The activity of cerebrospinal fluid (CSF) β-site APP cleaving enzyme (BACE) is a potential diagnostic biomarker for Alzheimer disease (AD).P values of all SNPs for CSF BACE were adjusted for multiple comparisons.A total of 340 non-Hispanic Caucasian participants from the Alzheimer’s Disease Neuroimaging Initiative cohort (ADNI) database were included in this study with quality-controlled CSF BACE and genotype data. Association of CSF BACE with the genetic variants of single nucleotide polymorphisms (SNPs) was assessed using PLINK under the additive genetic model. The ATP6V1H gene reached genome-wide significance for associations with CSF BACE (P = 4.88 × 10− 9). The minor allele (G) of rs1481950 was associated with higher CSF BACE activity. Although seven SNPs in SNX31, RORA, CDH23, RGS20, LRRC4C, MAPK6PS1 and LOC105378355 did not reach genome-wide significance (P < 10− 8), they were identified as suggestive loci (P < 10− 5).One SNP (rs1481950) in the ATP6V1H influencing human CSF BACE activity, which indicated that ATP6V1H gene may play some roles in the pathogenesis of neurodegenerative diseases such as AD.This study identified rs1481950 within The online version of this article (10.1186/s12881-018-0603-z) contains supplementary material, which is available to authorized users. Amyloid plaque which is composed primarily of amyloid-beta (Aβ) has been reported as an important pathological change of some neurodegenerative diseases including Alzheimer’s disease (AD) . Aβ is gadni.loni.usc.edu) cohort in order to discover genetic factors involved in BACE protein.The recent development of a sensitive assay for BACE in CSF makes it feasible to study the association between CSF BACE enzymatic activity and AD . Moreovewww.loni.ucla.edu/ADNI). The ADNI was launched in 2003 as a public-private partnership, led by Principal Investigator Michael W. Weiner, MD, VA Medical Center and University of California–San Francisco. ADNI includes more than 800 participants ranging in age from 55 to 90. All these individuals were recruited from over 50 sites across the United States and Canada, including approximately 200 healthy controls (HC), 400 patients diagnosed with mild cognitive impairment (MCI) and 200 patients diagnosed with early AD. The AD patients were followed for 2 years and others were followed for 3 years. Structural 1.5-T magnetic resonance imaging (MRI) collected the full sample. PIB and FDG positron emission tomography (PET) imaging of a subset, some other biological markers, and performance on neuropsychological or clinical assessments were collected at baseline and at follow-up visits in 6- to 12-month intervals. The CSF BACE and genome-wide genotyping in this study were respectively available on approximately half of the cohort and the full ADNI sample. Further information about ADNI can be found in previous publications and at www.adni-info.org [Data used in this study were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database , MCI group and AD group participants with available baseline CSF BACE samples and genotype data. To reduce the likelihood of population stratification effects in the GWAS, all the participants were restricted to non-Hispanic Caucasians. We also tested a multidimensional scaling (MDS) plot and found some genetic outliers  baseline of CSF BACE measures were calculated by observers blind to diagnostic information and subjects who had a value greater or smaller than 3-fold SD from the mean value were regarded as extreme outliers and removed from the analysis.Single nucleotide polymorphism (SNP) genotyping for more than 620,000 target SNPs was completed on all ADNI participants using the following protocol. A total of 7 mL blood of each participant was taken in EDTA containing Vacutainer tubes and genomic DNA was extracted using the QIAamp DNA Blood Maxi Kit  following the manufacturer’s protocol. EBV-transformed B lymphoblastoid cell lines were established. Genomic DNA samples were analyzed using the Human 610-Quad BeadChip  according to the manufacturer’s protocols .http://pngu.mgh.harvard.edu/purcell/plink/), release v 1.07,15 as described previously. Stringent QC assessment followed these criteria: minimum call rate for SNPs and individuals > 95%, minimum minor allele frequencies (MAF) > 0.05, Hardy-Weinberg equilibrium test P > 0.001. The restriction to SNPs with a MAF greater than 5% served to reduce the likelihood of false-positive results in the context of modest sample size to enhance statistical power [Stringent QC assessment was performed using the PLINK software package .To find the association of CSF BACE with the genetic polymorphism, a separate GWAS was performed using PLINK software under an additive genetic model. The thresholds of ectively . The anaP = 0.32470, MCI P = 0.26220, HC P = 0.82720, Total P = 0.81770) among the three diagnostic groups. For age , the results showed a linear correlation between age and CSF BACE activity in HC groups (P = 0.0063). As expected, the ε4 allele of APOE gene substantially increased the risk of AD with dose effects. Furthermore, CSF BACE enzymatic activity did not have statistical difference  among three diagnostic groups.The demographic information on the final set of 340 non-Hispanic Caucasian participants and summary statistics on the CSF BACE are showed in Table P < 10− 8. As shown in the Fig. ATP6V1H gene reached genome-wide significance (P = 4.88 × 10− 9). Moreover, all SNPs whose P values reached the level of P < 10− 5 for CSF BACE enzymatic activity and their annotation information are listed in Table P < 10− 5 as suggestive SNPs. Seven independent potential candidates (uncorrected P < 10− 5) were included . The Quantile-Quantile plot (QQ plot) is shown in Fig. P < 0.01. As the Fig. After adjusting for age, gender and APOEε4 status, one SNP  was identified associated with CSF BACE enzymatic activity at genome-wide significance levels of P = 4.88 × 10− 9, Beta: 22.48, 95% CI: 15.22 ~ 29.75). Further analysis found this linear association between genotype and CSF BACE activity in both AD  and MCI groups . However, in HC group, results did not show significant differences in CSF BACE activity among three genotype subgroups in both ANOVA (P = 0.06370) and multiple linear regression analysis .All these eight SNPs were analyzed further to examine possible interactions among baseline diagnosis and genotypes on associated CSF BACE enzymatic activity by a multiple linear regression test considering age, gender and APOEε4 status as covariates. For the top SNP (rs1481950), as indicated in Fig. P = 0.04389) and parahippocampal gyrus . Similarly to this result, in AD patients, results also showed that mutations of rs1481950 had significant correlations with the atrophy of middle temporal gyrus  and parahippocampal gyrus . In MCI patients, results showed that mutations of rs1481950 had correlations with the atrophy of hippocampus , entorhinal cortex  and parahippocampal gyrus .This study also tested relations between rs1481950 and AD-related phenotypes. In all samples, results showed that mutations of rs1481950 had significant correlations with the atrophy of middle temporal gyrus . It is worth noting that this statistical differences between gene polymorphism of ATP6V1H and CSF BACE activity existed in both AD (P = 0.0027) and MCI groups (P = 0.0003). These results indicated that the rs1481950 risk variant (G) in ATP6V1H might significantly increase the CSF BACE activity especially in the AD and MCI individuals.In this multicenter study, we identified a genome-wide significant association of a SNP (rs1481950) in the gene ATP6V1H. However, the roles of intronic regions which have been ignored all the time have attracted the attention of scientists. And more and more studies have proved that intronic region may play some important roles in controling the initiation and termination of transcription. Moreover, common molecular mechanisms for an intronic SNP to alter mRNA levels are to affect transcription, RNA elongation, splicing, or maturation [Rs1481950 is in an intronic region. Current studies haven’t found the exact mechanisms of impairing the expression of turation –22.+ transporting, lysosomal 50/57 kDa, V1 subunit H gene (ATP6V1H) at Chr8q11.2 encodes for the V1H subunit of vacuolar ATPase (V-ATPase) [ATP6V1H gene was mainly studied and discussed about its roles in diabetes in previous researches and the data showed that the down-regulation of its gene expression correlates with the presence of type-2 diabetes [ATP6V1H gene and AD, some studies about encoded protein and metabolic process of BACE indicate that mutations of ATP6V1H gene may contribute to the increased BACE activity. V-ATPase belongs to the rotary ATPase family and is a multiprotein membrane complex. The most important function of V-ATPase is to acidify intracellular compartments by using the energy gathered from ATP hydrolysis to pump protons [The ATPase, H-ATPase) , 24. ATPdiabetes . Though  protons . One of  protons . These s protons , 28. To The results also showed that mutations of rs1481950 had significant correlations with the atrophy of AD-related encephalic regions including middle temporal gyrus, parahippocampal gyrus, hippocampus and entorhinal cortex. Moreover, the results showed that mutations of rs1481950 had significant correlations with the CSF p-tau. These results indicated that mutations of rs1481950 may relate to AD and may influence the volume of AD-related encephalic regions by changing the metabolism of some AD-related proteins such as tau.In this study, we did not find any significant differences in CSF BACE activity among the three diagnostic groups. This aspect of our research is in line with two previous studies including an ADNI cohort study , 30. SomThere are several potential limitations of this study. First, the ADNI-1 sample was limited in sample size when CSF BACE data, different genotypes and diagnosis subgroups were taken into consideration, which makes effective sample size small for some tests. Thus, it will still be necessary to replicate these findings in a larger dataset. Second, recent studies also indicated that except CSF BACE, plasmatic BACE was also associated with AD . The assATP6V1H gene with the P value reaching genome-wide significance and seven suggestive SNPs with the P value lower than 10− 5. Rs1481950 risk variant (G) in ATP6V1H may increase the CSF BACE activity. Seven genes  were regarded as candidate genes. These results provide clues to some novel pathogenic genes associated with some BACE related diseases, such as AD. The in-depth discussion and study of these associations can help us to find the exact mechanisms of AD, which may indicate some new diagnostic methods and therapeutic directions.In summary, after a separate GWAS of CSF BACE, we found a top SNP (rs1481950) in Additional file 1:The MDS plot of samples. (TIF 8989 kb)Additional file 2:The correlation between gender and CSF BACE activity. (TIF 2480 kb)Additional file 3:The correlation between age and CSF BACE activity. (TIF 2436 kb)


