Presence and relevance of serum anti-C1P IgG antibodies in relapsing-remitting multiple sclerosis

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Szulc, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7825469/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract There is a growing interest in the role of sphingolipids in the background of multiple sclerosis (MS). The goal of this study was to evaluate the serum levels of antibodies against ceramide-1-phosphate (C1P) subclasses and their relationships with clinical status in MS. The study groups comprised 39 patients with relapsing-remitting MS (RRMS), 26 patients with other neurological diseases (OND) and 12 healthy subjects (HS). Anti-C1P IgG levels in serum were determined using ELISA test. Levels of anti-C18:0-C1P and anti-C24:1-C1P IgG were significantly increased (p = 0.0005; p < 0.0001, respectively) in RRMS compared to HS, while anti-C16:0-C1P and anti-C24:0-C1P IgG – significantly lower (p < 0.0001) in RRMS compared to OND. Validation by ROC and cluster analysis confirmed the ability of these anti-C1P IgG panels to discriminate between the study groups. In addition, level of anti-C24:1-C1P IgG was significantly lower (p = 0.0448) in RRMS patients with relapse than in those with remission. No relationships were found between levels of antibodies in the anti-C1P IgG panel in RRMS group and disease duration, degree of disability or Link index. These findings highlight the relevant role of C1P as a target and/or mediator of autoimmune response in MS and potential value of anti-C1P antibodies as biomarkers in differential diagnosis of this disease. Health sciences/Biomarkers Health sciences/Diseases Biological sciences/Immunology Health sciences/Neurology Biological sciences/Neuroscience biomarkers ceramide C1P lipid antigens multiple sclerosis sphingolipids Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Multiple sclerosis (MS) is a chronic polyphasic disease of the central nervous system (CNS) which causes multifocal damage to the brain and the spinal cord and results in multiple aspects of disability. The complex background of MS involves two major components: autoimmune-mediated inflammatory injury to the myelin sheath and neurodegeneration with progressive axonal loss. These two processes develop in parallel but with different dynamics throughout the disease and their interplay determines MS-related damage to CNS, with emerging clinical picture of this disorder 1 . Despite a substantial progress in understanding MS pathophysiology, some of its aspects have not been yet elucidated, including key elements of initiating immune-mediated inflammatory cascade in CNS or factors modulating highly variable course of the disease 2 . Thus, reliable markers are being extensively sought, which would allow to identify and predict activity and progression of the disease 3 . Investigations have long been focused mainly upon protein molecules as indices of immune activity or neuronal/axonal damage. However, with the advent of metabolomic approach, lipids have also gained attention in this field 4 – 7 . Sphingolipids (SL) seem to be promising candidates for the role of such biomarkers, as the main components of CNS myelin sheath 8 , but also relevant players in cellular signaling and cross-talk between inflammation and neurodegeneration 9 . There has been a solid evidence of accumulation of bioactive SL mediators, specifically ceramide (Cer), in different types of MS lesions and microstructurally altered CNS tissues. Cer and its glycosylated derivatives have been also revealed in the cerebrospinal fluid (CSF), serum, plasma and white blood cells of MS patients 7 , 10 – 13 . Previous research by the members of our team indicated a significant role of Cer derivatives in initiation and shaping of the autoimmune response, targeted at CNS antigens, a key element of MS background. The analysis of the cellular and exosomal profile of bioactive SL mediators in human oligodendroglioma (HOG) cells showed that exosomal Cer subspecies (C16:0-, C24:0- and C24:1-Cer) participate in the apoptosis process induced by pro-inflammatory Th1 cytokines 14 . Comparative assessment of the lipid profile in brain tissues collected during autopsy (demyelinating plaques in the specimens from MS patients vs. unaffected cerebral white matter in the specimens from the control group) allowed to establish specific SL markers that differentiated active and inactive chronic MS lesions 15 . Active lesions were characterized by a significant increase in the main subclasses of dihydroceramide (dhCer), while inactive ones – by decreased levels of dhCer, Cer and sphingomyelin (SM) subclasses and significantly elevated levels of hexosylceramides (HexCer) and ceramide-1-phosphates (C1P). These findings suggested a key element of 'pathological switch' in the metabolic pathways of Cer, driving alternately active or inactive phase of the disease. Furthermore, analysis of anti-Cer immunoglobulin G (IgG) in serum and CSF revealed elevated levels of their particular subclasses (anti-C16:0-, C18:0-, C18:1-, C24:0- and C24:1–Cer) in MS group in comparison with healthy subjects (HS) and those with other CNS disorders 16 . In addition, differences in anti-Cer IgG panels were observed between subgroups with different clinical types of MS [i.a. relapsing-remitting (RRMS) vs. progressive]. For selected anti-Cer IgG subclasses, significant correlations were found between their level in CSF and serum, as well as Link index. The results indicated the relevant role of Cer as a target for autoreactive humoral response, an essential element of MS pathology. Encouraged by the above findings, which supported the need for further research aimed at identification of SL putative biomarkers for MS activity and progression, we undertook the current study focused on particular Cer derivatives. Our main goal was to evaluate the serum level of IgG antibodies against selected C1P subclasses in MS subjects in comparison with the reference groups. We also aimed at investigating their relationships with clinical status. Materials and Methods Human specimens of serum Participants were recruited among the patients hospitalized at the Department of Neurology, Wroclaw Medical University. The following 3 groups were included in this study: i) patients with RRMS (n = 39), diagnosed according to McDonald’s criteria 17 ; ii) patients with other inflammatory disorders of the nervous system (I-OND, n = 13) that comprised chronic inflammatory demyelinative polyneuropathy (CIDP) or immune-mediated vasculitis, as well as iii) patients with non-inflammatory disorders of the nervous system (NI-OND, n = 13), including migraine, cerebrovascular disease and hydrocephalus. Exclusion criteria in RRMS and I-OND groups comprised: recent (within previous 3 months) short-term treatment with corticosteroids, any kind of long-term immunomodulatory or immunosuppressive treatment during at least previous 6 months, current infection and coexisting systemic diseases, in particular inflammatory ones. In the RRMS group, clinical data, such as duration of disease and degree of disability in Expanded Disability Status Scale (EDSS), have been determined based on medical records. The patients were divided into two subgroups, according to their clinical status: those in relapse – exacerbation of disease (RRMS-rel, n = 23) or in remission – a period of stable condition (RRMS-rem, n = 16). In addition, the blood samples were obtained from healthy subjects (HS, n = 12) at the Regional Center of Blood Donation and Blood Treatment in Wroclaw. Participation in the study was anonymous and voluntary. All the subjects gave informed written consent to participate in this study. The project was approved by the Bioethics Committee of Wroclaw Medical University (No. KB 337/2024). The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Approximately 15 ml of peripheral venous blood was collected from each participant. After coagulation the samples were centrifuged at 2000xg at room temperature for 10 min, and afterwards stored at -80°C prior to IgG preparation. Preparation of IgG antibodies from human sera The IgG antibodies were isolated from human serum by affinity chromatography on Protein G GraviTrap™ columns (GE Healthcare). Prior to chromatographic separations, serum samples (0.5 ml) were centrifuged for 10 min at 10,000xg at 4°C, then diluted 1:1 (v/v) with 20 mM phosphate buffer pH 7.0 and filtered through a 0.45 µm Millex®-HV filter. Columns containing 1 ml of packed gel were equilibrated with phosphate buffer and prepared in the described above way sera were loaded. Proteins not bound by protein G were eluted from the columns with phosphate buffer. Next, the column was washed with 12 column volumes of phosphate buffer and the wash fraction was collected. Elution of IgG, bound to the ligand, was performed with 0.1M Gly/HCl pH 2.7 and subsequently neutralized with 1M Tris/HCl pH 9.0. During the fractionation the amount of proteins in all fractions was monitored by measuring the absorbance at 280 nm on 96 well UV half-plate area plates in an Enspire reader. The fractions containing IgG only were concentrated on an Amicon® Ultra100K by ultracentrifugation at 4500xg at 4°C to a final volume of 250 µl. During this process, IgG samples were desalted and albumin residues were removed. The protein concentration of column fractions was measured using bicinchoninic acid (BCA) protein assay kit (Pierce) with bovine serum albumin (BSA) as a standard. Purified IgG antibodies were preserved with 0.02% NaN 3 , aliquoted and stored at -80°C prior to further analysis. SDS-PAGE and Western blotting analysis The electrophoretic separation was performed in a BIO-RAD® Tetra Cell Systems apparatus as described earlier 16 . Briefly, proteins (2 µg) were analyzed on 12% polyacrylamide gel in reducing conditions and either stained with Coomassie Brilliant Blue (CBB) or transferred into 0.45 µm Immobilon P. The separated proteins were detected with, produced in donkey, anti-human IgG (H + L), conjugated with alkaline phosphatase (AP), antibody (1:7500) (Sigma). The reaction was visualized with AP substrates, according to standard procedure. Synthesis of C1P derivatives C1P derivatives were synthesized in the Synthesis Unit of the Lipidomics Shared Resources at the Medical University of South Carolina, Charleston, SC, USA, as previously described 18 . Their purity was checked by the high performance liquid chromatography- tandem mass spectrometry (HPLC-MS/MS). ELISA assay ELISA plates (Immulon 1B, Thermo Fisher Scientific) were coated with selected C1P solutions in absolute ethanol (10 µg/ml; 0.1 ml /well). The plates were incubated overnight at room temperature to facilitate complete solvent evaporation. Next, the plates were washed three times with phosphate buffered saline (PBS; 0.2 ml/well). Nonspecific binding sites were blocked by adding 100 µl of 1% (w/v) BSA in PBS and incubating the plates for 1 hour at 37°C. After washing 5 times with PBS purified IgG (0.1 mg/ml of protein, 0.1 ml /well) from serum of MS patients and individuals from reference groups were added and the plates were incubated overnight at 4°C. In the next step the plates were washed again for 5 times with PBS and then incubated with horseradish peroxidase (HRP) conjugated anti-human IgG (GE Healthcare, 1:2000, 0.1 ml/well). The enzymatic activity was developed by adding peroxidase substrate (Sigma tablets). The colorimetric reaction was stopped by adding of 100 µl of 3N H 2 SO 4 . and the optical density (OD) was read at 492 nm in an Enspire reader. Statistical analysis Ordinary one-way Anova with post-hoc Tukey test was used to estimate differences in respect to age of examined subjects belonging to different groups. The U Mann-Whitney test was used to compare levels of serum IgG anti-C1P antibodies between patients with RRMS and reference groups (HS, OND) as well as between RRMS-rel and RRMS-rem subgroups. The diagnostic significance of immunoreactivities of these IgG antibodies with appropriate C1P antigens was analyzed using received operating characteristic (ROC) curves with clinical meaning considered as zero (values from 0-0.5); limited (values from 0.5–0.7); moderate (values from 0.7–0.9) and high (> 0.9). The Youden index method was used for the determination of cut off points. The IgG samples, for which the area under the curve (AUC) values were established as moderate or high, were subjected to a cluster analysis, a multivariate statistical method. Methods belonging to two different categories were used. A hierarchical Ward’s method was applied to determine the number of clusters. A non-hierarchical method, namely k-means clustering (k-means function of base R) from the selected anti-C1P IgG dataset was used for further analysis. The Spearman’s rank correlation was used to check the associations between measured levels of anti-C1P antibodies and clinical parameters such as disease duration, EDSS and Link index. The ROC analysis was performed using Statistica 13.3 software (StatSoft Inc., Tuls, OK, USA), cluster analysis was executed by OriginPro 2025b (OriginLab Corporation, Northampton, MA, USA). For the remaining statistical analysis GraphPad Prism 7.01 (GraphPad Software Inc., San Diego, CA, USA) was applied. Results Analysis of the study cohort Basic demographic data, including age and sex structure of the patients included in the study are presented in the Table 1 . One-way Anova followed by Tukey’s multiple comparisons test indicated no significant age differences between the study groups. In RRMS group, duration of the disease ranged from 1 to 108 months (mean 22 months) and EDSS score – from 1 to 4 (median 1.5) points. All the RRMS patients (n = 39) had magnetic resonance imaging (MRI) of the brain performed, with typical demyelinative lesions. In 32 patients the results of CSF testing were available, with confirmed presence of IgG oligoclonal bands and calculated Link index (increased value ≥ 0.7 in 20 cases). Table 1 Basic demographics of the study subgroups. Abbreviations: I-OND- inflammatory other neurological diseases; NI-OND- non-inflammatory other neurological diseases; rel- relapse; rem- remission; RRMS- relapsing-remitting multiple sclerosis; SD- standard deviation. Subgroup Number of patients Female/Male Age (years) Mean ± SD (Min-Max) RRMS-rel 23 16/7 32.2 ± 10.1 (28–65) RRMS-rem 16 6/10 33.1 ± 7.5 (21–48) I-OND 13 