Re-assessment of monoclonal antibodies against diclofenac for their application in the analysis of environmental waters.

The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is an important environmental contaminant occurring in surface waters all over the world, because, after excretion, it is not adequately removed from wastewater in sewage treatment plants. To be able to monitor this pollutant, highly efficient analytical methods are needed, including immunoassays. In a medical research project, monoclonal antibodies against diclofenac and its metabolites had been produced. Based on this monoclonal anti-DCF antibody, a new indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed and applied for environmental samples. The introduction of a spacer between diclofenac and the carrier protein in the coating conjugate led to higher sensitivity. With a test midpoint of 3 µg L-1 and a measurement range of 1-30 µg L-1, the system is not sensitive enough for direct analysis of surface water. However, this assay is quite robust against matrix influences and can be used for wastewater. Without adjustment of the calibration, organic solvents up to 5%, natural organic matter (NOM) up to 10 mg L-1, humic acids up to 2.5 mg L-1, and salt concentrations up to 6 g L-1 NaCl and 75 mg L-1 CaCl2 are tolerated. The antibody is also stable in a pH range from 3 to 12. Cross-reactivity (CR) of 1% or less was determined for the metabolites 4'-hydroxydiclofenac (4'-OH-DCF), 5-hydroxydiclofenac (5-OH-DCF), DCF lactam, and other NSAIDs. Relevant cross-reactivity occurred only with an amide derivative of DCF, 6-aminohexanoic acid (DCF-Ahx), aceclofenac (ACF) and DCF methyl ester (DCF-Me) with 150%, 61% and 44%, respectively. These substances, however, have not been found in samples. Only DCF-acyl glucuronide with a cross-reactivity of 57% is of some relevance. For the first time, photodegradation products were tested for cross-reactivity. With the ELISA based on this antibody, water samples were analysed. In sewage treatment plant effluents, concentrations in the range of 1.9-5.2 µg L-1 were determined directly, with recoveries compared to HPLC-MS/MS averaging 136%. Concentrations in lakes ranged from 3 to 4.4 ng L-1 and were, after pre-concentration, determined with an average recovery of 100%.

Adjuvant PD-1 Checkpoint Inhibition in Early Cutaneous Melanoma: Immunological Mode of Action and the Role of Ultraviolet Radiation.

Melanoma ranks as the fifth most common solid cancer in adults worldwide and is responsible for a significant proportion of skin-tumor-related deaths. The advent of immune checkpoint inhibition with anti-programmed death protein-1 (PD-1) antibodies has revolutionized the adjuvant treatment of high-risk, completely resected stage III/IV melanoma. However, not all patients benefit equally. Current strategies for improving outcomes involve adjuvant treatment in earlier disease stages (IIB/C) as well as perioperative treatment approaches. Interfering with T-cell exhaustion to counteract cancer immune evasion and the immunogenic nature of melanoma is key for anti-PD-1 effectiveness. Yet, the biological rationale for the efficacy of adjuvant treatment in clinically tumor-free patients remains to be fully elucidated. High-dose intermittent sun exposure (sunburn) is a well-known primary risk factor for melanomagenesis. Also, ultraviolet radiation (UVR)-induced immunosuppression may impair anti-cancer immune surveillance. In this review, we summarize the current knowledge about adjuvant anti-PD-1 blockade, including a characterization of the main cell types most likely responsible for its efficacy. In conclusion, we propose that local and systemic immunosuppression, to some extent UVR-mediated, can be restored by adjuvant anti-PD-1 therapy, consequently boosting anti-melanoma immune surveillance and the elimination of residual melanoma cell clones.

Beyond T cell toxicity - Intrathecal chemokine CXCL13 indicating B cell involvement in immune-related adverse events following checkpoint inhibition: A two-case series and literature review.

