Abstract: Patients with COVID-19 can have a variety of neurological symptoms, but the pathomechanism of CNS involvement in COVD-19 remains unclear. While routine cerebrospinal fluid (CSF) analyses in patients with neurological manifestations of COVID-19 generally show no or only mild inflammation, more detailed data on inflammatory mediators in the CSF of patients with COVID-19 are scarce. Here, we used mass spectrometry to study the proteome, Enzym-linkend immunoassays, semiquantitative cytokine arrays, autoantibody screening, and RNA profiling to study the neuroinflammation. We study the inflammatory response in paired CSF and serum samples of patients with COVID-19 (n=38). Patients with herpes simplex virus encephalitis (HSVE, n=10) and patients with non-inflammatory, non-neurodegenerative neurological diseases (n=28) served as controls. Proteomics on single protein level and subsequent pathway analysis showed similar yet strongly attenuated inflammatory changes in the CSF of COVID-19 patients compared to HSVE patients. CSF/serum indices of interleukin-6, interleukin-16 and CXCL10 together point at an origin from these inflammatory proteins from outside the central nervous system. When stratifying COVID-19 patients into those with and without bacterial superinfection as indicated by elevated procalcitonin levels, inflammatory markers were significantly higher in those with concomitant bacterial superinfection. RNA sequencing in the CSF revealed 101 linear RNAs comprising messenger RNAs, micro RNAs and t-RNA fragments being significantly differentially expressed in COVID-19 than in HSVE or controls. Our findings may explain the absence of signs of intrathecal inflammation upon routine CSF testing despite the presence of SARS-CoV2 infection-associated neurological symptoms. The relevance of blood-derived mediators of inflammation in the CSF for neurological post-COVID-19 symptoms deserves further investigation.

Background:While there have been advances in the therapy of systemic lupus erythematosus (SLE) in recent years, there have been no major new findings in SLE biomarkers [1, 2]. Type I interferon (IFN) plays a pivotal role in the pathogenesis of SLE [3]. In 2008, we first described CD169 / SIGLEC-1 (sialic acid-binding immunoglobulin-like lectin-1), an interferon-induced adhesion molecule on monocytes in SLE patients [4]. For over five years SIGLEC-1 has been routinely assessed in our clinic.Objectives:To evaluate and compare the diagnostic utility of the type I IFN induced SIGLEC-1 with established biomarkers in the initial diagnosis of the disease.Methods:We analyzed retrospectively 232 patients who were on suspicion of SLE at Charité University Hospital Berlin between October 2015 and September 2020. Patients underwent full clinical characterization, and biomarkers were determined in the routine laboratory. Based on the final diagnosis, we divided patients into two groups: A) initial diagnosis of SLE and B) Non-SLE mimicking condition.Results:In 76 patients (32.3 %) SLE was confirmed by fulfilling the EULAR / ACR 2019 classification criteria [5]. SIGLEC-1 was dramatically increased in patients with an initial diagnosis of SLE compared to patients without SLE (p<0.0001). For a threshold of 2500 molecule per monocyte, a sensitivity of 98.7 %, a specificity of 82.1 %, a negative predictive value (NPV) of 99.2 %, and a positive predictive value (PPV) of 72.8 % were calculated for SIGLEC-1. Adjusted to the prevalence of SLE in Germany (36.7 per 100,000 inhabitants [6]) NPV and PPV turned out to > 99.9 % and 0.2 %. We further aimed to compare not only the performance of the tests at a given cutoff but also across all possible measured values. Therefore, we conducted ROC curves analyses (see figure 1). The area under the curve (AUC) of SIGLEC-1 test was significantly higher than that of ANA test (AUC=0.88, p=0.031), C3 (AUC = 0.83, p=0.001), C4 (AUC=0.83, p=0.002), but not than that of the Anti-dsDNA ELISA (AUC=0.90, p=0.163).Conclusion:Our study shows that IFN activity is a hallmark at the onset of the disease and that the interferon biomarker SIGLEC-1 is valuable to rule out SLE in suspected cases.References:[1]Ostendorf L, Burns M, Durek P, Heinz GA, Heinrich F, Garantziotis P, Enghard P, Richter U, Biesen R, Schneider U et al: Targeting CD38 with Daratumumab in Refractory Systemic Lupus Erythematosus. N Engl J Med 2020, 383(12):1149-1155.