Mobilization failure may occur when the conventional hematopoietic stem cells (HSCs) mobilization agent granulocyte colony-stimulating factor (G-CSF) is used alone, new regimens were developed to improve mobilization efficacy. Multiple studies have been performed to investigate the efficacy of these regimens via animal models, but the results are inconsistent. We aim to compare the efficacy of different HSC mobilization regimens and identify new promising regimens with a network meta-analysis of preclinical studies.− Sca1+ Kit+ (LSK) cells/ml PB. Bayesian network meta-analyses were performed following the guidelines of the National Institute for Health and Care Excellence Decision Support Unit (NICE DSU) with WinBUGS version 1.4.3. G-CSF-based regimens were classified into the SD  group and the LD  group based on doses, and were classified into the short-term (2–3 days) group and the long-term (4–5 days) group based on administration duration. Long-term SD G-CSF was chosen as the reference treatment. Results are presented as the mean differences (MD) with the associated 95% credibility interval (95% CrI) for each regimen.We searched Medline and Embase databases for the eligible animal studies that compared the efficacy of different HSC mobilization regimens. Primary outcome is the number of total colony-forming cells (CFCs) in per milliliter of peripheral blood (/ml PB), and the secondary outcome is the number of LinWe included 95 eligible studies and reviewed the efficacy of 94 mobilization agents. Then 21 studies using the poor mobilizer mice model (C57BL/6 mice) to investigate the efficacy of different mobilization regimens were included for network meta-analysis. Network meta-analyses indicated that compared with long-term SD G-CSF alone, 14 regimens including long-term SD G-CSF + Me6, long-term SD G-CSF + AMD3100 + EP80031, long-term SD G-CSF + AMD3100 + FG-4497, long-term SD G-CSF + ML141, long-term SD G-CSF + desipramine, AMD3100 + meloxicam, long-term SD G-CSF + reboxetine, AMD3100 + VPC01091, long-term SD G-CSF + FG-4497, Me6, long-term SD G-CSF + EP80031, POL5551, long-term SD G-CSF + AMD3100, AMD1300 + EP80031 and long-term LD G-CSF + meloxicam significantly increased the collections of total CFCs. G-CSF + Me6 ranked first among these regimens in consideration of the number of harvested CFCs/ml PB . In addition, 7 regimens including long-term SD G-CSF + AMD3100, AMD3100 + EP80031, long-term SD G-CSF + EP80031, short-term SD G-CSF + AMD3100 + IL-33, long-term SD G-CSF + ML141, short-term LD G-CSF + ARL67156, and long-term LD G-CSF + meloxicam significantly increased the collections of LSK cells compared with G-CSF alone. Long-term SD G-CSF + AMD3100 ranked first among these regimens in consideration of the number of harvested LSK cells/ml PB .Considering the number of CFC and LSK cells in PB as outcomes, G-CSF plus AMD3100, Me6, EP80031, ML141, FG-4497, IL-33, ARL67156, meloxicam, desipramine, and reboxetine are all promising mobilizing regimens for future investigation.The online version contains supplementary material available at 10.1186/s13287-021-02379-6. Hematopoietic stem cell transplantation (HSCT) is a life-saving strategy for variety of severe disorders, including bone marrow failure after high-dose radiation and various hematological malignancies . PeripheGranulocyte colony-stimulating factor (G-CSF) is the most commonly used steady-state HSC mobilization agent in clinical practice. However, mobilization failure may occur when G-CSF is used alone . In addi− Sca1+ Kit+ (LSK) cells per milliliter of peripheral blood (/ml PB). Since aged mice were reported to have better mobilization efficiency compared with young mice and no significant difference was reported among mice younger than 3 months, we excluded studies using mice older than 12 weeks in meta-analysis to reduce heterogeneity [We searched Medline and Embase from inception to February 23, 2021, with the search term “stem cell mobilization” and a filter of “animals”. The titles and abstracts of retrieved citations were independently screened by two investigators (CXL and XL) for eligibility. Disagreements were resolved by full-text review and discussion with a third investigator (SNX). Preclinical studies that met the following criteria were included for review: (1) compared the efficacy of two or more different regimens in the mobilization of hematopoietic stem and progenitor cells (HSPCs) and (2) using any species of mice as experimental animals. As for network meta-analysis, the inclusion criteria were (1) using the poor mobilizer mice model-C57BL/6 mice as experimental animals  and (2) ogeneity . In addiFull text of all eligible studies was reviewed, and two investigators (CXL and XL) independently extracted data using predesigned data collection forms. Data was extracted on studies characteristics, animal’s characteristics, dosage of mobilization regimens, and efficacy outcomes. We chose the number of total CFCs per milliliter PB as primary outcome, and the number of LSK cells per milliliter PB as secondary outcome. The mean, standard deviation (SD) or standard error (SE) of each outcome are extracted directly from published text or from related graphs with Adobe Photoshop version CS3 via previously validated methods . The met., which could handle trials with multiple arms and rank treatments with additional code [We conducted network meta-analyses to compare the efficacy of multiple mobilization regimens simultaneously. Network plot for each outcome was obtained using Stata version 12.0. Bayesian network meta-analyses were performed with WinBUGS version 1.4.3 , employing the Markov Chain Monte Carlo (MCMC) approach and following the guidelines of the National Institute for Health and Care Excellence Decision Support Unit (NICE DSU) . We usednal code . Three cnal code . The pronal code .We identified 3826 records from database searches. After removing 835 duplicates, 2991 records were screened on title and abstract, and 2749 clearly irrelevant records were excluded. We retrieved the full text of the remaining 242 records for further assessment. We excluded 147 records for the reasons listed in the flow diagram  per milliliter of PB was reported as primary outcome in 17 studies and involved 43 mobilization regimens. The network graph of all comparisons in these 17 studies is shown in Fig. The number of LSK cells/ml PB was reported as primary outcome in 11 studies, which have evaluated the efficacy of 34 mobilization regimens with mice models. The network graph of all comparisons in these 11 studies is shown in Fig. −Sca-1+c-kit+Flt3− cells), SLAM LSK cells (Lin−Sca-1+c-kit+ CD48−CD150+ cells), or LT-HSCs compared with G-CSF alone. The truncated form of chemokine GROβ (tGROβ) plus AMD3100, Cobalt protoporphyrin IX (CoPP), mobilized higher levels of SLAM LSK cells than G-CSF.Although the number of CFCs and LSK cells are the most commonly used outcomes to evaluate HSPC mobilization efficiency, enriched cell subsets such as CFCs and LSK cells do not measure long-term reconstituting HSCs, and additional markers such as fms-like tyrosine kinase-3 (Flt3) and signaling lymphocyte activation molecule (SLAM) CD150 were used to identify LSK subsets and assess the mobilization of self-renewing HSCs and long-term HSCs (LT-HSCs) . The mobTo further assess the mobilization of long-term repopulating HSCs, in vivo transplantation experiments were performed in 49 studies. The characteristics and results of these 49 studies are reviewed in Supplementary Table This work reviewed the efficacy of 94 new HSC mobilization agents from 95 preclinical studies. In addition, we included 21 studies using the poor mobilizer model-C57BL/6 mice for network meta-analysis and compared the efficacy of 57 mobilization regimens. We identified several promising regimens with great HSC mobilization efficacy, including long-term SD G-CSF + Me6, long-term SD G-CSF + AMD3100 + EP80031, long-term SD G-CSF + AMD3100 + FG-4497, long-term SD G-CSF + ML141, long-term SD G-CSF + desipramine, AMD3100 + meloxicam, long-term SD G-CSF + reboxetine, AMD3100 + VPC01091, long-term SD G-CSF + FG-4497, Me6, POL5551, long-term SD G-CSF + AMD3100, long-term LD G-CSF + meloxicam, AMD3100 + EP80031, long-term SD G-CSF + EP80031, short-term SD G-CSF + AMD3100 + IL-33, and short-term LD G-CSF + ARL67156.To our best of knowledge, this study is the first network meta-analysis that compared the efficacy of different HSC mobilization regimens with data from preclinical studies. We provide a comprehensive summary of new mobilization agents that have been investigated in mice models. The efficacy of these agents alone or in combination with other agents was indirectly compared via network meta-analysis. Moreover, we ranked all of the investigated regimens based on their ability to mobilize HSCs into blood stream. We identified several promising agents and regimens that have the most potent mobilizing capacity. The majority of mobilization regimens that show great improvements over G-CSF are combined regimens containing both G-CSF and new mobilization agents. Although these regimens would be unlikely to reduce severe adverse events, they provide a perspective that the incorporation of new agents could reduce the incidences of G-CSF-related adverse events through reducing the doses of G-CSF that required to mobilization sufficient HSCs since they can synergistically enhance the G-CSF-mediated mobilization. In addition, we identified several agents showed superior mobilization potential than G-CSF even when used alone, such as Me6 and POL5551. It is worth further investigation that whether these agents could reduce mobilization-related toxicity compared with G-CSF.. and has been proved to have robust ability of mobilizing HSPCs [Among the new agents, EP80031, Me6, FG-4497, and ML141 significantly improved the efficiency of G-CSF-induced HSC mobilization. EP80031 is a synthetic octosaccharide mimicking the structure of heparan sulfate. A single dose of EP80031  could lead to rapid and prominent mobilization of hematopoietic stem and progenitor cells (HSPCs), and the combination of EP80031 with G-CSF and AMD3100 resulted in 3-fold increase in the number of LSK cells and total CFCs . In adding HSPCs . The comng HSPCs . FG-4497ng HSPCs . The addng HSPCs , 106. Inng HSPCs . ML141 ing HSPCs . Taking In addition, we established the favorable efficacy of G-CSF and AMD3100 in HSC mobilization, which has been verified by clinical trials. AMD3100, also known as plerixafor, is an antagonist of the chemokine receptor CXCR4 that could rapidly induce the mobilization of stem cells through antagonizing the interaction of CXCR4 and stromal cell-derived factor-1α (SDF-1α) . MultiplNevertheless, there are some limitations in this study. Firstly, we integrated evidences from animal models. It is suggested that HSC mobilization is evolutionarily conserved from mice to humans, so mice models also represent a valuable experimental system for investigating the efficacy and mechanisms of mobilization regimens , 81. EveIn summary, this study identified several promising mobilization agents and regimens that significantly increased the mobilization of HSCs compared with the conventional agent G-CSF alone. We think that our results can provide important perspectives for future researches.Additional file 1: Supplementary Table 1. Characteristics of the 95 studies included for review.Additional file 2: Supplementary Table 2. Risk of bias assessment using the SYRCLE tool.Additional file 3: Supplementary Table 3. Results about the mobilization of different LSK subsets.Additional file 4: Supplementary Table 4. Characteristics and results of in vivo transplantation experiments.


