Ligaments anatomy often show a huge anatomy variations between species and individuals. For example calcaneofibular ligaments (CFL) characterize the great variability of morphological shape or presence of additional bands. The aim of this study was to propose first anatomical classification of CFL concerning on human fetuses. We investigated thirty spontaneously-aborted human fetuses aged 18–38 weeks of gestation at death. Sixty lower limbs (30 left and 30 right) fixed in 10% formalin solution were examined. The morphological variability of CFL was assessed. Four types of CFL morphology were observed. Type I was characterized by a band shape. This was the most common type, occurring in 53% of all cases. Based on our study we are proposing a classification based on four morphological types of CFL. Types 2 and 4 are further divided into subtypes. Present classification may be useful to better understand the anatomical development of ankle joint. According to classical anatomy textbooks, the CFL origin is located on the tip of the lateral malleolus and courses posteroinferiorly and medially to insert onto the lateral surface of the calcaneus.The lateral collateral ligament complex (LCL) of the ankle joint consists of the calcaneofibular (CFL), anterior talofibular (ATFL) and the posterior talofibular ligaments (PTFL). Recent anatomical studies have described the relationship between the inferior band of ATFL and CFL and proposed the existence of a lateral fibulotalocalcaneal ligament (LFTCL) complex, which plays an essential role in maintaining lateral ankle stabilityAlthough many studies describe the anatomical types of CFL, these have all focused on adult human cadavers. Their classifications were based on the shape of the main band and the presence of additional fibers.One of the most common musculoskeletal injuries, particularly during sporting activities, is ankle sprain. The most common mechanism of injury is inversion of the foot. Although the most common ruptured ligament is the ATFL, 20% of sprains involve the CFL rupture combined with ATFL.The aim of this study was to determine the morphological variability of the CFL and thus establish the first classification of CFL types in human fetuses.The results can be compared with the spectrum of morphological variability in adults.Thirty spontaneously-aborted human fetuses  aged 18–38 weeks of gestation at death were examined. The fetuses were donated to the Chair of Anatomy and Histology before 1998.The study was performed in accordance with the legal procedures in force in Poland and in accordance with the Donation Corpse program for both adults and fetuses. The age of the fetuses was determined from the head and craniosacral measurements. Permission for the study was given by the Local Bioethics Commission (agreement no. RNN/242/22/KE).4. Dissection started with incision of the skin placed 2 cm above the lateral mallelous; the skin, subcutaneous and superficial fascia were removed towards the lateral ankle. After the fibularis longus muscle and fibularis brevis tendons were identified, the skin and subcutaneous tissue of the lateral compartment of the foot were removed. Following this, peroneal tendons were cut below the lateral mallelous and lifted to visualize CFL. The following characteristics were recorded:Morphological type of CFL.Length of CFL and additional bands if present.The thickness and width of the CFL, and any additional bands that were present,at three points; origin, in the middle and insertion.Sixty paired lower limbs of human fetuses were dissected by the same orthopedic surgeon. The protocol for the dissection of the lateral compartment of the leg and lateral site of the foot was described previouslyThe foot was positioned in a neutral position for all measures. Measurements were made with an electronic digital caliper . Each measurement was performed twice by independent researchers with an accuracy of up to 0.01 mm.http://statistica.io]. The Shapiro–Wilk test indicated that continuous variables had a non-normal distribution; therefore nonparametric tests were used. CFL types were compared using the Mann–Whitney test (for two groups) and the Kruskal–Wallis test by ranks with a dedicated posthoc test (more than two groups). A p-value of less than 0.05 was considered significant; this value was modified for multiple testing with Bonferroni correction. Results are presented as mean and standard deviation unless otherwise stated.Statistical analysis was performed using Statistica 13 software [TIBCO Software Inc. (2017). Statistica, Type I-band-shaped; the origin was located on the lateral malleolus and the insertion on the lateral surface of the calcaneus bone. It was present in 32 cases (53%) Fig. a.Type II-Y-shaped; present in 11 cases (18%). In three cases (5%), both arms of the ligament were attached to the lateral malleolus (subtype A) and in the remaining cases (13%), one was attached to the lateral malleolus and the other one to the talus (subtype B). In both subtypes, the insertion was located on the lateral surface of the calcaneus bone  . Two subtypes were recorded: subtype A with additional CFL originating on the lateral malleolus , and subtype B with presence of lateral talocalcaneal ligament originating on the talus bone . Both subtypes inserted on the lateral surface of the calcaneus bone  length in the whole group was 174.1 (± 31.1). No difference in CS length was observed between types .Thickness of main band at distal attachment point was bigger in type III than in other types.Width of main band in the center was bigger in types II and III than in type IV.Width of main band at origin in type III than in type IV.The differences between CFL types regarding the origin and distal attachment are given in Table This study presents the first systematic classification of the CFL in human fetuses; previous studies have presented a similar classification, but for adult cadavers.During the fourth week of embryonic development, the lower limb buds become visible for the first time, and 2 weeks later the distal portion of the limb bud develops into the digital plate. During this period, the plate is oriented in line with the long axis and the entire limb is externally rotated. The plantar surface of the digital plate is thus turned towards in a cranial direction. Then the digital plate adopts a fan-like shape appearing from the eighth-week notches. At the same time, during the eighth week of embryonic development, external rotation decreases, ending with the plantar surfaces being turned to each other and the feet in a position of equino-varus-adductus. Finally, at the end of the eleventh week of fetal life, the position of the feet is close to neutral. Joint cavities with synovial linings appear after 10 weeks of fetal life. There are two ways of developing the collateral ligaments of joints: by localized thickening of the joint capsule or as a loose tissue distant from the capsule or joint cavity. After ten weeks of gestation age, the CFL, ATFL and PTFL begin to form. ATFL arises as a thickening of the joint capsule, while the ATFL and CFL are distant from the capsule. At this stage, histological studies describe these ligaments as consisting of straight fibers. With further embryological development, the fibers in the ATFL and PTFL change direction, while those of the CFL simply elongate. Therefore, we believe that the presence of additional bands, and their shape, are determined from the beginning of fetal life. Indeed, no significant difference in CS length was found between types in the present study.5. In the plantigrade position, the ligament runs obliquely downwards deeply to fibular longus and fibularis brevis tendons and sheaths6. They also most completely cover the CFL, and about 1 cm of the ligament is uncovered7. The CFL inserts to the small tubercle at the posterior aspect of the lateral calcaneus8. An anatomically positioned CFL forms a 113° to 150° angle with the fibula9.The calcaneofibular ligament originates on the anterior part of the lateral malleolus. It is placed beneath the inferior band of the ATFL. Recent anatomical studies indicate that the anterior part of CFL and inferior part of the ATFL are covered by a fibrous layer; however, this is only superficial and easy to remove. After removing the fibers, the ATFL and CFL have independent attachments9 described he CFL as band-shaped ligament with a mean length of 35.8 mm; although the study mentioned the LTC ligament it did not give a precise frequency of occurrence9. Trouilloud et al.10 present a classification of three types of CFL ligament based on its relationship with the TCL, without describing the shape of the main band. Wiersma et al.11 propose two variants of CFL shape: a cord-like structure (66%) and a flat, fanning shape (34%). Kitsoulis et al.12 distinguished three types of CFL based on the presence of an accessory band: one band was present in 52 out of 72 cases, two bands in sixteen cases and three bands in four lower limbs. The most recent current classification was given by Pereira et.al.13 from a study of 47 adult human limbs, comprising single bundle, V-shape, Y-shape and double band CFL types13. It is important to note that these findings closely resembleour proposed classification; however, we propose expanding the Y-shape and double band categories, with the latter including subtypes based on origin point13.Although several studies describe morphological variations of the CFL, all are based on adult cadavers. Burks et al.15. Performing studies on a larger sample of adult lower limbs should confirm our classification of CFL types. Particularly noteworthy are the types 2 and 4 proposed by us. In our opinion, type 4a is a previously undescribed double CFL. Cases of a double band ligament, which in classical anatomy are described as a single band, are known in current anatomical literature for example the double fibular collateral ligament of the knee16. There are morphological similarities between types 2b and 4b due to the connection with the talus bone. The occurrence of an independent ligament attaching to the talus bone is described in the literature in 23–35% of cases13. Pereira et al.13 in his work on adults described both of the above-mentioned types and suggested that the Y-type, which corresponded to our type 2b, is formed by CFL and LTC blended into a single ligament. In our work, we proved that this assumption is wrong because these ligaments already have that shape during fetal life. In our opinion, these are two different morphological types, however, the Y-shape with attachment to the talus bone certainly increases the stability of the ankle joint. Another interesting fact is that the occurrence of LTC has always been associated with the occurrence of single band CFL.We proposed a first CFL classification based on human fetuses. Due to the fact that between the types there was no significant statistical difference regarding the cranio-sacral length, we believe that the type of CFL is already defined in fetal life. In our opinion, the shape of the CFL is determined from the beginning and it is observed in other human ligamentous structures, such as the ligamentum mucosum17. The inferior ATFL band is connected by arciform fibers to the CFL; this forms an extra-articular part creating the LFTCL. Interestingly, these fibers are visible during fetal growth. In contrast, the superior band of ATFL follows an intraarticular course. In ankle inversion sprains, this intraarticular part is ruptured most easily, followed by the CFL and inferior band of ATFL19.Recent anatomical literature indicates that the ATFL and CFL are components of the lateral collateral ligament complexThis study has some limitations. A larger sample size would have been desirable; however, it is the first classification of the ligament in human fetuses. In addition, all samples were obtained from a specific population from Lodz, Poland; as such more studies are needed to determine ligament types in larger populations. In addition, no calculation of the sample size was carried out. Nevertheless, our study is the first classification based on human fetuses. We believe that a systematic classification will be a valuable tool to improve knowledge about embryology of the ankle joint region.We distinguished four types of CFL in human fetuses. The most common was Type I, characterized by a band shape. The results of our study on the anatomical variation of CFL in fetuses will allow further comparisons with other studies focused on ankle joint development, including this performed on adult cadavers.


