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Growth differentiation factor 15 (GDF-15), neutrophil gelatinase-associated lipocalin (NGAL), and ADAMTS13 have previously been implicated in the pathophysiological processes of SAH. In the present study, we aim to examine their role in the early period of SAH and their relationship to primary and secondary outcomes. Serum samples were collected at five time periods after SAH (at 24 h (D1), at 72 h (D3), at 120 h (D5), at 168 h (D7) and at 216 h (D9), post-admission) and) were measured by using MILLIPLEX Map Human Cardiovascular Disease (CVD) Magnetic Bead Panel 2. We included 150 patients with SAH and 30 healthy controls. GDF-15 levels at D1 to D9 were significantly associated with a 3-month unfavorable outcome. Based on the ROC analysis, in patients with a good clinical grade at admission (WFNS I-III), the GDF-15 value measured at time point D3 predicted a 3-month unfavorable outcome (cut-off value: 3.97 ng/mL, AUC:0.833, 95%CI: 0.728–0.938, sensitivity:73.7%, specificity:82.6%, p < 0.001). Univariate binary logistic regression analysis showed that serum NGAL levels at D1-D5 and ADAMTS13 levels at D7-D9 were associated with MVS following SAH. GDF-15 is an early indicator of a poor 3-month functional outcome even in patients with mild clinical conditions at admission.

Due to the current burden of COVID-19 on public health institutions, increased migration and seasonal touristic traveling, there is an increased risk of epidemic outbreaks of measles, mumps and rubella (MMR). The aim of the present study was to analyze the epidemiological data on MMR immunization coverage and the number of measles cases in 2001–2019 in Croatia and a number of European countries. Results revealed a decreasing trend in vaccination in 2001–2019 throughout Europe. However, Croatia and Hungary still have the highest primary and revaccination coverage, compared to other analyzed countries. The highest number of measles cases was in 2017 in Romania. There was no significant correlation between the percentage of primary vaccination and the number of measles cases (r = −0.0528, p = 0.672), but there was a significant negative correlation between the percentage of revaccination and the number of measles cases (r = −0.445, p < 0.0001). In conclusion, the results of the present study emphasize the necessity to perform a full protocol of vaccination to reach appropriate protection from potential epidemic outbreaks. Furthermore, in the light of present migrations, documenting the migrants’ flow and facilitating vaccination as needed is of utmost importance to prevent future epidemics.

Chronic hepatitis C (CHC) infection is associated with increased TIM-3, PD-1 immune checkpoint receptors expression that inhibits adaptive T cells and increases NK cell cytotoxicity against T helper cells, both resulting T cell exhaustion. Elimination of the virus with direct-acting antivirals (DAAs) may modify host immune response via altering these immune checkpoint receptors’ expression. We conducted a prospective study to analyze changes in TIM-3, PD-1 and their ligands galectin-9, PD-L1 expression by peripheral blood T cell subpopulations, NK cell subpopulations, and monocytes by multicolor flow cytometry in 14 CHC patients successfully treated with 12 weeks of dasabuvir, ombitasvir, and paritaprevir/ritonavir plus ribavirin. Blood samples were collected before, at the end of treatment, and 12 and 24 weeks later. Sustained virological response (SVR) was associated with increased percentage of peripheral blood CD3+ T and CD8+ cytotoxic T lymphocytes and decreased percentage of NKbright cells. After DAA treatment, decreased TIM-3 expression by CD4+ T cells, by NKbright, and by NKT cells was found. Expression of immune checkpoint molecules’ ligand PD-L1 by NK cells and by regulatory T cells and galectin-9 by NK cells and monocytes also decreased significantly at SVR. Our data suggest that DAA treatment not only inhibits viral replication but may alter host adaptive and innate immune responses. A decrease in immune checkpoint molecules and their ligands expression both on adaptive and on innate immune cells may contribute to the recovery of exhausted adaptive immune responses and to sustained virological response. Electronic supplementary material The online version of this article (10.1007/s10238-020-00618-3) contains supplementary material, which is available to authorized users.

The spleen is the largest secondary lymphoid organ which is involved in the development of B cells and also in systemic (auto)immune responses. Using the recombinant human G1 domain-induced arthritis (GIA) model in splenectomized and control BALB/c mice, we investigated the role of the spleen in the induction and pathogenesis of autoimmune arthritis. Splenectomized mice developed GIA with a similar clinical picture to the control group. However, we observed significant alterations in the humoral and cellular immune responses in splenectomized mice. In the sera of the splenectomized mice, we found lower pro-inflammatory cytokine and anti-rhG1 IgM levels, but higher IL-4, anti-rhG1 IgG1 and anti-CCP and RF antibodies. The arthritis induction in the splenectomized group was associated with a significant expansion of activated helper T cells and an increase in the proportion of the circulating B1 and marginal zone B cell subsets. Importantly, immunization of the splenectomized mice with rhG1 induced the formation of germinal centers in the inguinal- and mesenteric lymph nodes (i/mLNs) which showed an active immune response to rhG1. Finally, both B and T cells from the mLNs of the splenectomized mice showed decreased intracellular Ca2+ signaling than those of the control group. Collectively, these findings indicate that the presence of the spleen is not critical for the induction of GIA, and in its absence the autoimmune arthritis is most likely promoted through the compensatory activity of the i/mLNs. However, our data implies the immunological role of the spleen in arthritis which could be further assessed in human RA.