Vaccines containing inactivated toxins confer protection by eliciting a neutralizing antibody response against bacterial toxins such as tetanus and diphtheria. At present, release of tetanus toxoid (TT) and diphtheria toxoid (DT)-containing vaccines relies on in vivo experiments showing the protective vaccine response. The aim of this study was to develop a reliable in vitro assay for TT vaccine antigen characterization with the potential of replacing in vivo potency experiments. To this end, we exploited that TT elicits a recall response in vaccinated donors: human peripheral blood mononuclear cells (PBMC) were stimulated with alum-adsorbed TT bulk antigen and low concentrations of TLR9 ligand; induction of TT-specific IgG was quantified via ELISpot after 5 days. Proof-of-concept was obtained using paired samples from donors before and after vaccination; anti-TT IgG was only detected in PBMC collected after booster vaccination; specificity was demonstrated with DT stimulation as control. Notably, when using PBMC from buffy coats, the specific response to TT was reproducible in 30% of cells; responsiveness correlated with higher numbers of switched memory B cells. Consecutive results showed that TT-specific IgG was also detectable when PBMC were stimulated with DTaP final vaccine product. Thus, the assay provides a viable means to test B-cell differentiation and induction of TT-specific IgG secretion using bulk antigen and final vaccine. However, prequalification of PBMC is required for reliable performance. Along with physicochemical and immunochemical methods, the functional assay could represent a complementary tool to replace in vivo potency assays in batch release of TT-containing vaccines. Vaccines confer safety and are the most effective means of protection against infectious disease as demonstrated by the success of vaccines against polio, smallpox, measles, diphtheria, and tetanus among others. Introduction of infant immunization against these pathogens or their disease-provoking toxins has led to an impressive reduction of global disease burden and improved survival of neonates and children worldwide.Clostridium tetani, which persist in soil, dust, saliva, or manure. The bacterial toxins affect the nervous system and infection leads to painful muscle spasms and ultimately to death. Tetanus toxoid (TT), e.g., tetanus toxin inactivated with formaldehyde, is the basis for tetanus vaccine formulations, which induce a strong and long-lasting neutralizing antibody response to the toxin4.Tetanus is a serious illness caused by exposure of a wound to the spores of the bacterium 6. Reflecting this, in Germany, >75% of adults have been vaccinated within last 10 years7.Booster vaccinations are required to acquire long-term protection against tetanus and to maintain immunity over a lifetime. Having established immunity in childhood, adolescents, and adults are recommended to receive booster vaccinations every 10 years at the latest8. Anti-TT IgG serum antibody titers above 0.1 IU/ml are considered protective11.TT-induced antibodies represent the correlate of protection for these vaccines, because they neutralize the bacterial toxin, and, thus, prevent disease manifestation. The antibodies are derived from the pool of long-lived plasma cells and memory B cells previously exposed to TT14. The currently employed in vivo models for batch release testing of TT vaccines are based on inducing in vivo humoral immunity and evaluated either by protection from tetanus toxin challenge or presence of tetanus toxin-specific antibodies16.The secondary immune response to antigen induces proliferation and differentiation of antigen-specific memory B cells into antibody-secreting cells, which is in vivo accompanied by a transient elevation in serum antibodies. This process has a key role in the neutralization and elimination of pathogens and their virulence factors17, Directive of the European parliament and of the council 2010/63/EU on the protection of animals used for scientific purposes18 or European Pharmacopoeia chapter (5.2.14) “Substitution of in vivo methods by in vitro methods for the quality control of vaccines”19. Subsequent method developments have focused on quantification of vaccine antigen, absence of toxin, and irreversibility of toxoid, the degree of adsorption of antigen to adjuvant in the final product as well as mass spectrometry-based analyses of protein content to detect inconsistencies in production and susceptibility to degradation23. However, to date, none of these methods can be used for potency testing of TT vaccines, which is classically associated with functional testing. In the present study, we describe a new assay that mimics the process of TT-induced specific antibody induction in vitro.In vivo potency assays used for consistency testing of vaccine batches were established decades ago. The need for replacement and reduction of in vivo methods for quality control of toxoid vaccines is well recognized and enforced by changes in the animal legislation including the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS No. 123)26, antigen-specific memory B cells need to be stimulated to undergo differentiation to antibody-secreting cells (ASC). Notably, a variety of protocols developed for detection and enumeration of vaccine and infection-induced antigen-specific memory B cells exist. They are usually based on 3–10-day cultures with addition of polyclonal stimuli, e.g., R848, CpG, pokeweed mitogen, Staphylococcus aureus Cowan strain I, CD40 ligand or anti-CD40 antibody, in combination with differentiation-supporting cytokines such as BAFF, IL-2, IL-6, and IL-1028. In contrast to previous approaches, the assay developed in this study avoids unspecific, polyclonal B-cell stimulation but makes use of the co-stimulatory potential of TLR9 ligand CpG to support antigen-driven terminal differentiation of memory B cells30.Although secretion of antibodies derived from activated plasmablasts can be quantified by seeding them on ELISpot membranes directly after isolationHere, we present original data demonstrating that eliciting a recall response via dual stimulation with B-cell receptor (BCR) antigen and TLR9 ligand can be applied to confirm the presence and test the functional integrity of vaccine antigen in TT-containing vaccine batches.To enable in vitro testing of the functional integrity of TT vaccine antigen, we established a human PBMC assay based on dual stimulation of B cells with TT bulk antigen and a low concentration of TLR9 ligand CpG ODN 2006. The assay principle is summarized in Fig. 4-adsorbed TT for 5 days. Stimulation of PBMC from donors before vaccination did not result in the generation of anti-TT IgG-secreting cells . The results confirmed the specificity of TT-specific antibody detection. However, cells from two freshly vaccinated donors with high levels of anti-TT IgG enzymatic reactivity also displayed a positive despite lower response to DT, while all other samples (before and after vaccination) showed a negative TT-specific IgG response   Fig. . Slight 6 PBMC when compared with reactive donors  of TT-specific memory B-cell counts with counts of switched memory B cells per 1 Mio PBMC  and TT-specific memory B cells (p < 0.0001) with the enzymatic activity of anti-TT IgG   Fig. .Fig. 4InSince it has repeatedly been claimed that alum is cell-toxic, we asked whether increasing amounts of antigen and adsorbed adjuvant would affect the results. The results showed that enzymatic activities derived from anti-TT IgG-secreting B cells produced in response to 0.000001 Lf/ml or 0.0025 Lf/ml TT were comparable and independent of the adsorption with alum Fig. . Howeverp < 0.0001) of anti-TT IgG enzymatic activity on an individual donor level   Fig. .p value, a decreased correlation coefficient and increased scattering of single values along the trend line when compared with the less-rigorous protocol at 37 °C.Next, we tested whether the assay detects heat alteration of TT. To this end, we used two different heat treatment protocols: TT was either incubated at 37 °C for 4 weeks or at 45 °C for 1 week. The results shown in Fig. The ultimate goal of this assay development is to test final vaccine product batches. Thus, we tested the response of reactive donor PBMC to final DTaP vaccine product. A drop-out DaP sample lacking TT was used as a control. The results obtained in these experiments showed a TT-specific response that was absent when PBMC were stimulated with the drop-out sample Fig. . These f31. These responses require expansion and subsequent differentiation of antigen-specific B cells into antibody-producing cells. Use of ELISpot is well established for detection of antigen-specific memory B cells and determining the frequency of pathogen and vaccine-specific B cells35. However, the published protocols focus on the enumeration of antigen-specific B cells and are therefore based on polyclonal stimulation , which was used for characterization of PBMC in Fig. It is well known that memory B cells enable rapid responses upon re-encounter with a known pathogen39. This percentage corresponded well with the number of TT-specific IgG-positive B cells we determined in buffy coat-derived PBMC. Using flow cytometric analysis we estimated the frequency of IgG memory B cells at 0.13%, which corresponds to 0.012% of total peripheral blood B cells  were unresponsive. The reasons for this finding remain to be clarified: no difference was identified based on years elapsed from previous vaccination, number of days post vaccination, switched memory B-cell counts or levels of anti-TT antibodies in plasma. Furthermore, we can only speculate whether unspecific responses to DT reflect elevated activation of antibody-secreting cells after vaccination.According to the literature, TT-specific plasma cell counts peak around day 7 after booster vaccinationlls Fig. . Notably7. Although we presumed that blood donors were likely to have received TT vaccination in the past, information on the individual history of vaccination and the time elapsed from the last (re)vaccination was not available. We reduced the impact of this variability on the performance of our assay by two measures: (i) to increase sensitivity we performed experiments using a high number of technical replicates with the aim of increasing the total amount of B cells analyzed per sample  PBMC samples fulfilling this criterion would have been repeatedly reactive  were isolated from buffy coats from healthy donors obtained from German Red Cross South transfusion center  or from healthy volunteers before and 7–26 days after booster DTaP vaccination, which occurred 5–16 years after their previous vaccination. Table PBMC were isolated by Pancoll gradient centrifugation . Before cryopreservation, isolated PBMC were centrifuged and resuspended in fetal calf serum (FCS) to the concentration 30 Mio/ml. Aliquots à 15Mio (500 µl of the cell suspension) per freezing vial  from each donor were prepared. Shortly before vial would be closed, mixed by inverting and placed into slowly cooling container  additional 500 µl of freezing media , 60% RPMI  and 20% DMSO ) were added to the cell aliquot in each vial. Freezing container with vials was immediately placed into −80°C freezer.l-glutamine (200 mM) , 1% penicillin/streptomycin  ) and 180 U/ml of DNAse I . Followed by immediate addition of 1 ml prewarmed (37 °C) HEK media. Cell suspension was placed for 1 hour in and 37 °C incubator. Afterwards, the cell suspension was centrifuged  and the supernatant was discarded. Cells were resuspended in 1 ml HEK media and counted using trypan blue  and Neubauer chamber.Required number of frozen vials of each donor was processed proportionally to the number of vials according to the following protocol. One vial containing 1 ml of cell suspension was placed into 37 °C water bath for 1 min. The cell suspension was transferred into 1 ml of prewarmed (37 °C) thawing medium prepared as 50% FCS, 50% HEK medium   at a final concentration of 0.002 µM. TT adsorbed to alum adjuvant from manufacturer #1 (lot# A and B) and manufacturer #2 (lot# C) were used at a concentration of 0.0025 Lf/ml, if not stated otherwise. Alum-adsorbed DT bulk antigen provided by manufacturer #1 was used at a final concentration of 0.0025 Lf/ml . AlPO4 obtained from manufacturer #1 was applied as control at a final concentration of 0.058 µg/ml AlPO4 , which is equivalent to the amount in Alum-adsorbed TT conditions (0.0025 Lf/ml). In the experiment, where TT adsorbed from two manufacturers was compared, TT adsorbed from manufacturer #2 was used at a concentration of 0.0025 Lf/ml, the adsorbent Al(OH)3 in final concentration of 0.26 µg/ml . DTaP and a DaP drop-out sample lacking TT