A small proportion of patients with clinical parkinsonism have normal transporter-single photon emission computed tomography (DaTSPECT) which is commonly defined as scans without evidence of dopaminergic deficits (SWEDD). A better understanding of SWEDD can improve the current therapeutic options and appropriate disease monitoring.We aimed to assess CSF biomarkers levels including α-synuclein (α-syn), amyloid βeta (Aβ1–42), total tau (t-tau), and phosphorylated tau (p-tau) in SWEDD and investigate the longitudinal alteration in the CSF profile.In total, 406 early-stage PD, 58 SWEDD, and 187 healthy controls (HCs) were entered into our study from PPMI. We compared the level of CSF biomarkers at baseline, six months, one year, and two years. Furthermore, the longitudinal alteration of CSF biomarkers was explored in each group using linear mixed models.p = 0.016) and two years (p = 0.006). Also, we observed a significant difference in the level of CSF Aβ1–42 between SWEDD and PD at one year (p = 0.012). Moreover, there was a significant difference in the level of CSF t-tau between SWEDD and PD subjects at one (p = 0.013) and two years (p = 0.017). Furthermore, there was a significant difference in the level of CSF p-tau between SWEDD and PD groups at two years visits (p = 0.030). Longitudinal analysis showed a significant decrease after one (p = 0.029) and two years (p = 0.002) from baseline in the level of CSF α-syn only in the PD group. Also, we observed that the level of CSF Aβ1–42 significantly increased after one year in SWEDD (p = 0.031) and decreased after two years from baseline in PD subjects (p = 0.005). Moreover, there was a significant increase in the level of CSF t-tau after two years (p = 0.036) and CSF p-tau after six months from baseline in SWEDD subjects (p = 0.011).There was no significant difference in the level of CSF α-syn Aβ1–42, t-tau, and p-tau between HCs and SWEDD at different time points. Investigating the level of CSF α-syn in PD and SWEDD showed a significant difference at one (This finding suggests a faster neurodegeneration process in PD patients compared to SWEDD at least based on these biomarkers. Future studies with longer follow-up duration and more sample sizes are necessary to validate our results. Globally, Parkinson’s disease (PD) is the second leading cause of disability among neurodegenerative disorders in older adults . During Most recently, dopamine transporter-single photon emission computed tomography (DaTSPECT) has been widely utilized for identifying the degeneration of presynaptic dopamine receptors in the nigrostriatal system . By thisCerebrospinal fluid (CSF) is a favorable source of CNS biomarkers for neurodegenerative disorders. The CSF α-synuclein (α-syn), amyloid βeta (Aβ)1–42, total tau (t-tau), and phosphorylated tau (p-tau) have been accounted as diagnostic, prognostic, and predictive biomarkers for PD . AccordiConsequently, we sought for investigating how the CSF levels of α-syn, Aβ1–42, t-tau, and p-tau varied and changed over the progression of PD and SWEDD in addition to investigating whether the level of differences in the biomarkers could be used for differentiating these two conditions.The participants were recruited from Parkinson’s Progression Markers Initiative . The study was approved by the institutional review board of all participating sites. Written informed consent was obtained from all participants before study enrolment. The study was performed based on relevant guidelines and regulations. PD was diagnosed according to Movement Disorder Society (MDS) criteria , and disAll participants completed motor and non-motor tests at the baseline visit assessed by PPMI staff. The H&Y staging and MDS-UPDRS were used for motor assessments. The Montreal Cognitive Assessments (MoCA) were performed to measure cognitive status, Letter Number Sequencing (LNS), and semantic fluency to assess executive function and working memory. Also, Epworth Sleepiness Scale (ESS) was applied to evaluate the patient's sleepiness. The olfaction function measured by the University of Pennsylvania Smell Identification Test (UPSIT) and Rapid eye movement (REM) sleep behavior disorder (RBD) was evaluated using REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ).CSF measures were obtained from the PPMI database for each patient via the following process. After testing cell count, total protein level, and glucose level, the frozen samples which were collected via standard lumbar puncture method were sent to PPMI Biorepository Core Laboratories and then transferred to the University of Pennsylvania for measurement of CSF total α-syn, Aβ1–42, t-tau, and p-tau. A commercial enzyme-linked immunosorbent assay kit  was used to assess CSF total α-syn . Also, EP-values < 0.05 were regarded as significant.All statistical analyses were performed using SPSS version 22 . The normality of the data was checked using the Kolmogorov-Smirnov test. The t-test and Mann–Whitney U-test were performed for comparing the parametric and non-parametric variables between every two groups. Furthermore, we used linear mixed models within each group to assess the longitudinal changes of CSF biomarkers. The model included time from baseline as a fixed effect factor, CSF biomarkers as a dependent variable and each subject as random effect. The Benjamini-Hochberg method was used to address the type I error due to the multiple comparisons. The clinical and demographical characteristics of the participants are detailed in p = 0.016) and two years (p = 0.006) but not at baseline (p = 0.122) and six months (p = 0.746) (p = 0.012) but not at other time points. Further analysis showed a significant difference in the level of CSF T-tau between SWEDD and PD subjects at one (p = 0.013) and two years (p = 0.017). Also, there was a significant difference in the level of CSF P-tau between SWEDD and PD groups at two years visits (p = 0.030).There was no significant difference in the level of CSF α-syn Aβ1–42, t-tau, and p-tau between HCs and SWEDD at different time points . Investi= 0.746) . We obsep = 0.029) and two years (p = 0.002) from baseline in the level of CSF α-syn only in the PD group not in SWEDD or HCs (p = 0.031) and decreased after two years from baseline in PD subjects (p = 0.005). The CSF T-tau and P-tau levels did not change during follow-up in the HCs and PD group, however, we detected a significant increase in the level of CSF T-tau after two years (p = 0.036) and CSF P-tau after six months from baseline in SWEDD subjects (p = 0.011).The linear mixed model analysis showed significant changes after one . The exact mechanism by which α-syn contributes to PD is not clearly elucidated yet; however, there are some mechanisms suggested. Some of the proposed mechanisms include mitochondrial dysfunction, endoplasmic reticulum (ER) stress, loss of proteostasis, synaptic impairment, cell apoptosis, and inflammation . In addiOur data demonstrated that the level of Aβ1–42 was significantly different between PD and SWEDD individuals after one year but this result is not applicable for other time points. This result can be another indicator of the different pathophysiology of PD and SWEDD. One of the studies suggested that the source of the different motor and cognition functions between PD and SWEDD is the difference in their white matter tracks. It was ultimately observed that three clusters in the cingulum bundle (CB) were different in PD in comparison with SWEDD and NC versus SWEDD . AnotherT-tau and p-tau were suggested as potential biomarkers for the detection of cognitive impairments in PD patients along with other CSF biomarkers like Aβ1–42 and α-syn . FurtherIn our study, we found no considerable difference in the levels of CSF biomarkers  between SWEDD individuals and HCs at baseline, 6-month, 1 year, and 2 years while patients with SWEDD had significantly lower cognitive function compared to HCs. This finding might show that the pathophysiology and cognitive decline observed in SWEDD individuals are carried out through other pathways and not the known mechanisms contributing to PD.During our longitudinal study, we observed that the level of the α-syn in PD patients differs significantly from baseline after one year and two years; however, the same data is not applicable to other groups. The level of Aβ1–42 considerably increased after one year in SWEDD individuals while it showed a decline in PD patients after two years from baseline. The level of p-tau and t-tau increased significantly after 6 months and two years in SWEDD individuals respectively.Our results suggest different proteinopathies in SWEDD and PD; however, there should be more investigations to confirm our findings including post-mortem studies. We found that there is not a significant difference in the levels of these biomarkers between SWEDD individuals and HCs, while the comparisons for PD and SWEDD were significant at several time points. All these findings suggest that the process of cognitive impairment in patients with SWEDD might be different from those with idiopathic PD. Also, the level of CSF biomarkers increased at some points in SWEDD while the results for PD patients mostly showed a decrease over time. This finding suggests a faster neurodegeneration process in PD patients compared to SWEDD at least on the basis of these biomarkers. Future studies with longer follow-up duration and more sample sizes are necessary to validate our results.Since the data in this paper were obtained from the PPMI database (ppmi.loni.usc.edu), it does not include any research involving human or animal subjects.We do not have any financial support for this study.FN & SK: Designed the study, analyzed the data, and wrote the paper; FN, MN & SK: collected data, analyzed and interpreted the data, and wrote the draft version of the manuscript. The manuscript was revised and approved by all authors.The authors declare no conflict of interest regarding the publication of this paper.