Disease-associated, high-affinity pathological autoantibody production is a well-described consequence of immune dysregulation affecting B cells in systemic sclerosis (SSc), including the distribution of B-cell subsets. We have previously shown that the increased relative frequency of CD19+CD27+IgD− switched memory B cells is associated with the severe form of SSc. This study sought to analyze memory B cell subsets using an extended range of markers for further subdivision based on CD19, IgD, CD27, CD38 and CD95 phenotype, to define relationship between the alterations of memory B cell subsets and the clinical features of SSc. Peripheral blood samples were obtained from 21 SSc patients, including 14 diffuse (dcSSc) and 7 limited (lcSSc) cutaneous SSc patients, with disease duration of 2.7 ( ± 1.6) years. After purification of CD19+ B cells, multiparametric flow cytometry was performed and the frequencies of CD19+IgD−CD27−CD38+ double negative (DN) 1, CD19+IgDloCD27+CD38+ unswitched, CD19+IgD−CD27+CD38+CD95− resting switched and CD19+IgD−CD27+CD38−CD95+ activated switched memory (ASM) B cells were determined, and correlated with clinical features of SSc. The dcSSc patients had a higher frequency of ASM B cells (p = 0.028) compared to lcSSc patients. The percentage of ASM B cells was elevated in anti-Scl-70 (anti-topoisomerase I) antibody positive patients compared to negative patients (p = 0.016). Additionally, the frequency of ASM B cells was also increased in patients with pulmonary fibrosis (p = 0.003) suggesting that patients with severe form of SSc have higher ASM B cell ratios. Furthermore, the ratio of DN1 B cells was decreased (p = 0.029), while the level of anti-citrate synthase IgG natural autoantibody was elevated (p = 0.028) in patients with active disease. Our observations on the increase of ASM B cells in dcSSc and in patients with pulmonary fibrosis may point to the association of this alteration with the severe form of the disease. Functionally the correlation of ASM B cells as effector memory-plasma cell precursors with anti-topoisomerase I antibody positivity could reflect their contribution to pathological autoantibody production, whereas the decrease of memory precursor DN B cells and the increase of anti-citrate synthase IgG autoantibody may have potential significance in the assessment of disease activity.

Objective Functional disturbances in regulatory T cells (Treg) have been described in autoimmune diseases, and their potential therapeutic use is intensively studied. Our goal was to investigate the influence of glucocorticoid hormone on the in vitro differentiation of Treg cells from thymic and splenic CD4+ T cells under different conditions to establish methods for generating stable and functionally suppressive iTregs for future use in adoptive transfer experiments. Methods Thymic and splenic CD4+ T lymphocytes were isolated from 3 to 4 week-old control and in vivo dexamethasone (DX) pretreated BALB/c mice using magnetic bead negative selection, followed by CD25 positive selection. The cells were cultured with anti-CD3/CD28 beads and IL-2 in the presence or absence of TGFβ and/or DX for 3–6 days. Multiparametric flow cytometry was performed using CD4, CD25, CD8, TGFβ (LAP) cell surface and Foxp3, IL-4, IL-10, IL-17 and IFNγ intracellular staining. Quantitative RT-PCR was performed to measure IL-10, TGFβ cytokine and Foxp3 mRNA levels. Results Differentiation of thymus-derived CD4+ cells in vitro into iTreg cells was most effective (24–25%) when anti-CD3/CD28 beads, IL-2, and TGFβ were present. Splenic CD4+ T cell expansion under same conditions resulted in a higher (44–45%) iTreg cell ratio that further increased (up to 50% Treg) in the presence of DX. Elevated immunosuppressive cytokine (IL-10 and TGFβ) production by iTregs could be measured both at protein and mRNA levels without elevation of Th1/Th2 or Th17 cytokine production. We got the highest iTreg ratio (74%) and TGFβ production when CD4+CD25+ splenic T cells were stimulated in the presence of TGFβ. In vivo 4 days DX pretreatment resulted in enhanced in vitro expansion and Foxp3 expression of thymus-derived iTregs and decreased differentiation of spleen-derived iTreg cells. In these Tregs the relative expression of IL-10 mRNA significantly decreased under all in vitro stimulation conditions, while TGFβ mRNA level did not change. Conclusion DX promotes the expansion of thymic and splenic Treg cells, and enhances Foxp3+ expression and the production of immunosuppressive cytokines IL-10 and TGFβ in vitro. In vivo pretreatment of mice with DX inhibited the immunosuppressive cytokine production of in vitro differentiated Treg cells. We hypothesize that patients receiving GC therapy may need special attention prior to in vitro expansion and transplantation of Treg cells.

Because of measles outbreaks there is a need for continuous monitoring of immunological protection against infection at population level. For such monitoring to be feasible, a cost-effective, reliable and high-throughput assay is necessary. Herein we describe an ELISA protocol for assessment of anti-measles antibody levels in human serum samples that fulfills the above criteria and is easily adaptable by various laboratories. A serum bank of anonymous patient sera was established (N > 3000 samples). Sera were grouped based on measles immunization schedules and/or changes in vaccine components since the introduction of the first measles vaccine in Hungary in 1969. Newly designed ELISA was performed by using Siemens BEP 2000 Advance System and data were confirmed using commercially available kits. Our indirect ELISA was compared to indirect immunfluoresence and to anti-measles nucleocapsid (N) monoclonal antibody-based sandwich ELISA. The results obtained are in high agreement with the confirmatory methods, and reflect measles vaccination history in Hungary ranging from pre-vaccination era, through the initial period of measles vaccination, to present. Based on measurement of 1985 sera, the highest ratio of low/questionable antibody level samples was detected in cluster ‘1978–1987’ (~25.4%), followed by cluster ‘1969–1977’ (~15.4%).Our assay is suitable for assessment of anti-measles immunity in a large cohort of subjects. The assay is cost-effective, allows high-throughput screening and has superior signal-to-noise ratio. This assay can serve as a first step in assessment of the effectiveness of all three components of the MMR vaccine.