BACKGROUND AND PURPOSE: This study was undertaken to raise awareness of a role of B cells in immune checkpoint inhibitor (ICI)-associated neurological immune-related adverse events (nirAE). METHODS: A systematic literature review was made, with case observations of a melanoma and a non-small cell lung cancer (NSCLC) patient who developed ICI-associated nirAE with cerebrospinal fluid (CSF) findings indicating B cell involvement. RESULTS: Two patients receiving ipilimumab/nivolumab for melanoma and chemotherapy/pembrolizumab for NSCLC developed nirAE in the form of myocarditis/myositis/myasthenia gravis overlap syndrome (triple M) and cerebellitis plus longitudinal transverse myelitis (c-LETM), respectively. Intrathecal inflammation with chemokine C-X-C motif ligand (CXCL13) elevation was present in both patients; the triple M case had acetylcholine receptor antibodies, antititin reactivity, altered CD4/CD8 T cell ratio in blood, and depressed programmed death-1 (PD-1) expression on CSF T cells; the c-LETM case showed intrathecal antibody production and plasma cells. Both patients insufficiently responded to first-line treatment. The NSCLC case improved upon administration of B cell-depleting therapy with rituximab, whereas the melanoma patient died before escalation therapy was initiated. Literature research revealed one additional ICI-associated LETM case with intrathecal CXCL13 elevation, three cases with ICI-associated aquaporin-4 antibody neuromyelitis spectrum disorder, and evidence of B cell-mediated toxicity based on antibody-mediated immune pathologies in ICI-associated immune-related adverse events. CONCLUSIONS: The case observations highlight the plethora of uncertainties in diagnosis and treatment of ICI-associated nirAE, exemplify the heterogeneity of immune mechanisms involved, and suggest a role of B cells, which may be underdiagnosed. Intrathecal CXCL13 may serve as a biomarker of B cell involvement in nirAE, supported by intrathecal immunoglobulin synthesis, presence of plasma cells, and/or recruitment of cognate immune cells.

Immunomodulatory Aspects of Therapeutic Plasma Exchange in Neurological Disorders-A Pilot Study.

Therapeutic plasma exchange (TPE) is used for drug-resistant neuroimmunological disorders, but its mechanism of action remains poorly understood. We therefore prospectively explored changes in soluble, humoral, and cellular immune components associated with TPE. We included ten patients with neurological autoimmune disorders that underwent TPE and assessed a panel of clinically relevant pathogen-specific antibodies, total serum immunoglobulin (Ig) levels, interleukin-6 (IL-6, pg/mL), C-reactive protein (CRP, mg/dL), procalcitonin (PCT, µg/L) and major lymphocyte subpopulations (cells/µL). Blood was collected prior to TPE (pre-TPE, baseline), immediately after TPE (post-TPE), as well as five weeks (follow-up1) and 130 days (follow-up2) following TPE. Pathogen-specific antibody levels were reduced by -86% (p < 0.05) post-TPE and recovered to 55% (follow-up1) and 101% (follow-up2). Ig subclasses were reduced by -70-89% (p < 0.0001) post-TPE with subsequent complete (IgM/IgA) and incomplete (IgG) recovery throughout the follow-ups. Mean IL-6 and CRP concentrations increased by a factor of 3-4 at post-TPE (p > 0.05) while PCT remained unaffected. We found no alterations in B- and T-cell populations. No adverse events related to TPE occurred. TPE induced a profound but transient reduction in circulating antibodies, while the investigated soluble immune components were not washed out. Future studies should explore the effects of TPE on particular cytokines and assess inflammatory lymphocyte lineages to illuminate the mode of action of TPE beyond autoantibody removal.

Serum Neurofilament Light Chain as Biomarker for Cladribine-Treated Multiple Sclerosis Patients in a Real-World Setting.

