Treatments to improve outcomes following severe traumatic brain injury (TBI) are limited but may benefit from understanding subacute-chronic brain protein profiles and identifying biomarkers suitable for use in this time. Acute alterations in the well-known TBI biomarkers glial fibrillary acidic protein (GFAP), αII-spectrin, and their breakdown products (BDPs) have been well established, but little is known about the subacute-chronic post-injury profiles of these biomarkers. Thus, the current study was designed to determine the extended profile of these TBI-specific biomarkers both in brain tissue and cerebral spinal fluid (CSF). Protein abundance was evaluated in brain tissue samples taken from regions of interest and in CSF at 24 h, 3 days, 7 days, 1 month, and 3 months following severe TBI in rats. Results showed increased full length GFAP (GFAP-FL) and GFAP-BDPs starting at 24 h that remained significantly elevated in most brain regions out to 3 months post-injury. However, in CSF, neither GFAP-FL nor GFAP-BDPs were elevated as a consequence of injury. Regional-specific reduction in αII-spectrin was evident in brain tissue samples from 24 h through 3 months. In contrast, SBDP-145/150 was robustly elevated in most brain regions and in CSF from 24 h through 7 days. Correlation analyses revealed numerous significant relationships between proteins in CSF and brain tissue or neurological deficits. This work indicates that TBI results in chronic changes in brain protein levels of well-known TBI biomarkers GFAP, αII-spectrin, and their BDPs and that SBDP-145/150 may have utility as an acute-chronic biomarker. Severe penetrating traumatic brain injury (TBI) from gunshot wounds is of concern to both military and civilians alike. Approximately 5,000 penetrating TBI cases were reported from 2000 to 2017 among military personnel –5 and thTraumatic brain injury results in a primary injury characterized by immediate destruction of brain tissue with hemorrhage followed by a complex secondary injury cascade during which there is an influx of intracellular calcium –13 and sThe increased presence of these proteins and their BDPs in brain tissues has been well described following acute TBI –27. HoweThe goal of the current study was to temporally define the abundance of GFAP, αII-spectrin, and their associated BDPs in specific brain tissue regions and CSF following TBI. Injuries were induced using a rat model of penetrating ballistic-like brain injury (PBBI), which mimics the permanent injury tract and temporary cavity generated from a penetrating ballistic round –32 and rGuide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition.Adult male Sprague-Dawley rats  weighing ~250–300 g were used in this study. All rats were singly housed with a 12 h normal light/dark cycle. Surgical procedures were performed under isoflurane anesthesia (2–5% delivered in oxygen). For terminal bio-sample collections at 24 hours (h), 3 days (d), 7 days, 1 month (m), and 3m following injury, rats were deeply anesthetized with 70 mg/kg ketamine and 6 mg/kg xylazine. Research was conducted under an approved animal use protocol in an AAALACi accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the The WRAIR PBBI model captures the injury trajectory and temporary cavity generated by energy dissipation from a high-energy bullet wound to the head . UnilateNeurological deficits were assessed using a modified testing paradigm  at 24 h g for 10 min at 4°C. The resulting cell free CSF was stored at −80°C until use. To increase the concentration of antigens in CSF, the entire collected volume of each CSF sample was concentrated with Amicon Ultra Centrifugal Filter Units with Ultracel-3 membranes  for 30 min at 14,000 × g at 4°C . Ipsilateral brain tissues were washed with 0.9% saline and dissected on ice to isolate the frontal cortex, striatum, hippocampus, and residual midbrain. All tissues were flash-frozen in liquid nitrogen. Concentrated CSF and flash-frozen brain tissues were stored at −80°C until use.A 4-cm midline incision was made from 0.5 cm anterior to the interauricular line. The atlanto-occipital dura mater was exposed by separating the nuchal muscles and CSF was collected with a 30-gauge syringe needle through the membrane. CSF was stored on ice, supplemented with 1xHALT protease/phosphatase inhibitor mix , and centrifuged at 1,400 Brain tissues were sonicated in 1xRIPA buffer containing 1xHALT protease/phosphatase inhibitors . Protein concentrations were determined using the BCA assay kit  and crude homogenates were stored frozen until use. All samples for western blotting were prepared in 1x LDS NuPAGE sample buffer and 250 μM DTT and denatured for 5 min at 95°C. Equivalent amounts of total protein  for tissue samples or 10 μL of concentrated CSF prepared as described above were loaded onto 12% (GFAP in tissue) or 4–12% bis-tris gels  for separation by SDS-PAGE . Proteins were transferred to PVDF membranes and blocked in 5% milk in 1xPBS (GFAP and αII-spectrin in tissues) or to nitrocellulose membranes and blocked with Odyssey Blocking Buffer – PBS . Individual membranes were probed with the following primary antibodies: GFAP , αII-spectrin , Calpain-II – Large Subunit , and washed with phosphate buffered saline containing 0.01–0.1% Tween 20 (PBST). For tissue GFAP and αII-spectrin blots, membranes were incubated with horseradish peroxidase (HRP)-linked secondary antibodies and bands were detected with Clarity Western ECL . Blots were visualized and quantified using the ImageQuant LAS4000 and ImageQuant TL v7.0 software . For calpain-II and GFAP and αII-spectrin blots of CSF, InfraRed (IR)-Dye labeled secondary antibodies were used to visualize bands on the Odyssey CLx imaging system  and bands were quantified using ImageStudio v5.2 software .Concentrated CSF samples were evaluated for GFAP content using an in-house assay developed for detection with the Mesoscale Discovery (MSD) platform . SamplesQ = 0.1%) were excluded from all data sets. For neuroscore, western blotting experiments, and ELISAs, significant injury effects were determined at each time point using a one-way ANOVA followed by Tukey's multiple comparisons test to compare experimental groups. Two-tailed Pearson correlation coefficients (r) were determined using all available data points from Western blot experiments with sham, probe, and PBBI rats. For all analyses, results were considered statistically significant when p < 0.05. All statistical analyses were performed using GraphPad Prism v6 .For tissue western blots, mean target band densities for probe and PBBI groups are presented as a percent of the sham control value (100%) calculated from individual membranes. For CSF western blots, target bands at each time point were normalized to the mean value of a positive control band generated from frontal cortex tissue lysates collected 24 h post-PBBI. Since variable volumes of CSF were used to concentrate proteins, these normalized band values were subsequently normalized by the original μL of CSF loaded for protein concentration. Concentrations of GFAP (ng/mL) obtained in ELISA experiments were also normalized by the original μL of CSF. For CSF western blots and ELISAs, any negative values that were generated  were set as ‘0’ for the purpose of data analysis. Sample sizes are detailed in the figure legends. Please note that 9–10 rats/group and time point were initiated on study, however, due to inability to collect CSF or insufficient collection volumes from all subjects, additional rats were injured for acute collections time points when necessary. All values are presented as mean ± SEM. Outliers as determined by ROUT analysis  and PBBI  resulted in significantly elevated cumulative neuroscore values compared to sham   at 50 kDa and its breakdown products (BDPs) from 37 to 48 kDa, the brain regions that directly encompass the injury trajectory  and adjacent distal regions  were examined by western blotting at 24 h, 3 days, 7 days, 1 month, and 3 months following injury Figure . QuantitAs with GFAP-FL, significant elevations of GFAP-BDPs following probe injury compared to sham were detected only in the frontal cortex and striatum at 3 days (3247%) or 7 days , respectively Figure . In contNext, the abundance of αII-spectrin and spectrin breakdown products (SBDPs) was assayed in the same manner as GFAP. Visual inspection of the western blots confirmed that full length αII-spectrin was detectable at 280 kDa as expected. After probe injury and PBBI, SBDP-145/150 was robustly evident, while SBDP-120 was minimally detected and poorly resolved Figure . QuantitConcomitant increases in SBDP-145/150 were also observed Figure . Probe iTo determine if calpain-II was differentially abundant after brain trauma, protein levels were assessed by western blot in the cohorts used to define GFAP, αII-spectrin, and respective BDP levels Figure . PromineCerebral spinal fluid samples were used to determine the abundance of GFAP, αII-spectrin, and their BDPs after injury. Total GFAP ELISAs (representing both GFAP-FL and GFAP-BDPs) indicated that neither probe nor PBBI resulted in significantly altered total GFAP at 24 h or 3 days after injury Figure . Total GWestern blotting of CSF indicated that αII-spectrin, SBDP-145/150, and SBDP-120 were present as late as 3 months after injury Figure . Quantitr = 0.579). Starting at 24 h, GFAP-BDPs in CSF correlated with these same cleavage products in the frontal cortex, striatum, and residual midbrain (r range: from 0.476 to 0.569). This observation persisted at 3 days with the frontal cortex, striatum, and hippocampus (r range: 0.511–0.797). Similar to the results for GFAP, the relationship between SBDP, but not αII-spectrin, in CSF and brain tissues was significant in many instances  and at 3d in the striatum, hippocampus, and residual midbrain (r range: 0.498–0.918). This correlation persisted at 7 days in the frontal cortex, striatum, and residual midbrain areas (r range: 0.507–0.777). CSF SBDP-120 was positively associated with values detected in the frontal cortex at 7 days post injury only (r = 0.560).To determine if CSF levels of GFAP, αII-spectrin, and their BDPs may have utility to predict abundance of the same protein levels in brain tissues, correlation analyses were performed using grouped data points from all three conditions as described in materials and methods. These correlations between specific protein levels in CSF and the corresponding protein levels in brain tissues were performed for each brain region and time point. GFAP-BDPs, but not GFAP-FL, revealed many significant positive correlations between CSF and tissues (Supplementary Table r range: 0.498–0.749) and 7 days (0.476–0.828), GFAP-FL and GFAP-BDP abundance in all brain regions correlated significantly with neurological deficits. Acute neuroscore values and brain αII-spectrin levels were negatively associated with one another throughout acute-chronic injury . In accordance with the gain of SBDPs in brain tissues, analysis indicated that these levels were positively associated with acute neurological deficits. This was overwhelmingly true of SBDP-145/150 detected at 24 h–7 days, wherein this correlation was significant among all four brain regions at 3 days, but was greatest with the residual midbrain (r = 0.742). Fewer instances of significance were observed with analysis of SBDP-120 levels, where the strongest correlation was with the residual midbrain at 7 days (r = 0.664).Additional correlations were performed to determine the relationship between acute neurological deficits and brain tissue protein abundance grouped from all three injury conditions. Overall, acute neuroscore values were positively associated with GFAP-FL and GFAP-BDPs in brain Table . At 3 daThis study provides a comprehensive