Aneurysmal subarachnoid hemorrhage (aSAH) is associated with activation of the inflammatory cascade contributing to unfavorable outcome and secondary complications, such as delayed cerebral ischemia (DCI). Both fatty acid-binding protein 3 (FABP3) and CXC-chemokine ligand 16 (CXCL-16) have been linked to vascular inflammation and cellular death. The authors aimed to assess the 30-day prognostic value of serum levels of FABP3 and CXCL-16 and explore their associations with DCI in aSAH patients.A total of 60 patients with aSAH were prospectively enrolled. Sampling for markers was done at 24 hours after the index event. FABP3 and CXCL-16 serum concentrations were determined by MilliPlex multiplex immunoassay method. The primary endpoint was unfavorable outcome at Day 30 based on the modified Rankin Scale.Both FABP3 and CXCL-16 levels were significantly elevated in patients with unfavorable outcome compared to those with favorable outcome after aSAH (FABP3: 2133 pg/mL, IQR: 1053-4567 vs. 3773, 3295-13116; p0.003 and CXCL-16: 384 pg/mL, 313-502 vs. 498, 456-62, p0.001). The area under the curve (AUC) for serum CXCL-16 levels as a predictor of unfavorable outcome at Day 30 was 0.747 (95% CI =0.622-0.871; p0.001). Based on binary logistic regression analysis, serum CXCL-16 with a cut-off level446.7 ng/L independently predicted Day 30 unfavorable outcome with a sensitivity of 81% and a specificity of 62%. Neither CXCL-16 nor FABP3 showed a significant correlation with DCI.Early FABP3 and CXCL-16 levels are significantly associated with poor 30-day outcome in patients with aSAH.

Plasma harvested from convalescent COVID-19 patients (CCP) has been applied as first-line therapy in the early phase of the SARS-CoV2 pandemic through clinical studies using various protocols. We present data from a cohort of 267 hospitalized, severe COVID-19 patients who received CCP. No transfusion-related complications were reported, indicating the overall safety of CCP therapy. Patients who eventually died from COVID-19 received CCP significantly later (3.95 versus 5.22 days after hospital admission) and had higher interleukin 6 (IL-6) levels (28.9 pg/ml versus 102.5 pg/ml) than those who survived. In addition, CCP-transfusion caused a significant reduction in the overall inflammatory status of the patients regardless of the severity of disease or outcome, as evidenced by decreasing C-reactive protein, IL6 and ferritin levels. We conclude that, CCP-transfusion is a safe and effective supplementary treatment modality for hospitalized COVID-19 patients characterized by better expected outcome if applied as early as possible. We also observed that, IL-6 may be a suitable laboratory parameter for patient selection and monitoring of CCP therapy effectiveness.

BackgroundIn children undergoing chemotherapy yearly influenza vaccination is recommended by treatment protocols. We investigated the relationship between cellular immunity and the antibody response to inactivated influenza vaccines.Methods25 patients (age: 2-18 years) undergoing chemotherapy for different malignancies participated in our study. Flow cytometric detection of peripheral blood lymphocyte subpopulations together with hemagglutination inhibition antibody titers were measured before and 21-28 days after vaccination. We examined the ratio and total numbers of CD3+, CD4+, CD8+ T cells, activated helper (CD3+CD4+CD25low), regulatory (CD3+CD4+CD25high), naive (CD3+CD45RA+) and memory (CD3+CD45RO+) T cells, CD56+NK, and CD3+CD56+ (NKT-like) cells. Relationships between specific antibody responses (seroprotection, seroconversion, geometric mean titer (GMT), geometric mean fold increase (GMFI)) and the ratios and counts of lymphocyte subpopulations were evaluated using one-way ANOVA and the paired sample t test after dichotomization according to age-related reference values.ResultsPatients with CD4+ lymphocyte levels in the normal age-specific range showed significantly better response regarding postvaccination GMT elevation for H1N1 and H3N2 strains (97.52 vs. 19.2, p=0.019, 80 vs. 14.43, p=0.021, respectively). GMFI results were significant only against B strain (2.69-fold vs. 1.23-fold, p=0.046). Prevaccination CD3+CD56+ (NKT-like) cells above predicted values according to age showed significant associations both in postvaccination GMT elevation (H1N1: 75.11 vs. 14.14, p=0.010; H3N2: 62.18 vs. 11.22, p=0.012; B: 22.69 vs. 6.67, p=0.043) and GMFI against all three strains (H1N1: 3.76-fold vs. 1.06-fold, p=0.015; H3N2: 2.74-fold vs. 1, p=0.013; B: 2.57-fold vs. 1, p=0.008). By one-way ANOVA, we found a positive relation between absolute lymphocyte cell count above 1000/µl and the postvaccination GMT elevation against H3N2 (12.81 vs. 56.56, p=0.032), and GMFI regarding H1N1 (1.22-fold vs. 3.48-fold, p=0.044).ConclusionsIn addition to verifying the predictive value of absolute lymphocyte count above 1000/µl, our results suggest an association between NKT-like cell counts and the specific antibody response against all three investigated influenza strains in highly immunosuppressed patients. Furthermore, prevaccination CD4+ lymphocyte levels in the normal age-specific range may influence seroresponse.

Purpose. Decreased expression of TLR homolog CD180 in peripheral blood B cells and its potential role in antibody production have been described in autoimmune diseases. Effectiveness of anti-CD20 therapy in neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) strengthens the role of B cells in the pathogenesis. Therefore, we aimed to investigate the CD180 expression of peripheral blood B cell subsets in NMOSD and MS patients and analyze the levels of natural anti-citrate synthase (CS) IgG autoantibodies and IgG antibodies induced by bacterial infections reported to play a role in the pathogenesis of NMOSD or MS. Methods. We analyzed the distribution and CD180 expression of peripheral blood B cell subsets, defined by CD19/CD27/IgD staining, and measured anti-CS IgM/G natural autoantibody and antibacterial IgG serum levels in NMOSD, RRMS, and healthy controls (HC). Results. We found decreased naïve and increased memory B cells in NMOSD compared to MS. Among the investigated four B cell subsets, CD180 expression was exclusively decreased in CD19+CD27+IgD+ nonswitched (NS) memory B cells in both NMOSD and MS compared to HC. Furthermore, the anti-CS IgM natural autoantibody serum level was lower in both NMOSD and MS. In addition, we found a tendency of higher anti-CS IgG natural autoantibody levels only in anti-Chlamydia IgG antibody-positive NMOSD and MS patients. Conclusions. Our results suggest that reduced CD180 expression of NS B cells could contribute to the deficient natural IgM autoantibody production in NMOSD and MS, whereas natural IgG autoantibody levels show an association with antibacterial antibodies.