The cerebrospinal fluid analysis has been employed for supporting multiple sclerosis diagnosis and ruling out the differential diagnoses. The most classical findings reflect the inflammatory nature of the disease, including mild pleocytosis, mild protein increase, intrathecal synthesis of immunoglobulin G, and, most typically, the presence of oligoclonal bands. In recent years, new biomarkers have emerged in the context of multiple sclerosis. The search for new biomarkers reflect the need of a better evaluation of disease activity, disease progression, and treatment efficiency. A more refined evaluation of disease and therapy status can contribute to better therapeutic choices, particularly in escalation of therapies. This is very relevant taking into account the availability of a greater number of drugs for multiple sclerosis treatment in recent years. In this review, we critically evaluate the current literature regarding the most important cerebrospinal fluid biomarkers in multiple sclerosis. The determination of biomarkers levels, such as chemokine ligand 13, fetuin A, and mainly light neurofilament has shown promising results in the evaluation of this disease, providing information that along with clinical and neuroimaging data may contribute to better therapeutic decisions. Relapsing-remitting multiple sclerosis (RRMS) in the most common form of the disease, in which symptoms resolve spontaneously. After tissue damage accumulates over years, patients often evolve to secondary progressive multiple sclerosis (SPMS), in which neurologic 3The diagnostic criteria of MS are based on clinical and paraclinical assessments emphasizing the need to demonstrate lesions in distinct central nervous system locations (dissemination in space), occurring in separate times (dissemination in time) and to exclude alternative diagnoses. Lesions may be detected either by clinical examination and magnetic resonance imaging (MRI).4 In the late 1950s, quantitative methods showing an elevated gamma globulin in CSF samples, but not in serum, were introduced.6 The development of electrophoretic separation of CSF proteins allowed the demonstration of different IgG fractions in the CSF of MS patients. These fractions were first shown in 1959, and were called oligoclonal bands.7 The most typical pattern found in MS shows at least two bands in CSF ad none in serum samples.8The cerebrospinal fluid (CSF) has been studied in for a very long time. The Lange colloidal gold curve, first studied in the diagnosis of syphilis in 1942, showed abnormal protein patterns in patients with MS; however, with low sensibility and specificity.8 For some decades, the usefulness of CSF analysis in MS remained restricted to diagnostic evaluation; however, in recent years, many new CSF biomarkers have been studied in MS. These biomarkers are CSF proteins whose CSF concentrations were correlated with clinical and therapeutic parameters in MS. The aim of this study is to critically review the existing literature on the new CSF biomarkers in MS.The sensitivity for detecting oligoclonal bands in MS patients with isoelectric focusing method is approximately 95% and the specificity is greater than 86%. The quantitative IgG index positivity is approximately 75% among oligoclonal bands positive cases.A critical review of the literature was conducted on MS and new CSF biomarkers. The search strategy used included the words “multiple sclerosis”, “cerebrospinal fluid”, and “biomarkers” to search PubMed/MEDLINE database, from January 1990 to March 2016.Each study found was critically analyzed, taking into account the articles referring to biomarkers with greatest potential for incorporation into clinical practice. The study findings were classified as three main axes: CSF biomarkers of disease activity, CSF biomarkers of clinical progression, and CSF biomarkers of therapeutic response.9 Reliable biomarkers reflecting subclinical disease are still lacking.The assessment of disease activity is useful to guide therapeutic interventions. At present, this assessment is based on clinical relapses, disability progression, and the appearance of new lesions in magnetic MRI.10Several CSF cytokines and chemokines have been studied as potential markers of MS activity. These molecules participate in the inflammatory response as modulators of cell recruitment and migration to the sites of inflammation. An increase in pro-inflammatory and a decrease in anti-inflammatory cytokines and chemokines occur during MS exacerbations.12 Interleukin 6 (IL-6)is another pro-inflammatory cytokine, and some studies showed a mild elevation of CSF IL-6 during MS exacerbations.14 Chemokine ligand 13 (CXCL13) is a B cell chemoattractant that participates in the formation of B cell follicles, which are important in MS pathophysiology. CXCL13 is increased in the CSF of patients with several MS forms and its levels seem to correlate with disease activity.16Cerebrospinal fluid tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine. Contradictory results with this biomarker were found in MS. Some investigators described an association between TNF CSF levels and MS activity, but this association was not reproduced by other authors.Cerebrospinal fluid CXCL13 appears to be a promising CSF biomarker in the evaluation of MS activity, although the number of studies is still small and the results are not robust enough to propose its use in routine clinical practice.17 This glycoprotein is increased in patients with SPMS and also in the CSF of patients with active RRMS, suggesting that elevated fetuin A is potentially useful as a marker of MS activity.19Fetuin A  is a glycoprotein found by proteomic analysis of CSF. It was associated with increased risk of conversion from clinically isolated syndrome to RRMS.20 In MS, the CSF NfL concentrations increase after relapses, reaching their peak at 2 weeks after the beginning of symptoms, remaining elevated for at least 15 weeks after an exacerbation.22 Cerebrospinal fluid NfL levels are associated with the presence of gadolinium enhancing MRI lesions.23 These data indicates that increased CSF NfL levels correlate with clinical and MRI parameters, being a potential biomarker of MS clinical activity.Neurofilaments (Nf) are the most important component of the axonal cytoskeleton in neurons and consist of three chains: light (NfL) with 68-70kDa, intermediate (NfI) with 145-160kDa, and heavy (NfH) with 200-220kDa. They provide structural support to neurons and regulate axon diameter. Neurofilaments are released in significant quantity following axonal damage or neuronal degeneration. In these situations, Nf is released into the interstitial fluid and into CSF.25Predicting MS clinical progression remains a great challenge. The difficulty reflects a great heterogeneity of the disease. Axonal degeneration accumulates over the course of the disease and is the primary cause of permanent neurological dysfunction in MS patients. The evaluation of axonal degeneration is based mainly on clinical parameters and MRI findings; however, these parameters are not highly precise. More effective biomarkers of axonal degeneration are still required.26 One study showed that CXCL13 continuously increases in progressive MS suggesting that CXCL13 correlates with clinical progression.27 However, more prospective studies are still necessary to assess the role of CXCL13 in MS prognostic evaluation.The presence of meningeal B-cell follicles is thought to play an important role in clinical progression of MS.28 Some studies showed that higher CSF NfL levels were associated with worse long term disability progression. On the contrary, other studies failed to show a correlation between CSF NfL, disease progression, and the risk of conversion to SPMS.31 Cerebrospinal fluid NfL appears to be a potential biomarker for neuronal damage and clinical progression in MS; nonetheless, more studies are still needed in this area.Cerebrospinal fluid levels reflect neuronal injury and are potentially correlated with axonal degeneration. High CSF NfL levels were positively correlated with higher MS severity score at long-term evolution. Also, higher NfL levels were associated with higher risk of conversion from RRMS to SPMS.35Markers of glial damage were also tested in the CSF of MS patients. Glial fibrillary acidic protein (GFAP) is a component of astrocytes filaments. Increased GFAP levels in CSF reflect astrocyte damage. Multiple sclerosis patients have increased CSF GFAP levels when compared to controls. Multiple sclerosis patients with worse ambulation or more severe disability have higher CSF GFAP levels than less disabled patients and controls, suggesting that CSF GFAP may be a biomarker of clinical progression and prognosis.More prospective studies correlating the levels of biomarkers of neuronal and glial degeneration with clinical and neuroimaging data are still necessary to more precisely establish their role in predicting progression of MS.36 Although a potential marker of acute demyelination, MBP does not seem to correlate with MS clinical progression.Myelin basic protein (MBP) is a component of central nervous system myelin. Myelin basic protein levels in CSF were previously tested as predictor of clinical progression. Cerebrospinal fluid MBP levels increase in acute demyelination; however, high MBP levels did not rise as the disease progressed.37Several MS therapeutic options have emerged in recent years. Many algorithms have been proposed in order to optimize the use of these drugs, most of them based on the occurrence of clinical relapses, clinical progression, and the appearance of new lesions in MRI. Some new parameters, such as grey matter disease activity and brain atrophy, were also evaluated. However, the parameters to assess the effectiveness of treatment are still imprecise. The contribution of measuring some CSF biomarkers in the assessment of therapeutic efficacy has been studied.38 Cerebrospinal fluid NfL levels were also tested in MS patients treated with natalizumab, with marked decrease during treatment.31 Cerebrospinal fluid NfL levels were also studied in patients treated with fingolimod, and reduced levels were observed after the treatment.39 Patients treated with fingolimod with no reduction in CSF NfL levels had clinical and MRI deterioration. These findings suggest that at least some MS treatments are associated with a progressive reduction in CSF NfL levels, and that a persistent increase in the levels of CSF NfL may indicate a poor therapeutic response. Further evidence is still needed but the existing data support the use of this biomarker in MS clinical trials.The CSF NfL levels were significantly reduced after immunosuppressive treatment with mitoxantrone.40 More studies with this and other drugs are required to assess the role of this biomarker in MS therapeutic response evaluation.The levels of CSF fetuin A were reduced in patients treated with natalizumab for 1 year.40 More studies assessing of the role of this biomarker during natalizumab and other MS treatments are still required.Osteopontin is a matrix protein that works as a pro-inflammatory cytokine. Cerebrospinal fluid osteopontin levels were reduced in patients during natalizumab treatment but it is unknown if these levels are predictive of natalizumab response.42 Other immunossupressive treatments are also able to reduce CXCL13 CSF levels.38 Future studies may address if this biomarker predicts therapeutic response, particularly in B-cell-targeting therapies candidates.43CXCL13 correlates with B-cell activity in MS and it is therefore a good biomarker candidate to evaluate the efficacy of B-cell-targeting therapies, such as rituximab and ocrelizumab. It was shown that CSF CXCL13 was reduced during rituximab therapy; however, its baseline level was not able to predict rituximab response.The recent studies with new biomarkers indicate that CSF analysis in MS may go beyond diagnosis. Cerebrospinal fluid new biomarkers may contribute in the assessment of disease activity, prognostic evaluation, and therapeutic monitoring of MS.28 Therefore, it is possible that in the near future these new CSF biomarkers will contribute to the clinical monitoring of MS patients.The existing data suggest that CXCL13, fetuin A, and NfL CSF levels are associated with disease activity. CXCL13, NfL, and GFAP seem to be correlate with prognosis and clinical progression. Neurofilaments intermediate, fetuin A, CXCL13, and osteopontin were associated with therapeutic response, suggesting they can contribute in the assessment of therapy effectiveness.Few points need to be more precisely established. First, it is not known yet how often these markers should be measured. Second, there is not yet a standard clinical interpretation of the results. Finally, a greater standardization in performing these techniques in different laboratories is still required. All these issues should be addressed in future clinical studies in this area.The better knowledge of new CSF biomarkers levels may refine the assessment of MS patients, supporting clinical decisions that may contribute to more favorable outcomes.A number of candidate cerebrospinal fluid biomarkers for multiple sclerosis have emerged in recent years. These biomarkers are cerebrospinal fluid proteins that have been shown to correlate with disease activity, disease progression, and treatment response. The future application of such biomarkers measures in clinical practice may provide a more refined and individualized evaluation of the disease, helping optimizing therapeutic decision making.