[2]Furie R, Rovin BH, Houssiau F, Malvar A, Teng YKO, Contreras G, Amoura Z, Yu X, Mok CC, Santiago MB et al: Two-Year, Randomized, Controlled Trial of Belimumab in Lupus Nephritis. N Engl J Med 2020, 383(12):1117-1128.[3]Ronnblom L, Leonard D: Interferon pathway in SLE: one key to unlocking the mystery of the disease. Lupus Sci Med 2019, 6(1):e000270.[4]Biesen R, Demir C, Barkhudarova F, Grun JR, Steinbrich-Zollner M, Backhaus M, Haupl T, Rudwaleit M, Riemekasten G, Radbruch A et al: Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus. Arthritis Rheum 2008, 58(4):1136-1145.[5]Aringer M, Costenbader K, Daikh D, Brinks R, Mosca M, Ramsey-Goldman R, Smolen JS, Wofsy D, Boumpas DT, Kamen DL et al: 2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus. Annals of the Rheumatic Diseases 2019, 78(9):1151-1159.[6]Brinks R, Fischer-Betz R, Sander O, Richter JG, Chehab G, Schneider M: Age-specific prevalence of diagnosed systemic lupus erythematosus in Germany 2002 and projection to 2030. Lupus 2014, 23(13):1407-1411.Disclosure of Interests:None declared

Background: Idiopathic inflammatory myopathies (IIM) are autoimmune diseases that mainly affect skeletal muscle, lung, skin and joints. IIM can be separated into dermatomyositis (DM), inclusion body myositis (IBM), antisynthetase syndrome (AS) and immune-mediated necrotizing myopathy (IMNM). Type I interferons (IFN) are known to play a crucial role in the etiopathogenesis of some of these entities such as DM.[1] Sialic acid binding Ig-like lectin 1 (SIGLEC1, CD169) is part of the type I IFN signature found in SLE and DM and is expressed on the cell surface of monocytes. Thus, analysis of SIGLEC1 expression by flow cytometry enables a straightforward assessment of the type I IFN signature. Its utility has been shown for juvenile and adult SLE and other rheumatic diseases but not in IIM.[2,3] The assessment of the type I IFN system in clinical practice is an unmet need and, in this context, SIGLEC1 might be useful. Objectives: To assess SIGLEC1 expression on monocytes by flow cytometry as a type I IFN biomarker in IIM Methods: Pediatric and adult patients with a clinical diagnosis of DM, AS, IMNM and IBM and at least one measurement of SIGLEC1 who have been treated at the Department of Rheumatology, Charité - Universitätsmedizin Berlin between 2015 and 2020 were included in this retrospective study. Control groups of healthy individuals (n=19) and SLE patients (n=30) were included. Disease activity was assessed by Physician Global Assessment (PGA) and Childhood Myositis Assessment Scale (CMAS). SIGLEC1 expression on monocytes was analyzed by flow cytometry. Cross-sectional analyses (n=74) were performed using Mann Whitney-U test (MWU) and two-level mixed-effects linear regression model was used for longitudinal analyses (n=26, 110 visits). This study was approved by the local ethics committee of the Charité - Universitätsmedizin Berlin. Results: 74 patients (adult/juvenile DM: n=21/n=17; AS: n=19; IMNM: n=8; IBM: n=9) were included. In cross-sectional analysis, SIGLEC1 expression was significantly upregulated in adult and juvenile DM patients with moderate to severe disease activity (PGA≥5) compared with adult/juvenile DM patients with no to moderate disease activity (PGA<5) (both p<0.001). In longitudinal analyses, SIGLEC1 correlated with disease activity in juvenile DM (SIGLEC1 vs. CMAS: betaST=-0.65; p<0.001) and adult DM (SIGLEC1 vs. PGA: betaST=0.52; p<0.001), better than Creatine Kinase (CK) (juvenile DM, CK vs. CMAS: betaST=-0.50; p<0.001; adult DM, CK vs PGA: betaST=0.17; p=0.149). In AS 42,1% of the patients showed elevated SIGLEC1 expression, while it was not upregulated in IMNM and only in two patients with IBM, who were concurrently positive for autoantibodies that affect the type I IFN system (see Figure 1). Conclusion: SIGLEC1 is a useful biomarker to identify an activated type I IFN system in IIM. Flow cytometry is used widely in laboratory medicine, which could facilitate the