evaluation of well-described TBI biomarkers  in brain tissue regions and CSF throughout acute-chronic (24 h to 3 months) injury progression. A representation of this timeline following PBBI is indicated for brain tissues Figure  as approGFAP and αII-spectrin proteolysis and subsequent BDP generation in brain tissue is well-established following TBI but has focused on the acute time frame –27. ThisCalpain- or caspase- mediated protein degradation is proposed to be a key factor in TBI mediated neurodegeneration. Generation of GFAP-BDPs and SBDP-145/150 are products of calpain-mediated proteolysis , whereasThis study indicates that chronic, progressive degradation occurs not only in brain regions containing the primary lesion as previously reported , 32, 45 This work corroborates and expands upon previous work from our laboratory, which defined the presence of GFAP, αII-spectrin, and their BDPs in a coronal brain section containing the primary injury lesion through 7 days after PBBI  with a fThe differential effects of penetrating injury alone compared to a penetrating ballistic-like injury were examined here by inclusion of both the probe injury and PBBI groups, respectively. While both injuries resulted in acute neurological deficits compared to sham rats, these deficits were of a greater magnitude following PBBI compared to probe injury. Greater elevation in the tissue abundance of GFAP, αII-spectrin, and their BDPs in PBBI compared to probe injured rats was also observed longitudinally across multiple brain regions. This is consistent with previous work that has indicated elevated SBDP-145/150 following more severe CCI . These dThe identification and validation of biofluid based biomarkers for use in informing individualized treatment plans and/or monitoring the efficacy of treatment over time holds great potential for improving TBI patient outcomes. Both GFAP-BPDs  and SBDPAcute neurological deficits correlated significantly with acute-chronic brain tissue levels of GFAP, αII-spectrin, and BDPs, however, these associations presented in a different pattern than the CSF correlations discussed above. While significant CSF correlations resulted primarily from GFAP-BDPs and SBDP-145/150, significant relationships between neuroscore values and brain tissue protein levels were more generalized and not as limited to BDPs specifically. This was especially true for GFAP. One explanation for this difference is that more severe injuries that cause greater neurological deficits are more likely to result in generalized injury induced alterations in protein abundance. Thus, in clinical practice, the assessment of neurological deficits with rating scales such as the NOS-TBI may have more benefit for classification of injury severity  and predThis study is the first to characterize acute through chronic profiles of the well-known TBI biomarkers GFAP, αII-spectrin, and their BDPs in distinct brain regions of interest, identify the presence of these proteins and their BDPs in CSF, and demonstrate significant correlations between the levels of BDPs present in CSF and brain tissues following PBBI in rats. These results indicate that a sustained period of reactive astrogliosis but finite period of axonal cytoskeletal degradation follow penetrating brain injury and that the detection of calpain generated BDPs in CSF may predict the underlying presence of these tissue proteins in real time. Additionally, the evidence for chronic proteolysis and astrogliosis presented in this study informs the design of drug treatment paradigms following penetrating TBI, indicating that prolonged treatments into subacute-chronic periods should be considered.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.KD, CC, AB, and DS designed the experiments and prepared the manuscript. KD, CC, and HH performed the experiments and conducted data analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Iberian), which were additionally affected by different maturation times and feeding types (acorn vs. fodder). Results showed that 20 volatile compounds were able to distinguish Iberian and non Iberian hams, and 16 of those had relevant sensory impact according to their odor activity values. 3-Methylbutanol, 2-heptanol and hexanal were among the most concentrated volatile compounds. In the case of non-volatile compounds, the concentrations of amino acids were generally higher in Iberian hams, and all the amino acids were able to distinguish Iberian from non Iberian hams with the exception of tryptophan and asparagine. A strong correlation of some amino acids with volatile compounds was found in the particular case of alcohols and aldehydes when only Iberian hams were considered. The high correlation values found in some cases proved that proteolysis plays an important role in aroma generation.The flavor of dry cured ham explains the high appreciation of this product and it determines consumer acceptance. Volatile compounds provide valuable information about the odor and sensory quality of dry cured hams. Since amino acids are the origin of some volatile compounds of dry cured ham, the volatile and amino acid compositions of forty-one dry cured hams from Spain and France were determined to establish associations between them. The samples included different pig breeds (non Iberian  The unique flavor of dry cured ham is the result of a long manufacturing process that produces changes in its aroma and taste. The dry cured ham aroma is markedly affected by the raw material and several parameters during the production process. Raw hams undergo several stages, such as salting with dry salt, washing, post-salting for salt equalization and ripening-drying. In the course of this long process, over 24 months in some cases, the temperature and humidity are controlled to reduce the risk of bacterial spoilage . Howeverpost-mortem process  results from lipid oxidation as well. Their concentrations were, in general, lower than those of saturated aldehydes but they contribute to aroma since their concentrations are higher than their OT in many of the samples. Concerning (E)-2-heptenal, the number of hams whose concentrations are higher than the corresponding OT is a little lower in SF than in the muscles. Its low OT (0.05 mg/kg) allows contributing to aroma with almond-like, green fruity and fatty sensory notes. The concentration of this compound has not allowed distinguishing non Iberian from Iberian hams, which could mean that these sensory notes would be of similar level in all the hams. The concentrations of (E)-2-octenal and (E)-2-nonenal are higher in SF but their low OTs (0.004 mg/kg and 0.15 mg/kg respectively) indicate that they contributes to the aroma in all the analyzed locations. Their concentrations allowed distinguishing Iberian from non Iberian hams, and they contribute to the pungent and fatty sensory descriptors of Iberian hams.The monounsaturated aldehydes [(p < 10–4). Authors [In addition to the branched aldehydes, the reaction between amino acids also produces aromatic aldehydes such as benzaldehyde, although the latter can also be formed during lipid oxidation. It contributes substantially to dry-cured ham aroma with a bitter almond sensory note because of its low OT as found by other authors  as well. Authors  found a E,E)-2,4-decadienal, octanal, (E)-2-heptenal, nonanal, (E)-2-octenal, decanal) are correlated with amino acids with diverse regression coefficients (adjusted-R2 ≥ 0.75).Benzaldehyde is another volatile well correlated with different amino acids in ST muscle . The rest of aldehydes  were found when the study was centered in the whole set of hams, in which the non Iberian hams mean more than 80%. However studying Iberian hams, whose maturation period is longer, many other relationships between amino acids and volatiles were found. Thus, p < 0.01)  .et al. [Lipid oxidation, by means of autoxidation or beta-oxidation of fatty acids, is mainly responsible for the production of methyl ketones. These compounds contribute to dry-cured ham aroma  and theyet al.  had alreThree main carboxylic acids were identified in the headspace of the samples. The acids, previously described by other authors ,35, are Furans have a significant impact on the aroma of dry-cured hams  because Esters are described in dry-cured hams at the end of the maturation process and it seems that NaCl concentration affects the ester production through the activation of esterases . Esters Sulfur compounds mainly originate from amino acids via Strecker degradation and have been associated to undesirable aromas for dry-cured hams. The presence of these kinds of compounds in outer muscles has been already reported . Only a vs. non Iberian ham). p < 0.05). The selected volatile compounds were those with the codes 5–6, 13, 17–18, 21, 24–27, 29–31, 34, 36–38, 40–42  × (Piétrain × Large White), (Duroc or Landrace) × (Landrace or Large White or Landrace × Large White) and Landrace × Large White crossbreed sows mated with several genetic types, eight hams were from Iberian pigs—100% Iberian pig or Iberian × Duroc—Jersey with a minimum of 75% Iberian pig , and two hams were from Gascon and Basque pigs although crossed with Large White and other genetic types. The hams were processed by local manufacturers using the traditional method of each geographical origin, some of them being described in regulations [The samples in this study were forty-one dry cured hams from Iberian breeds and other commercial lines and crosses, including hams from France and Spain. ulations ,48,49,50biceps femoris (BF), semimembranosus (SM) and semitendinosus (ST) muscles and subcutaneous fat (SF)  .Three grams of the minced hams were placed in 20 mL glass vials, tightly capped with a PTFE septum, and left for 10 min at 40 °C to allow equilibration of the volatiles in the headspace. The septum covering each vial was then pierced with a solid-phase micro-extraction (SPME) fiber coated with Carboxen/PDMS/DVB . This fiber was exposed to the headspace for 180 min. The temperature and time were automatically controlled in a Combipal  by the software Workstation v.5.5.2 .When the adsorption process was completed, the fiber was inserted into the injector port of the GC for 5 min at 260 °C with the purge valve off (splitless mode). The compounds were separated in a DB-WAX column  installed on a Varian 3900 gas chromatograph (Varian) equipped with a flame ionization detector. The carrier gas was hydrogen. The oven temperature was held at 40 °C for 4 min and programmed to rise at 1 °C/min to a temperature of 91 °C, and then to rise at 10 °C/min to a final temperature of 201 °C, where it was held for 10 min to eliminate the memory effect. Each sample was analyzed in three replicates. The content of each volatile compound was calculated from the FID area and expressed as area units. A solution of 4-methyl-2-pentanol (1.2 mg/kg) was used as external standard in order to standardize the results of all the analyses. Thus, the quantitative result (mg/kg) of each volatile compound was computed by relating the peak area of the volatile compound to the area of external standard but taking into account the sample weight and the response factor of each volatile. The identification of volatile compounds by GC-MS  was carrConcentrations in the range of 0.1–5.0 mg/kg and 0.5–20 mg/kg were analyzed under the analytical conditions described above. These two ranges allowed analyzing the recovery of volatile compounds at two different concentration levels. The absolute response factors of the standard compounds were calculated as the slopes of the linear regressions obtained from the ratio of total peak area as a function of concentration . Relative response factors were obtained as the ratio of the absolute response factor of each compound to that of the