Multiple cytokines have been implicated in aneurysmal subarachnoid hemorrhage (aSAH), but tumor necrosis factor superfamily 14 (LIGHT/TNFSF14) and oncostatin-M (OSM) have not been previously explored.The primary objective of this study was to examine the relationship between TNFSF14 and OSM levels and survival. Our secondary goal was to investigate a potential association between these markers and the incidence of delayed cerebral ischemia (DCI).We consecutively recruited 60 patients with a clinical diagnosis of aSAH. LIGHT/TNFSF14 and OSM serum concentrations were determined by ELISA. The primary endpoint was survival at Day 30, while development of DCI was assessed as secondary outcome.Patients had significantly higher levels of both markers than the control group (median of LIGHT: 18.1 pg/ml vs. 7 pg/ml; p = 0.01; median of OSM: 10.3 pg/ml vs. 2.8 pg/ml, p 0.001). Significantly lower serum level of LIGHT/TNFSF14 was found in nonsurviving patients (n = 9) compared with survivors (n = 51; p = 0.011). Based on ROC analysis, serum LIGHT/TNFSF14 with a cutoff value of7.95 pg/ml predicted 30-day survival with a sensitivity of 71% and specificity of 78% (Area: 0.763; 95% CI: 0.604-0.921, p = 0.013). In addition, it was also a predictor of DCI with a sensitivity of 72.7% and a specificity of 62.5% (AUC: 0.702; 95% CI: 0.555-0.849, p = 0.018). Based on binary logistic regression analysis, LIGHT/TNFSF14 was found to be independently associated with 30-day mortality, but not with DCI.In this cohort, a higher serum level of LIGHT/TNFSF14 was associated with increased survival of patients with aSAH.

Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10–6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3&minus, /&minus, ) the spleen&rsquo, s histological structure is fundamentally changed, therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3&minus, mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3&minus, and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3&minus, mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3&minus, mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures, in the sera, we found less anti-CCP-IgG2a, IL-17 and IFN&gamma, but more IL-1&beta, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3&minus, mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.

Our goal was to explore immunoserological relations between IgM and IgG isotypes of natural autoantibodies (nAAbs) and pathogen (or vaccine)‐induced or disease‐related antibodies in systemic autoimmune diseases (SAD); systemic sclerosis (SSc), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA). The found significant associations between IgG isotype nAbs and specific humoral antibodies may underscore the immune response‐inducible nature of the diseases investigated, and may corroborate the notion that nAbs can act as a mediator between the innate‐like and the adaptive arms of the immune system .The relationship between protective anti‐dsDNA IgM and the IgM isotype of anti‐F4 and anti‐CS may provide immuno‐serological evidence for the beneficial roles of nAAbs in SLE patients., Summary Infection or vaccine‐induced T cell‐dependent immune response and the subsequent high‐affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease‐specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine‐induced and disease‐related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme‐linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti‐CS) and F4 fragment (anti‐F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n = 92), rheumatoid arthritis (n = 73) and systemic sclerosis (n = 157) disease groups. Anti‐measles IgG and anti‐dsDNA IgG/IgM autoantibodies were measured using commercial and in‐house indirect ELISA tests. In all SAD groups the anti‐measles IgG‐seropositive cases showed significantly higher anti‐CS IgG titers (P = 0·011). In anti‐dsDNA IgG‐positive SLE patients, we detected significantly higher levels of anti‐CS and anti‐F4 IgG nAAbs (P = 0·001 and

In Hungary, between February 2017 and July 2019, 70 confirmed measles cases were reported, raising questions about the adequacy of population-level immunity. Although the assumed vaccination coverage is ≥99%, in a recent study, we detected potential gaps in the anti-measles humoral immunity. In Hungary, according to a decree by the Ministry of Public Welfare, beginning from 2021, the healthcare provider should conduct a serosurvey of anti-measles protection levels of healthcare professionals. To facilitate the compliance with this requirement, we developed a quick ‘three-in-one’ or ‘triple’ MMR (measles, mumps and rubella) indirect ELISA (IgG); an assay format that is currently not available commercially. High throughput applicability of the ‘three-in-one’ ELISA was verified using 1736 sera from routine laboratory residual samples, using an automated platform (Siemens BEP 2000 Advance). Assay verification was performed by comparing the full antigen repertoire-based ‘target’ assay with in-house ‘control’ assays using recombinant viral antigen coatings, and by validated commercially available kits. Indirect immunofluorescence was used as an independent reference method. Data were analysed using OriginLab, IBM SPSS, RStudio and MedCalc. In case of measles, we combined our current results with previously published data (Ntotal measles = 3523). Evaluation of anti-mumps and anti-rubella humoral antibody levels was based on the measurement of 1736 samples. The lowest anti-measles seropositivity (79.3%) was detected in sera of individuals vaccinated between 1978 and 1987. Considering the antigen-specific seropositivity ratios of all samples measured, anti-measles, -mumps and -rubella IgG antibody titres were adequate in 89.84%, 91.82% and 92.28%, respectively. Based on the virus-specific herd immunity threshold (HIT) values (HITMeasles = 92–95%, HITMumps = 75–86%, HITRubella = 83–86), it can be stated that regarding anti-measles immunity, certain age clusters of the population may have inadequate levels of humoral immunity. Despite the potential gaps in herd immunity, the use of MMR vaccine remains an effective and low-cost approach for the prevention of measles, mumps and rubella infections.

