Prognostic relevance of the C‐X‐C motif chemokine ligand 13 and interleukin‐8 in predicting the transition from clinically isolated syndrome to multiple sclerosis

The initial phase of multiple sclerosis (MS), often known as clinically isolated syndrome (CIS), is a critical period for identifying individuals at high risk of progressing to full‐blown MS and initiating timely treatment. In this study, we aimed to evaluate the prognostic value of C‐X‐C motif chemokine ligand 13 (CXCL13) and interleukin‐8 (IL‐8) as potential markers for CIS patients' conversion to MS. Our study encompassed patients with CIS, those with relapsing–remitting MS (RRMS), and control subjects, with sample analysis conducted on both cerebrospinal fluid (CSF) and serum. Patients were categorized into four groups: CIS–CIS (no MS development within 2 years), CIS–RRMS (conversion to RRMS within 2 years), RRMS (already diagnosed), and a control group (CG) with noninflammatory central nervous system disorders. Results showed significantly elevated levels of CXCL13 in CSF across all patient groups compared with the CG (p < 0.0001, Kruskal–Wallis test). Although CXCL13 concentrations were slightly higher in the CIS–RRMS group, statistical significance was not reached. Similarly, significantly higher levels of IL‐8 were detected in CSF samples from all patient groups compared with the CG (p < 0.0001, Kruskal–Wallis test). Receiver operating characteristic analysis in the CIS–RRMS group identified both CXCL13 (area under receiver operating characteristic curve = .959) and IL‐8 (area under receiver operating characteristic curve = .939) in CSF as significant predictors of CIS to RRMS conversion. In conclusion, our study suggests a trend towards elevated CSF IL‐8 and CSF CXCL13 levels in CIS patients progressing to RRMS. These findings emphasize the importance of identifying prognostic markers to guide appropriate treatment strategies for individuals in the early stages of MS.

K E Y W O R D S biomarkers, clinically isolated syndrome, CXCL13, IL-8, multiple sclerosis

| INTRODUCTION
The initial phase of multiple sclerosis (MS) typically manifests as clinically isolated syndrome (CIS), which constitutes the first clinical episode bearing resemblance to MS. Predominantly observed in young adults, CIS impacts the optic nerve, brainstem, or spinal cord.Specifically, CIS is characterized by an acute or subacute occurrence of neurological symptoms, persisting for a duration exceeding 24 h (Thouvenot, 2015).The subsequent progression of the disease post-CIS exhibits significant variability.While around a third of patients exhibit a benign trajectory marked by minimal disability, another half develops secondary progressive MS (Miller et al., 2012).Within the CIS stage, the identification of patients at heightened risk of MS development stands as a pivotal objective.The potential progression of the disease can significantly influence the formulation of treatment strategies.
The burgeoning focus among MS specialists on treating CIS prompts an inquiry into the prospective ramifications of forecasting the transition from CIS to MS.The potential impact of such prognostication lies in its capacity to substantially influence clinical practice.By identifying individuals at an augmented risk of conversion, clinicians can adopt a preemptive therapeutic approach, intervening at an earlier stage in the disease continuum.Early identification of patients poised for progression allows for timely and targeted interventions, potentially mitigating the severity of subsequent MS manifestations.The strategic incorporation of predictive markers into clinical decision-making processes may, therefore, usher in a paradigm shift towards more proactive and personalized management strategies for CIS.Consequently, numerous prognostic markers, such as chitinase 3 like 1 and chitinase 3 like 2, neurofilament light (NfL) chains, and interleukins IL-2, IL-6, and IL-10, have been subject to investigation for their predictive value (Ferreira-Auesta et al., 2021;Petržalka et al., 2022).
Furthermore, understanding and foreseeing the conversion from CIS to MS have broader implications for resource allocation in healthcare systems.Anticipatory interventions may lead to optimized resource utilization, reducing the burden on healthcare infrastructure associated with the management of advanced MS cases.Additionally, early identification offers the prospect of enhancing patient outcomes, ameliorating quality of life, and minimizing the societal and economic impact of fullfledged MS.
Cerebrospinal fluid (CSF) analysis, in conjunction with the assessment of the patient's clinical state and magnetic resonance imaging (MRI) results, holds a crucial role in MS diagnosis.A defining feature of CSFrelated alterations is the presence of limited oligoclonal bands (OCBs), observable in the vast majority of MS patients (Deisenhammer et al., 2019).The role of B cells in connection with MS and its progression has been substantiated through the scrutiny of B cell-associated biomarkers in CSF (Arneth, 2019;Bhargava et al., 2022).Among these markers, one such example is C-X-C motif chemokine ligand 13 (CXCL13), a chemotactic molecule that attracts B cells, which are deemed pivotal in MS pathogenesis.CXCL13 is generated within ectopic lymphoid follicles, linked to inferior MS outcomes.Elevated CXCL13 levels might aid in predicting the conversion of patients with CIS to full-fledged MS (Alvarez et al., 2013;Hartung et al., 2014).
An additional noteworthy marker tied to MS activity is interleukin-8 (IL-8), produced by various cell types such as monocytes, lymphocytes, granulocytes, astrocytes, and epithelial cells.IL-8 is an inflammatory chemokine primarily responsible for attracting and activating neutrophilic granulocytes, basophilic granulocytes, and specific lymphocyte subsets.Notably, IL-8 also exerts robust angiogenic effects (Bielekova et al., 2012).Research has demonstrated significantly raised levels of CSF IL-8 in MS patients (Matejčíkov a et al., 2015).
Our study aimed to assess the significance of CXCL13 and IL-8 as prognostic indicators in patients diagnosed with CIS, in comparison with a control group.The evaluation occurred at the juncture when the initial clinical manifestations of the disease were registered.

