Clostridioides difficile infection (CDI) and inflammatory bowel disease (IBD). The engraftment of donor bacteria is essential for the effectiveness of FMT, which to some extent depends on the matching of donors and recipients. However, how different types of donor-derived bacteria affect FMT efficacy has not been fully dissected. We recruited two longitudinal IBD cohorts of 103 FMT recipients and further analyzed 1,280 microbiota datasets from 14 public CDI and IBD studies to uncover the effect of donor-derived microbiota in recipients. We found that two enterotypes, RCPT/E and RCPT/B , consistently exist in both CDI and IBD patients. Based on a time-course-based multi-cohort analysis of FMT fecal samples, we observed the interplay between recipient and donor-derived microbiota during FMT, in which the FMT outcome was significantly associated with the enterotype and microbiota distance between donor and recipient after FMT. We proposed a new measurement, the ratio of colonizers to residents after FMT (C2R), to quantify the engraftment of donor-derived bacteria in the recipients, and then constructed an enterotype-based statistical model for donor-recipient matching, which was validated by both cross-validation and an additional IBD FMT cohort (n = 42). We believe that with the accumulation of FMT multi-omics datasets, machine learning-based methods will be helpful for rational donor selection for improving efficacy and precision FMT practices.Fecal microbiota transplantation (FMT) is a promising treatment for microbiota dysbiosis associated diseases, such as  Clostridioides difficile infection (CDI) and inflammatory bowel disease (IBD) are the two most notorious. CDI, caused by the antibiotic resistant gram-positive pathogen C. difficile, has a high incidence of ~20% recurrence and results in 30,000 deaths in the US per year.1 Meanwhile, IBD affects over 7 million people globally, with symptoms of persistent diarrhea, abdominal pain, and cramping in the gut.2 Although CDI and IBD are distinct entities, they are still highly similar in many ways.3–5 Recent studies found that both diseases frequently undergo disruption of the gut microbiota.7 In view of the harmfulness of the two diseases and the defects of conventional therapeutic methods, there is an emerging need for microbial therapy targeting their disordered microbiota.Gastrointestinal dysbiosis is closely related to a variety of health problems in humans, of which 9 In FMT, the gut microbiota is transplanted from healthy donors to patients to treat diseases that do not respond to traditional medicine. This technique has proven effective in many microbiota-related metabolic,10 infectious,11 and inflammatory diseases, including recurrent CDI and IBD. For recurrent CDI, FMT breaks the recurrence cycle and effectively cures up to 85% of patients.11 Similarly, the transmitted donor microbes are beneficial for relieving inflammation in IBD patients. In a meta-analysis of 122 patients, the remission rate of IBD after FMT was 45%, which was significantly higher than that in the placebo group (20%).12 Unfortunately, although FMT is a feasible solution for treating the two diseases, it is ineffective or associated with relapse in a certain proportion of patients. To improve the efficacy of FMT in precision medicine, understanding the mechanism underlying its effectiveness against the two diseases and the reasons for FMT failure in some cases is necessary.Fecal microbiota transplantation (FMT) is one of the alternative therapies used enthusiastically in clinical practice in recent years.13 which may be because more microbial species would have more opportunities for engraftment. This donor-dependent effect on FMT efficacy was also reported in many other clinical trials of gastrointestinal disorders, and such donors were recognized as “super-donors”.14 Even though super-donors may be a solution for improving the efficacy of FMT, such donors are not easy to identify, and may not fit all.15 Therefore, leveraging donor-recipient matching to improve the engraftment of the donor microbiota in the recipient has been taken into consideration and is thought to be essential for FMT success.16 Recently, many studies reported that the donor’s microbes were more easily engrafted when certain species were already present in the recipient.18 However, how donor-recipient matching and engrafted donor microbes contribute to FMT success has not been well elucidated.19 Considering that different types of microbial disorders may take place in different diseases, more studies on the principles of effective donor selection and the patterns of donor-recipient matching are still needed.Screening an effective donor and matching them to a suitable recipient are considered crucial for improving the success rate of FMT. High microbial richness in the donor’s microbiota was found to be associated with the response of IBD patients,Here, we firstly recruited a cohort of 61 patients with mild to severe IBD symptoms who completed a full FMT trial to investigate the relationships between their gut microbiota signatures and the FMT response rate. We performed a comprehensive analysis of 1,440 longitudinal microbiota samples from our study and public cohorts of CDI and IBD patients treated with FMT. Next, we explored the gut microbiota signatures through enterotype clustering across the two diseases, quantified donor-derived bacteria that colonized the recipients and tested the contributions of these bacteria to the response rate of FMT. Enterotype matching between recipients and donors that affected the response rate was identified, applied in a clinically accessible machine learning model with rigorous cross-validation, and finally validated in an independent cohort of IBD patients (n = 42) who were recruited in this study and treated with FMT.We enrolled 102 IBD patients with mild to severe symptoms with at least a 3-month follow-up evaluation for treatment effectiveness , as CDI is also frequently and effectively treated by FMT.To validate the donor selection model, we further recruited a validation cohort of 42 IBD patients by applying different donor-recipient matching strategies. The disease severity of these patients showed no significant difference from that previously measured . Longitudinal fecal samples of these patients before and after FMT as well as from their corresponding donors were collected and sequenced using the same experimental procedure.Bacteroides , and hereafter, they are denoted as RCPT/E and RCPT/B, respectively . In addition, the two enterotypes were pervasive in both CDI and IBD patients with a separate preference , implying enterotype is a distinct and reliable typing method for microbiota-related diseases.To make the characteristics of gut microbiota dysbiosis in the 15 IBD and CDI cohorts comparable, we first unified the processing and analysis methods for all microbiome datasets . After strict quality control, we retained the gut microbial profiles of 322 patients who received FMT , and for each disease, we clustered the patients into different enterotypes. Patients were divided into two enterotypes regardless of disease category  . The two enterotypes in different diseases were consistently dominated by Enterobacteriaceae and 36 As shown in To validate the consistency of these two enterotypes in CDI and IBD, we combined all the datasets of the two diseases and performed enterotype clustering using multiple approaches (partitioning around medoids (PAM) and Dirichlet multinomial mixtures (DMM))Bacteroides) were markedly differentiated across enterotypes  tended to be present in the upper gastrointestinal tract, featuring lower pH and higher oxygen levels,37 and they were enriched in RCPT/E instead of RCPT/B , except for the factor “corticosteroids history” . It indicates that some medications, like corticosteroids, may contribute to shaping patients’ enterotype as RCPT/B. We next explored the microbiota diversity between the two enterotypes, and found that alpha diversity was significantly lower in RCPT/E than in RCPT/B and healthy donors regardless of disease category  , indicatf RCPT/B , which hConsidering the two types of gut microbiota dysbiosis among the patients suffering from CDI and IBD, we investigated the improvement of the recipient’s gut microbiota after FMT from the perspective of enterotype-based analysis. After FMT, the alpha diversity of the recipient’s gut microbiota increased significantly over time , where both enterotypes exhibited a significant increase  and approximated the level of healthy donors  , which illustrated the effect of FMT donors on modulating microbial community structure.We next analyzed the variation in dominant bacteria in the two enterotypes of recipients and donors. In contrast to alpha diversity, the relative abundance of the dominant bacteria in recipients after FMT decreased toward the level of the donor, especially for RCPT/E . MoreoveTo quantify the donor’s contribution to modulating recipients’ microbial community, we calculated the Bray-Curtis dissimilarity (BC distance) among these samples and performed principal coordinates analysis (PCoA) . Recipients (before and after FMT) and donors formed three distinct clusters along principal coordinate 1 (PC1). After FMT, the recipients were closer to the donors along PC1 than to themselves before FMT . We then calculated the BC distance between each recipient and its corresponding donor over time. The distance in both enterotypes decreased during the first few weeks after FMT and reached a minimum at approximately two months , potentially reflecting the engraftment and bloom of donor-derived bacteria.To explore the underlying links between donor-derived bacteria and FMT efficacy, we further focused on recipients with known FMT outcomes (response/failure). We found that the recipients with different outcomes clearly formed two groups based on the microbial BC distance to their corresponding donors and themselves before FMT  . We compared the cumulative abundance of these taxa between the two potential sources (donors and patients before FMT)  were also classified as residents in the respective patients , which in turn reduced the C2R. In addition, several taxa in donors were negatively correlated with Clostridioides in recipients after FMT , indicating the significance of selecting suitable donors to reduce pathogenic residents in patients.Using the abundances of all residents and colonizers, we calculated the C2R for each recipient, and found that the C2R was significantly elevated in the response group compared to the failure group  , which w40) on these microbiota datasets (see Methods). The microbial profiles of donors and patients before FMT were used as potential sources in the input to FEAST, and the fold change was calculated using the probability from the two potential sources to mimic the C2R. As shown in 19 and an IBD study,41 and found that a similar transmission trend was reproduced based on the residents (e.g. Fusobacterium and Enterobacteriaceae) and the colonizers  in the 16S rRNA datasets . In addition, the C2R was higher in the response group compared to that in the failure group . Taken together, these findings demonstrated the robustness and scalability of the C2R.To further