internal standard (4-methyl-2-pentanol). Fully deodorized edible oil was the matrix for the assessment of the odor threshold values; the absence of volatile compounds in the matrix was checked by the SPME-GC procedure described above. The sensory assessment was carried out in a test room arranged for evaluating sensory characteristics. Five assessors with a large experience of odor recognition in fat food products carried out the evaluation. Three samples were presented to the assessors following the triangle test whose results were statistically analyzed. Each sample (15 mL) was kept in standardized glasses at 29 °C ± 2 °C for 15 min and then tested. The samples were diluted until none of the assessors was able to classify the samples by odor intensity. The odor activity values (OAVs) of the volatile compounds, defined as the ratio of the concentration to the odor threshold , were caGC-O was applied to assess the aroma notes corresponding to ham volatile compounds. It was performed with a GC (Varian 3900) connected to an olfactory port . Purified helium (purity > 99.99%) was used as the carrier gas at a constant flow rate of 3.8 mL/min. The inlet pressure was 170 kPa, and the inlet system was in split/splitless mode. The oven temperature program was that already described in a previous section. The effluent of the GC column was split 1–10 to the detector and the sniffing port, respectively.vs. Rt) is the result of merging the information from the individual analyses of the assessors. The olfactory detection was performed during the chromatographic separation by three assessors with a large experience in odor recognition and sniffing of dry-cured hams. Damp air was continuously passed through the head of the port during operation; the flow rate was 30 mL/min. Elution of each aroma compound through the sniffing port was recorded by writing the beginning and end of the entire sensation of any odorant as well as its odor properties. The final aromagram  of standard amino acids obtained from Sigma Chemical: L-Ala, L-Arg HCl, L-Asn, L-Asp, L-Cys, L-Glu, L-Gln, Gly, L-His HCl, Pro (4-OH), L-Ile, L-Leu, L-Lys HCl, L-Met, L-Phe, L-Pro, L-Ser, L-Thr, L-Trp, L-Tyr and L-Val. Creatine and creatinine were analyzed by using the method described by Mora et al. .Univariate and multivariate algorithms have been used by means of Statistica 8.0 . An ANOVA allowed selecting the volatile compounds and amino acids that better characterize the samples by breed. Correlation was used to determine the relationship between sensory attributes and chemical compounds while the first screening of the relationship between those two set of variables was carried out by principal component analysis (PCA).p < 0.05) in their concentration depending on the combined effect of breed, feeding and curing process. Sixteen out of these 20 compounds have a sensory impact on dry cured ham aroma given that their concentrations were higher than their odor threshold. The amino acids composition of Iberian and non Iberian hams were also different for all the amino acids excepting tryptophan and asparagine. In this case, the concentration values were higher in Iberian hams in all cases probably due to their longer curing process compared to the other samples. Carnitine analyses showed, on the contrary, lower concentration values for Iberian hams due to the degradation of this compound during the processing [2 > 0.70) when only Iberian hams were including in the data set, the highest correlation coefficients corresponding to alcohols and aldehydes. The branched alcohols and aldehydes, typically produced from amino acid degradation [The results comparing the volatile composition of Iberian and non Iberian hams showed that 20 volatile compounds had significant differences (ocessing . The corradation , gives a


We discuss a case where medically optimal investigations of health problems in a donor-conceived child would require their egg donor to participate in genetic testing. We argue that it would be justified to contact the egg donor to ask whether she would consider this, despite her indicating on a historical consent form that she did not wish to take part in future research and that she did not wish to be informed if she was found to be a carrier of a ‘harmful inherited condition’. We suggest that we cannot conjecture what her current answer might be if, by participating in clinical genetic testing, she might help reach a diagnosis for the donor-conceived child. At the point that she made choices regarding future contact, it was not yet evident that the interests of the donor-conceived child might be compromised by her answers, as it was not foreseen that the egg donor’s genome might one day have the potential to enable diagnosis for this child. Fertility consent forms tend to be conceptualised as representing incontrovertible historical boundaries, but we argue that rapid evolution in genomic practice means that consent in such cases is better seen as an ongoing and dynamic process. It cannot be possible to compel the donor to aid in the diagnosis of the donor-conceived child, but she should be given the opportunity to do so. Genomic testing is blurring the boundaries between clinical tests and research. Children with undiagnosed genetic conditions are now frequently offered the chance to access genome sequencing via hybrid clinical research initiatives such as the 100,000 Genomes Project, aiming to establish why health problems have arisen.Progress in fertility treatments is also generating new ethical issues. The practice of egg donation began in the early 1980s, involving obtaining eggs from a donor woman, fertilising them with sperm and implanting the embryos created in the uterus of a recipient woman.A child born as a result of egg donation presented with features suggestive of a rare genetic condition. Routine NHS diagnostic genetic tests were unable to identify a genetic cause for their difficulties, but the child was potentially eligible for further diagnostic testing in the context of the 100,000 Genomes Project. This involves trio genome sequencing, where the genome sequence of a child is compared with the genome sequences of both of their biological parents. Samples from the biological father and the child were readily available.The fertility clinic that had facilitated the egg donation was asked whether it would be possible to approach the egg donor regarding potential participation in the 100,000 Genomes Project, with the aim of finding a diagnosis for the child that had been conceived using her donated egg. The fertility clinic declined to contact the egg donor on the basis that she had indicated on her consent form that she did not wish to be contacted about future research. She had also indicated that she did not wish to be notified if it was found that she had a previously unsuspected genetic disease or if she was found to be a carrier of a ‘harmful inherited condition’. This meant that the donor-conceived child could not have trio genome testing, reducing the chance of finding a genetic diagnosis for their difficulties .We argue that it would be ethically acceptable for the fertility clinic to contact the egg donor in order to explore the possibility of trio genome testing for the child conceived from her donated egg. We discuss our rationale for thinking that contact would be ethically justifiable in this situation.At the core of this case is the question of what constitutes valid consent, or indeed valid refusal of consent, and how far it can take us. Did the questions asked of the egg donor at the time of donation encompass the issue of future contact to facilitate diagnosis for a child born as a result of the donation? Even if they did, what weight should we give to the ticking of boxes on a form many years ago—should we follow the choices apparently made at that point or is it acceptable to consider that the situation may have evolved such that current reality might supersede those previous abstract choices?The concepts of consent that often imbue medical practice have largely been shaped around decisions with relatively limited potential consequences, such as whether or not to take a particular medication, or whether or not to have surgery. Professional guidance such as the GMC document ‘Consent: patients and doctors making decisions together’There is little professional guidance as to how we should approach the notion of consent in situations where by virtue of a near infinite number of possible outcomes or because of unpredictable evolution in technology, no one can truly understand, retain and weigh all the relevant information in the depth that we might historically consider necessary for valid consent. ‘Broad consent’ is gaining increasing traction as a feasible option, coming to the fore in situations where providing specific information regarding all possible implications of a decision would be overwhelming or impossible, for example, for biobank participantsDid the questions asked of the egg donor at the time of donation sufficiently cover the possibility of future contact to help make a diagnosis for any children born as a result of the donation? Details as to exactly what discussions she had when she donated her eggs are impossible to obtain from medical records—written consent forms provide some idea, but cannot perfectly reflect the nuances of the consent conversation and to what extent particular decisions were context-dependent. The egg donor indicated that she did not want to be contacted about future research, but in the genomic era, the distinction between clinical and research testing is becoming increasingly artificial. If ‘research testing’ is the only avenue left to achieve a clinical diagnosis, we think that this contact can legitimately be viewed as more in the realms of healthcare than research, though made more complex by the fact that any resulting medical benefits would pertain to the donor-conceived child, rather than the egg donor. Innovative technologies transgress ‘research’ and ‘clinical treatment’ categories, representing a space where both may co-exist,Similarly, the egg donor indicated that she did not wish to be contacted if it was found that she had a previously unsuspected genetic disease or if she was found to be a carrier of a ‘harmful inherited condition’. But in this case, the issue is that her biological child has a likely genetic disorder that needs explanation—as a side effect of testing, it might transpire that the egg donor is a carrier of a ‘harmful inherited condition’, but this would not be the intention of doing the test. Should we cling on to the fact that the egg donor ticked a box on a form saying that she did not want to find out information on carrier status as sufficient reason not to contact, if the primary reason for testing would be to diagnose the donor-conceived child, not to determine the egg donor’s carrier status? There are also potential medical benefits to the donor that should be considered: for some X-linked conditions, it is possible that she would benefit from medical interventions or screening herself.Even if we consider that the egg donor’s choices covered the possibility of contact in this instance and indicated a preference against it, what weight should this have some years down the line? Should she be given an opportunity to consider the choice again? Recent research indicates that participants in the 100,000 Genomes Project sometimes have an inaccurate recollection of whether or not they chose to be informed of, for example, additional findings .In other cases where potentially relevant theoretical decisions have been made some years ago, good practice would involve checking that the decision still holds. For example, if a patient has made an advanced decision to refuse a