Background and purpose: The goal of this study was to determine the prevalence and incidence of neuromyelitis optica spectrum disorder (NMOSD) in Hungary based on the 2015 International Panel of NMO Diagnosis (IPND) criteria. Methods: A retrospective population-based cohort study was conducted of 6.4 million Hungarians (age ≥ 16 years) between 1 January 2006 and 31 December 2016. Possible NMOSD patients were selected via multistage re-evaluation from multiple sources. Crude and sex- and serostatus-specific prevalence (per 100 000 persons) and incidence rates (per 1 000 000 person-years) from 2006 to 2015 were estimated and age-adjusted rates were determined. Results: Of 2262 study candidates, 154 NMOSD patients (age ≥ 16 years) with onset until 31 December 2016 were identified based on 2015 IPND criteria. The prevalence analysis on 1 January 2016 included 123 NMOSD living cases, resulting in a prevalence of 1.91 [95% confidence interval (CI) 1.52–2.28] per 100 000 persons. The 101 incident cases emerging from the observed 76 394 288 person-years provided an incidence rate of 1.32 (95% CI 1.08–1.61) per 1 000 000 person-years. Age-adjusted prevalence was 1.87 (95% CI 1.56–2.23) per 100 000 persons and incidence was 1.20 (95% CI 0.98–1.46) per 1 000 000 person-years. Conclusions: In this first report of a large population-based epidemiological study from an Eastern European Caucasian population using robust case validation, a greater prevalence and incidence of NMOSD was found compared to previous large studies in Caucasian populations.

Altered expression and function of the Toll-like receptor (TLR) homologue CD180 molecule in B cells have been associated with autoimmune disorders. In this study, we report decreased expression of CD180 at protein and mRNA levels in peripheral blood B cells of diffuse cutaneous systemic sclerosis (dcSSc) patients. To analyze the effect of CD180 stimulation, together with CpG (TLR9 ligand) treatment, on the phenotype defined by CD19/CD27/IgD/CD24/CD38 staining, and function (CD69 and CD180 expression, cytokine and antibody secretion) of B cell subpopulations, we used tonsillar B cells. After stimulation, we found reduced expression of CD180 protein and mRNA in total B cells, and CD180 protein in B cell subpopulations. The frequency of CD180+ cells was the highest in the CD19+CD27+IgD+ non-switched (NS) B cell subset, and they showed the strongest activation after anti-CD180 stimulation. Furthermore, B cell activation via CD180 induced IL-6 and natural autoantibody secretion. Treatment with the combination of anti-CD180 antibody and CpG resulted in increased IL-6 and IL-10 secretion and natural autoantibody production of B cells. Our results support the role of CD180 in the induction of natural autoantibody production, possibly by NS B cells, and suggest an imbalance between the pathologic and natural autoantibody production in SSc patients.

Summary Our previous studies showed that anti-citrate synthase (anti-CS) immunoglobulin (Ig)M natural autoantibodies are present in healthy individuals without previous antigen stimulation, but no studies have investigated their presence in the pericardial fluid (PF). Therefore, we detected the natural anti-CS IgG/M autoantibody levels in plasma and PF of cardiac surgery patients and investigated their relationship with cardiovascular disease-associated bacterial pathogens. PF and blood samples of 22 coronary artery bypass graft (CABG) and 10 aortic valve replacement (AVR) patients were tested for total Ig levels, natural autoantibodies and infection-related antibodies using enzyme-linked immunosorbent assay (ELISA) and Luminex methods. The B cell subsets were measured by flow cytometry. The total Ig subclass levels were four to eight times lower in PF than in plasma, but the natural anti-CS IgM autoantibodies showed a relative increase in PF. The frequency of CD19+ B lymphocytes was significantly lower in PF than in blood (P = 0·01), with a significant relative increase of B1 cells (P = 0·005). Mycoplasma pneumoniae antibody-positive patients had significantly higher anti-CS IgM levels. In CABG patients we found a correlation between anti-CS IgG levels and M. pneumoniae, Chlamydia pneumoniae and Borrelia burgdorferi antibody titres. Our results provide the first evidence that natural autoantibodies are present in the PF, and they show a significant correlation with certain anti-bacterial antibody titres in a disease-specific manner.

Abstract: Introduction: The role of autoimmune responses against central nervous system (CNS) antigens in encephalitis presenting with non-classified neurologic or psychiatric symptoms has been appreciated in the past decade. Paraneoplastic limbic encephalitis has a poor prognosis and is most commonly associated with lung, ovarium, and testicular neoplasms, leading to immune reactions against intracellular antigens (anti-Hu/ANNA1, anti-Ri/ANNA2, anti-CV2/CRMP5 and anti-Ma2/Ta). In contrast, the recently described autoimmune encephalitis subtypes present with a broad spectrum of symptoms, respond to autoimmune therapies well and usually associate with autoantibodies against neuronal cell surface receptors (NMDAR, GABABR, AMPAR) or synaptic proteins (LGI1, CASPR2). Aim: Our aim is to bring to awareness the increasing number of autoimmune encephalitis patients requiring neurologic, psychiatric and intensive care and to emphasize the significance of detecting various autoantibodies in diagnosing patients. Method: In the past 6 years, our laboratory received 836 autoimmune encephalitis diagnostic test requests from a total of 717 patients. Serum and cerebrospinal fluid (CSF) samples were analysed with indirect immunofluorescence using a BIOCHIP consisting of cell lines transfected with 6 different receptor proteins. Results: IgG autoantibodies against receptor proteins were present in 7.5% of patients. The frequency of positive samples was the following: NMDAR > LGI1 > GABABR > CASPR2. Conclusion: Detecting autoantibodies facilitates the diagnosis of autoimmune encephalitis in an early stage. Patients diagnosed early can be effectively treated with plasmapheresis and immunosuppressive drugs. The efficiency of therapies can be monitored by autoantibody detection. Therefore, the diagnostic immune laboratory plays an important role in proper diagnosis and in the prevention of rapidly progressing symptoms. Orv Hetil. 2018; 159(3): 107–112.