validate the applicability of the C2R, we first performed source tracking analysis using a state-of-the-art method . The two donor enterotypes had similar microbial diversity  and age distribution  . We next measured the response rate for each enterotype of recipients with different donor types. As shown in Since the engraftment of donor-derived bacteria was significantly associated with FMT outcome, we wanted to know which enterotype of donors was more effective for CDI or IBD patients. We first clustered the donors’ gut microbiota profiles following the same approach as that used for patients. The donors could be divided into two enterotypes, DONOR/P and DONOR/B, named according to their dominant taxa, To determine why DONOR/P was more effective against RCPT/B than DONOR/B, we first compared the microbial BC distances of RCPT/B to the two donor enterotypes. As shown in To illustrate the engraftment superiority of distantly related donors in RCPT/B, we obtained the bacteria enriched in each of the two donor enterotypes  and calculated the cumulative abundance of these bacteria. As shown in To evaluate the application of enterotype-based donor-recipient matching in FMT, we built a machine learning model to match patients with suitable donors, and validated it using both cross-validation and an additional FMT cohort with 42 newly recruited IBD patients . A randoWe further compared the EDS model against three alternative models, in which the input features included microbial profiles from only patients, only donors, or both. Consistent with the EDS model, random forest model was also used for prediction in the three alternative models with same parameters. Through 500 iterations of 5-fold cross-validation, we found that the EDS model outperformed all three alternative models  , indicatAlthough model performance was evaluated using cross-validation, we sought to validate it by recruiting an additional validation cohort of IBD patients n = 42, . We appl11 and metabolic disorders.10 However, how to choose a suitable donor for patients has not been fully elucidated. In this study, we applied a conceptual framework to the human gut microbiota to obtain more insights into donor-recipient matching and thus built an enterotype-based model for donor selection. By combining our two recruited cohorts treated with FMT and additional 14 public FMT cohorts, we identified the existence of two robust enterotypes (RCPT/E and RCPT/B) in both CDI and IBD patients. We demonstrated that the engraftment of donor-derived microbes was significantly associated with FMT outcome and discovered that DONOR/P was more suitable for treating RCPT/E patients. By leveraging enterotype-based classification of the gut microbiota in donors and recipients, we proposed an EDS model for selecting suitable donors for patients, which was confirmed through statistically rigorous cross-validation and an independent IBD cohort, achieving a response rate of 93.3%.By restructuring the recipient gut microbiota with donor fecal samples, FMT has been used to treat many diseases, such as infections42–44 researchers focused on one disease and neglected the commonalities in the microbial interactions of FMT. In this study, we introduced enterotype to find the microbial similarity between the two diseases. In the light of enterotype, patients of the two diseases can be divided into two conservative enterotypes (RCPT/E and RCPT/B), which exhibit similar microbial compositions  cohort, transplanted microbes were found to engraft and colonize the patients’ gut for months,45 which is in line with our finding on the reduced BC distance between recipients and donors over time, implying the engraftment and bloom of the donor-derived microbiota in the recipients’ intestines. Engraftment of the donor-derived microbiota in recipients may help improve patients’ gut microecology and health. For example, a significant increase in short-chain fatty acid-producing microbes, particularly Eubacterium, was observed in FMT-treated CDI patients.47 The engraftment of some species in Actinobacteria, was found to be linked with better FMT outcomes in IBD patients.41 However, because of restrictions in sample size and analytical methods, the quantitative correlation between the engraftment of donor-derived bacteria and FMT outcomes has not been clearly elucidated. In this study, we illustrated a significant association between donor-derived bacterial engraftment and FMT outcomes by introducing a new quantitative measurement: the ratio of colonizers to residents after FMT (C2R). Our findings demonstrate that the engraftment of donor-derived microbes in patients after FMT can be used as an indicator for FMT success.In this study, we found that both alpha diversity and BC distance in recipients’ gut microbiota resemble those of the donors’ after FMT. In a previous metagenomic analysis of 19 CDI patients, nearly 40% of the strains from donors were found in post-FMT patients, and the abundances of the strains were significantly enhanced.48 Donors selected according to these criteria may have a healthy gut microbiome, but it is unlikely to be suitable for all patients with different clinical and microbiota backgrounds.16 For example, previous studies found that the colonization and therapeutic effect of a transplanted microbiota from the same donor differed greatly among patients.49 One solution is to find beneficial microbes for disease curation, such as Akkermansia for IBD,50 which is transplanted into patients with a designed beneficial microbiota formulation. However, the outcomes of this strategy in CDI trials were vague,52 probably because certain beneficial bacteria cannot survive or fulfill their function without a complete microbial community. In this study, we proposed a new EDS model for evaluating donor-recipient matching and improving FMT efficacy mainly for moderate and severe patients in our cohort. The two enterotypes (RCPT/E and RCPT/B) used in the EDS model have been confirmed by several recent studies. In a study of 57 patients who suffered from CDI, for example, gut microbiota samples were clustered into two enterotypes, with Bacteroidaceae and Enterobacteriaceae being the two most abundant taxa.42 Similarly, in the Meta-HIT cohort43 and HMP cohort,44 the enterotype Bacteroides was found in the majority of IBD patients, whereas Enterobacteriaceae was predominant in the other IBD patients. Considering that FMT is a therapy used to restructure the recipient’s gut microbial community through bacterial transmission and engraftment, recipients with similar community structures may be good matches for the same kind of donor. Our study found that patients with different enterotypes (RCPT/E and RCPT/B) responded best to different donor selection guidelines. For RCPT/E patients, there was little difference in response rate when selecting donors with different enterotypes, particularly in CDI. For RCPT/B patients, donors dominated by Bacteroides were not suitable for transplantation. However, distantly related donors, such as DONOR/P, were more effective, which showed a high possibility of engrafting more diverse bacteria. A meta-analysis of 319 patients with IBD showed that receiving fecal bacteria from a close related donor  was less effective than from a distant donor, with a 16% decrease in clinical remission.53 And certain species in the dominant taxon in DONOR/B (e.g. Bacteroides) were associated with poor outcomes in human clinical studies in IBD patients,41 further suggesting that DONOR/P may be an optimal option for treating IBD patients with RCPT/B.Currently, FMT donor screening criteria mainly relate to biosafety issues to ensure that donors do not pose any risk of transmissible adverse factors, such as pathogens or diseases.54 By analyzing two metagenomic datasets (CDI and IBD) , we found that two enterotypes (RCPT/E and RCPT/B) were prevalent in patients with IBD or CDI. And our proposed C2R was scalable and robust in the metagenomic datasets, where colonizers and residents were consistent at a higher resolution. Second, our meta-analysis was limited by the inability to adequately control for the factors that may potentially influence gut microbiome and FMT efficacy, including biological factors , dietary factors55 and medicines.48 Future clinical studies with comprehensive design to measure the effect of these factors would contribute to the standardization of FMT.This study still has several limitations. First, our work mainly focused on 16S rRNA amplicon sequencing data, which is limited in the resolution of taxonomic assignment and function inference. Metagenomic sequencing can provide enough phylogenetic resolution to strain level, which would be great helpful for engraftment estimation.Our study reveals the consistency of two enterotypes between CDI and IBD, and based on enterotype matching between recipients and donors, we propose an EDS model to improve FMT outcomes. This study also provides a quantitative measurement to bridge the engraftment of donor-derived microbes and FMT outcomes, which may be the foundation for precision FMT. With the accumulation of FMT multi-omics datasets, we believe that more robust machine learning-based methods will be developed to improve the clinical treatment of intestinal diseases.56We enrolled a cohort of IBD patients treated with FMT. This clinical trial was registered as NCT01793831 and NCT01790061, and approved by the Second Affiliated Hospital of the Nanjing Medical University Institutional Ethical Review Board. A part of this clinical trial has been published previously.Patients with IBD were recruited based on our inclusion criteria (see details in Supplementary Methods). All patients who had poor response to convention therapy, for example, 5-aminosalicylic acid, corticosteroids, immunomodulators, and willing to receive FMT were included. Corticosteroids were required to taper off at least 1 week before FMT, and other medications were stopped except 5-aminosalicylic acid prior to the first FMT. Probiotics or antibiotics were not suggested after FMT. The baseline characteristics of eligible participants were recorded, including age, gender, diagnosis, Mayo score/Harvey–Bradshaw index, and relationship with the corresponding donor (Supplementary Table S1). All subjects underwent laboratory examinations  before FMT. Endoscopy and total abnormal magnetic resonance imaging were used to assess the condition of the patients when necessary. Due to histology or endoscopy, which can accurately reflect disease severity, was not practical for each patient, Mayo score/Harvey–Bradshaw index was used to evaluate disease activity.57 The FMT procedure, involving the preparation of washed microbiota suspension,58 the step-up FMT strategy and FMT delivery methods, was the same as the procedure published previously.56 3–5 Units of fresh fecal bacteria (1 U = 1 × 1013 cells) in suspension  according to patients’ conditions58 was delivered through one of the three delivery ways: endoscopic, nasojejunal tube or transendoscopic enteral tubing.56 The decision on the delivery method was made based on the patient’s characteristics and medical application conditions. Prior to transplantation, no antibiotics or/and colon washing were specifically ordered for bowel