particular treatment, but at some point later, the treatment becomes a medically appropriate option, we would expect the patient’s medical team to revisit the decision with the patient if they still have the capacity to decide.We can stick to a rigid notion of boxes ticked on a consent form as a binding decision that makes any further discussion impossible. But in light of evidence that people may not recollect what choices they made when consenting to complex interventions,The question then arises as to what purpose written consent forms do serve. We think that they have value in that they may indicate preferences, highlight issues that need consideration and give insight into what discussions have previously taken place. We are not arguing that we should ignore them, but we dispute the idea that they should be taken as gospel. The wishes documented on a consent form are relevant to decision-making about subsequent actions, but they need to be considered in context and potentially weighed with and against other factors. For example, we might weigh the egg donor having ticked a box saying she did not want to be contacted about future research, against the likelihood that she meant pure research as opposed to clinical research initiatives, and the likelihood that if she knew the medical benefit that might entail to the donor-conceived child she would make the same decision.Rose v Secretary of State for Health in 2002, when the High Court ruled that Article 8 of the European Convention on Human Rights included a right to know details about one’s identity, including information about biological parents.Another issue raised by this case is how the rights and interests of the egg donor interact with those of the child conceived from her donated egg. At a molecular level, they share half of their genetic information, so decisions regarding genetic testing in one inevitably impinge on the other. The possibility of future contact is embedded into the donor ‘contract’—when donor-conceived children reach 18, they can ask for their donor’s name, date of birth and last known address (provided that the donation was made after April 2005).In the case that we describe, the rights of the donor-conceived child and of the egg donor are potentially in conflict. On the one hand, if the egg donor participates in genetic testing, it may help reach a diagnosis for the donor-conceived child, which would help them access better information and support regarding their genetic condition. On the other hand, the impact on the egg donor is more uncertain. She may perceive the contact as an unwelcome intrusion and an invasion of her privacy. She may not want to know that a child was conceived with her donated egg or that the child has a potential genetic condition, and that if she has other children they may also have a chance of being affected. However, it is also possible that the egg donor will find this information useful, for example, if she has gone on to have a child with a currently undiagnosed genetic condition, where information on the donor-conceived child could help all parties reach a diagnosis. She may also want to help the donor-conceived child reach a diagnosis for altruistic reasons. When she donated an egg, she had to accept the possibility of contact in the future from adults born because of her donation. We acknowledge that there is a difference in learning about the existence of a donor-conceived small child relative to the existence of an 18-year-old, but we argue that in this case, the age of the child does not make a significant difference to the ethical acceptability of contacting her. Putting off the contact until the age of 18 would delay a clear benefit to the child of improved diagnostic testing, based on an uncertain premise that the egg donor would accept finding out retrospectively that her donation had led to the existence of a child, but would be unwilling to learn this more contemporaneously. Moreover, the egg donor would not be contacted directly by the donor-conceived child in this circumstance, but by the fertility clinic that facilitated the donation.In order to facilitate appropriate medical care for the donor-conceived child, we think it would be justifiable for the fertility clinic to approach the egg donor to ask the question of whether she would consider participating in genetic testing to help reach a diagnosis for the child born from her donation. Refusing to contact her based on a consent form that referred to her alone fails to consider that the interests of the donor-conceived child are inevitably dependent on the decision taken.So far, we have argued that it may be appropriate to contact the egg donor to facilitate genetic testing for the donor-conceived child. This is both in view of the benefit that her genetic information could provide in diagnosing the child, and because it is not clear that she ever expressed a view regarding what she would have wanted to happen in the instance that by providing a DNA sample she could help the medical care of the donor-conceived child. Here, we consider whether there are any additional issues that we need to consider by virtue of the fact of her being an egg donor.The way that egg donors are conceptualised by the healthcare community is unclear: previous research suggests that in some situations egg donors may be vulnerable to mistreatment or may be seen as ‘spare parts’, rather than being conceptualised as patients in their own right.The question arises of how and whether this usefully informs decision-making regarding whether to contact the egg donor in the case we describe. We consider that it is important to consider these concerns in making contact with the egg donor and to be aware that contact from the fertility clinic is likely to be very unexpected. It is essential to consider carefully how much information to provide in any initial contact, especially in light of evidence that some egg donors who feel positively about having donated attribute this feeling at least in part to a hope that their donation has led to the birth of a healthy child.These concerns make the situation more complex, but we do not think that this complexity should preclude the egg donor having an opportunity to make a choice as to whether to participate in genetic testing to help reach a diagnosis for the donor-conceived child. We recognise that it is important to have high standards for consent in the area of egg donation, but we argue that ongoing dialogue and clarification may be an acceptable element of that; high standards for consent does not mean conjecturing what the donor’s response might be to a question she was never meaningfully asked. For example, it would be possible to write to the egg donor in very general terms, explaining that genetic technology has advanced significantly since her donation and that occasionally it is helpful to have access to samples from biological parents in order to guide healthcare of any donor-conceived children. Of course, this may lead to her correctly conjecturing that a child was born from her egg donation who has a health problem, but there would still be room for her to retain uncertainty as to whether this is the case.Instinctively, the decision to contact the egg donor may feel uncomfortable. We argue that this discomfort arises because we continue to rely on a narrow model of consent that is unsuited to technology such as genomic medicine and egg donation. There is an understandable concern that stepping away from written consent forms as being definitive risks being exploitative: does this mean that anything goes? Patient consent has never been the only factor that matters in medical decision-making—other issues come into play such as balancing benefits and risks, and aiming to use resources responsibly and fairly. For narrower interventions, these other factors tend to influence the decision of a clinician to offer an option in the first place, with patient consent being the final element that determines whether the option happens. For broader interventions, maybe we need to consider all these factors concurrently, with consent viewed as an ongoing process rather than the single moment that allows things to happen (or not). We need to move away from the idea that the way to solve all our problems is to be imaginative enough to think of all the possible outcomes of a choice and make sure that they are all down as boxes on a consent form. This might work for more tractable, time-locked decisions such as whether to start a statin or whether to get your knee replaced, but it is unsuited to more complex interventions occurring over many years, where the consequences may evolve as technology alters.In summary, consent plays a crucial role in medical decision-making, but we need to update how we think about it in light of progressing technology. It needs to be appropriate for the dynamic and shifting nature of complex interventions, especially those that occupy the space between clinical care and research. We are not suggesting that consent does not matter or that it no longer has a role, but we need to embed consent throughout medical decision-making, being updated and clarified where necessary, rather than seeing it as a once-and-for-all rubber stamp at the end of a process from which there is no going back. In the case we describe, we think that the fertility clinic should contact the egg donor to ask whether she would consider having genetic testing aiming to reach a diagnosis for the donor-conceived child. We consider that the questions that the donor was asked at the time of egg donation did not sufficiently encompass this possibility for us to be sure what she would want now, some years down the line. The potential benefit of this course of action to the donor-conceived child also needs to be given weight. We recognise the need for scrupulous consent practices in egg donation and the importance of avoiding coercion. However, we argue that in this instance, it would be ethically justifiable, and in keeping with high standards of consent, for the fertility clinic to contact the egg donor to facilitate medical care of the donor-conceived child.