were provided by manufacturer #1 and used for stimulation at a final concentration of 0.0025 Lf/ml of TT (4000-fold dilution). Wherever possible, each condition was performed in six or eight technical replicates. The final volume of the stimulated cell suspension was 1 ml per well in a 48-well plate. Cells were subsequently incubated in 37 °C, 5% CO2 for 5 days.PBMC were seeded into 48-well plate  2 × 10g, 6 min, 20 °C) and cell were washed carefully twice with 1 ml of HEK media. After the supernatants were discarded, the cells in each well were resuspended in 400 µl of HEK media. In all, 200 µl of the cell suspension of single well was transferred in single TT coated ELISpot well. For detection of total IgG secretion—190 µl from the other portion of stimulated PBMC was discarded and 400 µl of HEK media was added. From this 40 times diluted cell suspension , 200 µl of cells were seeded on anti-human IgG coated ELISpot plate. Cells were placed in 37 °C incubator with 5% CO2 for 4 hours. The cells were subsequently discarded and plate was washed three times with PBS. Biotinylated anti-IgG detection mAbs MT78/145 clone  at a concentration of 0.001 mg/ml in PBS supplemented with 10% FCS were added to the wells and incubated for 2 hours at RT. The plates were washed three times with PBS with 0.05% Tween 20  before streptavidin conjugated with Alkaline-phosphatase  diluted 1:1000 in PBS with 10% FCS was added and incubated for 1 hour at RT. Plates were washed three times with PBS with 0.05% Tween 20 and three times with PBS. AP-conjugate Substrate kit  was added to the wells for 2–5 min. The reaction was stopped by rinsing the plates with tap water. The plates were dried overnight in dark. Evaluation of plates was done with iSpot ELISpot reader  with EliSpot Reader v.7.0  within 7 days from development.MultiScreen HTS IP Durapore PVDF ELISpot plates  were coated with 10 Lf/ml TT  or with 6.25 ng/ml anti-human IgG  in PBS overnight at 4 °C. Before cell seeding, the ELISpot plates were washed three times with PBS and block with HEK media for at least 30 min at the room temperature (RT). Cells after 5 days of incubation in 48-well plate were centrifuged  if an enzymatic activity was ≥200 in at least one of six replicate wells stimulated with CpG and TT and (2) if the highest value of the enzymatic activity from the wells treated with CpG and TT was higher than that obtained in all wells that were stimulated with CpG only.6 cells per well in 1 ml of HEK media supplemented with R848 (1 µg/ml)  and 10 ng/ml IL-2  and cultured in 37 °C with 5% CO2. On day 5 PBMC were washed and seeded in twofold serial dilutions in duplicates  and incubated for 4 hours on ELISpot plates coated with TT 38. The coating and developing procedures were performed as described above. Frequency of TT-specific memory B cells was determined per 1 × 106 of PBMC.PBMC were plated in 48-well plate at a concentration of 2 × 10g, 6 min, RT). Cells were stained with 50 µl of mixture of anti-human CD19-Pc7 , anti-human CD27-BV421 , anti-human IgM-PerCP Cy5.5  and PBS with 0.5% FCS for 30 min, 4 °C, dark. Afterwards 150 µl PBS with 0.5% FCS was added and the samples were centrifuged , supernatant discarded. The cells were resuspend in 100 µl of PBS, followed by adding 100 μl of 4% PFA   and the suspension was well mixed. The samples were kept in fridge (4 °C) overnight. Next day, the samples were washed before the measurement and cells were resuspended in PBS with 0.5% FCS. Samples were measured on a BD LSRII SORP flow cytometer with BD FACS Diva software version 8.0.1  and analyzed with Kaluza Analysis Software . The gating strategy is shown in Supplementary Fig. For B-cell count and B-cell subpopulations determination, 200,000 PBMC were transferred into 96 U-well plate . After PBS was added up to volume of 200 µl, the plate was centrifuged . TT-specific B cells in buffy coat PBMC were then characterized with an antibody panel consisting of anti-human CD19-Pc7 , anti-human CD27-BV711 , anti-human IgM-BV605 , anti-human IgA  and with TT purchased from AJ vaccines, Denmark, biotinylated with EZ-Link Sulfo-NHS-LC-Biotin  and fluorescently labeled with streptavidin-BV421 . Samples were measured on a FACS Aria™ Fusion . The gating strategy is depicted in Supplementary Fig. Levels of anti-TT IgG antibodies in plasma of were evaluated by human anti-TT IgG EIA Kit  according to the protocol provided by the manufacturer.P values are depicted in the respective graphs, ns non-significant.Statistical analysis of results was performed using GraphPad Prism 8.4.2 . Data were analyzed using two-tailed Wilcoxon matched-pairs signed rank test, two-tailed Mann–Whitney test and Spearman correlation. Further information on research design is available in the Supplementary InformationReporting Summary


While elevations of CSF p-Tau/Aβ40 can occur in amyloid-β (Aβ) negative (Aβ-) individuals, the factors associated with these elevations and their role in neurodegeneration and cognitive decline are unknown. We aim to explore factors associated with elevated tau in CSF, and how these elevated tau are related to neurodegeneration and cognitive decline in the absence of Aβ positivity.We recently reported that CSF phosphorylated tau  as well as vascular risk factors in 149 cognitively unimpaired and 52 impaired individuals who were presumably not on the Alzheimer’s disease (AD) pathway due to negative Aβ status on both CSF and PET. Subgroups had 18F-fluorodeoxyglucose (FDG) PET and adjusted hippocampal volume (aHCV), and longitudinal measures of CSF, aHCV, FDG PET, and cognition data, so we examined CSF p-Tau/Aβ40 associations with these measures as well.We examined relationships between CSF p-Tau/Aβ40 was associated with older age, male sex, greater WMH, and hypertension as well as a pattern of hippocampal atrophy and temporoparietal hypometabolism characteristic of AD. Lower CSF Aβ42/Aβ40, higher WMH, and hypertension but not age, sex, Aβ PET, APOE-ε4 status, body mass index, smoking, and hyperlipidemia at baseline predicted CSF p-Tau/Aβ40 increases over approximately 5 years of follow-up. The relationship between CSF p-Tau/Aβ40 and subsequent cognitive decline was partially or fully explained by neurodegenerative measurements.Elevated CSF p-Tau/AβThese data provide surprising clues as to the etiology and significance of tau pathology in the absence of Aβ. It seems likely that, in addition to age, both cerebrovascular disease and subthreshold levels of Aβ are related to this tau accumulation. Crucially, this phenotype of CSF tau elevation in amyloid-negative individuals share features with AD such as a pattern of metabolic decline and regional brain atrophy.The online version contains supplementary material available at 10.1186/s13195-021-00913-5. Amyloid-β (Aβ) plaques and tau neurofibrillary tangles are the core features of Alzheimer’s disease (AD) . AccordiWhile PET provides spatial information on where tau deposits, CSF measurement of phosphorylated tau (p-Tau) provides complementary, although not interchangeable –21, info40 ratio reduced measurement error likely related to individual differences in CSF production rather than pathology and improved associations with AD biomarkers compared to using CSF p-Tau alone , p = 0.003). None of these significant risk factors  were related to CSF Aβ42/Aβ40 or CSF Aβ40, and only age was related  to the non-ratio CSF p-Tau measurement  and HTN ; HTN and WMH were also positively associated .At baseline, elevated CSF p-Tau/Aβ42/Aβ40 predicted  subsequent ΔCSF p-Tau/Aβ40  and HTN  were associated with subsequent ΔCSF p-Tau/Aβ40  of individuals with diabetes limits interpretation. When all the significant risk factors  were entered into a single model, CSF Aβ42/Aβ40  and HTN  were related to ΔCSF p-Tau/Aβ40  with the non-ratio ΔCSF p-Tau measurement . After FWE correction (p<0.05), significant negative associations remained in bilateral posterior cingulate/precuneus, right inferior temporal, middle temporal, and inferior parietal regions  and ΔPACC   and aHCV . aHCV was correlated with FDG SUVR . Elevated CSF p-Tau/Aβ40 predicted subsequent FDG SUVR decrease , but did not predict change in aHCV . Conversely, FDG SUVR also predicted subsequent aHCV decrease .Among these significant risk factors related to CSF p-Tau/Aβ40, FDG SUVR (metaROIs), aHCV, ΔFDG SUVR, and ΔaHCV after excluding 5 individuals who developed to Aβ+ at follow-up . The results were substantially the same , aHCV, ΔFDG SUVR, and ΔaHCV  than CU individuals (0.09 (0.46)). CSF p-Tau/Aβ40 , FDG SUVR (metaROIs) , and aHCV  all predicted subsequent cognitive decline (ΔPACC). The baseline non-ratio CSF p-Tau and CSF Aβ40 measures were not associated with ΔPACC  annual ΔPACC of CI individuals (−0.56 (1.47)) was faster  related to longitudinal PACC decline when all the predictors were entered into one model , as well as a pattern of neurodegeneration that is characteristic of AD . In longitudinal analyses, lower CSF Aβ42/Aβ40 and CSF p-Tau/Aβ40 at baseline, lower CSF Aβ42/Aβ40 predicted subsequent longitudinal CSF p-Tau/Aβ40 increase but not vice versa. There was thus no countervailing evidence that tau deposition drives changes in Aβ in these data or in a previous report from our laboratory [Although there was no relationship between CSF Aβboratory . These fboratory , 22, andboratory –55, thes40 increases in the absence of abnormal Aβ pathology whereas they had no association with CSF Aβ42/Aβ40 or Aβ40 alone, suggesting vascular risk factors may be closely linked to CSF p-Tau/Aβ40 independent of Aβ pathology. ADNI data have previously been explored to examine some of the relationships between tau and the variables we measured, though usually not in Aβ- individuals. For example, a few studies based on the ADNI cohort reported significant associations of CSF p-Tau or plasma p-Tau with WMH [One of the key findings in this study was that white matter lesions and hypertension were related to both baseline and longitudinal CSF p-Tau/Aβwith WMH , 37, 56 with WMH . In addiwith WMH , global with WMH , and hypwith WMH , 59 werewith WMH  or both with WMH , and higwith WMH  or both with WMH .Age is a well-known risk factor for tau accumulation, and it was also related to these vascular factors in our data. In a multivariable model, age was the only variable associated with tau. However, both WMH and HTN, but not age, were related to longitudinal tau increases. Together, these findings suggest that both age and cerebrovascular risk factors may be related to tau increases in the absence of abnormal Aβ pathology.A surprising result of this study was the association between tau and regional cerebral hypometabolism in a pattern typically associated with AD despite the absence of measurable Aβ. In patients with clinical and biomarker evidence of AD, patterns of tau deposition are highly correlated with glucose metabolism while Aβ deposition is not . Althoug40 predicted metabolic decline but not progressive atrophy). Previous work is inconsistent, with some studies of Aβ- ADNI participants failing to find associations between CSF p-Tau and hippocampal or cortical atrophy [40 ratio [Further evidence that elevated tau is related to neurodegeneration is found in its relationship with hippocampal volume. In fact, all three variables–tau, glucose metabolism in typically affected AD regions, and hippocampal volume–were related to one another at baseline and to a large extent in longitudinal relationships (although baseline p-Tau/Aβ atrophy , 25, whi atrophy . It is d40 ratio  may playIn addition to its relationship with neurodegeneration, tau was also related to cognitive decline, providing further evidence that it is not benign when Aβ is low. These “downstream” pathways appear to be related as tau affects cognition through both glucose metabolism and hippocampal atrophy. Previous studies have not been in agreement that elevated CSF p-Tau in Aβ- individuals is linked to significant cognitive decline , 65, alt181/Aβ40, but it would be helpful to validate the findings in other samples and with other phosphorylation sites [181/Aβ40 ratio introduces a confounder that could account for these associations, we think that this is unlikely since CSF Aβ40 alone was not associated with any of the factors we investigated. Therefore, we believe that these findings reinforce earlier evidence [181 measurements due to individual variability in CSF production and due to the limited dynamic range in this group, and this variability can be attenuated by adjusting for Aβ40. This noise reduction appears to facilitate the observation of subtle associations between tau and other risk factors at early stages of abnormality. An additional limitation of this study was that some Aβ- subgroups we