Hyperacute serum (HAS) is a blood derivative product that promotes the proliferation of various cell types and controls inflammation in vitro. The aim of this study is to investigate the regenerative potential of different formulations of HAS, including lyophilized and hyaluronic acid combined versions, to obtain a stable and standardized therapeutic in osteoarthritis (OA), which may be able to overcome the variability limitations of platelet-rich plasma (PRP). Primary human osteoarthritic chondrocytes were used for testing cellular viability and gene expression of OA-related genes. Moreover, a co-culture of human explants of cartilage, bone and synovium under inflammatory conditions was used for investigating the inflammatory control capacities of the different therapeutics. In this study, one formulation of lyophilized HAS achieved the high cell viability rates of liquid HAS and PRP. Gene expression analysis showed that HAS induced higher Col1a1 expression than PRP. Cytokine quantification from supernatant fluids revealed that HAS treatment of inflamed co-cultures significantly reduced levels of IL-5, IL-15, IL-2, TNFα, IL-7 and IL-12. To conclude, lyophilized HAS is a stable and standardized therapeutic with high potential in joint regeneration.

Background Osteopontin (OPN) is a proinflammatory biomarker, and neurofilament light chain (NFL) levels reflect axonal damage. Resting-state functional MRI (rs-fMRI) defines brain networks during wakeful rest. Objective To examine, if levels of OPN and NFL are associated on the long term with (i) lesion evolution, (ii) changes in normal-appearing white matter (NAWM) microstructure and (iii) functional connectivity in multiple sclerosis (MS). Methods Concentration of NFL and OPN in the blood and CSF were related to MRI findings 10.3 ± 2.8 years later in 53 patients with MS. NFL was examined by Simoa method, OPN by ELISA. Lesion volume in the brain and cervical spinal cord was examined by 3D FLAIR images. Voxel-wise images of fractional anisotropy (FA), axial diffusivity (AD), mean diffusivity (MD), and radial diffusivity (RD) were examined by tract-based spatial statistics corrected for gender, age and lesion volume. Metabolites were examined by single-voxel MR-spectroscopy in the NAWM. Fifty-five default mode network connections were examined by rs-fMRI corrected for gender, age, MS subtype and current therapy as covariates. Results While NFL in paired serum and CSF positively correlated (p = 0.019), there was no correlation between serum and CSF OPN. Higher OPN levels in the CSF but not in the serum showed association with increased brain WM lesion volume (p = 0.009) in 10.3 ± 2.8 years. Higher OPN in the CSF was associated with reduced FA, increased MD, and reduced RD in different NAWM areas 10.3 ± 2.8 years later. Higher OPN in the serum and CSF were associated with increased connectivity strength between the medial prefrontal cortex (MPFC) and other regions except with inferior parietal lobule. NFL in the CSF and in the serum was associated with decreased connectivity strength except for ventral MPFC-hippocampal formation. Neither serum OPN nor NFL at the time of the MRI were associated with functional connectivity changes. Conclusion While serum NFL levels reflects CNS production, OPN in serum and CSF may have different cellular sources. OPN within the CSF but not in the serum may forecast development of lesions and microstructural abnormalities in 10 years, indicating the detrimental role of CNS inflammation on the long-term. Although both OPN and NFL in the CSF were associated with functional connectivity changes in 10 years, NFL was associated with decreased strength possibly indicating general axonal loss. In contrast, the positive association of OPN levels in the CSF with increased connectivity strength in 10 years may point to adaptive re-organization due to inflammatory WM lesions and microstructural changes.

Background Based on the phenotypic and functional characteristics unconventional T-lymphocytes such as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells link the innate and adaptive immune responses. Up to now data are scarce about their involvement in pulmonary disorders including chronic obstructive pulmonary disease (COPD). This study explores simultaneously the frequencies of iNKT and MAIT cells in the peripheral blood and sputum of stable and exacerbating COPD patients. Methods By means of multicolor flow cytometry frequencies of total iNKT and MAIT cells and their subsets were enumerated in peripheral blood and sputum samples of healthy controls, and COPD patients. In addition, gene expression of TCR for iNKT, MAIT cells, and CD1d, MR1 were assessed by qPCR in the study cohorts. Results Percentages of total iNKT and MAIT cells were dramatically dropped in blood, and reduced numbers of iNKT cells were observed in the sputum of COPD patients. Furthermore decreased DN and increased CD4+ iNKT subsets, while increased DN and decreased CD8+ MAIT subpopulations were measured in the blood of COPD patients. Reduced invariant TCR mRNA levels in COPD patients had confirmed these previous findings. The mRNA expression of CD1d and MR1 were increased in stable and exacerbating COPD patients; however both molecules were decreased upon antibiotic and systemic steroid treatments. Conclusions Our results support the notion that both invariant T-cell populations are affected in COPD. Further detailed analysis of invariant T cells could shed more light into the complex interactions of these lymphocyte groups in COPD pathogenesis. Electronic supplementary material The online version of this article (10.1186/s12931-017-0671-1) contains supplementary material, which is available to authorized users.

