Effective antibiotic therapy of cerebral infections such as meningitis or ventriculitis is hindered by low penetration into the cerebrospinal fluid (CSF). Because continuous infusion of meropenem and vancomycin and routine therapeutic drug monitoring (TDM) have been proposed to optimize antimicrobial exposure in ventriculitis patients, an individualized dosing strategy was implemented in our department. We present a retrospective analysis of meropenem and vancomycin concentrations in serum and CSF in the first nine ventriculitis patients treated with continuous infusion and TDM-guided dose optimization aiming at 20–30 mg/L. Median initial dosing was 8.8 g/24 h meropenem and 4.25 g/24 h vancomycin, respectively, resulting in median serum concentrations of 21.3 mg/L for meropenem and 24.5 mg/L for vancomycin and CSF concentrations of 3.4 mg/L for meropenem and 1.7 mg/L for vancomycin. Median CSF penetration was 15% for meropenem and 7% for vancomycin. With initial dosing, all but one patient achieved CSF concentrations above 1 mg/L. Dose adjustment according to TDM ensured sufficient CSF concentrations in all patients within 48 h of treatment. Given the limited penetration, continuous infusion of meropenem and vancomycin based on renal function and TDM-guided dose optimization appears a reasonable approach to attain sufficient CSF concentrations in ventriculitis patients.  Acute subarachnoid hemorrhage, intraventricular bleeding, tumors of the brain stem, and other acute intracranial pathologies may require insertion of external ventricular drains to manage hydrocephalus and monitor intracranial pressure. However, introduction of a plastic catheter into the cerebrospinal fluid (CSF) space holds the potential for intracerebral infections such as ventriculitis and/or meningitis. Ventriculitis is the most frequent complication in these patients with significant morbidity, prolonged ICU and hospital length of stay, and higher costs . The spectrum of pathogens in ventriculitis is characterized mainly by common skin microorganisms, which are mostly Gram-positive . The InfRecent data show a highly variable penetration of vancomycin and meropenem into the CSF in ventriculitis patients ,9,10,11.The present study retrospectively analyses the first nine patients suffering from ventriculitis, who received continuous infusion of meropenem and vancomycin based on renal function and TDM-guided dose optimization at our department. The objective was to evaluate meropenem and vancomycin concentrations in serum and CSF.Overall, 11 patients, all suffering from subarachnoid hemorrhage, have been identified as having received continuous dosing and routine TDM. Two of these had to be excluded, because microbiological analysis retrieved other sources of infection  with consecutive changes in antibiotic treatment. Median duration of antibiotic therapy was 13 days . All patients underwent operative revision of the external ventricular drain within the first few hours after diagnosis and at the start of antibiotic therapy, which included removal of the drain and replacement with a new external ventricular drain. An overview of patient characteristics is given in Initial dosing yielded a median dose of 8.8 g/24 h  meropenem and 4.25 g/24 h  vancomycin for the first 24 h A. This eCSF/Cserum ratio was 0.15  for meropenem and 0.07  for vancomycin. CSF/Cserum ratios as well as Cserum < 20 mg/L, resulting in insufficient CSF concentrations. For vancomycin, only patient 6 showed an insufficient CSF concentration. After dose adjustment, however, target CSF concentration was achieved in all patients. Median Cc is the volume of the central compartment, and kbc, kcb, kpc, and kcp are distribution constants, which represent the transition between the three compartments. A schematic representation of the structural model is shown in cb), 0.1642 , 14.6389 , and 13.7958 . For vancomycin, using the present pharmacokinetic model parameter adjustment failed to mimic vancomycin distribution between compartments.Values estimated in the first simulation using the model developed for intermittent application did not correlate with the observed values in the present study. We therefore added a bolus of 1 g of meropenem or vancomycin , set the infusion rate [R(t)] of the drug in relation to the volume of the central compartment, and fitted the model to the study data. These changes lead to the following equation:Measured creatinine clearance is a good predictor of meropenem clearance calculated by the pharmacokinetic model with a rank correlation coefficient according to Spearman of ρ = 1 . The adjThis retrospective analysis was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics committee of Goethe University, Frankfurt am Main, Germany (# 4/17).Patients included were diagnosed with ventriculitis secondary to subarachnoid hemorrhage, received continuous infusion of meropenem and vancomycin, and received TDM measurements between January 2016 and March 2017.http://www.eucast.org/clinical_breakpoints, accessed on 16 December 2015). All patients received an initial bolus of 1 g meropenem and 1 g vancomycin as 60 min infusion prior to continuous infusion. Initial dosing of meropenem and vancomycin was calculated by an algorithm published by Pea et al.  and adapBlood and intraventricular CSF samples were taken 24 h and 72 h after the start of continuous infusion. Further sampling was performed as needed . For traInfection and renal function parameters, fluid intake, and urinary excretion were extracted from the patients’ medical record. Creatinine serum concentrations were measured daily, and creatinine clearance was calculated using the Cockcroft–Gault equation .The analyses were performed in the laboratory of the Pharmacy Department of the Hospital of Heidenheim. Serum and CSF concentrations of meropenem were analyzed using a validated high-performance liquid chromatography (HPLC) assay with ultraviolet detection . The assStatistical analysis was conducted using R version 3.2.4 . Besides standard methods for descriptive statistics  we used a robust local regression method, where outliers are iteratively identified and down-weighted .Total and cumulative doses, serum, and CSF concentrations as well as distribution constants are given as median . The first step of the statistical analysis was to compare measured concentrations of meropenem and vancomycin with simulated values using the pharmacokinetic model and calculation developed by Blassmann et al. . This waHaving adjusted the distribution constants for each individual patient, meropenem doses for three different CSF target concentrations were estimated. These estimates were plotted to create a nomogram for initial dosing of meropenem until TDM results are retrieved. In this study, we retrospectively evaluated vancomycin and meropenem concentrations in serum and CSF of ventriculitis patients receiving continuous infusion and routine TDM. We found that all but one patient achieved CSF concentrations of meropenem and vancomycin above 1 mg/L with initial dosing based on renal function. Dose optimization according to TDM ensured sufficient CSF concentrations in all patients within 48 h. However, there was considerable variation in serum and CSF concentrations as well as resultant CSF/serum ratios. These findings are concordant with those derived from previous studies in which researchers have also described large interindividual variability . TherefoPenetration of the blood–CSF barrier for most antibiotic drugs occurs, if at all, slowly, resulting in much lower concentrations in the CSF as compared with serum concentrations. Drug as well as disease factors, such as the presence of meningeal inflammation and the integrity of the blood–CSF barrier, are important to consider when devising dosing strategies in patients with intracerebral infection . During cp and kpc were not adjusted, because there were no measured values for the peripheral compartment. Median penetration into the CSF in this study was higher than that reported in ventriculitis patients with intermittent infusion  [Higher than standard doses of meropenem and vancomycin were needed to reach target serum concentrations of 20–30 mg/L. Recommended daily doses for meropenem are 6 g in adults , while w vs. 3%) . For van vs. 3%)  However,The present study was not designed for efficacy evaluation. More work is required to better understand pharmacodynamic targets at the site of infections for patients with ventriculitis. It is unclear which pharmacokinetic–pharmacodynamic target should be used to optimize the outcome for intracerebral infections . Thus, wThese data show that continuous infusion of vancomycin and meropenem based on renal function and TDM-guided dose optimization ensured sufficient CSF concentrations within 48 h of treatment. The results suggest that this approach appears feasible and effective in cases of ventriculitis, given the limited penetration of meropenem and vancomycin into CSF. However, data are sparse and higher than standard doses are necessary. This approach should be evaluated with a larger number of patients in a prospective study in the future.