The presence in cerebrospinal fluid of antibodies to EV peptides at higher levels than non-AFM controls supports the plausibility of a link between EV infection and AFM that warrants further investigation and has the potential to lead to strategies for diagnosis and prevention of disease. P = 0.023, 0.0001, and 0.0028, respectively). Six of 14 CSF samples (43%) and 8 of 11 sera (73%) from AFM patients were immunoreactive to an EV-D68-specific peptide, whereas the three control groups were not immunoreactive in either CSF  or sera .Acute flaccid myelitis (AFM) has caused motor paralysis in >560 children in the United States since 2014. The temporal association of enterovirus (EV) outbreaks with increases in AFM cases and reports of fever, respiratory, or gastrointestinal illness prior to AFM in >90% of cases suggest a role for infectious agents. Cerebrospinal fluid (CSF) from 14 AFM and 5 non-AFM patients with central nervous system (CNS) diseases in 2018 were investigated by viral-capture high-throughput sequencing (VirCapSeq-VERT system). These CSF and serum samples, as well as multiple controls, were tested for antibodies to human EVs using peptide microarrays. EV RNA was confirmed in CSF from only 1 adult AFM case and 1 non-AFM case. In contrast, antibodies to EV peptides were present in CSF of 11 of 14 AFM patients (79%), significantly higher than controls, including non-AFM patients (1/5 [20%]), children with Kawasaki disease (0/10), and adults with non-AFM CNS diseases (2/11 [18%]) ( While there has been a pronounced decrease in poliomyelitis cases worldwide due to global polio eradication efforts , a similThe cause of AFM remains elusive. An infectious agent is only rarely detected in cerebrospinal fluid (CSF). In the initial CDC investigation, the presence of EV RNA by reverse transcription-PCR (RT-PCR) was demonstrated in the CSF from only 1 of 55 children with confirmed AFM. However, more than 40% of children with AFM had PCR evidence of EV RNA in respiratory or fecal samples, suggesting a possible role for EV in AFM , 7. In tP = 0.0098, <0.0001, and <0.0001, respectively)   . Whereas10.1128/mBio.01903-19.1TEXT S1Text S1, DOCX file, 0.1 MB.Supplemental appendix showing AFM and non-AFM control (NAC) sample characteristics and comparisons. Download Copyright © 2019 Mishra et al.2019Mishra et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Virologic testing was performed on all CSF samples from AFM and NAC cases, as well as fecal and respiratory specimens . EV-A71 De novo assembly yielded approximately 271,000 assembled contiguous sequences (contigs) and 9.7 million unique sequence reads (singletons). NCBI nucleotide BLAST analysis enabled assignment of 10,623 contigs and 4.9 million singletons to viruses. The control CSF specimens containing wild poliovirus type 1 yielded 65 and 29 reads for poliovirus, with genome recovery up to 60.7%. EV-A71 and E-25  identified by PCR and Sanger sequencing were also detected by VirCapSeq-VERT. We did not detect reads for viruses other than EV in CSF samples. For samples AFM-07 and non-AFM-16, VirCapSeq-VERT produced 16,994 and 58,206 EV reads, respectively, comprising 99.0% and 74.8% of their genomes , Babesia microti (babesiosis), Borrelia burgdorferi (Lyme disease), Borrelia miyamotoi, Ehrlichia chaffeensis (human monocytic ehrlichiosis), Rickettsia rickettsii (Rocky Mountain spotted fever), Heartland virus, and Powassan virus  were tested for IgM and IgG antibodies to EV and WNV using AFM-SeroChip-1. Sera were available for testing from only a subset of these 14 subjects . CSF samP = 0.023, 0.0001, and 0.0028, respectively).High-density peptide microarrays were used to detect immunoreactivity against a comprehensive EV capsid proteome. We identified an 18-aa region (7 overlapping 12-mer peptides from each EV species)  in the V10.1128/mBio.01903-19.2FIG S1FIG S1, TIF file, 2.2 MB.Immunoreactivity against EV VP1 conserved peptide sequences of EV-A, EV-B, EV-C, and EV-D in serum samples of patients with AFM, non-AFM controls (NAC), Kawasaki disease controls (KDC), and adult CNS disease controls (AC). Download Copyright © 2019 Mishra et al.2019Mishra et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the  and serum .Although 70% (18/26) of both AFM and control sera were immunoreactive with at least one VP1 EV-D conserved peptide , we idenhttps://www.cdc.gov/acute-flaccid-myelitis/afm-surveillance.html]).Since the summer of 2014, >560 cases of AFM have been confirmed in the United States. The uncertainty underlying the cause for the majority of AFM cases precludes important public health interventions such as development of diagnostics, specific therapy, and preventive measures. Growing evidence suggests that infections with EV, including EV-D68 and EV-A71, may contribute to AFM . A potenThe potential link to EV, combined with the inability to directly detect its nucleic acids, encouraged us to search for indirect evidence of EV infection in CSF by testing for the presence of EV-specific antibodies. Since EV infection is common in children, traditional immunoassays such as enzyme-linked immunosorbent assay (ELISA) are impeded by cross-reactivity. Virus neutralization assays are considered the “gold standard” but are resource intensive and impractical to implement for >100 types of EV. Accordingly, we employed a microarray system that can simultaneously screen thousands of peptides from all known human EVs with high resolution.Across the entire EV capsid proteome tiled with 160,000 peptides, the conserved EV VP1 peptides were consistently immunoreactive in CSF of 79% 11/14) of children with AFM, the majority of whom had no EV RNA in CSF. In comparison, NAC patients without evidence of EV RNA in any specimen and KDC patients did not have antibodies to EV peptides in CSF. This conserved peptide region reflects broad EV immunoreactivity, suggesting an association with EV infection and AFM. It is also consistent with EV pathogenesis in mouse models, wherein EVs, including EV-D68, can cause paralytic illness /14 of ch.The demonstration that antibodies to EV peptides are intrathecally produced, such as using an intrathecal ratio of CSF to sera, would provide stronger evidence of CNS infection. We do not have access to sufficient numbers of simultaneously collected CSF and serum specimens to exclude trafficking of EV antibodies from the peripheral circulation into the CNS. However, we consider this unlikely to explain the presence of antibodies to EV peptides in CSF of AFM patients. Of 16 control patients who had antibodies to EV peptides in serum , only 2 adults and 1 child also had antibodies against EV peptides in CSF. The child with antibodies against EV peptides in CSF had an active CNS infection, with E-25 detected in the same CSF sample. One of the 2 adults had multiple sclerosis, and the other had idiopathic epilepsy. Furthermore, in a study of unexplained encephalitis in Gorakhpur, India, using the same peptide array platform, only 3 of 77 children with antibodies to EV in serum also had antibodies to EV in CSF. Two of these 3 children had diagnoses of EV meningoencephalitis based on symptoms and EV RNA (coxsackievirus or echovirus) in their CSF .We demonstrated immunoreactivity to EV-D68-specific peptides (located within the VP1 C terminus) in CSF from 43% of AFM patients and no immunoreactivity in any of the CSF samples from NAC, KDC, and AC control groups. Based on structural homology, the VP1 C terminus of the EV-D68 strain has been predicted to form neutralization immunogens and conformational epitopes for putative neutralizing antibodies , 14. TheOur serological findings warrant further investigation into the association of AFM with enteroviruses, including EV-D68. Reports of respiratory illness consistent with viral infection typically occurred several days before onset of weakness in the majority of AFM cases. Our inability to detect EV-D68 RNA in the CSF of AFM patients may reflect lack of virus shedding from CNS parenchyma into the intrathecal compartment. Alternatively, CSF specimens may have been collected after virus was cleared from the CNS.An important limitation of our study is that controls were not optimally matched to the AFM cases with respect to age, year, or season of collection. Although we tried to test for specificity of findings through use of three control groups, prospective studies with appropriately matched controls from the same EV outbreak season will be critical to testing their validity.While other etiologies of AFM continue to be investigated, our study provides further evidence that EV infection may be a factor in AFM. In the absence of direct detection of a pathogen, antibody evidence of pathogen exposure within the CNS can be an important indicator of the underlying cause of disease. Our findings warrant additional investigation, including testing more specimens, developing confirmatory assays, and designing optimally controlled prospective studies. These initial results may provide avenues to further explore how exposure to EV may contribute to AFM as well as the development of diagnostic tools and treatments.https://www.cdc.gov/acute-flaccid-myelitis/afm-cases.html). Classification criteria for a confirmed AFM case are acute flaccid limb weakness and magnetic resonance imaging evidence of a predominantly gray matter lesion that spans at least one spinal segment (https://www.cdc.gov/acute-flaccid-myelitis/hcp/case-definition.html) .In 2018, CDC received specimens from 381 patients suspected to have AFM (on.html) . Suspecton.html)  and an EFor investigation using VirCapSeq-VERT and peptide microarray approaches, we selected a subset of samples (based largely on available CSF volumes) from AFM cases with onset in 2018, including 13 children and 1 adult. Three groups of controls  are inclThe VirCapSeq-VERT capture probe library comprises approximately 2 million oligonucleotides that tile the coding regions of genomes for all viral taxa that contain at least one virus known to infect vertebrates . Total nTo determine the efficiency of VirCapSeq-VERT in EV genome recovery, we quantitated the amount of EV template in CSF by quantitative reverse transcription-PCR (qRT-PCR). A one-step qRT-PCR assay that targeted a conserved region within the 5′ untranslated region (UTR) was performed on all CSF samples. Ten microliters of total nucleic acid extract was reverse transcribed using Superscript III (Life Technologies). The qRT-PCR assay was performed using 2 μl of cDNA, EV-F primer (TCCTCCGGCCCCTGAATGYGGCTAAT), EV-R primer (GGAAACACGGWCACCCAAAGTA), and EV-probe (6-carboxyfluorescein [FAM]-GCAGCGGAACCGACT-MGB) in a Bio-Rad Touch-CFX 96 real-time PCR instrument at 95°C for 2 min followed by 45 cycles of qPCR at 95°C for 15 s and 60°C for 1 min.Samples were diluted in binding buffer  and hybridized on the AFM array overnight (16 h) at 4°C on the flat surface. After incubation, arrays were washed 3 times for 10 min each on a Little Dipper processor  with 1× TBST  at room temperature. Secondary antibodies were diluted to 1:5,000 in binding buffer at a concentration of 2 μg/ml. Secondary antibody incubation was done in a plastic Coplin jar  for 3 h at room temperature with gentle shaking on a rocker shaker. First, arrays were incubated with Alexa Fluor 647 AffiniPure goat anti-human IgG that was Fcγ fragment specific , followed by three 10-min washes. Then arrays were incubated with AffiniPure goat anti-human IgM that was Fc5μ fragment specific . Incubation with secondary antibody was followed by three 10-min washes with 1× TBST at room temperature and spin-drying. The array slides were scanned on a NimbleGen MS 200 microarray scanner (Roche) at a 2-μm resolution, with excitation wavelengths of 635 and 537 nm simultaneously. In the images recorded with the laser scanner, the relative fluorescent unit (RFU) signals for all the probes were extracted using Roche Sequencing Solutions image extraction software. The RFU signals for all the probes were extracted from the images using Roche Sequencing Solutions image extraction software. The RFU signals were converted into intensity plots after quantile normalization, background and spatial correction, and deconvolution for redundant peptides. An epitope was considered reactive when the signal intensity for at least three contiguous 12-mer peptides was above the threshold. The signal threshold was defined for each reactive epitope by calculating the mean plus 3 standard deviations of the signal intensity for the same epitope in the negative-control samples.The array data, in the form of relative fluorescence signal intensities from the scanned images (arbitrary units [AU]), were spatially and background corrected and quantile normalized. Signal data points were filtered to retain only immunoreactive peptides that showed the mean +2 standard deviations . A region was considered immunoreactive when the signal intensity for at least three contiguous 12-mer peptides was above the threshold. The signal threshold was defined for each immunoreactive epitope by calculating the mean +2 standard deviations of the signal intensity for the same epitope in negative-control samples , 7. ImmuFor the EV VP1 conserved peptide and EV-D68-specific peptide sequences, comparison of immunoreactivity signal intensity between AFM, non-AFM, Kawasaki disease, and adult CNS groups were evaluated using the Wilcoxon test for paired samples .