preparation. However, it is not practical to exclude the following patients with potential bowel preparation: the patients with previous antibiotic treatment for intestinal infections or infections beyond gut, or patients underwent colonic endoscopy for evaluation. Clinical response was evaluated based on the criteria in the Supplementary Methods at 3 months after FMT or the last medical visit (≥ 3 months).The donors mainly came from Chinese fmtBank (China Microbiota Transplantation System) and patients’ relatives or friends who met our strict criteria (see Supplementary Methods).59 These samples were used to extract microbial DNA for sequencing. The V4–V5 region of the bacterial 16S rRNA gene was amplified using Phusion High-Fidelity PCR Master Mix with HF buffer  and then sequenced on the Illumina MiSeq platform  using standard Illumina sequencing protocols.61 Only patients with complete pre- and post-transplant samples and donor samples were included in downstream analysis.Stool samples were collected from patients and donors in sterilized Eppendorf tubes and stored at −80°C.62 we manually excluded studies with fewer than 20 human fecal samples, missing sequence data, and unclear metadata . Most metadata were downloaded from the NCBI SRA database or supplementary files provided in the original publications, but some were acquired after communicating with the authors by e-mail. To maintain uniformity,63 we focused on only the 16S rRNA gene amplicon sequencing data of CDI and IBD patients, which included most studies and samples in the searched hits. Fourteen suitable studies were found, and their corresponding characteristics are listed in Supplementary Table S2. These cohorts in these studies were used as discovery cohorts in downstream analysis.We used PubMed and the NCBI BioProject to search for studies that included fecal metagenomic sequence data of human FMT patients and donors . To guarantee the robustness of our meta-analysis,64 Only merged reads were used in downstream analyses. After that, the software AfterQC was used to perform quality control with default parameters.65 To ensure consistency with previous studies, operational taxonomic units (OTUs) were selected against the 13–8 Green Genes release and clustered at a 97% identity using QIIME pick_otu_closed_ref with the following parameters: -strand both, -id 0.97.66 Samples with fewer than 2500 OTU counts were removed. We then collapsed OTUs to the genus level with QIIME summarize and adopted standard library size normalization in log-space. All statistical analyses were performed on these normalized genus-level relative abundance data.Sequence data were downloaded from the SRA database using the accessions listed in Supplementary Table S2. When paired-end reads were available, they were merged using FLASH with default parameters.67To evaluate the effect of potential confounders related to different FMT cohorts, we applied an ANOVA-type analysis and compared the amounts of mean variance explained for each genus. The explanations were compared across these factors, including FMT status and batch effect (study). Variance estimates were obtained for ranks to account for the non-Gaussian distribution of the microbiota abundance data. To further eliminate the potential batch effect, we also employed a blocked version of the Wilcoxon test in the coin package in R by treating ‘study’ as a blocking factor in all analyses unless otherwise specified.68 To decrease noise, a genus was discarded if its average abundance across all samples was below 1%. Samples were clustered by partitioning around medoids (PAM), and the optimal number of clusters was estimated using the Calinski-Harabasz index. Samples were projected into two dimensions and visualized through principal coordinates analysis (PCoA) by the “dudi.pco” function in the ade4 package in R. Cluster dissimilarity was measured by the “adonis” function in the vegan package in R. The dominant taxon in each enterotype was identified based on the significance level, fold change and relative abundance between enterotypes. Enterobacteriaceae was identified as the dominant taxon in the RCPT/E, because it was abundant and significantly enriched in this cluster . Four of the top five differential genera in the RCPT/E  were from Enterobacteriaceae. To make the dominant taxon comparable between RCPT/E and RCPT/B, the most abundant and significantly differential genus in Enterobacteriaceae  was used to represent the dominant genus in the subsequent analyses.We first clustered the gut microbiota of the patients by following a previously published tutorial.68 and Dirichlet multinomial mixtures (DMM) combined approach.36 In the PAM separate approach, samples from patients with the two diseases (CDI and IBD) were clustered using PAM separately. In the combined approach, all samples from patients with the two diseases were mixed and clustered by PAM and DMM. Clustering results were obtained with the three approaches, and consistency was measured as the number of common individuals in the same clusters and common marker genera afterward.The consistency of the two enterotypes was assessed by three approaches: the PAM separate approach, PAM combined approach,66 Bray-Curtis distance (BC distance) was calculated by vegan in R with the genus abundance. Principal coordinates analysis (PCoA) of the pairwise BC distance matrix of all samples was performed using the ade4 package in R. We also compared the variation in BC distance between the patient and the corresponding donor over time, and the trend was fitted with the “ggline” function in R. We also narrowed down the patients to those with clear FMT outcomes and compared their BC distances between the two enterotypes. Corresponding FMT outcomes were assessed according to original studies and criteria listed in the Supplementary Methods. The cumulative abundance of response-enriched or response-depleted was calculated based on the sum of significant bacteria with FMT outcomes .To analyze the dynamics of the patient microbiota, we measured the alpha and beta diversities of the patients and the donors. Alpha diversity was calculated based on the relative abundance of non-collapsed OTUs by QIIME alpha_diversity with the Shannon metric.40 The microbiota profiles of donors and recipients before FMT were used as the sources, and the microbiota profiles of recipients after FMT were used as corresponding sinks.To quantify the ratio of residents and colonizers in the patients after FMT, we calculated the C2R for each patient. We first calculated the distance (log fold change) of each taxon between the recipient after FMT and the patient or donor before FMT. Two categories, residents and colonizers, were distinguished based on the distance. Residents were classified as having a shorter distance between the recipients before FMT and after FMT. Colonizers were classified as having a shorter distance between the recipients after FMT and the donors before FMT. We then summed the abundance of all taxa in the two categories separately. Finally, the ratio of the summed abundance of the two categories was defined as the C2R. FEAST was used as a source tracking tool to validate our C2R with default parameters.68 DONOR/P and DONOR/B denoted the two enterotypes identified in the donors, respectively. To compare the engraftment of donor-derived taxa, we first filtered out donor-specific taxa which were significantly differed between the two enterotypes (DONOR/P and DONOR/B) . And then cumulative abundance was calculated in the recipients before and after FMT by summing up the abundance of these differential taxa. To eliminate the difference in sample size, we used the sample fraction (from 0 to 1) and depicts cumulative abundance against it in To explore the enterotype matching between recipients and donors in FMT, the enterotype clustering of donors was analyzed as that performed in the recipients .To predict the FMT outcomes for each patient before FMT, we built an enterotype-based donor selection (EDS) model by the random forest package in R. The input features of the enterotype-based donor selection (EDS) model included enterotypes of recipients and donors before FMT and their corresponding microbial profiles, like microbial diversity and abundance of certain taxa (mean abundance > 1%). In the EDS model, random forest classifiers were trained to select features and predict FMT outcomes for the two enterotypes separately. We used the predicted value from the EDS model to evaluate the degree of matching between patients and donors. For a new patient waiting for a suitable FMT donor, we first distinguished their enterotype based on the distance to two enterotypes’ medoids and applied the EDS model to predict FMT outcomes for each potential donor .To compare the performance of the EDS model with three alternative models without the enterotype feature, we shuffled the discovery cohorts by 5-fold cross-validation with 500 iterations. The alternative models used the same machine learning classifier (random forest) as the EDS model. The input features of the alternative models included only the microbial profiles from patients, donors, or both. We measured the mean area under the ROC curve (AUROC) of each model based on 500 iterations of 5-fold cross-validation using the ROCR package in R.To validate performance, we used the EDS model trained on the discovery cohorts analyzed above for selection in the validation cohort, which formed the EDS selection group. The matching degree of each patient-donor pair in the EDS group was predicted by the EDS model. A higher matching degree means a higher probability that the patient-donor pair would achieve a response after transplantation. The optimal threshold of the matching degree was determined based on the mean of Youden index and the point closest to the top-left part of the ROC curve.To evaluate the sample size in our analysis, two calculation formula (“pwr.t.test” and “pwr.chisq.test” function) from the pwr package in R were applied. At 80% power and 5% significance level, averaged sample size is 121 per group with mean values and standard deviation of the response and failure groups at the genus-level. For the key conclusion “DONOR/P is more suitable for RCPT/B than DONOR/B in FMT”, the power was at 0.90 (CDI and IBD patients combined) and 0.80 (IBD patients only) with 5% significance level and “pwr.chisq.test” function.Unless otherwise stated, statistical analyses were performed and figures were created in R (version 3.6) with the ggplot2 package. We performed univariate analyses on a blocked version of the Wilcoxon test with the coin package in R. Wilcoxon test in the main text represents Wilcoxon rank-sum test (Mann–Whitney U test). All p values resulting from multiple hypothesis testing in all analyses were adjusted with the Benjamini-Hochberg false discovery rate using the p-value package in R. The adjusted p value is referred to as the q value in the main text. Descriptions of sample size are available in the main text and accompanying figure legends, where n typically reflects the number of patients. Individual data points are shown where possible, and the error bars in the bar plots represent as the 95% confidence interval of the mean.Click here for additional data file.