Serum neurofilament light chain (sNfL) is an intensely investigated biomarker in multiple sclerosis (MS). The aim of this study was to explore the impact of cladribine (CLAD) on sNfL and the potential of sNfL as a predictor of long-term treatment response. Data were gathered from a prospective, real-world CLAD cohort. We measured sNfL at baseline (BL-sNfL) and 12 months (12Mo-sNfL) after CLAD start by SIMOA. Clinical and radiological assessments determined fulfilment of "no evidence of disease activity" (NEDA-3). We evaluated BL-sNfL, 12M-sNfL and BL/12M sNfL ratio (sNfL-ratio) as predictors for treatment response. We followed 14 patients for a median of 41.5 months (range 24.0-50.0). NEDA-3 was fulfilled by 71%, 57% and 36% for a period of 12, 24 and 36 months, respectively. We observed clinical relapses in four (29%), MRI activity in six (43%) and EDSS progression in five (36%) patients. CLAD significantly reduced sNfL (BL-sNfL: mean 24.7 pg/mL (SD ± 23.8); 12Mo-sNfL: mean 8.8 pg/mL (SD ± 6.2); p = 0.0008). We found no correlation between BL-sNfL, 12Mo-sNfL and ratio-sNfL and the time until loss of NEDA-3, the occurrence of relapses, MRI activity, EDSS progression, treatment switch or sustained NEDA-3. We corroborate that CLAD decreases neuroaxonal damage in MS patients as determined by sNfL. However, sNfL at baseline and at 12 months failed to predict clinical and radiological treatment response in our real-world cohort. Long-term sNfL assessments in larger studies are essential to explore the predictive utility of sNfL in patients treated with immune reconstitution therapies.

Revisiting the Role of the CXCL13/CXCR5-Associated Immune Axis in Melanoma: Potential Implications for Anti-PD-1-Related Biomarker Research.

CXCL13 is a potent chemoattractant cytokine that promotes the migration of cells expressing its cognate receptor, CXCR5. Accordingly, T follicular helper cells and B cells migrate towards B cell follicles in lymph nodes, where the resulting spatial proximity promotes B cell/T cell interaction and antibody formation. Moreover, effector cells of the CXCL13/CXCR5-associated immune axis express PD-1, with corresponding circulating cells occurring in the blood. The formation of so-called ectopic or tertiary lymphoid structures, recently detected in different cancer types, represents an integral part of this axis, particularly in the context of its emerging role in anti-tumor defense. These aspects of the CXCL13/CXCR5-associated immune axis are highlighted in this review, which focuses on cutaneous malignant melanoma. Specifically, we elaborate on the role of this important immune axis as a possible ancillary target of immune checkpoint inhibition with anti-PD-1 antibodies in different therapeutic settings and as a potential source of predictive biomarkers regarding treatment efficacy.

Humoral Response to SARS-CoV-2 Antigen in Patients Treated with Monoclonal Anti-CD20 Antibodies: It Is Not All about B Cell Recovery.

Anti-CD20 therapies decrease the humoral response to SARS-CoV-2 immunization. We aimed to determine the extent of the humoral response to SARS-CoV-2 antigens in correlation with peripheral B-cell dynamics among patients with central nervous system inflammatory disorders treated with anti-CD20 medications. We retrospectively included patients receiving anti-CD20 therapy after antigen contact who were divided into responders (>7 binding antibody units (BAU)/mL) and non-responders (<7 BAU/mL). In participants with first antigen contact prior to therapy, we investigated the recall response elicited once under treatment. We included 80 patients (responders n = 34, non-responders n = 37, recall cohort n = 9). The B-cell counts among responders were significantly higher compared to non-responders (mean 1012 cells/µL ± SD 105 vs. mean 17 cells/µL ± SD 47; p < 0.001). Despite very low B-cell counts (mean 9 cells/µL ± SD 20), humoral response was preserved among the recall cohort (mean 1653 BAU/mL ± SD 2250.1) and did not differ significantly from responders (mean 735 BAU/mL ± SD 1529.9; p = 0.14). Our data suggest that peripheral B cells are required to generate antibodies to neo-antigens but not for a recall response during anti-CD20 therapy. Evaluation of B-cell counts and pre-existing SARS-CoV-2 antibodies might serve as biomarkers for estimating the immune competence to mount a humoral response to SARS-CoV-2 antigens.

The CXCL13/CXCR5 Immune Axis in Health and Disease-Implications for Intrathecal B Cell Activities in Neuroinflammation.