Cryptococcus neoformans is a major human central nervous system (CNS) fungal pathogen causing considerable morbidity and mortality. In this study, we provide the widest view to date of the yeast transcriptome directly from the human subarachnoid space and within cerebrospinal fluid (CSF). We captured yeast transcriptomes from C. neoformans of various genotypes in 31 patients with cryptococcal meningoencephalitis as well as several Cryptococcus gattii infections. Using transcriptome sequencing (RNA-seq) analyses, we compared the in vivo yeast transcriptomes to those from other environmental conditions, including in vitro growth on nutritious media or artificial CSF as well as samples collected from rabbit CSF at two time points. We ranked gene expressions and identified genetic patterns and networks across these diverse isolates that reveal an emphasis on carbon metabolism, fatty acid synthesis, transport, cell wall structure, and stress-related gene functions during growth in CSF. The most highly expressed yeast genes in human CSF included those known to be associated with survival or virulence and highlighted several genes encoding hypothetical proteins. From that group, a gene encoding the CMP1 putative glycoprotein (CNAG_06000) was selected for functional studies. This gene was found to impact the virulence of Cryptococcus in both mice and the CNS rabbit model, in agreement with a recent study also showing a role in virulence. This transcriptional analysis strategy provides a view of regulated yeast genes across genetic backgrounds important for human CNS infection and a relevant resource for the study of cryptococcal genes, pathways, and networks linked to human disease. Cryptococcus neoformans is an encapsulated yeast which became a major cause of meningoencephalitis during the AIDS pandemic  reC. neoformans within the central nervous system (CNS), since these are the most frequent clinical stage and site at which clinicians encounter C. neoformans and Cryptococcus gattii in the patient. Our previous work demonstrated that we can identify cryptococcal transcriptomes directly from the subarachnoid space of an animal model, the immunosuppressed rabbit , also described as Cryptococcus tetragattii  occurred in males. The mortality was 42% for cryptococcal meningitis at 72 days in this cohort. The range of CSF quantitative yeast counts from patients was from 104 to 107 CFU/ml. Those with higher CSF yeast counts (106 to 107 CFU/ml) generally yielded the most RNA and represented specimens with the highest sequencing read counts.Cryptococcal yeast cells were isolated from cerebrospinal fluid (CSF) collected from patients in two hospitals in Gaborone and Francistown, Botswana, in 2015. Samples represented the initial lumbar puncture of 34 individual patients with neurological symptoms or signs, prior to receiving any antifungal therapies. All patients had advanced HIV infection with CD4 counts of <100 μl. Of the 34 isolates, 31 were typed as ragattii . The C. MATa mating type, while the remaining 23 isolates are MATα. In fact, PMH1056 is only the fourth VNI MATa isolate reported to date from either patients or the environment worldwide (in vivo versus yeast peptone dextrose broth (YPD) exposure and did not find any enrichment for known virulence genes or pathways on these chromosomes  of all genes for all 31 hCSF samples. “Ranked median” shows values representing the rank of the gene expressed in each hCSF sample. The last column shows the medians of the gene ranks out of all 31 hCSF samples. Download Table S2, XLSX file, 2.5 MB.Gene expression ranking of all 31 good-quality Copyright © 2021 Yu et al.2021Yu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the CMP1, CIG1, ENA1, RIM101, CDA1, CQS1, and RCK2) have previously been associated with virulence or survival within a cryptococcal host model system and at least 2 (FKS1 and EF3) are essential for C. neoformans growth. Notably, multiple cell wall-associated genes are found on this list of genes most highly expressed in the CSF. The cryptococcal cell wall provides foundational yeast cell structure and integrity for the yeast, is critical for the attachment of virulence factors such as the polysaccharide capsule, melanin, and phospholipase, and represents the interface for host interactions. We observed that several genes associated with cell wall metabolism are highly expressed, including 1,3-beta-glucan synthase (FKS1), alpha-1,3-glucan synthase, glucan 1,3-beta-glucosidase, and endo-1,3(4)-beta-glucanase. Another cell wall-associated, highly expressed gene, CDA1, encodes a major chitin deacetylase that accounts for all of the chitosan produced during vegetative growth to maintain cell integrity and aids in bud separation for the yeast cell  associated with Rim101  ligase, omega-6 fatty acid desaturase (delta-12 desaturase), and stearoyl-CoA desaturase (delta-9 desaturase), are involved in fatty acid metabolism. We had previously shown that FAS1 (fatty acid synthase) is required for basic cryptococcal survival  signal transduction pathway, is critical to respond to host conditions, such as by capsule production and increased iron uptake, as well as for growth inside the infected host  without a predicted function, which supports further study for their role in pathogenesis and potentially as drug targets. Taken together, these observations show that Cryptococcus genes highly expressed in the CNS are important to the yeast.Other notable functions include several proteins linked to environmental responses or interactions. For instance, other highly expressed genes, including  factors . The quoxpressed . A predihe brain , and is C. gattii VGIV isolates to identify their highly expressed genes  expression profiles of three ed genes . After coformans  and 16 a10.1128/mBio.02313-21.3TABLE S3C. gattii hCSF sample. Genes marked with colors are the highly expressed genes overlapping between C. neoformans and C. gattii isolates. The NRH5051 column is grey because this sample was removed due to the low total read count. Download Table S3, XLSX file, 0.4 MB.List of genes sorted by expression level for each Copyright © 2021 Yu et al.2021Yu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To complement our analysis of quantitative expression rankings, we also attempted to provide a global view of gene functions that appear important to the yeast under CNS stress. Therefore, we carried out a gene set enrichment analysis (GSEA) to identify functional categories enriched in genes that are highly expressed in CSF. This analysis showed that the most significantly enriched functional terms included those involved in metabolism of carbohydrates and lipids and ion binding and metabolism , similarin vivo and in vitro conditions. We compared expression profiles for the hCSF condition to four additional conditions: rabbit CSF (rCSF), artificial CSF (aCSF), capsule-inducing medium (CAP), and rich medium (YPD). After filtering out low-quality samples , we analyzed 10 aCSF, 11 hCSF, 16 rCSF, 28 CAP, and 29 YPD samples for these condition comparisons. A principal-component analysis (PCA) of gene expression shows separation between samples from different conditions  are compared to those grown in YPD, the in vivo CSF strains form a separate and more dispersed cluster than the more uniform YPD samples (P < 0.001) between the in vivo CSF conditions and YPD showed significant enrichment for functions including transport, carbohydrate metabolism, fatty acid metabolism, and lipid processing  being upregulated within the subarachnoid space. We also found 10 genes specifically associated with oxidative and nitrosative stress both up- and downregulated between conditions  cycle, the pentose phosphate pathway, glycolysis, and complex sugar metabolism being upregulated . We chose day 1 to understand the initial stress of yeasts entering the subarachnoid space and day 4 as the time when the quantity of yeasts in the CSF seems to stabilize and is similar to that found in human CSF. Samples isolated from rCSF at day 1 and day 4 postinoculation cluster based on time within the rabbit host. Samples collected at day 4 cluster tightly compared to those collected at day 1, highlighting a converging rCSF expression profile over exposure time at the body site (P < 0.001) over 72 h, and of these, 41 genes are annotated as hypothetical proteins, 10 genes are annotated as transporters, 4 are implicated in carbohydrate metabolism, 5 encode protein processing genes, and 7 are responsive to stress  conditions. Module analysis found that isolates grown in YPD upregulate sterol metabolism, glycolysis, the TCA cycle, and oxidative phosphorylation compared to C. neoformans isolated from the limited-nutrient (stress) environments of in vivo CSF and the subarachnoid space. However, within in vivo CSF, C. neoformans upregulates fatty acid degradation and sugar transport, indicative of the increased nutrient transport required in this nutrient limited environment (in vivo CSF]). Comparison of isolates from rabbit versus human CSF in vivo revealed that C. neoformans upregulates pathways involved in mitogen-activated protein kinase (MAPK) signaling, actin regulation, and heat shock response within the rabbit model, perhaps reflective of the suddenly increased body temperatures of rabbits and acute nature of the rabbit infection compared to human disease. Isolates from human CSF upregulate the RIM pathway , RAS1 signaling, HOG1 signaling, and glycogen metabolism . This is suggestive of yeast growth arrest as the yeast attempts to equilibrate and adapt to its new site of infection  and stress  was recently characterized a putative mannoprotein, and loss of this gene resulted in an attenuated cryptococcal strain in mice    and B an < 0.01) . However the CSF . Thus, ain vivo growth and control conditions for a diverse set of isolates spanning the VNI, VNBI, and VNBII