examined were very small, such as APOE-ε4 carriers, which limited our ability to examine covariates and other risk factors. Finally, tau PET is a relatively late addition to ADNI, so we did not include this because of minimal longitudinal tau data. Nevertheless, CSF p-Tau may have been an advantageous measure for the scientific questions in this study because it probably reflects earlier tau pathology than PET [This study has several limitations. Our findings were based on tau measured using CSF p-Tauon sites . Furtherevidence  that thethan PET , 21.The elevation of p-Tau in CSF in our Aβ- subjects is consistent, at least nosologically, with the disorder that has been described as PART; however, in the absence of neuropathology we cannot confirm this diagnosis. These data provide surprising clues as to the etiology and significance of tau pathology in aging. It seems likely that, in addition to age, both cerebrovascular disease and subthreshold levels of Aβ are related to this tau accumulation. Whether cerebrovascular factors alone or in concert with low levels of Aβ can drive tau pathology is uncertain, but the literature is replete with evidence for a relationship between tau and cerebrovascular disease. Crucially, evidence of this tau pathology occurs early, as CSF probably reflects tau abnormalities before PET scans do, and shares features with AD such as a pattern of metabolic decline and regional brain atrophy. Further longitudinal follow-up of these individuals will be critical for determining whether these processes are Alzheimer’s-independent. However, our data indicate that this phenotype of Aβ- CSF tau elevation is similar to AD, which implies that it may represent an amyloid-independent pathway to AD, a pathophysiology that mimics AD, or an important, and very early, interaction between Aβ and vascular disease that underlies neurodegeneration and cognitive decline.Additional file 1. Supplemental material can be found at online.


Clostridium difficile infections and chronic gastrointestional infections. However, the risks of FMT and the selection process of suitable donors remain insufficiently characterized. The eligibility rate for screening, underlying microbial basis, and core ethical issues of stool donors for FMT are yet to be elucidated in China.Faecal microbiota transplantation (FMT) is an effective therapy for recurrent The potential stool donors were screened from December 2017 to December 2019 with the help of an online survey, clinical assessments, and stool and blood testing. Bioinformatics analyses were performed, and the composition and stability of gut microbiota in stool obtained from eligible donors were dynamically observed using metagenomics. Meanwhile, we build a donor microbial evaluation index (DoMEI) for stool donor screening. In the screening process, we also focused on ethical principles and requirements. Of the 2071 participants, 66 donors were selected via the screening process (3.19% success rate). Although there were significant differences in gut microbiota among donors, we found that the changes in the gut microbiota of the same donor were typically more stable than those between donors over time.DoMEI provides a potential reference index for regular stool donor re-evaluation. In this retrospective study, we summarised the donor recruitment and screening procedure ensuring the safety and tolerability for FMT in China. Based on the latest advances in this field, we carried out rigorous recommendation and method which can assist stool bank and clinicians to screen eligible stool donor for FMT.The online version contains supplementary material available at 10.1186/s12934-021-01705-0. Clostridium difficile infection (rCDI) . Additionally, the qualified donors showed high gut microbial richness  were generated for the 66 potential donors. Due to the presence of gut harmful and beneficial microbial taxa, as well as microbial richness, low DoMEI scores (< 10 points) were observed in six donors  . However, it was apparent that samples collected from the same donor at different time points were generally close to each other in Non-metric multidimensional scaling (NMDS) ordination . These results indicate that the gut microbiota within each donor overtime was typically more stable than that between donors. Our findings also suggested that the faeces sampled and sequenced at present are likely to be similar to those taken after a few months.To further quantify the community similarity over time, we calculated the Bray–Curtis dissimilarity from one sampling period to the next for each donor. The dissimilarity values within one donor ranged from 0.2 to 0.4, fluctuating around the mean value. In particular, some donors  remained quite stable with little variation over time  reported that only 3% of potential candidates successfully passed four necessary screening stages . This isUnlike blood donation, stool donation and FMT are not well known and accepted by the general public in China. The initial challenges for stool donor screening, including lack of knowledge of FMT and embarrassment of donation, are primary barriers to participating in the online pre-screening survey. Moreover, we observed that the largest proportion of donor candidates were excluded in the online survey , primarily due to social history (smoking and drinking), logistic issues, and unqualified BMI. Social history included smoking and drinking, which modify gut microbiota that provide a protective effect against gastrointestinal disorders or cancers .Appropriate faecal donor age criteria remain inconsistent in other guidelines, studies, and stool banks. Generally, the recognised donor age ranged from 16 to 65 years  and indiWhen this screening method was used, additional assessment scales were supplemented for the actual evaluation of lifestyle and mental health in the process of stool donor screening. The purpose of using the lifestyle questionnaire survey was to obtain information on health status, dietary habits, and physical activity. Diet plays a key role in shaping the gut microbiome, with several experiments demonstrating that dietary alterations can result in temporary and large microbial changes within 24 h . HabituaChanges in gut microbiome composition have been reported in various mental diseases, including depression and anxiety , screeniH. pylori infections in China are relatively high (40–60%), especially in young people, compared with other developed countries. H. pylori infections are closely related to the occurrence of gastric cancer; however, most infected people do not experience symptoms or complications. With this high incidence in China, H. pylori may be the preferred stool donor screening test for gastrointestinal pathogens. In our online pre-screening survey, we found that 44 of 166 participants tested positive for H. pylori.We excluded FMT donors that might have carried any gastrointestinal pathogens. The rate of Substantial evidence has verified that SARS-CoV-2 can be found in faeces by viral detection in biopsy specimens and stool . The isoCampylobacter coli (0.014%). Campylobacter has been the most commonly reported major human bacterial enteropathogen since 2005 in the European Union. When transmitted to humans, Campylobacter coli may particular cause gastroenteritis [Fusobacterium and Ruminococcus gnavus [Akkermansia muciniphila, unclassified Ruminococcaceae, Ruminococcus spp and Bifidobacterium [Beyond the clinical examination and faecal pathogen cultivation, we further extended the assessment of donors to include wider faecal microbiota and microbial richness using shotgun metagenomics for each candidate donor. Understanding and extending the assessment of donor community composition for FMT has been appealed recently. In practice, we built and applied the scoring DoMEI and pathogen assessments for all candidate donors since 2017, taking into consideration the published metagenomic data of donors and healthy subjects. It is necessary to accept re-evaluation when candidate donors who had low DoMEI scores (<10 points) or a high abundance of pathogens (>0.01%). Thanks to DoMEI scores, we found six candidates had different influencing factors on faecal microbiota and microbial richness. For example, the Donor 40 had a quite low DoMEI score (8 points) and also carried a relatively high abundance of pathogen nteritis . This innteritis , low levs gnavus , high reacterium , 32. In acterium . A subseacterium .Our results showed little intra-donor variation of gut microbiota, and supported the view that a single assessment of faecal microbiota of donors is likely sufficient to represent the microbial compositions in subsequent months . AlternaThe accompanying ethical, social, and regulatory challenges must be addressed as part of an appropriate regulatory policy response to FMT to foster its safe and effective implementation. Concerns have been raised that whether a donor’s religious background, particularly where this entails special dietary requirements, should be considered when selecting and screening donors and allocating faecal microbiota to recipients . For exaIncreasing studies in human microbiome research have demonstrated that personal microbiomes contain enough distinguishing features to identify an individual over time based on their past travel experiences and sexual habits . A notab2. Individuals who were active smokers and drinkers were excluded because these factors perturb the gut microbiome [Interested donors were recruited via local and internet media  in Xiamen and Guangzhou, China. Donor candidates filled out an online pre-screening form to document the general health information, such as health status, occupation, disease history, medication history, family history, and risk of infectious disease. Empirically, it is suggested that potential FMT donors should be ≥ 18 and ≤ 40 years old and have a body mass index (BMI) between > 18.5 and < 28 kg/mcrobiome . As it iThis evaluation was performed by a trained physician and nurse and supervised by a senior internal medicine specialist  should be blended with normal saline (60%) and pharmaceutical grade glycerol (15%), and placed in an automatic stirring and separation machine . This machine is performed in 4 °C in order to preserve the microbial samples while processing the faeces. The process of blending was performed in an anaerobic chamber to prevent obligate anaerobic bacteria from exposure to oxygen. After blending, the stool mixture was aliquoted into individual sterile cryotolerant pots in a biological safety cabinet. The specifications, appearance, quantity, and weights of all the products were checked thoroughly. All samples were immediately frozen at − 80 °C. We carefully labelled each sample with the information and the date. The label is identified by a special number which only accessible by the authority and can be traced in the event of potential illness developing upon received by the recipients.® dsDNA HS Assay kit . For shotgun metagenomics, DNA was fragmented to an average insert size of 350 bp using a Bioruptor NGS sonicator  and further selected using VAHTS™ DNA Clean Beads . Metagenomic libraries were generated using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina. Library size and quality were assessed using an HS-DNA chip on an Agilent Bioanalyzer 2100 . Library concentrations were measured by quantitative PCR with KAPA Illumina Library Quantification Kits  on an ABI 7300 Plus machine . Each library was sequenced with 150 bp paired-end reagents on Illumina platforms . Amplicon metagenomics was adopted to explore the gut bacterial stability of the frequently donating donors over time. Samples were consecutively  collected from 16 donors from March 2018 to August 2019. They were then amplified using the 16S V4 primer set: 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′), following the methods reported [Faecal samples from each donor were collected to obtain the gut microbiota information. Total DNA was extracted from the samples weighing 0.25 g, using the QIAamp Fast DNA Stool Mini Kit . The resulting DNA yield and quality were checked with Qubitreported , and seqhttps://huttenhower.sph.harvard.edu/kneaddata), set at default parameters. Taxonomic profiles were generated by MetaPhlan2 [For shotgun metagenomics, raw sequencing data and human genome reads were trimmed using KneadData tool .Additional file 5:Table S2. Reasons for candidate donor exclusion .Additional file 6:Table S3. Reasons for candidate donor exclusion .Additional file 7: Table S4. Reasons for candidate donor exclusion (Stage 4 – Blood screening).Additional file 8:Table S5. Summary of samples, sampling days, number of OTUs, and the count of samples in phylum, class, order, family, genus.