6/7 42.5 ± 18.9 (22–83) NI-OND 13 7/6 41.3 ± 13.8 (19–64) Analysis of Protein G-isolated and purified from human serum IgG antibodies The IgG antibodies present in human serum were isolated by affinity chromatography on Protein G Gravi Trap columns. A representative preparation of IgG antibodies from 0.5 ml of serum is shown on Fig. 1 . In a typical elution profile obtained during the isolation of IgG from RRMS-rel patient two peaks were visible (Fig. 1 A). The first one − Fr. 1 − represented the proteins unbound by protein G. The second one, Fr. 3, corresponded to four isotypes of IgG (IgG 1 , IgG 2 , IgG 3 and IgG 4 ), bound by protein G. The obtained fractions were subjected to qualitative and quantitative analysis. The CBB staining (Fig. 1 B) obtained as a result of electrophoretic separation of samples, collected during the isolation of serum IgG from both RRMS-rel and RRMS-rem, indicated that in both cases the products obtained as a Fr. 3 were IgG antibodies (lanes 4 and 8). This is evidenced by two characteristic bands with a molecular mass of 50 and 25 kDa, corresponding to the heavy and light chain of immunoglobulin G, as appeared under these conditions. In the remaining lanes (lanes 1–3 and 5–7), distinct bands with a molecular mass of about 65 kDa were observed, originating from other proteins found in the serum. Presumably, these bands indicate the presence of albumin, which constitutes up to 65% of all serum proteins, and perhaps the heavy chain of immunoglobulin M, constituting about 5 to 10% of serum immunoglobulins. Quantitation of proteins in the various fraction demonstrated high and comparable total protein amounts in Fr. 1 and residual protein amounts in Fr. 2. The preparation resulted in the recovery of 5.6 ± 0.9 mg of purified IgG (Fr. 3), a predominant class of antibody in serum, which corresponded to 14.3% ± 2.4 of all serum proteins (Supplemental Table 1S). The absence of IgG in Fr. 1 and 2 (Fig. 1 C; lanes 2 and 3, respectively) and the presence of the purified IgG antibodies was confirmed by Western blotting (Fig. 1 C, lane 4). This analysis indicated the high efficiency of the IgG purification method used. Altered levels of serum anti-C1P IgG in RRMS patients The Protein G-purified IgG antibodies from serum of MS patients (Fr. 3) were evaluated for the presence of selected C1P subclasses binding activity, using an ELISA (Fig. 2 and Supplemental Fig. 1S). The binding activity of serum IgG to C16:0-C1P was significantly higher in RRMS patients (median value: 0.4535) in comparison to HS group (median value: 0.34) and significantly lower in relation to OND group (median value: 0.7725), with significance of p = 0.0425 and p < 0.0001, respectively (Fig. 2 A). The binding activity of serum IgG to C18:0-C1P in RRMS patients (median value: 0.7715) was significantly increased in comparison to HS group only (median value: 0.4635) with significance of p = 0.0005 (Fig. 2 B). In turn, there were no significant differences in the binding activity of serum IgG to C18:1-C1P in RRMS individuals in relation to both reference groups (Fig. 2 C). In contrast, RRMS patients manifested lower level of anti-C24:0-C1P serum IgG (median value: 0.2725) in comparison to OND group (median value: 0.924) with significance of p < 0.0001 (Fig. 2 D). The binding activity of serum IgG to C24:1-C1P represented the pattern similar to C16:0-C1P. Interestingly, serum IgG derived from RRMS patients exhibited the highest reactivity for C24:1-C1P among all C1P subclasses examined (median value: 1.001) in comparison to HS group (median value: 0.4915, p < 0.0001), while the level of anti-C24:1-C1P IgG was significantly decreased in RRMS in comparison to OND group (median value: 1.25; p = 0.0183) (Fig. 2 E). The presented above results, obtained by ELISA test, are summarized in Table 2 . Table 2 The levels of reactivity of selected anti-C1P serum IgG in RRMS patients in comparison to healthy subjects (HS) and individuals with other neurological diseases (OND). Significant differences compared to reference groups were indicated as follows: a-HS, b- OND. Abbreviations: C1P- ceramide-1-phosphate; SD- standard deviation. Serum IgG Group RRMS N = 39 HS N = 12 OND N = 26 MEAN ± SD MEAN ± SD MEAN ± SD Anti-C16:0-C1P 0.4745 ± 0.21 p = 0.0425 a p < 0.0001 b 0.3534 ± 0.1224 0.7838 ± 0.3031 Anti-C18:0-C1P 0.7512 ± 0.2753 p = 0.0005 a 0.4853 ± 0.1202 0.7789 ± 0.2951 Anti-C18:1-C1P 0.3478 ± 0.1289 p = 0.0136 a p < 0.0001 b 0.2555 ± 0.08237 0.7749 ± 0.3728 Anti-C24:0-C1P 0.3192 ± 0.1579 p < 0.0001 b 0.3878 ± 0.2019 0.9394 ± 0.3448 Anti-C24:1-C1P 0.9526 ± 0.3096 p < 0.0001 a p = 0.0183 b 0.4265 ± 0.2061 1.17 ± 0.4374 Next, we reanalyzed the data considering the division of RRMS patients into subgroups in relapse and in remission (RRMS- rel and RRMS -rem) and OND patients – into subgroups with inflammatory or other etiology of disease (I-OND, NI-OND) (Supplemental Fig. 1S). The Mann Whitney test showed significantly higher levels of serum IgG derived from both RRMS subgroups against most C1P subclasses (except of C24:0-C1P) compared to HS, particularly significant for IgG anti-C18:0-C1P and C24:1-C1P (p = 0.0024 and p < 0.0001, Supplemental Fig. 1S, panel B & E, respectively). The level of IgG anti-C24:1-C1P in RRMS-rel subgroup was significantly lower than in RRMS-rem (p = 0.0448), as well as lower in both RRMS subgroups in relation to NI-OND only (p < 0.0001 and p = 0.0004, Supplemental Fig. 1S, panel E, respectively). The level of IgG anti-C24:0-C1P in both RRMS subgroups was significantly lower than in I-OND and NI-OND (p < 0.0001, Supplemental Fig. 1S, panel D). The above results in details are included in Supplemental Table 1S. ROC analysis In order to validate ELISA results, we performed ROC analysis for all serum anti-C1P IgG levels in RRMS patients, as presented in Table 2 . The results of ROC curve analysis for anti-C1P IgG data sets with the AUC value higher than 0.700 are presented in Fig. 3 . The cut-off points, determined by the Youden index method, are shown in Table 3 . Table 3 Summary of receiver operating characteristic (ROC) curves for serum anti-C1P IgG levels in the study groups. Abbreviations: AUC- area under the curve; C1P- ceramide-1-phosphate; HS- healthy subjects; OND- other neurological diseases; RRMS- relapsing-remitting multiple sclerosis. Antibody Examined groups AUC AUC with 95% confidence interval Youden index Cut off point Sensitivity Specificity P value Anti-C16:0-C1P RRMS vs. HS 0.653 0.506–0.800 0.369 0.523 0.436 0.933 0.0417 RRMS vs. OND 0.798 0.682–0.914 0.526 0.701 0.654 0.872 0.0000 Anti-C18:0-C1P RRMS vs. HS 0.781 0.661–0.904 0.564 0.702 0.564 1.000 0.0000 RRMS vs. OND 0.532 0.385–0.679 0.154 0.952 0.385 0.769 0.6682 Anti-C18:1-C1P RRMS vs. HS 0.569 0.381–0.758 0.400 0.297 0.667 0.733 0.4718 RRMS vs. OND 0.153 0.047–0.258 0.654 0.619 0.000 0.346 0.0000 Anti-C24:0-C1P RRMS vs. HS 0.409 0.222–0.595 0.123 0.17 0.923 0.200 0.3371 RRMS vs. OND 0.942 0.886–0.998 0.769 0.801 0.769 1.000 0.0000 Anti-C24:1-C1P RRMS vs. HS 0.769 0.603–0.936 0.595 0.727 0.795 0.800 0.0015 RRMS vs. OND 0.654 0.508–0.800 0.372 1.182 0.577 0.795 0.0386 Anti-C16:0-C1P IgG level indicated a moderate value in differentiating RRMS group from OND group (Fig. 3 B), with sensitivity 65.4% and specificity of 97.2%, respectively (proposed cut off point: 0.701, AUC = 0.798). Anti-C18:0-C1P IgG level showed moderate discriminating value between RRMS and HS groups (Fig. 3 C), with sensitivity and specificity of 56.4% and 100%, respectively (proposed cut off point: 0.702, AUC = 0.781). The level of anti-C24:0-C1P IgG had a high discriminating value between RRMS group and OND group, with sensitivity of 76.9% and specificity of 100%, respectively (proposed cut off point: 0.801, AUC = 0.942) (Fig. 3 F). The level of anti-C24:1-C1P IgG exhibited a moderate value in differentiating between RRMS group and HS group, with sensitivity of 79.5% and specificity of 80% (proposed cut off point: 0.727, AUC = 0.769) (Fig. 3 G). Cluster analysis Based on the results of ROC analysis, serum anti-C1P IgG levels with AUC equal or greater than 0.769 were selected to perform cluster analysis. The number of expected clusters was pre-determined by using the hierarchical Ward’s method (Supplemental Fig. 2S). Relative level of anti-C16:0-, anti-C18:0-, anti-C24:0- and anti-C24:1-C1P serum IgG was subsequently used to perform k-means clustering to categorize the samples examined into 3 clusters (Fig. 4 A). Cluster 1 comprised 34 subjects: all healthy ones; 16 patients with RRMS (including 9 ones from RRMS-rel subgroup) and 6 individuals with OND (containing 5 from I-OND subgroup). Cluster 2 included 23 RRMS patients (14 from RRMS-rel, 9 from RRMS-rem subgroup) and 9 OND ones (5 from I-OND and 4 from NI-OND subgroup). Cluster 3 consisted of OND patients only, with the majority belonging to NI-OND subgroup (8 out of 11) (Supplemental Fig. 3S). Significant differences in anti-C1P IgG panels between the study groups (RRMS vs. HS: anti-C18:0- and anti-C24:1-C1P IgG; RRMS vs. OND: anti-C16:0- and anti-C24:0-C1P IgG) (Fig. 2 ), with discriminatory ability of these IgG subsets, confirmed by ROC analysis (Fig. 3 , Table 3 ), together with similar patterns of relative levels of anti-C16:0- / anti-C24:0-C1P, as well as anti-C18:0- /anti-C24:1-C1P IgG (Supplemental Fig. 4S), supported the notion that anti-C1P IgG panels might differentiate pathological from physiological condition but also reflect distinct biological processes (i. e. neuroinflammation vs. neurodegeneration) with different degrees of sensitivity. To check this hypothesis, a k-means analysis was subsequently performed to divide the appropriate IgG samples into 2 clusters. Relative levels of anti-C18:0- and anti-C24:1C1P serum IgG were subjected to k-means clustering to differentiate RRMS from HS group (Fig. 4 B). Cluster 1 comprised 28 subjects (12 HS and 16 RRMS, almost equally distributed between RRMS-rel and RRMS-rem subgroups) whereas cluster 2 was represented by 23 RRMS patients (mainly RRMS-rel subgroup) (Supplemental Fig. 5S). In turn, relative levels of anti-C16:0- and anti-C24:0-C1P serum IgG were subjected to k-means clustering to differentiate RRMS from OND group (Fig. 4 C). Cluster 1 comprised 45 subjects: all RRMS patients (n = 39) and 6 OND ones (mainly from I-OND subgroup), whereas cluster 2 was composed of 20 OND subjects, mainly from NI-OND subgroup (Supplemental Fig. 6S). Correlation analysis between selected serum anti-C1P IgG derived from RRMS patients and clinical parameters Next, we examined whether there are any correlations between anti-C1P IgG titers (as reflected by OD at 492 nm) in RRMS group and MS-related clinical parameters. The levels of anti-C16:0-C1P, anti-C18:0-C1P, anti-C18:1-C1P, anti-C24:0-C1P and anti-C24:1-C1P IgG in RRMS did not correlate with disease duration (Supplemental Fig. S7, panels A, D, G, J and M) or EDSS score (panels B, E, H, K and N), accordingly. No associations were found between the levels of these antibodies and Link index, either (Supplementary Fig. S7, panels C, F, I, L and O, respectively). The summary of this analysis is displayed in Table 4 . Table 4 Correlation of serum anti-C16:0-C1P, anti-C18:0-C1P, anti-C18:1-C1P, anti-C24:0-C1P, anti-C24:1-C1P IgG levels in RRMS group with clinical parameters. Abbreviations: C1P- ceramide-1-phosphate; EDSS- Expanded Disability Status Scale; RRMS- relapsing-remitting multiple sclerosis. Serum IgG Clinical parameter Spearman’s correlation coefficient predictor (r value) P value Anti-C16:0-C1P Disease duration -0.1753 0.2857 EDSS 0.04531 0.7842 Link index -0.1286 0.4759 Anti-C18:0-C1P Disease duration -0.2746 0.0906 EDSS 0.06427 0.6975 Link index 0.0565 0.7548 Anti-C18:1-C1P Disease duration -0.251 0.1232 EDSS 0.08361 0.6128 Link index -0.2063 0.2494 Anti-C24:0-C1P Disease duration -0.1884 0.2506 EDSS -0.01253 0.9396 Link index -0.2496 0.1613 Anti-C24:1-C1P Disease duration -0.1706 0.2992 EDSS 0.03706 0.8228 Link index -0.1914 0.2859 Discussion Cer products have been suggested to be involved in the main mechanisms underlying MS pathology, including those - recently highlighted - driven by B cells. Activation of B cells together with cytokine stimulation promote tissue penetration and destabilization of membrane lipids in CNS, resulting in damage to myelin sheath. SL (in particular Cer derivatives) are essential for maintenance of lipid membranes integrity, so changes in their metabolism and proportion correspond with dynamic processes of demyelination and repair and their pathophysiological consequences 19 . Although in the aforementioned studies there is convincing evidence for Cer abnormalities in MS, it relies mainly on preclinical models and their translation into human clinical context, with clear identification of Cer related mechanisms of action and potentially relevant MS biomarkers, still remains a challenge 20 4–7 . Based on results of our previous studies 14 – 16 and scarce literature data 10 , 12 , 13 , 21 , 22 , here we focused on particular Cer derivative – C1P, aiming to investigate its putative role as a target and/or mediator of autoimmune response in MS. C1P, a major metabolite of Cer, can be formed through a stereospecific reaction of phosphorylation, due to direct action of Cer kinase (CerK), while the reverse reaction (conversion of C1P to Cer) is mediated by C1P phosphatase. An appropriate balance between these two molecules is required to ensure cells and tissues homeostasis through regulation of relevant metabolic or signaling pathways. Thus, coordinated action of CerK and C1P phosphatases may have crucial importance for maintaining this balance. Its disturbances with a shift towards accumulation either Cer or C1P may result in metabolic dysregulation, supposedly involved in the background of autoimmune diseases 23 , neurodegenerative disorders 24 , cardiovascular illnesses 25 or cancer 26 . It has been demonstrated that C1P exerts pro-inflammatory properties; however, the underlying mechanisms have not been yet fully elucidated. One of the major function of C1P is its capability to activate cytosolic phospholipase PLA 2 (cPLA 2 ), which mediates release of arachidonic acid 27 - the substrate used for production of eicosanoids, such as prostaglandin E 2 (PGE 2 ). This is aligned with the fact that MS patients exhibit higher PGE 2 expression/activity in CSF 28 , lymphocytes 29 as well as demyelinative lesions in brain tissue 30 . A sharp increase of this pro-inflammatory mediator was observed in active stage of MS 31 , and experimental studies with the use of cyclooxygenase (COX) inhibitors, which oppose C1P action by preventing PGE 2 synthesis, suggested their beneficial effect as potential options in MS therapy 32 . Other pro-inflammatory activities affected by C1P include: activation of degranulation in mast cells 33 , stimulation of phagocytosis in neutrophils 34 , as well as stimulation of macrophage migration 35 . C1P also increases transport of P-glycoprotein, which regulates the permeability of the blood brain barrier via COX-2/PGE 2 signaling 36 . On the other hand, it has been shown that C1P might also act as an anti-inflammatory agent, under specific conditions. Anti-apoptotic effects of C1P, promoting cell survival, are related with its ability to block activity of serine palmitoyltransferase 37 , acid sphingomyelinase 38 and TNF-α 39 . Furthermore, it promotes macrophage chemoattractant protein-1 (MCP-1) release in different types of cells 35 . Taken together, C1P seems to act on its own rights, executing regulatory effects and modulating cell functions depending on circumstances. In one of the previous reports by our team members, an increase in C1P subspecies (mainly C18:1-C1P and C24:1-C1P) was demonstrated in inactive MS lesions in comparison with specimens representing other CNS disorders or normal brain tissue. On the contrary, active MS plaques were characterized by a significant decrease of C1P subspecies 15 . The studied autopsy samples from MS subjects represented advanced stage of disease, associated with chronic inflammation and presumably extensive neurodegenerative component. In the present study we focused on earlier phase of the disease (i. e. RRMS), when active immune-mediated inflammation is a predominating mechanism of CNS injury - therefore antibodies as markers of autoimmune humoral response were chosen as the subject of investigation. The main findings presented here included elevated levels of nearly all analyzed anti-C1P IgG in serum of RRMS patients in comparison to HS, with the highest significance for anti-C24:1-C1P and anti-C18:0-C1P (Fig. 2 E and B). This striking consistency in the results from both studies, despite differences in MS stages and studied material (brain tissues vs. serum), indeed suggests the relevant role of C1P in MS pathology. It can be hypothesized that each stage of MS is associated with the concerted action of Cer derivatives, with their changing interactions in this pathological cross-talk. It should be mentioned that the level of IgG anti-C24:1-C1P was significantly lower in both RRMS subgroups in relation to NI-OND only (Supplemental Fig. 1S, panel E), whereas the level of IgG anti-C24:0-C1P in both RRMS subgroups was significantly lower than in both OND subgroups (Supplemental Fig. 1S, panel D). Overall, these results suggest that increased humoral response against long-chain C1P subspecies is particularly specific for MS and it does not just reflect overall neuronal injury or dysregulated immune-mediated inflammation in CNS. Importantly, the reliability of the above findings was supported by eliminated impact of immunosuppressive/immunomodulatory drugs, as the patients were included in the study prior to such treatment or after appropriate wash-out period. There is an evidence that fatty acid chain length of Cer can determine biophysical properties of this SL mediator 40 . In physiological conditions, medium chain Cer (C16 and C18) are known to be enriched in cerebral gray matter, while long chain ones (C24) - in white matter and myelin sheath 41 . Considering distribution of Cer derivatives in the brain, presence of the medium chain ones would reflect neuronal injury 42 , while the long chain ones – oligodendrocytes degradation 14 . The long chain Cer (C24) are also known to be the major Cer subspecies present in serum/plasma whereas C1P constitute only 0.15% of all SL components 43 . However, it should be taken into account that the pattern of anti-Cer IgG in body fluids, revealed by our previous study 16 reflected metabolic perturbations of Cer, supposed to occur due to processes involved in MS background. The anti-Cer IgG alterations in serum (unlike in the CSF) were observed only for Cer containing medium chains (C16:0-, C18:0- and C18:1-Cer) with no change for the long chain ones 16 . Current findings revealed altered patterns of IgG against C1P in serum of RRMS patients. Here we found that anti-C16:0-C1P (Fig. 2 A), anti-C18:0-C1P (Fig. 2 B) and anti-C24:1-C1P (Fig. 2 E) serum IgG levels were increased in RRMS patients in comparison with HS group. In turn, levels of IgG against C16:0-C1P (Fig. 2 A), C24:0-C1P (Fig. 2 D) and C24:1-C1P (Fig. 2 E) were decreased in RRMS in relation to OND (including distinguished subgroup of inflammatory ones). In addition, the results of ROC analysis indicated that the determination of anti-C18:0-C1P and anti-C24:1-C1P serum IgG levels might be useful for discriminating RRMS from HS group (Fig. 3 , panel C and G), whereas anti-C16:0-C1P and anti-C24:0-C1P serum IgG levels – for discriminating RRMS from OND group (Fig. 3 , panel B and F). Furthermore, the usefulness of the above anti-C1P IgG subsets in distinguishing pathological from physiological conditions, as well as disorders with different etiology, was confirmed by means of multivariate statistical method of cluster analysis (Supplemental Fig. 5S and 6S). Despite somehow subjective selection of variables for clustering and the number of clusters (justified by the results of initial analysis), the findings from this multivariate technique support the concept that panels/sets of anti-C1P IgG may be of greater discriminating value than single immunological parameters (Fig. 4 ). Such a panel including anti-C16:0-C1P, anti-C18:0-C1P, anti-C24:0-C1P and anti-C24:1-C1P IgG might be considered for further investigation towards potential clinical implications - the use of anti-C1P IgG as biomarkers in differential diagnosis at MS onset, especially in atypical cases. As a result of more precise analysis within RRMS group, the level of anti-C24:1-C1P IgG was found to be significantly lower in those with relapse than those in remission (Supplemental Fig. 1S, panel E). These findings may indicate that alterations of Cer metabolism (revealed by presence of corresponding IgG) are associated with changing phases of disease activity. Few attempts have been made to use anti-Cer antibodies as biomarkers of activity in other disorders, including those of the nervous system. In leprosy, presence of anti-Cer antibodies was found to be related to extent of nerve sheath damage and particular bacterial strains as pathogenic factors 44 . In the rare autoimmune condition, encephalomyeloradiculoneuropathy (EMRN), accumulation of species-specific Cer (mainly long chain ones) was demonstrated in CSF and presence of anti-neutral glycolipids antibodies – in CSF and serum. Profile of these antibodies allowed to differentiate patients with ERMN from the reference group with Parkinson disease, and their titers decreased after effective immunomodulatory treatment. Dysregulation of glycolipids metabolism and its impact upon invariant natural killer T cell development, as well as abnormal complement activation, were suggested to play a relevant role in EMRN pathophysiology 41 , 45 . Furthermore, in non-small-cell lung cancer (NSCLC) anti-Cer antibodies were supposed to be produced by the tumor and its microenvironment, and to neutralize pro-apoptotic effect of Cer. Significantly elevated levels of anti-Cer antibodies were revealed in serum and bronchial wash fluid samples from NSCLC group (in comparison with HS) and they showed a tendency (though not significant) for prolonged overall survival 46 . Evaluation of activity in MS has been so far based on clinical assessment and radiological indices (new and/or contrast-enhanced lesions in MRI). However, there is an ongoing search for specific and reliable body fluid biomarkers of disease activity 8 . Their clinical applications would include i.a. differentiating relapse from pseudorelapse (temporary aggravation of symptoms) or defining active phase in progressive stage of the disease, and in long term follow-up – monitoring of MS course and response to treatment. In view of our previous and current findings, Cer derivatives and antibodies against them seem indeed promising candidates for the role of such biomarkers. Although no relationships were found in our study group between immunological measures and MS-related clinical data (Supplemental Fig. S7), potential clinical utility of these findings should not be discredited. The group was characterized by a relatively short MS duration and mild disability, and marked individual differences in disease course in MS populations must be taken into account. Perhaps in a larger group of patients and in prospective observation these relationships would appear more relevant. Indeed, a small sample size and focusing only on RRMS should be considered as limitations of our study, as its results may not accurately reflect abnormalities in SL metabolic pathways in different types and stages of the disease. Another limitation is associated with the fact that anti-C1P IgG presence was determined only in serum and not in CSF. Nevertheless, in view of long-term course of MS and need for regular follow-up, serum/plasma biomarkers are much desired due to their better availability. Based on experiences with other body fluid biomarkers, their serum level may adequately reflect their production in CNS 8 . There is also some evidence that peripheral blood may contribute to formation of immune complexes due to selective transport and the blood-brain barrier (BBB) leakage 47 . To our best knowledge, this study for the first time focuses on the presence and meaning of anti-C1P antibodies in serum of MS patients and provides evidence of their potential relevance. The results shed some light on the perturbation on SL metabolism (especially significance of balance between Cer and C1P) in the background of neurological diseases, specifically MS. Encouraged by these findings, further investigation is planned with the use of IgG against particular C1P subclasses, analyzed parallel in blood and CSF, in larger groups of MS patients and with prospective observation of immunological and clinical parameters. Hopefully, these studies will lead to a better understanding of the role of this intriguing phosphosphingolipid in pathophysiology of MS, with emerging clinical implications. Conclusions The serum levels of IgG anti-C1P subspecies (especially long-chain ones) differed significantly between the patients with RRMS and healthy subjects, as well as the patients with other neurological diseases. Differences were also found between subgroups of RRMS patients with relapse or with remission. These findings indicate the relevant role of C1P as a target and/or mediator of autoimmune response in MS. Potential value of anti-C1P antibodies as biomarkers in differential diagnosis of MS and monitoring disease activity warrants further investigations. Abbreviations AP Alkaline Phosphate AUC Area Under Curve BBB Blood Brain Barrier BCA Bicinchoninic Acid BSA Bovine Serum Albumin C1P Ceramide-1-Phosphate CBB Coomassie Brilliant Blue Cer Ceramide CerK Ceramide Kinase CIDP Chronic Inflammatory Demyelinative Polyneuropathy CNS Central Nervous System COX-2 Cyclooxygenase-2 cPLA 2 ­ Cytosolic Phospholipase A 2 CSF Cerebrospinal Fluid EAE Experimental Autoimmune Encephalomyelitis EDSS Expanded Disability Status Scale ELISA Enzyme-Linked Immunosorbent Assay EMRN Encephalomyeloradiculoneuropathy HexCer Hexosylceramide HOG Human Oligodendroglioma HPLC-MS/MS High Performance Liquid Chromatography tandem mass spectrometry HRP Horseradish Peroxidase HS Healthy Subjects IgG Immunoglobulin G I-OND Inflammatory Other Neurological Diseases MCP-1 Macrophage Chemoattractant Protein-1 MRI Magnetic Resonance Imaging MS Multiple Sclerosis NI-OND Non- Inflammatory Other Neurological Diseases NSCLC Non-Small-Cell Lung Cancer OD Optical Density OND Other Neurological Diseases PBS Phosphate Buffered Saline PC Principal Component PGE 2 ­ Prostaglandin E 2 rel Relapse rem Remission ROC Received Operating Characteristic RRMS Relapsing Remitting Multiple Sclerosis SD Standard Deviation SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis SL Sphingolipid SM Sphingomyelin Declarations Acknowledgments The authors are grateful to Mrs. Malgorzata Cyrul (Wroclaw Medical University) for centrifugation, aliqoting and labeling serum specimens. The authors also appreciate Dr. Karol Marschollek (Wroclaw Medical University) consultations regarding ROC analysis. Funding This research was supported in part by Wroclaw Medical University (SUBK.C220.24.081) to J.C.-L., by the National Science Centre (NCN, Krakow, Poland; Preludium 23, Award no. UMO-2024/53/N/NZ3/01093) to A.J.-A. and by the Lipidomics Shared Resource, Holling Cancer Center, Medical University of South Carolina (P30 CA 138313) to Z.M.S. Author contributions J.C.-L.: investigation, resources, clinical data curation and funding acquisition. A.J.-A.: investigation, methodology, data curation, formal analysis, visualization and review. Z.M.S: investigation, methodology, review and editing. J.G. and S.B.: supervision and writing-review. A.P.-D.: conceptualization, investigation, resources, clinical data curation, formal analysis, writing-original draft preparation, review and editing. 