To determine risk factors and management for the development of a postoperative cerebrospinal fluid (CSF) leak after an endoscopic endonasal surgery (EES) for pituitary adenomas.The clinical data of 400 patients who underwent EES for resection of pituitary adenomas from December 2018 to November 2019 in the Department of Neurosurgery of Beijing Tiantan Hospital were retrospectively reviewed. Age, gender, body mass index (BMI), tumor size, Knosp grade, suprasellar extension grade, sellar floor erosion grade, repeated transsphenoidal surgery, intraoperative CSF leak, use of pedicled nasoseptal flap and lumbar drain were collected and analyzed.p = 0.024), sellar floor erosion grade  and intraoperative CSF leak  were factors associated with an increased postoperative CSF leak rate.Postoperative CSF leak occurred in 14 of 400 patients (3.5%). Age, gender, BMI, tumor size, Knosp grade and repeated transsphenoidal surgery were not risk factors for CSF leak. Suprasellar extension grade (≥B 6.0% vs. <B 1.4%; Higher suprasellar extension grade, higher sellar floor erosion grade and intraoperative CSF leak were risk factors for postoperative CSF leak after endoscopic treatment of pituitary adenoma. Strict skull base reconstruction including use of a pedicled nasoseptal flap and perioperative lumbar drainage may avoid postoperative CSF leak. Pituitary adenoma (PA) is one of the common primary neoplasms of the central nervous system. It makes up approximately 10%–15% of all intracranial tumors . EndoscoThe management to prevent CSF leak after EES of pituitary adenomas is gradually improving. In 2006, the use of a vascular pedicled flap from the nasal septum mucoperiosteum was introduced, which has significantly optimized the skull base reconstruction technique . It reduIn our study, we comprehensively analyzed the risk factors of CSF leak after EES for pituitary tumor surgery. Based on these risk factors, we initially formulated a scheme to prevent CSF leak after EES for the resection of pituitary tumors. These risk factors include age, gender, BMI, tumor size, Knosp grade, suprasellar extension grade, sellar floor erosion grade, lumbar drain, repeated transsphenoidal surgery and intraoperative CSF leak.To determine risk factors and management for the development of a postoperative cerebrospinal fluid (CSF) leak after an endoscopic endonasal surgery (EES) for pituitary adenomas.Patients with pituitary adenoma who underwent EES between December 2018 and November 2019 in the Department of Neurosurgery of Beijing Tiantan Hospital affiliated to Capital Medical University were selected as the research subjects. All patients were treated by the same team, Neurosurgery Oncology 3 Ward. All patients' medical records and operative notes were reviewed in detail. All patients were followed up for at least 3 months.Accurately recorded the following information about the patients: age, gender, body mass index (BMI), tumor size, Knosp grade, suprasellar extension grade, sellar floor erosion grade, repeated transsphenoidal surgery, intraoperative CSF leak, use of pedicled nasoseptal flap and lumbar drain. Postoperative CSF leak was defined as a definite CSF leak within one month after EES. Laboratory tests indicated that fluid from the nose contained cerebrospinal fluid components (a definite CSF leak).2 as the cutoff for our analysis was based on the definition of overweight by the National Health, Family Planning Commission of the People's Republic of China. In this study, lumbar drain refers to the placement of a drainage tube before postoperative CSF leakage occurs. Lumbar drain is usually placed immediately after surgery or the day after surgery.Tumor size is represented by the longest distance of anteroposterior, transverse and vertical diameters. The determination of the Knosp grade of the cases is based on the 0-IV grade classification proposed by Professor Knosp  Figure . Knosp gp value <0.05 was considered statistically significant.To measurement variables, independent t-tests were used to compare the two groups of patients with and without CSF leaks. Chi-square tests were used to categorical variables. All independent variables that showed a significant correlation with dependent variables were placed in a multiple logistic forward stepwise regression. Multivariate logistic regression for predictors of postoperative CSF leak was conducted finally. The analyses were performed using SPSS , and a A total of 421 pituitary adenoma patients who underwent EES were screened for inclusion in our study. 20 patients were excluded due to missing data and 1 patient was excluded for a serious complication . Finally, 400 patients were included for analysis. The clinical characteristics of patients are detailed in 2 (17.2–42.5 kg/m2). Among those patients, 257 (64.25%) were overweight or obese (BMI ≥ 24), 143 (35.75%) of healthy weight (BMI < 24). There were 326 (81.5%) patients underwent EES for the first time and 74 (18.5%) patients underwent EES again.There were 191 female patients and 209 male patients. The average patient age at surgery was 48.5 years (11–82 years). The average patient BMI was 25.6 kg/mp = 0.204)  .Of the 400 patients enrolled for analysis, fourteen patients occurred postoperative CSF leak (3.5%). p = 0.009).There were 107 patients with CSF leakage during the operation. Patients with intraoperative CSF lake were more likely to develop postoperative CSF lake  and sellar floor erosion (p = 0.042) were more likely to develop postoperative CSF leak (p = 0.024). Patients with sellar floor erosion grades III and IV had a significantly higher leakage rate than those with less than III grades . Patients with Knosp less than grade 3 appeared to have a lower rate of CSF leak, but this was not statistically significant   .p = 0.119).In 57 of 400 patients, pedicled nasoseptal flap was used for skull base reconstruction. Of these 57 patients, 4 (7.0%) patients developed postoperative CSF leakage. Leakage rates were relatively low in patients who did not use a nasoseptal flap (2.9% [10/343]). But there is no statistically significant difference (p < 0.0001).A total of 41 patients had a lumbar drain placed after operation (10.3%). Patients who underwent postoperative lumbar drain had a higher rate of CSF leakage compared with did not underwent postoperative lumbar drain . Patients with intraoperative CSF leak were 3.75 times more likely to have a CSF lake when compared with those without intraoperative CSF leak. Patients with a suprasellar extension grade ≥ B were 4.29 times more likely to have a postoperative CSF leak when compared with those with a suprasellar extension grade < B (Multiple logistic regression analysis showed that intraoperative CSF leak and high grade of suprasellar extension were significantly associated with postoperative CSF leak (rade < B .EES is the preferred first-line treatment for pituitary adenomas as skull base tumors. CSF leak is one of the most common postoperative complications after EES for pituitary adenomas. According to literature reports, the incidence of CSF leak after EES for pituitary adenomas ranges from 2.6% to 12.1% , 11–17. It is reported that increased intracranial pressure in overweight and obese patients can place additional strain on skull base reconstruction, leading to increase the risk of postoperative CSF leak . It is pConsistent with reports in the literature , 18, ourIntraoperative CSF leak flow strongly affects postoperative CSF leak rate, as reported by Di Perna et al . High flFor the first time, we introduced the effect of suprasellar extension grades and sellar floor erosion grades on CSF leak after EES. The Hardy–Wilson classification was used for the assessment of suprasellar extension grades and sellar floor erosion grades , 3. BaseWith advances in the development of vascularized flaps in EES, pedicled nasoseptal flaps have been increasingly employed for skull base reconstruction. A systematic review found that vascularized flaps were associated with a lower rate of postoperative CSF leaks . EspeciaLumbar drainage is often used in the perioperative period to reduce intracranial pressure and prevent postoperative CSF leaks following EES for skull base lesions , 24. A pFree tissue grafts, vascularized flaps, gasket sealing and lumbar drains are most commonly used to prevent postoperative CSF leaks . Based oHigher suprasellar extension grade, higher sellar floor erosion grade and intraoperative CSF leak were risk factors for postoperative CSF leak after endoscopic treatment of pituitary adenoma. Rigorous skull base reconstruction including use of a pedicled nasoseptal flap and perioperative lumbar drainage may avoid postoperative CSF leak.This study is a retrospective study, and there is obvious selection bias in the two risk factors of nasoseptal flap and lumbar drainage. These two risk factors require further prospective studies to clarify their impact on postoperative CSF leakage. In addition, to avoid the impact of surgeons' experience on the study due to years of operation, we only counted cases for one year. Therefore, the number of cases is relatively small. Finally, factors such as reconstruction technique, intraoperative CSF leak flow, and history of previous radiation treatment were not accounted for in our analysis.


Therefore, in the current study, we investigated whether BLT2 plays a role in the production of G-CSF in lipopolysaccharide/ovalbumin (LPS/OVA)-induced steroid-resistant neutrophilic asthma. The data showed that BLT2 critically mediated G-CSF production, contributing to the progression of neutrophilic airway inflammation. We also observed that 12-lipoxygenase (12-LO), which catalyzes the synthesis of the BLT2 ligand 12(S)-HETE, was also necessary for G-CSF production. Together, these results suggest that the 12-LO-BLT2-linked signaling network is critical for the production of G-CSF, contributing to the development of neutrophilic airway inflammation. Our findings can provide a potential new target for the therapy of severe neutrophilic asthma.Granulocyte colony-stimulating factor (G-CSF) has been suggested to be closely associated with neutrophilic asthma pathogenesis. However, little is known about the factors regulating the production of G-CSF in neutrophilic asthma. We previously reported that a leukotriene B Neutrophilic asthma is considered to be a poorly controlled disease, showing severe pathological symptoms ,2. WhileColony-stimulating factors (CSFs) were previously defined as regulators of the generation of myeloid populations but were later demonstrated to also regulate the survival, proliferation, and function of myeloid cells, which are functions closely related to inflammation . Among C4 (LTB4) interacts with two distinct receptors, BLT1 and BLT2 [4 and is expressed mainly on immune cells, such as leukocytes [4 and is expressed ubiquitously, including in airway epithelial and mast cells [S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a lipid mediator derived from arachidonic acid by the enzymatic action of 12-lipoxygenase (12-LO) [S)-HETE played critical roles in asthmatic airway inflammation [Eicosanoids such as leukotrienes (LTs) and prostaglandins (PGs) are well-defined lipid metabolites that were reported to be associated with asthma pathology ,14,15. Aand BLT2 . The majukocytes ,18,19. Ost cells ,21. BLT2 (12-LO) . Recentlammation ,26,27,28ammation ,30,31. HIn the present study, we examined whether BLT2 was involved in G-CSF production in lipopolysaccharide/ovalbumin (LPS/OVA)-induced steroid-resistant severe neutrophilic airway inflammation. The data showed that BLT2 critically mediated the production of G-CSF, thereby contributing to the progression of neutrophilic airway inflammation. We also observed that 12-LO was located upstream of BLT2, mediating G-CSF production. Collectively, our results suggest that a 12-LO-BLT2-linked network mediates the production of G-CSF, thus contributing to neutrophilic airway inflammation. The present study can provide a potential new target for the therapy of severe neutrophilic asthma.Escherichia coli serotype O55:B5), and ovalbumin were purchased from Sigma-Aldrich . The 12-LO inhibitor baicalein and BLT2 antagonist LY255283 were purchased from Enzo Life Sciences, Inc.  and Cayman Chemical , respectively. Antibodies against BLT2, 12-LO, and GAPDH were obtained from Enzo Life Sciences, Inc., Invitrogen , and Santa Cruz Biotechnology, Inc. , respectively. Neutralizing monoclonal antibodies against G-CSF and the IgG1 isotype control were obtained from R&D Systems .Dexamethasone, dimethyl sulfoxide (DMSO), LPS  as reported previously [8-week-old wild-type (WT) female C57BL/6 mice were obtained from Orient Bio . BLT2eviously . AnimalsMice were anesthetized with 5% isoflurane before the treatment. Then, the mice were sensitized with 10 µg of LPS plus 75 µg of OVA in 20 µL of phosphate-buffered saline (PBS) by intranasal administration. During the challenges, 50 µg of OVA in 20 µL PBS was administered intranasally (i.n.). To test for steroid resistance, dexamethasone (1 mg/kg) was injected intraperitoneally (i.p.) 1 h before each challenge. For the inhibition experiments, 100 µL of LY255283 (10 mg/kg), baicalein (20 mg/kg), or vehicle control (mixture of 8:2 DMSO/PBS) was administered i.p. 1 h before each challenge. For the neutralization of G-CSF, an anti-G-CSF antibody (5 mg/kg) or control IgG antibody in PBS (100 µL) was injected i.p. 1 h before each challenge. Negative control (NC) mice were not injected or treated during the experiment.Western blotting was performed as previously described . Mouse lS)-HETE ELISA kit was purchased from Enzo Life Sciences Inc., and the levels of 12(S)-HETE in BALF were quantified following the manufacturer’s instructions.G-CSF and MPO were measured using bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assay (ELISA) kits  in accordance with the manufacturer’s instructions. The 12 at 500 rpm for 5 min and stained by a Diff-Quik staining kit .Bronchoalveolar lavage (BAL) cells were separated from BALF by centrifugation at 1000× TM Plus slides. After deparaffinization, they were rehydrated and then blocked for 1 h with buffer  at RT. Then, the slides were incubated with antibodies against BLT2, MPO, and G-CSF conjugated with Alexa Fluor 488, 594, and 647, respectively, using antibody conjugation kits  at 4 °C. After washing three times in PBS, they were incubated with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The slides were processed with washing in PBS and mounted. The images were observed by confocal laser scanning microscopy .Lung tissues were fixed in 10% formalin for 10 days and then embedded in paraffin. The sections of lung tissues (4.0–4.5 μm thick) were adhered on Superfrost Plus glass slides . After deparaffinization, they were stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) staining solution. The images were observed through a DP71 digital camera  using a BX51 