Donor specific transfusions have been the basis of tolerance inducing protocols since Peter Medawar showed that it was experimentally feasible in the 1950s. Though trials of cellular therapies have become increasingly common in solid organ transplantation, they have not become standard practice. Additionally, whereas some protocols have focused on cellular therapies as a method for donor antigen delivery—thought to promote tolerance in and of itself in the correct immunologic context—other approaches have alternatively focused on the intrinsic immunosuppressive properties of the certain cell types with less emphasis on their origin, including mesenchymal stem cells, regulatory T cells, and regulatory dendritic cells. Regardless of intent, all cellular therapies must contend with the potential that introducing donor antigen in a new context will lead to sensitization. In this review, we focus on the variety of cellular therapies that have been applied in human trials and non-human primate models, describe their efficacy, highlight data regarding their potential for sensitization, and discuss opportunities for cellular therapies within our current understanding of the immune landscape. Presently, solid organ allotransplantation is hampered by poor outcomes due to the nonspecific effects of immunosuppressive medications, especially calcineurin inhibtors , 2. IndeIn a distinct vein, there has long been an interest in the ability to specifically modify the immune response to donor antigens. This research has focused on utilizing donor derived biological products across a number of cell types and preparations to condition the immune system to accept the allograft and is based, conceptually, on the work of Sir Peter Medawar . In spitIn the present manuscript, we will review the progress made in the application of donor specific cellular therapies starting with an examination of early evidence. We will further describe the multiple cellular strategies currently being investigated in clinical trials including the combination of donor specific transfusion (DST) with novel immunosuppressive regimens, modified cellular therapies, and specific cell type based therapies. Finally, we will examine the future landscape of donor directed therapies and the unmet research need. Few experiments in transplantation are as well-known as those of Sir Peter Medawar. In his seminal set of studies, Dr. Medawar built on the work of Dr. Ray Owen  and demoAdditionally, they utilized a mix of donor cells from the “testis, kidney, and splenic tissue” . Here, tThough his experiments were conceptually very important, the clinical reality of early transplantation was more complex and the application of cellular products of as treatment remains elusive . As adulvs. 56%). Despite a high degree of donor responsiveness as measured by mixed-lymphocyte reaction (MLR), patients who received DST had lower rates of acute rejection in the first 3 months using similar immunosuppression (44% vs. 82%). From these experiments, it was difficult to determine the exact differentiating factor which caused sensitization in some and tolerogenic effects in others. However, the authors did show that all patients who became sensitized with DST had also received multiple other blood transfusions. In this instance, the treatment was standardized  but the context, potentially measured by the amount of 3rd party  blood transfusions, may have modified the ultimate immune response to donor antigen. Concomitantly, experimentation in mouse models showed similar results with improved skin graft survival with DST but not with 3rd party transfusion  mismatched recipients, whereas it had little effect in fully haploidentical recipients . This wade novo DSA after transplant, though it is unclear what clinical significance these antibodies have.One area where DST continues to be investigated is in conjunction with costimulation blockade. Early murine experiments showed indefinite islet graft survival when DST with small lymphocytes (with or without T-cell depletion) was combined with anti-CD154  or when rd party grafts. Multiple findings from this first study are mechanistically interesting. First, they found that the ablation of Treg via an anti-CD25 antibody prior to ECDI-treated cell infusion abrogated tolerance induction but late (120 days after transplantation) depletion of Treg did not. Additionally, they showed that the PD-1 axis was necessary for tolerance induction.Another distinct line of investigations has attempted to deliver donor antigen itself in a more tolerogenic manner. Beginning with multiple early studies in mice, Luo and colleagues have shown that pre-treating cellular infusions (in this case splenocytes) with ethylene carbodiimide (ECDI) leads to long term tolerance in a high proportion of mice undergoing islet transplantation into the kidney capsule . Of notevia suppression of IFN-α signaling and subsequent expansion of MDSCs  model of islet cell transplantation, again, using splenocytes as the donor cell source supplemented by culture-expanded donor B cells from the peripheral blood. This lead to long term tolerance in a high proportion of recipients . Multiplin vitro context, potentially via downregulation of costimulatory molecules CD80/CD86 and ICAM-1. Intriguingly, T cells exposed to these DCs became stably tolerized to the antigens presented as these cells could not be restimulated after co-culture with these MICs . Terness and colleagues initially observed that DCs treated with mitomycin C become tolerogenic in an ese MICs . Furtherese MICs . They fuese MICs . They alese MICs . Recentlese MICs . Howevervs. host disease (GVHD), especially with the use of HLA-mismatched donors (Another area where a form of DST continues to be investigated is in chimerism protocols. Early murine models showed tolerance could be achieved by the depletion of the recipient immune system followed by concomitant hematopoietic stem cell transplant (HSCT) and solid organ transplant from the same donor . Within d donors . Studiesd donors , and thed donors  were encd donors .In a more targeted way, the same group as above also performed combined HSCT and kidney transplant in five patients using a non-myeloablative regimen consisting of cyclophosphamide, thymic irradiation, and anti-CD2 monoclonal antibody, and a short course of cyclosporine. Four out of five patients were able to be weaned off all immunosuppression with stable graft function. Interestingly, the second patient on this protocol developed acute humoral rejection with early graft loss (day 10) in spite of a negative crossmatch, though none of the patients developed GVHD . Other gvia culture methods that take advantage of the specific propensity of MSCs to adhere to plastic  and showed good safety    may modivia the instant blood mediated inflammatory reaction  led clinicians to abandon these therapies. A shift towards specific cell type based therapies has been productive in improving the field’s ability to manufacture and deliver consistent therapies but has yet to revolutionize the way in which we approach immunosuppression. Modified cellular therapies are on the horizon and represent an exciting development that is grounded in long standing understanding of mechanisms of the immune response. Combinations of DST with costimulation blockade have emerged as a promising approach. However, questions remain regarding sensitization, long-term outcomes, and the pool of patients appropriate for costimulation blockade-based therapy. Hematopoietic stem cell transplant in conjunction with solid organ transplant is less clinically applicable due to the morbidity involved with conditioning, even in the absence of high rates of GVHD. Regardless of the cellular therapies pursued, there are key characteristics that these therapies must possess to be most useful (summarized in Further phase 1 studies should continue to follow the example of the ONE study and others who attempt to gain as much information as possible with the most parsimonious control group. Additionally, further investigations into modifications of donor cells may be one way to overcome the significant hurdle that is sensitization. Transplantation is the original precision medicine, with HLA matching and crossmatch testing ensuring that organs implanted function appropriately and for the longest possible time. Cellular therapies ought to consider this precision in their development given the cost and potential for adverse events. In the future, the extent to which a therapy can inhibit a donor specific response relative to its overall immunosuppressive effect may become the most important metric, rather than a simplistic view which preferences only the inhibition of rejection episodes.BS drafted the manuscript. JO performed critical review and revision of the manuscript. CN performed critical review and revision of the manuscript. XL performed critical review and revision of the manuscript and oversaw the drafting process. All authors contributed to the article and approved the submitted version.BS was supported by NIH grant R38 AI140297.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Background: Hemorrhagic transformation (HT) is a common complication of intravenous thrombolysis with alteplase. Cardiac troponin has been found to be associated with poor prognosis and cognitive impairment in acute ischemic stroke. But studies on the relationship between troponin and HT after thrombolysis are scarce.Methods: This study retrospectively analyzed thrombolytic patients from June 2015 to June 2021 in the Second Affiliated Hospital of Wenzhou Medical University. Cardiac troponin I were measured on admission and on following days to determine the presence of elevation and dynamic changes. HT within 24–36 h after treatment was identified by cranial computed tomography (CT). Besides, a score on the modified Rankin Scale (mRS) > 2 at discharge was defined as unfavorable outcome. Univariate analysis was used to explore the factors related to the troponin elevation on admission and troponin dynamic changes. Multivariate logistic regression model was used to investigated the association between troponin elevation on admission, troponin dynamic changes and HT after thrombolysis, respectively.Results: Troponin levels on admission were measured in 377 patients, and follow-up assay was performed in 292 patients (77.5%). 