| Ethical statement
The study was carried out in accordance with the Helsinki Declaration of 1964Declaration of (2013 revision) revision).This study was approved by the ethics committee of Palacký University in Olomouc.All patients signed an informed consent form for a lumbar puncture for diagnostic reasons; no healthy volunteers were involved.

| Patients and sampling
In the prospective study conducted at the Department of Neurology, Faculty of Medicine and Dentistry, Palacký University and University Hospital Olomouc from 2018 to 2022, CSF and serum samples were collected from patients with CIS and relapsing-remitting MS (RRMS).Lumbar puncture was performed as part of routine diagnostic procedures.Over a 2 year time span, some patients progressed to RRMS (CIS-RRMS), while others remained in CIS (CIS-CIS).Disability was assessed using Kurtzke's (1961) (Thompson et al., 2018).Patients were treated with platform therapy.
The patient cohort was subsequently categorized into four distinct groups.The first group, labelled CIS-CIS, consisted of individuals who did not progress to MS within 2 years following the collection of CSF samples.The second group, designated CIS-RRMS, encompassed patients who transitioned to RRMS within the same 2 year period following CSF collection.The third group, denoted as RRMS, comprised patients for whom marker analysis was conducted at the point of confirmed disease diagnosis.The fourth and final group constituted the control group.The control group comprised patients with noninflammatory central nervous system (CNS) involvement, such as headaches, back pain, and vertigo, for whom lumbar puncture was indicated for differential diagnostic purposes, in order to exclude pathological processes in the CNS.The control cohort comprised individuals matched for age and sex patients and did not exhibit signs of inflammatory, structural, or haemorrhagic lesions during MRI.
CSF was acquired through a lumbar puncture procedure using an atraumatic needle with patients positioned in a seated posture.The puncture was targeted at the L4/5 intervertebral space.A volume of 10 mL of CSF was collected from each patient into a sterile tube devoid of any additives.The sample underwent initial evaluation for cell count and qualitative cytology, followed by centrifugation (at 1100 g for 10 min at 4 C) for further biochemical and immunological analysis.Samples were not subjected to freezing and were promptly dispatched for analysis.
The paired samples of CSF and blood were analysed immediately after collecting.The following laboratory tests were performed: basic biochemical examination (total protein and glucose), cytological examination (cell count and qualitative cytology), determination of albumin quotient (Q Alb) to assess the status of the blood-CSF barrier, quantitative determination of immunoglobulin concentrations (IgG, IgM, and IgA), isoelectric focussing to determine OCBs, and determination of interleukins.Normal ranges of individual analytes were verified and published by the accredited Laboratory for CSF, Neuroimmunology, Pathology and Special Diagnostics, Prague (according to ISO 15189; see Laboratory Manual at www.likvor.cz).