microscope . A subjective scale from 0 to 3 was used to evaluate the degree of lung inflammation . Briefly−/− and WT mice. GraphPad Prism 8.0  was used for the statistical analyses. The results are presented as the mean ± SD. A p-value of < 0.05 indicated statistical significance.Statistical analyses were performed with a one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. A two-way ANOVA followed by Bonferroni’s post hoc test was done to compare the data obtained from BLT2First, we established a murine model for steroid-resistant neutrophilic airway inflammation as described previously with some modifications ,32. MiceRecent studies showed that G-CSF had a critical role in the recruitment of neutrophils into airways in severe asthma . To inveSince previous studies suggested a mediating role of BLT2 in neutrophilic airway inflammation ,30,31, w−/− mice were tested. As expected, we found that BLT2−/− mice showed the suppression of inflammation and mucus secretion in the airways -HETE, which is a ligand for BLT2, we examined whether 12-LO activity was also necessary for the production of G-CSF. The levels of 12(S)-HETE in BALF were suppressed by the administration of the 12-LO inhibitor, baicalein -HETE, which is a ligand for BLT2, was necessary for the production of G-CSF. Taken together, our results point to BLT2 as a potential therapeutic target in G-CSF-associated neutrophilic airway inflammation.In this study, we demonstrated the critical mediatory role of BLT2 in the production of G-CSF in steroid-resistant neutrophilic airway inflammation. The results showed that the blockade of BLT2 by antagonist treatment or genetic ablation suppressed the production of G-CSF, thus alleviating neutrophilic inflammation in the murine model. We also found that 12-LO, an enzyme that synthesizes 12 ,34,35,36In addition to G-CSF, interleukin-17 (IL-17) and interleukin-1β (IL-1β) have been reported to be strongly related to severe neutrophilic asthma ,42,43,444 and its receptor BLT1 played roles in the production of G-CSF in our experimental conditions. Quite interestingly, no reduction in the levels of G-CSF in BALF was detected by treatment with the BLT1 antagonist U75302 (4 in BALF (data not shown). The reason why BLT1 did not mediate the production of G-CSF in the present study is not clear, but we suspect that it may be due to the different cell types targeted by LTB4-BLT1 and 12(S)-HETE-BLT2 signaling. BLT1 is highly expressed in leukocytes [In addition to BLT2, BLT1 was also reported to play a role in mediating the recruitment of neutrophils in inflammatory responses . Therefot U75302 . We alsoukocytes , whereasukocytes . TherefoIn summary, we have shown that BLT2 played a critical mediating role in the production of G-CSF in steroid-resistant neutrophilic lung airway inflammation. We also found that the blockade of BLT2 suppressed the production of G-CSF, thus alleviating neutrophilic inflammation in the murine model. In support of the mediatory role of BLT2 in the production of G-CSF, the synthesis of its ligands by 12-LO was also shown to be necessary for mediating the production of G-CSF. Together, our results suggest that the 12-LO-BLT2 cascade is critical for the production of G-CSF, thus contributing to the progression of neutrophilic airway inflammation (as summarized in


The ability of kinematic alignment (KA) to consistently restore trochlea anatomy in total knee arthroplasty (TKA) is unknown despite recreation of constitutional anatomy being its rationale for use. The purpose of this study was to assess if alignment choice in TKA effects the ability to restore the native trochlea groove.One hundred and twenty-two consecutive patients undergoing robotic-assisted TKA using the MAKO image-based robotic platform had simulated femoral components placed according to kinematic, mechanical and functional alignment principals. Implant position and trochlea restoration between groups were compared. Restoration was assessed by shift  and depth relative to the native groove from three consistent points; full extension (0°), mid-flexion (30°–40°) and deep flexion (70°–80°).p ≤ 0.001). In full extension, FA most closely restored the trochlear groove depth in all three positions of flexion.Three hundred and sixty-six alignment options were analysed. Femoral alignment was significantly different between groups. Of KA, 13.1% compared to 3.3% of FA plans were outside safe coronal boundaries. The trochlear groove was translated the most by MA compared to KA and FA (full extension, MA 7.84 ± 1.99 mm lateral to the native groove, KA 6.40 ± 2.43 mm and FA 6.88 ± 1.74 mm, Alignment philosophy led to significant differences in trochlea groove recreation. A kinematically placed femoral component led to positioning considered unsafe in over 13% of cases. A functionally placed femoral component most closely restored trochlea depth in all three positions of flexion. As many as 50% of patients experience ongoing symptoms following total knee arthroplasty (TKA) . These fIn order to improve function and satisfaction in TKA, alignment philosophy’s that restore constitutional alignment have been proposed. Insall et al. first described mechanical alignment (MA) as a technique for TKA . In 2008The ability of KA to consistently restore native trochlea anatomy in TKA is unknown despite recreation of constitutional anatomy being its rationale for use. Previous work concluded that neither MA or KA restored the sulcus orientation, although a KA groove more closely resembled the native state . HoweverThe purpose of this study was to assess if alignment philosophy effects the ability to restore the native trochlea groove when using a standard femoral implant. The hypothesis was that a kinematically placed femur would best restore the native trochlea groove but result in a significant number of femoral components being placed outside safe limits for rotational alignment.Data collection and analysis were carried out in accordance with MR004 Reference Methodology from the Commission Nationale de l'Informatique et des Libertés .This was a prospective matched cohort study of 169 consecutive patients presenting to a single centre that underwent robotically assisted TKA using the MAKO robotic platform and Triathlon Implant  between March 2020 and February 2022.Trochlea groove recreation was assessed in patients undergoing robotic-assisted TKA using a FA philosophy. Patients with trochlea dysplasia, intra articular femur fracture, stage 4 patellofemoral arthritis according to the IWANO classification  or previThe MAKO system of anatomical landmark registration has been previously described . A 3D moImplant position outside following thresholds were defined as unsafe and are based on previous literature and guidelines , 16. LimAll measurements were made using PACS digital radiographic software  and measured by the same evaluator (J.S). The hip-knee-ankle HKA angle, the lateral distal femoral angle (LDFA) and the medial proximal tibial angle (MPTA) were measured pre post-operatively using previously described techniques .The deepest point of trochlea groove on the prosthesis was compared to the deepest point of the native trochlea on pre-operative CT scan using a calibrated tool that allows measurements in 1 mm increments. Restoration was assessed on the MAKO implant planning interface by measuring translation  on coronal slices and depth on sagittal slices Fig. .Fig. 2TrThe position of the prosthetic trochlear was compared to the patients native bony trochlear in three locations on the femoral prosthesis. The first location was the earliest axial slice where the prosthetic sulcus was visible (start of the trochlea groove), the second was the point at which the anterior flange met the anterior chamfer on the sagittal profile of the prosthesis and the final position was the last axial slice where the prosthesis groove was visible . During ten consecutive TKA’s, the flexion range at which the patella engaged each of these three positions was recorded and named according to the flexion range that the patella engaged the prosthesis Fig. ; full exPatients were grouped based on alignment philosophy: MA, KA, FA and compared. For the first 100 cases, measurements were recorded twice by each investigator to compare inter-rate reliability and confirm accuracy of measurements.The intraclass correlation coefficient (ICC) was calculated to measure the reliability (intra and inter observer) of trochlea groove over-/under-stuffing and translation measurements between the two observers in 100 consecutive knees in each group. The intraclass coefficient measurements for trochlea depth and translation were above 0.75 (0.935 and 0.896 respectively), indicating a high degree of intra and inter observer reliability of the two measurements.Planning software was used in a mode that adds 2 mm of cartilage thickness to the subchondral bony surface on the CT scan—an assumption previously described in the use of KA . The femFor the FA, implant positioning is adjusted within limits to achieve an overall alignment and soft tissue laxity goal. Following the assessment of laxity intra-operatively, virtual gaps are assessed to determine if the original plan will deliver a balanced knee. Final adjustments to the femoral component position aim to minimise deviations from the constitutional groove as much as possible. For KA, the distal femur and proximal tibia resections were planned parallel to the native joint line accounting for a 2-mm cartilage thickness, and a similar symmetric posterior condylar (PCA) resection was performed which set femoral rotation parallel to the PCA. Resection depths are based on the thickness of the implant . For MA,P > 0.05). Normally distributed data was analysed using analysis of variance (ANOVA) and skewed data using Kruskal–Wallis test. Post hoc analysis was performed using Bonferroni’s test. Significance was set at p < 0.05 for all tests. statistical analysis was performed using SPSS .Baseline characteristics were described using mean and standard deviation for continuous measures. Data distribution and equality of variances were tested using Shapiro–Wilk and Levine’s test respectively with a significance level set at Following exclusions, a total of 122 patients were available for a matched comparative analysis. Cohort characteristics are summarised in Table . Majority of these occurred by placement of femoral component in more than 6° of valgus. For femoral rotation using the TEA as reference, KA placed 3.3% outside of a safe zone limit of 3° ER to 6° IR, with 25% being placed in more than 3° IR . Groove translation difference between FA and MA in full extension was also significantly different, mean difference − 0.96 mm p = 0.002. In mid-flexion, there was a significant difference in groove translation between KA and MA mean − 0.75 mm, p = 0.003, but not between KA and MA. Case-by-case comparison and distribution of groove translation between alignment philosophy’s is shown in Fig. Trochlear groove translation between alignment philosophy’s is summarised in Table p = 0.002). In flexion, all philosophies had a mean under-stuffing compared to the native groove. Under-stuffing was greatest with KA followed by MA and FA (p <  = 0.001). Case-by-case comparison and distribution of groove depth between alignment philosophy’s is shown in Fig. A comparison of trochlear groove depth comparisons between alignment philosophy’s is summarised in Table Choice of alignment philosophy led to significant variations in trochlear groove restoration. MA resulted in the greatest translation shift and KA created the most under-stuffing overall. FA and KA were equally consistent in reproducing the trochlear groove in terms of translation; however, KA resulted in femoral coronal component positioning that is considered unsafe in 13.2% of cases, compared to FA in 3.7% of cases. A KA philosophy also resulted in a femoral component that was IR beyond 3° relative to the TEA in more than 25% of cases compared to 3.3% with a FA philosophy. These findings confirm our hypothesis that a KA placed femoral component restores the constitutional trochlea groove alignment but frequently results in unsafe coronal or axial implant positioning. These findings may explain why patellofemoral complications are the most common reason for revision in KA TKA –24.In the current study, more than 13% of patients in the KA group had a femur positioned in excess of 6° valgus. This is the upper limit of valgus considered safe using the prosthesis used in this study. Importantly, only 3% of FA aligned femurs were outside the safe range, none of which were in valgus. Aligning the femoral component according to the LDFA results in kinematically aligned femur increasing when the valgus obliquity of the distal femur increases . This reThe most common cause of revision following KA TKA are patellofemoral problems (instability and pain) –24. FemoLittle data is published regarding the effect of alignment philosophy on groove translation in TKA. Previously, Rivière et al. examined the effect of KA in 10 arthritic knees, demonstrating the prosthetic groove was 2 mm lateral in extension, neutral with the native groove at 40° flexion and ended medial at 90° flexion . In the Data examining the effect of alignment philosophy on over- and under-stuffing of the trochlea is also limited. Previously, a study of ten arthritic knees demonstrated that KA led to a mean under-stuffing of 4 mm in extension, 4–5 mm under-stuffing in mid-flexion but no under-stuffing in 100° flexion. In the current study, all alignment philosophy’s had a tendency to slightly overstuff the trochlear in full extension whilst under-stuffing through mid-flexion flexion. In deep flexion, FA resulted in a component flush with the native groove, whilst MA and KA resulted in statistically more under-stuffing (1.0 mm and 2.2 mm respectively). These findings demonstrate that trochlea depth restoration is difficult with the use of a standard design implant. Differences larger than 2 mm have been used to define over-stuffing or under-stuffing , 29; howThis study has weakness’s. Firstly, all comparisons were simulated and performed in silico. However, the purposes of this study were to examine the recreation of the native trochlea groove which can be done using the CT-based planning to within 1 mm and 1° accuracy , 15, meaAlignment choice has a significant effect on the ability to restore the constitutional trochlea in TKA when using a standard femoral component. MA translates the groove furthest from the native anatomy. KA and FA have a similar ability to restore the constitutional trochlear groove; however, KA requires unsafe coronal implant positioning in at least 13% of cases and an internally rotated femoral component beyond 3° in more than 25% of cases. These findings have implications for the function of the patellofemoral joint, implant design, and choice of alignment philosophy in TKA.


