Effect of Third and Fourth mRNA-Based Booster Vaccinations on SARS-CoV-2 Neutralizing Antibody Titer Formation, Risk Factors for Non-Response, and Outcome after SARS-CoV-2 Omicron Breakthrough Infections in Patients on Chronic Hemodialysis: A Prospective Multicenter Cohort Study.

The aim of this study is to determine the effect of repeated vaccinations on neutralizing SARS-CoV-2 IgG antibody titers, evaluate risk factors for immunological non-response, and to report breakthrough infections in chronic hemodialysis patients. METHODS: A prospective, multi-center cohort study in 163 chronic hemodialysis patients was conducted. Antibody titers were measured three months after second, third, and fourth (10 pts) booster vaccinations. SARS-CoV-2 neutralizing antibody titers in BAU/mL and % inhibition were divided into three categories (<216, 216-433, >433 and <33, 33-66, and >66%). Somers's test, paired t-test, and univariable and multivariable logistic regression analysis were applied to evaluate differences in antibody levels and search for risk factors for vaccination failure defined as neutralizing titers <50% and/or need for repeated booster vaccinations. Furthermore, we report on a case series to describe characteristics of patients after four vaccinations (n = 10) and breakthrough infections (n = 20). RESULTS: Third dose boosters resulted in higher proportions of patients with neutralizing antibody levels >66% as compared to after the second dose (64.7% after second dose vs. 88.9% after third dose, p = 0.003), as well as in a respective increase in neutralizing titer levels in % from 68 ± 33% to 89 ± 24 (p <0.001). The proportion of patients with IgG-titers below 216 BAU/mL decreased from 38.6 to 10.5% (p ≤ 0.001). Age (p = 0.004, OR 1.066, 95% CI 1.020-1.114) and presence of immunosuppressive medications (p = 0.002, OR 8.267, 95% CI 2.206-30.975) were identified as major risk factors for vaccination failure. Repeated booster vaccinations ≥4 times were effective in 8 out of 10 former low-responders (80%) without any side effects or safety concerns. Breakthrough infections showed a clinically mild course but were associated with prolonged viral shedding on PCR-testing ranging 7-29 (mean 13) days. CONCLUSIONS: Third and fourth mRNA-based booster vaccinations resulted in higher and longer lasting SARS-CoV-2 antibody levels as compared to after two dosages. The presence of immunosuppressive medication and repeat vaccinations are major potentially modifiable measures to increase antibody levels in non-or low-responders. Breakthrough infections with SARS-CoV-2 Omicron were associated with prolonged viral shedding but clinically mild disease courses.

Long-term trajectories of SARS-CoV-2 neutralizing antibodies and predictive value of first dose vaccination-induced IgG-antibodies in hemodialysis patients.

PURPOSE: The predictive value of antibody titers after the first SARS-CoV-2 vaccination and long-term trajectories of antibody titers in hemodialysis patients are unknown. METHODS: SARS-CoV-2 IgG antibodies and their neutralizing effect six weeks after the first and second vaccination were analysed in 30 hemodialysis patients. IgG titers served to classify participants as responders or non-responders and to calculate sensitivity, specificity, and accuracy. Associations between potential risk factors and post-vaccine non-response were analysed by Mann-Whitney-U test and Chi-Squared test. Long-term follow-up analysis (ANOVA) on the evolution of neutralizing IgG-titers was performed in 24 participants 94 and 135 days after the second immunization. RESULTS: IgG antibodies ≥ 1 AU/L (mean 9 ± 20 AU/L) after the first dose were found in 20 patients (66.7%). After the second dose only two participants (6.7%) remained sero-negative and 16.6% showed neutralizing levels below 30%, whereas 25 patients showed IgG antibodies with the high neutralizing activity of 86 ± 18%. Positive IgG antibodies 6 weeks after the first vaccination predicted vaccination effectiveness after two cycles with a specificity of 100%, sensitivity of 76%, and accuracy of 87%. Even low-dose immunosuppressive therapy increased the relative risk for non-response after the first and second dose 1.9 (95% CI 0.8-4.6) and 4.9 (95% CI 1.0-23.8) times, respectively. Over a period of about 4.5 months IgG titers slowly declined by 51% from baseline or by 0.45 AU/mL per day, respectively. CONCLUSION: Two cycles of SARS-CoV-2 vaccination-induced high seroconversion rates comparable to the general population. Immunosuppressive medication is a major risk factor for vaccination non-response. Mounted IgG antibodies showed a high neutralizing capacity as evidence of protective effectiveness. IgG antibodies after the first dose may serve to predict later vaccination outcome. Patients on dialysis display a more rapid decline in antibody titers on long-term follow-up compared to healthy controls.