| CXCL13 and IL-8 analysis
The concentration of CXCL13 in CSF was quantified utilizing a sandwich Enzyme-Linked Immuno Sorbent Assay technique, employing the Euroimmun CXCL13 ELISA CE kit (Euroimmun, Lübeck, Germany), with a catalogue number Equation 6811-9602-L.The analysis was executed in compliance with the manufacturer's guidelines, where values below 20 pg/mL were deemed within the normal range.
To measure IL-8 levels in CSF, an enzyme-labelled solid-phase chemiluminescent sequential immunometric assay was employed, specifically, the IMMULITE ® 1000 IL-8 (Euroimmun, Lübeck, Germany) kit, catalogue number LK8P1.The analysis was conducted following the manufacturer's protocol, with CSF concentrations of up to 62 pg/mL considered as falling within the normal range.

| Statistical analysis
The Shapiro-Wilks normality test showed that the data do not have a normal distribution.Therefore, the data were expressed as median, minimum, and maximum values, and independent samples were compared using the Kruskal-Wallis test.Graphically, the distribution of data is shown in box graphs.The distribution of the measured values is shown in a box graph.The horizontal line in the box shows the median value, the lower edge of the box the value of the first quartile (25th percentile), and the upper edge the value of the third quartile (75th percentile).The terminals show the maximum and minimum measured values.Outliers (values that are more than 1.5 times the interquartile range from the quartiles) are plotted in circles.Extremes (values that are more than three times the interquartile range from the quartiles) are plotted with asterisks.All tests were performed at a significance level of 0.05.IBM SPSS Statistics for Windows, Version 23.0 statistical software was used for statistical processing, Armonk, NY: IBM Corp.
In this study, receiver operating characteristic (ROC) analysis was employed to assess the diagnostic performance of the tested method.ROC curves were constructed to evaluate the trade-off between sensitivity and specificity across different threshold values.The optimal threshold point was determined to maximize the diagnostic accuracy.The area under the ROC curve (AUC) was calculated to quantify the overall discriminatory power of the method.ROC analysis was used to aid in the evaluation and selection of the most suitable diagnostic criteria for our study.

| Patients and controls
The study encompassed 124 patients, who were categorized into four distinct groups, comprising 37 individuals in the CIS-CIS group (consisting of 7 males and 30 females, with a mean age of 34.0), 14 patients in the CIS-RRMS group (comprising 2 males and 12 females, with a mean age of 35.0), 18 patients in the RRMS group (comprising 4 males and 14 females, with a mean age of 38.0), and 55 individuals in the control group (comprising 9 males and 46 females, with a mean age of 33.0).These groups exhibited homogeneity with regard to both gender and age.Detailed data are presented in Tables 1 and 2.
All patients underwent examinations for CXCL13 in CSF, IL-8 in CSF, CSF cell count, qualitative CSF cytology, OCB in CSF of IgG, and the Expanded Disability Status Scale.A summary of the data is presented in Tables 3  and 4. At the time of collection, patients exhibited no clinical or laboratory indications of neuroinfection, and there were no ongoing systemic inflammatory processes.