genotypes. While in many countries, clinical isolates of C. neoformans are often dominated by the VNI genotype, along with the less common VNII genotype  revealed a critical requirement of gluconeogenesis for yeast survival in the lung; however, PCK1 does not appear to be important for growth in the CSF of rabbits. In contrast, the enzyme pyruvate kinase, encoded by PYK1, which is required for glycolysis, is essential for survival in the subarachnoid space , a quorum-sensing gene, is highly upregulated in human CSF, which suggests that yeast cells may sense other yeast cells. However, in the rabbit subarachnoid space, the qsp1Δ mutant survives similarly to the wild-type yeast  shown in a recent study to be a downstream target of the C. neoformans F-box protein, Fbp1, and to encode a mannoprotein  anchors in C. neoformans such as Cmp1. For instance, an inhibitor of the Gwt1 enzyme, APX2039, has extremely potent anticryptococcal activity both in vitro and in vivo  Samples were collected from rabbit CSF (rCSF). CSF yeast cells (1 × 109) were inoculated intracisternally into 2- to 3-kg New Zealand White rabbits. Rabbits received 5 mg/kg hydrocortisone acetate intramuscularly 1 day prior to inoculation and for the duration of the experiment. Each yeast strain was inoculated into three individual rabbits. rCSF was withdrawn (1 to 2 ml) after 24 and 96 h of infection in the rabbit subarachnoid space. rCSF samples from each animal containing the same strain were pooled and centrifuged to pellet the cells, and the cell pellets were stored at −80°C. (iii) Yeast cells (1 × 105 CFU/ml) were grown for 24 h in yeast peptone dextrose broth (YPD), centrifuged to collect the cell pellet, and stored at −80°C. (iv) Capsule-inducing medium (CAP) was prepared using diluted Sabouraud broth in 50 mM MOPS  as reported in reference 4 to 107 CFU of yeasts per ml of CSF was directly withdrawn with a lumbar puncture as standard of care, and CSF remaining after clinical tests was utilized. The hCSF samples were centrifuged, and the cell pellets were stored at −80°C until RNA isolation was performed. In addition to the 31 C. neoformans isolates, RNA-seq was performed for three patient isolates that were subsequently identified as Cryptococcus gattii VGIV  and therefore excluded from the downstream analysis. Both Duke and Botswana institutional review board (IRB) approvals supported this study.RNA was isolated from five additional conditions for a subset of the isolates. (i) The first was artificial CSF (aCSF) with 1 × 10cmp1Δ mutant were obtained from a genome-wide Cryptococcus deletion library , and 3′ flanking sequence; the neomycin (NEO) drug-resistance cassette (AD2334/AD2335) amplified from pJAF1  with BWA-MEM v0.7.17 . A maximum-likelihood phylogeny was estimated using 96,943 segregating SNP sites present in one or more isolates and ambiguous in a maximum of 10% of isolates with RAxML v8.2.12 with the GTRCAT model and rapid bootstrapping (https://github.com/broadinstitute/funpipe).Whole-genome sequence was generated in a previous study  or was n GCA_0001245.3 wit v0.7.17  variant-trapping . Chromosg for 15 min. The aqueous phase was collected and mixed with an equal volume of 80% ethanol and immediately applied to a column from the Qiagen RNeasy minikit. The column was then centrifuged at 16,200 × g for 1 min. The remaining steps for RNA isolation were performed following the manufacturer’s guidelines (Qiagen).Sterile glass beads (1 to 3 mM) were added to the cell pellet prior to freezing or immediately after lyophilization. For RNA extraction, the frozen pellets were lyophilized and vortexed to a fine powder. The yeast cells were lysed in 1 ml of TRIzol (Invitrogen) followed by incubation at room temperature for 5 min. Then, 200 μl of chloroform was added, and the tubes were shaken for 30 s followed by incubation for 3 min at room temperature. The samples were centrifuged at 9,600 × in vivo (human and rabbit) and artificial CSF conditions were adapted using the TagSeq protocol (C. neoformans var. grubii H99 (CNA3) (C. gattii IND107 genome (GCA_000835755.1), following the above process to evaluate the gene expression profiles. NRH5051 was removed from the analysis of most highly expressed genes due to the very low read count. The median of the gene expression rank was then calculated from the other two samples.Libraries were constructed from total RNA using two methods. The samples from YPD and CAP conditions were adapted using the Illumina TruSeq protocol and sequenced on a HiSeq 2500 system to generate paired 101-base reads. All protocol  in whichprotocol , the reaprotocol  to the g9 (CNA3) , excludi9 (CNA3) . The raw9 (CNA3) . For VGIC. neoformans genes in human CSF, we first sorted all genes based on the normalized expression levels for each human CSF sample. We then calculated the median of the rank number for each gene across all human CSF samples. The rank of all gene expressions was determined by sorting the median rank number of the genes.To identify the most highly expressed https://gsea-msigdb.github.io/gseapreranked-gpmodule/v6/index.html) (v6.0.12) tool on a predefined ranked list of the genes. The ranked gene list was determined by aggregating the sorted gene list of all human CSF samples using the aggregateRanks function in the R package RobustRankAggreg (v.1.1) (Cryptococcus H99 Gene Ontology (GO) terms from vEUpathDb (accessed August 2020)   by apply (v.1.1) . The ranst 2020)  were usein vivo (hCSF and rCSF) versus YPD, hCSF versus rCSF, and rCSF (1 day) versus rCSF (4 day). The differentially expressed genes (DEGs) between the three condition groups were determined by implementing the negative binomial generalized linear models with Fisher’s exact tests (exactTest functions in the edgeR package)     at the Fv.1.0.7) . To assely 2021) . This enGuide for the Care and Use of Laboratory Animals  for 24 h, centrifuged, and washed twice in phosphate-buffered saline (PBS). The cells were resuspended in PBS and quantified using a T4 cell counter (Nexcelom). Equal numbers of female and male CD-1 mice (Charles River Laboratories) were infected with approximately 5 × 104 CFU per mouse via intranasal aspiration while under isoflurane anesthesia. Mice were monitored daily and observed for acute and chronic adverse symptoms. Mice were sacrificed on day 14. The brain and left lung were homogenized in 1 ml PBS for 25 s using two steel beads and a Mini-Beadbeater 16 apparatus (Biospec Products). The homogenized tissues were serially diluted, and 100 μl from each dilution was plated onto YPD containing 100 μg/ml chloramphenicol. The plates were incubated for 3 days at 30°C. Colonies were counted, and the tissue burden (CFU per gram of tissue) was determined. Fungal burden data were log10 transformed and evaluated using t tests for unpaired means . A P value of ≤0.05 was considered statistically significant.Wild-type (CM026), 8 CFU. For assessing fitness and virulence of Cryptococcus mutants, these animals were infected and cisternal taps were performed on days 3, 7, and 10 followed by enumeration of CFU in the CSF. The time series fungal burden data were then assessed by using a repeated-measures analysis of variance (ANOVA) via the aov (stats v. 4.0.2) function of the R package.New Zealand White male rabbits weighing 2 to 3 kg were treated with hydrocortisone acetate (2.5 mg/kg) by intramuscular injections daily starting 1 day prior to yeast inoculation. Animals were sedated with ketamine and xylazine and inoculated intracisternally with 0.3 ml of 1 × 10in vivo (human CSF and rabbit CSF) versus YPD, human CSF versus rabbit CSF, and rabbit CSF at day 1 versus rabbit CSF at day 4. ModuleDiscoverer (MODifieRDev v.0.1.3) was employed to identify regulatory modules from significant DEGs (Network analysis was performed with genes that were significantly differentially expressed in the following comparisons: ant DEGs . Module ant DEGs .PRJNA694643. RNA-seq data are available in the GEO database under accession no. GSE171092, and data for C. gattii VGIV isolates are available at accession no. PRJNA715187.Whole-genome sequence data for NRH5081, NRH5084, and NRH5076 can be accessed via


Data on the quantification of the potentially neurotrophic adipo-myokine METRNL (Meteorin-like protein) in human cerebrospinal fluid (CSF) are lacking and migration of this secreted protein across the blood–brain barrier (BBB) is uncertain. In the present pilot study, METRNL concentrations were quantified by ELISA in paired serum and CSF samples of 260 patients  undergoing neurological evaluation. METRNL was abundant in serum (801.2 ± 378.3 pg/mL) and CSF (1007.2 ± 624.2 pg/mL) with a CSF/serum ratio of 1.4 ± 0.8. Serum METRNL levels were significantly correlated (rho = +0.521) to those in CSF. CSF METRNL concentrations were significantly correlated (rho = +0.480) with albumin CSF/serum ratios. The CSF/serum ratios of METRNL and albumin were positively correlated in Reibergram analysis (rho = 0.498), indicating that raising CSF concentrations of METRNL are mediated by increasing BBB dysfunction. The CSF concentrations of METRNL strongly increased in a stepwise manner along with increasing BBB dysfunction from grade 0 to grade 3 and with rising CSF cell count. CSF/serum ratio of METRNL also increased from grade 0 (1.2 ± 0.7) to grade 3 (3.0 ± 0.2). Furthermore, CSF levels were positively correlated with age. In conclusion, METRNL is a secreted and neurotrophic myokine that crosses over the BBB. CSF concentrations of METRNL increase with BBB dysfunction. Thee leptin ,6,7,8 sumuscle–brain axis [myokines” [adipo-myokines” such as myonectin, myostatin, IL-6, IL-8, IL-15, FGF-21, irisin, and sclerostin [Similarly, the concept of a ain axis ,10 has byokines” ,10,11 orlerostin . The muslerostin , many oflerostin ,14. Exer-/- mice are viable, but have a dysregulated cytokine production and are highly susceptible