The presence of ≥3 oligoclonal bands (OCB) in the cerebrospinal fluid (CSF) without corresponding bands in serum represents a definite pathological pattern, whereas the clinical significance of 1–2 CSF bands (borderline pattern) is poorly investigated.We screened 1986 consecutive CSF and serum samples which were collected over a four-year time period and had results of isoelectric focusing (IEF) available. Of patients with borderline OCB we reviewed individual medical charts for assessment of clinical diagnoses. Where feasible, IEF was replicated and results of follow-up samples were obtained. IEF was performed using polyacrylamide gel followed by immunoblotting and IgG-specific antibody staining. Additionally, we performed a systematic literature review of the diagnostic specificity of OCB using different cut-offs for CSF-restricted bands.Out of 253 patients with borderline OCB, 21.7% had an inflammatory neurological disease (IND) of the central nervous system, comprising 4% multiple sclerosis patients, and 14.2% had a peripheral IND, whereas the remaining 64.1% of patients showed non-inflammatory diseases. Frequency of one or two CSF bands without corresponding serum bands did not differ between the disease groups. In a subgroup of 100 patients IEF was repeated. Of those, 73% were OCB negative, while no sample was positive. In 26 patients IEF results were available of a follow-up sample collected after a median of 27 months. Of those, 4 (15.4%) turned positive. Systematic literature review revealed a diagnostic specificity of OCB of 97% and 92% using a cut-off ≥3 and ≥2 CSF bands in patients with mainly non-inflammatory neurological diseases.The clinical significance of one or two CSF-restricted bands is moderate and, hence, indicates a possible but not reliable proof of intrathecal B-cell activity. Sample re-testing, introduction of an additional diagnostic category, e.g. “possible intrathecal IgG synthesis”, and follow-up lumbar puncture might be possible options to address this scenario. Detection of intrathecal IgG synthesis is part of the routine cerebrospinal fluid (CSF) work-up . IsoelecTherefore, we aimed i) to establish the frequency and disease associations of single or double CSF bands (borderline OCB pattern) in a large number of patients, ii) to perform IEF replication experiments in order to assess reliability and reproducibility of borderline OCB, iii) to determine OCB status in follow-up CSF samples. We also performed a systematic review of literature in order to determine diagnostic specificity of OCB in published cohorts of healthy subjects or patients with non-inflammatory neurological diseases considering different cut-offs for OCB positivity.We have stored the results of CSF and serum sample analyses which were performed for routine diagnostic purposes from patients with mainly neurological diseases in a computerized database at the Neuroimmunology Laboratory of Medical University of Innsbruck–a reference laboratory for Western Austria. In a time period of four years IEF was carried out in 1986 paired CSF and serum samples and results were registered as negative, positive, or borderline. OCB negative status was defined as no bands in CSF and serum, or as bands in the CSF identical to those in serum. OCB positivity was defined as three or more bands in CSF without corresponding bands in serum. Borderline OCB pattern was defined as one or two bands in CSF without corresponding bands in serum.a), two sharp bands in CSF without corresponding bands in serum (type b), and faint bands only in CSF that were not clearly distinguishable from artefacts (type c).Results of borderline OCB pattern were reviewed by a blinded neurologist trained in OCB assessment (FD) and further stratified into three sub-patterns : one shaIndividual medical charts of patients with borderline OCB were reviewed to obtain clinical diagnoses . When moIEF was repeated in a subset of patients with available CSF and serum samples (n = 100). These samples were stored at -20°C until re-analysis. Furthermore, in another subset of patients (n = 26), IEF results of a follow-up sample were available.Detection of OCB was performed by isoelectric focusing and subsequent immunoblotting using IgG-specific antibody staining as previously described by Keir et al. apart from using polyacrylamide instead of agarose gel .3PO4 for the anode and 1N NaOH for the cathode. The samples were run for 2 hours . After that, gels were mechanically blotted on nitrocellulose membranes over 20 minutes. Membranes were then placed in blocking solution  for 30 minutes and washed three times with 0.9% NaCl. For immunolabelling, membranes were incubated for 1 hour with goat anti-human IgG  diluted 1:2000 in 50 ml diluent . Rinsing with tap water for ten times was followed by one wash in diluent for 5 minutes. Thereafter, membranes were incubated with horseradish peroxidase-labelled rabbit anti-goat IgG  diluted 1:1000 in 50 ml diluent for 1 hour. Another rinsing with tap water for ten times was followed by one wash in 0.9% NaCl for 5 minutes. Staining was performed by using 25 mg of 3-amino-9-ethylcarbazole diluted in 10 ml ethanol and 50 ml acetate buffer. After adding 50 μl of 30% hydrogen peroxide, membranes were incubated for 15 minutes. After development of the red-brown bands, membranes were washed with distilled water and air-dried.15 μl of CSF and serum (diluted in Aqua dest. to achieve an IgG concentration of 3 mg/dl as appropriate) were applied to polyacrylamide gel (7.5%) covering a pH range of 3–10. Isoelectric focusing was carried out using 1N Hst September 2018 was performed and returned 252 references. All abstracts were screened and only original articles written in English were considered. Abstracts that primarily did not deal with the diagnostic performance of OCB in the CSF of neurological disease controls were excluded. Only studies were considered which detected OCB by means of IEF followed by IgG-specific immunofixation/ -blotting, specified a cut-off for positive CSF OCB, provided patients’ clinical diagnoses allowing allocation to disease categories and presented results for diagnostic specificity   or healt2 test. Comparisons of non-parametric data such as IgG index between groups was performed by Kruskal-Wallis or Mann-Whitney U test. A p-value <0.05 was considered statistically significant.Statistical analysis was performed using SPSS 25.0 . Frequency distributions were analysed by χWith regard to the systematic review of studies, results of studies using the same cut-off to define OCB positivity (i.e. either ≥2 and ≥3 CSF-restricted bands) were combined in order to calculate diagnostic sensitivity and specificity.No ethical vote was needed because this is an anonymous retrospective analysis of existing data that were obtained in routine diagnostic procedures and used for quality assurance purposes within this study.Out of 1986 CSF and serum sample pairs, 330 (16.6%) showed borderline OCB pattern. Samples were excluded, when available clinical information was not sufficient to allocate patients to a disease group  or when particular requirements defining non-inflammatory neurological diseases, i.e. CSF WBC count, were not met. This resulted in a total of 253 patients with borderline OCB pattern who were finally available for statistical analysis .th-95th percentile: 18.9–75.1) with a balanced sex ratio . Overall, 55 (21.7%) patients showed IND, whereas 36 (14.2%) were classified as PIND, 89 (35.2%) as NIND, 52 (20.6%) as SC and 21 (8.3%) as NND group. Demographic and main CSF findings of each disease group are shown in a occurred in 81 (32.0%), b in 41 (16.2%) and c in 131 (51.8%) of cases. Frequency of sub-pattern did not differ between disease groups  were similar across borderline OCB sub-patterns.Of this cohort, median age was 45.3 years  did not significantly change between original and re-IEF (p = 0.137), however, there was a significant shift to OCB negative status , whereas in 27 samples a borderline OCB pattern was reproduced. In the latter case, an identical sub-pattern occurred in 10 and a different sub-pattern in 17 samples. No borderline OCB pattern tested positive OCB when IEF was repeated. Distribution of the sub-patterns  was associated with reproducibility of OCB. The frequency of reproducible borderline OCB pattern was similar between the disease groups , the other two patients belonged to the NIND group (type b at baseline).In 26 patients, results from a follow-up IEF were available. Six (23.1%) patients belonged to the IND group, whereas 3 (11.5%) were classified as PIND, 12 (46.2%) as NIND, 2 (7.7%) as SC and 3 (11.5%) patients as NND. In total, follow-up samples of 4 (15.4%) patients tested OCB positive, 9 (34.6%) showed again borderline OCB pattern, and 13 (50%) were OCB negative . The median time interval between baseline and the follow-up IEF was overall 27 months; in the OCB positive group 33 months as opposed to 22.5 months in the remaining patients. Two of the four patients who turned OCB positive at follow-up belonged to the IND group . Four studies applying a cut-off of ≥3 CSF bands comprised 1002 patients with solely non-inflammatory diseases. Combined evaluation of these studies revealed a diagnostic specificity of 92% using a cut-off of ≥2 CSF bands, whereas specificity was 97% by a cut-off of ≥3 CSF bands. Diagnostic sensitivity for MS was 87% and 90% applying a cut-off of ≥2 and ≥3 CSF bands, respectively. The main findings are summarized in The detection of OCB in CSF is the gold standard to prove intrathecal IgG synthesis indicating sustained inflammation within the CNS compartment supporting the diagnosis of a variety of neurological diseases, for example multiple sclerosis (MS) . HoweverThe majority of studies on the diagnostic accuracy of OCB (including those cited by published CSF guidelines ) used a First, we reviewed clinical diagnoses of patients with borderline OCB. We observed that 78% of samples were assigned to diseases other than inflammatory CNS diseases, while an IND was found in only 22% of cases. Furthermore, IND did not occur more frequently in one of the OCB sub-pattern, i.e. the diagnostic value of one or two CSF band(s) was similar. A closer look on the clinical diagnoses within the IND group reveals that about half of patients suffered from chronic inflammatory diseases, which are typically associated with intrathecal IgG synthesis (e.g. MS), whereas the remaining showed predominantly acute inflammatory disorders which are not necessarily associated with OCB (e.g. meningitis).c contributing approximately 50% of borderline OCB, one might argue that non-linearity of the pH gradient used for IEF is the cause [Second, we performed replication experiments in a subgroup of patients which revealed that the majority of samples were OCB negative when re-tested. In contrast, borderline OCB pattern was reproduced in only 27%, exactly the same sub-pattern in 10% of patients. To consider various factors that might have influenced reproducibility of IEF results, we compared the frequency of disease groups, CSF storage time and amount of  IgG between samples with reproducible and non-reproducible borderline OCB pattern in the second run and did not find any significant differences. It is not clear how this low reproducibility can be explained. Generally, most IEF results are unambiguous, i.e. either negative–without any CSF bands–or definitely positive–with numerous bands in the CSF. Miscounting e.g. one of ten bands results in a small relative error without changing the