| Group comparison
Following the statistical analysis, markedly elevated levels of IL-8 were observed in all examined patient groups when compared with the control group (CIS-CIS: median 51.40 vs. 36.60pg/mL; CIS-RRMS: median 55.85 vs. 36.60pg/mL; RRMS: 55.70 vs. 36.60pg/mL; p < 0.0001, Kruskal-Wallis test).No statistically significant distinctions were noted among the individual patient groups, as the levels of IL-8 in these groups were comparable, as illustrated in Figure 1.A summary of the data is presented in Table 3.
Significantly elevated levels of the CXCL13 marker were also observed in all groups when compared with the control group (CIS-CIS: median 16.82 vs. .10pg/mL; CIS-RRMS: median 32.30 vs. .10pg/mL; RRMS: median 5.37 vs. .10pg/mL; p < 0.0001, Kruskal-Wallis test).Notably, within the CIS-RRMS group, the concentration of CXCL13 was higher in comparison with the other groups (CIS-CIS median 16.82 pg/mL; CIS-RRMS median 32.30 pg/mL; RRMS median 5.37 pg/mL).However, these differences did not attain statistical significance, as indicated by Figure 2. A summary of the data is presented in Table 3.

| ROC analysis
In the CIS-RRMS group, ROC analysis identified both CXCL13 (AUC = .959)and IL-8 (AUC = .939)in CSF as significant predictors for the conversion from CIS to RRMS.The optimal cut-off value for IL-8 in CSF was determined to be 45.6 pg/mL, as calculated by the Youden's J statistic, which maximizes the sum of sensitivity (.9) and specificity (.836).For CXCL13 in CSF, the optimal cut-off value was .210pg/mL, also determined by Youden's J statistic, with sensitivity of .95 and specificity of .873.This test showed higher sensitivity.Alternatively, if a higher cut-off value (1.795) were selected, both sensitivity and specificity would be more balanced, both approximately at .9, with sensitivity = .900and specificity = .909.The data are summarized in Table 4 and depicted in Figure 3.

| Correlation analysis
The results of the correlation analysis are summarized in Table 5.

| CIS-CIS group
Correlation analysis for patients within the CIS-CIS group provides the following information: Q albumin and CSF IL-8 Correlation coefficient (r) = 0.383, indicating a moderate positive linear correlation between levels of Q albumin and CSF IL-8.This suggests that there is a certain relationship between these two variables.
p-value = 0.025, which is lower than the standard significance level of 0.05, meaning that the correlation is statistically significant.There is a probability of less than 5% that this correlation was obtained by chance.

OCBs CSF IgG and CSF CXCL13
Correlation coefficient (r) = 0.447, indicating a moderate to strong positive linear correlation between OCBs T A B L E 3 Interleukin-8 (IL-8), C-X-C motif chemokine ligand 13 (CXCL13), oligoclonal band (OCB) (IgG), cell count, cerebrospinal fluid (CSF) protein, IgG index, and Q albumin in patients in the study groups.in the CSF and levels of CXCL13.This means there is a significant relationship between these two variables.p-value = 0.008, which is lower than the standard significance level of 0.05, suggesting that the correlation is statistically significant.There is a probability of less than 1% that this correlation occurred by chance.

CSF protein and CSF IL-8
Correlation coefficient (r) = 0.342, indicating a moderate positive linear correlation between CSF protein concentration and IL-8 levels.This means there is a mild to moderately strong relationship between these two variables.p-value = 0.048, which is lower than the standard significance level of 0.05, suggesting that the correlation is statistically significant.There is a probability of less than 5% that this correlation occurred by chance.

| CIS-RRMS group
Correlation analysis for patients within the CIS-RRMS group provides the following information: Q albumin and CSF IL-8 Correlation coefficient (r): The value of 0.578 indicates a moderately strong positive linear correlation between Q albumin levels and CSF IL-8.This means there is a relatively strong relationship between these two variables, with an increase in one being associated with an increase in the other.
p-value: The p-value of 0.030 is lower than the standard significance level of 0.05, suggesting that the correlation is statistically significant at the 0.05 level.There is a probability of less than 5% that this correlation occurred by chance.