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07:50:53","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2853948,"visible":true,"origin":"","legend":"","description":"","filename":"AntiC1PAbsinMSSupplementalMaterialSciRepformatted.docx","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/e310a2a03d967d36133cdcdb.docx"},{"id":95226959,"identity":"be4b6b01-cc85-4bd8-898d-64893175dc36","added_by":"auto","created_at":"2025-11-05 16:31:56","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155609,"visible":true,"origin":"","legend":"","description":"","filename":"580571f377b2401a9752233fc5fd4ce71enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/d9bfa192d17839443351ee7b.xml"},{"id":95227825,"identity":"edfaf59a-1ef3-4ca8-8e51-c82c41c2d071","added_by":"auto","created_at":"2025-11-05 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07:50:53","extension":"html","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":170395,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/0e7ac32f42a3957471155b13.html"},{"id":95227945,"identity":"7a13b9e4-5094-4441-ad88-d72329eaa758","added_by":"auto","created_at":"2025-11-05 16:33:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":243917,"visible":true,"origin":"","legend":"\u003cp\u003ePurification of IgG antibodies from human serum. Fractionation of human serum on Protein G GraviTrap\u003csup\u003eTM\u003c/sup\u003e column (panel \u003cstrong\u003eA\u003c/strong\u003e). Amount of\u0026nbsp; 0.5 ml of serum, diluted 1:1 with 20 mM phosphate buffer, pH 7.0, was applied on the column (Fr. 0- load). This fraction was recycled 4 times (tubes [1-4]- Fr. 1- flow trough) and washed with phosphate buffer (tubes [5-16]- Fr. 2- wash). Bound IgGs were eluted with 0.1 M Gly/HCl buffer, neutralized with 1M Tris/HCl, pH 9.0 (tube 17- Fr. 3- elution) and concentrated on an Amicon® Ultra100K. Products present in the above fractions were analyzed on 12% SDS-PAGE and stained with 0.25% Coomassie Brillant Blue R-250 (panel \u003cstrong\u003eB\u003c/strong\u003e) or subjected to Western blotting analysis and immunostained with anti-human IgG (panel \u003cstrong\u003eC\u003c/strong\u003e). \u003cstrong\u003eLane 1-\u003c/strong\u003e Fr.0, \u003cstrong\u003elane 2-\u003c/strong\u003e Fr. 1, \u003cstrong\u003elane 3-\u003c/strong\u003e Fr. 2, \u003cstrong\u003elane 4- \u003c/strong\u003eFr. 3. The molecular mass of the reference standards is shown on the left side.\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/acd815d6b7d3989a16b30f3a.jpg"},{"id":95177542,"identity":"50a88d3c-dfab-4777-8a89-47ea757c9a1d","added_by":"auto","created_at":"2025-11-05 07:50:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":459092,"visible":true,"origin":"","legend":"\u003cp\u003eReactivity of purified IgGs purified from human serum against: \u003cstrong\u003eA\u003c/strong\u003e) C16:0-C1P, \u003cstrong\u003eB\u003c/strong\u003e) C18:0- C1P, \u003cstrong\u003eC)\u003c/strong\u003e C18:1- C1P, \u003cstrong\u003eD\u003c/strong\u003e) C24:0- C1P and \u003cstrong\u003eE\u003c/strong\u003e) C24:1- C1P by ELISA test. All boxes represent the 25th–75th percentile while horizontal lines inside of the boxes point out the median (50th percentile). Whiskers extend from the boxes indicate the range of the data - minimum and maximum values, accordingly. Differences between groups of nonparametric data were determined by the Mann Whitney test using GrapPad Prism 7.01. RRMS (n=39), HS (n=12), OND (n=26). Abbreviations: HS- healthy subjects; OND- other neurological diseases; RRMS- relapsing remitting multiple sclerosis.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/2b167dd3299cf6565406e3d6.jpg"},{"id":95226818,"identity":"779e7be6-c9b6-4b23-a9ee-451b9827a1a3","added_by":"auto","created_at":"2025-11-05 16:31:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":388171,"visible":true,"origin":"","legend":"\u003cp\u003eReceiver operating characteristic (ROC) curves for selected serum IgG anti-C1P antibodies with area under the curve (AUC) higher than 0.700. Abbreviations: C1P- ceramide-1-phosphate; HS- healthy subjects; RRMS- relapsing remitting multiple sclerosis.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/b117f3444fe27e765c24b01a.jpg"},{"id":95226254,"identity":"45183c08-d80f-4ddf-bac4-3b3bca716e93","added_by":"auto","created_at":"2025-11-05 16:30:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":495873,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component (PC) analysis summarizing and visualizing information in a dataset differentiating RRMS, HS and OND group (panel \u003cstrong\u003eA\u003c/strong\u003e) as well as RRMS from HS group only (panel \u003cstrong\u003eB\u003c/strong\u003e) and RRMS from OND group only (panel \u003cstrong\u003eC\u003c/strong\u003e), containing observations described by four correlated quantitative variables: anti-C16:0-C1P, anti-C18:0-C1P, anti-C24:0-C1P anti-C24:1-C1P IgG levels (panel \u003cstrong\u003eA\u003c/strong\u003e) as well as by two correlated quantitative variables: anti-C18:0-C1P and anti-C24:1-C1P IgG levels (panel \u003cstrong\u003eB\u003c/strong\u003e) and anti-C16:0-C1P and anti-C24:0-C1P IgG levels (panel \u003cstrong\u003eC\u003c/strong\u003e). Abbreviations: HS- healthy subjects; OND- other neurological diseases; RRMS- relapsing remitting multiple sclerosis.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/ec67919804cc4c9e91a8ffca.jpg"},{"id":105223408,"identity":"d0a2eb50-467b-4ef9-8757-157edf612993","added_by":"auto","created_at":"2026-03-23 16:05:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2864671,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/558e2123-23fd-4289-b110-05db44590447.pdf"},{"id":95177548,"identity":"850ce566-8135-4482-a713-83124729e9cb","added_by":"auto","created_at":"2025-11-05 07:50:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2853948,"visible":true,"origin":"","legend":"","description":"","filename":"AntiC1PAbsinMSSupplementalMaterialSciRepformatted.docx","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/c097261e785384c05f797e36.docx"},{"id":95177546,"identity":"9784f733-ea6e-463a-a674-6631db24e73c","added_by":"auto","created_at":"2025-11-05 07:50:53","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15801,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7825469/v1/9defc80df743d448be40a7ab.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Presence and relevance of serum anti-C1P IgG antibodies in relapsing-remitting multiple sclerosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple sclerosis (MS) is a chronic polyphasic disease of the central nervous system (CNS) which causes multifocal damage to the brain and the spinal cord and results in multiple aspects of disability. The complex background of MS involves two major components: autoimmune-mediated inflammatory injury to the myelin sheath and neurodegeneration with progressive axonal loss. These two processes develop in parallel but with different dynamics throughout the disease and their interplay determines MS-related damage to CNS, with emerging clinical picture of this disorder\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Despite a substantial progress in understanding MS pathophysiology, some of its aspects have not been yet elucidated, including key elements of initiating immune-mediated inflammatory cascade in CNS or factors modulating highly variable course of the disease\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Thus, reliable markers are being extensively sought, which would allow to identify and predict activity and progression of the disease\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Investigations have long been focused mainly upon protein molecules as indices of immune activity or neuronal/axonal damage. However, with the advent of metabolomic approach, lipids have also gained attention in this field\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Sphingolipids (SL) seem to be promising candidates for the role of such biomarkers, as the main components of CNS myelin sheath\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, but also relevant players in cellular signaling and cross-talk between inflammation and neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. There has been a solid evidence of accumulation of bioactive SL mediators, specifically ceramide (Cer), in different types of MS lesions and microstructurally altered CNS tissues. Cer and its glycosylated derivatives have been also revealed in the cerebrospinal fluid (CSF), serum, plasma and white blood cells of MS patients\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePrevious research by the members of our team indicated a significant role of Cer derivatives in initiation and shaping of the autoimmune response, targeted at CNS antigens, a key element of MS background. The analysis of the cellular and exosomal profile of bioactive SL mediators in human oligodendroglioma (HOG) cells showed that exosomal Cer subspecies (C16:0-, C24:0- and C24:1-Cer) participate in the apoptosis process induced by pro-inflammatory Th1 cytokines\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Comparative assessment of the lipid profile in brain tissues collected during autopsy (demyelinating plaques in the specimens from MS patients vs. unaffected cerebral white matter in the specimens from the control group) allowed to establish specific SL markers that differentiated active and inactive chronic MS lesions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Active lesions were characterized by a significant increase in the main subclasses of dihydroceramide (dhCer), while inactive ones \u0026ndash; by decreased levels of dhCer, Cer and sphingomyelin (SM) subclasses and significantly elevated levels of hexosylceramides (HexCer) and ceramide-1-phosphates (C1P). These findings suggested a key element of 'pathological switch' in the metabolic pathways of Cer, driving alternately active or inactive phase of the disease.\u003c/p\u003e\u003cp\u003eFurthermore, analysis of anti-Cer immunoglobulin G (IgG) in serum and CSF revealed elevated levels of their particular subclasses (anti-C16:0-, C18:0-, C18:1-, C24:0- and C24:1\u0026ndash;Cer) in MS group in comparison with healthy subjects (HS) and those with other CNS disorders\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In addition, differences in anti-Cer IgG panels were observed between subgroups with different clinical types of MS [i.a. relapsing-remitting (RRMS) vs. progressive]. For selected anti-Cer IgG subclasses, significant correlations were found between their level in CSF and serum, as well as Link index. The results indicated the relevant role of Cer as a target for autoreactive humoral response, an essential element of MS pathology.\u003c/p\u003e\u003cp\u003eEncouraged by the above findings, which supported the need for further research aimed at identification of SL putative biomarkers for MS activity and progression, we undertook the current study focused on particular Cer derivatives. Our main goal was to evaluate the serum level of IgG antibodies against selected C1P subclasses in MS subjects in comparison with the reference groups. We also aimed at investigating their relationships with clinical status.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eHuman specimens of serum\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eParticipants were recruited among the patients hospitalized at the Department of Neurology, Wroclaw Medical University. The following 3 groups were included in this study: i) patients with RRMS (n\u0026thinsp;=\u0026thinsp;39), diagnosed according to McDonald\u0026rsquo;s criteria\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; ii) patients with other inflammatory disorders of the nervous system (I-OND, n\u0026thinsp;=\u0026thinsp;13) that comprised chronic inflammatory demyelinative polyneuropathy (CIDP) or immune-mediated vasculitis, as well as iii) patients with non-inflammatory disorders of the nervous system (NI-OND, n\u0026thinsp;=\u0026thinsp;13), including migraine, cerebrovascular disease and hydrocephalus.\u003c/p\u003e\u003cp\u003eExclusion criteria in RRMS and I-OND groups comprised: recent (within previous 3 months) short-term treatment with corticosteroids, any kind of long-term immunomodulatory or immunosuppressive treatment during at least previous 6 months, current infection and coexisting systemic diseases, in particular inflammatory ones. In the RRMS group, clinical data, such as duration of disease and degree of disability in Expanded Disability Status Scale (EDSS), have been determined based on medical records. The patients were divided into two subgroups, according to their clinical status: those in relapse \u0026ndash; exacerbation of disease (RRMS-rel, n\u0026thinsp;=\u0026thinsp;23) or in remission \u0026ndash; a period of stable condition (RRMS-rem, n\u0026thinsp;=\u0026thinsp;16).\u003c/p\u003e\u003cp\u003eIn addition, the blood samples were obtained from healthy subjects (HS, n\u0026thinsp;=\u0026thinsp;12) at the Regional Center of Blood Donation and Blood Treatment in Wroclaw.\u003c/p\u003e\u003cp\u003eParticipation in the study was anonymous and voluntary. All the subjects gave informed written consent to participate in this study. The project was approved by the Bioethics Committee of Wroclaw Medical University (No. KB 337/2024). The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines.\u003c/p\u003e\u003cp\u003eApproximately 15 ml of peripheral venous blood was collected from each participant. After coagulation the samples were centrifuged at 2000xg at room temperature for 10 min, and afterwards stored at -80\u0026deg;C prior to IgG preparation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePreparation of IgG antibodies from human sera\u003c/h3\u003e\n\u003cp\u003eThe IgG antibodies were isolated from human serum by affinity chromatography on Protein G GraviTrap\u0026trade; columns (GE Healthcare). Prior to chromatographic separations, serum samples (0.5 ml) were centrifuged for 10 min at 10,000xg at 4\u0026deg;C, then diluted 1:1 (v/v) with 20 mM phosphate buffer pH 7.0 and filtered through a 0.45 \u0026micro;m Millex\u0026reg;-HV filter. Columns containing 1 ml of packed gel were equilibrated with phosphate buffer and prepared in the described above way sera were loaded. Proteins not bound by protein G were eluted from the columns with phosphate buffer. Next, the column was washed with 12 column volumes of phosphate buffer and the wash fraction was collected. Elution of IgG, bound to the ligand, was performed with 0.1M Gly/HCl pH 2.7 and subsequently neutralized with 1M Tris/HCl pH 9.0. During the fractionation the amount of proteins in all fractions was monitored by measuring the absorbance at 280 nm on 96 well UV half-plate area plates in an Enspire reader. The fractions containing IgG only were concentrated on an Amicon\u0026reg; Ultra100K by ultracentrifugation at 4500xg at 4\u0026deg;C to a final volume of 250 \u0026micro;l. During this process, IgG samples were desalted and albumin residues were removed. The protein concentration of column fractions was measured using bicinchoninic acid (BCA) protein assay kit (Pierce) with bovine serum albumin (BSA) as a standard. Purified IgG antibodies were preserved with 0.02% NaN\u003csub\u003e3\u003c/sub\u003e, aliquoted and stored at -80\u0026deg;C prior to further analysis.