Gekko geckos are capable to crawl on the steep even on upside-down surfaces. Such movement, especially at great altitude, puts them at high risks of incidentally dropping down and inevitable body or head impactions, though they may trigger air-righting reaction (ARR) to attenuate the landing shocks. However, the air-righting ability (ARA) in Gekko geckos is not fully developed. The implementation of ARR in some geckos is quite slow; and for those without tails, the ARR is even unobservable. Since ARA is compromised in Gekko geckos, there must be some other mechanisms responsible for protecting them from head injuries during falls. In this study, we looked into a Gekko gecko's brain to study its internal environment and structure, using the magnetic resonance imaging (MRI) technique. The results showed that the brain parenchyma was fully surrounded by the cerebrospinal fluid (CSF) in the skull. A succulent characteristic was presented, which meant the intracalvarium was significantly occupied by the CSF, up to 45% in volume. Then a simplified three-dimensional finite element model was built, and a dynamic simulation was conducted to evaluate the mechanical property of this succulent characteristic during the head impactions. These implied the succulent characteristic may play certain roles on the self-protection in case of head impaction, which is adaptable to the Gekko gecko's locomotion and behavior. Gekko geckos turn up by rotating their tail [Gekko geckos, because the relevant air-righting reflex did not mature, developmentally speaking, into a complete central program. The published data [Gekko geckos may possess certain characteristics to prevent themselves especially their head from injury caused by impacting the ground while falling down. To disclose the potential mechanism underlying the self-protection in head impaction, it is necessary to look into the intracalvarium structure and material of the animal's head.Many animals in nature are able to crawl, climb, or run on different inclined surfaces, such as walls, ceilings, branches, and leaves, at a certain height above the earth. Falling down, as a common experience, is somehow unavoidable. To avoid the body being injured when falling and impacting with the ground, animals have developed various abilities, such as air-righting abilities (ARA). Cats try to make their feet to attach on the ground at first to reduce the impact forces acting on the body by turning up the body upside-down through the vertebra and tail –4. Gekkoeir tail . Preventeir tail , 7, catseir tail –4, rats eir tail , 9, rabbeir tail , frogs [eir tail , and geceir tail . Howevereir tail . ActuallGekko gecko's head has been rarely investigated, since its significance of antishock characteristic is much lower than the former. The woodpecker's head can stand high-frequency shocks that are introduced from its drumming beak during the daily forage, while the gecko's head is only in the risk of one sudden head shock caused by an incident dropping. The underlying mechanism of head protection should be different. Here, we took a close look at the intracranial structures by the magnetic resonance imaging (MRI) technique. Then a simplified mathematical model was built to qualitatively evaluate the corresponding mechanical property.Not like a woodpecker's head, which has been studied thoroughly for decades –15, a GeGekko gecko lizards were brought from Nanning, Guangxi Province, China, and habituated to the study colony for two months before the experiments. The mean temperature and relative humidity were 25°C and 65%, respectively, which were close to the values for the natural ambiance of Gekko geckos. Adult Gekko geckos weighted 40–70 g were selected for the MRI study.The The entire study was carried out in accordance with the Guide of Laboratory Animal Management Ordinance of China and approved by the Jiangsu Association for Laboratory Animal Science .The intracalvarium morphology was investigated by two ways, the qualitative observation after the surgical anatomy and the quantitative measurement using MRI.After the surgical anatomy, we found that the gecko's brain parenchyma  is surroFor MRI investigation, the animal was anaesthetized by the intraperitoneal injection of 0.4% sodium pentobarbital in a 0.75% NaCl solution. A dose of 0.75 ml/100 g body weight was administered. After the pain reflex had disappeared, the gecko was fixed to a custom-designed fixture . It is not difficult to distinguish the brain parenchyma and the CSF by gray level of the MRI image. Then, the surface integral and the volume integral were conducted for the brain parenchyma and the CSF, respectively.Based on the MRI image sequence, the distribution and the volume of the CSF in the skull were evaluated with the help of the open source software ImageJ  of the CSF [The gecko brain was simplified into concentric spheres filled with liquid interval . The parrenchyma , 17 is q the CSF  is arounThe plan model was introduced to present the mechanics of the head impact cushioning. The contact target surface (ground) is defined as a rigid body in the model. The elasticity modulus and passion ratio of skull and brain parenchyma are set to be 15 GPa and 0.3 GPa, respectively. The Young's modulus for the CSF was set at 2.436 GPa, similar processing as the biomechanical study on the hemolymph in insects .The finite element model was built and simulated using the ABAQUS/Explicit , which is always chosen to solve nonlinear dynamical problems such as impact and explosion. Considering the symmetrical structure, the finite element model was built as a half-plane strain model. The initial contact between the ground and the skull, between the CSF and the skull, and the brain and the CSF were established using the contact-pair tool in ABAQUS/Explicit. All the structures were meshed into a quadrilateral, and the CPE4R elements were employed throughout. The model was built based on the fundamental principles of continuum mechanics, and the simplification on the fluid of CSF also had precedents , which gThe simulation showed the pressure distribution around the brain was varying during the interaction between the head and the ground. When the two objects got stuck, strain built up. Along the vertical direction, the compressive stress was increasing that caused positive pressure on the brain, while along the lateral direction, the tensile stress was increasing that caused negative pressure on the brain .The CSF accounts for around 45% of a gecko brain. Different proportion of the fluid may affect the pressure limits caused by falling and head impact. Additionally, the height of falling could be another factor. Here, we simulated two proportions of the CSF, 45% and 22%, given two different falling heights, 1 m and 0.5 m, respectively. The comparison is shown in The MRI investigation has disclosed the succulent characteristic of a gecko's head. The intracalvarium of the head is full of the CSF, up to 45% in volume. Animal skulls contain a space between the brain and the skull's vascular tissue, called a subarachnoid cavity. The cavity houses the CSF. It is said that CSF can only provide cushioning from minor bumps and jostling. In the instances of strong vibrations or blows, CSF will allow excessive movement of the brain, potentially resulting in bruising and concussions. One factor of the antishock mechanism in the woodpecker is that it has relatively little CSF , 20, theAccording to the comparison in Gekko geckos qualitatively from several aspects. First, the fluid spreads the impulse across a wide area, allowing the material to absorb more of the impact. Second, the hydrodynamic drag gradually slows down the motion of the brain parenchyma caused by inertia after head impact. Thirdly, the good liquidity of the CSF attenuates the positive pressure in the intracalvarium. All those imply that the succulent characteristics of a gecko's head may play an important role in the self-protection in the head impaction, which is worth further studying and supplements to the behavioral and bionic application studies in Gekko geckos [The finite element model and simulation developed in here is quite simple, which need further investigation using more fidelity models, such as considering the complex cortical bone property , 25. Howo geckos .ARA is one of the important abilities for animals who move on high. It has been shown that ARA probably starts developmentally as a reflex and within days/weeks matures into CPG . From anGekko gecko's brain qualitatively and quantitatively, in order to understand their mechanism underlying the self-protection in head impaction. This study was also necessary in order to discover possible alternative mechanisms, besides the air-righting abilities, responsible for protecting animals moving on high from head injuries in falling. The succulent characteristic of a Gekko gecko's brain was disclosed. By means of surgical anatomy, it was shown that the Gekko gecko's brain parenchyma was fully surrounded by the CSF, while by means of the MRI techniques, it was shown that the intracalvarium was significantly occupied by the CSF, up to 45% in volume. The three-dimensional finite element model and its simulation on the impactions due to falling from different heights showed that the succulent characteristic contributed to the positive pressure decreasing during the head impaction but also to the negative pressure increasing. The former is beneficial to the self-protection in head impactions while the latter is not. Though the decreased positive pressure showed clear advantages over the increased negative pressure when falling from great height, at the point of view of the change rate in pressures, it needs further detail and elegant comparative studies to draw comprehensive conclusions. To our certain knowledge, to date, there has been no systematic interspecific or intraspecific comparative studies on how the CSF affects the mechanical property of the head and the consequence in self-protection. As one step further of this research, a fidelity finite element modeling study and physical model validation involving the morphology of the brain, the CSF and the skull, and their mechanical properties are demanded.This study investigated the internal environment and structure of a