overall positive OCB result whereas miscounting one of few bands has a larger relative effect and might lead to a false positive or negative OCB result. Previous studies investigating inter-observer agreement revealed good agreement with respect to the presence of OCB, but only poor agreement with respect to number of CSF bands ,18 incluhe cause . Also, the cause . Irregulhe cause  which diThird, we were able to observe in a small subgroup of patients whether OCB status changed over time. Out of 26 patients, who had another lumbar puncture after a median time of 27 months, 4 (15.4%) turned OCB positive. This observation confirms a previous small study showing that out of 27 patients with a single CSF band, only a minority (33%) developed OCB positivity (defined as ≥2 CSF bands) during follow-up  . There aThere are several limitations of the study. Due to the retrospective nature of the study inclusion of patients depended on availability of clinical data, and selection of samples for replication experiments was based on availability of samples. Furthermore, follow-up samples were collected on a clinical routine basis and thus, were only available in a small subgroup of patients. A potential bias due to CSF storage and additional thawing cycle on reproducibility of borderline OCB cannot be ruled out, even though we did not observe a difference between storage time of samples with reproducible and non-reproducible OCB patterns. It has been shown that IgG are stable even in case of long-term storage  , but theWe showed that not only a single CSF band, as reported previously , but alsAt this point we also want to state that we detected OCB by IEF on polyacrylamide gel followed by immunoblotting and–labelling (as described above). Even when adhering to the recommendations on how to perform OCB detection ,3, severApart from cut-off considerations, borderline OCB might already indicate intrathecal IgG synthesis in some individual cases. Borderline OCB may be connected to the maturation of the humoral immune response. From the initial production of IgG with a wide range of affinity, this response leads to the selection of a few plasma cell clones that secrete high-affinity IgG, i.e. oligoclonal IgG . This prAltogether, we suggest rethinking the currently widely used cut-off of ≥2 CSF bands, as the clinical significance of 1–2 CSF bands is moderate and current evidence  shows thS1 Checklist(PDF)Click here for additional data file.S1 TableAIDS, acquired immune deficiency syndrome; CNS, central nervous system; St.p., status post.(PDF)Click here for additional data file.S2 Tablea, b and c are shown for each disease group (sub-pattern with the highest frequency are marked bold). Abbreviations: IND, inflammatory neurological disease; NIND, non-inflammatory neurological disease; NND, no neurological disease; OCB, oligoclonal bands; PIND, peripheral inflammatory neurological disease; SC, symptomatic control.In the whole cohort (n = 253), frequency of original OCB sub-pattern (PDF)Click here for additional data file.S3 TableAbbreviations: IEF, isoelectric focusing; IgG, immunoglobulin G; OCB, oligoclonal bands; Q, quartile.Quartiles of IgG index were separated by values of 0.44, 0.50 and 0.56. IgG index was calculated as (CSF IgG/ serum IgG)/ . (PDF)Click here for additional data file.S4 TableAbbrevations: IND, inflammatory neurological disease; N, number; OCB, oligoclonal bands; PIND, peripheral inflammatory neurological diseae; SC, symptomatic control.(1) Studies that included only patients with NIND and SC are shown and used for specificity calculation. (2) Studies that included patients with NIND/SC and IND/PIND are shown. The N of patients with IND/PIND are substracted from the total N of controls, but not from the N of OCB negative patients. This approach results in a "Best OCB" scenario, where excluded IND/PIND are considered OCB positive. (3) Studies that included patients with NIND/SC and IND/PIND are shown. The N of patients with IND/PIND are substracted from the total N of controls and from the N of OCB negative patients. This approach results in a "Worst OCB" scenario, where excluded IND/PIND are considered OCB negative. (4) Studies from (1) and (2) are combined. (5) Studies from (1) and (3) are combined. (PDF)Click here for additional data file.S5 TableAbbreviations: CSF, cerebrospinal fluid; N, number; OCB, oligoclonal bands.(PDF)Click here for additional data file.


Classical swine fever (CSF) remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. The causative agent is CSF virus, it is highly contagious, with high morbidity and mortality rates; as such, it is an OIE-listed disease. Owing to a nationwide policy of vaccinations of pigs, CSF is well-controlled in China, with large-scale outbreaks rarely seen. Sporadic outbreaks are however still reported every year. In order to cope with future crises and to eradicate CSF, China should strengthen and support biosecurity measures such as the timely reporting of suspected disease, technologies for reliable diagnoses, culling infected herds, and tracing possible contacts, as well as continued vaccination and support of research into drug and genetic therapies. This mini-review summarizes the epidemiology of and control strategies for CSF in China. Pestivirus in the Flaviviridae family. It is highly contagious and causes disease that can be acute  or chronic. Disease progression is dependent on a number of factors, such as strain virulence, host factors, and secondary pathologies. Typically though the acute disease is characterized by high fever, inappetence, and general weakness followed by neurological deterioration, petechial hemorrhages of the skin, and splenic infarction , is a farction , 2. Thesfarction , 4.Because of its worldwide distribution and its immense economic impact on the porcine industry globally –7, CSF iwww.oie.int). In areas with dense wild boar populations CSF tends to become endemic whereas it is often self-limiting in small, less dense populations. There has however been a disturbing trend of recurrence in some countries that had declared CSF eliminated  (Domestic pigs and wild boars are the known reservoirs for CSFV . Since iBelgium) , 15. ParBelgium) .China has the largest pig breeding industry in the world, accounting for more than half of global production along with ~40 million sows and 7 billion fattening pigs . AccordiA major challenge facing China is preventing the sporadic outbreaks of CSF on the smaller and medium pig farms , 25. LarHaemophilus parasuis, swine pasteurellosis, Streptococcosis, swine enzootic pneumonia, paratyphoid, colibacillosis, toxoplasmosis, and eosporophilosis , pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), swine influenza viruses (SIV), and often secondary infection such as philosis . Some caphilosis , 30. Thinst CSFV , 32. Basnst CSFV . These cChina and surrounding countries, especially countries of Southeast Asia, have long been the epidemic areas . BroadlyThere is a high degree of variation among the prevalent strains of CSFV in China. In the 1990s, the main epidemic strains of CSFV in mainland of China belonged to subtypes 1.1, 2.1, 2.2, and 2.3 . Subtypeet al as 2.1d are now reclassified into subgenotype 2.1g  and full-length E2 gene sequence (1119 nt), phylogenetic analysis showed that the currently prevalent subgenotype 2.1 can be further divided into 10 sub-subgenotypes (2.1a~2.1j), and the isolates previously identified by Peng ype 2.1g . Accordiype 2.1g .In summary, all four subgenotypes existed before 2008; 2.1 was the most predominant, followed by 1.1, 2.2, and 2.3 which were geographically scattered. Under pressure of the C strain vaccine (1.1), the prevalence of subgenotype 1.1 gradually decreased, and subgenotypes 2.2 and 2.3 gradually withdrew from the epidemic areas, leaving subgenotype 2.1, which is the most phylogenetically distant from the vaccine strain, the dominant CSFV strain in China . The epiIn the more than 60 years since the safe and effective attenuated vaccine was developed in 1954 and used in China , CSF hasE2 (gp55), the envelope glycoprotein is where most of the antigenic epitopes of CSFV are concentrated, it is highly immunogenic and induces neutralizing antibodies . The mutMany studies have shown that vaccination has exerted an influence on the evolution of classical swine fever virus . In receTo further investigate viral gene variation, we compared the amino acid and nucleotide sequences of the E2 gene of the C strain vaccine with those of epidemic strains isolated from different regions in China from 2010 to 2015. We found 79.4–99.0% nucleotide homology and 78.0–97.9% amino acid homology .In general, the homology of nucleotide and amino acid sequences between the isolates and C strain is about 80%, except for some isolates that were very similar to the C strain. Given that the % homology of E2 and other major antigenic proteins between the isolates and the C strain is quite different, does this indicate that the vaccine is failing to provide effective immune protection for pigs? Wang et al.  studied There are two strategies for CSF control in the world: preventive immunization and comprehensive culling. For most countries that have no endemic CSF, such as the United States, Canada, Brazil, Chile, South Africa, and the EU countries, culling is used to control CSF. In China, large-scale culling is not feasible, for the present prophylactic vaccination is the best way to reduce the CSF disease burden. The C strain vaccine is widely used in China, but in addition to the problem of being unable to distinguish naturally infected pigs from immunized pigs, use of the C strain vaccine poses other practical problems such as immunization optimization, immunosuppression, vaccine quality, and of course availability and compliance. For example, antibody levels of sows may be above 90%, but the antibody titers of nursery pigs is uneven. Chinese scientists have been working hard to develop new gene-labeled and E2 subunit vaccines for many years and these will be powerful tools for CSF eradication , 81. A rThe biosecurity levels in large-scale pig farms are constantly improving as the Chinese government gives more priority to CSF eradication policies. Listed below are some specific conditions that need to be pursued if the goal of CSF eradication by the end of 2020 is to be met. The conditions are: (1) Cooperative prevention and control. In addition to monitoring and documenting CSFV infection rates and antibody levels, we should also closely monitor the other important swine diseases that are often coinfections with CSFV  , 82, 83;The development of CSFV antigen and antibody detection technologies are important for the prevention and control of CSF. For example, epidemiological investigation and real-time monitoring of antibody levels in immunized pigs are indispensable steps in the process of eradication. Currently there are many diagnostic methods, among which the diagnosis of clinical symptoms is the most direct. But even professional veterinarians are prone to misjudgment in the diagnosis of clinical symptoms and pathological changes. Therefore, to get more reliable results, immunology and molecular biology methods are commonly used to determine levels of CSFV infection as follows . The mosin vitro and in vivo viral challenge assays have demonstrated that replication of CSFV and CSFV-associated pathologies and mortality in these pigs is effectively limited (Depending on vaccination alone though, may not be sufficient to eradicate CSF and the development supplemental