to LPS in a sepsis model [Meteorin-like protein has been discussed as a novel adipo-myokine secreted by adipocytes and muscle cells ,16. Exeris model .muscle–brain axis with a translational impact .Interestingly, METRNL has been suggested to act as a neurotrophic factor with therapeutic potential in neural development . However-n = 260) with various diseases who underwent neurological evaluation, including lumbar puncture in a single and tertiary care centre;to quantify the protein concentrations of the neurotrophic adipo-myokine METRNL by ELISA in paired serum and CSF samples from a well- characterized cohort of patients  and lipid parameters  were measured by standard methods at the Institute of Clinical Chemistry and Laboratory Medicine, University Hospital of Giessen, Germany. Total cell count, total protein, albumin, glucose, lactate, ferritin, and immunoglobulins G, A, and M were measured in serum and CSF by using standard techniques in the Neurochemical laboratory of the Department of Neurology, University Hospital of Giessen, Germany. The basal anthropometric and laboratory characteristics were published earlier by our group c and lip,7,8. CSFThe collected paired samples of human serum and cerebrospinal fluid were stored at −20 °C in aliquots of 150 µL. Serum and CSF levels of METRNL were measured in duplicate by high-sensitive enzyme-linked immunosorbent assays (ELISA) purchased from R & D Systems, Wiesbaden, Germany (Human Meteorin-like/METRNL DuoSet ELISA). The assay range was 15.6–1000 pg/mL.SPSS 27.0. METRNL serum concentrations did not follow a Gaussian distribution. Non-parametric numerical parameters were analyzed by Mann–Whitney U-test (for 2 unrelated samples) and Kruskal–Wallis test (>2 unrelated samples). Correlation analysis was performed by using the Spearman rho test (non-parametric parameters). Partial correlation analysis was applied to control for possible covariates. A p-value below 0.05 (two-tailed) was considered as statistically significant.For statistical analysis, data were exported into the statistical software program A total of 260 patients, 107 males (41.2%) and 153 females (58.8%), were included (mean age 50.5 ± 17.5 years) in the study . Of the We were able to detect METRNL by ELISA both in the serum and CSF. The concentrations of METRNL could successfully be measured in serum and CSF of all patients in duplicate. In serum, METRNL concentrations ranged from 203.1 pg/mL to 2685.1 pg/mL. The mean value was 801.2 pg/mL with a standard deviation of 378.3 pg/mL. In CSF, METRNL ranged from 230.0 to 4275.3 pg/mL. The mean CSF concentration was similar to the respective serum levels with 1007.2 ± 624.2 pg/mL. For the first time in the literature, we are able to provide data that are suitable to calculate a specific CSF/serum ratio for METRNL. This ratio ranged from 0.2 to 6.7 (mean: 1.4 ± 0.8) .p = 0.031), whereas there was a non-significant trend (p = 0.052) to higher METRNL concentrations in CSF (1071.2 pg/mL ± 603.4 pg/mL) when compared to females . Patients with hypertension had significantly higher METRNL concentrations in CSF (1117.5 pg/mL ± 673.1 pg/mL) than normotensive subjects (929.6 pg/mL ± 575.2 pg/mL) (p = 0.023)  (= 0.023) . There w= 0.023) .p = 0.011) but not with leukocyte count. METRNL concentrations in CSF were correlated significantly and positively with several markers of BBB dysfunction  such as total protein , albumin , immunoglobulins , IgG , IgA ), and lactate .p < 0.001) significantly increased with increasing BBB dysfunction in a stepwise manner from grade 0 to grade 3 (p < 0.001) in a stepwise manner along with increasing blood–brain barrier (BBB) dysfunction. There was no significant difference between the subgroups of neurological disease entities. These data indicate, for the first time, that CSF METRNL concentrations are dependent on the integrity of the BBB and that leakage of the BBB increases the respective CSF concentrations. Furthermore, higher counts of CSF immune cells were associated with elevated CSF and serum METRNL concentrations (METRNL concentrations in CSF ( grade 3 . Accorditrations . In thisSpearman rho test. The serum METRNL concentrations were significantly (p < 0.001) correlated (rho = +0.521) with the respective CSF concentrations. This indicates that individuals with higher serum METRNL levels overall have a significant trend to higher CSF concentrations. In more detail, the calculated albumin CSF/serum ratio (as a common and widely used predictor of BBB permeability) was significantly associated with CSF METRNL concentrations  . This finally supports the role of BBB permeability for the migration of METRNL over the BBB into the central nervous system. Since BBB permeability increases with age, the observed correlation between age and CSF METRNL concentrations is reasonable and relevant from a clinical point of view.< 0.001) B. This i albumin C. The CSAdipo-myokines are of physiological relevance in the context of the metabolic syndrome (MetS). The MetS is characterized by obesity, high triglycerides and low HDL cholesterol, hypertension, and insulin resistance/type 2 diabetes mellitus. Since these clinical parameters were available in the patients, we tested whether METRNL was correlated to single entities of the MetS. Neither serum nor CSF METRNL levels were correlated to BMI, triglycerides, HDL cholesterol, LDL cholesterol, or the occurrence of type 2 diabetes mellitus. As mentioned above , CSF MET−3), resistin (4.4 × 10−3), adipsin (27 × 10−3), RBP4 (8.2 × 10−3), progranulin (16.3 × 10−3), clusterin (29.6 × 10−3), PEDF (42.3 × 10−3), or CTRP-3 (110 × 10−3). Based on these calculations, the high basal permeability of the BBB for METRNL explains the high concentrations of this adipo-myokine in CSF, which are similar to the respective serum concentrations. It is of high clinical interest why the myokine METRNL has such a high BBB permeability when compared to classical adipokines mentioned above. One explanation could be the fact that METRNL is a relatively small peptide that does not undergo an assembly into higher order molecular structures  as it was described for adiponectin or CTRP-3. Since the expression and secretion of METRNL strongly increases during and after muscle contraction or exercise [myocyte–brain axis.The present data clearly indicate for the first time that there exists a very high grade of both basal and inducible permeability of the blood–brain barrier (BBB) for METRNL. The basal permeability was proven by the highly significant correlation between serum and CSF METRNL concentrations and by specific calculation of the relatively high CSF/serum ratio of 1.4 ± 0.8. When compared to other adipokines ,7,8, thiexercise ,23,24,25Reibergrams. Finally, a detailed laboratory grading system (0–3) was used for a specific description of the BBB. We found a highly significant increase in CSF METRNL levels along with increasing grades of BBB dysfunction and with rising CSF cell count . If BBB permeability increases , METRNL permeability also increases in parallel. Taken together, these results are novel and prove that METRNL fulfils the basal criteria of a novel and putative mediator of the muscle–brain axis.Moreover, the inducible permeability of the BBB for METRNL was shown by the highly significant correlation of the widely used albumin CSF/serum ratio with CSF METRNL concentrations. In addition, the albumin CSF/serum ratio was also significantly correlated with the METRNL CSF/serum ratio as shown in our classical In general, a putative autochthonous production of METRNL in cells of the central nervous system such as microglial cells has to be considered; however, data are extremely scarce. Activated microglial cells play a role in cytotoxic and inflammatory processes of neurodegenerative diseases as well as during aging. We remember that CSF METRNL levels were associated with increasing age in the present study. Although cells of the central nervous system might be able to express METRNL, a significant autochthonous production in CSF by ependymal/microglial cells is not probable on the basis of the present data and the CSF/serum ratios. A relevant autochthonous production could not explain the clear correlations of CSF levels with the serum concentrations and the albumin ratio. As a first step to detect any implications of METRNL concentrations for neurological diseases, we investigated 7 disease groups/entities. The limitations of the observational character of this study have to be kept in mind. METRNL serum or CSF concentrations were not altered in disease subgroups. However, future prospective studies are necessary to prove or to exclude the role of serum or CSF METRNL concentrations as a biomarker for disease. The limitation of this pilot study is given by the wide variety of neurological disease subgroups. The results are encouraging, but it is necessary to conduct prospective and confirmatory studies on more homogenous cohorts of neurological patients on the one hand, and on cohorts of patients with specific diseases (such as multiple sclerosis) on the other hand.muscle–brain axis.The present study provides, for the first time, a systematic quantification and biostatistical analysis of paired serum and CSF concentrations of the neurotrophic adipo-myokine METRNL. This work could provide a basis for the development of human reference ranges and for the establishment of disease-specific biomarkers. The blood–brain barrier was shown to be permeable for METRNL under basal and under inducible conditions when an increasing blood–brain barrier dysfunction causes increasing CSF METRNL concentrations. METRNL represents a novel and promising neurotrophic adipo-myokine and a putative mediator of the