Allobates femoralis during tadpole transport and subsequent homing. To elicit exploration, we reduced resource availability experimentally by simulating desiccated deposition sites. We found that tadpole transport is strongly directed towards known deposition sites and that frogs take similar direct paths when returning to their home territory. Frogs move faster during tadpole transport than when homing after the deposition, which probably reflects different risks and costs during these two movement phases. We found no evidence for exploration, neither during transport nor homing, and independent of the availability of deposition sites. We suggest that prospecting during tadpole transport is too risky for the transported offspring as well as for the transporting male. Relying on spatial memory of multiple previously discovered pools appears to be the predominant and successful strategy for the exploitation of reproductive resources in A. femoralis. Our study provides for the first time a detailed description of poison frog movement patterns during tadpole transport and corroborates recent findings on the significance of spatial memory in poison frogs. When these frogs explore and discover new reproductive resources remains unknown.Animals relying on uncertain, ephemeral and patchy resources have to regularly update their information about profitable sites. For many tropical amphibians, widespread, scattered breeding pools constitute such fluctuating resources. Among tropical amphibians, poison frogs (Dendrobatidae) exhibit some of the most complex spatial and parental behaviors—including territoriality and tadpole transport from terrestrial clutches to ephemeral aquatic deposition sites. Recent studies have revealed that poison frogs rely on spatial memory to successfully navigate through their environment. This raises the question of when and how these frogs gain information about the area and suitable reproductive resources. To investigate the spatial patterns of pool use and to reveal potential explorative behavior, we used telemetry to follow males of the territorial dendrobatid frog  In a dynamic environment resource availability changes in time and space which has major influences on animal movement decisions . AnimalsMost amphibians depend on aquatic sites for breeding, making this taxon particularly suitable for investigating how animals deal with varying availability, stability and distribution of resources. Amphibians exhibit a great diversity of reproductive strategies ranging from explosive breeders that gather at large ponds for synchronized spawning, to prolonged breeders with terrestrial clutches that use small widespread pools for tadpole development . TropicaAmeerega trivittata and A. femoralis  exhibit a variety of complex spatial behaviors such as territoriality, tadpole transport, and offspring provisioning . The comemoralis ; Ranitomriabilis ; R. retiticulata ; Colostenamansis ; Dendrob auratus ; D. leuctrionica ; for revtrionica . When trtrionica . Recent  auratus ) and relemoralis ) and to emoralis . Furtheremoralis . Togethe auratus ) or prio pumilio ). HoweveAllobates femoralis is a small territorial poison frog with predominantly male tadpole transport  common throughout Amazonia and the Guiana Shield  which thield  . All nec in 2012  that wer in 2012 . Occasio in 2012 .To determine male territories, we continuously scanned the area for calling males during our study. All frogs were caught in transparent plastic bags, photographed and individually identified by their unique ventral coloration pattern  using thA. femoralis clutches are difficult to find and the timing when the male picks up the tadpoles is variable , we focused our search effort on finding frogs already transporting tadpoles, mostly by searching around known deposition sites. Sampling was done every day between 07:00 and 13:00 h as tadpole transport mainly occurs in the morning , relocating each individual and recording their position every 30–60 min. We followed each frog until all tadpoles had been deposited and the male had returned to his territory, where the tag was removed. We assumed that the frogs had returned when they approached locations at which they had previously displayed territorial behavior. During tracking we tried to minimize disturbance by carefully approaching the signal source while searching with the transceiver until the frog was visually spotted or the origin of the signal could be narrowed down to less than 1 m. We approached individual frogs from different directions so as to not influence or bias their movement in any direction. Occasional disturbances during tracking only influenced our measurements of the directionality of long distance movements minimally, because A. femoralis responds to disturbance by immediately hiding in the leaf litter rather than fleeing over longer distances . We caught tagged individuals when they had not moved for more than a day to check for possible issues such as skin injuries—in a single case we immediately removed the tag. In one case without movement for more than two days but without apparent injuries we also removed the tag to minimize any potential long-term effects on behavior.Before the release, we equipped transporting males with a transponder attached to a waistband . The entemoralis . We fittemoralis . Both paWe handled and visualized spatial data in the GIS software ArcGIS10 (ESRI) after projecting , and all analyses were performed in the statistical software R version 3.2.0 . We spliWe created a generalized linear mixed model (GLMM) to test the effects of tadpole presence (TT vs. HT) on the movement speed of frogs . As response variable, we used the average speed per TT and HT, as explanatory variable “tadpoles present” (yes/no), and individual frog ID as random factor to account for repeated trajectories of the same individuals.A. femoralis, with tadpole transport happening predominately in the morning, low activity around noon, high calling activity during the afternoon, and high calling activity and most female-male interactions  in the evening . For each tadpole carrier, we recorded all potential tadpole deposition sites visited , and whether tadpole deposition occurred at the available pools or not. To test whether frogs explored further pools after the complete deposition of their offspring, we recorded for each location whether there were tadpoles still present on their back or not. If frogs moved to further pools without tadpoles on their back, we considered this to be exploratory behavior. In addition, we calculated the average number of deposition sites the frogs visited  per tadpole transport. We excluded trajectories with only one final deposition site and no detailed TT.To investigate whether frogs explored the surrounding area by taking additional detours, we estimated the precision of the frogs’ orientation towards the upcoming pools during TTs, and towards the territory during the HTs. To estimate navigation precision, we calculated three different parameters: (1) the straightness coefficient (SC) of the trajectory, (2) the average angular deviation between the ideal orientation angle and consecutive tracking locations, and (3) the average normal distance of tracking locations from the straight-line path. The SC is defined as the ratio between the straight-line distance to the respective goal and the actual path distance. SC ranges from 0 to 1, with 1 indicating a perfectly straight trajectory. The angular deviations were measured as the absolute angular difference between the ideal direction  and the actual direction of the individual frog’s movement (angle from each tracking location to the next one). For the distance of the frog’s movement from the straight-line path, we calculated the perpendicular deviation of each tracking location from the straight path. While the SC takes into account the entire trajectory at once, the average angular deviations describe the movement decisions from location to location, and the distance from the straight-line path assesses the frog’s position in relation to the straight line for every location. We calculated the mean angular deviation and distance from the straight-line path by averaging all values per TTs and HTs in order to have three precision measurements per TTs and HTs.We tested for significant goal-directed orientation using absolute angles calculated with the “as.ltraj” function from the package “adehabitatLT”  per TTs In addition, we tested whether the precision during TTs differed (for example resulting from olfactory cues from the water), when tadpole carriers approached an available deposition site  or an unavailable pool site (removed or desiccated). Therefore, we compared the precision of TTs  of frogs that were heading towards available and non-available pools. We used different GLMMs with the “SC” , “average angular deviation”  and “average distance to the straight-line path”  as response variable. For all three models, we used “heading towards an available vs. non-available pool” as explanatory variable and individual frog ID as a random effect.Finally, we investigated potential differences in precision along the tracked HTs between frogs that either did or did not encounter a site with a non-available pool  during prior tadpole transport. For the analysis, we used different linear mixed models (LMM), with “average angular deviation” and “average distance from the straight-line path” as response variable and a GLMM  with “SC” as response variable. For all three models, we used “removed pool encountered during prior tadpole transport” (yes/no) as explanatory variable and the individual frog ID as random factor to account for repeated trajectories of the same individuals.All the full models but one contained only a single explanatory variable, which was a binary factor. We compared these models with the corresponding null (intercept) model based on the second-order form of Akaike’s information criterion . No difAll models were calculated using the R packages “lme4”  or “glmmn = 15 TTs from 10 individuals ; n = 22 HTs from 16 individuals, . Other trajectories were excluded because some individuals had either not moved further than 5 m for two days (n = 1), had an injury (n = 1), were predated on by a spider (n = 1), or the entire tadpole transport took place inside their territory (n = 1). All averaged values  were estimated by first averaging per trajectory section (TT and HT), followed by calculating the overall average from all TTs and HTs.During the study period, we captured 67 individual males a total of 658 times. 