implementation of SIGLEC1 into clinical routine. References: [1]Gallay L, Mouchiroud G, Chazaud B. Interferon-signature in idiopathic inflammatory myopathies: Current Opinion in Rheumatology 2019; 31 :634–42. doi:10.1097/BOR.0000000000000653 [2]Rose T, Grutzkau A, Hirseland H, et al. IFNalpha and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus. Ann Rheum Dis 2013; 72 :1639–45. doi:10.1136/annrheumdis-2012-201586 [3]Stuckrad SL von, Klotsche J, Biesen R, et al. SIGLEC1 (CD169) is a sensitive biomarker for the deterioration of the clinical course in childhood systemic lupus erythematosus. Lupus 2020;:961203320965699. doi:10.1177/0961203320965699 Figure 1. SIGLEC1 expression on monocytes in IIM subgroups and control groups; in IIM subgroups, patients with low disease activity (PGA<5) are marked in blue, patients with high disease activity (PGA≥5) are marked in red; mAb/cell, monoclonal antibodies bound per cell Disclosure of Interests: None declared

Autoantibodies to interferon(IFN)-α and IFN-ω (type I IFNs) were recently reported as causative for severe COVID-19 in the general population. Autoantibodies against IFN-α and IFN-ω are present in almost all patients with Autoimmune-Polyendocrine-Syndrome Type 1 (APS-1) caused by biallelic deleterious or heterozygous dominant mutations in AIRE. We therefore hypothesized that autoantibodies against type I IFNs also predispose patients with APS-1 to severe COVID-19. We prospectively studied six patients with APS-1 between April 1st, 2020 and April 1st, 2021. Biobanked pre-COVID-19 sera of APS-1 subjects were tested for neutralizing autoantibodies to IFN-αand IFN-ω. The patients ́ sera ability to block recombinant human IFN-α and IFN-ω was assessed by assays quantifying phosphorylation of signal transducer and activator of transcription 1 (STAT1) as well as infection-based IFN-neutralization assays. We describe four patients with APS-1 and pre-existing high titers of neutralizing autoantibodies to IFN-α and IFN-ω who contracted SARS-CoV-2, yet developed only mild symptoms of COVID-19. None of the patients developed dyspnoea, oxygen requirement or high temperature. All infected patients with APS-1 shared female sex and age younger than 26 years. Clinical penetrance of neutralizing autoantibodies against type I IFNs for severe COVID-19 is not complete.

Almost 40% of stroke patients have a poor outcome at 3 months after the index event. Predictors for stroke outcome in the early acute phase may help to tailor stroke treatment. Infection and inflammation are considered to influence stroke outcome. In a prospective multicenter study in Germany and Spain, including 486 patients with acute ischemic stroke, we used multivariable regression analysis to investigate the association of poor outcome with monocytic HLA-DR (mHLA-DR) expression, interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide-binding protein (LBP) as markers for immunodepression, inflammation and infection. Outcome was assessed at 3 months after stroke via a structured telephone interview using the modified Rankin Scale (mRS). Poor outcome was defined as a mRS score of 3 or higher which included death. Furthermore, a time-to-event analysis for death within 3 months was performed. Three-month outcome data was available for 391 patients. Female sex, older age, diabetes mellitus, atrial fibrillation, stroke-associated pneumonia (SAP) and higher National Institute of Health Stroke Scale (NIHSS) score as well as lower mHLA-DR levels, higher IL-6 and LBP-levels at day 1 were associated with poor outcome at 3 months in bivariate analysis. Furthermore, multivariable analysis revealed that lower mHLA-DR expression was associated with poor outcome. Female sex, older age, atrial fibrillation, SAP, higher NIHSS score, lower mHLA-DR expression and higher IL-6 levels were associated with shorter survival time in bivariate analysis. In multivariable analysis, SAP and higher IL-6 levels on day 1 were associated with shorter survival time. SAP, lower mHLA-DR-expression and higher IL-6 levels on day one are associated with poor outcome and shorter survival time at 3 months after stroke onset. www.clinicaltrials.gov, NCT01079728 , March 3, 2010.