| Controls
Correlation analysis for patients within the CONTROLS group provides the following information:

CSF protein and CSF IL-8
Correlation coefficient (r): The value of 0.303 indicates a low positive linear correlation between total protein in CSF and levels of CSF IL-8.This means there is some weak relationship between these two variables, with an increase in one being associated with an increase in the other.
p-value: The p-value of 0.025 is lower than the standard significance level of 0.05, suggesting that the correlation is statistically significant at the 0.05 level.There is a probability of less than 5% that this correlation occurred by chance.

| DISCUSSION
The diagnosis of MS requires a comprehensive approach and is based on the evaluation of the patient's clinical condition and MRI findings.However, MRI is not an infallible diagnostic tool, especially at the CIS stage.CSF examinations play an irreplaceable role in the diagnosis of MS.Many studies have recently been conducted that focus on the use of new prognostic markers such as serum and CSF NfL, serum NfH, CSF chitinase 3 like 1, CSF chitinase 3 like 2, CSF IL-2, and CSF IL-2/IL-6.They may be beneficial in the diagnosis and prognosis of MS (Eikelenboom et al., 2011;Ferreira-Auesta et al., 2021;Modvig et al., 2015;Petržalka et al., 2022;Sapko et al., 2020).NfL chains, for instance, have shown F I G U R E 3 Receiver operating characteristic (ROC) analysis and clinically isolated syndrome (CIS)relapsing-remitting multiple sclerosis (RRMS) prediction.Statistically significant predictors for CIS-RRMS include interleukin-8 (IL-8) cerebrospinal fluid (CSF) and C-X-C motif chemokine ligand 13 (CXCL13) CSF.In both cases, the AUC is greater than .9,indicating excellent discriminatory ability.The optimal cutoff value for IL-8 CSF is 45.6 pg/mL (determined by the Youden's J statistic, where the sum of sensitivity and specificity is the highest; sensitivity = .9;specificity = .836).The optimal cut-off value for CXCL13 CSF is .210pg/mL (determined by the Youden's J statistic, where the sum of sensitivity and specificity is highest; sensitivity = .95;specificity = .873).
promise in assessing disease progression (Cai & Huang, 2018).In our research, we focussed on the use of the markers CXCL13 and IL-8 in assessing CIS to MS conversion.Considering CSF biomarkers, such as CXCL13 and IL-8, there is a need to elucidate their significance in enhancing the prediction of MS progression.