\u003c/p\u003e\n\u003ch3\u003eSDS-PAGE and Western blotting analysis\u003c/h3\u003e\n\u003cp\u003eThe electrophoretic separation was performed in a BIO-RAD\u0026reg; Tetra Cell Systems apparatus as described earlier\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Briefly, proteins (2 \u0026micro;g) were analyzed on 12% polyacrylamide gel in reducing conditions and either stained with Coomassie Brilliant Blue (CBB) or transferred into 0.45 \u0026micro;m Immobilon P. The separated proteins were detected with, produced in donkey, anti-human IgG (H\u0026thinsp;+\u0026thinsp;L), conjugated with alkaline phosphatase (AP), antibody (1:7500) (Sigma). The reaction was visualized with AP substrates, according to standard procedure.\u003c/p\u003e\n\u003ch3\u003eSynthesis of C1P derivatives\u003c/h3\u003e\n\u003cp\u003eC1P derivatives were synthesized in the Synthesis Unit of the Lipidomics Shared Resources at the Medical University of South Carolina, Charleston, SC, USA, as previously described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Their purity was checked by the high performance liquid chromatography- tandem mass spectrometry (HPLC-MS/MS).\u003c/p\u003e\n\u003ch3\u003eELISA assay\u003c/h3\u003e\n\u003cp\u003eELISA plates (Immulon 1B, Thermo Fisher Scientific) were coated with selected C1P solutions in absolute ethanol (10 \u0026micro;g/ml; 0.1 ml /well). The plates were incubated overnight at room temperature to facilitate complete solvent evaporation. Next, the plates were washed three times with phosphate buffered saline (PBS; 0.2 ml/well). Nonspecific binding sites were blocked by adding 100 \u0026micro;l of 1% (w/v) BSA in PBS and incubating the plates for 1 hour at 37\u0026deg;C. After washing 5 times with PBS purified IgG (0.1 mg/ml of protein, 0.1 ml /well) from serum of MS patients and individuals from reference groups were added and the plates were incubated overnight at 4\u0026deg;C. In the next step the plates were washed again for 5 times with PBS and then incubated with horseradish peroxidase (HRP) conjugated anti-human IgG (GE Healthcare, 1:2000, 0.1 ml/well). The enzymatic activity was developed by adding peroxidase substrate (Sigma tablets). The colorimetric reaction was stopped by adding of 100 \u0026micro;l of 3N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. and the optical density (OD) was read at 492 nm in an Enspire reader.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eOrdinary one-way Anova with post-hoc Tukey test was used to estimate differences in respect to age of examined subjects belonging to different groups.\u003c/p\u003e\u003cp\u003eThe U Mann-Whitney test was used to compare levels of serum IgG anti-C1P antibodies between patients with RRMS and reference groups (HS, OND) as well as between RRMS-rel and RRMS-rem subgroups. The diagnostic significance of immunoreactivities of these IgG antibodies with appropriate C1P antigens was analyzed using received operating characteristic (ROC) curves with clinical meaning considered as zero (values from 0-0.5); limited (values from 0.5\u0026ndash;0.7); moderate (values from 0.7\u0026ndash;0.9) and high (\u0026gt;\u0026thinsp;0.9). The Youden index method was used for the determination of cut off points.\u003c/p\u003e\u003cp\u003eThe IgG samples, for which the area under the curve (AUC) values were established as moderate or high, were subjected to a cluster analysis, a multivariate statistical method. Methods belonging to two different categories were used. A hierarchical Ward\u0026rsquo;s method was applied to determine the number of clusters. A non-hierarchical method, namely k-means clustering (k-means function of base R) from the selected anti-C1P IgG dataset was used for further analysis.\u003c/p\u003e\u003cp\u003eThe Spearman\u0026rsquo;s rank correlation was used to check the associations between measured levels of anti-C1P antibodies and clinical parameters such as disease duration, EDSS and Link index.\u003c/p\u003e\u003cp\u003eThe ROC analysis was performed using Statistica 13.3 software (StatSoft Inc., Tuls, OK, USA), cluster analysis was executed by OriginPro 2025b (OriginLab Corporation, Northampton, MA, USA). For the remaining statistical analysis GraphPad Prism 7.01 (GraphPad Software Inc., San Diego, CA, USA) was applied.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of the study cohort\u003c/h2\u003e\u003cp\u003eBasic demographic data, including age and sex structure of the patients included in the study are presented in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. One-way Anova followed by Tukey\u0026rsquo;s multiple comparisons test indicated no significant age differences between the study groups. In RRMS group, duration of the disease ranged from 1 to 108 months (mean 22 months) and EDSS score \u0026ndash; from 1 to 4 (median 1.5) points. All the RRMS patients (n\u0026thinsp;=\u0026thinsp;39) had magnetic resonance imaging (MRI) of the brain performed, with typical demyelinative lesions. In 32 patients the results of CSF testing were available, with confirmed presence of IgG oligoclonal bands and calculated Link index (increased value\u0026thinsp;\u0026ge;\u0026thinsp;0.7 in 20 cases).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBasic demographics of the study subgroups. Abbreviations: I-OND- inflammatory\u003c/p\u003e\u003cp\u003eother neurological diseases; NI-OND- non-inflammatory other neurological diseases;\u003c/p\u003e\u003cp\u003erel- relapse; rem- remission; RRMS- relapsing-remitting multiple sclerosis; SD- standard\u003c/p\u003e\u003cp\u003edeviation.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubgroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber of patients\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFemale/Male\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAge (years) \u003c/p\u003e\u003cp\u003eMean \u0026plusmn; SD\u003c/p\u003e\u003cp\u003e(Min-Max)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRRMS-rel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16/7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32.2 \u003cb\u003e\u0026plusmn;\u003c/b\u003e 10.1\u003c/p\u003e\u003cp\u003e(28\u0026ndash;65)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRRMS-rem\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6/10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33.1 \u003cb\u003e\u0026plusmn;\u003c/b\u003e 7.5\u003c/p\u003e\u003cp\u003e(21\u0026ndash;48)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-OND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6/7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e42.5 \u003cb\u003e\u0026plusmn;\u003c/b\u003e 18.9\u003c/p\u003e\u003cp\u003e(22\u0026ndash;83)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNI-OND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7/6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41.3 \u003cb\u003e\u0026plusmn;\u003c/b\u003e 13.8\u003c/p\u003e\u003cp\u003e(19\u0026ndash;64)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of Protein G-isolated and purified from human serum IgG antibodies\u003c/h2\u003e\u003cp\u003eThe IgG antibodies present in human serum were isolated by affinity chromatography on Protein G Gravi Trap columns. A representative preparation of IgG antibodies from 0.5 ml of serum is shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In a typical elution profile obtained during the isolation of IgG from RRMS-rel patient two peaks were visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The first one \u0026minus; Fr. 1 \u0026minus; represented the proteins unbound by protein G. The second one, Fr. 3, corresponded to four isotypes of IgG (IgG\u003csub\u003e1\u003c/sub\u003e, IgG\u003csub\u003e2\u003c/sub\u003e, IgG\u003csub\u003e3\u003c/sub\u003e and IgG\u003csub\u003e4\u003c/sub\u003e), bound by protein G. The obtained fractions were subjected to qualitative and quantitative analysis. The CBB staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) obtained as a result of electrophoretic separation of samples, collected during the isolation of serum IgG from both RRMS-rel and RRMS-rem, indicated that in both cases the products obtained as a Fr. 3 were IgG antibodies (lanes 4 and 8). This is evidenced by two characteristic bands with a molecular mass of 50 and 25 kDa, corresponding to the heavy and light chain of immunoglobulin G, as appeared under these conditions. In the remaining lanes (lanes 1\u0026ndash;3 and 5\u0026ndash;7), distinct bands with a molecular mass of about 65 kDa were observed, originating from other proteins found in the serum. Presumably, these bands indicate the presence of albumin, which constitutes up to 65% of all serum proteins, and perhaps the heavy chain of immunoglobulin M, constituting about 5 to 10% of serum immunoglobulins. Quantitation of proteins in the various fraction demonstrated high and comparable total protein amounts in Fr. 1 and residual protein amounts in Fr. 2. The preparation resulted in the recovery of 5.6 \u0026plusmn; 0.9 mg of purified IgG (Fr. 3), a predominant class of antibody in serum, which corresponded to 14.3% \u0026plusmn; 2.4 of all serum proteins (Supplemental Table\u0026nbsp;1S). The absence of IgG in Fr. 1 and 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC; lanes 2 and 3, respectively) and the presence of the purified IgG antibodies was confirmed by Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, lane 4). This analysis indicated the high efficiency of the IgG purification method used.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAltered levels of serum anti-C1P IgG in RRMS patients\u003c/h2\u003e\u003cp\u003eThe Protein G-purified IgG antibodies from serum of MS patients (Fr. 3) were evaluated for the presence of selected C1P subclasses binding activity, using an ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplemental Fig.\u0026nbsp;1S).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe binding activity of serum IgG to C16:0-C1P was significantly higher in RRMS patients (median value: 0.4535) in comparison to HS group (median value: 0.34) and significantly lower in relation to OND group (median value: 0.7725), with significance of p\u0026thinsp;=\u0026thinsp;0.0425 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe binding activity of serum IgG to C18:0-C1P in RRMS patients (median value: 0.7715) was significantly increased in comparison to HS group only (median value: 0.4635) with significance of p\u0026thinsp;=\u0026thinsp;0.0005 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In turn, there were no significant differences in the binding activity of serum IgG to C18:1-C1P in RRMS individuals in relation to both reference groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eIn contrast, RRMS patients manifested lower level of anti-C24:0-C1P serum IgG (median value: 0.2725) in comparison to OND group (median value: 0.924) with significance of p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eThe binding activity of serum IgG to C24:1-C1P represented the pattern similar to C16:0-C1P. Interestingly, serum IgG derived from RRMS patients exhibited the highest reactivity for C24:1-C1P among all C1P subclasses examined (median value: 1.001) in comparison to HS group (median value: 0.4915, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while the level of anti-C24:1-C1P IgG was significantly decreased in RRMS in comparison to OND group (median value: 1.25; p\u0026thinsp;=\u0026thinsp;0.0183) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The presented above results, obtained by ELISA test, are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe levels of reactivity of selected anti-C1P serum IgG in RRMS patients in comparison to healthy subjects (HS) and individuals with other neurological diseases (OND). Significant differences\u003c/p\u003e\u003cp\u003ecompared to reference groups were indicated as follows: a-HS, b- OND. Abbreviations: C1P- ceramide-1-phosphate; SD- standard deviation.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eSerum IgG\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRRMS\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;39\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHS\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;12\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOND\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;26\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMEAN \u0026plusmn; SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMEAN \u0026plusmn; SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMEAN \u0026plusmn; SD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-C16:0-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.4745 \u0026plusmn; 0.21\u003c/p\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0425\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.3534 \u0026plusmn; 0.1224\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.7838 \u0026plusmn; 0.3031\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-C18:0-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.7512 \u0026plusmn; 0.2753\u003c/p\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0005\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.4853 \u0026plusmn; 0.1202\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.7789 \u0026plusmn; 0.2951\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-C18:1-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.3478 \u0026plusmn; 0.1289\u003c/p\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0136\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.2555 \u0026plusmn; 0.08237\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.7749 \u0026plusmn; 0.3728\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-C24:0-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.3192 \u0026plusmn; 0.1579\u003c/p\u003e\u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.3878 \u0026plusmn; 0.2019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.9394 \u0026plusmn; 0.3448\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-C24:1-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.9526 \u0026plusmn; 0.3096\u003c/p\u003e\u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0183\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.4265 \u0026plusmn; 0.2061\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.17 \u0026plusmn; 0.4374\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNext, we reanalyzed the data considering the division of RRMS patients into subgroups in relapse and in remission (RRMS- rel and RRMS -rem) and OND patients \u0026ndash; into subgroups with inflammatory or other etiology of disease (I-OND, NI-OND) (Supplemental Fig.