The chemokine C-X-C- ligand 13 (CXCL13) is a major B cell chemoattractant to B cell follicles in secondary lymphoid organs (SLO) that proposedly recruits B cells to the cerebrospinal fluid (CSF) during neuroinflammation. CXCR5, the cognate receptor of CXCL13, is expressed on B cells and certain T cell subsets, in particular T follicular helper cells (Tfh cells), enabling them to follow CXCL13 gradients towards B cell follicles for spatial proximity, a prerequisite for productive T cell-B cell interaction. Tfh cells are essential contributors to B cell proliferation, differentiation, and high-affinity antibody synthesis and are required for germinal center formation and maintenance. Circulating Tfh cells (cTfh) have been observed in the peripheral blood and CSF. Furthermore, CXCL13/CXCR5-associated immune activities organize and shape adaptive B cell-related immune responses outside of SLO via the formation of ectopic lymphoid structures in inflamed tissues, including the central nervous system (CNS). This review summarizes the recent advances in our understanding of the CXCL13/CXCR5 immune axis and its role in vaccination, autoimmunity, and infection with a special focus on its relevance for intrathecal B cell activities in inflammatory CNS diseases.

Long-term immunological consequences of anti-CD20 therapies on humoral responses to COVID-19 vaccines in multiple sclerosis: an observational study.

Background: Anti-CD20 therapies induce pronounced B-cell depletion and blunt humoral responses to vaccines. Recovery kinetics of anti-CD20 therapy-mediated cellular and humoral effects in people with multiple sclerosis (pwMS) are poorly defined. Objective: To investigate the duration of the anti-CD20 treatment-induced effects on humoral responses to COVID-19 vaccines. Methods: This retrospective observational study included pwMS who had discontinued anti-CD20 therapy for ⩾12 months and remained without immunomodulation. We retrieved demographics and laboratory parameters including B-cell counts and immunoglobulin (IgG, IgM, IgA) levels prior to anti-CD20 commencement (baseline) and longitudinally after anti-CD20 treatment discontinuation from electronic medical records. Humoral responses to SARS-CoV-2 vaccines were compared with a population of 11 pwMS with ongoing anti-CD20 medication (control cohort). Results: A total of 24 pwMS had discontinued anti-CD20 therapy for a median of 34 months (range: 16-38 months). Antibody responses to COVID-19 vaccines were available in 17 (71%). Most individuals (n = 15, 88%) elicited a measurable antibody response [mean: 774 BAU/ml (±SD 1283 BAU/ml)] to SARS-CoV-2 immunization on average 22 months (range: 10-30 months) from the last anti-CD20 infusion, which was higher compared with the population with ongoing anti-CD20 therapy (n = 11, mean: 12.36 ± SD 11.94 BAU/ml; p < 0.00001). Significantly increased antibody levels compared with the control cohort were found among pwMS who were vaccinated >18 months after treatment discontinuation (19-24 months: n = 2, p = 0.013; 25-36 months: n = 9; p < 0.001). The interindividual kinetics for B-cell reconstitution were heterogeneous and mean B-cell counts approached normal ranges 18 months after treatment discontinuation. There was no correlation of B-cell repopulation and vaccine responses. Mean total IgG, IgM, and IgA levels remained within the reference range. Conclusion: Anti-CD20-induced inhibition of humoral responses to COVID-19 vaccines is transient and antibody production was more pronounced >18 months after anti-CD20 treatment discontinuation. The immunological effect on B-cell counts appears to wane by the same time.

Recall response to COVID-19 antigen is preserved in people with multiple sclerosis on anti-CD20 medications - A pilot study.