Regulation of spontaneous breathing is highly complex and may be influenced by drugs administered during the perioperative period. Because of their different pharmacological properties we hypothesized that midazolam and s-ketamine exert different effects on the variability of minute ventilation (MV), tidal volume (TV) and respiratory rate (RR).Patients undergoing procedural sedation (PSA) with propofol and remifentanil received a single dose of midazolam  or s-ketamine . We used non-invasive impedance-based respiratory volume monitoring to record RR as well as changes in TV and MV. Variability of these three parameters was calculated as coefficients of variation.TV and MV decreased during PSA to a comparable extent in both groups, whereas there was no significant change in RR. In line with our hypothesis we observed marked differences in breathing variability. The variability of MV , TV , and RR  was significantly reduced in patients receiving midazolam. In contrast, variability remained unchanged in patients receiving s-ketamine . After termination of PSA breathing variables returned to baseline values.While midazolam reduces respiratory variability in spontaneously breathing patients undergoing procedural sedation, s-ketamine preserves variability suggesting different effects on the regulation of spontaneous breathing. The respiratory centers (located at the medulla and pons) receive inputs from not only peripheral and central chemoreceptors, but also from many other receptors like those sensing pain, or reacting to stretch or irritant agents within the lungs . In addise curve . In contse curve –8.Breathing variability is defined as the extent to which respiratory parameters such as respiratory rate (RR), tidal volume (TV) and minute ventilation (MV) fluctuate over time . It has During anesthesia and procedural sedation, various drugs with potential effects on the regulation of breathing are administered. For instance, it is acknowledged that midazolam can impair MV mainly through a decrease in TV and to a lesser extent through a decrease in RR . In contIn this study, we investigated the effects of a small single dose of midazolam or s-ketamine on RR, TV, and MV as well as their variability in patients undergoing procedural sedation with propofol and remifentanil. We hypothesized that respiratory variability is impaired more after administration of midazolam than after administration of s-ketamine. To test our hypothesis we studied respiratory variables as well as their variability with a non-invasive impedance-based respiratory volume monitor as described previously . We indeThis prospective, observational, single-center cohort study was approved by the research ethics committee of Amsterdam UMC . We included 20 patients undergoing procedural sedation and analgesia (PSA) for pulmonary vein isolation for drug-refractory paroxysmal atrial fibrillation, between October 2019 and June 2020. Written informed patient consent was obtained from all participants. Patients younger than 18 years of age at the time of inclusion or with an allergy for adhesives were excluded.2 > 94%.All patients were seen at the pre-operative clinic. PSA was conducted in accordance with the Dutch guidelines and local protocols for the administration of PSA outside the operating room. All patients received propofol  and remifentanil  via target-controlled infusion. In addition, patients received a single dose of either midazolam  or s-ketamine  simultaneously with initiation of the target-controlled infusion of propofol and remifentanil. Both drugs are included in our local protocols as additional drugs to be used during PSA. The dosage of propofol, remifentanil and midazolam or s-ketamine was titrated to the desired clinical effect by a certified registered nurse anesthetist specialized in PSA with a supervising anesthetist. Supplemental oxygen was administered when necessary to maintain SpOAfter induction of PSA and after the single dose of midazolam or s-ketamine, the cardiologist accessed the femoral vein with a Seldinger technique and introduced a trans-septal catheter into the left atrium. Focal catheter ablation with radiofrequency current was applied to achieve pulmonary vein isolation. A 3-D electro-anatomical mapping system was used to navigate this catheter .2), heart rate, electrocardiography and capnography were monitored continuously while non-invasive blood pressure was measured intermittently .Pulse oximetry  were continuously measured before, during and after PSA using an impedance-based superficial respiratory volume monitor  with a thoracic electrode , 24. ResThis study was performed in accordance with the strengthening the reporting of observational studies in epidemiology (STROBE) guidelines .For sample size calculation, variability of MV was defined as the primary outcome. It was calculated that 10 subjects are required per group in order to detect a 25% difference in variability between the midazolam group and the s-ketamine group; the α value was 0.05 and the power (1 – β) was 0.80. Data were analyzed using SPSS  and R . Normally distributed data are presented as mean ± standard deviation (SD) and non-parametric data as median with interquartile range (IQR). Variability over time of RR, TV and MV were calculated as the coefficient of variation, defined as the ratio of the SD and the mean. Variability of RR, TV and MV were calculated before PSA, during PSA and after PSA, over time periods lasting 30 min each.Differences between groups were analyzed using the unpaired Student’s t-test. Changes within groups were analyzed with the paired Student’s t-test. Significance was assumed with a two-sided p-value of < 0.05.2, duration of sedation or dosage of other drugs between the two groups (with exception of midazolam or s-ketamine), respectively.We included 20 patients undergoing PSA with propofol and remifentanil. Ten patients received an additional administration of midazolam while the other 10 received an additional administration of s-ketamine. Demographics of the two groups are given in Table A representative registration of the respiratory variability of one patient from the midazolam group and one patient from the s-ketamine group is shown in Fig. In both groups, we observed a decrease in TV and MV during PSA when compared to baseline. The mean decrease of MV was – 31.4% ± 13.9% (p < 0.0001) in the midazolam group, and – 41.1% ± 24.4% (p = 0.002) in the s-ketamine group. The mean decrease of TV was – 16.9% ± 28.6% (p = 0.07) in the midazolam group, and – 32.0% ± 21.4% in the s-ketamine group (p=0.003). There was no significant change in RR during PSA; mean difference – 2.5 ± 3.9 breaths/min (p = 0.073) in the midazolam group, and – 2.4 ± 3.9 breaths/min (p = 0.084) in the s-ketamine group. All three variables increased again after termination of PSA and almost returned to baseline within one hour. There was no significant difference in RR, TV and MV between patients receiving midazolam or s-ketamine, see Fig. In contrast, we observed a significant difference between both groups with regard to the variability of the three respiratory variables. In the group receiving midazolam, the variability of all variables significantly decreased during PSA when compared to baseline; MV 0.23 ± 0.07 vs. 0.11 ± 0.05 (p = 0.011), TV 0.26 ± 0.06 vs. 0.14 ± 0.08 (p = 0.003), RR 0.19 ± 0.05 vs. 0.13 ± 0.04 (p = 0.011). Variability remained unchanged in the group receiving s-ketamine; MV 0.23 ± 0.08 vs. 0.27 ± 0.16 (p = 0.182), TV 0.23 ± 0.05 vs. 0.25 ± 0.11 (p = 0.390), RR 0.18 ± 0.03 vs. 0.23 ± 0.07 (p = 0.129). Variability of MV, TV, and RR during PSA was significantly lower in the group receiving midazolam than in the group receiving s-ketamine , see Fig. In spontaneously breathing patients undergoing procedural sedation with propofol and remifentanil, the variability of RR, TV, and MV was significantly reduced by a single dose of midazolam whereas they remained unchanged when patients received s-ketamine, respectively. These observations suggest that midazolam and s-ketamine differ in their effects on spontaneous respiration.For the interpretation and extrapolation of our results some methodological aspects should be considered. All measurements were performed in spontaneously breathing patients before, during and after PSA with propofol and remifentanil. While one group received a single dose of midazolam, the other received a single dose of s-ketamine in a non-randomized fashion. Also, midazolam or s-ketamine was administered concomitantly with induction of PSA. Thus, although unlikely, we cannot exclude that other results would have been recorded in patients receiving only midazolam or s-ketamine, without propofol and remifentanil. Second, we administered a single small dose of midazolam or s-ketamine and we cannot extrapolate our conclusions to higher or repeated doses. Further studies are required to establish the dose-response relationship. Third, a non-invasive impedance-based technique was used to measure respiratory volume variables before, during and after PSA. Previous studies reported acceptable accuracy of this technique , 26–28. A and glycine-mediated pathways [In the present study, we hypothesized that midazolam and s-ketamine have different effects on the variability of respiratory variables during PSA. Consistent with this hypothesis, we observed marked differences between the midazolam and the s-ketamine group. While the variability significantly decreased with midazolam, no changes were observed with s-ketamine. Our observations may be explained by the different pharmacological pathways of midazolam and s-ketamine. While benzodiazepines alter the control of breathing through gamma-amino butyric acid (GABA)pathways , 5, s-kepathways . It is apathways , 29. In pathways .It is likely that respiratory variability has physiological functions also during anesthesia and procedural sedation. For example, episodes of deep breathing may recruit dependent alveolar regions that otherwise would collapse. High respiratory variability is associated with lower organ failure score in the intensive care unit , increasIn conclusion, the variability of breathing is reduced by midazolam but not by s-ketamine. We suggest that midazolam and s-ketamine affect regulation of breathing in a different way.