\u0026nbsp;1S). The Mann Whitney test showed significantly higher levels of serum IgG derived from both RRMS subgroups against most C1P subclasses (except of C24:0-C1P) compared to HS, particularly significant for IgG anti-C18:0-C1P and C24:1-C1P (p\u0026thinsp;=\u0026thinsp;0.0024 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Supplemental Fig.\u0026nbsp;1S, panel B \u0026amp; E, respectively). The level of IgG anti-C24:1-C1P in RRMS-rel subgroup was significantly lower than in RRMS-rem (p\u0026thinsp;=\u0026thinsp;0.0448), as well as lower in both RRMS subgroups in relation to NI-OND only (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and p\u0026thinsp;=\u0026thinsp;0.0004, Supplemental Fig.\u0026nbsp;1S, panel E, respectively). The level of IgG anti-C24:0-C1P in both RRMS subgroups was significantly lower than in I-OND and NI-OND (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Supplemental Fig.\u0026nbsp;1S, panel D). The above results in details are included in Supplemental Table\u0026nbsp;1S.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eROC analysis\u003c/h2\u003e\u003cp\u003eIn order to validate ELISA results, we performed ROC analysis for all serum anti-C1P IgG levels in RRMS patients, as presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results of ROC curve analysis for anti-C1P IgG data sets with the AUC value higher than 0.700 are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The cut-off points, determined by the Youden index method, are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of receiver operating characteristic (ROC) curves for serum anti-C1P IgG levels in the study groups. Abbreviations: AUC- area under the curve; C1P- ceramide-1-phosphate; HS- healthy subjects; OND- other neurological diseases;\u003c/p\u003e\u003cp\u003eRRMS- relapsing-remitting multiple sclerosis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntibody\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExamined groups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAUC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAUC with 95% confidence interval\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eYouden\u003c/p\u003e\u003cp\u003eindex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCut off point\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSensitivity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSpecificity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAnti-C16:0-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRRMS vs. HS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.653\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.506\u0026ndash;0.800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.369\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.523\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.436\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.933\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.0417\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eRRMS vs. OND\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.798\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.682\u0026ndash;0.914\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.526\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.701\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.654\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0.872\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAnti-C18:0-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eRRMS vs. HS\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.781\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.661\u0026ndash;0.904\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.564\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.702\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.564\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e1.000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRRMS vs. OND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.532\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.385\u0026ndash;0.679\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.154\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.952\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.385\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.769\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.6682\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAnti-C18:1-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRRMS vs. HS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.569\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.381\u0026ndash;0.758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.297\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.667\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.733\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.4718\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRRMS vs. OND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.153\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.047\u0026ndash;0.258\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.654\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.619\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.346\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.0000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAnti-C24:0-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRRMS vs. HS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.409\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.222\u0026ndash;0.595\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.923\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.3371\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eRRMS vs. OND\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.942\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.886\u0026ndash;0.998\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.769\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.801\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.769\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e1.000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAnti-C24:1-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eRRMS vs. HS\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.769\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.603\u0026ndash;0.936\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.595\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.727\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.795\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0.800\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.0015\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRRMS vs. OND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.654\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.508\u0026ndash;0.800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.372\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.795\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.0386\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAnti-C16:0-C1P IgG level indicated a moderate value in differentiating RRMS group from OND group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), with sensitivity 65.4% and specificity of 97.2%, respectively (proposed cut off point: 0.701, AUC\u0026thinsp;=\u0026thinsp;0.798). Anti-C18:0-C1P IgG level showed moderate discriminating value between RRMS and HS groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with sensitivity and specificity of 56.4% and 100%, respectively (proposed cut off point: 0.702, AUC\u0026thinsp;=\u0026thinsp;0.781). The level of anti-C24:0-C1P IgG had a high discriminating value between RRMS group and OND group, with sensitivity of 76.9% and specificity of 100%, respectively (proposed cut off point: 0.801, AUC\u0026thinsp;=\u0026thinsp;0.942) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The level of anti-C24:1-C1P IgG exhibited a moderate value in differentiating between RRMS group and HS group, with sensitivity of 79.5% and specificity of 80% (proposed cut off point: 0.727, AUC\u0026thinsp;=\u0026thinsp;0.769) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCluster analysis\u003c/h2\u003e\u003cp\u003eBased on the results of ROC analysis, serum anti-C1P IgG levels with AUC equal or greater than 0.769 were selected to perform cluster analysis. The number of expected clusters was pre-determined by using the hierarchical Ward\u0026rsquo;s method (Supplemental Fig.\u0026nbsp;2S). Relative level of anti-C16:0-, anti-C18:0-, anti-C24:0- and anti-C24:1-C1P serum IgG was subsequently used to perform k-means clustering to categorize the samples examined into 3 clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Cluster 1 comprised 34 subjects: all healthy ones; 16 patients with RRMS (including 9 ones from RRMS-rel subgroup) and 6 individuals with OND (containing 5 from I-OND subgroup). Cluster 2 included 23 RRMS patients (14 from RRMS-rel, 9 from RRMS-rem subgroup) and 9 OND ones (5 from I-OND and 4 from NI-OND subgroup). Cluster 3 consisted of OND patients only, with the majority belonging to NI-OND subgroup (8 out of 11) (Supplemental Fig.\u0026nbsp;3S).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSignificant differences in anti-C1P IgG panels between the study groups (RRMS vs. HS: anti-C18:0- and anti-C24:1-C1P IgG; RRMS vs. OND: anti-C16:0- and anti-C24:0-C1P IgG) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), with discriminatory ability of these IgG subsets, confirmed by ROC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), together with similar patterns of relative levels of anti-C16:0- / anti-C24:0-C1P, as well as anti-C18:0- /anti-C24:1-C1P IgG (Supplemental Fig.\u0026nbsp;4S), supported the notion that anti-C1P IgG panels might differentiate pathological from physiological condition but also reflect distinct biological processes (i. e. neuroinflammation vs. neurodegeneration) with different degrees of sensitivity. To check this hypothesis, a k-means analysis was subsequently performed to divide the appropriate IgG samples into 2 clusters. Relative levels of anti-C18:0- and anti-C24:1C1P serum IgG were subjected to k-means clustering to differentiate RRMS from HS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Cluster 1 comprised 28 subjects (12 HS and 16 RRMS, almost equally distributed between RRMS-rel and RRMS-rem subgroups) whereas cluster 2 was represented by 23 RRMS patients (mainly RRMS-rel subgroup) (Supplemental Fig.\u0026nbsp;5S). In turn, relative levels of anti-C16:0- and anti-C24:0-C1P serum IgG were subjected to k-means clustering to differentiate RRMS from OND group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Cluster 1 comprised 45 subjects: all RRMS patients (n\u0026thinsp;=\u0026thinsp;39) and 6 OND ones (mainly from I-OND subgroup), whereas cluster 2 was composed of 20 OND subjects, mainly from NI-OND subgroup (Supplemental Fig.\u0026nbsp;6S).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCorrelation analysis between selected serum anti-C1P IgG derived from RRMS patients and clinical parameters\u003c/h2\u003e\u003cp\u003eNext, we examined whether there are any correlations between anti-C1P IgG titers (as reflected by OD at 492 nm) in RRMS group and MS-related clinical parameters. The levels of anti-C16:0-C1P, anti-C18:0-C1P, anti-C18:1-C1P, anti-C24:0-C1P and anti-C24:1-C1P IgG in RRMS did not correlate with disease duration (Supplemental Fig. S7, panels A, D, G, J and M) or EDSS score (panels B, E, H, K and N), accordingly. No associations were found between the levels of these antibodies and Link index, either (Supplementary Fig. S7, panels C, F, I, L and O, respectively). The summary of this analysis is displayed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCorrelation of serum anti-C16:0-C1P, anti-C18:0-C1P, anti-C18:1-C1P, anti-C24:0-C1P, anti-C24:1-C1P IgG levels in RRMS group with clinical parameters. Abbreviations: C1P- ceramide-1-phosphate; EDSS- Expanded Disability Status Scale; RRMS- relapsing-remitting multiple sclerosis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSerum IgG\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eClinical parameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpearman\u0026rsquo;s correlation coefficient predictor (r value)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAnti-C16:0-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDisease duration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.1753\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.2857\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEDSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.04531\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7842\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLink index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.1286\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.4759\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAnti-C18:0-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDisease duration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.2746\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0906\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEDSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.06427\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.6975\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLink index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0565\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7548\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAnti-C18:1-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDisease duration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.251\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.1232\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEDSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.08361\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.6128\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLink index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.2063\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.2494\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAnti-C24:0-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDisease duration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.1884\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.2506\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEDSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.01253\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.9396\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLink index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.2496\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.1613\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAnti-C24:1-C1P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDisease duration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.1706\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.2992\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEDSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.03706\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.8228\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLink index\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.1914\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.2859\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCer products have been suggested to be involved in the main mechanisms underlying MS pathology, including those - recently highlighted - driven by B cells. Activation of B cells together with cytokine stimulation promote tissue penetration and destabilization of membrane lipids in CNS, resulting in damage to myelin sheath. SL (in particular Cer derivatives) are essential for maintenance of lipid membranes integrity, so changes in their metabolism and proportion correspond with dynamic processes of demyelination and repair and their pathophysiological consequences\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although in the aforementioned studies there is convincing evidence for Cer abnormalities in MS, it relies mainly on preclinical models and their translation into human clinical context, with clear identification of Cer related mechanisms of action and potentially relevant MS biomarkers, still remains a challenge \u003csup\u003e20 4\u0026ndash;7\u003c/sup\u003e. Based on results of our previous studies\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and scarce literature data\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, here we focused on particular Cer derivative \u0026ndash; C1P, aiming to investigate its putative role as a target and/or mediator of autoimmune response in MS.\u003c/p\u003e\u003cp\u003eC1P, a major metabolite of Cer, can be formed through a stereospecific reaction of phosphorylation, due to direct action of Cer kinase (CerK), while the reverse reaction (conversion of C1P to Cer) is mediated by C1P phosphatase. An appropriate balance between these two molecules is required to ensure cells and tissues homeostasis through regulation of relevant metabolic or signaling pathways. Thus, coordinated action of CerK and C1P phosphatases may have crucial importance for maintaining this balance. Its disturbances with a shift towards accumulation either Cer or C1P may result in metabolic dysregulation, supposedly involved in the background of autoimmune diseases\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, neurodegenerative disorders\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, cardiovascular illnesses\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e or cancer\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIt has been demonstrated that C1P exerts pro-inflammatory properties; however, the underlying mechanisms have not been yet fully elucidated. One of the major function of C1P is its capability to activate cytosolic phospholipase PLA\u003csub\u003e2\u003c/sub\u003e (cPLA\u003csub\u003e2\u003c/sub\u003e), which mediates release of arachidonic acid\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e - the substrate used for production of eicosanoids, such as prostaglandin E\u003csub\u003e2\u003c/sub\u003e (PGE\u003csub\u003e2\u003c/sub\u003e). This is aligned with the fact that MS patients exhibit higher PGE\u003csub\u003e2\u003c/sub\u003e expression/activity in CSF\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, lymphocytes\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e as well as demyelinative lesions in brain tissue\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. A sharp increase of this pro-inflammatory mediator was observed in active stage of MS\u003csup\u003e31\u003c/sup\u003e, and experimental studies with the use of cyclooxygenase (COX) inhibitors, which oppose C1P action by preventing PGE\u003csub\u003e2\u003c/sub\u003e synthesis, suggested their beneficial effect as potential options in MS therapy \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOther pro-inflammatory activities affected by C1P include: activation of degranulation in mast cells\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, stimulation of phagocytosis in neutrophils\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, as well as stimulation of macrophage migration\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. C1P also increases transport of P-glycoprotein, which regulates the permeability of the blood brain barrier via COX-2/PGE\u003csub\u003e2\u003c/sub\u003e signaling\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOn the other hand, it has been shown that C1P might also act as an anti-inflammatory agent, under specific conditions. Anti-apoptotic effects of C1P, promoting cell survival, are related with its ability to block activity of serine palmitoyltransferase\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, acid sphingomyelinase\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and TNF-α\u003csup\u003e39\u003c/sup\u003e. Furthermore, it promotes macrophage chemoattractant protein-1 (MCP-1) release in different types of cells\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Taken together, C1P seems to act on its own rights, executing regulatory effects and modulating cell functions depending on circumstances.\u003c/p\u003e\u003cp\u003eIn one of the previous reports by our team members, an increase in C1P subspecies (mainly C18:1-C1P and C24:1-C1P) was demonstrated in inactive MS lesions in comparison with specimens representing other CNS disorders or normal brain tissue. On the contrary, active MS plaques were characterized by a significant decrease of C1P subspecies\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The studied autopsy samples from MS subjects represented advanced stage of disease, associated with chronic inflammation and presumably extensive neurodegenerative component. In the present study we focused on earlier phase of the disease (i. e. RRMS), when active immune-mediated inflammation is a predominating mechanism of CNS injury - therefore antibodies as markers of autoimmune humoral response were chosen as the subject of investigation. The main findings presented here included elevated levels of nearly all analyzed anti-C1P IgG in serum of RRMS patients in comparison to HS, with the highest significance for anti-C24:1-C1P and anti-C18:0-C1P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and B). This striking consistency in the results from both studies, despite differences in MS stages and studied material (brain tissues vs. serum), indeed suggests the relevant role of C1P in MS pathology. It can be hypothesized that each stage of MS is associated with the concerted action of Cer derivatives, with their changing interactions in this pathological cross-talk.\u003c/p\u003e\u003cp\u003eIt should be mentioned that the level of IgG anti-C24:1-C1P was significantly lower in both RRMS subgroups in relation to NI-OND only (Supplemental Fig.\u0026nbsp;1S, panel E), whereas the level of IgG anti-C24:0-C1P in both RRMS subgroups was significantly lower than in both OND subgroups (Supplemental Fig.\u0026nbsp;1S, panel D). Overall, these results suggest that increased humoral response against long-chain C1P subspecies is particularly specific for MS and it does not just reflect overall neuronal injury or dysregulated immune-mediated inflammation in CNS. Importantly, the reliability of the above findings was supported by eliminated impact of immunosuppressive/immunomodulatory drugs, as the patients were included in the study prior to such treatment or after appropriate wash-out period.\u003c/p\u003e\u003cp\u003eThere is an evidence that fatty acid chain length of Cer can determine biophysical properties of this SL mediator\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In physiological conditions, medium chain Cer (C16 and C18) are known to be enriched in cerebral gray matter, while long chain ones (C24) - in white matter and myelin sheath\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Considering distribution of Cer derivatives in the brain, presence of the medium chain ones would reflect neuronal injury\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, while the long chain ones \u0026ndash; oligodendrocytes degradation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The long chain Cer (C24) are also known to be the major Cer subspecies present in serum/plasma whereas C1P constitute only 0.15% of all SL components\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. However, it should be taken into account that the pattern of anti-Cer IgG in body fluids, revealed by our previous study\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e reflected metabolic perturbations of Cer, supposed to occur due to processes involved in MS background. The anti-Cer IgG alterations in serum (unlike in the CSF) were observed only for Cer containing medium chains (C16:0-, C18:0- and C18:1-Cer) with no change for the long chain ones\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Current findings revealed altered patterns of IgG against C1P in serum of RRMS patients. Here we found that anti-C16:0-C1P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), anti-C18:0-C1P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and anti-C24:1-C1P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) serum IgG levels were increased in RRMS patients in comparison with HS group. In turn, levels of IgG against C16:0-C1P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), C24:0-C1P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and C24:1-C1P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) were decreased in RRMS in relation to OND (including distinguished subgroup of inflammatory ones). In addition, the results of ROC analysis indicated that the determination of anti-C18:0-C1P and anti-C24:1-C1P serum IgG levels might be useful for discriminating RRMS from HS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel C and G), whereas anti-C16:0-C1P and anti-C24:0-C1P serum IgG levels \u0026ndash; for discriminating RRMS from OND group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel B and F). Furthermore, the usefulness of the above anti-C1P IgG subsets in distinguishing pathological from physiological conditions, as well as disorders with different etiology, was confirmed by means of multivariate statistical method of cluster analysis (Supplemental Fig.\u0026nbsp;5S and 6S). Despite somehow subjective selection of variables for clustering and the number of clusters (justified by the results of initial analysis), the findings from this multivariate technique support the concept that panels/sets of anti-C1P IgG may be of greater discriminating value than single immunological parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Such a panel including anti-C16:0-C1P, anti-C18:0-C1P, anti-C24:0-C1P and anti-C24:1-C1P IgG might be considered for further investigation towards potential clinical implications - the use of anti-C1P IgG as biomarkers in differential diagnosis at MS onset, especially in atypical cases.\u003c/p\u003e\u003cp\u003eAs a result of more precise analysis within RRMS group, the level of anti-C24:1-C1P IgG was found to be significantly lower in those with relapse than those in remission (Supplemental Fig.\u0026nbsp;1S, panel E). These findings may indicate that alterations of Cer metabolism (revealed by presence of corresponding IgG) are associated with changing phases of disease activity.\u003c/p\u003e\u003cp\u003eFew attempts have been made to use anti-Cer antibodies as biomarkers of activity in other disorders, including those of the nervous system. In leprosy, presence of anti-Cer antibodies was found to be related to extent of nerve sheath damage and particular bacterial strains as pathogenic factors\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In the rare autoimmune condition, encephalomyeloradiculoneuropathy (EMRN), accumulation of species-specific Cer (mainly long chain ones) was demonstrated in CSF and presence of anti-neutral glycolipids antibodies \u0026ndash; in CSF and serum. Profile of these antibodies allowed to differentiate patients with ERMN from the reference group with Parkinson disease, and their titers decreased after effective immunomodulatory treatment. Dysregulation of glycolipids metabolism and its impact upon invariant natural killer T cell development, as well as abnormal complement activation, were suggested to play a relevant role in EMRN pathophysiology\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Furthermore, in non-small-cell lung cancer (NSCLC) anti-Cer antibodies were supposed to be produced by the tumor and its microenvironment, and to neutralize pro-apoptotic effect of Cer. Significantly elevated levels of anti-Cer antibodies were revealed in serum and bronchial wash fluid samples from NSCLC group (in comparison with HS) and they showed a tendency (though not significant) for prolonged overall survival\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEvaluation of activity in MS has been so far based on clinical assessment and radiological indices (new and/or contrast-enhanced lesions in MRI). However, there is an ongoing search for specific and reliable body fluid biomarkers of disease activity\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Their clinical applications would include i.a. differentiating relapse from pseudorelapse (temporary aggravation of symptoms) or defining active phase in progressive stage of the disease, and in long term follow-up \u0026ndash; monitoring of MS course and response to treatment. In view of our previous and current findings, Cer derivatives and antibodies against them seem indeed promising candidates for the role of such biomarkers.