39 patients (10.3%) had troponin elevation on admission, and 66 patients (22.6%) had troponin dynamic changes comprising rising and falling pattern. The pre-existing heart disease, renal insufficiency and higher stroke severity are related to both troponin elevation on admission and the subsequent troponin dynamic changes. After adjusting the potential confounding factors, logistic regression model showed that patients with troponin elevation on admission had insignificant trend to develop HT , while patients with troponin dynamic changes had significantly higher risk of HT . Compared to the troponin elevation, a statistically stronger association was present between rising troponin dynamic changes and unfavorable outcome .Conclusion: Troponin dynamic changes are associated with HT after thrombolysis. Serial measurements are quite necessary in thrombolytic patients with risk factors associated with troponin dynamic changes . Intravenous thrombolysis with alteplase effectively improves functional outcome in patients with acute ischemic stroke (AIS)  and, curCardiac troponins, a specific marker for myocardial injury, are recommended by the American Heart Association (AHA)/American Stroke Association (ASA) for routine detection in all patients with AIS . TroponiTherefore, the aim of our study was to investigate troponin and its association with HT after thrombolysis in patients with AIS. In view of the dynamic changes of troponin levels in the course of AIS , we adopThis study was a single-center observational study in the Second Affiliated Hospital of Wenzhou Medical University. We retrospectively analyzed the consecutive patients with AIS who received intravenous thrombolysis in our hospital from June 2015 to June 2021. Patients treated with 0.9mg/kg (maximum 90mg) alteplase within 4.5 h of onset were included. Patients with the following conditions were excluded: (1) troponin levels were not measured on admission, (2) cranial computed tomography (CT) was not repeated within 24–36 h, (3) comorbid ACS, identified mainly by symptoms and electrocardiographic findings, and (4) thrombolysis with low-dose alteplase or interruption of thrombolysis for reasons other than HT. This study was approved by the Ethics Committee of the Second Affiliated Hospital and Yuying Children’s Hospital of the Wenzhou Medical University. Written informed consent was obtained from participants or their guardians.We collected data regarding the patient’s demographics , vascular risk factors, comorbidities, clinical examination findings, blood test results, cranial CT, echocardiography, electrocardiogram and therapeutic measures. Vascular risk factors comprised hypertension, diabetes, hyperlipidemia, and smoking, while comorbidities comprised atrial fibrillation (AF), coronary artery disease (CAD), previous stroke. Clinical examination findings consisted of blood pressure on admission and stroke severity assessed using the National Institutes of Health Stroke Scale (NIHSS) score. Blood test results included troponin, glucose, HbA1c, platelet, international normalized ratio (INR), creatinine, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C). Treatment measures involved ongoing antithrombotic therapy, onset-to-treatment time (OTT), and bridging therapy. Stroke etiology was evaluated according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification .Cardiac troponin I levels were measured with AccuTnI + 3 assay on chemiluminescnet analyzer Access2 . The first blood sample was taken preceding treatment in the emergency department, and the majority of patients underwent retesting on the following days. For this assay, the lower limit of detection was 12 ng/L and the 99th percentile of the upper reference limit (URL) was 34 ng/L. Values above the URL were considered troponin elevation. Troponin dynamic changes were defined as a rise or fall of more than 20% in troponin levels and at least one troponin I value above the URL on serial measurements .All patients received routine cranial CT examination at admission and reviewed within 24-36 h after treatment. HT was defined as bleeding observed on follow-up CT, which was not observed on first CT examination. According to the European Cooperative Acute Stroke Study (ECASS) criteria, we classified HT into hemorrhagic infarction (HI-1 or-2) and parenchymal hemorrhage (PH-1 or-2) . All CT t-test or U-test as appropriate, while differences between categorical variables in two or more groups were performed by Pearson’s chi-square test or Fisher’s exact probabilities test as appropriate.Normally distributed continuous variables were presented as mean ± SD, skewed continuous variables as median (interquartile range), and categorical variables as absolute values (percentages). For univariate analysis, differences between continuous variables in two groups were performed by p < 0.1 in the univariate analysis. Subgroup analysis was executed to further investigate the relationship between troponin and HT after thrombolysis in different types of patients. All statistical tests were two-tailed, and p < 0.05 was considered statistically significant. All analyses were performed using SPSS 25.0  and figures were drawn with the use of Excel software 2019 .Univariate and multiple logistic regression models were used to analyze the association between troponin elevation on admission, troponin dynamic changes, and HT after thrombolysis, respectively. For the multiple model, model 1 was adjusted for age and sex, and model 2 was adjusted for age, sex and other variables with p < 0.05, A total of 408 patients with acute ischemic stroke were treated with thrombolysis from June 2015 to June 2021 in our hospital. Based on the protocol , 31 patip < 0.05, p < 0.05, p < 0.001).Patients with troponin elevation on admission had more comorbid AF and CAD, cardiac dysfunction (lower ejection fraction), renal insufficiency (higher serum creatinine) and higher stroke severity (higher NIHSS scores) than those without troponin elevation , but patients with falling changes developed only HI rather than PH.Excellent inter-rater agreement for HT (κ = 0.94) and its further classification (κ = 0.84) was observed. Eventually, HT was identified in 61 patients (16.2%), comprising 19 (5%) with HI-1, 16 (4.2%) with HI-2, 15 (4.0%) with PH-1, and 11 (2.9%) with PH-2. Univariate analysis showed that patients with HT had a higher proportion of troponin elevation on admission, troponin dynamic changes and AF as well as higher NIHSS score, INR, and lower ejection fraction , rather than troponin elevation, were associated with unfavorable outcome at discharge when confounding factors were considered  is a common adverse reaction of thrombolytic therapy, and the main mechanism involves blood-brain barrier disruption . In a smCompared with troponin elevation on admission, the association between troponin dynamic changes and HT after thrombolysis was stronger. Rising troponin dynamic changes may also reflect stroke-heart syndrome caused by HT. It is reported that nearly 90% of HT after thrombolysis occurs within 24 h after treatment , while tWe found prognostic significance of troponin for unfavorable outcome at discharge in patients with AIS. A recent meta-analysis provided compelling evidence that troponin elevation can predict all-cause mortality in AIS . PreviouThe study still has limitations. Firstly, previous study has found that approximate 25% AIS patients with troponin elevation of at least 4-fold URL have acute coronary culprit lesions . As for Overall, we found that troponin dynamic changes including both rising and falling pattern were independently associated with HT after thrombolysis. Rising troponin dynamic changes were also prone to adverse outcomes at discharge. Patients with advanced age, premorbid heart disease, renal insufficiency, higher NIHSS score, and troponin elevation on admission tend to present troponin dynamic changes. Continuous monitoring for troponin levels in thrombolytic patients with relevant risk factors is warranted.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of The Second Affiliated Hospital and Yuying Children’s Hospital of the Wenzhou Medical University. The patients/participants provided their written informed consent to participate in this study.ZH and ZC conceived and designed the study. ZC and ZZ analyzed the data. ZC, ZZ, and XH assisted in data interpretation. ZC wrote the manuscript. All authors acquired the data, participated in revising the article, and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Ten well-trained female sprinters  performed an all-out 400 m indoor sprint. Normalized sEMG signals were recorded bilaterally from the selected lower limb muscles. The two-way ANOVA (curve × side) revealed no statistically significant interaction. However, the main effect analysis showed that normalized sEMG signals significantly increased in subsequent curves run for all the studied muscles: gastrocnemius medialis (p = 0.003), biceps femoris (p < 0.0001), gluteus maximus (p = 0.044), tibialis anterior (p = 0.001), and vastus lateralis (p = 0.023), but differences between limbs were significant only for the gastrocnemius medialis (p = 0.012). The results suggest that the normalized sEMG signals for the lower limb muscles increased in successive curves during the 400 m indoor sprint. Moreover, the gastrocnemius medialis of the inner leg is highly activated while running curves; therefore, it should be properly prepared for high demands, and attention should be paid to the possibility of the occurrence of a negative adaptation, such as asymmetries.The purpose of this cross-sectional study was to analyze changes in normalized surface electromyography (sEMG) signals for the  The 400 m race is considered to be one of the most demanding track and field running events . It is rThe times achieved in the curved track sections are significantly slower than those that are straight, due to slower sprint velocities . This isgastrocnemius medialis than for the right leg muscle during the first curve of the 200 m sprint for elite female sprinters in the innermost lane. However, no statistically significant differences were found between the other studied muscles of the lower limbs . The authors speculated that the gastrocnemius medialis is the muscle that manages the movement and the distribution of force during sprinting around curves. Yet, it should be noted that just the first curve of the 200 m dash was evaluated. Considering that the 400 m sprint is an extremely demanding event for the athlete, and the fact that fatigue affects the patterns of muscle activity [However, thus far, only a few studies have considered this issue ,12,13. Tactivity ,15, it cgastrocnemius medialis, biceps femoris, gluteus maximus, tibialis anterior, vastus lateralis) between subsequent curved sections of the track during a 400 m indoor sprint. These muscles were selected because their activation is most often analyzed in sprint studies [gastrocnemius medialis will be higher for the left (inside) than for the right leg (outside) while running curved sections of the 400 m race, but the magnitude of this difference will decrease in successive curves. Additionally, it was expected that the normalized sEMG signals of all studied muscles will increase across the 400 m sprint.No studies have investigated changes in lower limb muscle activity patterns while elite female sprinters have been running curved sections of the indoor 400 m race. Therefore, the purpose of this study was to analyze changes in normalized sEMG signals of selected lower limb muscles  and was performed in accordance with the ethical standards of the Declaration of Helsinki, 2013.A sample size estimation using G*Power software  showed that to detect an effect size of 0.57 [The evaluations were carried out over two trials, 48 h apart (Monday and Wednesday), on an indoor synthetic four-lane track with IAAF certification (Certified Facility by World Athletics as Class 2 ). The figastrocnemius medialis, biceps femoris, gluteus maximus, tibialis anterior, vastus lateralis. An eight-channel Noraxon TeleMyo 2400 Wireless system  was used for the measurements and analysis of the biopotentials from the studied muscles. The whole procedure, including maximum isometric voluntary contraction (MVIC) assessment, electrode placement, and normalization of the sEMG signals to the percent of MVIC, was carefully replicated as described elsewhere [The sEMG signals were recorded bilaterally for the following lower limb muscles: lsewhere .2). Partial eta squared values were classified as small (0.01 to 0.059), moderate (0.06 to 0.137), and large (>0.137). Post hoc comparisons using Bonferroni correction were conducted to locate the differences between mean values when a main effect or interaction was found. For pairwise comparisons, effect sizes were determined by Hedges’ g, which was interpreted