CIS-CIS
CXCL13 CSF (pg/mL) IL-8 CSF (pg/mL) CXCL13, a chemokine associated with B cell recruitment, and IL-8, an inflammatory cytokine, may provide valuable insights into the underlying pathophysiological processes.Their measurement in CSF can potentially refine the prognostic accuracy by offering a more nuanced understanding of the immune and inflammatory responses within the CNS.Integrating these markers into predictive models may contribute to a more comprehensive and accurate assessment of the disease course, aiding in prognostication and informing therapeutic decisions in the clinical management of MS.IL-8 is a chemoattractant, a cytokine produced by many tissue cells and leucocytes.It attracts and activates neutrophilic granulocytes and other immune cells in inflammatory areas (Baggiolini & Clark-Lewis, 1992).In CSF, its levels are elevated during inflammatory processes of the CNS.IL-8 is responsible for opening the blood-brain barrier allowing immune cells to migrate to the CNS (Bielekova et al., 2012).Neutrophils are highly variable population concerning to their involvement in MS aetiopathogenesis.Upon activation, they could release proinflammatory acting cytokine IL-1, several proteases, myeloperoxidase, and reactive oxygen species, could damage the blood-brain barrier, could destroy myelin, present autoantigens, and release neutrophil extracellular traps which, particularly, are known to contribute either to inflammation activation or inflammation resolution.Neutrophils seem to be more involved in the MS initiation than the maintenance (De Bondt et al., 2020;Woodberry et al., 2018).In our study, we identified heightened IL-8 levels in patients diagnosed with CIS and MS, thereby affirming the presence of ongoing inflammatory processes.Previous investigations, including those by Matejčíkov a et al. (2015Matejčíkov a et al. ( , 2017)), have detected increased CSF levels of IL-8 coinciding with the initial clinical manifestations of MS, while concurrently observing decreased IL-8 levels in the serum.Studies by Bartosik-Psujek and Stelmasiak (2005) and those by Stelmasiak et al. (2000) have further substantiated significant rises in CSF IL-8 levels during relapse episodes.
Within our cohort, IL-8 levels consistently displayed elevation across all groups.Specifically, for the CIS-RRMS group, an ROC analysis was conducted, and it revealed that CSF IL-8 served as a noteworthy predictor for the transition from CIS to RRMS, with an AUC of .939.The optimal threshold for IL-8 in CSF was identified as 45.6 pg/mL, as determined by Youden's J statistic, which maximizes the amalgamation of sensitivity (.9) and specificity (.836).
These observations imply that CSF IL-8 may prove to be a suitable indicator for forecasting disease prognosis.According to our observation, Rossi et al. (2015) reported that heightened CSF IL-8 levels were linked to an increased risk of transition from CIS to RRMS and a heightened frequency of relapses during the initial 2 years of the study.
CXCL13 is a chemokine that exerts chemotactic effects on B cells, which play a crucial role in the pathogenesis of MS.This chemokine is produced by macrophages and follicular dendritic cells, and monitoring changes in its concentration can provide insights into the condition of MS patients.CXCL13 interacts with the C-X-C chemokine receptor type 5, acting as a chemoattractant that guides B cells to secondary lymphatic organs.Furthermore, CXCL13 promotes cytokine secretion, facilitating humoral immune responses and the migration of plasma cells and B cells to the CSF (Bielekova et al., 2012;Legler et al., 1998;Rupprecht et al., 2009).Elevated CXCL13 levels are commonly observed in patients with neuroborreliosis (Hartung et al., 2014).Numerous studies have investigated the levels and prognostic significance of CXCL13 in patients with CIS and MS.For instance, a study by Khademi et al. (2011) associated CXCL13 with disease exacerbation and poor prognosis in RRMS.High CXCL13 levels were predictive of the conversion from CIS to MS (Rossi et al., 2015).In a prospective study by Brettschneider et al. (2010), CXCL13 was found to be relevant in CIS for predicting conversion to MS, underlining its role in the inflammatory cascade linked to the intrathecal B cell response.Lepennetier et al. (2019) confirmed CXCL13 as a reliable marker and its utility in predicting disease activity in MS and diagnosing lyme neuroborreliosis and CNS lymphoma.Additionally, Ferraro et al. (2015) established a correlation between CSF CXCL13 levels and markers of CNS inflammation, suggesting that CXCL13 levels were associated with earlier conversion to MS and a more aggressive disease course.A study by DiSano et al. (2020) emphasized the predictive value of the CXCL13 index in comparison with OCBs and CSF NfL in patients with clinically and radiologically isolated syndrome and MS.The CXCL13 index demonstrated significant increases in MS patients with active disease, surpassing both OCB and CSF NfL in terms of sensitivity, specificity, and the identification of future disease activity (DiSano et al., 2020).
Our study findings indicate elevated CXCL13 levels in patients with CIS and MS.Within our cohort, we observed a tendency towards higher CXCL13 levels in patients transitioning from CIS to RRMS.In the CIS-RRMS group, ROC analysis identified CXCL13 in CSF as a highly significant predictor for the transition from CIS to RRMS, with an impressive AUC value of .959.The optimal cut-off value for CXCL13 in CSF was determined to be .210pg/mL, based on Youden's J statistic, providing a sensitivity of .95 and specificity of .873.Notably, this test demonstrated notably high sensitivity.Alternatively, if a higher cut-off value of 1.795 were chosen, both sensitivity and specificity would be more evenly balanced, each approximately at .9, with sensitivity = .900and specificity = .909.
Analysing the correlation results reveals notable patterns among different patient groups.In the CIS-CIS group, there is a moderate to strong positive correlation between Q albumin and CSF IL-8 and OCBs of IgG in CSF and CSF CXCL13, as well as CSF protein concentration and CSF IL-8.These correlations demonstrate statistical significance.Similarly, within the CIS-RRMS group, a moderately strong positive correlation is evident between Q albumin and IL-8, reaching statistical significance.Meanwhile, in the control group, a low positive correlation exists between total protein in CSF and levels of CSF IL-8, with this correlation being statistically significant.Conversely, no significant correlations were observed in the RRMS group based on the correlation analysis.A significant correlation between Q albumin and IL-8 could indicate a disruption of the blood-CSF barrier in the early stages of the disease.IL-8 may be transgressing through the compromised blood-CSF barrier.The findings align with the results reported by Matejčíkov a et al. (2017).
These findings are in line with previous research underlining the significance of CSF CXCL13 and CSF IL-8 as biomarkers in patients with MS.CXCL13 levels in CSF and CSF IL-8 thus hold the potential to serve as an additional tool for identifying patients at a heightened risk of transitioning to MS, thereby assisting clinicians in determining the need for early intervention.Nevertheless, further investigation involving a larger patient cohort is imperative to validate these observations.