408 captures were associated with male territorial behavior and were used to calculate territory centers . We obsen = 14, 10 individuals) frogs moved an average of 141.73 m  and were tracked for an average of 17.62 h . Tracked TT  on average, adding up to an average distance of 87.71 m  for the whole TT. The elapsed time until all tadpoles were deposited was 5.55 h  on average. The speed during tracked TTs was on average 10.16 m/h, reaching a maximum of 17.91 m/h . Speed during homing was 7.22 m/h on average with a maximum of 22.16 m/h  and were never observed to visit further pools after depositing all their tadpoles. In 25 of the 28 TTs, successful deposition of tadpoles was recorded. The remaining three individuals either lost the tadpoles overnight (n = 1), were predated on by a spider (n = 1) or disappeared during tracking (n = 1). From the 49 pool visits recorded, deposition occurred in 29 cases  and no deposition took place in 20 cases . On average, the first observed pool visited by each frog (n = 28) was 41.07 m (range = 2.1–98.33 m) away from the territory center. During the tracked TTs, male frogs visited an average of 2.4 depositionssites per transport event . However, we cannot exclude that frogs visited other deposition sites before we encountered them.We recorded 49 pool visits during 28 TTs  including all artificial and natural, available and removed/dry pools. Frogs carried on average 8.5 tadpoles  moved directly to and between potential deposition sites with an average straightness coefficient of 0.83 (sd = 0.13), an average angular deviation of 24.52° and an average linear deviation of 2.9 m (sd = 4.02) from the straight-line path. On their way back to the home territory, frogs  reached an average straightness coefficient of 0.87 (sd = 0.12) and moved with an average angular deviation of 32.28° from the ideal path and an average linear deviation of 2.04 m (sd = 2.03).Frog movement was strongly directed towards potential deposition sites and the home territory for TTs and HTs, respectively see . We onlyMovement precision during TTs: None of the models with SC, average angular deviation or average distance to the straight-line path as response variable and heading towards an available or unavailable pool as explanatory variable improved the AICc compared to their corresponding null model. Thus, we could not find any difference in the frog’s movement behavior when encountering a removed or available pool .p = 0.9). Overall, this indicates that there is no difference during homing between frogs that did or did not encounter an unavailable deposition site during tadpole transport , but there was no difference between the levels of the explanatory variable  on the precision or speed of movement.In our study, we quantified movements of tadpole-transporting frogs, but we did not observe any exploratory behavior of A. femoralis are mostly found in their territories, calling to attract females and repel competing males  Sensitive learning phases during the juvenile stage are common in vertebrates  they could show that flight approaches to feeders that were primarily guided by spatial memory were of shorter duration than approaches that included object-based cues, indicating that there is a short-term energy advantage to the spatial-memory strategy  managed to find an available pool for deposition even if they had previously encountered a removed or dry pool. Furthermore, we recorded one male visiting a natural pool site that never held water during the entire study period and could only have been known as a potential deposition site from previous years. These findings suggest that male frogs rely predominantly on their spatial memory to find deposition sites and do not invest time and energy in exploring further pools during tadpole transport. The availability of suitable breeding pools can change rapidly in the tropics as a result of sudden, heavy rainfalls or fast desiccation due to strong solar radiation. Nevertheless, resource availability seems to be sufficiently stable for frogs to rely on memory-based orientation strategies. Previously unavailable pools might turn into available breeding resources after one heavy rainfall, whereas some pool locations remain the same not only for the entire breeding season, but for several years at a time. Thus, frogs may remember previously visited pools as having been available in the past and to be at least in principal able to contain water. The best strategy might be to remember previous sites and visit them repeatedly, even at the risk that they might have vanished since the last visit. Many nectar-feeding species use similar strategies and primarily use spatial information to relocate flowers over object-based cues e.g., . In a ststrategy . RelyingA. femoralis mainly uses suitable pools based on spatial memory  or callamanders ), might on sites .A. femoralis remember the location of up to six different pool sites  and a dipole antenna made of flexible coated wire.Click here for additional data file.10.7717/peerj.3745/supp-3Figure S2Trajectory map showing movement patterns of frogs homing back to their territory after tadpole transport. Red asterisks represent the territory centers of tracked carriers and colored lines show different tracking events. Squares represent the cross-array of thirteen artificial tadpole deposition sites, blue squares representing available pools and gray crossed squares the removed deposition sites. Blue circles represent four potential natural pools, which were visited by tadpole carriers during tracking. Contour lines (1 m) and the Arataye River are drawn in light gray.Click here for additional data file.10.7717/peerj.3745/supp-4Figure S3Histogram showing the range of movement speed during tadpole transport from one tracking location to the next one (m/h).Click here for additional data file.10.7717/peerj.3745/supp-5Figure S4Histogram showing the range of movement speed during homing from one tracking location to the next one (m/h).Click here for additional data file.10.7717/peerj.3745/supp-6Table S1File contains Click here for additional data file.10.7717/peerj.3745/supp-7Table S3Each row represents a specific tracking event. Columns show the frog ID, number of tadpoles, tracked distance, total time of the tracked path (h), average speed (m/h) across the entire TTs, number of deposition sites visited (in parentheses the number of available (a) and non-available (n) deposition sites), total estimated tadpole transport distance, straight-line distance  and a straightness coefficient.Click here for additional data file.10.7717/peerj.3745/supp-8Table S4Each row represents a specific tracking event. Columns show the tracked distance during homing, straight line distance (from the last deposition site to the territory center), duration of homing, whether homing took more than one day (overnight = 1), average speed and the straightness coefficient.Click here for additional data file.10.7717/peerj.3745/supp-9Table S5Summarized data of the average angular deviation, average distance from the straight-line path and SC for the tadpole transport of frogs which encountered only available pools during tadpole transport (A) and frogs which encountered non-available pools (N), minimum, maximum, 1st and 3rd quartile, median and mean.Click here for additional data file.10.7717/peerj.3745/supp-10Table S6Summarized data of the average angular deviation, average distance from the straight-line path and SC for the homing trajectories of frogs which encountered only available pools during tadpole transport (A) and frogs which encountered non-available pools (N), minimum, maximum, 1st and 3rd quartile, median and mean.Click here for additional data file.


Pulmonary Surfactant reduces surface tension in the terminal airways thus facilitating breathing and contributes to host’s innate immunity. Surfactant Proteins (SP) A, B, C and D were recently identified as inherent proteins of the CNS. Aim of the study was to investigate cerebrospinal fluid (CSF) SP levels in hydrocephalus patients compared to normal subjects.CSF SP A-D levels were quantified using commercially available ELISA kits in 126 patients . 60 patients without CNS pathologies served as a control group. Hydrocephalus patients were separated in aqueductal stenosis , acute hydrocephalus without aqueductal stenosis  and idiopathic normal pressure hydrocephalus . Furthermore, six patients with pseudotumor cerebri were investigated.SP A—D are present under physiological conditions in human CSF. SP-A is elevated in diseases accompanied by ventricular enlargement  in a significant manner . SP-C is also elevated in hydrocephalic conditions  and in Pseudotumor cerebri . SP-B and SP-D did not show significant alterations.The present study confirms the presence of SPs in human CSF. There are significant changes of SP-A and SP-C levels in diseases affecting brain water circulation and elevation of intracranial pressure. Cause of the alterations, underlying regulatory mechanisms, as well as diagnostic and therapeutic consequences of cerebral SP’s requires further thorough investigations. Surfactant proteins (SPs) are part of the pulmonary surfactant, a thin layer covering the alveolar surface serving three main purposes (i) decreasing the surface tension at the air-tissue interface to prevent the collapse of the small airways at the end of expiration (ii) facilitating the clearance of airborne pathogens and (iii) regulating the local innate and adaptive immune response ,2. The pSurfactant protein A (SP-A) and surfactant protein D (SP-D) help to maintain the physicochemical properties of the surfactant layer. Furthermore, both molecules are opsonins, facilitating the elimination of invading pathogens and dead cells in the lungs and other organs ,5. SP-A The hydrophobic proteins SP-B and SP-C strongly interact with phospholipids, thus forming and stabilizing the pulmonary surfactant layer –8. Lack Recently our group detected surfactant proteins as inherent proteins of the CNS . The disCSF net flow comprises of a pulsatile convective flow of different frequencies (e.g. heart cycle and breathing), diffusion and active transport across barriers of the CNS . Since STherefore, the aim of the present study was to analyze differences in CSF-SP levels between normal subjects and patients suffering from idiopathic normal pressure hydrocephalus (NPH), aqueductal stenosis (AQS), acute hydrocephalus without aqueductal stenosis (acute HC w/o AQS) and pseudotumor cerebri (PC).CSF specimens of 126 patients were examined. All patients or caregivers gave their written informed consent for the scientific use of CSF-samples and analysis of clinical and radiological data. The study was approved by the local ethics committee (Ethikkommission Universität Leipzig Az 330-13-18112013). Specimens of 60 subjects without conclusive proof of neurological pathologies specimens were used as control group. Those specimens were obtained during the diagnostic workup that necessitated CSF examination by lumbar puncture . Furthermore CSF samples were obtained from 66 patients that underwent diagnostic workup and treatment of hydrocephalus or pseudotumor cerebri. Hydrocephalus patients were categorized into the following pathophysiological entities after the review of patient records and brain imaging: aqueductal stenosis (AQS), acute hydrocephalus without aqueductal stenosis (acute HC w/o AQS) and normal pressure hydrocephalus.rd ventricle in cranial imaging were classified as