BACKGROUND: Antibody responses to SARS-CoV-2 vaccination are impaired in people with multiple sclerosis (MS) under anti-CD20 therapies. It is however unclear, whether patients who received the basic immunization prior to anti-B cell medication start respond to the COVID-19 booster dose, once B cells are depleted. AIM: To investigate the humoral response to recall antigen by COVID-19 booster vaccines in people with MS (pwMS), who recently started an anti-CD20 therapy compared to people with long-term B cell depletion. METHODS: We enrolled 15 pwMS who had received booster vaccination on anti-CD20 therapy. Of these, 11 had established anti-CD20 medications and were therefore vaccinated during a continuous state of B cell depletion (CD20-vaccine cohort). Four pwMS had received the basic immunization prior to anti-CD20 therapy commencement and only the booster dose (vaccine-CD20-vaccine cohort) under conditions of B cell depletion. We assessed SARS-CoV-2 specific antibody responses after booster vaccination among both groups and evaluated accompanying B cell numbers and proportions from the peripheral circulation. RESULTS: The booster dose of SARS-CoV-2 vaccination elicited measurable antibody responses in 18% of individuals from the CD20-vaccine cohort compared to 100% from the vaccine-CD20-vaccine cohort. Antibody-levels were significantly higher among patients from the vaccine-CD20-vaccine cohort compared to the CD20-vaccine cohort (mean 951.25 ± 1137.96 BAU/ml, vs mean 12.36 ± 11.94 BAU/ml; mean difference 938 BAU/ml (95% CI: 249-1629 BAU/ml), p <0.0001). Among the vaccine-CD20-vaccine cohort, the booster immunization led to augmentation of spike antibody levels in 75% despite concomitant B cell depletion, and values increased by 3.8 - 9.4-fold compared to basic immunization. We observed no correlation of B cell kinetics and SARS-CoV-2 antibody levels. CONCLUSION: Our study suggests that antibody production to recall COVID-19 antigens is preserved in pwMS despite concomitant anti-CD20 therapy. If corroborated in bigger cohorts, this could have implications in the management of individuals about to start B cell medications.

Pre-Existing Humoral Immunological Memory Is Retained in Patients with Multiple Sclerosis Receiving Cladribine Therapy.

Cladribine (CLAD) is a lymphodepleting agent approved for active relapsing multiple sclerosis (MS). The impact of CLAD on the adaptive humoral immune system has not sufficiently been studied. This study aimed to assess the influence of CLAD treatment on specific antibody titers to common pathogens. We included 18 MS patients treated with CLAD. Serum IgG antibody levels to measles, mumps, rubella, hepatitis B and varicella zoster virus (VZV), as well as diphtheria and tetanus toxins, were measured prior to the initiation of treatment and at 12 and 24 months after first CLAD administration. Moreover, specimens were longitudinally analyzed regarding absolute blood concentrations of IgG and main lymphocyte subsets. No reduction in antibody levels against measles, mumps, rubella, VZV, hepatitis B, diphtheria toxin and tetanus toxin associated with CLAD treatment was observed. Loss of seroprotection occurred in <1%. We found no significant impact of CLAD on absolute serum IgG levels. Absolute lymphocyte counts were significantly reduced at the end of each treatment year (p < 0.00001 and p < 0.000001). This study suggests that CLAD does not interfere with the pre-existing humoral immunologic memory in terms of pathogen-specific antibody titers.

Tetravalent Influenza Vaccine Is Not Associated With Neuroaxonal Damage in Multiple Sclerosis Patients.

Background: Efficacy of vaccines and disease activity linked to immunization are major concerns among people with multiple sclerosis (pwMS). Objective: To assess antibody responses to seasonal influenza antigens and vaccine-associated neuroaxonal damage utilizing serum neurofilament light chain (sNfL) in pwMS receiving dimethyl fumarate (DMF). Methods: In this prospective study, the 2020/2021 seasonal tetravalent influenza vaccine was administered to 20 pwMS treated with DMF and 15 healthy controls (HCs). The primary endpoints were responder rate of strain-specific antibody production (seroconversion or significant (4-fold) increase in influenza-antibody titers for ≥2/4 strains) at 30 days post-vaccination and changes in sNfL levels. Results: All patients treated with DMF fulfilled the responder criteria for immunization compared with 53% of the controls. However, higher proportions of HCs already had influenza-antibody titers ≥1:40 at baseline (53% vs. 41%, p = 0.174). sNfL levels were comparable among both groups at baseline and did not increase 34 days after vaccination. In addition, no clinical or radiological disease reactivation was found. Conclusion: DMF-treated patients mount an adequate humoral immune response to influenza vaccines. Within the limits of the small cohort investigated, our data suggest that influenza immunization is not associated with clinical or subclinical disease reactivation.