Two-year overall survival (OS) and PFS  with MSD were significantly better at 58% compared with 50%, p ≤ 0.001, and 51% vs 47%, p = 0.029, with a HD. Relapse at 2 years was lower with a HD 23% than with MSD 29% (p = 0.016). Non relapse mortality (NRM) was higher with HD in the first 6 months post-transplant [HR 2.59 (1.5–4.48) p < 0.001] and was also higher at 2 years being 30% for HD and 20% for MSD, p ≤ 0.001. The incidence of acute GVHD grade II-IV and III–IV at 100 days was comparable for MSD and HD, however, chronic GVHD at 2 years was significantly higher with MSD being 44% vs 32% for HD (p < 0.001). After multivariable analysis, OS and primary graft failure were significantly worse for HD particularly before 6 months [HR 1.93(1.24–3.0)], and HR [3.5(1.5–8.1)]. The median age of HD 37 (IQR 30–47) years was significantly lower than sibling donors 56 (IQR 49–62 years) p < 0.001. However, there was no effect on NRM, relapse or PFS. This data set suggests that a MSD donor remains the preferred choice in MDS over a haplo donor. Transplants with haploidentical donors result in satisfactory long-term outcome, justifying it’s use when no better donor is available.Myelodysplastic syndromes (MDS) are the second common indication for an Allo-HCT. We compared the outcomes of 1414 matched sibling (MSD) with 415 haplo-identical donors (HD) transplanted with post-transplant cyclophosphamide (PTCy) as GVHD prophylaxis between 2014 and 2017. The median age at transplant with MSD was 58 and 61 years for HD. The median time to neutrophil engraftment was longer for HD being 20 vs 16 days for MSD ( The myelodysplastic syndromes are a heterogenous cluster of clonal stem cell disorders that occur in the older adult manifesting as either bone marrow failure and or a progression towards acute leukemia. Allogeneic hematopoietic cell transplantation  is the only option that offers the potential for long-term disease-free survival in 30–50% of recipients –4. Both All patients provided informed consent for data registration, according to the Declaration of Helsinki. This study was approved by the Chronic Malignancies Working Party of EBMT. Data were retrieved from the EBMT registry for sibling and mismatched family donor transplants performed between 2014 and 2017. Within the mismatched family donor transplants all patients received post-transplant cyclophosphamide as GVHD prophylaxis.9/L for three consecutive days and platelet engraftment as a platelet count >20 × 109/L for seven consecutive days without transfusion support. Primary graft failure (PGF) was defined as failing to reach neutrophil >0.5 × 109/L in the first 28 days post-transplant or documentation of autologous reconstitution by chimerism analysis in the absence of relapse . The use of HD resulted in poorer OS [HR = 1.93 ] particularly in the first 6 month due to a higher NRM  than MSD whereas this was similar after the first 6 months HR 0.93 . PFS was similar [HR = 1.16 ], as was relapse [HR = 0.56 ]. Disease status, utilizing the WHO classification, with either excess blasts or AML at transplant did not influence OS, NRM, PFS or relapse. For recipients treated prior to a transplant, the absence of CR was associated adversely with OS [HR = 1.31 ], but not PFS [HR = 1.16 ] or relapse [HR = 1.04 ]. Both untreated and patients not in CR experienced increased NRM HR 1.45 (95% CI 0.99–2.11) p = 0.056 and 1.41 (95% CI 1.03–1.93) p = 0.0315 respectively. Surprisingly, untreated patients had a lower risk of relapse [HR = 0.52 ] when compared to those who received treatment (whether in CR or not in CR). PB as a stem cell source was not associated with higher NRM [HR = 1.25 ]. The use of donor other than female to male was associated with lower NRM [HR 0.7 ], and higher relapse [HR = 1.37 ] (Table p < 0.001). Causes of death overall, within six and between 6 and 24 months and multivariate analysis are summarized in supplementary table The results of multivariable analysis are summarized in Table p = 0.026)] Table p = 0.5)], PFS [HR = 1.04 ] or NRM [HR = 1.08 ] in either HD or MSD groups (Table Increasing donor age (sibling or haplo) by decade in general appeared not to influence overall survival in the first 6 months but increasing donor age had a detrimental effect on OS after 6 months [HR = 1.19 (95%CI (1.02–1.4); With the limited availability of MSD and increased feasibility of performing HD transplants  comparisIn comparison with MSD, as expected, the relapse rate with HD is relatively lower. This may be due to a higher graft versus leukemia effect attributed to HD, as has also recently been shown by Robin et al- relapses being comparable between mismatched MUD, HD and cord blood transplantation . In our In the current data set, the NRM was significantly higher with HD versus MSD, although there has been a substantial fall in rates of NRM (30%) as compared to previous CMWP EBMT- reports (36–40%). Within the HD cohort graft rejection rates were high at 10% which may significantly contribute to the increased early NRM due to infection, in the first 6 months. NRM was independently predicted by refractory disease, CMV seropositivity of patient and donor recipient sex mismatch  but did not depend on recipient age. This is in discordance with previous reports - where Hp < 0.001, given the older median age of recipients, the younger HD are likely to be offspring rather than siblings. We hypothesis that the lower survival with younger HD after 6 months may be due their higher graft failure. Robinson et al, similarly described in acute leukemia that the older recipient (>55 years) with a younger offspring donor combination experienced higher graft failure, NRM and lower overall survival compared to the older recipient with a matched MSD donor [The cohort described here were older with a median age of 58 for sibling and 60 for Haplo recipients which is expected for MDS. Within this spectrum, recipient age was not predictive of overall outcomes in both HD and MSD cohorts, unlike previous studies , 18. TheSD donor . Other sSD donor , 15 Our The existing literature is inconsistent, as another study showed that in recipients >55 years with older MSD, tend to have an increased NRM, and lower PFS and overall survival thus having overall outcomes comparable to Haplo transplants from a younger mismatched family donor . This stThe study suffers from various drawbacks in that there was inadequate data on cytogenetic risk to inform more precisely the outcomes with IPSS or R-IPSS scores. The incidence of donor directed antibodies also would have helped interpret the non-engraftment data. Importantly valuable information on the kinship of the donors is missing. Additionally, the platforms for transplanting MSD were different to that of the uniform PTCy platform for HD and may influence outcomes. We also do not have data on immune- reconstitution and CMV reactivation, particularly for HD transplants. It could be hypothesized that the HD transplants were at higher risk of NRM in first 6 months probably due to a patient selection bias (higher risk disease) and delay in time to transplantation. Maybe as physicians now are likely to go for earlier HD transplants this may need to be re-evaluated in the future.These data suggest that matched siblings are the optimal donor in MDS, however in their absence, despite the higher early NRM and primary graft failure, HD transplantation is a reasonable option. Studies in MDS, that include donor kinship to confirm whether a mismatched sibling donor may be preferable to a younger mismatched offspring donor are needed.Supplementary Table 1: Comparison of engraftment by BM or PB for Haplo donorSupplementary table 2- causes of deathSupplementary table 3- Multivariate analysis for various factors affecting a- OS, b- NRM for patients > or =40 years of ageSupplementary figure 1Supplementary figure 2


Cannabis sativa plant, is substantiated more and more. We aimed to determine the pharmacokinetic behavior of CBD after a single dose via intranasal (IN) and intrarectal (IR) administration in six healthy Beagle dogs age 3–8 years old, and compare to the oral administration route (PO). Standardized dosages applied for IN, IR and PO were 20, 100, and 100 mg, respectively. Each dog underwent the same protocol but received CBD through a different administration route. CBD plasma concentrations were determined by ultra-high performance liquid chromatography-tandem mass spectrometry before and at fixed time points after administration. Non-compartmental analysis was performed on the plasma concentration-time profiles. Plasma CBD concentrations after IR administration were below the limit of quantification. The mean area under the curve (AUC) after IN and PO CBD administration was 61 and 1,376 ng/mL*h, respectively. The maximal plasma CBD concentration (Cmax) after IN and PO CBD administration was 28 and 217 ng/mL reached after 0.5 and 3.5 h (Tmax), respectively. Significant differences between IN and PO administration were found in the Tmax (p = 0.04). Higher AUC and Cmax were achieved with 100 mg PO compared to 20 mg IN, but no significant differences were found when AUC (p = 0.09) and Cmax (p = 0.44) were normalized to 1 mg dosages. IN administration of CBD resulted in faster absorption when compared to PO administration. However, PO remains the most favorable route for CBD delivery due to its more feasible administration. The IR administration route is not advised for clinical application.The therapeutic potential of cannabidiol (CBD), a non-psychtropic component of the  Cannabis sativa plant  and intrarectal (IR) administration in healthy Beagle dogs and compare this to the more widely used oral administration route (PO). The plasma CBD concentrations were evaluated over a period of 60 h post-administration. We hypothesized that CBD delivered via IN administration would avoid first-pass liver effect and CBD delivered via IR administration would partially avoid liver metabolization and therefore higher plasma concentrations and subsequent exposure would be achieved compared to the PO administration route.The purpose of this study was to determine the pharmacokinetic behavior of CBD after a single dose t-test exact solution with a non-central t-distribution, taking none-detection in one dog into account , 3–8 years of age, weighing an average of 12 kg  were included in a randomized crossover study. A sample size of 6 was found to be the minimum sample size based on a power analysis with the following settings: the smallest relevant difference of 5 , based  account , with a www.randomizer.org). Following a two-week wash-out period, each dog underwent the same protocol but received CBD through a different administration  route. The first blood sample (T0) was taken 10 min before the CBD administration.The dogs were randomly