\u003c/p\u003e\u003cp\u003eAlthough no relationships were found in our study group between immunological measures and MS-related clinical data (Supplemental Fig. S7), potential clinical utility of these findings should not be discredited. The group was characterized by a relatively short MS duration and mild disability, and marked individual differences in disease course in MS populations must be taken into account. Perhaps in a larger group of patients and in prospective observation these relationships would appear more relevant.\u003c/p\u003e\u003cp\u003eIndeed, a small sample size and focusing only on RRMS should be considered as limitations of our study, as its results may not accurately reflect abnormalities in SL metabolic pathways in different types and stages of the disease. Another limitation is associated with the fact that anti-C1P IgG presence was determined only in serum and not in CSF. Nevertheless, in view of long-term course of MS and need for regular follow-up, serum/plasma biomarkers are much desired due to their better availability. Based on experiences with other body fluid biomarkers, their serum level may adequately reflect their production in CNS\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. There is also some evidence that peripheral blood may contribute to formation of immune complexes due to selective transport and the blood-brain barrier (BBB) leakage\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo our best knowledge, this study for the first time focuses on the presence and meaning of anti-C1P antibodies in serum of MS patients and provides evidence of their potential relevance. The results shed some light on the perturbation on SL metabolism (especially significance of balance between Cer and C1P) in the background of neurological diseases, specifically MS. Encouraged by these findings, further investigation is planned with the use of IgG against particular C1P subclasses, analyzed parallel in blood and CSF, in larger groups of MS patients and with prospective observation of immunological and clinical parameters. Hopefully, these studies will lead to a better understanding of the role of this intriguing phosphosphingolipid in pathophysiology of MS, with emerging clinical implications.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe serum levels of IgG anti-C1P subspecies (especially long-chain ones) differed significantly between the patients with RRMS and healthy subjects, as well as the patients with other neurological diseases. Differences were also found between subgroups of RRMS patients with relapse or with remission. These findings indicate the relevant role of C1P as a target and/or mediator of autoimmune response in MS. Potential value of anti-C1P antibodies as biomarkers in differential diagnosis of MS and monitoring disease activity warrants further investigations.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Alkaline Phosphate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAUC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Area Under Curve\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBBB\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Blood Brain Barrier\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBCA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Bicinchoninic Acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBSA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Bovine Serum Albumin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC1P \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ceramide-1-Phosphate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCBB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Coomassie Brilliant Blue\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCer \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ceramide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCerK \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ceramide Kinase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCIDP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Chronic Inflammatory Demyelinative Polyneuropathy\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCNS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Central Nervous System\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCOX-2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cyclooxygenase-2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ecPLA\u003csub\u003e2\u003c/sub\u003e\u0026shy;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cytosolic Phospholipase A\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCSF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cerebrospinal Fluid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEAE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Experimental Autoimmune Encephalomyelitis\u003c/p\u003e\n\u003cp\u003eEDSS\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Expanded Disability Status Scale\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eELISA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Enzyme-Linked Immunosorbent Assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEMRN \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Encephalomyeloradiculoneuropathy\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHexCer \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hexosylceramide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHOG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Human Oligodendroglioma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHPLC-MS/MS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;High Performance Liquid Chromatography tandem mass spectrometry\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHRP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Horseradish Peroxidase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Healthy Subjects\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIgG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Immunoglobulin G\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eI-OND\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Inflammatory Other Neurological Diseases\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMCP-1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Macrophage Chemoattractant Protein-1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMRI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Magnetic Resonance Imaging\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Multiple Sclerosis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNI-OND \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Non- Inflammatory Other Neurological Diseases\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNSCLC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Non-Small-Cell Lung Cancer\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Optical Density\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOND \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Other Neurological Diseases\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Phosphate Buffered Saline\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Principal Component\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePGE\u003csub\u003e2\u003c/sub\u003e\u0026shy; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Prostaglandin E\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003erel \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Relapse\u0026nbsp;\u003c/p\u003e\n\u003cp\u003erem \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Remission\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Received Operating Characteristic\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRRMS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Relapsing Remitting Multiple Sclerosis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Standard Deviation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSDS-PAGE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis\u003c/p\u003e\n\u003cp\u003eSL \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sphingolipid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Sphingomyelin\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Mrs. Malgorzata Cyrul (Wroclaw Medical University) for centrifugation, aliqoting and labeling serum specimens. The authors also appreciate Dr. Karol Marschollek (Wroclaw Medical University) consultations regarding ROC analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported in part by Wroclaw Medical University (SUBK.C220.24.081) to J.C.-L., by the National Science Centre (NCN, Krakow, Poland; Preludium 23, Award no. UMO-2024/53/N/NZ3/01093) to A.J.-A. and\u0026nbsp;by the Lipidomics Shared Resource, Holling Cancer Center, Medical University of South Carolina (P30 CA 138313) to Z.M.S.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.C.-L.: investigation, resources, clinical data curation and funding acquisition. A.J.-A.: investigation, methodology, data curation, formal analysis, visualization and review. Z.M.S: investigation, methodology, review and editing. J.G. and S.B.: supervision and writing-review. A.P.-D.: conceptualization, investigation, resources, clinical data curation, formal analysis, writing-original draft preparation, review and editing. M.P.: conceptualization, investigation, data curation, formal analysis, visualization, writing-original draft preparation, review and editing as well as project coordination and supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplemental material for this article can be found in the online version of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDobson, R. \u0026amp; Giovannoni, G. 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The complex relationship between oligoclonal bands, lymphocytes in the cerebrospinal fluid, and immunoglobulin G antibodies in multiple sclerosis: Indication of serum contribution. \u003cem\u003ePloS one\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, e0186842. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0186842\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0186842\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"biomarkers, ceramide, C1P, lipid antigens, multiple sclerosis, sphingolipids","lastPublishedDoi":"10.21203/rs.3.rs-7825469/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7825469/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere is a growing interest in the role of sphingolipids in the background of multiple sclerosis (MS). The goal of this study was to evaluate the serum levels of antibodies against ceramide-1-phosphate (C1P) subclasses and their relationships with clinical status in MS.\u003c/p\u003e\u003cp\u003eThe study groups comprised 39 patients with relapsing-remitting MS (RRMS), 26 patients with other neurological diseases (OND) and 12 healthy subjects (HS). Anti-C1P IgG levels in serum were determined using ELISA test. Levels of anti-C18:0-C1P and anti-C24:1-C1P IgG were significantly increased (p\u0026thinsp;=\u0026thinsp;0.0005; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively) in RRMS compared to HS, while anti-C16:0-C1P and anti-C24:0-C1P IgG \u0026ndash; significantly lower (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in RRMS compared to OND. Validation by ROC and cluster analysis confirmed the ability of these anti-C1P IgG panels to discriminate between the study groups. In addition, level of anti-C24:1-C1P IgG was significantly lower (p\u0026thinsp;=\u0026thinsp;0.0448) in RRMS patients with relapse than in those with remission. No relationships were found between levels of antibodies in the anti-C1P IgG panel in RRMS group and disease duration, degree of disability or Link index.\u003c/p\u003e\u003cp\u003eThese findings highlight the relevant role of C1P as a target and/or mediator of autoimmune response in MS and potential value of anti-C1P antibodies as biomarkers in differential diagnosis of this disease.\u003c/p\u003e","manuscriptTitle":"Presence and relevance of serum anti-C1P IgG antibodies in relapsing-remitting multiple sclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 07:50:48","doi":"10.21203/rs.3.rs-7825469/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-14T13:47:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-04T15:19:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87463758461457425603109732609220836456","date":"2025-12-20T08:09:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-10T16:49:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308647329370632707016969810971701591212","date":"2025-12-05T13:49:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36463772275918051619409690121564886241","date":"2025-11-30T15:15:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"226100063092423214754195710211148820531","date":"2025-10-31T13:56:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213758823298516597778767527401272968487","date":"2025-10-27T08:47:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-24T03:39:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-14T17:59:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-11T14:04:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-11T14:03:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-10T09:43:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d12c17ac-11f1-41af-abde-29789a7b6897","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57307225,"name":"Health sciences/Biomarkers"},{"id":57307226,"name":"Health sciences/Diseases"},{"id":57307227,"name":"Biological sciences/Immunology"},{"id":57307228,"name":"Health sciences/Neurology"},{"id":57307229,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-03-23T16:02:45+00:00","versionOfRecord":{"articleIdentity":"rs-7825469","link":"https://doi.org/10.1038/s41598-026-43823-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-19 15:58:05","publishedOnDateReadable":"March 19th, 2026"},"versionCreatedAt":"2025-11-05 07:50:48","video":"","vorDoi":"10.1038/s41598-026-43823-y","vorDoiUrl":"https://doi.org/10.1038/s41598-026-43823-y","workflowStages":[]},"version":"v1","identity":"rs-7825469","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7825469","identity":"rs-7825469","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}