The CXCL13/CXCR5-chemokine axis in neuroinflammation: evidence of CXCR5+CD4 T cell recruitment to CSF.

BACKGROUND: C-X-C chemokine ligand 13 (CXCL13) is frequently elevated in cerebrospinal fluid (CSF) in a variety of inflammatory central nervous system (CNS) diseases, has been detected in meningeal B cell aggregates in brain tissues of multiple sclerosis patients, and proposedly recruits B cells into the inflamed CNS. Besides B cells also follicular helper T (Tfh) cells express the cognate receptor C-X-C chemokine receptor type 5 (CXCR5) and follow CXCL13 gradients in lymphoid tissues. These highly specialized B cell helper T cells are indispensable for B cell responses to infection and vaccination and involved in autoimmune diseases. Phenotypically and functionally related circulating CXCR5+CD4 T cells occur in blood. Their co-recruitment to the inflamed CSF is feasible but unresolved. METHODS: We approached this question with a retrospective study including data of all patients between 2017 and 2019 of whom immune phenotyping data of CXCR5 expression and CSF CXCL13 concentrations were available. Discharge diagnoses and CSF laboratory parameters were retrieved from records. Patients were categorized as pyogenic/aseptic meningoencephalitis (ME, n = 29), neuroimmunological diseases (NIMM, n = 22), and non-inflammatory neurological diseases (NIND, n = 6). ANOVA models and Spearman's Rank-Order correlation were used for group comparisons and associations of CXCL13 levels with immune phenotyping data. RESULTS: In fact, intrathecal CXCL13 elevations strongly correlated with CXCR5+CD4 T cell frequencies in the total cohort (p < 0.0001, r = 0.59), and ME (p = 0.003, r = 0.54) and NIMM (p = 0.043, r = 0.44) patients. Moreover, the ratio of CSF-to-peripheral blood (CSF/PB) frequencies of CXCR5+CD4 T cells strongly correlated with CXCL13 levels both in the total cohort (p = 0.001, r = 0.45) and ME subgroup (p = 0.005, r = 0.50), indicating selective accumulation. ME, NIMM and NIND groups differed with regard to CSF cell counts, albumin quotient, intrathecal IgG, CXCL13 elevations and CXCR5+CD4 T cells, which were higher in inflammatory subgroups. CONCLUSION: The observed link between intrathecal CXCL13 elevations and CXCR5+CD4 T cell frequencies does not prove but suggests recruitment of possible professional B cell helpers to the inflamed CSF. This highlights CSF CXCR5+CD4 T cells a key target and potential missing link to the poorly understood phenomenon of intrathecal B cell and antibody responses with relevance for infection control, chronic inflammation and CNS autoimmunity.

Role and Relevance of Cerebrospinal Fluid Cells in Diagnostics and Research: State-of-the-Art and Underutilized Opportunities.

Cerebrospinal fluid (CSF) has recently experienced a revival in diagnostics and research. However, little progress has been made regarding CSF cell analysis. For almost a century, CSF cell count and cytomorphological examination have been central diagnostic parameters, with CSF pleocytosis as a hallmark finding of neuroinflammation and cytology offering valuable clues regarding infectious, autoimmune, and malignant aetiologies. A great deal of information, however, remains unattended as modern immune phenotyping technologies have not yet been broadly incorporated into routine CSF analysis. This is a serious deficit considering the central role of CSF cells as effectors in central nervous system (CNS) immune defence and autoimmune CNS processes, and the diagnostic challenges posed by clinically overlapping infectious and immune-mediated CNS diseases. Here, we summarize historical, specimen-intrinsic, methodological, and technical issues determining the state-of-the-art diagnostics of CSF cells and outline future perspectives for this underutilized window into meningeal and CNS immunity.