The contrast sensitivity function (CSF) describes how sensitivity (1/contrast threshold) to narrow-band stimuli varies with spatial and/or temporal frequency. It provides a fundamental characterization of spatial or temporal vision, and reflects the combined effects of limiting factors from the optics of the eye all the way to the response properties of cortical neurons. As the boundary between the visible and invisible narrow-band visual stimuli, the CSF correlates better with real-world daily visual functions than visual acuity (VA). This Research Topic, consisting of a collection of 13 fundamental and clinical oriented research articles, extends our knowledge related to the CSF.Camillo et al. found that the contrast and temporal frequency tunings of mouse V1 neurons are inseparable. The divisive contrast-gain normalization model provided a good fit to their data at high temporal frequencies, but not at low temporal frequencies. The results suggest that different normalization mechanisms exist in the mouse visual cortex and may underlie the different relationships between temporal frequency and spatial contrast tunings at different temporal frequencies.How stimulus contrast at different spatial or temporal frequencies affects the response properties of the visual system is the core of the CSF. Negishi and Shinomori found that weak color (low saturation) had a stronger inhibitory effect on contrast sensitivity than strong color (high saturation), and the interaction between chromatic and achromatic signals happened in V1. Contrast processing can also be improved by perceptual learning. Xi et al. measured ERP contrast response functions before and after 10 days of perceptual learning. They found that training significantly improved VA and contrast sensitivity, and led to changes at different stages of visual processing.Using psychophysics and functional MRI, Rosenkranz et al. validated the performance of qCSF, a Bayesian adaptive method for efficient CSF measurement . They found that among all the measures of visual function, the CSF correlated best with anatomic features of the retina. Li et al. evaluated the effects of different myopia control lenses on the CSF and VA in a group of myopic children. They found that the lenses with concentrically arranged aspherical lenslets had the least influence on the CSF and VA. Min et al. evaluated effects of Eyetronix Flicker Glass (EFG) treatment on children with amblyopia on the CSF and VA, and concluded that the EFG can be an additional choice for amblyopia therapy. Cheng et al. showed that 0.01% atropine had no significantly detrimental effect on the CSF in myopic adults, suggesting that 0.01% atropine might be safely used for myopia control. Finally, Stalin et al. assessed visual functions and skiing performance in elite skiers with visual impairments, and found that the CSF did not add any predictive value on skiing performance.The CSF is an important functional vision assessment in the clinic. A series of studies on various clinical populations further demonstrated its importance. Tardif et al. examined this putative idea and concluded that the Campbell-Robson chart did not accurately quantify the CSF. Xiong et al. provided a method to simulate visual quality of patients with low vision based on clinical measures of VA and CSF. Their method successfully predicted reading performance and text visibility under a wide range of low vision conditions. Xu et al. developed a Bayesian adaptive method (qVFM) to estimate the contrast sensitivity map over the visual field. They demonstrated that the qVFM method could accurately and efficiently evaluate the contrast sensitivity visual field map in normal observers. Finally, because current CSF tests are too boring to sustain young children's interest, Elfadaly et al. developed a gamified, child-friendly CSF assessment and showed that it successfully assessed CSF in young children.In clinical settings, it is important to measure the CSF quickly, accurately and reliably. Some of the submitted works explored new methods for efficient CSF measurement. The Campbell-Robson chart is widely used to illustrate the CSF as the boundary between the visible and invisible narrow band visual stimuli, and this border might be used to estimate the CSF. However, Altogether this collection of articles emphasizes the importance of measuring the CSF to assess visual function in both basic research and clinical settings. It presents some methods to perform and improve those measures, and considers their interpretation and implications.All authors contributed to manuscript writing and revision, read, and approved the submitted version.This work was supported by the National Natural Science Foundation of China (NSFC81600764 to FH) and the Department of Human Resources and Social Security of Zhejiang Province .PB and Z-LL hold equity shares in Adaptive Sensory Technology, Inc. and have patents related to the qCSF technology. 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.


TREM2 gene plays a pivotal role in microglial activation, has been identified as a risk factor for AD. In this work, we aimed to assess the interrelationships of soluble TREM2 (sTREM2) level in cerebrospinal fluid (CSF), minimal depressive symptoms (MDSs), and CSF amyloid markers. The linear regression analyses were conducted on 796 cognitively unimpaired participants from the CABLE (Chinese Alzheimer’s Biomarker and LifestylE) study. Causal mediation analyses with 10,000 bootstrapped iterations were used to test the mediation effects. In addition, similar statistical analyses were performed in subgroups stratified by sex, age, and APOE ε4 carrier status. In total subjects, MDSs were associated with lower CSF sTREM2 levels (p < 0.0001), lower CSF amyloid markers (p < 0.0001), and poorer cognitive performance . The influence of MDSs on CSF amyloid markers was partially mediated by CSF sTREM2 . And we found that the sTREM2-amyloid pathway partially mediated the effects of MDSs on cognition. Of note, exploratory subgroup analyses showed that the above influences of CSF sTREM2 were pronounced in the APOE ε4 (−) group. These results suggest that early depression is associated with amyloid pathology, which might be partly mediated by microglial activation, especially in the absence of APOE ε4.The effects of microglial activation on the associations between depression and Alzheimer’s disease (AD) are still unclear.  Alzheimer disease (AD) is the most common cause of dementia, and nearly all patients had accompanied neuropsychiatric symptoms in the very early stage of the disease . DepressTREM2 is an immune-related gene expressed in microglia, promoting microglial phagocytosis, chemotaxis, and survival [Previous studies have reported that activation of immune mediators is an important regulator of the AD pathology, and dysfunction of immune-microglia molecular network is one pathogenesis of AD  as well survival , 10 and survival . The solsurvival , 13. In survival , lower asurvival , and gresurvival . Togethesurvival , suggestAPOE ε4 status; (3) to assess whether the association of MDS with amyloid pathology was mediated by CSF sTREM2. To achieve these aims, we used MDS to explore these associations since it represents an early manifestation of depression, and all these analyses were performed in non-demented participants since they were at the preclinical stage of AD, which allowed us to investigate very early inflammatory changes associated with early stages of depression.Therefore, we aimed: (1) to analyze the differences in CSF sTREM2 between participants with MDS and normal participants; (2) to explore the interrelationship of MDS with CSF sTREM2 and CSF amyloid markers, as well as its interaction with age, sex, and All non-demented participants in this study were included from the Chinese Alzheimer’s Biomarker and Lifestyle (CABLE) cohort, which was an ongoing large and independent cohort focusing primarily on the biomarkers and risk factors of AD. Participants in the CABLE study were recruited from Qingdao Municipal Hospital, Shandong, China. All eligible participants were required to be 40–90 years old and to be of Han Chinese origin. Participants with a history of major neurological disorders, major psychological disorders, malignant tumors, or genetic disorders were excluded. In this study, additional exclusion criteria also included the medical history and medication records of depression. All enrolled participants were required to undergo comprehensive clinical, neuropsychological, psychosocial, and psychiatric evaluations, and their biological sample (blood and CSF sample) were collected by doctors with standardized training. The CABLE database was approved by the ethic committee of Qingdao Municipal Hospital and was accorded with the Helsinki Declaration. All the participants provided written informed consent.Depressive symptoms were measured using the 17-item version of the Hamilton Depression Rating Scale. Eligible participants were classified into normal group (absence of depressive symptoms) and MDS group. Our definition of MDSs in participants was consistent with its definition as a Hamilton Depression Rating Scale score ≥1 and ≤7 by previous studies , 19. Theg for 10 min and stored in freezers at −80 ˚C. Before testing CSF samples, the thaw/freezing cycle was controlled within 2 cycles. CSF AD biomarkers  and CSF sTREM2 were measured on the microplate reader . CSF sTREM2 measurements were conducted using the enzyme-linked immunosorbent assay (ELISA) kit , and CSF AD biomarker measurements were done with other ELISA kits . All ELISA measurements were tested by experienced technicians who were blinded to the basic information of patients. CSF samples and standards were measured in duplicate, and the means of duplicates were used for the statistical analyses. The within-batch precision values were <5% for all proteins and the inter-batch coefficients of variation were <15%. The ratio of Aβ42 to Aβ40 was used to assess the levels of pathologic species while accounting