P = 0.02). Furthermore, for individuals with positive brain amyloid by PET (n = 263), CSF pT217/T217 was more strongly correlated with the amount of amyloid (Spearman’s ρ = 0.69) than Aβ42/Aβ40 . In two independent cohorts of participants with symptoms of AD dementia (n = 55 and n = 90), CSF pT217/T217 and pT205/T205 were better correlated with tau PET measures than CSF p-tau181 concentration. These findings suggest that CSF pT217/T217 and pT205/T205 represent improved CSF biomarkers of amyloid and tau pathology in AD.Cerebrospinal fluid (CSF) amyloid-β peptide (Aβ)42/Aβ40 and the concentration of tau phosphorylated at site 181 (p-tau181) are well-established biomarkers of Alzheimer’s disease (AD). The present study used mass spectrometry to measure concentrations of nine phosphorylated and five nonphosphorylated tau species and phosphorylation occupancies (percentage phosphorylated/nonphosphorylated) at ten sites. In the present study we show that, in 750 individuals with a median age of 71.2 years, CSF pT217/T217 predicted the presence of brain amyloid by positron emission tomography (PET) slightly better than Aβ42/Aβ40  tests are used in the clinical diagnosis of Alzheimer’s disease. This study found that CSF tau phosphorylation at two sites (T217 and T205) is a better test for Alzheimer’s disease pathology than currently available tests. AD brain pathology is associated with a lower concentration of Aβ42 and a lower ratio of Aβ42:Aβ40 in the CSF, probably due to sequestration of Aβ42 into amyloid plaques3, but higher concentrations of CSF total tau (t-tau) and p-tau181 (refs. 5). Importantly, CSF t-tau and p-tau181 concentrations increase around the time of amyloid plaque deposition, when no neurofibrillary tangles are detected via tau PET, suggesting that elevated CSF t-tau and p-tau181 concentrations may reflect a response to amyloid plaques rather than neurofibrillary tangle burden7.AD is characterized by the aggregation of Aβ into amyloid plaques and the hyperphosphorylation and accumulation of tau into neurofibrillary tangles, which begins a decade or more before the onset of dementia symptoms. These neuropathological features can be visualized and quantified in living individuals using PET with radiotracers binding to amyloid and tau11. Several studies have demonstrated that CSF and/or plasma p-tau217 and p-tau231 concentrations are strongly associated with imaging and clinical measures of AD20. Immunoassays are widely used to measure concentrations of different p-tau species including p-tau181, p-tau217 and p-tau231, but they employ different antibodies and assay conditions, so in comparative studies it is often unclear whether differences in biomarker associations are related to differences in the analyte or the assay22. In contrast, mass spectrometry (MS) enables simultaneous measurement of nonphosphorylated and phosphorylated tau species and has high specificity. MS has identified p-tau217, p-tau205 and a species of the microtubule-binding region of tau (MTBR-tau243) as candidate AD biomarkers24, revealed the degree of phosphorylation at different tau sites in response to AD25 and enabled comparisons between CSF p-tau species that demonstrate the order in which sites are phosphorylated over the course of AD26.Most studies of CSF p-tau have examined only p-tau181, but tau is phosphorylated at many different sites and is truncated in the CSF and plasmaIn the present study of older individuals with and without cognitive impairment, MS was used to evaluate 24 different measures of CSF tau: the concentrations of nine phosphorylated and five nonphosphorylated tau peptides, and the phosphorylation occupancy (percentage phosphorylated:nonphosphorylated) at ten sites. This comprehensive evaluation allowed analysis of which CSF tau measures are most strongly associated with amyloid plaques and neurofibrillary tangles as measured by PET, regional brain volumes, clinical status (cognitively unimpaired or impaired) and dementia severity.APOE) ε4 allele and 16% were cognitively impaired as defined by a Clinical Dementia Rating (CDR) of ≥0.5, which includes mild cognitive impairment (MCI) and AD dementia (Extended Data Table n = 55)  at Washington University in St. Louis, MO, USA. The Knight ADRC amyloid PET cohort included 750 individuals with a median age of 71.2 years (interquartile range (IQR) 65.3–76.1 years); 55% were female, 90% self-identified as white, 39% carried at least one apolipoprotein E  compared with pT217/T217 (3.0%), pT181/T181 (5.8%) and pT205/T205 (9.1%) (see Appendix n = 750) Knight ADRC amyloid PET cohort  and the fold difference was largest for CSF p-tau231 concentration (7.79-fold) (Extended Data Table P = 0.02) and p-tau217 concentration (AUC = 0.95 (0.93–0.96), P < 0.0001; Extended Data Fig. The relationships of the CSF measures with amyloid PET status (positive or negative according to established cut-offs) were evaluated in the large (ρ = 0.76 (95% CI 0.73–0.79), which was almost significantly higher than Lumipulse Aβ42/Aβ40 (ρ = −0.74 (−0.77 to −0.71), P = 0.08) and was significantly higher than p-tau217 concentration (ρ = 0.71 (0.67–0.74), P < 0.0001) (Supplementary Tables ρ = 0.69 (0.62–0.75)), whereas the correlation between Lumipulse Aβ42/Aβ40 and amyloid PET Centiloid was only modest (ρ = −0.42 (−0.52 to −0.32), P < 0.0001; Supplementary Table The correlations of CSF measures with continuous amyloid PET Centiloid values were examined Figs.  and 2. CThe overlapping Knight ADRC tau PET cohort was used to evaluate the relationships between CSF measures and tau PET status  and the concentrations of four p-tau species  that were strongly associated with amyloid PET status also distinguished tau PET status with an AUC ≥ 0.89 , p-tau205 concentration (AUC = 0.87 (0.81–0.93)), pT217/T217 (AUC = 0.83 (0.76–0.90)) and p-tau217 concentration (AUC = 0.80 (0.72–0.88)) ), p-tau205 (ρ = 0.51 (0.43–0.58), pT217/T217 (ρ = 0.47 (0.39–0.55)) and pT205/T205 (ρ = 0.47 (0.39–0.55)) had among the highest correlations with the tau PET summary measure (Supplementary Tables n = 125), CSF pT205/T205 had the highest correlation with the tau PET summary measure (ρ = 0.72 (0.63–0.80)), which was not superior statistically to p-tau205 concentration (ρ = 0.65 (0.54–0.74)), but was superior to pT217/T217 (ρ = 0.55 (0.42–0.67), P = 0.02) and p-tau217 concentration (ρ = 0.48 (0.33–0.60), P = 0.0002)   or cognitively impaired ) was predicted by biomarkers with and without the covariates of age, sex and years of education , CSF pT217/T217 (ρ = 0.43 (0.37–0.48)) and pT205/T205 (ρ = 0.39 (0.33–0.45)) had some of the highest correlations with CDR-SB (Supplementary Table ρ = 0.65 (0.54–0.74)), CSF pT217/T217 (ρ = 0.50 (0.41–0.59)) and pT205/T205 (ρ = 0.50 (0.40–0.59)), whereas correlations with Lumipulse Aβ42/Aβ40 were lower (ρ = −0.23 (−0.34 to −0.12); Supplementary Table Correlations of amyloid PET, tau PET and CSF biomarkers with dementia severity as measured by the CDR sum of boxes (CDR-SB) were also examined in the large Knight ADRC amyloid PET cohort Fig. . The taun = 55; Supplementary Table ρ = 0.77 (0.63–0.86)) compared with Aβ42/Aβ40 (ρ = −0.58 (−0.73 to −0.37), P = 0.06; Supplementary Table ρ = 0.71 (0.55–0.82) and/or pT205/T205 (ρ = 0.67 (0.50–0.80)) had higher correlations with a tau PET summary measure compared with pT181/T181 (ρ = 0.42 (0.18–0.62), P = 0.006 and P = 0.04) or Lumipulse p-tau181 concentration (ρ = 0.44 (0.20–0.63), P = 0.003 and P = 0.07; Supplementary Table To evaluate the performance of these measures in a more clinically relevant cohort, CSF tau measures were examined in cognitively impaired (CDR ≥ 0.5) individuals from the Knight ADRC tau PET cohort ) was better correlated with amyloid PET Centiloid than CSF Aβ42/Aβ40 (ρ = −0.19 (−0.38 to 0.02), P = 0.0007; Extended Data Fig. ρ = 0.76 (0.65–0.83)) and/or pT205/T205 (ρ = 0.72 (0.60–0.81)) had higher correlations with a tau PET measure for the Braak I–IV region compared with pT181/T181 (ρ = 0.59 (0.43–0.71), P = 0.002 and P = 0.14) and CSF p-tau181 concentration by immunoassay (ρ = 0.42 (0.23–0.57), P = 0.0004 and P = 0.02, respectively; Extended Data Fig. ρ = 0.59 (0.43–0.71)) and pT205/T205 (ρ = 0.66 (0.52–0.76)) were better correlated with a tau PET measure for the Braak V–VI region compared with pT181/T181 (ρ = 0.44 (0.25–0.59), P = 0.004 and 0.02) and p-tau181 concentration by immunoassay (ρ = 0.23 (0.02–0.42), P = 0.0003 and P = 0.0007; Extended Data Fig. The CSF tau measures were further examined in participants with symptomatic AD (MCI or AD dementia) from the BioFINDER-2 cohort (= 0.55 0.8–0.68) wIn the present study, we examined the relationships of 24 CSF tau MS measures and 5 CSF Lumipulse automated immunoassay measures with amyloid PET, tau PET, regional brain volumes, clinical status and dementia severity. The CSF measure that best distinguished amyloid PET status was not Aβ42, Aβ42/Aβ40 or even p-tau217 concentration, but pT217/T217. Moreover, CSF pT217/T217 was strongly correlated with amyloid PET Centiloid in amyloid PET-positive individuals in whom the correlation between Aβ42/Aβ40 and amyloid PET Centiloid was only modest. Overall, although six of the ten sites investigated  were strongly associated with amyloid PET status, only CSF pT217/T217 was also strongly associated with tau PET status. In contrast, CSF pT205/T205 was not strongly associated with amyloid status, but, in amyloid PET-positive individuals, pT205/T205 had the highest correlations with the tau PET summary measure, regional tau PET measures and regional brain volumes. In amyloid PET-positive individuals, the tau PET summary measure best distinguished cognitively impaired from cognitively unimpaired individuals, but it was not statistically superior to CSF pT217/T217 or pT205/T205. Overall, these data demonstrate that CSF pT217/T217 is a superior biomarker of amyloid plaque burden and pT205/T205 is a promising biomarker of neurofibrillary tangle burden in individuals with brain amyloidosis.27. However, our study found that CSF pT217/T217 was superior to Aβ42/Aβ40 in reflecting amyloid plaque burden and was also strongly associated with tau PET status, demonstrating that a single measure may reflect the presence of both amyloid and tau (A and T). Notably, CSF pT217/T217 was not well correlated with tau PET in amyloid PET-positive individuals, suggesting that the association of pT217/T217 with tau PET status could be partially driven by the high amyloid levels in individuals who are tau PET positive. Concentrations and/or phosphorylation occupancies associated with sites T111, T231, T153, S208 and T181 were also highly associated with amyloid PET, but unlike T217 they were not strongly associated with tau PET, suggesting that these biomarkers may more specifically reflect amyloid (A). Our findings agree with the conclusions of other studies that p-tau217, p-tau181 and p-tau231 change early in AD in response to amyloid31. In contrast, CSF pT205/T205 was only modestly associated with amyloid PET, but was strongly associated with tau PET in amyloid PET-positive individuals, indicating that pT205/T205 is a relatively specific biomarker of tau (T). CSF pT205/T205 had the highest correlations with brain volumes, suggesting that pT205/T205 may also be a biomarker of neurodegeneration (N). The relationships between CSF tau measures and other biomarkers probably reflect the timing of biomarker changes in AD: previous work suggests that T217 is phosphorylated early in AD around the time of amyloid plaque deposition, whereas T205 is phosphorylated later and closer to neurofibrillary tangle accumulation and symptom onset26. The present study demonstrates the complexity of the relationship between tau phosphorylation and AD pathology, which may defy simple categorization.The amyloid–tau–neurodegeneration (ATN) framework has posited that CSF Aβ42 or Aβ42/Aβ40 is a measure of amyloid plaques (A) and that CSF p-tau  is a measure of neurofibrillary tangles (T)32. MS enables assessment of measures such as phosphorylation occupancy, which may be difficult to evaluate via immunoassay. In the present study, CSF pT217/T217 had slightly but significantly higher performance compared with p-tau217 in prediction of amyloid PET status, which could be meaningful when accuracy is highly valued . It is possible that phosphorylation occupancies attenuate interindividual variability unrelated to AD pathology which affects the concentrations of both phosphorylated and nonphosphorylated tau species34. Notably, the CSF biomarker assays that are currently available for clinical AD diagnosis do not include p-tau217, p-tau205, pT217/T217 