Objective Autoantibody detection is crucial for the early diagnosis of autoimmune encephalitis (AIE) since prompt therapy can determine the disease outcome. Here, we report a single‐center 6‐year retrospective study of autoantibody testing in AIE in the Hungarian population. Methods Serum and/or cerebrospinal fluid (CSF) autoantibody tests were performed using cell‐based indirect immunofluorescence assay for AIE diagnosis. Samples were provided by neurology clinics as part of a nationwide program. Test results were analyzed for samples received during the period from 2012 to 2018. Results We tested 1,247 samples from 1,034 patients with suspected AIE. Autoantibodies were present in 60 patients (5.8% of total). The distribution of patients with different autoantibodies by age and sex was as follows: NMDAR (70%), mostly in young females, LGI1 (15%) in middle‐aged males, GABABR (12%) in elderly males, and Caspr2 (7%) in males. Long‐term follow‐up was conducted in 30 patients with repeated test requests, of which 17 remained positive, and 13 switched to negative. Conclusion We report the most comprehensive clinical laboratory study of autoantibody testing in AIE in the Hungarian population. Our results show that the frequency of different autoantibody types in AIE corresponds to the data described in the literature., We report the most comprehensive clinical laboratory study of autoantibody testing in AIE in the Hungarian population. Our results show that the frequency of different autoantibody types in AIE corresponds to the data described in the literature. In agreement with the international recommendations, our data support that for AIE diagnosis both serum and CSF samples should be tested.

Background Composition of pericardial fluid (PF) may reveal immunological processes influencing oxidative stress and microcirculation of different tissues of the heart and may play a role in the course of myocardial infarction, atherosclerosis, and aortic stenosis. Patients and methods We investigated lymphocyte populations, cardiovascular markers and immunoglobulin composition in PF and blood samples of patients undergoing CABG operation and compared them to those who had aortic valve surgery. Results The amount of CD8 + T, NK, memoT and activated T-cytotoxic cells were elevated in PF compared to blood, but naiveT and activated T-helper cell ratio were lower in PF. Amount of activated T-helper cells and regulatory T-lymphocytes were elevated in CABG participants in both PF and blood. INKT cells represented the only regulatory lymphocyte population reaching significantly higher concentration in PF than in blood. IL-6 and MCP1 level were elevated in PF compared to blood and MCP1 plasma level was markedly elevated in CABG group. Conclusions Our study describes a comprehensive immunological analysis of PF in humans for the first time. We showed that the investigated lymphocyte populations and cardiovascular markers in PF have significantly different distribution compared to blood, and lymphocyte populations show different compartmentization in coronary disease and aortic stenosis.

Background B cell activation is an early event in the development of systemic sclerosis (SSc) as transcriptome profiling identified local B-cell activation in early diffuse cutaneous (dc) SSc skin biopsies. Autoantibody production is a widely investigated function of B cells in SSc but less attention has been devoted to the role of innate immune molecules and their receptors, like Toll-like receptors (TLRs). The classical B cell activation downstream of BcR and CD19 co-receptor engagement involves phosphatidylinositol-3 kinases (PI3K) signaling pathway that integrates the effects of multiple co-stimulatory receptors. Objectives Our goal was to examine PI3K signaling by investigating the mRNA expression of 92 pathway related genes in B cells from early dcSSc patients. Akt is a central element of PI3K pathway, but it can also converge into innate receptor mediated pathways such as mTOR and MAPK. Thus for functional relevance we also analyzed the phosphorylation of Akt, S6 and NF-κB in B cells from early dcSSc patients. Methods Twenty-one patients with early dcSSc were enrolled with disease duration of 2.0 (±1.2) years based on the date of the first non-Raynaud’s symptom. 30% of patients received immunosuppressive therapy. Peripheral blood CD19+ B cells were purified from dcSSc patients and age- and sex-matched healthy controls (HC, n=15). mRNA expression of 92 B cell specific PI3K pathway related genes was measured using a Taqman qPCR array and was validated by individual qPCR. Isolated B cells were activated through BcR using anti-IgG/M antibody or anti-CD180 antibody, and their combination followed by flow cytometric analysis of phospho-Akt (S473), phospho-S6 (S235/S236) and phospho-NF-κB p65 (S529) positive cells. Results Analyzing the expression of 92 PI3K pathway associated genes we found altered expression of molecules playing a role in alternate B cell activation and innate signaling. The expression of both IL-4 receptor and osteopontin (SPP1) was upregulated in B cells of untreated dcSSc patients, but became downregulated upon immunosuppressive therapy. Innate molecules like TLR4 and complement component 3 remained highly upregulated and the downregulation of CD180 was not inhibited by immunosuppressive treatment, thus may preserve the activated state of B cells in dcSSc. We aimed to model B cell activation via TLR4 and CD180 but LPS and endogenous ligands of TLR4 failed to activate isolated B cells. Since there are not known ligands of CD180, we investigated the effects of anti-CD180 itself and also its combination with BcR mediated stimulation of B cells. Analyzing the phosphorylation of the PI3K pathway-associated molecules revealed an impaired responsiveness of dcSSc B cells to anti-CD180 alone, and the combination of anti-CD180 and anti-Ig induced different phosphorylation patterns of Akt, S6 and NF-kB molecules in dcSSc and healthy controls (HC). Conclusion Analysis of B cells from early dcSSc patients revealed that B cells possess molecular changes in PI3K pathway that involves innate immune components. Gaining new insight into innate B-cell activation in SSc could be helpful for finding new therapeutic targets. References [1] Bhattacharyya S, et al. Adv wound care (2017) 6:356–369. [2] Chaplin JW, et al. J Immunol (2011) 187:4199–209. Acknowledgement This work was supported by GINOP-232-15-2016-00050 and EFOP 361-16-2016-00004 grants. Disclosure of Interests None declared