for individual differences in amyloid production. The t-tau/Aβ42 and p-tau/Aβ42 ratios were used to predict cerebral Aβ deposition and cognitive decline because they were considered as better predictors than those expressed alone [CSF samples were collected by lumbar puncture into 10 ml polypropylene tubes, and they were sent to the lab within 2 h. All CSF samples were centrifuged at 2000 × APOE ε4 genotyping was conducted with restriction fragment length polymorphism technology.The blood samples were used to extract DNA using QIAamp DNA Blood Mini Kit . The extracted DNA was amplified by the polymerase chain reaction with forward primers 5′-ACGGCTGTCCAAGGAGCTG-3′ (rs429358) and 5′-CTCCGCGATGCCGATGAC-3′ (rs7412). The extracted DNA was then separated and stored at −80 °C until the U test (for continuous variables).Extreme values of CSF biomarkers were excluded (outside three SDs). We evaluated the normality of distribution for continuous variables using Shapiro–Wilk test, and those that did not follow a normal distribution were normalized by the Box-Cox transformations via “car” package of R software and were standardized by z-scale. The differences in baseline characteristics were tested by 1-42, tau, and p-tau181), and cognition (dependent variables) in total participants. Sex, age, education, and APOE ε4 status were added as covariates in all MLR models. In addition, we further extended our analyses into subgroups stratified by sex, age (<65 years or ≥65 years), and APOE ε4 status  to analyze the differences between normal group and MDS group.Data Analyses with Bootstrap-coupled ESTimation (DABEST) were used to compare the differences in CSF sTREM2 levels . These Dwww.processmacro.org) to perform these multiple mediator models [Mediation analyses of the single mediator (“mediate” package in R 4.1.0 software) were used to assess whether CSF sTREM2 could modulate the association between MDSs and amyloid pathology based on the method proposed by Baron and Kenny . For eacr models . With thp value < 0.05 was considered statistically significant. The “dabestr”, “lm”, “mediate”, and “car” packages in R version 4.1.0 software and IBM SPSS Statistics 23 were used to perform the above analyses. The R version 4.1.0 software and GraphPad Prism version 9.00 were used for the above figure preparation.A two-sided APOE ε4 carrier proportion of 17.5%. Individuals with MDSs tended to be older and they were more likely to have lower levels of CSF sTREM2, poorer cognitive performance, and greater amyloid deposition  subgroups, but not in the APOE ε4 carrier subgroup .We found that participants with MDSs had a significantly lower CSF sTREM2 than those in the normal group  Fig. . To invep = 4.34 × 10−5), and a similar association existed in male (p = 0.0097), female (p = 0.0012), late-life (p = 0.0001), and APOE ε4 non-carrier (p = 3.16 × 10−5) subgroups, but not in mid-life and APOE ε4 carrier subgroups (p > 0.05). Considering a significant association between depressive symptoms and anxiety symptoms (p < 2 × 10−16), we further added anxiety symptoms as a covariate in our model. Consistent with previous findings, we found the association between MDSs and CSF sTREM2 remained significant after controlling for anxiety symptoms or/and cognitive scores , Aβ42/40 , Tau/Aβ42 , and Ptau/Aβ42 . In subsample analyses, the above associations remained significant , which also existed in female , mid-life , and APOE ε4 non-carrier subgroups , rather than male, late-life, and APOE ε4 carrier subgroups  (Table p > 0.05). Overall, these findings demonstrated that MDSs were significantly related with cerebral amyloid deposition and cognitive impairment in the early stage of AD.We used MLR models to explore the associations of MDSs with CSF AD biomarkers and cognition. In these models, MDSs were found associated with Aβ42 (nt Table . As for 5) Table . HoweverAPOE ε4 non-carrier (Fig. APOE ε4 non-carrier subgroup (Fig. Based on the preceding results, we then tested the main hypothesis around whether CSF sTREM2 was involved in the association between MDSs and amyloid pathology. We performed mediation analyses on CSF Aβ42, and the ratios of Aβ42/40 and Ptau/Aβ42 rather than the Tau/Aβ42 ratio because it did not show a significant association with CSF sTREM2 Table . Resultsier Fig. , late-liier Fig. , male (Fier Fig. , and femier Fig.  subgroupier Fig.  and APOEoup Fig.  (ie, MDSAPOE ε4 status may modulate this association. We also found that the association of MDSs with amyloid pathology was partially mediated by CSF sTREM2. Of note, this mediation effect further led to cognitive decline. These findings support the hypothesis that lower CSF sTREM2 related to microglial activation is associated with abnormal amyloid pathology and worsened cognition in MDS participants (Fig. This is the largest-scale study to assess the interrelationships between MDSs, CSF sTREM2, CSF amyloid biomarkers, and cognition among non-demented adults. Our primary result showed that the MDS group had significantly lower levels of CSF sTREM2 than the normal group. There was a strong association between MDSs and lower levels of CSF sTREM2, and the interaction analyses showed that Our findings are consistent with a recent longitudinal study which showed that the levels of CSF sTREM2 were significantly lower in MDD individuals than controls , suggestEmerging evidence has demonstrated that epidemiological sex differences in depression and dementia are well characterized, and to be specific, both diseases are more common in females than males . PreviouSeveral studies suggested that the incidence of depression has reached its peak in people aged 80 years or older , and latAPOE ε4 allele is thought to be implicated in microglial responses and it has been identified as a risk factor for depression and AD [APOE ε4 on CSF sTREM2, and the association between MDSs and CSF sTREM2 was only significant in APOE ε4 non-carriers. To our knowledge, this has not been reported before. Multiple hypotheses may explain these observations. There is an over-activated microglial response that may be presented prior to the onset of depressive symptoms due to the role of APOE ε4 in systemic inflammatory response [APOE ε4 complexes may disturb depression-sTREM2 pathway in the presence of APOE ε4 [n and AD , 39. In response . At this APOE ε4 , 41, as  APOE ε4 . HoweverSeveral biological mechanisms have been proposed to explain how depressive symptoms affect CSF sTREM2 and amyloid pathology. Firstly, persistent chronic stresses with depressive-like condition have been implicated in impaired microglial function and suppressed hippocampal neurogenesis , and theThere are two strengths of this study. First, the use of non-demented participants made our results highly sensitive to the preclinical stage of AD. Second, we were the first to reveal the impact of sTREM2 on the relationship between depression and amyloid pathology. However, some limitations also need to be taken into account in this study. Firstly, depression in this study was restricted to MDSs, which may limit the interpretability of associated results with depression. Secondly, the question that whether CSF sTREM2 represents a direct measure of TREM2 or a soluble form of TREM2 is still controversial. Thirdly, all our findings are based on CSF data that are less accurate than PET imaging for measuring cerebral neurodegeneration. Last, although our models suggested a mediation analysis best described the association between depression and amyloid pathology, we cannot conclude the causality of this association due to the limitation of mediation analysis and cross-sectional data that we use.In conclusion, our results showed that CSF sTREM2 is a key mediator in the associations of depression with amyloid pathology and cognitive function. This provided clinical implications for the treatment of depressive individuals in the prodromal stage of AD, since targeting sTREM2 may reduce amyloid deposition and improve cognitive function. Together, our results indicated that TREM2 could serve as a promising target for future AD treatment.Supplementary materials


Borrelia-specific antibodies. In most cases, the presence of intrathecally produced Borrelia-specific antibodies is determined by using an enzyme-linked immunosorbent assay (ELISA). The edge effect is a known phenomenon in ELISAs and can negatively influence the assay reproducibility and repeatability, as well as index calculations of sample pairs which are tested in the same run. For LNB diagnostics, an index calculation is used for which the relative amounts of Borrelia-specific antibodies in CSF and serum are measured to calculate a CSF/serum quotient, which is needed to calculate the Borrelia-specific antibody index (AI). The presence of an edge effect in an ELISA used for LNB diagnostics may thus have implications. In this study, we investigated the intra-assay variation of the commercial Enzygnost Lyme link VlsE/IgG ELISA used for LNB diagnostics and showed the presence of an edge effect. Minor adaptations in the ELISA protocol decreased this effect. The adapted protocol was subsequently used to test 149 CSF-serum pairs of consecutive patients received in a routine diagnostic laboratory. By simulation, we showed that, if the standard protocol would have been used, then the edge effect for this study population could have resulted in 15 (10.1%) false-pathological and two (1.3%) false-normal Borrelia-specific IgG AIs. Thus, the observed edge effect can lead to inaccurate LNB diagnoses. Our study underlines that the edge effect should be investigated when ELISAs are implemented in routine diagnostics, as this phenomenon can occur in any ELISA.The diagnosis of Lyme neuroborreliosis (LNB) is based on neurological symptoms, cerebrospinal fluid (CSF) pleocytosis, and intrathecally produced  The annual incidence rate of Lyme neuroborreliosis (LNB), a