AQS [24 patients who revealed narrowing or obstruction of the Sylvian aqueduct with corresponding enlargement of the lateral ventricles and the 3d as AQS .20 Patients with the classic triad of symptoms of idiopathic NPH  and typical morphological criteria in brain imaging  were included after standardized clinical and radiological examination ,14,15,16Quantification of surfactant protein concentrations was performed using enzyme-linked immunosorbent assays (ELISA) according the manufacturers manual. Commercially available enzyme-linked immunosorbent assay kits  were used to quantify the amount of SP-A , SP-B , SP-C  and SP-D  in CSF samples. The analysis was performed using a microplate spectrophotometer (ELISA-reader) at a wavelength of 450 nm and a reference wavelenght of 405 nm for measuring the absorbance. Surfactant protein concentration in ng/ml CSF was calculated by comparison between standard series and the determined values of antigen concentration (protein concentration) according to the manufacturers manual. CSF concentrations of SP-A, SP-C and SP-D lay well above the detection limit of the ELISA kits. Detection limits according the manufacturers manual were as follows; SP-A: 18.27 pg/ml (0.0183 ng/ml), SP-C: 0.126 ng/ml and SP-D: 2.55 ng/ml. CSF concentrations of SP-B of most samples were slightly below the detection limit of the ELISA kit according the manufacturers manual (SP-B: 0.62 ng/ml).Routine CSF laboratory data  were obtained to rule out infection or other inflammatory and autoimmune diseases. Patients with CSF infection were excluded.Statistical analysis was performed using SPSS Version 22. Data was tested for normality using Shapiro-Wilks test. Differences between groups were analyzed using analysis of variance on ranks with a Dunnett post-hoc analysis. Correlation between age and the concentration of SPs was calculated using a Spearman Rho correlation since SP values were not normally distributed. Significance level was set to 0.05.CSF samples of 126 patients were analyzed for Surfactant proteins A, B, C and D. Routine CSF examinations as described above were also performed. An overview of patient demographics is given in Control patients with an age ranging from 14 days to 84 years  were investigated. Under normal conditions, Surfactant proteins A, B, C and D are present in CSF independent from gender. SP-B concentrations in the majority of CSF samples (n = 109) were slightly below the guaranteed detection limit of the ELISA kit. 17 CSF samples revealed SP-B concentrations within the detection range . Those specimens with values above the detection range are summarized graphically in In routine CSF examination, only total CSF protein concentration showed a significant reduction in AQS and VIIP patients (p = 0.01) compared to controls. Compared to control, SP-A values were significantly elevated in all types of acute CSF circulation disturbance . SP-A levels in NPH were slightly elevated compared to controls, but the difference did not reach statistical significance (p = 0.069) . CompareAdditionally, all SP values were normalized to total CSF protein concentration. However, 29 specimens had to be excluded due to missing total protein concentrations and small sample volume, thus resulting in a reduced total of samples of the different groups . Results of the normalized SPs are shown in SP-A showed a correlation to the age of the subjects in the control group only , it might be that a similar mechanism exists in the brain [Concerning the CNS, we recently identified the epithelium of the choroid plexus and the ependymal cells as cellular sources of SP-A and SP-C he brain . Assuminhe brain ,34.Interestingly, CSF SP-A levels showed a significant increase in correlation with age–exclusively in the control group. Since SP-A CSF levels are increased in all disease subgroups, it seems likely that the pathophysiological alterations of CSF SP-A obscure the identifed association with age. Furthermore, the small sample size of disease subgroups also contributes in dimishing this phenomenon. Arani and coworkers were able to demonstrate in vivo that the stiffness of the brain increases in older age . IncreasIn addition to AQS and acute HC without AQS SP-C levels were also elevated in patients suffering from pseudotumor cerebri. Pseudotumor cerebri is characterized by an elevated intracranial pressure but absence of ventricular enlargement ,39. A coInterestingly SP-A was only increased in pathologies with ventricular enlargement but did not reveal elevation in pseudotumor cerebri. This might indicate that dilation of the inner CSF spaces is the primary stimulus for SP-A secretion, probably succeeding periods of increased intracranial pressure without ventriculomegaly yet having occurred.The results of our SP-B measurements in the different groups seem inconsistent. 17 of 126 CSF samples revealed SP-B concentrations within the detection range of the ELISA system, 109 samples revealed concentrations below the guaranteed detection limit. The majority of detectable SP-B levels were found in the control group (12 out of 60 controls). In our previous study we were able to demonstrate production of SP-B in the human CNS and its abundance in CSF with western blot and ELISA. Considering the issue of the detection limit of the used ELISA system and the small sample size, further studies must be stressed to investigate the importance of SP-B for CSF homeostasis.SP-D was detected in all CSF samples. A trend of increased SP-D was observable in the pseudotumor cerebri group, but the difference did not reach statistical significance compared to our control cohort. Since the number of patients suffering from pseudotumor cerebri in our study was comparatively small (n = 6) but the difference in SP-D CSF levels compared to controls almost achieved statistical significance we hypothesize that SP-D alteration might play a role in pathophysiology of pseudotumor cerebri. To corroborate this hypothesis further studies with a larger cohort of pseudotumor cerebri patients are necessary.Normalising the SP concentrations to the absolute CSF protein content led mainly to a significant reduction of the sample group for NPH (n = 11), but also for the other entities. The normalization further substantiates that SP concentrations in AQS are highly elevated compared to controls. However, due to the small sample size of other pathological entities, only trends to SP elevation were found in PC patients. The results of the normalization additionally indicate that the production of SPs does not simply follow the (etiology dependent) trend of total CSF protein content. Down regulation of total CSF protein might be a counterregulatory measure to improve reabsorption of CSF while surfactant proteins are maintained or even elevated to support CSF flow and lower surface tension at the brain-CSF interface. Furthermore it demonstrates that surfactant proteins are not present within the CSF due to a passive filtration of serum proteins across the BBB.The major shortcomings of the study are the small size of the control group and the lack of follow-up surfactant protein levels after therapy (e.g. after shunt placement in NPH patients) to evaluate therapeutic effects on the cerebral surfactant system. Obtaining such data will be an ethical problem that can only be solved with randomised study protocols justified when more data become available.The identification of altered SP concentration in CSF as pathophysiological mechanism, counterregulatory effect or pure epiphenomenon is difficult. Further investigations require studies focusing e.g. on animal models and on the clinical progress of a particular condition over time  in correlation to changing SP concentrations in the CSF.The present study demonstrated the presence of Surfactant Proteins A, B, C and D under physiological conditions in human CSF. SP-A and SP-C are elevated in CSF samples of patients suffering from conditions with altered CSF dynamics. Elevated SP-A secretion may predominantly be caused by ventricular enlargement, whereas SP-C might predominantly be elevated due to increased intracranial pressure. These hypotheses require further investigations of underlying pathomechanisms and signaling cascades. Our findings, however, suggest a potential use of SP-A and SP-C as diagnostic markers in diseases with ventriculomegaly and elevated intracranial pressure.


Thus, disease staging using standard diagnostic methods may not accurately mirror the invasion of the brain by parasites. The progression of human African trypanosomiasis from the early hemolymphatic stage to the late meningoencephalitic stage is of critical diagnostic importance as it determines the choice of potentially toxic drug regimens. Current diagnostic criteria involving analysis of cerebrospinal fluid (CSF) for parasites and/or pleocytosis are sensitive, but recent evidence suggests that specificity may be poor.Trypanosoma brucei rhodesiense, using 1H nuclear magnetic resonance (NMR) spectroscopy.We used an untargeted global metabolic profiling approach for the discovery of novel candidate stage-diagnostic markers in CSF from patients infected with Metabolic markers did not distinguish between early and late-stage cases but were associated with neuroinflammatory responses and the presentation of neurological disturbances. In particular, increased concentrations of 3-hydroxybutyrate and alanine and reduced concentrations of mannose and urea were discriminatory for the presentation of daytime somnolence and gait ataxia.CSF metabolite concentrations provide markers for neuroinflammatory responses during central nervous system (CNS) invasion by trypanosomes and are associated with the presentation of neurological disturbances independently of disease stage determined by current criteria. This suggests that applying a dichotomous-stage diagnosis on the basis of CSF pleocytosis does not accurately reflect the biological changes occurring as parasites invade the CNS and has implications for biomarker discovery strategies. Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. After an initial hemolymphatic infection, parasites penetrate the central nervous system (CNS), leading to meningoencephalitis, which is fatal if untreated [T. b. rhodesiense infection, treatment requires the use of the trivalent arsenical melarsoprol, with serious neurological damage ensuing in up to 10% of treatments and a fatal reactive encephalopathy in up to 5% of treatments. In T. b. gambiense infection, melarsoprol treatment of the late stage has been largely replaced with the better-tolerated eflornithine and nifurtimox combination therapy, but this treatment still carries significant side effects in up to 2% of treatments and is logistically challenging to deliver [Human African trypanosomiasis (HAT) is caused by infection with the tsetse fly–transmitted hemoflagellates ntreated . Progres3 [T. b. gambiense infection, it has been demonstrated that moderate pleocytosis of the CSF, from 5 cells/µL to 20 cells/µL, may still be effectively treated with drugs developed for early stage infection [T. b. rhodesiense HAT, neuroinflammatory responses symptomatic of parasite invasion of the brain have been detected in cases that were also effectively treated with early stage drugs [Because of the hazards involved with treatment of the late stage of infection, an accurate diagnosis of stage is essential. Currently this involves detection of one or both of the parasites in the cerebrospinal fluid (CSF) or a pleocytosis of the CSF >5 cell/mmnfection . In T. bge drugs . These cge drugs , 8. TakeT. b. rhodesiense HAT cases and in experimental animal models to discover disease diagnostic markers and understand pathophysiological processes [T. b. gambiense [T. b. rhodesiense HAT, using proton nuclear magnetic resonance (1H NMR) spectroscopy, a high-throughput and robust method that is optimal for analyzing multiple metabolites simultaneously within complex biological mixtures [Metabolic profiling has been shown to be a powerful tool in providing key insights into the complex dynamic interplay between metabolic processes of both the host and the parasite, where parasite-related perturbations in host metabolic pathways may highlight different mechanisms involved in either acute or chronic infection . We haverocesses , 11. A sambiense . We now mixtures , 14, incmixtures . CytokinThe aim of this study was to determine the relationship of small-metabolite markers to current staging criteria, neuroinflammatory processes, and the presentation of neurological symptoms as a strategy to discover novel diagnostic markers and understand the physiological processes involved in progression of HAT.Human CSF samples were collected from adult patients with suspected HAT only, following standard diagnostic methods  and apprDetails of study sites, patient recruitment, and diagnostic and treatment protocols have been presented elsewhere , 11. A t1H NMR spectroscopy. Samples were prepared in 1 batch in a randomized order. Prior to acquisition, an aliquot of 280 µL per CSF sample was diluted with 260 µL of distilled water and 60 μL of Bruker urine buffer  in microfuge tubes. Samples were centrifuged for 1 minute at 14 000×g, and 570 µL per sample was transferred into 5-mm NMR tubes. 1H NMR data were acquired (over 3 days) on a Bruker Avance 600 MHz (14.1 T) NMR spectrometer with a BB probe head and refrigerated SampleJet autosampler , using Topspin software . Metabolic data were acquired as described elsewhere [A total of 46 CSF samples from patients with HAT underwent metabolic screening via lsewhere  with thelsewhere . Spectralsewhere , 17. SpeInterferon γ (IFN-γ), interleukin 6 (IL-6), and interleukin 10 (IL-10) concentrations were measured using a solid-phase sandwich enzyme-linked immunosorbent assay , as described previously . BiologiFollowing data processing, spectral NMR files were analyzed using principal component analysis (PCA) and orthogonal partial least squares discriminatory analysis (OPLS-DA) . PCA is an unsupervised pattern-recognition model used to evaluate overall variance in the data set, while OPLS-DA is a supervised modeling method that maximizes differences between groups while minimizing within-group variation and was used to compare profiles for patients with early infection to those for patients with late-stage infection . In bothU tests, followed by Benjamini-Hochberg false-discovery rate adjustment [Metabolite integrals were further analyzed via univariate nonparametric tests, using Mann-Whitney justment  to correDemographic and clinical details of patients with HAT recruited to this study are presented in Inflammatory and regulatory cytokine concentrations were also measured in a subset of CSF samples. These were selected purely on the basis of having sufficient residual volume to allow quadruplicate enzyme-linked immunosorbent assays and hence may be regarded as a random sample (comprising 8 of 11 early stage cases and 19 of 31 late-stage cases). Progression from early to late-stage infection was associated with significant increases in IL-6, IFN-γ, and IL-10 concentrations .1H NMR pulse program, a common method for detecting and quantifying a wide range of complex biological molecules. Using this technology, we identified the peaks of 27 endogenous metabolites, comprised largely of sugars, amino acids, amines, and organic acids, with relatively low levels of lipid and protein resonances (Supplementary Table 1). Two exogenous metabolites were also detected , which were removed from all spectra prior to analyses.The metabolic profiles in CSF samples collected from patients with HAT were screened using a standard 1-dimensional myo-inositol, and 3-hydroxyisovalerate.To discover metabolite patterns that may be associated with disease state, multivariate analysis of the NMR peak integral data was undertaken using PCA, followed by OPLS-DA. Results from multivariate analyses of NMR data demonstrated that the inherent variability within the metabolic phenotype of CSF samples was not associated with diagnostic stage classification of HAT. PCA models using either complete spectra  or integPCA and OPLS-DA were used to investigate association between the CSF metabolic markers and the presence or absence of neurological symptoms in patients, including somnolence, gait impairment, tremor, and impairment of consciousness as measured by the Glasgow coma scale score. Differences in metabolic phenotypes were observed between somnolent and nonsomnolent patients, demonstrated by partial vertical separation between 2 groups along the first component in both models, although particularly in OPLS-DA . Examinamyo-inositol than those with no gait impairment, whereas somnolent patients had decreased levels of acetone in their CSF than alert patients. Additionally, increases in 3-HB and alanine concentrations and decreases in mannose and urea concentrations were significant for both somnolent patients and those with gait abnormalities. No significant differences in metabolite concentrations were found when the presence of tremor or an abnormal Glasgow coma scale score were the grouping variables.Nonparametric analyses of individual metabolite concentrations confirmed results observed in multivariate analyses, with significant differences observed between patients presenting with and those presenting without somnolence and gait impairment, as shown in myo-inositol, and urea, with IL-10 being additionally positively correlated with 3-HB and lactate. Interestingly, mannose was found to be significantly negatively correlated with all 3 cytokines, as well as with white blood cell count.CSF concentrations of IL-10, IL-6, and IFN-γ were significantly increased in patients with late-stage infection, compared with those with early stage infection . We ther1H NMR metabolic profiling approach was used to characterize the small-metabolite profiles in CSF samples for patients with HAT in different stages of disease progression. It was anticipated that these would inform discovery programs for new diagnostic biomarkers and provide candidate metabolic pathways associated with the neuropathogenesis of this disease. However, no metabolites were detected that could distinguish early from late-stage cases. Instead, a subset of metabolites was found to be discriminatory for patients exhibiting neurological signs of infection. In particular, patients presenting with both daytime somnolence and gait abnormalities exhibited higher levels of 3-HB and alanine and reduced levels of mannose and urea. Additionally, patients presenting with either of these symptoms also exhibited higher CSF concentrations of lactate and lower levels of myo-inositol and acetone. These differences appear to be independent to those of plasma metabolite profiles previously described [myo-inositol levels were higher.In this study, an untargeted escribed , where amyo-inositol, and urea, all negatively associated with neurological disturbance, were found to be also significantly negatively correlated with CNS IL-6 and IL-10 responses. Similarly, 3-HB and lactate, which were positively associated with neurological disturbance, were also positively correlated with the CSF IL-10 concentration. This represents the first evidence linking neurological symptoms, the immune response, and metabolite changes in the CNS during infection.The mechanisms underpinning these metabolite changes are not defined and are likely to be complex and related to the activation of the cellular immune response in the CNS. In this respect, the relationships demonstrated between metabolites discriminatory for neurological symptoms and CSF inflammatory cytokines provide further evidence for the role of immunological disturbance in HAT neuropathogenesis. Progression in HAT has previously been shown to be associated with increases in levels of inflammatory cytokines, as well as the counterinflammatory cytokine IL-10 , as was myo-inositol concentrations in CSF have been shown to be discriminatory for other neuroinflammatory diseases, including meningitis [Changes observed with ketone bodies such as 3-HB and acetone, alanine, and/or ningitis , 24 and ningitis , 26, butningitis . The larningitis , 29.T. b. gambiense, where metabolic signatures were only discriminatory between early and an advanced late stage of disease defined by CSF white blood cell concentrations of >20 cells/µL [The lack of clear discrimination of disease stage by CSF metabolites is consistent with our recent observations of kynurenine pathway activation in the CNS  and withcells/µL . Thus, mT. b. rhodesiense–infected patients who were correctly identified as having early stage infection, based on current diagnostic criteria , and yet who presented neurological sequelae, including impairment of consciousness [While a range of CSF cytokines , chemokiiousness . Thus, fiousness , 34.In conclusion, we have used metabolic profiling to reveal subtle changes in CSF during disease progression in HAT. CSF metabolites correlated with neuroinflammation and the presentation of neurological disturbances independently of disease stage. This suggests that the currently used dichotomous-stage diagnosis based on CSF pleocytosis does not accurately reflect the biological changes occurring as parasites invade the CNS and indicates the potential of metabolic profiling in new biomarker discovery strategies.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Figure_S1Click here for additional data file.Supplementary Figure_S2Click here for additional data file.Supplementary TableClick here for additional data file.


Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells.Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4 Clostridium tetani, a ubiquitous bacterium in soil and the environment can lead to tetanus, a disease characterized by progressive muscle spasms and up to a 10% mortality rate [Infection with ity rate . After 1ity rate , 4. Five+ T helper (Th) responses to the vaccine [Key components of a successful vaccination include development and maintenance of sufficient titers of neutralizing antibodies , and CD4 vaccine . Indeed, vaccine , 8. It h vaccine , 10.ex vivo analysis of human TT responses have been limited, and hampered by a lack of well-defined epitopes and assay systems.Surprisingly, in light of its prominent role in vaccinology and basic immunology alike, the exact nature of the human Class II restricted TT epitopes has not been thoroughly analyzed despite several studies having defined epitopes –16. SeveMycobacterium tuberculosis [Previous studies of epitope biology, in the contexts of erythropoietin (EPO) and the timothy grass (Phl p) allergens, identified HLA Class II binding promiscuity (capacity to bind multiple HLA class II molecules) as a good predictor of immunogenicity –19. Baserculosis . Additiorculosis .ex vivo responses of human PBMC [ex vivo TT-specific human CD4+ T cells and associated memory Th subsets.Here, we developed a TT-specific megapool consisting of previously identified epitopes, and 24 new epitopes, based on predicted HLA binding capacity. Independently, we recently reported the development of a new Activation Induced Marker (AIM) assay, which allows detecting man PBMC . AccordiClostridium tetani infection at any given time in their life, were excluded. A subset of these donors, either originally vaccinated with DTwP or DTaP in infancy, received recently a booster vaccination with Tdap. The remaining donors had not, to the best of our knowledge, received recent booster immunizations in the last 4 years. This study was performed with approvals from the Institutional Review Board at La Jolla Institute for Allergy and Immunology .We recruited of 36 healthy adults from San Diego, USA . All parClostridium tetani toxin (TT) sequence (Acc. No. P04958) by combining two different selection processes. A first selection approach identified 40 peptides by searching the Immune Epitope Database (IEDB) [Peptides were derived from the e (IEDB)  for "Hume (IEDB)  to selechttp://tools.iedb.org/mhcii/). The median of the predicted IEDB consensus percentile scores of the seven alleles was estimated for each peptide and the peptides with the median percentile score < 20.0 were selected as the top predicted epitope candidates–a total of 124 peptides. We next determined the sequence overlap of these 124 peptides with the peptides identified from the IEDB (above). Of the 44 peptides reported in the IEDB, 37 (84%) peptides nested (or largely overlapped) with one or more of the predicted 124 epitope candidates. Therefore, the second selection approach identified 81 predicted peptides that were unique from the IEDB panel. Accordingly, from both selection approaches a total of 125 peptides were synthesized as crude material on a small (1mg) scale by A and A  and tested as described below. The 125 peptides are listed in In the second selection approach, we selected peptides based on the epitope candidate prediction protocol described previously . As per in vitro expansion, 2 x 106 cells/ml were cultured in the presence of 5 μg/ml Tetanus Toxoid (TT)  in RPMI media (Omega Scientific) supplemented with 5% human AB serum (Gemini Bioscience), GlutaMAX (Gibco), and penicillin/streptomycin (Omega Scientific). Every 3 days, 10 U/ml IL-2 in media was added to the cultures.PBMC  were isolated from collected blood or leukapheresis by density gradient centrifugation, according to the manufacturer’s instructions. Cells were cryopreserved in liquid nitrogen suspended in FBS containing 10% (vol/vol) DMSO. For PBMCs 4 cells/well with either individual peptides (10 μg/ml), peptide pools (5 μg/ml) or TT antigen (5 μg/ml). PHA (10 μg/ml) was used as a positive control and medium containing 0.13% DMSO (corresponding to the percentage of DMSO in the pools/peptides) was used as a negative control. ELISPOT plates were then incubated for 24 h. Consistent with previous studies [6 PBMC, p ≤ 0.05 by Student’s t-test or by a Poisson distribution test, and stimulation index ≥ 2. Criteria for peptide positivity were identical except with a threshold of 20 SFCs per 106 PBMC was utilized. Cumulative SFC was calculated by sorting the epitopes according to their total SFC or according to the consensus percentile for binding prediction, and then calculating the cumulative sum of responses for each peptide or consensus percentile from highest to lowest. The percentage of cumulative response was calculated by dividing each individual value with the total.After 14 days of culture with TT, the response to peptides, peptide pools, or whole antigens was measured by IFNγ and IL-5 dual ELISPOT as previously described . Briefly studies , to be c+ T cells for TCR-dependent upregulation of OX40 and CD25 and/or PD-L1 after an optimal time of 18–24 h of culture. This method exhibits increased sensitivity and is one of the most robust methods for identification of Ag-specific cells in humans. Briefly, cryopreserved PBMCs were thawed and 1x106 cells/condition were immediately cultured together with TT peptide pools (2 μg/ml), Dengue virus peptide pool (1 μg/ml), TT (5 μg/ml), or PHA  as a positive control in 5% human serum (Gemini Bioproducts) for 24 hours. To determine the memory phenotype of responding T cells staining for CD45RA and CCR7 were performed.For the Activation Induced Marker (AIM) assay we used a protocol described elsewhere . This asFor intracellular cytokine staining (ICS), PBMCs were incubated with TT peptide pool for 2h. After 2h, BFA  was added for an additional 4h. Cells were then washed, stained for extracellular markers for 30 min, and then washed, fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin (Sigma) and stained for intracellular IFNγ and IL-4. A combination of PMA and Ionomycin (1 μg/ml) was used as positive control. Samples for both AIM and ICS were acquired on a BD LSRII Flow Cytometer and analyzed using FlowJo X Software. All flow cytometry mAb reagents are shown in HLA typing was performed either at LJI or by an American Society for Histocompatibility and Immunogenetics-accredited laboratory at Murdoch University. HLA typing was performed for Class I  and Class II  using locus-specific PCR amplification on genomic DNA. Primers used for amplification employed patient-specific barcoded primers. Amplified products were quantitated and pooled by subject and up to 48 subjects were pooled. An unindexed (454 8-lane runs) or indexed (8 indexed MiSeq runs) library was then quantitated using Kappa universal QPCR library quantification kits. Sequencing was performed using either a Roche 454 FLX+ sequencer with titanium chemistry or an Illumina MiSeq using a 2 x 300 paired-end chemistry. Reads were quality-filtered and passed through a proprietary allele calling algorithm and analysis pipeline using the latest IMGT HLA allele database as a reference. Potential HLA-epitope restriction odds ratios and relative frequencies were calculated using the RATE program .in vitro stimulation of PBMCs for 14 days with whole antigen. Accordingly, PBMCs were incubated with whole TT antigen for 14 days in the presence of IL-2, followed by testing 13 pools of approximately 10 peptides each. Positive pools were deconvoluted to identify individual epitopes.To define the patterns of T cell responses and identify TT derived T cell epitopes, we utilized a strategy we successfully applied to several other systems , 21–23, 6 detected and frequency of associated positive responses are listed in Overall, T cell responses were analyzed using this strategy for a total of 20 individuals . Of thesThe top 28 epitopes  accounteConversely, a total of 19 epitopes were recognized in at least 2 out of the 20 donors tested, corresponding to a frequency of recognition of 10% . Only foTo correlate HLA types with T cell responses and to facilitate the design of tetramer reagents to detect antigen-specific T cells, we used the RATE program  to calcuThis analysis allowed inferring potential restrictions for the 14 of the main epitopes. All of these 14 restrictions were promiscuous restrictions (the epitope was inferred to be restricted by multiple HLAs), thus confirming and extending previous results that suggested that dominant epitopes are often associated with promiscuous HLA restriction , 23. It + T cells, and/or predicted to bind HLA molecules by a recently described prediction protocol [As stated above, the set of peptides tested were either already described in the literature as being recognized by human CD4protocol . InteresWe further found that 38.5% of the total response, was accounted for by 24 of the novel identified epitopes , thus unAs mentioned above, some of the PBMC donations analyzed were derived from recent Tdap boosted donors. Accordingly we plotted separately responses from donors boosted with the Tdap vaccine within the previous 6 months, as detailed in The acellular DTaP vaccination has been reported to induce greater responses of the Th2 subset compared to the cellular DTwP formulation, and this Th2 bias is maintained in 4-6-year-olds boosted with Tdap, and it extends to the TT component , 31. HerWe examined the polarization of T cell responses based on ratio of IFNγ /IL-5 production per epitope tested  as a funWe recently reported a strategy to generate pools of large numbers of potential epitopes for use in cellular assays and avoid toxicity associated with high amount of DMSO required to achieve solubility of such large numbers of peptides , 33. Herex vivo AIM assay  along with the DMSO stimulation control . Furtherin vitro re-stimulation, the AIM assay showed no differences in TT-specific memory CD4+ T cells between Tdap boosted and not boosted donor cohorts using either a TT-megapool (-CCR7+) and effector  memory subsets. Specifically  was phased out in favor of the less reactive acellular form of the vaccine (DTaP). Previous studies highlighted how this different vaccine was associated, in the newborn and up to 9 years of age, with a polarization switch towards Th2 responses. The DTwP vaccine induced a predominant Th1 response, while the DTaP vaccine induced a predominant Th2 response , 31, 36.ex vivo [Hereby, we also performed sequential lyophilization to develop pools of large number of epitopes (Megapools) . By the ex vivo , 37, 38.+ T cells using well-defined antigenic stimuli is highly desirable in human translational immunology. We used a TT megapool, which, in combination with the AIM assay, permitted reproducible detection of TT-specific CD4+ T cells in healthy blood donors. Ex vivo phenotypic analysis of defined T cell populations is critical, since in vitro culture may alter composition and functional properties of the populations of interest. Subsequent phenotypic characterization showed a significant difference in the level of T cell effector versus central memory compartments between the recently boosted and not recently boosted donor cohorts.Characterization of antigen-specific CD4Taken together, these results validate the combination of a megapool approach and the AIM assay as a general tool to study TT-specific responses in humans.S1 TableAge, priming vaccination, booster vaccination, time since last boost, and the number of samples taken for each cohort is shown. Age is expressed as the mean ± standard error for the entire group.(XLSX)Click here for additional data file.S2 TablePeptide sequence, start position, peptide set, no. of donors tested, no. of donors positive for IFNγ / IL-5 or any of the two cytokines, as well as individual donor responses. Table is sorted according to Total SFC.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableShown are the HLA restriction of the dominant epitopes represented by the epitope sequence, start position, restricting alleles, number of donors that responded (R+) or did not respond (R-) to a given peptide, number of donors expressing (A+) or not expressing (A-) given HLAs, the response frequency, odds ratio and the p-value associated associated with its significance estimated by Fisher's exact test.(XLSX)Click here for additional data file.