or pT205/T205. Although immunoassays are more widely available than specialized MS assays, MS may potentially provide unique and better performing measures than immunoassays.An advantage of MS over immunoassays is that different protein species can be specifically and simultaneously identified36. It is unknown whether other CSF tau measures such as pT217/T217 or pT205/T205 perform similarly across racial and ethnic groups, and further studies of these measures in diverse and clinically relevant cohorts are needed.The study cohorts consisted of participants enrolled in research studies and, therefore, the findings are most relevant to research studies and clinical trials and less generalizable to clinical populations. The cohorts were relatively young (early to mid-70s). Older cohorts, especially clinical cohorts, would be expected to have a higher prevalence of neurological comorbidities, which could complicate biomarker relationships. The cohorts included relatively few individuals with symptomatic AD and/or moderate or high levels of tau pathology. In addition, minoritized populations were not well represented in the study cohorts and studies have found racial differences in CSF t-tau and p-tau181 concentrationsTechnical limitations include that tau was immunopurified for analysis, which may result in some species of tau not being recovered well. Data for some CSF tau measures (particularly those related to p-tau231) did not meet quality control criteria for a subset of the samples and were thus excluded from analyses. The variance for p-tau231 measures was higher than for p-tau217 measures, which could have resulted in underestimating the performance of p-tau231 measures. Measures associated with p-tau231 had the largest fold-change between amyloid PET-negative and amyloid PET-positive individuals, and between tau PET-negative and tau PET-positive individuals, but the higher variance led to reduced associations of p-tau231 measures with outcomes. A limitation of the comparisons between CSF Aβ42/Aβ40 and tau measures is that CSF Aβ peptides were measured with immunoassays rather than MS, and it is possible that MS measurements of CSF Aβ42/Aβ40 could perform better. Overall, it is important to consider that the observed relationship between a biomarker and AD depends not only on the biological relationship, but also on the technical characteristics of the assay.38. Comprehensive evaluation of corresponding tau species in blood may yield improved AD biomarkers that are more accessible to researchers and patients. In particular, plasma pT217/T217 is highly correlated with CSF pT217/T217 (ref. 14) and a recent head-to-head study of ten plasma tau assays  found that plasma pT217/T217, as measured by MS, had the best performance in predicting amyloid PET status and progression to AD dementia17. Studies of the correspondence of plasma pT205/T205, tau PET and clinical AD symptoms are needed. Overall, the present study demonstrates that different CSF tau species represent different aspects of AD, and that pT217/T217 and pT205/T205 may be superior to the CSF biomarkers of AD currently in widespread use.Future directions for this work include evaluating longitudinal trajectories of CSF tau measures to define how these measures change over the time course of sporadic AD, and whether they predict progression from cognitive normality to symptomatic AD. It will also be important to evaluate these CSF tau measures in non-AD dementias, including primary tauopathies39, the CDR-SB and the Mini-Mental State Examination. Individuals with a CDR ≥ 0.5 were considered to have a dementia syndrome, and the probable etiology of the dementia syndrome was formulated by clinicians based on clinical features in accordance with standard criteria and methods40. APOE genotype was determined as previously described41. Sex and race were self-identified. Participants included in the present study underwent a clinical assessment and lumbar puncture (LP) within 2 years of an amyloid PET scan (amyloid PET cohort). An overlapping cohort was selected in which participants underwent both an amyloid PET and a tau PET scan within 2 years of an LP (tau PET cohort). Previous work has demonstrated that the relationship between CSF biomarkers and amyloid PET is stable if CSF is collected up to 6 years before or 2 years after amyloid PET42. If more than one CSF sample from a participant met the criteria, the most recently obtained sample was included. No data points were excluded from analyses and outliers were not removed.Participants in the Knight Alzheimer Disease Research Center (ADRC) at Washington University in St. Louis were community-dwelling volunteers enrolled in studies of memory and aging. Most participants were recruited from memory clinics or self-referred due to interest in dementia. Individuals were excluded from enrollment if they were diagnosed with a non-AD dementia at their initial assessment , had conditions that might interfere with study procedures ) or had medical issues that might affect long-term participation . All procedures were approved by the Washington University Human Research Protection Office (HRPO) and written informed consent was obtained from each participant or their legally authorized representative when appropriate (protocol no. 201109100). Participants received compensation for time spent undergoing procedures, as approved by the HRPO. All participants underwent a comprehensive clinical assessment that included a detailed interview of a collateral source, a neurological examination of the participant, the CDR12. All participants included underwent an LP and cognitive testing within 2 years of both an amyloid PET scan and a tau PET scan. All participants gave written informed consent and ethics approval was granted by the Regional Ethical Committee in Lund, Sweden.Participants in the Swedish BioFINDER-2 confirmatory cohort (NCT03174938) included individuals diagnosed with MCI due to AD or AD dementia. Details on recruitment, exclusion and inclusion criteria have been described previouslyFor studies in the Knight ADRC cohorts, 20–30 ml of CSF was collected via LP at approximately 8am after overnight fasting. CSF was collected in a 50-ml poly(propylene) tube via a gravity drip using an atraumatic Sprotte 22-gauge spinal needle. The entire sample was gently inverted to disrupt potential gradient effects and centrifuged at low speed to pellet any cellular debris. CSF was then aliquoted into poly(propylene) tubes and stored at −80 °C. Aβ42, Aβ40, t-tau and p-tau181 concentrations were measured with a single lot of reagents for all samples with an automated immunoassay platform  according to the manufacturer’s specifications.12. Aβ42, Aβ40, t-tau and p-tau181 concentrations were measured using fully automated Elecsys immunoassays43.For the BioFINDER-2 confirmatory cohort, CSF was collected as previously described25). Briefly, tau was immunopurified by incubating 450 µl of CSF with tau1 and HJ8.5 antibodies covalently attached to Sepharose beads at room temperature for 4 h. Immunopurified tau was digested for 16 h at 37 °C with 400 ng of trypsin (Promega). AQUA peptides (Life Technologies) were added to a final sample concentration of 5 fmol per labeled phosphorylated peptide and 50 fmol per labeled unmodified peptide. Samples were subjected to liquid chromatography–tandem HRMS (LC-MS/HRMS) analysis on a nanoAcquity ultra-performance LC system (Waters) coupled to an Orbitrap Tribrid Eclipse mass spectrometer (Thermo Fisher Scientific) operating in parallel reaction monitoring mode. MS/HRMS transitions were extracted using Skyline v.22.2.2.278 . Data were aggregated using Tableau v.2022.2.2 (Tableau Software) to calculate concentrations and phosphorylation occupancies. All assays and data extraction steps were performed by operators blinded to any clinical or biomarker information.All samples from an individual were run in the same batch. CSF tau was immunopurified and digested, then phosphorylated and nonphosphorylated peptides were quantified using high-resolution MS (HRMS) as previously described and detailed in Appendix 18F]AV45 (florbetapir) or [11C]Pittsburgh Compound B (PiB). An overlapping cohort additionally underwent tau PET with [18F]AV1451 (flortaucipir). Both amyloid PET and tau PET scans were performed in coordination with a 3-T structural MRI scan. T1-weighted MRIs were processed using Freesurfer 5.3 to generate ROIs used for the processing of PET data. Estimates of regional volumes were adjusted for intracranial volume using a regression approach. Data from the 30- to 60-min post-injection window for PiB, the 50- to 70-min window for florbetapir or the 80- to 100-min window for flortaucipir were converted to SUVRs using the cerebellar gray as a reference and partial volume corrected using a geometric transfer matrix44. Values from the following regions were averaged together to represent mean cortical SUVR for florbetapir or PiB: bilateral orbitofrontal, medial orbitofrontal, rostral middle frontal, superior frontal, superior temporal, middle temporal and precuneus. Amyloid PET positivity was previously defined as a mean cortical SUVR > 1.42 for PiB and >1.19 for florbetapir45. Mean cortical SUVRs were converted to Centiloid units to combine data from the two tracers46. Values from the bilateral entorhinal cortex, amygdala, lateral occipital cortex and inferior temporal cortex regions were averaged together as a summary measure of tau PET47. Tau PET positivity was defined as a tau PET summary measure >1.52, based on Gaussian mixture modeling  and later change (Braak V–VI region) were calculated. The cut-off for positivity in the Braak I–IV region was SUVR > 1.32 (ref. 48).Participants in BioFINDER-2 underwent amyloid PET using [31. Measured values for many biomarkers were not normally distributed and therefore nonparametric analyses were performed. The significance of differences by biomarker status (amyloid PET or tau PET status) were evaluated with Wilcoxon’s rank-sum tests for continuous variables and χ2 or Fisher’s exact test for categorical variables. ROC analyses were used to evaluate the correspondence of CSF biomarker measures with amyloid PET status, tau PET status or clinical status (cognitively unimpaired (CDR = 0) or cognitively impaired (CDR > 0)). Cut-offs that best distinguished amyloid PET or tau PET status were found based on the highest combined sensitivity and specificity (Youden’s index). Differences between ROC AUCs were evaluated using DeLong tests49. Spearman’s correlations were used to evaluate the continuous relationships of CSF biomarker measures with amyloid PET Centiloid, the tau PET summary measure or the CDR-SB. For partial Spearman’s correlations with amyloid PET, tau PET and brain volumes, analyses included covariates of age and sex; for correlations with CDR-SB, analyses included covariates of age, sex and years of education. Comparisons between Spearman’s correlations were performed by bootstrapping. When multiple measures were compared, the significance was adjusted using the Benjamini–Hochberg procedure50. Analyses were replicated in the subcohort of individuals with no missing data. Statistical analyses were implemented using SAS 9.4. Plots were created with GraphPad Prism v.9.2.0. All P values were from two-sided tests and results were deemed statistically significant at P < 0.05.No statistical methods were used to predetermine sample sizes but our sample sizes are similar to or larger than those used for similar studies50.For visualization of the associations between CSF measures and regional tau PET or brain volumes, partial Spearman’s correlations, including age and sex, were calculated with partial.r from the R psych toolbox v.2.1.9. The ggseg package v.1.6.5 was used to visualize correlations and results from the left hemisphere are shown. The significance of correlations was adjusted for multiple comparisons using the Benjamini–Hochberg procedureFurther information on research design is available in the Supplementary InformationSupplementary Fig. 1, Tables 1–32 and Appendix 1.Reporting Summary