as ≤0.20 “small”, 0.21–0.8 “medium”, and >0.80 “large”. Statistical significance was set at p < 0.05.All statistical analyses were performed using SPSS . The Shapiro–Wilk test was used to verify the normality of the sample data. Two-way ANOVAs with repeated measures were performed to analyze differences in normalized sEMG signals between curved sections and lower limbs during the 400 m sprint (4 curves × 2 sides (right limb vs. left limb)) for each muscle. Effect sizes for main effects and interactions were determined by partial eta squared .p = 0.925; η2 = 0.017). The simple main effect analysis showed that running curves  and the lower limbs  had a statistically significant effect on normalized sEMG signals. Post hoc tests indicated a significantly higher normalized sEMG signal for the left gastrocnemius medialis than for the right leg muscle, while running the first and fourth curves . In addition, there was a significantly higher normalized sEMG signal for the right gastrocnemius medialis during the fourth curve in comparison to the first one .A two-way ANOVA revealed that there was no statistically significant interaction between running curves and the right or left lower limbs . The simple main effect analysis showed that running curves  had a statistically significant effect on normalized sEMG signals, but the lower limbs did not . Post hoc tests revealed a significantly higher normalized sEMG signal for the left biceps femoris from the second and fourth curves in comparison to the first one .The two-way ANOVA revealed that there was no statistically significant interaction between running the curves and the right or left lower limbs . The simple main effect analysis showed that running curves  had a statistically significant effect on normalized sEMG signals, but the lower limbs did not . However, post hoc tests did not show any significant differences.The two-way ANOVA revealed that there was no statistically significant interaction between running curves and the right or left lower limbs . The simple main effect analysis showed that running curves  had a statistically significant effect on normalized sEMG signals, but the lower limbs did not . However, post hoc tests did not show any significant differences.The two-way ANOVA revealed that there was no statistically significant interaction between running curves and the right or left lower limbs . The simple main effect analysis showed that running curves  had a statistically significant effect on normalized sEMG signals, but the lower limbs did not . Post hoc tests indicated a significantly higher normalized sEMG signal for the right vastus lateralis while running the fourth curve in comparison to the third one .The two-way ANOVA revealed that there was no statistically significant interaction between running curves and the right or left lower limbs  than in the right (outside) limb while running the curved sections. However, we did not confirm that the magnitude of this difference will decrease over successive curves. Our results show a higher normalized sEMG signal for the left gastrocnemius medialis at each curve, and on the first and fourth curves, this was significantly higher than for the right leg. These results are partially in contrast to those reported by Iwanska et al. [gastrocnemius medialis was significantly greater than that for the right leg muscle during curve sprinting on the inside lane (lower radius), which was not noticed during sprinting on the outside lane (greater radius).These results confirm part of our hypothesis that the normalized sEMG signals of the a et al. . Those aa et al. ,18,19. Ta et al.  partialla et al.  may alsobiceps femoris, gluteus maximus, tibialis anterior, vastus lateralis), a trend of higher normalized sEMG signals of the left than the right limb was observed, but they were not statistically significant. Pietraszewski et al. [gastrocnemius medialis muscle. Therefore, these results could confirm suggestions by Pietraszewski et al. [gastrocnemius medialis muscle of the inner limb manages the distribution of forces generated by the remaining muscles.For the remaining analyzed muscles  was not investigated. Moreover, the sEMG signals measured during high-speed movement might be influenced by, for example, electrode shift, changes in the conductivity of the muscle tissue, and crosstalk from other muscles . Lastly,gastrocnemius medialis muscle of the inner leg were significantly greater than those for the outer leg, and this pattern was maintained throughout the whole race. Coaches and athletes should bear in mind that the gastrocnemius medialis of the inner leg is more highly activated while running curves; thus, this could lead to a motor learning effect, and morphological and physiological adaptations in muscle structure and function [In conclusion, the normalized sEMG signals of the lower limb muscles increased in successive curves on the 400 m indoor sprint track. Moreover, the normalized sEMG signals for the function , leading


To study the presence of liver‐expressed antimicrobial peptide 2 (LEAP2) in human cerebrospinal fluid (CSF) and to measure its concentrations in neurological disorders.We identified the presence of LEAP2 in human CSF by chromatographic analysis and a LEAP2‐specific enzyme immunoassay. We measured LEAP2 concentrations in the CSF of 35 patients with neurological disorders.SD, 9.32 ± 3.76 ng/ml) were significantly higher (p < .05) than those in the other four groups .CSF LEAP2 concentrations in the bacterial meningitis group , an antimicrobial peptide, in human CSF, and is the first to measure CSF LEAP2 in neurological disorders. CSF LEAP2 is increased in patients with bacterial meningitis, suggesting its biological significance in the pathology of CNS infections. These patients were admitted to our hospital between April 2014 and March 2020, and CSF was examined immediately after admission. Patients with CIDP, GBS, and MS were diagnosed according to European Federation of Neurological Societies/Peripheral Nerve Society clinical criteria  by the hydrogen peroxide method  prepared as described elsewhere , serum glucose, C‐reactive protein (CRP), CSF protein, and CSF cell count were analyzed by Spearman's correlation coefficient test. Data are expressed as means ± 33.1To identify the presence of LEAP2 in human CSF, we analyzed LEAP2‐immunoreactive substances in the CSF by RP‐HPLC. ELISA analysis of human CSF samples showed two immunoreactive peaks Figure . Their e3.2CSF LEAP2 concentrations in the bacterial meningitis group were significantly higher than those in the other four clinical groups Figure . CSF LEA3.3SD, 18.35 ± 7.15 ng/ml) .The serum LEAP2 concentrations in the 12 healthy subjects ranged from 6.89 ng/ml to 28.84 ng/ml (mean ± 4In this study, we first verified the presence of LEAP2 in human CSF by RP‐HPLC combined with ELISA. LEAP2 has an N‐terminal methionine whose thiomethyl residue is readily oxidized under conventional conditions Davies, . We hypoLEAP2 penetrates the negatively charged bacterial membrane because of its characteristic strong basicity and two disulfide bonds, causing holes or pores that eventually lead to bacterial cell death. This behavior affects the membranes of bacteria more preferentially than those of eukaryotes (Townes et al., In this study, we showed that patients with bacterial meningitis had significantly higher CSF LEAP2 concentrations than those with other neurological disorders and a control group. CSF LEAP2 concentrations positively correlated with inflammatory parameters in the CSF and serum. The CSF LEAP2 concentration in the patient with Listeria meningitis declined during the recovery phase. Elevated CSF LEAP2 concentrations appear to reflect bacterial infection in the CNS.The patient with bacteremia had a normal CSF LEAP2 concentration but an elevated serum LEAP2 concentration, while the reverse was true in the patient with Listeria meningitis. Another antimicrobial peptide, LL‐37, was produced in astrocytes and microglial cells in a rat model of pneumococcal meningitis (Brandenburg et al., Inflammation produces a variety of anorectic substances that reduce food intake (Roxburgh and McMillan, Clinical guidelines recommend that when bacterial meningitis is suspected, empirical therapy should be commenced before CSF tests and CNS imaging are performed (Young and Thomas, Limitations of this study include the lack of serum samples from patients with pneumococcal meningitis and the small number of CSF samples obtained during the course of routine clinical examination.Considering LEAP2’s functions as an antimicrobial peptide and an antagonist of the ghrelin receptor, elevated CSF LEAP2 concentrations in bacterial meningitis suggest that LEAP2 may act against local bacterial infection or acute inflammation. Thus, increased LEAP2 levels during bacterial infection may be applicable as a potential biomarker. Furthermore, the presence of LEAP2 in the human CSF may suggest the existence of a peptidergic innate immune system in the CNS. This study should contribute to better understanding the host defense in bacterial meningitis.5CSF LEAP2 is increased in patients with bacterial meningitis, suggesting its biological significance in the pathology of CNS infections.6We confirm that we have read the Journal's position on issues concerning ethical publication and affirm that this report is consistent with those guidelines.The authors declare that they have no competing interests.Katsuya Sakai contributed to study concept and design, analysis and interpretation of data, drafting of the manuscript, and statistical analysis. Masamitsu Nakazato, Kazutaka Shiomi, Hitoshi Mochizuki, Md Nurul Islam, Hiroki Nabekura, Ryota Tanida, and Hideyuki Sakoda contributed to critical revision of the manuscript for important intellectual content.https://publons.com/publon/10.1002/brb3.2111.The peer review history for this article is available at