Evolution of SARS-CoV-2-Neutralizing Antibodies after Two Standard Dose Vaccinations, Risk Factors for Non-Response and Effect of a Third Dose Booster Vaccination in Non-Responders on Hemodialysis: A Prospective Multi-Centre Cohort Study.

The aim of this investigation was to determine the effect of SARS-Cov-2 vaccination in hemodialysis patients, search for risk factors for non- or low-response, and to measure the effect of a third booster vaccination in non- or low-responders. Methods SARS-CoV-2 IgG antibodies and the virus-neutralizing capacity were measured 4-5 weeks after a full standard vaccination in 95 chronic hemodialysis patients and 60 controls. IgG titers > 30 AU/mL served to classify participants as responders. Multivariable binary logistic regression analysis was used to search for risk factors of reduced vaccination success. Patients with vaccination failure were offered a third booster dosage. Results 82.1% of the patient cohort as compared to 98.3% of the healthy control group were able to mount SARS-CoV-2 titers above 30 AU/mL after two standard vaccine doses. Mean IgG antibody titers were lower in hemodialysis patients than controls (78 ± 35 vs. 90 ± 20 AU/mL, p = 0.002). Multivariable binary logistic regression analysis showed age and immunosuppressive medication as major risk factors for vaccination failure with a decreased probability of successful vaccination of -6.1% (95% CI -1.2 to -10.9) per increase in age of one year and -87.4% (95% CI -98.0 to -21.5) in patients on immunosuppressive therapy (crude odds ratio for vaccination failure for immunosuppressive therapy 6.4). Ten out of 17 patients with non-response to vaccination were offered a third dose. Booster vaccination after the second dose induced an increase in effective antibody titers of >30 AU/mL in seven out of ten patients 4-5 weeks later (70%). Conclusion Standard SARS-CoV-2 vaccination schemes are highly effective in mounting protective neutralizing IgG antibodies in chronic hemodialysis patients. Nevertheless, response to vaccination is diminished as compared to a healthy control group. Major risk factors for vaccination failure are older age and immunosuppressive therapy. In non- or low-responders to standard vaccination a third booster vaccination was able to induce effective antibody titers in about 70% of patients, indicating that a third booster vaccination might be preferable to decreasing immunosuppressive therapy.

Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers.

BACKGROUND: Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)). METHODS: Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (ß2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-ß2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human ß2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human ß2GPI or after CL-micelle absorption. RESULTS: Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM aß2GPI and aCL. Anti-CL and anti-ß2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and ß2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized ß2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-ß2GPI humoAbs. CONCLUSIONS: The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.

Antiphospholipid antibody profiling: time for a new technical approach?

Detection of anti-phospholipid (aPL) antibodies for state-of-the art diagnosis of antiphospholipid syndrome(APS) still remains a laboratory challenge due to the great diversity of aPL antibodies and their relevance with regard to the diagnostic criteria. According to the recently revised classification criteria for APS, several enzyme-linked immunosorbent assays (ELISAs) should be performed simultaneously in routine laboratories for the detection of aPL antibodies. Therefore, new approaches to aPL profiling have been proposed recently to provide information regarding diagnosis and eventually outcome in APS patients. Multiplex analysis could meet the increasing demand for cost-efficient detection and profiling of aPL antibodies. Multi-line immunodot assays or bead-based multiplex techniques candidate as alternatives to assess several aPL antibodies simultaneously employing different solid-phases for bound/free separation of reactants. Particularly, multi-line immunodot assays present an alternative to ELISA for aPL antibody detection and profiling in APS patients. The use of hydrophobic membranes as solid-surface by this technique appears to offer a distinct solid-phase reaction environment for the assessment of aPL antibodies. This article reviews novel developments in the field of laboratory diagnostics of APS with special emphasis on multiplex assays.