manifestation of the nervous system, was 2.6  per 100,000 Dutch inhabitants in 2010  and serum followed by an antibody index (AI) calculation as described by Reiber and Peter  between inner (lower OD) and outer (higher OD) wells of an ELISA plate and are referred to as the “edge effect”  and recombinant VlsE from B. afzelii, Borrelia garinii, and Borrelia burgdorferisensu stricto (B. burgdorferi), which was subsequently divided over 30 wells. The remaining wells were tested with the negative kit control (human serum without antibodies to B. burgdorferi) , here referred to as the Enzygnost IgG ELISA, was investigated. The Enzygnost IgG ELISA consists of a 96-well plate holder harboring 12 strips, each consisting of 8 wells coated with a mixture of deactivated  stricto . In thisdorferi) .In a second experiment, the intra-assay variation of the Enzygnost IgG ELISA was further investigated. The second experiment was identical to the first except for an additional strip filled with distilled water, which was placed in the fifth column of the 96-well plate holder . Both exIn a third experiment, the intra-assay variation of the Enzygnost IgG ELISA was assessed by using a different ELISA robot. This experiment resembled the first; however, a single dilution (1:231) of the positive kit control was tested in 6 strips (48 wells) that were placed in the first 6 columns of a 96-well plate holder . The negTo assess the intra-assay variation of the Enzygnost IgG ELISA, the intra-assay coefficients of variation (CV) of the OD values of the positive kit control were calculated by using the formula CV =  × 100%. The CVs were calculated for each column, row, and plate. The negative kit control was not included in any of the analyses performed.post hoc test (https://CRAN.R-project.org/package=PMCMRplus). Raw P values of <0.05 were interpreted as statistically significant; however, to account for the multiple statistical analyses in this study, the Benjamini-Hochberg procedure (BH) was applied. Therefore, the false-discovery rate (FDR) was set at the level of 5.0%, and less than one false-positive result was allowed in the list of rejections  for manual testing or testing with the automated BEP III system , each CSF-serum pair should be tested in consecutive wells, column-wise, starting with the serum sample; and (ii) for testing with the automated BEP 2000 system , all serum samples of the patients to be tested should be tested first in consecutive wells and column-wise, followed by all the corresponding CSF samples .For use in our validation study (see next paragraph), the standard protocol was adapted in the following two ways before the patient samples were tested: (i) two strips filled with distilled water were added in each run, one located before the first strip, the other located after the last strip; and (ii) all CSF-serum pairs were tested row-wise in consecutive wells. For all other steps, the standard protocol was used .Borrelia-specific CSF/serum IgG quotient, we followed the instructions of the manufacturer  the CSF and serum concentration must lie within the measurement range, and (ii) the concentration quotient must lie between 1.0 and 3.0 . In this validation study, all consecutive patients were included from whom a CSF-serum pair, taken less than 24 h apart, was sent to the laboratory of the Diakonessenhuis Hospital, Utrecht, the Netherlands, in the period between August 2013 and June 2016. Until the start of this study in 2017, leftover material from these patients (both CSF and serum) had been stored at −20°C and/or −80°C. Prior to the start of this study, all samples had been freeze-thawed once to aliquot for use in multiple assays for the larger validation study and were stored at −20°C until use. Patients for whom an insufficient amount of CSF and/or serum was available were excluded. In total, 156 consecutive patients were included in this study, and the CSF and serum pairs of 149 of them were tested by using the Enzygnost IgG ELISA. Seventy-one (47.7%) of the 149 patients were male, and their mean age was 52.0 years (95% CI: 49.4 to 54.7). Twenty-nine (19.5%) patients had pleocytosis (≥5 leucocytes/μl) based on the CSF cell counts that had been determined at the time of the lumbar puncture in the past.To investigate the possible consequences of using the standard protocol for the measurement of All CSF and serum pairs used in this study were anonymized and, according to the rules of our hospital, approval of the local ethics committee was not necessary as the main goal of our study was assay validation for which leftover material could be used. We did, however, obtain approval for this study from the hospital board.Borrelia-specific IgG AI. For instance, if the CSF of a CSF-serum pair is tested in a well located in a certain position in the plate that structurally leads to higher OD values than a well located elsewhere in the same plate, then this could result in a higher concentration, a higher Borrelia-specific CSF/serum IgG quotient, and a higher Borrelia-specific IgG AI. This could thus also affect the interpretation of the AI. To gain insight into the consequences thereof, we used the Enzygnost IgG results of the 149 consecutive patients that were obtained by using the adapted protocol. In this adapted protocol, CSF and serum of a CSF-serum pair were tested in such a way to minimize the intra-assay variation of the standard protocol of the manufacturer. By using these Enzygnost IgG results, we constructed so-called simulated Enzygnost IgG results that could have been obtained when the standard protocol would have been used, and either CSF or serum of a CSF-serum pair was tested in a well that was subjected to the intra-assay variation. To construct these simulated Enzygnost IgG ELISA results, the raw OD values of either the CSF or the serum of the CSF-serum pairs (obtained by using the adapted protocol) were subjected and adjusted to the intra-assay variation found in the first experiment. Therefore, we determined a factor that reflected the magnitude of the intra-assay variation. As the intra-assay variation was most prevalent between the columns in the first experiment, we based the factor on the largest difference in median (raw) OD value of the positive kit control found between any of the two columns. This factor was calculated by dividing the highest median OD value of any of the four columns by the lowest median OD value of any of the four columns. By using this factor, two scenarios were investigated. In the first scenario, the CSF of a CSF-serum pair was simulated as if it had been tested by using the standard protocol in a well in the column that displayed the highest median OD value. Therefore, the raw OD value of the CSF, obtained by using the adapted protocol, was adjusted through multiplication by the factor reflecting the magnitude of the intra-assay variation. In this scenario, the raw OD value of the serum, obtained by using the adapted protocol, was not adjusted. In the second scenario, the serum of a CSF-serum pair was simulated as if it had been tested by using the standard protocol in a well in the column that displayed the highest median OD value. Therefore, the raw OD value of the serum, obtained by using the adapted protocol, was multiplied by the factor reflecting the magnitude of the intra-assay variation. In this scenario, the raw OD value of the CSF, obtained by using the adapted protocol, was not adjusted. Subsequently, in both scenarios, the raw OD values were corrected, the concentrations were calculated, the simulated Borrelia-specific CSF/serum IgG quotients were determined, and, hence, the simulated Borrelia-specific IgG AIs were calculated according to the instructions of the manufacturer   , 14. By To visualize the intra-assay variation of the Enzygnost IgG ELISA, heat maps of the OD values obtained in the first and second experiment were constructed, which showed that higher OD values were found in the outer wells than those in the inner wells  and B. T5–Further analyses showed that the OD values in the wells in the outer columns (columns 1 and 4 in the first experiment and column 1 in the second experiment) were higher than those in the wells in the inner columns  and B. IThe third experiment was similar to the first experiment except for the use of a different automated ELISA instrument and the number of wells (48 instead of 30) filled with the positive kit control . Again, Borrelia-specific IgG production was established. For 107 (71.8%) of the 149 patients, no detectable levels of Borrelia-specific IgG in the CSF were found, and, therefore, no AIs were calculated  could be tested in an outer well, and the other material could be tested in an inner well . Consequlculated . Of thosBorrelia-specific IgG AI  of the 149 patients, an AI was calculated. The majority of them  had a normal c IgG AI  of whom ilutions . For 11 ific IgG , and 6  by the lowest median OD value  . This reBorrelia-specific IgG AI values greater than or equal to 1.5 increased from 11 to 26 . Ideally, these CSF-serum pairs should be repeated in a next ELISA run by using different dilutions  OD values and might not indicate disease. The relationship between the OD values (plotted on the y axis) and the (log-transformed) concentrations (plotted on the x axis) is described by a sigmoid-shaped curve. When the OD values of both materials are either located in the lower left corner or the upper right corner of the curve, small differences between the OD values will result in larger differences between the concentrations, as is reflected by an almost horizontal line in the sigmoid-shaped curve in both corners. Thus, when the OD value of the serum is located in the lower left part of the curve on the horizontal line , or when the OD value of the CSF is located in the upper right part of the curve on the horizontal line , the difference in