Donor/recipient size matching is paramount in heart transplantation. Body weight, height, body mass index, body surface area, and predicted heart mass (PHM) ratios are generally used in size matching. Precise size matching is important to achieve better clinical outcomes. This study aims to determine the donor/recipient ascending aortic diameter (AAoD) ratio as a metric for donor selection and its effect on postoperative clinical outcomes in heart transplant patients.n = 96), and Group B, AAoD > 0.8032 (n = 265). A propensity score-matched (PSM) study was performed to equalize the two groups comprising 77 patients each in terms of risk. A Cox regression model was developed to identify the independent preoperative variables affecting the primary end-point. The primary endpoint was all-cause in-hospital mortality.We retrospectively reviewed all consecutive patients who underwent heart transplantation from January 2015 to December 2018. A cutoff value of 0.8032 for the donor/recipient AAoD ratio  was determined for predicting in-hospital mortality. The patients were divided into two groups based on the cutoff value. Group A, AAoD < 0.8032 , and diabetes  were found to be independent predictors of in-hospital mortality. Group A had higher 3-year mortality than Group B (P = 0.022). The surgery time was longer and postoperative RBC, plasma, and platelets transfusion were higher in Group A (P < 0.05). Although not statistically significant the use of continuous renal replacement therapy (P = 0.054), and extracorporeal membrane oxygenation (P = 0.086), was realatively higher, and ventilation time (P = 0.079) was relatively longer in Group A.A total of 361 patients underwent heart transplantation during the given period. On the multivariate analysis, donor/recipient PHM ratio [HR 16.907, 95% confidence interval (CI) 1.535–186.246, The donor/recipient AAoD ratio is a potential metric for patient matching and postoperative outcomes in heart transplantation. A donor/recipient AAoD ratio > 0.8032 could improve post-heart transplantation outcomes and donor heart utilization. Despite significant advances in the treatment of end-stage heart failure, heart transplantation (HTx) remains the definitive and effective therapeutic option. As of 2017, 144,230 cases of orthotopic heart transplantation have been registered worldwide  and was performed in accordance with the national program for deceased-donor organ donation in China  nationa. WrittenOur cohort incorporated all recipients of orthotopic heart transplantation at our institution from 1 January 2015 to 31 December 2018. A total of 361 patients were included, and all grafts were procured from donors after brain death. Patients who underwent cardiac retransplantation or multiorgan transplantation were excluded. Demographic and clinical characteristics of all heart transplant donors and recipients were collected retrospectively from electronic medical records, subsequent visits, or contact by our center. Preoperative echocardiography data were used to measure the recipient AAoD, and the donor AAoD was measured 3 weeks after operation . In thisen bloc removal of the superior vena cava with extra length of the inferior vena cava, aorta, and pulmonary artery. The organ was then prepared by marking the upper-left and lower-right pulmonary vein orifices and trimming the left atrium. A uniform preservation method was applied to all donor hearts during transport, consisting of 1 L of cold (4°C) histidine–tryptophan–ketoglutarate (HTK) solution. In addition, 500 ml of HTK solution was perfused before implantation.Donor heart removal was accomplished with 2/day for children), and prednisone was used for all recipients. Following endomyocardial biopsy, acute cellular rejection exceeding grade 2R according to the ISHLT criteria (Interleukin-2 receptor antagonist (basiliximab) and methylprednisolone were used for induction therapy. The first dose of basiliximab  was administered intravenously 2 h prior to the operation. The second dose was administered intravenously 4 days after transplantation. This medication was followed by a standard triple-drug immunosuppression regimen that included tacrolimus, mycophenolate mofetil, and prednisone. Maintenance immunotherapy consisting of tacrolimus , mycophenolate , including an intra-aortic balloon pump, extracorporeal membrane oxygenation, and continuous renal replacement therapy (CRRT) in the early postoperative periods, and short-term postoperative complications, such as acute rejection, neurological complications, renal complications, hepatic dysfunction, and respiratory complications.J = sensitivity + specificity - 1). The ROC curve revealed a best cutoff value of 0.8032   with the donor/recipient AAoD ratio < 0.8032 and Group B (n = 265) with the donor/recipient AAoD ratio > 0.8032. Finally, propensity score matching was performed across key variables including data from the recipients and donors except AAoD and AAoD ratio to balance the distinction of baseline variables. A 1:1 match was performed with 0.02 tolerance and each group consisting of 77 patients. A univariate analysis for all-cause mortality was conducted using the Cox regression model. The candidate variables associated with a univariate effect on prognosis  were then analyzed by stepwise multivariate regression with a probability of 0.05 and an elimination probability of 0.1. All tests were two-tailed with a significance level of P < 0.05. The time to event analysis was estimated by the Kaplan-Meier method and the difference between the groups by log-rank test. Statistical analysis was performed with SPSS Version 21.0  and MedCalc Version 19.0.4 .Unless otherwise stated, the continuous variables conforming to a normal distribution were expressed as a mean ± standard deviation and analyzed with a two-sample 2, respectively. The number and percentage of patients with the following medical condition are as follows: hypertension, 39, 25.32%; diabetes, 26, 16.88%; hyperlipidemia, 9, 5.84%; history of smoking, 63, 40.91%; history of cardiac surgery, 45, 29.22%; history of alcohol use, 43, 27.92%; peripheral vascular disease, 5, 3.25%; stroke, 5, 3.25%; preoperative dialysis, 3, 1.95%; chronic obstructive pulmonary disease (COPD), 5, 3.25%; chronic liver disease, 9, 5.84%; and preoperative mechanical support, 5, 3.25%. The diagnoses for patients who required transplantation were cardiomyopathy , coronary artery disease , valvular heart disease , congenital heart defects , and others . A total of 134 donors were men (87.01%), and the average age and weight were 33.22 ± 12.63 years and 62.32 ± 11.36 kg, respectively. The standardized mean difference of all variables included in From January 2015 to December 2018, 361 patients underwent heart transplantation in Wuhan Union Hospital. The baseline characteristics of the total cohort and PSM cohort are given in P = 0.001), donor weight , donor/recipient PHM ratio , donor/recipient AAoD ratio < 0.8032 , diabetes mellitus , and COPD . After multivariate Cox regression analysis, only three independent variables were significant that affected the postoperative in-hospital mortality (P = 0.021), donor/recipient AAoD ratio < 0.8032 , and diabetes .We performed a univariate Cox regression analysis for the correlation between various preoperative variables and postoperative in-hospital mortality and found six variables that significantly affect the postoperative mortality : BMI  and 3 years   .P = 0.027). RBC transfusion , plasma transfusion , and platelets transfusion  were significantly higher in Group A than in Group B. The number of patients who required postoperative dialysis in Group A was borderline statistically significantly higher than that in Group B . Meanwhile, even though not statistically significant the ECMO use  was relatively higher and ventilation time  was relatively longer in group A than in group B.We then compared the postoperative outcomes of these two groups . The resThe major novel findings of our study are as follows: the donor/recipient AAoD ratio is an important preoperative variable for in-hospital mortality in heart transplantation with a cutoff value of 0.8032. Survival analysis and secondary outcome comparison showed that donor/recipient AAoD ratio > 0.8032 results in a higher survival rate, low transfusion rate, shorter ventilation and surgery time, and less use of CRRT and ECMO. Thus, a donor/recipient AAoD ratio of >0.8032 may contribute to better survival and fewer postoperative complications in heart transplantation.With the rapid development of HTx in China and worldwide, the imbalance between organ demand and donation is gradually emerging. More transplant recipients can benefit when donor characteristics are also used to determine the optimal donor/recipient match and make correct and fast organ allocation strategies. As listed in the 36th annual report of the ISHLT , the topP < 0.001), while patients had no risk of increased mortality when donors were undersized for other metrics. They found that 32% of donors turned down for heart size would have been accepted if the PHM cutoff value of 0.86 was used. The 36th adult heart transplantation report of ISHLT (P = 0.021), which is in accordance with previous studies. Another study by Gong et al. (P = 0.007) but not TBW (P = 0.49), which was confirmed by multivariate analysis predicting that undersizing by PHM but not TBW is a predictor of PGD . They concluded that size matching by PHM is clinically more appropriate than size matching by TBW  as reflected by the donor/recipient body weight ratio of 0.98 ± 0.36 in P = 0.021), diabetes , and AAoD ratio  (which is the basis of our tow group categorization), other factors did not influence the survival. The similar risk groups, including similar donor/recipient weight ratio (P = 0.751), BMI (P = 0.309), recipient age (P = 0.470), and donor age (0.146) . Simultaneous inclusion of donor and recipient data seems to be a better approach than taking the factors of the donor or recipient alone into account. We believe the AAoD ratio should be considered complementary rather than an alternative to already available size-matching metrics until further research clarifies these results.The size of the recipient heart chambers and part of the ascending aorta changes immediately after transplantation, but other large blood vessels, body weight, and BSA remain the same. Therefore, the research is focused on these size-matching metrics that can predict whether the newly implanted heart best matches the explanted heart or adequately satisfies body perfusion demand. Some research studies have already been done on these size-matching metrics, as discussed above. In our study, we wanted to use the echocardiographic measurements of the AAoD of donors and recipients to predict the outcomes of HTx and attempted to find a new size-matching metric for donor selection. In our institution, donors were selected based on body weight (donor-to-recipient TBW (0.146) , should Each donor heart during procurement retains a part of the ascending aorta for anastomosis during transplant. Therefore, by measuring the AAoD before and after the operation, we directly get the AAoD of the recipient and donor, respectively. AAoD might be influenced by the choice of measurement method, such as echocardiography or cardiac computed tomography. To obtain consistent data, we measured the AAoD 1 cm above STJ at end-diastole using the leading edge-to-leading edge convention from the parasternal and suprasternal approaches by two-dimensional echocardiography.The size of the aorta changes with age, gender, body weight, and BSA , 21, 22.A donor/recipient AAoD ratio of <0.8032 was associated with high mortality of HTx recipients not only in the hospital but also during the follow-up, along with notable postoperative complications during hospitalization. The surgery time was significantly longer in Group A. Group A had higher RBC, platelets, and plasma transfusion than Group B. Subramaniam et al.  reportedOur study has some limitations. The single-center, retrospective, and observational design limited the ability to correct all confounding factors and selection bias; however, we used PSM to match most of the confounders. Donor information was limited and needed to be further tracked and completed. Moreover, the specific mechanism of the AAoD ratio affecting prognosis remains uncertain, and the relationship between a lower AAoD ratio and preload and afterload/blood volume needs to be clarified in future studies. Finally, the follow-up time was not sufficient to evaluate long-term outcomes. High-quality, multicenter, large-sample prospective studies are required to further verify the conclusions.In conclusion, HTx clinicians often face difficulty in donor selection, with the need to balance the risk of post-HTx mortality and worsening end-stage heart failure. The availability of a new potential variables facilitate the graft allocation and help in real-time decision-making. The donor/recipient AAoD ratio is a novel potential metric for donor selection and a ratio >0.8032 could improve donor heart utilization and post-HTx outcomes.