Background: The recombinant human G1(rhG1)-induced mouse arthritis (GIA) model resembles human rheumatoid arthritis in many aspects1. Similarly, to RA, GIA is also a T cell dependent disease: T cell activation and apoptosis has been shown to regulate arthritis severity2. ZAP-70 kinase is a key molecule of T cell receptor signaling, in its absence T cell differentiation and activation is seriously hindered3. In preliminary experiments, we found that partial ZAP-70 deficiency (in ZAP-70+/- heterozygous mice) ameliorated autoimmune arthritis in the GIA model, supported by both in vivo (decreased severity and milder arthritis) and in vitro tests (decreased T cell proliferation, IL-4 and IL-6 production). Objectives: In the present work we wanted to test the hypothesis that the observed changes in GIA in partial ZAP-70 deficient mice are due to complex alterations in T cell apoptosis. Methods: T cells were isolated from healthy and arthritic BALB/c and ZAP-70 heterozygous (ZAP-70+/-) knockout mice, then stimulated in vitro for 72 hours with anti-CD3/anti-CD28-coated beads. After stimulation, cells were lysed and the homogenates were subjected to Western-bloting using antibodies against activated Caspases-3,-8, and -9, respectively, and other apoptotic markers (cytochromeC), and some important regulator molecules (Bcl-2, Bim and Cbl). Results: Overall, as expected, we have seen decreased tyrosine phosphorylation in the T cells of arthritic ZAP-70+/- mice compared to the controls. The levels of cleaved (activated) Caspase-8 and -9 were comparable in ZAP-70+/- samples to the controls, however, we detected elevated levels in case of Caspase-3. There was slightly lower Cytochrome C release and Bcl-2 and Bim levels in the activated T cells of ZAP-70+/- mice. Importantly, there was markedly less Cbl-b in the T cells of arthritic ZAP-70+/- mice. Conclusion: Our results show the complex changes in the apoptotic pathways of activated T cells caused by the partial deficiency of ZAP-70. Impaired T cell activation through the T cell receptor complex, as shown by decreased phosphorylation, accompanied by slightly increased apoptosis sensitivity of T cells (stronger Caspase-3 signal) might explain the less severe arthritis seen in ZAP-70+/- mice. Furthermore, based on these results Cbl-b (a negative regulator of T cell activation) might serve as an important early molecule interacting with ZAP-70 and thus fine regulate T cell activation and apoptosis in autoimmune arthritis. References [1] Glant TT, Radacs M, Nagyeri G, Olasz K, Laszlo A, Boldizsar F, Hegyi A, Finnegan A, Mikecz K. Arthritis Rheum. 63(5):1312-21., 2011 [2] Hanyecz A, Olasz K, Tarjanyi O, Nemeth P, Mikecz K, Glant TT, Boldizsar F. Biomed Res Int. 2014:942148., 2014 [3] Kadlecek TA, van Oers NSC, Lefrancois L, Olson S, Finlay D, Chu DH, Connolly K, Killeen N, Weiss A. J Immunol. 161:4688-4694; 1998 Acknowledgement: Funding: OTKA K101493; EFOP-3.6.1.-16-2016-00004; GINOP 2.3.2-15-2016-00050; KA-2015-23 to K.O.; BO/00086/12/5 to F.B.; 716/180/2014/KIF to K.O.; KA-POSTDOK-12-05 to K.O. Disclosure of Interests: None declared

T cells play an essential role in the pathogenesis of both human rheumatoid arthritis (RA) and its murine models. A key molecule in T cell activation is ZAP-70, therefore we aimed to investigate the effects of partial ZAP-70 deficiency on the pathogenesis of recombinant human G1(rhG1)-induced arthritis (GIA), a well-established mouse model of RA. Arthritis was induced in BALB/c and ZAP-70+/&minus, heterozygous mice. Disease progression was monitored using a scoring system and in vivo imaging, antigen-specific proliferation, cytokine and autoantibody production was measured and T cell apoptotic pathways were analyzed. ZAP-70+/&minus, mice developed a less severe arthritis, as shown by both clinical picture and in vitro parameters (decreased T cell proliferation, cytokine and autoantibody production). The amount of cleaved Caspase-3 increased in arthritic ZAP-70+/&minus, T cells, with no significant changes in cleaved Caspase-8 and -9 levels, although expression of Bim, Bcl-2 and Cytochrome C showed alterations. Tyrosine phosphorylation was less pronounced in arthritic ZAP-70+/&minus, T cells and the amount of Cbl-b&mdash, a negative regulator of T cell activation&mdash, decreased as well. We hypothesize that the less severe disease seen in the partial absence of ZAP-70 might be caused by the decreased T cell activation accompanied by increased apoptosis.

Serological testing is a tool to predict protection against later infection. This potential heavily relies on antibody levels showing acceptable agreement with gold standard virus neutralization tests. The aim of our study was to investigate diagnostic value of the available serological tests in terms of predicting virus neutralizing activity of serum samples drawn 5-7 weeks after onset of symptoms from 101 donors with a history of COVID-19. Immune responses against Receptor Binding Domain (RBD), Spike1 and 2 proteins and Nucleocapsid antigens were measured by various ELISA tests. Neutralizing antibody activity in serum samples was assessed by a cell-based virus neutralization test. Spearman correlation coefficients between serological and neutralization results ranged from 0.41 to 0.91 indicating moderate to strong correlation between ELISA test results and virus neutralization. The sensitivity and specificity of ELISA tests in the prediction of neutralization were 35-100% and 35-90% respectively. No clear cut off levels can be established that would reliably indicate neutralization activity. For some tests, however, a value below which the sample is not expected to neutralize can be established. Our data suggests that several of the ELISA kits tested may be suitable for epidemiological surveys 1-2 months after the infection, estimating whether a person may have recently exposed to the virus. Sensitivities considerably superseding specificity at the cut-off values proposed by the manufacturers suggest greater potential in the identification of insufficient antibody responses than in confirming protection. Nevertheless, the former might be important in assessing response to vaccination and characterizing therapeutic plasma preparations.