CSF biomarkers determined AT status and indicated synaptic damage (neurogranin). The AD patients (n = 294) had higher CSF noradrenaline and adrenaline concentrations, but lower dopamine concentrations compared to the cognitively unimpaired (n = 113). AD patients in the MCI and dementia stage of the disease had similar CSF catecholamine concentrations. In the CSF neurogranin positively associated with noradrenaline and adrenaline but not with dopamine. Adjusted regression analyses including AT status, CSF neurogranin, age, gender, and APOEε4 status verified the findings. In restricted analyses comparing A+T+ patients to A−T− cognitively unimpaired, the findings for CSF adrenaline remained significant (p < 0.001) but not for CSF noradrenaline (p = 0.07) and CSF dopamine (p = 0.33). There were no differences between A+T+ and A−T− cognitively unimpaired. Thus, we find alterations in CSF catecholamines in symptomatic AD and the CSF adrenergic transmitters to increase simultaneously with synaptic damage as indexed by CSF neurogranin.Noradrenergic and dopaminergic neurons are involved in cognitive functions, relate to behavioral and psychological symptoms in dementia and are affected in Alzheimer’s disease (AD). Amyloid plaques (A), neurofibrillary tangles (T) and neurodegeneration (N) hallmarks the AD neuropathology. Today, the AT(N) pathophysiology can be assessed through biomarkers. Previous studies report cerebrospinal fluid (CSF) catecholamine concentrations in AD patients without biomarker refinement. We explored if CSF catecholamines relate to AD clinical presentation or neuropathology as reflected by CSF biomarkers. CSF catecholamines were analyzed in AD patients at the mild cognitive impairment (MCI;  Alzheimer’s disease (AD) is the most common cause of dementia . Along wBehavioral and psychological symptoms in dementia (BPSD) are associated with changes in catecholamine transmission . NoradreDopaminergic neurons originate from the substantia nigra and the ventral tegmental area (VTA). While the substantia pars compacta (SNpc) form pathways associated with movement, the VTA is, among other things, associated with reward and cognitive functions, such as memory , 6. AlteBiomarkers now allow in vivo determination of the AD AT(N) status , 26. ThuWe aimed to explore if CSF catecholamine concentrations relate to the AD clinical diagnosis, AD clinical stage, or AD neuropathology, including synaptic damage as reflected by CSF biomarkers. CSF catecholamines were analyzed in clinically diagnosed AD patients at the mild cognitive impairment (MCI) or dementia stage with cognitively unimpaired as reference. CSF measures were applied to secondarily determine AT(N) state. As cognitive impairment in AD seems related to synaptic damage , the assThe study-setup was cross-sectional, including memory clinic patients with a clinical presentation of AD or mixed AD and cerebrovascular disease at different disease stages (MCI and dementia) and cognitively unimpaired Table .Table 1Cn = 176) and St. Olav University Hospital (n = 143), with a clinical presentation of probable or possible AD or AD mixed with cerebrovascular disease according to the core clinical NIA-AA 2011 criteria [The study included patients admitted to the memory clinics at Oslo University Hospital , phosphorylated-tau181 (P-tau), and total-tau (T-tau), in CSF were analyzed at Akershus University Hospital (AHUS) by enzyme-linked immunosorbent assays  and β-amyloid 1–42 Fujirebio Europe, Gent, Belgium) [The patients underwent a diagnostic lumbar puncture, all before 11 AM, CSF for research purposes was obtained in cryotubes after the routine samples were collected. The cryotubes were centrifuged at 2000×Belgium) . CSF mean = 113). These underwent the same cognitive tests as the memory clinic patients and those with normal results at baseline were initially included. The majority was also tested after two years, and those with abnormal test results at follow-up were excluded. Patients were also excluded if they had sequela after stroke, Parkinson’s disease, or other neurodegenerative diseases likely affecting cognition (at baseline). CSF was obtained in conjunction with spinal anesthesia at the daytime surgery . Time of sampling was missing for two patients. The first drops of CSF were discarded and CSF thereafter obtained in cryotubes until a maximum of 12 ml. Further details are previously described for this cohort [.Patients undergoing elective surgery for gynecological, orthopedic, or urological problems were recruited as a cognitively unimpaired control group  by use of reagents from Chromsystems (#5000). An internal standard, 3,4-dihydroxybenzylamine (DHBA) was added to all CSF samples and CSF samples run after the calibration standards. The coefficients of variation (CV) were 4.7, 6.2, and 8.3% for noradrenaline, adrenaline, and dopamine, respectively, as calculated by a CSF pooled sample injected for every tenth injection. Further details are described in a previous publication .The analyses of CSF neurogranin were performed at the Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden using an in-house ELISA, based on the NG2 and NG36 antibodies for all samples. Duplicate measures were run with the same batch of reagents. Run acceptance followed accurate criteria and the CV of the duplicate measures were 5.0%. For further details, please see ref. .IBM SPSS version 25  was used for the statistical analyses.T-tests or one-way ANOVA were employed for the continuous variables, Pearson’s χ2 for the categorical variables.Parametric tests were applied as the sample size was relatively large and the sample distributions were found to approximate the normal distribution when visually inspected. Regression analyses were performed with noradrenaline, adrenaline, or dopamine as dependent variables, respectively. In addition to diagnoses, independent variables were included based on biological grounds and comprised Aβ42, P-tau, neurogranin as well as age, gender, and APOEɛ4 genotype.r = 0.84, p < 0.001) and in the cognitively unimpaired . N (T-tau) was therefore not included in the regression analyses as P-tau (T) is regarded more AD specific. Neurogranin is previously shown to correlate with P-tau and T-tau [r > 0.8, p < 0.001 for all groups). Therefore, in the multiple regression analyses only neurogranin was included as a marker of AD related synaptic dysfunction. CSF neurogranin data were not available for 91 memory clinic patients and one cognitively unimpaired. These were excluded from analyses, including CSF neurogranin, but included for other analyses.For analyses including both patients and cognitively unimpaired, Aβ42 and P-tau were dichotomized according to each laboratory’s own cut-offs. P-tau and T-tau were highly correlated both in the patients  and the Data Protector Officer at our institution approved the study.n = 54) and dementia stage (n = 240) and cognitively unimpaired (n = 113). The cognitively unimpaired were slightly older (p = 0.007) and were more educated (p < 0.001) than the AD patients. The cohorts are further described in Table r = 0.55, p < 0.001). The correlation was similar within the AD patients  and cognitively unimpaired . CSF dopamine concentrations did not correlate with neither CSF noradrenaline nor CSF adrenaline .The CSF catecholamine concentrations were analyzed in AD patients at the MCI . The CSF catecholamine concentrations were altered in symptomatic AD. The AD patients had higher concentrations of noradrenaline and adrenaline in CSF than the cognitively unimpaired (p = 0.001 and p < 0.001 n = 294 vs. n = 113). In contrast, the AD patients had lower CSF dopamine concentrations relative to the cognitively unimpaired . The other co-variates age, gender, APOEε4—and CSF Aβ42 positivity were not significant in these analyses. Corresponding analyses of CSF dopamine confirmed the negative association between an AD clinical diagnosis and lower CSF dopamine. Neither CSF neurogranin nor any of the other predictors  were significantly associated with CSF dopamine . As expected, the AD dementia patients had a higher score on the NPI-Q scale than the MCI patients  , 63 and LC NFT pathology in asymptomatic individuals is associated with alterations in stress-responses, microglia, and hippocampus tyrosine hydroxylase activity and alterations in α2 adrenergic receptor expression . In younA strength of the current study is the fairly large sample size, as the current study included more patients than previous studies. The patients were well defined, as they were recruited from specialized memory clinics and underwent a comprehensive clinical assessment with available biomarkers allowing secondary biomarker refinement. The cognitive unimpaired group went through the same comprehensive assessment.CSF biomarkers that have shown high accuracy , 64 wereStudy limitations also include that CSF neurogranin measurements were missing in 91 patients and one cognitive unimpaired. This may have weakened the association found between the CSF catecholamines and neurogranin. That the CSF core biomarkers were analyzed in two different laboratories prevented certain statistical analyses. Furthermore, only cognitively unimpaired and clinical AD patients with and without AT pathology were included in the study. Other pathologies also affect the catecholamine neurons or share traits such as neuroinflammation with AD, but the specificity for AD could here not be assessed. The time of CSF sampling was not matched between the AD patients and the cognitively unimpaired, but all samples were collected during daytime. As this was a cross sectional study including patients with MCI and mostly mild dementia, an evaluation of cognitive changes over time in relation to the catecholamines was not feasible.CSF catecholamine concentrations are altered in symptomatic AD from the early phase of the disease. CSF noradrenaline and adrenaline concentrations were higher among AD patients but their temporal dynamics may be non-linear inferring low prognostic and diagnostic value of these transmitters. The two adrenergic transmitters may increase with synaptic damage in symptomatic AD as they positively associated with CSF neurogranin, suggesting similar temporal dynamics but should be confirmed in follow-up studies. This, and the role of the LC in early AD pathology, would be interesting to pursue in future studies. In contrast to CSF noradrenaline and adrenaline, CSF dopamine was lowered in clinical AD and did not relate to the AD neuropathological markers. This indicate CSF dopamine to relate to AD clinical presentation rather than AD neuropathology.Supplementary Table 1.


The original figures and figure legends, were mislabeled with “0.16 ppm thyme oil”. They should have been labeled with the correct label “0.16 ppb thyme oil” in all instances. The correct figures and figure legends appears below.In the original article, there was a mistake in Throughout the Supplementary Material “0.16 ppm thyme oil” should be corrected to “0.16 ppb thyme oil” in all instances. The correct files appear below.In the original article, there was a mistake in the Throughout the text it was mistakenly written that “0.16 ppm thyme oil” was utilized for experiments. It should be written “0.16 ppb thyme oil” for all experiments. The original article has been updated.In the original article, there was an error. . It was mistakenly written “0.06 ppm thymol” instead of “0.06 ppb thymol”: “We estimate that 0.16 ppb thyme oil used in this experiment may contain approximately 0.06 ppb (60 ppb) thymol (37% thymol in 0.16 ppb thyme oil corresponds to ~ 60 ppb)”.In addition, in the original article, there was an error4. Materials and Methods, 4.4. Honey Bee Diet Preparation: “Post-injection, honey bees were housed in modified deli containers for the duration of the study. Bees in the control group were fed 50% sucrose syrup only, whereas bees in treatment groups were fed sucrose syrup containing one of the following additives: 0.16 ppb thyme oil (Body wonders), Fumagilin-B®, fumagillin dicyclohexyl ammonium  at manufacturer’s recommended dose of 25 ppm or a higher dose of 75 ppm, or clothianidin at the field relevant sublethal concentration of 1 ppb or near lethal dose of 10 ppb . Thyme oil contains 10% - 64% thymol (~37% average) depending on the plant species, geographical sources, and harvest season, which may affect the volatile composition of the plant . We estimate that 0.16 ppb thyme oil used in this experiment may contain approximately 0.06 ppb thymol (37% thymol in 0.16 ppb thyme oil corresponds to ~ 0.06 ppb). For clothianidin treatments we utilized the commercially available Poncho® 600, which contains 48% of the active ingredient clothianidin. A working stock of 1000 ppb clothianidin was prepared by 1:10 serial dilutions in 50% sucrose solution, which was further diluted in 50% sucrose to prepare 1 ppb  and 10 ppb  clothianidin solutions. Honey bees were fed sucrose solution either alone or with additives using cage feeders that were made by putting two holes on each side of a 1.5 mL centrifuge tube; sucrose solution was checked daily and refilled as needed throughout the study.”A correction has been made to The authors apologize for these errors and state they do not change the scientific conclusions of the article in any way.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.