The meninges, formed by dura, arachnoid and pia mater, cover the central nervous system and provide important barrier functions. Located between arachnoid and pia mater, the cerebrospinal fluid (CSF)-filled subarachnoid space (SAS) features a variety of trabeculae, septae and pillars. Like the arachnoid and the pia mater, these structures are covered with leptomeningeal or meningothelial cells (MECs) that form a barrier between CSF and the parenchyma of the optic nerve (ON). MECs contribute to the CSF proteome through extensive protein secretion. In vitro, they were shown to phagocytose potentially toxic proteins, such as α-synuclein and amyloid beta, as well as apoptotic cell bodies. They therefore may contribute to CSF homeostasis in the SAS as a functional exchange surface. Determining the total area of the SAS covered by these cells that are in direct contact with CSF is thus important for estimating their potential contribution to CSF homeostasis.Using synchrotron radiation-based micro-computed tomography (SRµCT), two 0.75 mm-thick sections of a human optic nerve were acquired at a resolution of 0.325 µm/pixel, producing images of multiple terabytes capturing the geometrical details of the CSF space. Special-purpose supercomputing techniques were employed to obtain a pixel-accurate morphometric description of the trabeculae and estimate internal volume and surface area of the ON SAS.In the bulbar segment, the ON SAS microstructure is shown to amplify the MECs surface area up to 4.85-fold compared to an “empty” ON SAS, while just occupying 35% of the volume. In the intraorbital segment, the microstructure occupies 35% of the volume and amplifies the ON SAS area 3.24-fold.We provided for the first time an estimation of the interface area between CSF and MECs. This area is of importance for estimating a potential contribution of MECs on CSF homeostasis.The online version contains supplementary material available at 10.1186/s12987-023-00423-6. The optic nerve (ON) is an extension of the telencephalon and as such represents a white matter tract of the central nervous system (CNS). The ON can be divided into an intracranial portion, an intracanalicular portion, and an intraorbital portion, as shown in Fig. The ON SAS, just as the SAS of the brain, is bridged with a variety of trabeculae, septae, vela and pillars that extend into the arachnoid layer. Their distribution is heterogeneous along the ON: the bulbar segment contains a complex network of delicate trabeculae. The intraorbital segment contains trabeculae combined with broader septae that divide the SAS into intertrabecular fluid spaces, whereas in the canalicular portion, septae are replaced with pillars. Transmission electron microscopy (TEM) studies of the trabeculae in the bulbar segment demonstrated that the trabeculae are covered with a layer of arachnoid cells and that trabeculae are connected by thin cytoplasmic extensions, thus forming a multitude of single intertrabecular fluid spaces within the trabecular structure .The dura mater harbors lymphatic capillaries that were demonstrated by TEM as well as immunohistochemistry. TEM studies show tracer particles within lymphatic clefts after injection of tracer into the SAS .Just as the ventricles, the cisterns and the cranial subarachnoid space, the SAS of the optic nerve is filled with CSF. CSF enters from the pituitary cistern via the optic canal into the ON SAS that ends blind behind the lamina cribrosa in a cul-de-sac fashion . CSF offArachnoid and pia mater are covered by meningothelial cells (MECs), also known as leptomeningeal cells . They arWith the MECs representing a potentially important functional exchange surface, the total area they cover is of high importance for investigating their effect on the CSF. The aim of this study is to provide a quantitative estimation of the interface area of CSF and MECs and assess a methodology to characterize compartmentalization of the ON SAS. Such quantitative analysis is of relevance to understand both CSF dynamics and pathophysiology of several disorders such a papilledema in the setting of idiopathic intracranial hypertension, axonal loss in normal tension glaucoma, among other diseases.In accordance with Swiss ethics regulations , a healthy human optic nerve specimen (about 6 mm x 8 mm x 10 mm) from an 85-year-old male was harvested post-mortem. Osmium tetroxide was employed for sample fixation.Helvetios supercomputer of the Swiss Federal Institute of Technology Lausanne .Using synchrotron radiation-based micro computed tomography (SRµCT), we acquired datasets of the ON SAS structure at the TOMCAT beamline of the Swiss Light Source . Two regions of interest of approximately 6 mm x 6 mm × 0.8 mm were imaged: Dataset1 and Dataset2. Dataset1 was obtained from a bulbar segment of the ON, and Dataset2 was acquired from an intraorbital ON segment. Overview scans with a field of view of 1.6 cm × 1.6 cm × 1.4 cm at 6.5 µm/pixels were carried out to determine the optimal imaging locations. The specimen was then imaged in-axis at 0.375 µm/pixel. Overall, we produced 71 tiles of 2560 × 2560 × 2160 pixels quantized  at 16bits. To facilitate image registration, a tile overlap of 20% was imposed. The total size of the acquired data was more than 2 terabytes (TBs). Image processing was carried out on the  TB.Tile registration was carried out with a novel phase-correlation algorithm exploiting the enhanced stability of wavelet frames . Due to A hard threshold on the grayscale image would either miss the thin trabeculae (under-segmentation) or coalesce the coarsest pillars (over-segmentation). To mitigate this issue, and suppress imaging artifacts, we digitally enhanced the image with 3D continuous curvelets sampled in Fourier space , coverinConnected components are manipulated to remove cavities in the foreground of the SAS image segment. A morphological closing allowed us to correctly capture the ON. The CSF foreground is obtained by retaining the main connected component of the inverted SAS + ON foreground. The SAS (segment) foreground, capturing only the microstructure, is obtained by a morphological closing of the CSF, intersected with the SAS + ON segment foreground  and α-synuclein thus help to maintain CSF homeostasis. The quantitative effect of MEC functions is likely to depend on their number and is therefore related to the total area they cover in the SAS. Interestingly, elevated CSF pressure has shown to decrease endocytotic activity of MECs and to cause proliferation and cell growth of meningothelial cells in vitro [MECs were demonstrated in vitro to exercise a multitude of functions. They secrete Lipocalin-type prostaglandin D synthetase (L-PGDS) , 34 and in vitro . As meniin vitro . More deThe subarachnoid space of the optic nerve does not only contain trabeculae but also septae that display a different geometry. This work is focused on the anatomy and geometry of the trabeculae at two locations of the retrobulbar portion of the subarachnoid space only and is, therefore, not representative for the whole SAS of the ON. Future work will include the analysis of septae, which were not present in the considered specimen.A second limitation of the study is related to the possible morphological changes triggered by aging. The geometrical features of the ON specimen under examination may substantially differ from the ones of a younger subject. Furthermore, according to previous histological studies , inter-iThirdly, fixation of the tissues involved pinching the ON to get a stable grip and to avoid shrinkage of the tissue. This, in turn, has caused structural deformations that are visible in both datasets. We partially overcame this limitation by discarding the circular section featuring the most prominent deformations. However, as with all fixation procedures, we cannot entirely exclude the possibility of fixation artifacts.Fourthly, while segmentation yielded high quality results for most of the dataset, over-segmentation in the ON SAS image segment upper region of the sample is apparent  . We did The current study provides, for the first time, quantitative data of the volume and surface of the trabeculae in the retrolaminar portion of the subarachnoid space of the optic nerve. These data can serve as the basis for flow modeling in the ON SAS. Trabeculae do not only serve a mechanical function, but also augment the overall MEC surface area, which may be of importance for the MECs’ possible contribution to maintain homeostasis of the optic nerve.Additional file 1: Figure S1. Fixation-generated spurious artifacts in the grayscale slices, featuring strong intensity gradients and creating manifolds in the CSF (left). Issue mitigation during image enhancement and image segmentation (right) of CSF (red). Figure S2. High-angle views from within the grayscale images (intensities mapped from translucent blue to opaque red) showing the multi-scale features of the ON SAS microstructure. Figure S3. Image cuts in the z-direction (top) and x-direction (bottom) of Dataset1. Extended CSF (green) is obtained by engulfing the ON SAS microstructure through a morphological closing operation. Figure S4. Circular sectors (red) in Dataset1 (top) and Dataset2 (bottom) where CSF volume and ON SAS area have been ignored due to the structural deformation occurred during the manipulation of the specimen. Movie S5. Slice-by-slice movies of trabecular thickness and trabecular separation of the ON SAS foreground, both for Dataset1 and Dataset2. Blue corresponds to 2μm or less and red corresponds to 160μm. Movie S6. Overview movies of 3D rendering of opaque isosurfaces of trabecular thickness and trabecular separation at the ON SAS foreground/background interface, both for Dataset1 and Dataset2. Blue corresponds to 2μm or less and red corresponds to 160μm. Movie S7. Closeup movies showing detailed morphometry of the microstructure in the ON SAS, both for Dataset1 and Dataset2. Blue corresponds to 2μm or less and red corresponds to 160μm. Movie S8. Slice-by-slice movies of the stitched signals for both datasets, alongside with image segmentation of the ON SAS trabeculae (yellow) and CSF (red). Movie S9. Volume-rendered close-up of the stitched signal of Dataset1, following the CSF over the ON section.


