 20) than in genera (n = 8), implying the importance of microbial metabolism.In order to further identify the significantly different taxa and predicted pathways between UC patients and the healthy controls without UC, we calculated the ORs based on the common genera and pathways in each study. By quantifying the ORs, a total of 52 genera and 74 pathways had significant ORs for the UC patients  sensitivity and specificity of the 9 individual studies for detection based on the cross-validation set using the common genus were 83.5% ± 7.4% and 82.9% ± 11.7% (area under the concentration-time curve [AUC] = 88.5% ± 7.6%), respectively, after feature selection. Meanwhile, we plotted the probability distribution based on the cross-validation results of the RF model for each study and found that values of UC patients and HCs were clearly separable and significantly (P < 0.001) different in both common genera  and pathways . More importantly, when pooling all studies, we obtained good performance based on genera (AUC = 0.90) and pathways (AUC = 0.86) for classification of UC (PRJNA438164) with favorable generalization ability on both genera (AUC = 0.89) and pathways (AUC = 0.87) as well . The improvements in AUC values based on genera and pathways selected by feature selection are shown in Subsequently, we built classification models with the widely used and robust random forest (RF) algorithm based on genera or pathways with significant ORs for UC and to further refine important genera and pathways. As expected, we obtained RF models with comparable levels of performance, except for those in the on of UC  and B anviation S sensitivSix representative microbial characteristics  from meta-analysis functioned as indicators to build a donor-recipient matching model by AHP. The pairwise comparison matrix of the criteria according to the experts' suggestion is shown in Table S5. The value of the consistency ratio was less than 0.1, which was within acceptable limits. Finally, the results of AHP showed that α-diversity was the most important indicator, with a weight of 28%. The overall α-diversity of donors was also higher than that of patients. Beneficial taxa and pathways had weights of 24% and 22%, respectively. The weights of indictors, including Bray-Curtis distances, harmful taxa, and harmful pathways were lower than 13% (Table S5).PRJEB33851 study and 7 patients from the PRJNA438164 study (PRJEB33851 (P1 and P14) and PRJNA438164 (P4 and P5).For verification of the model, we downloaded and analyzed sequencing data of two previous FMT clinical trials. Using the six gut microbial indicators of patients and donors as inputs, the model returned rank order for each patient  using microbial data sets from two FMT clinical trials for UC. More encouragingly, treatments of patients who received FMTs from donors who were recommended by the matching model were all effective in clinical practice. However, we must be aware of that this matching model as an exploratory hypothesis requires more samples, especially prospective trials for verification. Taken together, this study adopting the strategy of meta-analysis and an analytic hierarchy process developed and verified a donor-recipient matching model to optimize donor selection for patients with UC in FMT clinical studies and application. Moreover, we hypothesize that leveraging of public microbial data sets to build donor-recipient matching models for donor selection is necessary and feasible to ensure that FMT has higher effectiveness and successfully translates into clinical impact.We performed a systematic literature search of the PubMed, Web of Science, and EMBASE databases up to May 2020 using the following terms: “ulcerative colitis (UC) and fecal microbiota and human and 16S rRNA” according to the “Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)” guidelines  Fig. 1AFig. 1A. 44–48–http://qiime.org/)   using thme.org/) .4 permutations in the vegan package  correction. All statistical analyses were conducted in R v.3.5.3 , Shannon diversity, and evenness J based on rarefied ZOTU tables . Signifi package . envfit models were trained for individual studies and the data set combining all studies at the common genus or pathway levels to test whether a mixture of featured taxa can predict UC. We evaluated model performance using 5-fold cross-validation and scored the predictive power in a receiver operating characteristic (ROC) analysis. To further select important bacteria taxa and pathways, we trained random forest models based on genera and pathways by two-step feature selection. First, we calculated the AUC values of individual genera and pathways. Then, on the basis of the rank of the AUC values, we executed step-by-step feature selection to generate final model . The genera and pathways that overlapped in meta-analysis and ROC analyses were recognized as the more important features for donor selection in downstream analyses.Based on results obtained from the meta-analysis on bacterial communities in this study, a total of 6 metrics with significant differences between UC patients and HCs or with significant importance for differentiating UC patients and HCs in RF models were recognized as the criteria of the donor-recipient matching model for donor selection. These 6 metrics were assigned to diversity indicators (α-diversity and distance), individual taxon indicators , and predicted pathway indicators . The following are the detailed statements of these indicators.Diversity indicators included subindicators of α-diversity that considered the donor-specific ZOTUs , the cum(i) α-diversity indicators. The α-diversity indicators were calculated as shown in α-diver represents the richness of donor-specific ZOTUs, ObsDonor indicates the bacterial richness of the donor, ObsDonor∩patient represents the number of ZOTUs shared between donor and patient, and Abuα-diverObs is the cumulative relative abundance of the donor-specific ZOTUs.(ii) Distance indicator. The distance indicator was calculated as shown by P,i and SD,i represent the count of the ith OTU in the patient and donor communities, respectively.Donor, the ratio of the number of beneficial genera that the patient lacks and are detected in the donor to the number of beneficial genera that the patient lacks  and harmful taxa (defined as ORs of <1). The absence of a beneficial taxon indicator constitutes Ratio.btnt lacks , and thent lacks . Similar patient , and the patient .(iii) Absence of beneficial taxa. The absence of beneficial taxa was calculated as shown by Donor is the ratio of the number of beneficial genera that the patient lacks and are detected in the donor (Genera.bDonorpatient_absence) to the number of beneficial genera that the patient lacks (Genera.bpatient_absence), and Abu.btDonor represents the cumulative relative abundance of Genera.bDonorpatient_absence.(iv) Presence of harmful taxa. The presence of harmful taxa was calculated by Donor is the ratio of the number of harmful genera that are detected in both the patient and donor (Genera.hDonorpatient_presence) to the number of genera that are detected in the patient (Genera.hpatient_presence), and Abu.htDonor represents the cumulative relative abundance of Genera.hDonorpatient_presence.Donor, the ratio of the number of beneficial pathways that the patient lacks and are detected in the donor to the number of beneficial pathways that the patient lacks  and harmful pathways (defined as ORs of <1). The absence of a beneficial pathway indicator consists of Ratio.bpnt lacks  and the nt lacks . Similar patient  and the  patient .(v) Absence of beneficial pathways. The absence of beneficial pathways was calculated as shown by Donor is the ratio of the number of pathways that the patient lacks and are detected in the donor (Pathway.bDonorpatient_absence) to the number of pathways that the patient lacks (Pathway.bpatient_absence), and Abu.bpDonor represents the cumulative relative abundance of Pathway.bDonorpatient_absence.(vi) Presence of harmful pathways. The presence of harmful pathways was calculated as shown by Donor is the ratio of the number of pathways that the patient lacks and are detected in the donor (Pathway.hDonorpatient_presence) to the number of pathways that the patient lacks (Pathway.hpatient_presence), and Abu.hpDonor represents the cumulative relative abundance of Pathway.hDonorpatient_presence.(vii) Calculation of the score of the donors. The scores of the donors were calculated as shown by ii ∈  represents the weight of the six indicators calculated according to the judgment matrix, and Weight (i) i ∈  represents the weight ranking of each donor calculated according to the judgment matrix for each indicator. A pseudocode is provided in Fig. S1.PRJNA438164 (PRJEB33851 (Sequencing data from two previous clinical studies, NA438164  and PRJEJEB33851 , on trea