Die juvenile Dermatomyositis (jDM) ist eine seltene Autoimmunerkrankung, klinisch charakterisiert durch typische Hautveränderungen und Muskelschwäche. Bei einigen Patienten können myositisassoziierte (MAA) oder myositisspezifische Antikörper (MSA) nachgewiesen werden. Diese sind bedeutsam für Krankheitsspektrum und Prognose.

Introduction SIGLEC1 (sialic acid-binding Ig-like lectin 1, CD169) is a monocytic adhesion molecule induced by interferon – α. In adult systemic lupus erythematosus (SLE), SIGLEC1 correlates cross-sectionally and longitudinally with disease activity. The aim of this work was to examine whether SIGLEC1 also reflects the disease activity in paediatric SLE. Methods Over a period of 29 months the disease activity was clinically evaluated using SLEDAI (SLE-Disease Activity Index-2000). In 28 consecutive paediatric SLE patients (mean age 16 years, range 3–38 years, 86% female, 14% male), the number of SIGLEC1 molecules per CD14 +on blood monocyte was quantified using flow cytometry. At the same time, the level of anti-ds DNA-antibody titer (ELISA) and the concentration of complement factors C3 and C4 (nephelometry) were determined. The association between SIGLEC1, C3, C4 and ds DNA-antibody with SLEDAI was estimated using a mixed linear model to model the repeated measurement of parameters within a patient. The cut-off for the change in SIGLEC1 between two consecutive visits to predict minimal clinical improvement or worsening in SLEDAI was chosen on the maximum Youden Index. Results The density of SIGLEC1 molecules on the surface of monocytes based on two visits, correlated with the SLEDAI (128 determinations, betaST 0.22, p<0.012), but not with the C3 (108 determinations, betaST 0.03, p=0,80), the C4 (106 determinations, betaST −0.06, p<0.58) and the anti-dsDNA-antibodies (104 determinations, betaST 0.06, p=0.61). SIGLEC1 is a more change-sensitive biomarker than the conventional laboratory parameters C3, C4 and ds-DNA-Ab.Patients with an increase in SIGLEC1 of >1120 molecules/monocyte between two visits show a higher probability (OR=7.0, p<0.001) of minimal clinical worsening (SLEDAI more/equal 2 points). Patients who show a decrease >2902 SIGLEC1 molecules/monocyte have a higher chance (OR=6.3, p=0.004) to have a clinical improvement (SLEDAI more/equal 2 points). Conclusion This prospective cohort study showed for the first time the significant relationship between the routinely measured interferon biomarker SIGLEC1 and the disease activity in paediatric SLE patients. Thus, SIGLEC1 represents a potential marker for activity monitoring in this disease.

BACKGROUND:Chronic inflammatory demyelinating polyneuropathy (CIDP) is presented by a large heterogeneity of clinical phenotypes. Around 50% of patients suffer from typical CIDP and show better therapy response than atypical variants. The goal of our study was to search for cellular immunological differences in typical versus atypical CIDP in comparison to controls.METHODS:We evaluated 26 (9 typical, 17 atypical) patients with mainly active-unstable CIDP using clinical and immunological examinations (enzyme-linked immunospot assay ELISPOT, fluorescence-activated cell sorting FACS) in comparison to 28 healthy, age-matched controls (HC). Typical or atypical CIDP measurements were compared with HC using Kruskal-Wallis test.RESULTS:Atypical CIDP patients showed increased frequencies of T cell subsets, especially CD4+ effector memory T cells (TEM) and CD4+ central memory T cells (TCM) as well as a tendency of higher T cell responses against the peripheral myelin antigens of PMP-22, P2, P0 and MBP peptides compared to typical CIDP. Searching for novel auto-antigens, we found that T cell responses against P0 180-199 as well as MBP 82-100 were significantly elevated in atypical CIDP patients vs. HC.CONCLUSIONS:Our results indicate differences in underlying T cell responses between atypical and typical CIDP characterized by a higher peripheral myelin antigen-specific T cell responses as well as a specific altered CD4+ memory compartment in atypical CIDP. Larger multi-center studies study are warranted in order to characterize T cell auto-reactivity in atypical CIDP subgroups in order to establish immunological markers as a diagnostic tool.