antiviral strategies are needed. Anti-CSFV therapies such as capsid-targeted virus inactivation , RNA-hyd limited , and a r limited . Interes limited , 98. If The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intrathecal inflammation, compartmentalized in cerebrospinal fluid (CSF) and in meningeal infiltrates, has fundamental role in inflammation, demyelination, and neuronal injury in cerebral cortex in multiple sclerosis (MS). Since the exact link between intrathecal inflammation and mechanisms of cortical pathology remains unknown, we aimed to investigate a detailed proteomic CSF profiling which is able to reflect cortical damage in early MS.We combined new proteomic method, TRIDENT, CSF analysis, and advanced 3T magnetic resonance imaging (MRI), in 64 MS patients at the time of diagnosis and 26 controls with other neurological disorders. MS patients were stratified according to cortical lesion (CL) load.P < 0.0001), free hemoglobin (Hb) (P < 0.05), haptoglobin (P < 0.0001), and fibrinogen (P < 0.01) were detected. By contrast, CSF levels of sCD14 were significantly (P < 0.05) higher in MS patients with low CL load. Furthermore, CSF levels of sCD163 positively correlated (P < 0.01) with CSF levels of neurofilament, fibrinogen, and B cell‐related molecules, such as CXCL13, CXCL12, IL10, and BAFF.We identified 227 proteins differently expressed between the patients with high and low CL load. These were mainly related to complement and coagulation cascade as well as to iron homeostasis pathway . Accordingly, in the CSF of MS patients with high CL load at diagnosis, significantly higher levels of sCD163 (Intrathecal dysregulation of iron homeostasis and coagulation pathway as well as B‐cell and monocyte activity are strictly correlated with cortical damage at early disease stages. Accumulating neuropathological evidences indicate that multiple sclerosis (MS) is not only characterized by the presence of white matter (WM) demyelinated lesions, readily detectable by conventional magnetic resonance imaging (MRI), but also by focal and diffuse cortical grey matter (GM) demyelination.Compartmentalized inflammation within the meninges of MS patients is spatially related to subpial demyelination, which accounts for about 70% of GM demyelination in progressive MS,γ, CXCL12, IL6, IL8, and IL10, has been associated with a higher GM lesion load both at the time of diagnosis and at death.In a recent combined ex vivo and in vivo study, compartmentalized inflammation in meningeal infiltrates and cerebrospinal fluid (CSF) was found significantly associated with increased subpial demyelination.Several lines of evidence support the notion that regional differences in CSF flow might facilitate focal or diffuse intrathecal trapping of immune cells and stasis of inflammatory molecules, which may diffuse through the pial surface toward the inner cortical layers, in turn mediating a detrimental effect in the adjacent GM.Taken together, these studies suggest a crucial role of CSF/meningeal compartmentalized, intrathecal immune response in the pathogenesis of cortical lesions (CLs). Spurred by the above evidences, using advanced proteomic analysis of CSF combined with advanced MRI techniques, we have attempted to identify more specific molecular pathways involved in early MS‐specific tissue damage at the time of the diagnosis.Sixty‐four consecutive treatment‐naïve relapsing‐remitting MS patients from the MS Centre of Verona University Hospital  were enrolled at diagnosis between March 2015 and February 2016. All the MS patients had a confirmed diagnosis of MS according to Revised McDonald criteria and underwent neurological evaluation, including Expanded Disability Status Scale (EDSS)In each MS patient, MRI was performed at least 2 months after the last relapse using a Philips Achieva 3T MRI Scanner and the following image sets were acquired: (1) 3D T1‐weighted Turbo Field Echo (TFE); (2) 3D Double Inversion Recovery (DIR); (3) 3D Fluid‐Attenuated Inversion Recovery (FLAIR). Optimized parameters of each sequence were set as previously reported.Cerebrospinal fluid samples of MS patients were obtained at least 2 months after the last relapse and within 1 week of the MRI , according to Consensus Guidelines for CSF and Blood Biobanking.The levels of neurofilament light chain (NF‐L) in CSF were measured using Human NF‐light enzyme‐linked immunosorbent assays (ELISA) kit  according to procedures previously optimized.The levels of CSF sCD14 , haptoglobin , free‐hemoglobin (Hb) (Abcam ab157707), and fibrinogen total antigen  were measured in duplicate by ELISA assays according to manufacturer's instructions (Table https://david.ncifcrf.gov), and gene ontology analyses and protein–protein interactions using STRING software as described in detail in Data Six CSF samples obtained from three MS patients representative of the MSlow group and three MS patients representative of the MShigh patients, according to the immunoassay (BioPlex) CSF profiling as described above Table , were seU tests were used to test differences in MRI, EDSS, and proteomic data between MS and control groups or between MSlow and MShigh groups. Pearson correlation coefficients were calculated to analyze the strength of correlation between clinical, MRI, and CSF proteomic data. False discovery rate (FDR), with a significance level of 0.05, was adopted to correct for multiple testing problem. Statistical analysis was performed using GraphPad PRISM‐8‐Software.Nonparametric Mann–Whitney 3) was characterized by about 12‐fold higher numbers and volume of CLs compared to MSlow group   and disease duration . As expected, the female/male ratio showed higher number of females in both groups . The median EDSS was 2.0 in both MSlow group (range = 1.0–4.0) and MShigh group (range = 0.0–5.0),  elevated in MS compared to controls (Table P < 0.01) in the CSF of MShigh compared to MSlow group, whereas only interferon α 2 (IFNα2) and INFλ2 were significantly higher in MSlow than MShigh (P < 0.01) . Conversely, increased sCD163 was found in the CSF of MShigh in comparison with MSlow patients  and MShigh (P < 0.01), compared to controls. Haptoglobin levels in the CSF were significantly elevated in the MShigh group (P < 0.001), compared to the MSlow and control groups; lastly, CSF fibrinogen levels were significantly higher in MSlow (P < 0.05) and MShigh (P < 0.001) compared to controls, and in MShigh compared to MSlow (P < 0.01)  in the CSF of MSlow compared to MShigh patients  and numbers , while an opposite correlation was observed for sCD14   and negatively with sCD14  , IL10 , CXCL13 , and CXCL12  , haptoglobin , and fibrinogen   with the levels of BAFF (r = 0.4), IL10 (r = 0.5), CXCL13 (r = 0.5), CXCL12 (r = 0.5), and TNF (r = 0.5)  with high protein levels of IFNλ2  and with CSF fibrinogen levels   molecules involved in transport and macrophage uptake of free‐Hb. Haptoglobin binds Hb with very high affinity, thus representing an efficient buffering mechanism for free‐Hb liberated from damaged erythrocytes.On the contrary, we found elevated CSF levels of sCD14, an innate immune receptor expressed on monocyte, in MS patients with low CL load. The dissimilar sCD14/sCD163 levels detected in the CSF of patients with different degree of GM damage at the time of diagnosis suggest that an altered balance of monocyte profile and activation that may possibly be linked with different amounts of GM injury. To date, discordant data are available on the correlation between blood CD14 expression and disease activity.+ cells in destruction or phagocytosis of neurons and axons.The correlation found between CSF levels of sCD163 and NF‐L may reflect the activity of CD163β release.γ chain fibrinogen molecule with the CD11b/CD18 receptor induces the activation of numerous intracellular pathways, including the activation of microglia with the production of a pro‐inflammatory milieu (cytokines and chemokines), able to recruit within the CNS monocytes and myelin antigen‐specific Th1 cells, and mediate axonal and neuronal damageIn addition, we found a pronounced increase of fibrinogen, normally absent in healthy brain, in the CSF of MS patients at early stage of the disease with high GM lesion load. Fibrinogen deposition was previously found associated with blood brain barrier disruption, neuroinflammation, and neurodegeneration in MS and several other neurological conditions.Studies on experimental autoimmune encephalomyelitis models suggested that upon BBB disruption, fibrinogen together with prothrombin may enter the CNS and local activation of thrombin may induce fibrin deposition since early stage of the diseaseThis study corroborates our previous suggestions that high CL load is associated with profound CSF inflammatory changes. In particular, CSF abundance of B cell‐linked inflammatory mediators, such as CXCL13, CXCL12, CXCL10, BAFF, IL6, IL10, GM‐CSF, and TNF, supports the hypothesis of a prominent role of B cells in the pathogenesis of CLs.In addition, the CSF correlations found between B cell‐related molecules and macrophage markers may imply complex immunity interactions evocative of immunologic reactions occurring in the subcapsular sinus of lymph nodes or in the marginal zone of the spleen. These data suggest that CSF and meningeal infiltrating B cells might intrathecally interact with macrophages recognizing, internalizing, and retaining antigens through a variety of receptors including toll like receptors, C‐type lectin receptors but also scavenger receptors such as CD163.This work may have some limitations: the proteomic analysis was performed only on a representative subset of MS patients. Considering that the applied mass spectrometry analysis was not quantitative, we performed the data validation with quantitative techniques (ELISA or Luminex experiments) involving a larger number of patients.We suggest for the first time that intrathecal altered balance of complement/coagulation cascade, iron uptake, and innate response, in combination with dysregulated B‐cell immunity, may have a direct role in cortical damage since earliest MS stages and, possibly, in disease progression. Combined evaluation of CSF sCD14/sCD163 balance and of Hb/haptoglobin/fibrinogen levels, as well as of B‐cell activity biomarkers at the time of diagnosis may therefore represent a useful tool that, together with clinical and MRI assessment, may help to predict specific disease immunophenotypes.The local Ethic Committee approved the study . Written informed consent was obtained from each patient.RM and MC contributed to the conception and design of the study; RM, SH, FF, DM, SR, and MC and MC contributed to the acquisition and analysis of data; RM, SH, DM, SM, HL, and MC contributed to interpretation of the results; RM, SH, DM, FF, SR, VM, FL, AV, RN, RR, SM, HL, and MC contributed to drafting and revision of the text.The author(s) declare(s) that there is no conflict of interest.Table S1. Clinical parameters at the diagnosis of the control group.Click here for additional data file.Table S2. Clinical and MRI parameters at the diagnosis of the MS patients examined for CSF TRIDENT proteomic analysis.Click here for additional data file.Table S3. List of proteins and respective accession numbers detected by TRIDENT methodology followed by LC–MS/MS analysis.Click here for additional data file.Table S4. List of the 10 main pathways as key players differentiating the MShigh and MSlow patientsClick here for additional data file.Data S1. Supplementary materials and methods.Click here for additional data file.