Caprapyrenaica) is an ecologically and economically relevant medium-sized emblematic mountain ungulate. Diseases participate in the population dynamics of the species as a regulating agent, but can also threaten the conservation and viability of vulnerable population units. Moreover, Iberian ibex can also be a carrier or even a reservoir of pathogens shared with domestic animals and/or humans, being therefore a concern for livestock and public health. The objective of this review is to compile the currently available knowledge on (1) diseases of Iberian ibex, presented according to their relevance on the health and demography of free-ranging populations; (2) diseases subjected to heath surveillance plans; (3) other diseases reported in the species; and (4) diseases with particular relevance in captive Iberian ibex populations. The systematic review of all the information on diseases affecting the species unveils unpublished reports, scientific communications in meetings, and scientific articles, allowing the first comprehensive compilation of Iberian ibex diseases. This review identifies the gaps in knowledge regarding pathogenesis, immune response, diagnostic methods, treatment, and management of diseases in Iberian ibex, providing a base for future research. Moreover, this challenges wildlife and livestock disease and wildlife population managers to assess the priorities and policies currently implemented in Iberian ibex health surveillance and monitoring and disease management.Iberian ibex ( Disease can be defined as damage or deterioration affecting normal functions, including responses to extrinsic and intrinsic factors such as nutrition, climate, toxic agents, micro- and macroparasites, and congenital defects, alone or combined Wobeser . WildlifCapra pyrenaica) is a medium-sized mountain ungulate, formerly endemic to the Iberian Peninsula but recently introduced in the northern Pyrenees in France   molecular techniques to detect genetic material of Finally, the non-specific increases of acute-phase proteins and oxidative stress induced by sarcoptic mange have been proposed to diagnose and monitor sarcoptic mange in wildlife  ovis (formerly named Neisseria ovis), Listeria monocytogenes, Mycoplasma other than M. conjunctivae , Staphylococcus aureus, or Rickettsia-like spp.  was first reported almost 25 years ago  , although the confirmation through microbiological culture diagnostic techniques was not specified. This same study did not achieve to identify M. bovis through immunofluorescence in the lungs and lymph nodes of 175 Iberian ibexes . Hence, the reason to monitor and survey these three diseases in Iberian ibex is rather their relevance in domestic livestock, where they are included in eradication programs, and the risk for zoonosis and transmission among domestic animals and wildlife from a One Health approach  , a tick-transmitted orthonairovirus. It is a zoonotic disease that can cause potentially lethal systemic hemorrhagic disease in humans, with a mortality rate ranging between 3 and 30% of the humans infected Ergonul . DomestiChlamydophila spp., Coxiella burnetii , Leptospira pomona, Mycoplasma agalactiae, M. arginine and M. mycoides subspecies mycoides , Neospora caninum, pestivirus , Salmonella abortusovis, Schmallenberg virus, and Toxoplasma gondii has been investigated and/or reported , Corynebacterium pyogenes (most likely opportunistic), Mannheimia glucosida, Mannheimia haemolytica, Moraxella bovis, Mycoplasma ovipneumoniae, Pasteurella multocida, other hemolytic Pasteurellaceae or Mannheimia spp. or viruses as parainfluenza-3 virus (pneumovirus), and syncytial respiratory virus (respirovirus)  nodosus, Fusobacterium necrophorum, Arcanobacterium (Trueperella) pyogenes, and Treponema spp.  pyogenes and/or Staphylococcus aureus subsp. anaerobius can cause suppurative lesions. These bacteria are normally present in the skin and mucosae and can enter the body through wounds, producing multiple abscesses in the inner organs such as the lymph nodes, lungs, liver, kidneys, and/or central nervous system, among others, which can cause death  intended for individual parenteral use is a major animal welfare, environmental, and health concern. Since domestic livestock has been identified as the origin of sarcoptic mange outbreaks in Iberian ibex, surveillance and control of this disease in small ruminant livestock sympatric with Iberian ibex are probably more efficient management options.The review of other diseases reported in Iberian ibex raises doubts about the rationale for the diseases included in the health surveillance of this species, such as tuberculosis or brucellosis, since Iberian ibex seems not to be a reservoir for these diseases and even rarely if ever affected by them. Instead, other diseases that might be relevant in the health surveillance of Iberian ibex populations deserve further research and monitoring to elucidate their potential effects in the population dynamics of the species.


The understanding of the pathophysiology of multiple sclerosis (MS) has evolved alongside the characterization of cytokines and chemokines in cerebrospinal fluid (CSF) and serum. However, the complex interplay of pro- and anti-inflammatory cytokines and chemokines in different body fluids in people with MS (pwMS) and their association with disease progression is still not well understood and needs further investigation. Therefore, the aim of this study was to profile a total of 65 cytokines, chemokines, and related molecules in paired serum and CSF samples of pwMS at disease onset.Multiplex bead-based assays were performed and baseline routine laboratory diagnostics, magnetic resonance imaging (MRI), and clinical characteristics were assessed. Of 44 participants included, 40 had a relapsing–remitting disease course and four a primary progressive MS.There were 29 cytokines and chemokines that were significantly higher in CSF and 15 in serum. Statistically significant associations with moderate effect sizes were found for 34 of 65 analytes with sex, age, CSF, and MRI parameters and disease progression.In conclusion, this study provides data on the distribution of 65 different cytokines, chemokines, and related molecules in CSF and serum in newly diagnosed pwMS. HoweStudies have shown that cytokines play a significant role in the autoimmune nature of MS, with both Th1  and Th17  cell-driven inflammation being linked to the disease –16. In rAs peripheral inflammation is also believed to play a crucial part in MS pathogenesis , as blooThe main aim of our study was to quantify concentrations of 65 cytokines, chemokines, and related molecules in paired serum and CSF samples of newly diagnosed and untreated pwMS. Our secondary study aims subsumed identifying possible links between these concentrations and clinical or MRI progression and their correlation with routine laboratory parameters.22.1In a prospective research project with a nationwide scope in Austria, people diagnosed with multiple sclerosis (pwMS) were recruited at the Clinical Department of Neurology, Medical University of Innsbruck, with the aim of identifying biomarkers that are associated with disease progression, with a specific emphasis on progression into primary or secondary progressive disease course. People newly diagnosed with MS according to the 2017 revised McDonald Criteria , aged 18The study consisted of a single simultaneous collection of CSF and serum samples and regular clinical and radiological investigations. The first was performed during baseline visit for routine diagnostic purpose in people with suspected MS. Furthermore, at baseline, clinical, demographic, and radiological data were assessed. CSF as well as serum samples were stored immediately at -80°C until further use. Study participants underwent regular clinical examinations at least every 6 months.The herein collected parameters included the disability status as indicated by the Expanded Disability Status Scale (EDSS), disease history, including date and type of first symptom, relapse history, and treatment history. Additionally, data of sex, age, and ethnicity of the person were collected at baseline. Relapses were defined as subacute neurological deteriorations typical for demyelinating events occurring in the absence of infections or fever and with a time gap of at least 30 days to the last relapse . We defiRadiological data subsumed number of cerebral and spinal lesions showing hyperintensity in T2-weighted images and number of cerebral and spinal lesions showing contrast enhancement (CE) in T1-weighted images. We defined radiological progression as an increase in T2-hyperintense or in CE lesions in the follow-up MRI scan compared to the baseline MRI scan.2.2The primary aim of our study was to quantify CSF and serum concentrations of a broad panel of cytokines, chemokines, and related molecules in the abovementioned population of pwMS. Secondary study aims were to reveal possible associations of these concentrations and the occurrence of relapses, EDSS progression, or MRI progression as defined above. Furthermore, we aimed to investigate correlations between these concentrations and routine laboratory parameters.2.3Magnetic resonance imaging was performed centrally at the Department of Neuroradiology, Medical University of Innsbruck, Austria, at a field strength of 1.5 or 3 T. Scans with axial slices with a maximum thickness of 5 mm were obtained. The assessment of the above mentioned radiographic characteristics  was performed by raters (AG and PP) experienced in MRI diagnostics and blinded for any further information .2.4Routine laboratory data were assessed at the Neuroimmunology Laboratory, Department of Neurology, Medical University of Innsbruck, Austria, and performed for routine diagnostic purpose. It comprises CSF counts of white blood cells (WBC) and RBC, CSF, and serum albumin, immunoglobulin (Ig) G, A and M in CSF and serum, and oligoclonal IgG bands (OCB), which were analyzed as previously published –26. By d2.5Levels of 65 cytokines, chemokines, and related molecules were measured using commercially available immunoassays  according to the manufacturer’s instructions. Therefore, samples stored at -80°C were thawed on ice and then used immediately. Magnetic beads were washed with 25 µl universal assay buffer, and 25 µl of standards, 25 µl of undiluted serum, or undiluted CSF samples were added and incubated for 120 min at room temperature (RT) on a shaker at 500 rounds per minute (rpm). After washing, 25 µl of a detection antibody mixture was added and incubated for 30 min at RT on a shaker. After washing twice, 50 µl streptavidin-phycoerythrin solution was incubated for 30 min on a shaker at RT and washed afterwards. Then, 120 µl reading buffer was added into each well, and after a 5-min incubation period while shaking at RT fluorescence, intensity was measured on a Luminex MAGPIX instrument . In samples where analytes were not detectable or their concentrations exceeded the highest standard, the values of the lowest or highest standards were used for statistical analyses. A concise list of all