| CONCLUSION
Based on the analysis of the collected data, it appears that serum IL-8 may not be a suitable prognostic marker for the conversion from CIS to MS.Conversely, the CSF IL-8 and CSF CXCL13 markers show greater promise in this regard.We observed an inclination towards higher CSF IL-8 and CSF CXCL13 levels in patients who transitioned from CIS to RRMS.These findings are consistent with previously published studies that have highlighted the prognostic significance of IL-8 and CXCL13 in patients with CIS.
Considering the evidence from our results and those of other studies, it is conceivable that CSF IL-8 and CSF CXCL13 hold potential as an indicator of inflammatory activity in individuals with MS, and its inclusion in routine clinical practice should be contemplated.
Nonetheless, these findings should be validated through further investigation involving a larger patient cohort.

F
I G U R E 1 Comparisons of interleukin-8 (IL8).Comparison of IL-8 (pg/mL) levels in the CSF between the CIS-CIS, CIS-RRMS, RRMS, and controls.The distribution of measured values was represented using box plots (the horizontal line inside the box represents the median, the lower edge of the box represents the first quartile, the upper edge represents the third quartile, whiskers indicate the maximum and minimum measured values, and if outliers were found in the dataset, they are plotted as circles and asterisks).CIS, clinically isolated syndrome, CSF, cerebrospinal fluid; RRMS, relapsingremitting multiple sclerosis.F I G U R E 2 Comparisons of C-X-C motif chemokine ligand 13 (CXCL13).Comparison of CXCL13 (pg/mL) levels in the CSF between the CIS-CIS, CIS-RRMS, RRMS, and controls.The distribution of measured values was represented using box plots (the horizontal line inside the box represents the median, the lower edge of the box represents the first quartile, the upper edge represents the third quartile, whiskers indicate the maximum and minimum measured values, and if outliers were found in the dataset, they are plotted as circles and asterisks).CIS, clinically isolated syndrome, CSF, cerebrospinal fluid; RRMS, relapsingremitting multiple sclerosis.
Expanded Disability Status Scale by experienced neurologists affiliated with the Department of Neurology, Faculty of Medicine and Dentistry, Palacký University and University Hospital Olomouc.Diagnosis was established in accordance with the revised McDonald diagnostic criteria the group in terms of age.
Note:The table summarizes the characteristics of patient groups and healthy controls, including information on age.The comparison between the patient groups and the control group revealed no significant differences in terms of age ( p = 0.429).Abbreviations: CIS, clinically isolated syndrome; RRMS, relapsing-remitting multiple sclerosis.TA B L E 2 Characteristics of the group in terms of gender.