concentration will be larger, which will result in an increased CSF/serum quotient as well as an increased AI. Therefore, AIs should be interpreted with care.The Borrelia-specific CSF/serum IgG quotient. Consequently, for 17 (40.5%) of the 42 patients for whom an AI was calculated by using the adapted protocol, a different set of dilutions was used in at least one of the two scenarios in which the standard protocol was simulated (data not shown). Analyses of the XY-plots showed that the distribution of the OD values of CSF and serum used for the index calculation were comparable between the three protocols  show the distribution of the OD values of the 149 CSF-serum pairs  to F. Inrotocols  to F. Forotocols . The OD rotocols  to F. Sirotocols .Borrelia-specific IgG in CSF-serum pairs  and for 9 out of 149 (for whom the serum was simulated to be tested in an outer well) CSF-serum pairs. In practice, by using the standard protocol, more dilutions would have been needed to obtain valid results for these cases.In this study, we showed that the impact of the edge effect on CSF-serum pairs could be reduced by some minor adaptations to the standard protocol of the manufacturer , which, The edge effect found in this study could be caused by differences in temperature across the plate, with lower temperatures in the center of the plate than in the edges, as has been described previously , 7, 15. Borrelia-specific CSF/serum IgG quotients and, hence, the simulated Borrelia-specific IgG AIs. All CSF results, or all serum results, were subjugated to this simulated worst-case edge effect. Both the false-pathological AI values and the false-normal AI values were located close to the AI cutoff value of 1.5. This value, which is established by the manufacturer, is most likely based on the use of wells that perform similarly. Yet our study demonstrates that the AI cutoff value is too low for those cases in which the CSF is subjected to the edge effect  and the serum is not . Likewise, the AI cutoff value is too high for those cases in which the serum is subjected to the edge effect  and the CSF is not .We demonstrated that the observed edge effect found in the Enzygnost IgG ELISA could have an impact on LNB diagnostics, especially when CSF is tested in an outer well and serum in an inner well. The magnitude of this impact was shown by calculating simulated Enzygnost IgG ELISA results by using the results of 149 CSF-serum pairs that were part of a larger validation study . Depending on which sample (either CSF or serum) was placed in an outer well, either 15 (10.1%) of the 149 CSF-serum pairs resulted in a false-pathological AI, or 2 (1.3%) resulted in a false-normal AI. The number of false-pathological and false-normal results obtained through simulation was based on a worst-case scenario. That is, the edge-effect factor that was used to calculate the simulated CSF or serum results was based on selecting the two columns for which the difference between the median OD values was largest. Subsequently, these simulated CSF or serum results were used to calculate the simulated Our study suggests that by using the adapted protocol, false-pathological and false-normal AIs caused by the edge effect can be avoided. A study of Wang and Cheng, in which the concentration of the monoclonal antibody bevacizumab was measured in plasma of beagle dogs by using a similar platform (ELISA), also demonstrated an edge effect, and they showed that exclusion of the wells at the edges decreased this effect . Our stu


Sub-optimal sensitivity and specificity in current allograft monitoring methodologies underscore the need for more accurate and reflexive immunosurveillance to uncover the flux in alloimmunity between allograft health and the onset and progression of rejection. QSant—a urine based multi-analyte diagnostic test—was developed to profile renal transplant health and prognosticate injury, risk of evolution, and resolution of acute rejection. Q-Score—the composite score, across measurements of DNA, protein and metabolic biomarkers in the QSant assay—enables this risk prognostication. The domain of immune quiescence—below a Q-Score threshold of 32—is well established, based on published AUC of 98% for QSant. However, the trajectory of rejection is variable, given that causality is multi-factorial. Injury and subtypes of rejection are captured by the progression of Q-Score. This publication explores the clinical utility of QSant across the alloimmunity gradient of 32–100 for the early diagnosis of allograft injury and rejection. It is not surprising that advances in immunosuppression treatment for kidney transplant recipients have not had a more dramatic impact on the improvement of long-term allograft survival . A pivotThere is a critical unmet need to uncover this early stage-shift of acute rejection when injury is mild and potentially reversible. This requires the diagnostic to independently detect rejection, without the testing trigger being a change in graft function ,8; also A non-invasive, urine biomarker based multi-analyte diagnostic test, QSant was developed and clinically validated —to addren = 364) was to dichotomize an immunosuppressed renal transplant population into a two-class model—stable (STA) and acute rejection (AR)—with two independent validation cohorts (n = 162) to prognosticate rejection risk. Additionally, there was an independent prediction cohort (n = 91) to assess Q-Scores in borderline acute rejection (bAR), ad-mixed cases of AR and chronic allograft injury  and BK viral nephritis with and without the presence of tubulitis.The intent of the initial  Q-Score This study demonstrates the clinical utility of serial monitoring via QSant in prospectively collected longitudinal patient urine samples. A review of a large dataset of contemporaneous kidney allograft biopsy and QSant data, inclusive of real-world data (RWD) from 11 US kidney transplant centers, show that the Q-Score offers a dynamic view into the changing state of allograft injury and rejection in response to alterations in the patients’ immunosuppression regimen. Early detection of rejection, specifically in the 32–55 range, when graft function and biopsy may yet only be minimally perturbed, promulgates early and personalized care pathways for allograft rejection. Thus, precision monitoring with the Q-Score has the potential to not only improve a transplant recipient’s’ quality of life but to also alleviate the financial burden on national health care budgets, by minimizing unnecessary biopsies, untimely transplant loss, incidence of chronic dialysis, and overall morbidity.n = 162) and prediction (n = 91) data from the Yang cohort, which were collected between 2010 and 2018 );(ii)An increasing gradient, delineating patients moving into the mature AR domain (3% [1/32]);(iii)A decreasing gradient, delineating patients moving into the immunoquiescent domain, putatively in response to immunomodulation following assessment of chronic/active/mixed rejection (22% [7/32]). Albeit a small sample size (16% [5/32]), the evolving and resolving nature of higher-grade acute rejection (Q-Score > 55) in response to immunomodulation is exemplified here.Serial monitoring by QSant across a subset of 32 patients  over a period of two consecutive quarters (timepoints 1 and 2), captures the essence of the alloimmunity flux (on test) . It is wThe observed alloimmunity flux lends to the suggested QSant monitoring protocol  with a dn = 162). For a dichotomized cohort of STA versus AR, the cut-point of 32 was recapitulated ; specificity and sensitivity were established at 94% and 99%, respectively, with an AUC of 98%   and for-cause  biopsies. The protocol biopsies are enriched for sub-clinical rejection, 95% (37/39) whereas for cause biopsies enrich for AR at 54.9% (28/51). Published biopsy correlation data [p < 0.0001) and tubulitis  scores.Following the observations in the RWD study, a re-analysis was performed on the Yang cohort, and performance was characterized across the combined validation sets ( 0.0001) . To addr 0.0001) , demarcaThe etiology behind alloimmune rejection in renal transplantation is complex. This is further complicated by the differential health of the renal parenchyma of donor kidneys and the presence of comorbidities post-transplantation. The interplay of these factors lead to a dynamic spectrum of rejection inclusive of similar and disparate cellular and molecular mechanisms of ABMR and TCMR. The interplay of the individual component markers of QSant, that correlate with both the inflammation and tubulitis injury of acute rejection, highlights its clinical relevance for immunosuppression titration and serial of monitoring to evaluate the resolution of acute rejection. The quantitative nature of the Q-Score provides specific and detailed insights into early perturbations due to alloimmune injury in different compartments of the transplant kidney, which is not possible in other existing (blood) diagnostic assays, that only provide a single threshold for the presence or absence of acute rejection.We highlight in this study that the evaluation of the health of an allograft has to be able to involve accurate assessment of the state of immune quiescence (Q-Score < 32), where immunosuppression minimization could be supported, as well as understand the phase-shift of alloimmune injury through early stages of molecular and cellular rejection where histological injury and clinical graft dysfunction are still lagging indicators  (Q scoreIn conclusion, the Q-Score offers the potential to be used as an immune-monitoring tool to guide the use of immunosuppression, with the ultimate goal of controlling subclinical intra-graft inflammation and thus prolonging graft survival. Further assessment revealed that the QSant has the potential to differentiate higher-grade AR episodes from borderline as well as sub-clinical rejection.