Acute ischemic stroke induces systemic complications via activation of sympathetic autonomic nervous system. While cardiovascular complications3 are well known, CNS injury-induced immunodepression2,3,4 has recently moved into scientific focus. To date, no direct evidence of increased muscle sympathetic nerve activity (MSNA) has been shown in acute cerebral ischemia. In this pilot study, we sought to determine whether MSNA recording is feasible within emergency setting of acute stroke care. We measured MSNA in seven patients (m:f 5:2; age range 47–74 yrs.) within 48 hr of onset of acute ischemic stroke and following a rehabilitation period (3,5-7 months). During hospitalization, clinical signs (Centers for Disease control and Prevention; CDC) and blood infection parameters were monitored on a daily basis. All patients showed a moderate neurological deficit (National Institutes of Health Stroke Scale; NIH-SS 5–9 pts.) and marked degree of disability (modified Ranking Scale; mRS 3–4 pts.). No patient developed infectious complications (CDC-criteria), however, three patients showed elevated serum CRP-levels. There was no overall trend in MSNA, neither burst incidence (per 100 heartbeats) nor burst area (per min), within acute phase compared to follow-up. However, patients with elevated CRP-levels post-stroke (n = 3) revealed decreased MSNA indices (burst area in three, burst incidence in two) in acute stroke. Patients with normal CRP-levels showed unchanged or increased MSNA indices. Our study shows that MSNA recording is possible even in acute setting of stroke care. Our preliminary data suggests, that increased MSNA following acute stroke is linked to immunodepression and elevated markers of systemic infection.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare disease of the peripheral nervous system, characterized by gradual increasing weakness of the limbs, with more than 50% of patients experiencing marked disability 1. The underlying pathophysiological mechanism of CIDP remains unknown; however, studies have shown activated T cells in the circulation of CIDP patients 2-4. In addition, patient studies suggest a key role for autoreactive T cell responses against peripheral myelin antigens such as P0, P1, P2 and peripheral myelin protein PMP-22 5, 6. Mechanism-of-action studies in other chronic autoimmune diseases have shown that the T cell memory compartment influences antigen responses by showing up-regulation of CD4+ or CD8+ T effector memory (TEM) cells 7, 8. Recently, published data from a randomized, placebo-controlled clinical trial demonstrated the long-term efficacy and safety of intravenous immunoglobulin (IVIg) treatment in CIDP patients 9. However, the underlying mechanism of action of IVIg in the treatment of CIDP remains unclear 10, 11. The aim of this study was to investigate the course of autoreactive T cell responses against the two peripheral myelin antigens P2 and PMP-22 in addition to the frequency of memory T cell subsets during IVIg treatment in CIDP patients 12. In an observational trial of previously IVIg-treated patients (maintenance), previously untreated patients (treatment-naive) and controls (n = 48), IVIg treatment-naive patients (n = 18) were evaluated clinically prior to the first IVIg treatment (baseline) and at 4-week intervals after IVIg treatment initiation by using the adjusted Inflammatory Neuropathy Cause and Treatment (INCAT) disability score, the Medical Research Council (MRC) sum score and walking distance to assess the clinical status 12. In addition, a blood sample was provided for analysis. Peripheral blood monocytes (PBMCs) were isolated from blood samples from treatment-naive patients (n = 18) at baseline and at follow-up (at least 6 months after IVIg treatment initiation, mean 20 months). For comparison, PBMCs were extracted from blood samples from CIDP patients (n = 16) receiving IVIg as a maintenance therapy (mean 33 months). Additionally, patients with non-immune neuropathy or healthy individuals acted as controls (n = 14). In order to quantify