measured 65 molecules was published by Bauer et al. ), and all participants gave written informed consent prior to inclusion.2.7For statistical analyses, the software packages GraphPad Prism  and IBM SPSS  were used. Within-group comparisons between CSF and serum samples were calculated using the Wilcoxon signed-rank test for paired samples. P-values were corrected for multiple comparisons using a false discovery rate significance criterion of 1% based on the Benjamini, Krieger, and Yekutieli correction.The PANTHER  classification system with the PANTHER overrepresentation test (released 20221013) and the GO gene ontology database  were useCorrelations were evaluated by Spearman’s rank correlation tests and Spearman’s rho correlation coefficients were classified according to Cohen  as weak The association of CSF:serum ratios of cytokines/chemokines with sex and clinical or MRI progression was analyzed using the Mann–Whitney test. Effect sizes were calculated according to Cohen (1988)  and clasThe statistical power of correlation coefficients was calculated using G*Power 3.1 software for 44 participants and α = 0.05 . Only co3Overall, a total of 44 predominantly female (64%) pwMS with a median age of 31 years were included in the study. At baseline, 40 showed a relapsing–remitting (RR) and four a primary progressive (PP) disease course. The median disease duration, defined as time between first symptom and baseline visit, was 41 days. The vast majority of pwMS (77%) showed a monofocal disease onset. According to the inclusion criteria, no study participant received DMT. Patients showed a “minimal disability” as indicated by a median EDSS score of 1.0. At baseline, in all pwMS, cerebral MRI was performed with a median of 10 hyperintense lesions in T2-weighted images, while in 37 people with a spinal MRI, a median of one T2 hyperintense lesion was revealed. In total, the criteria of “Dissemination in space” (DIS) was fulfilled in 43 people. A total of 26 people showed at least one CE lesion whether in cerebral or in spinal MRI and therefore fulfilled the criteria of “Dissemination in time” (DIT) at baseline.CSF analysis was available in all 44 pwMS with a median WBC count of 7.5/µl, resulting in an elevated WBC in 30 (68.2%) pwMS. The median RBC count was 0, with no CSF sample exceeding an RBC count of 500/µl. The median QAlb was 4.3, and blood–brain-barrier dysfunction as indicated by elevated QAlb was detected in only two (4.6%) pwMS. The median IgG index was 0.84, in 36 people (81.8%) the IgG index was above 0.7, and all participants showed positive OCB.At the median follow-up visit of 25 months after baseline, one individual with a previously diagnosed RR form of MS progressed into a secondary progressive (SP) MS. Eight pwMS experienced a progression in their EDSS score, and 14 experienced at least one relapse event. As a result, 11 pwMS required initiation of a moderately effective DMT and 15 required a highly effective DMT by the last follow-up. 3.13.2Cytokines or chemokines classified as related with peripheral inflammation (increased serum concentrations compared to CSF) had an overrepresentation of chemokines. They were found to at least moderately correlate with many routine laboratory characteristics and clinical parameters as well as with some MRI baseline findings, as shown in When analyzing CSF inflammation (defined as CSF:serum ratio >1), various cytokines and chemokines were associated with routine laboratory characteristics. Notably, negative correlations were found between CSF white blood cell count and several CSF cytokines . Additionally, TWEAK was the only analyte that showed at least moderate associated with an increase in EDSS, while MCP3 was correlated with the occurrence of spinal MRI lesions.3.3With regard to peripheral inflammation, CD40L (R = 0.31↑) was the only protein that showed a positive association with a progressive disease course at follow-up and MDC (R = 0.30↑) was the only molecule associated with an EDSS progression at the last follow-up. Furthermore, higher baseline serum levels of APRIL were predictive of a MRI progression after 1 year (R = 0.40↑).In the CSF, high baseline levels of GROα (R = 0.30↑) were linked with higher relapse activity at the last follow-up. Also, two CSF cytokines, IL-2 and IL8, had a strong impact on MRI progression after 1 year. At baseline, a dissemination in time on MRI was linked to higher CSF levels of IL22 (R = 0.30↑).A higher EDSS score at baseline and a longer disease duration were both predictors for EDSS progression after 1 year, and a higher age at baseline was an additional predictor for a progressive MS disease course. Older age at baseline and more spinal T2 lesions, especially when these are contrast-enhancing lesions, were predictors of MRI progression after 1 year.4In the present study, we characterized the immune profile of pwMS by measuring a broad panel of 65 cytokines, chemokines, and related molecules in CSF and serum. Here, we could demonstrate significantly higher levels of 15 cytokines, chemokines, and related molecules in serum compared to CSF and higher levels of 29 of these proteins vice versa.The pathophysiology of MS is a complex interplay of mechanisms leading to demyelination as well as axonal and astrocytic damage , 37. A kEspecially Th1 cytokines  have been described as important drivers for inflammation in pwMS , 40–42. However, also chemokines trigger inflammation, by supporting lymphocyte, macrophage, and monocyte infiltration in lesions and plaques , 45. TheOn the other hand, also shown by gene ontology and PANTHER classification system analysis, many in serum increased cytokines and chemokines have a role in inflammatory response, immune response, migration of neutrophils, lymphocytes, and granulocytes and interestingly in the ERK1 and ERK2 cascade. ERK1/2 are the final components of the mitogen-activated protein kinase (MAPK) signaling cascade, involved in different cellular functions such as cell proliferation, differentiation, adhesion, migration, and survival . In micrA further interesting observation are significantly higher levels of macrophage and granulocyte colony stimulating factors  in CSF than in serum. Recently, a lively research interest concerning the role of the cytokine GM-CSF has evolved . For insAmong others, APRIL and BAFF are known to be involved in B-cell survival and proliferation. The importance of B-cell activity in the pathogenesis of MS is emphasized by the success of B-cell depleting therapies , 58. As Taking into account our correlation analyses between serum and CSF cytokine and chemokine levels, the positive correlation of, e.g., CD40L and IL-23, has to be pointed out. This may be of special interest, as blood samples are less invasive and easier to collect and therefore could potentially be taken serially. Nevertheless, it has to be pointed out that these correlations are only weak and only for CD40L and IL-23 were they found to be statistically significant. This is also in line with a recent study investigating CSF and serum levels of 36 cytokines and chemokines in MS patients, revealing a significant correlation of CSF and serum levels in only three cases  .In our study, disability worsening determined by EDSS worsening correlated with serum MDC levels, while inflammatory activity detected by relapse activity did so with CSF GROα levels. The macrophage-derived chemokine MDC is a chemokine produced by monocyte-derived dendritic cells and macrophages and is upregulated by the Th2-related cytokine IL-4 or IL-5 . This chIn our study, a progressive course of MS was found to be associated with higher levels of serum CD40L but not with cytokine markers of CSF inflammation. Although in our study only very few participants had a progressive course of MS, our finding is consistent with those of another study which observed higher serum soluble CD40L levels in people with active MS compared to those with an inactive disease . CD40L iMRI data provided in our cohort has to be assessed with caution, as exclusively patients at first diagnosis have been included who usually presented due to clinical episodes of high disease activity. Probably, this may have also influenced MRI scans, especially in terms of presence and number of CEL. For instance, in our cohort only MDC and VEGFA correlated with cerebral T2 lesions while MCP3 did so in terms of spinal T2 and CE lesions, while IL-22 correlated with fulfilling the radiological criteria of dissemination in time at baseline. Prima vista, this seems to contradict recent findings of higher levels of IL-12p40, TNFα, TNFβ, and IL-10 in patients with lesional inflammatory activity defined as presence of CEL or clinical relapses . NeverthThere are some limitations of this study. For the broad panel of 65 measured chemokines and cytokines, our study had probably a too low number of participants and was therefore underpowered. Consequently, results that were not significant in statistical tests cannot be interpreted in contrast to those that show significant differences between groups. Furthermore, to avoid random findings due to the measurement of such a high number of proteins, we corrected for multiple testing. Moreover, our radiological follow-up period was not as long as the clinical follow-up duration and both were relatively short. A further limitation of our study may be seen in the small number of people with a primary progressive disease course at the last follow-up. Indeed, it has been demonstrated by others that higher inflammatory cytokine profiles were described in people with RRMS compared to those with a progressive disease course, indicating a higher level of inflammatory activity , 80. The5In this study, we provide data of concentrations and distribution of 65 cytokines, chemokines, and related molecules in CSF and serum of people suffering from MS. These findings may also provide an important basis for the selection of cytokines or chemokines for further studies.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethical committee of the Medical University of Innsbruck ; ethical approval number: AN3041 257/4.8 393/5.4 (4319a. The patients/participants provided their written informed consent to participate in this study.KB: investigation, data curation, formal analysis, writing—original draft preparation; AB: investigation, data curation, formal analysis, writing—original draft preparation; DR: investigation, data curation, writing—reviewing and editing; MA: writing—reviewing and editing; RB: writing—reviewing and editing; AZ: writing—reviewing and editing; ML: writing—reviewing and editing; LH: writing—reviewing and editing; AG: investigation, writing—reviewing and editing; PP: investigation, writing—reviewing and editing; SW: writing—reviewing and editing; TB: writing—reviewing and editing; FP: writing—reviewing and editing; FD: writing—reviewing and editing; HH: writing—reviewing and editing; MR: conceptualization, supervision, visualization, data curation, formal analysis, writing—original draft preparation. All authors contributed to the article and approved the submitted version.