allocated to a 3-way crossover design by the principal investigator (DP), using an online randomization program . The total volume was fixed at 1 mL and was divided equally over the two nostrils. During the intranasal delivery, dogs were held in sternal recumbency with the head and neck gently dorsoflexed and were kept in this position for ~0.5 min after intranasal administration.For the IN administration, a polyethylene glycol (PEG):sodium chloride (NaCl) 0.9% (50:50) solution containing 20 mg of synthetic CBD -3-methyl-6-prop-1-en-2-yl-1-cyclohex-2-enyl]-5-pentylbenzene-1,3-diol) per dog was given ® synthesized CBD suppositories 100 mg, CB21 Pharma s.r.o., Prague, Czech Republic) was gently administered in each dog manually in the rectum.For the IR administration, dogs were first taken outside for a walk to avoid defecation during and after administration. Thereafter, a suppository containing 100 mg of CBD  was administered together with a small amount (≅10 g) of highly digestible commercial canned food .For the oral administration, a tablet containing 100 mg of CBD (Cannamedwww.CBDepot.eu), and the IN dose was a self-developed formulation using analytical standard dissoluted in PEG.The tablets for oral administration and suppositories for rectal use (100 mg), according to the manufacturers' specification, were commercially available (vena jugularis 10 min before CBD administration (T0) and at 15, 30, 45 min and 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, and 60 h after all routes of CBD administration. Blood samples were immediately transferred into tubes containing ethylenediamine tetraacetic acid and the plasma was immediately separated by centrifugation at 3,500 rpm for 5 min at 2°C. The plasma was then stored at −80°C until analysis.Blood samples (2 mL) were collected from the Adverse reactions during and after CBD administration were recorded. Attention was given to sneezing and reverse sneezing, coughing, head shaking, snorting and licking during IN administration, nausea, vomiting and salivation during PO administration and defecation after IR administration.2O was obtained via a Milli-Q water purification system . Standards of CBD (10 mg/mL in EtOH) and internal standard CBD-d3 (100 μg/mL) were purchased from Sigma-Aldrich . All solvents were of analytical grade. Acetonitrile was purchased from Thermo Fisher Scientific Inc. . Formic acid was obtained from VWR™ .Ultrapure HOne hundred microliter of the plasma sample was spiked with 25 μL of internal standard (CBD-d3) solution (400 ng/mL) and vortex mixed (±15 s). Next, 275 μL of acetonitrile was added to the samples and again vortex mixed (±15 s). After mixing, the samples were centrifuged . Thereafter, the liquid layer was transferred to an autosampler vial. Finally, an aliquot  was injected into the ultra-high-performance liquid chromatography with tandem mass spectrometry (LC—MS/MS) system.2O and 0.1% formic acid in methanol, respectively. The following program was applied: 0 min , 0–2 min (linear gradient to 100% B), 2–4 min (100% B), 4–4.1 min  and 4.1–8 min . Flow rate was set at 0.4 mL/min. The LC eluent was interfaced to a Xevo TQ-XS triple quadrupole mass spectrometer  with ion source heated electrospray ionization operating in positive ionization mode. Acquisition was performed in selected reaction monitoring mode. For CBD and internal standard, the following two most intense product ions were followed: CBD: mass-to-charge ratio 315.08 > 193.00/122.96 and CBD-d3: mass-to-charge ratio 318.12 > 196.03/122.96. The LC-MS/MS analytical methods were validated using matrix-matched calibrator and quality control samples, based on blank plasma of untreated dogs. The limit of quantification (LOQ) was 1 ng/mL. The LC-MS/MS analyses were conducted in accordance with the international guidelines  in combination with a guard column of the same type . The gradient elution programme consisted of two mobile phases (A and B). Mobile phase A and B were 0.1% formic acid in ultrapure Hidelines –35.0−inf); maximal plasma concentration (Cmax) and time to maximal plasma concentration (Tmax); terminal elimination half-life; elimination rate constant and mean residence time. Total body clearance and volume of distribution after IN and PO administration were not corrected for their respective bioavailabilities. The relative bioavailability of the IN administration when compared with the commercial oral product was calculated according to the following formula:Non-compartmental analysis was performed on the plasma concentration-time profiles using Phoenix 8.4 . All dosing groups were included in the analysis, except for the IR administration data because of the low plasma concentrations (around LOQ). The following pharmacokinetic parameters were calculated: area under the plasma concentration-time curve, from 0 to infinity  and the IN administration (20 mg) and to facilitate comparison of the systemic exposure between the administration routes, the AUC and CThe statistical analysis was conducted in R version 4.0.2 (“Taking off again”). Significance was set at α ≤ 0.05. A Wilcoxon signed rank test was used to compare the IN and PO administration routes.0−inf after IN and PO CBD administration before dose normalization was 61.31 and 1376.03 ng/mL*h and after normalization to 1 mg dosages was 3.06 and 13.76 ng/mL*h, respectively. The maximal plasma CBD concentration (Cmax) after IN and PO CBD administration before dose normalization was 27.96 and 216.76 ng/ml and 1.39 and 2.16 ng/mL after dose normalization, reached at 0.5 and 3.5 h (Tmax), respectively. Significant differences between IN and PO administration routes were found in the Tmax (p = 0.04) but no significant differences were found in the AUC normalized for dose (p = 0.09) and Cmax normalized for dose (p = 0.44). The different pharmacokinetic parameters after PO and IN administration are shown in Mean ± SD plasma CBD concentrations after PO, IN and IR administration were determined at 17 time points over 60 h post administration and are displayed in Due to the very low (below LOQ) plasma CBD concentrations  obtainedTwo dogs showed sneezing and 1 dog showed head shaking after IN administration. These symptoms were seen immediately after IN administration and disappeared over a period of 30 s. All 6 dogs showed good compliance with PO administration, where the tablets were spontaneously ingested together with the small amount of highly digestible food. The IR administration of the CBD suppository went uneventful. No signs of nausea, vomiting or salivation were seen during and after PO administration and no defecation was observed during and after the IR administration.via IN administration would bypass liver metabolization largely and via IR administration partially and therefore higher plasma concentrations would be achieved, in comparison with the PO administration route. However, this hypothesis was not confirmed, since the oral administration route still showed a numerically higher mean exposure and maximal concentration normalized for dose when compared to the IN and IR administration routes albeit not statistically significant.To the best of our knowledge, this is the first study examining the pharmacokinetic profile of CBD in healthy dogs after IN and IR administration. The pharmacokinetic parameters were compared with the more widely used oral route of administration. The plasma CBD concentrations after IR administration were below the LOQ and were therefore not used in our analysis. We hypothesized that CBD delivered max after IN administration was reached significantly faster (0.49 h) compared with the PO administration (3.5 h), but on the other hand, there was no significant difference between the mean AUC normalized for dose and mean Cmax normalized for dose when comparing IN and PO administrations. It is worth to mention that a substantial proportion of the oromucosal delivered dose of CBD may actually be absorbed through the gastrointestinal tract  used and the high lipophilicity of CBD. It is believed that PEG-only formulations are associated with a higher CBD permeation   , which mg P 6.3) . Furtherg P 6.3) . Also, imax and AUC than oral microencapsulated oil beads and CBD-infused transdermal cream  and a mean AUC of 1,297 ng-h/mL. This is possibly due to the use of an infused soft chew treat made with a glycerol/starch/fiber base which should be easily digestible and appears to deliver approximately two and a half times the concentration. As in our study, other research groups also used a higher oral dose in dogs (10 mg/kg) and a mean Cmax was reached, between 578 and 1,868 ng/mL, respectively  and 6,180 ng/mL*h (10 mg/kg of microencapsulated oil) in healthy dogs. CBD has a high affinity for lipids and low water solubility  , had a bioavailability of ~67% . IntestiP 7.4) . The useP 7.4) , thus usThe most frequently observed adverse effect associated with the IN route was short sneezing in two dogs and head shaking in one dog. Sneezing or snorting reaction and head shaking during or after intranasal administration have also been reported in three other studies, where dogs received IN diazepam , IN midaThe major limitation of this study was the lack of an IV route group, which could have provided some more consistent pharmacokinetic data. There are also potential limitations regarding the IN route, including spilling of the drug due to sneezing and swallowing of a part of the dose administered.The IN, IR and PO single administration of 20, 100, and 100 mg CBD, respectively, was well-tolerated by all of the dogs. PO remains the most favorable route for CBD delivery due to its more feasible administration. Nevertheless, IN administration of CBD provided a faster blood absorption when compared to the PO and IR CBD administration. These findings encourage the use of IN CBD in veterinary medicine as a possible alternative when PO route is not possible. The IR administration route is not advised for clinical application.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Ethical Committee of the Faculties of Veterinary Medicine and Bioscience Engineering, Ghent University (EC 2018-42).DP and SB were responsible for conception of the study, writing–original draft, and approval of the submitted manuscript. RT and MD were responsible for conception of the study, acquisition of the data, data analysis and interpretation, pharmacokinetic evaluation, and writing–review and editing of the manuscript. BB was responsible for statistical analysis and writing–review and editing of the manuscript. MC, LH, IC, PM, AF, and JS were responsible for writing–review and editing of the manuscript. All authors contributed to the article and approved the submitted version.JS was employed by company CBDepot. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.