frequencies of interferon (IFN)-γ-producing T cells directed against the peripheral myelin antigens PMP-22 and P2 (autoreactive T cell response), cryopreserved (and subsequently thawed) PBMCs were assessed by enzyme-linked immunospot (ELISPOT) analysis. In addition, flow cytometric analysis was performed using freshly isolated PBMCs to quantify T memory subsets. Response to treatment was defined as an improvement of 2 or more points on the MRC sum score in two different muscle groups 13, an improvement of 1 point or more on INCAT disability score (except for the changes in upper limb function from 0 to 1) 9 or an improvement of the walking distance of more than 50% compared to baseline results to also cover patients with a dominant sensory atactic syndrome 12. Baseline demographics were not significantly different between responders and non-responders, particularly with regard to sex, age, previous treatment, time since diagnosis, diagnosis or clinical severity. IVIg responders showed significantly higher autoantigen-specific T cell responses against peripheral myelin antigens PMP-22 and P2 (PMP-2232–51 and PMP-22120–133 as well as P214–25 and P261–70) at baseline compared to IVIg non-responders, maintenance therapy patients and controls. Maintenance therapy patients showed levels of IFN-γ responses similar to that of controls, those with other neuropathies and to non-responders. Analysing T memory compartments at baseline, IVIg responders (n = 10) showed increased frequencies of CD4+ central memory T cells (TCM; CD4+45RA–CCR7+) and effector/memory T cells (TEM; CD4+45RA–CCR7–) compared to controls and to the maintenance group. In contrast, non-responders (n = 8) did not differ from control groups. CD8+ memory T cells showed increased TEM frequencies in responders compared to non-responders and by trend to other groups. For CD8+ TCM, non-responders differed significantly from other groups (maintenance and healthy control group) 12. In order to investigate the long-term effect of IVIg on autoreactive T cell responses, treatment-naive CIDP patients were investigated longitudinally prior to treatment (baseline) and after repeated IVIg infusions (follow-up, mean 20 months). Data showed a significant reduction in IFN-γ-specific T cell responses for peripheral myelin antigens (PMP-2232–51 and PMP-22120–133 as well as for P261–70) over time in treatment responders. In contrast, treatment non-responders, who had no increased T cell response at baseline, did not differ in IFN-γ-specific T cell responses following IVIg treatment over time. Further analysis of T memory subsets found no statistical difference for CD4+ T cell subsets between baseline and follow-up. In contrast, CD8+ TEM were reduced significantly at follow-up 12. Our data demonstrate that treatment with IVIg on a long-term basis reduces the autoreactive T cell response against peripheral myelin antigens which may be influenced by altered maintenance of CD8+ and CD4+ effector/memory T cell subsets towards a more anti-inflammatory immune status. Therefore, the assessment of such antigen-specific T cell responses may also serve as a biomarker to predict responsiveness to IVIg, warranting confirmation in a greater multi-centre cohort trial. J. K., C. M. and A. M. thank Claudia Conert and Viola Kohlrautz for technical assistance as well as Siegfried Kohler, Lena Ulm, Jos Göhler and Hendrik Harms. The authors would also like to thank Meridian HealthComms Ltd for providing medical writing services. The study was funded by a research grant from Octapharma and supported by the Deutsche Forschungsgemeinschaft (German Research Foundation, NeuroCure Cluster of Excellence, Exc 257). J. K. has received honoraria for activities with Grifols, and CSL Behring. C. M. and A. M. have no conflicts of interest to report.

Einleitung: Die Bronchoskopie konnte bisher in kleinen Versuchstieren wie der Maus aufgrund der Größenverhältnisse nicht angewendet werden. Intratracheale und -bronchiale Applikationen erfolgten in vivo ausschließlich ohne direkte Sichtkontrolle.