Neurological complications associated with influenza in season 2017/18 in Austria- a retrospective single center study.

BACKGROUND: Neurological complications associated with influenza (NCI) are rare events in adults with seasonal influenza. Information about the characteristics of neurological complications and the burden of disease has been limited to case reports, mainly during the pandemic 2009. Influenza-associated encephalopathy/encephalitis (IAE) is one of the most severe and frequently reported NCI, mostly caused by influenza A. Isolated case reports exist about NCI caused by influenza B. OBJECTIVES: The aim of this single center retrospective study is the better understanding of the frequency and the characteristics of NCI in adults in season 2017-2018, depending on the influenza subtype A or B. STUDY DESIGN: We reviewed 874 adult patients with laboratory confirmed influenza admitted to the Christian Doppler University Hospital Salzburg, Austria from December 2017 until March 2018 looking for NCI. RESULTS: 37 (4 %) of the 874 patients with confirmed influenza had NCI. 4 (11 %) had influenza A and 33 (89 %) had influenza B. IAE was the most frequent complication diagnosed in 24 (65 %) patients, of whom all but one had influenza B and 3 (13 %) had neurological residuals. Moreover 6 (16 %) had isolated epileptic seizures, 2 (5 %) had acute inflammatory demyelinating polyneuropathy (AIDP), and 5 (14 %) were classified as having infection-associated stroke. CONCLUSIONS: We report an incidence of 4 % for NCI and a high frequency of IAE caused by subtype B. Therefore, we recommend considering both influenza A and B as an etiologic factor of encephalopathy and other neurological disease in adults.

Chemokine CXCL13 in serum, CSF and blood-CSF barrier function: evidence of compartment restriction.

BACKGROUND AND PURPOSE: Elevation of the chemokine CXCL13 in CSF frequently occurs during active and acute CNS inflammatory processes and presumably is associated with B cell-related immune activation. Elevation levels, however, vary a lot and "leaking" of CXCL13 from blood across dysfunctional brain barriers is a possible source. The aim was to clarify the relation between CXCL13 concentrations in CSF, CXCL13 concentrations in serum and blood-CSF barrier (BCSFB) function for a correct interpretation of the intrathecal origin of CXCL13. METHODS: We retrospectively analyzed CXCL13 of banked CSF/serum samples (n = 69) selected from patient records and categorized the CSF CXCL13 elevations as CXCL13 negative (< 30 pg/ml), low (30-100 pg/ml), medium (101-250 pg/ml), or high (> 250 pg/ml). CXCL13 concentrations in CSF and serum and the corresponding CSF/serum CXCL13 quotients (Qcxcl13) were compared to CSF/serum albumin quotients (QAlb) as a measure for BCSFB function. The CXCL13 negative category included two subgroups with normal and dysfunctional BCSFB. RESULTS: Serum CXCL13 concentrations were similar across categories with median levels around 100 pg/ml but differed between individuals (29 to > 505 pg/ml). Despite clear evidence in serum, CXCL13 was detectable only at trace amounts (medians 3.5 and 7.5 pg/ml) in CSF of the two CXCL13 negative subgroups irrespective of a normal or pathological QAlb. Moreover, we found no association between CSF and serum CXCL13 levels or between QAlb and CSF CXCL13 levels in any of the CSF CXCL13-delineated categories. CXCL13 apparently does not "leak" from blood into CSF. This implies an intrathecal origin also for low CSF CXCL13 levels and a caveat for analyzing the Qcxcl13, because higher serum than CSF concentrations arithmetically depress the Qcxcl13 resulting in misleadingly low CSF/serum quotients. CONCLUSION: We demonstrated that CXCL13 does not cross from blood into CSF, not even during severe BCSFB dysfunction. CSF CXCL13 elevations therefore most likely always are CNS-derived, which highlights their relevance as indicator of inflammatory CNS processes. We recommend data should not be corrected for BCSFB permeability (QAlb) and not to calculate CSF/serum quotients for CXCL13 as these may introduce error.

Beyond LNB: Real life data on occurrence and extent of CSF CXCL13 in neuroinflammatory diseases.

To evaluate occurrence and extent of CSF CXCL13 elevations beyond Lyme neuroborreliosis, we investigated CXCL13 in an unselected patient cohort with neuroinflammatory disease. From March 2016 to March 2017, 180 in-patients with CSF pleocytosis were categorized into following groups: pyogenic CNS infections, aseptic meningoencephalitis, neuroimmunological diseases, and reactive pleocytosis. We provide evidence that CXCL13 elevation occurs at variable extent in the majority of neuroinflammatory diseases. The exact role of CXCL13 in CSF is elusive, but the broad occurrence in neuroinflammation points at CNS immune activation, which is not exclusive for LNB.

Cerebrospinal fluid CXLC13 indicates disease course in neuroinfection: an observational study.

BACKGROUND: The chemokine CXCL13 is an intensively investigated biomarker in Lyme neuroborreliosis (LNB). Its role in other neuroinfections is increasingly recognized but less clear. OBJECTIVE: To determine the significance of CXCL13 in established central nervous system (CNS) infections other than LNB by matching cerebrospinal fluid (CSF) CXCL13 elevations with severity of the disease course. METHODS: We investigated 26 patients with bacterial (n = 10) and viral (n = 16; tick-borne encephalitis, n = 6; varicella zoster infection, n = 10) neuroinfections of whom CSF CXCL13 levels were available twice, from lumbar punctures (LP) performed at admission and follow-up. As outcome classification, we dichotomized disease courses into "uncomplicated" (meningitis, monoradiculitis) and "complicated" (signs of CNS parenchymal involvement such as encephalitis, myelitis, abscesses, or vasculitis). CXCL13 elevations above 250 pg/ml were classified as highly elevated. RESULTS: Eight of 26 patients (31%) with both bacterial (n = 4) and viral (n = 4) neuroinfections had a complicated disease course. All of them but only 3/18 patients (17%) with an uncomplicated disease course had CSF CXCL13 elevations > 250 pg/ml at the follow-up LP (p < 0.001). At admission, 4/8 patients (50%) with a complicated disease course and 3/18 patients (17%) with an uncomplicated disease course showed CXCL13 elevations > 250 pg/ml. All four patients with a complicated disease course but only one with an uncomplicated disease course had sustained CXCL13 elevations at follow-up. Patient groups did not differ with regard to age, time since symptom onset, LP intervals, type of infections, and anti-pathogen treatments. CONCLUSION: Our study revealed pronounced CXCL13 elevations in CSF of patients with severe disease courses of bacterial and viral neuroinfections. This observation indicates a role of CXCL13 in the CNS immune defense and points at an additional diagnostic value as biomarker for unresolved immune processes leading to or associated with complications.

From natalizumab to fingolimod in eight weeks - Immunological, clinical, and radiological data in quest of the optimal switch.

Natalizumab (NZB) discontinuation during a treatment change is associated with recurrence of disease activity in a significant proportion of multiple sclerosis (MS) patients. The immunological basis why disease reactivation occurs in selected patients is unresolved. In search of a prognostic biomarker for a safe and effective transition from NZB to fingolimod, we monitored five parameters related to pharmacokinetic and pharmacodynamic effects of the two drugs in 12 MS patients until six months on fingolimod. Clearance of free and cell-bound NZB, re-expression of alpha-4, and fingolimod-mediated changes on CD8+ and CD4+ T cell subsets showed pronounced interindividual variability. Higher frequencies of memory CD8+ T cells after six months on fingolimod were the sole association with disease reactivation. None of the investigated parameters thus had potential as prognostic biomarker for the outcome of the switch. Our findings rather support the thesis of broad interindividual differences in the immunopathogenesis of MS.

Corrigendum: Human Cerebrospinal Fluid Promotes Neuronal Viability and Activity of Hippocampal Neuronal Circuits In Vitro.

[This corrects the article on p. 54 in vol. 10, PMID: 26973467.].