Antiphospholipid antibodies induce translocation of TLR7 and TLR8 to the endosome in human monocytes and plasmacytoid dendritic cells.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by thromboembolic events and/or fetal loss in the presence of antiphospholipid antibodies (aPLs). The mechanisms underlying the pathogenicity of aPLs are still poorly understood. Here we show that 3 human monoclonal aPLs as well as IgG fractions from patients with the APS increase mRNA expression of the intracellular toll-like receptor (TLR) 7 in plasmacytoid dendritic cells and TLR8 in monocytes. Simultaneously they induce the translocation of TLR7 or TLR8 from the endoplasmic reticulum to the endosome. These effects depend on the uptake of aPLs into the endosome, subsequent activation of endosomal NADPH oxidase, and generation of superoxide. As a consequence cells are dramatically sensitized to ligands for TLR7 and TLR8. This observation delineates a novel signal transduction pathway in innate immunity originating from the endosome. Because the overexpression of TLR7 can also be detected in plasmacytoid dendritic cells from patients with the APS ex vivo, our results provide an explanation for proinflammatory and procoagulant effects of aPLs. Because inappropriate expression of TLR7 has been implicated in the development of systemic autoimmunity, these findings may also be relevant for the understanding of autoimmunity.

Single-step autoantibody profiling in antiphospholipid syndrome using a multi-line dot assay.

INTRODUCTION: Diagnosis of antiphospholipid syndrome (APS) still remains a laboratory challenge due to the great diversity of antiphospholipid antibodies (aPL) and their significance regarding APS-diagnostic criteria. METHODS: A multi-line dot assay (MLDA) employing phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL), and beta2-glycoprotein I (ß2 GPI) was used to detect aPL, immunoglobulin G (IgG) and immunoglobulin M (IgM) in 85 APS patients, 65 disease controls, and 79 blood donors. For comparison, anti-CL and anti-ß2 GPI IgG and IgM were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of agreement of both methods was good for anti-CL IgG, moderate for anti-CL IgM, very good for anti-ß2 GPI IgG, and moderate for anti-ß2 GPI IgM (kappa = 0.641, 0.507, 0.803 and 0.506, respectively). The frequency of observed discrepancies for anti-CL IgG (1.75%), anti-CL IgM (3.93%), anti-ß2 GPI IgG (1.75%), and anti-ß2 GPI IgM (0.87%) was low (McNemar test, P < 0.05, not-significant, respectively). Sensitivity, specificity, positive (+LR) and negative (-LR) likelihood ratios for at least one positive aPL antibody assessed by ELISA were 58.8%, 95.8%, 14.1, and 0.4, respectively, and for at least three positive aPl IgM and/or one positive aPL IgG by MLDA were 67.1%, 96.5%, 19.3, and 0.3, respectively. The frequency of IgM to PI, PS and CL, and combination of three or more aPL IgM detected by MLDA was significantly higher in APS patients with cerebral transient ischemia (P < 0.05, respectively). CONCLUSIONS: The novel MLDA is a readily available, single-step, sensitive diagnostic tool for the multiplex detection of aPL antibodies in APS and a potential alternative for single aPL antibody testing by ELISA.

Effect of autologous blood transfusion on cerebral cytokine expression.

BACKGROUND: Autologous blood transfusion (ABT), for example, by means of cell saver equipment, is used to reduce the need for allogenic blood transfusion in patients with high perioperative blood loss. This study investigated the effect of blood/extracorporal surface interaction during withdrawal and retransfusion of shed autologous blood on cerebral inflammation in rats. Rats subjected to hypotension with cerebral ischemia served as positive controls. METHODS: Eighty-eight male Sprague-Dawley rats were anesthetized with sevoflurane, instrumented, and randomly assigned to the following groups: sham-operation (SHAM), autologous blood withdrawal/transfusion only (ABT), or bilateral carotid artery occlusion and autologous blood withdrawal/transfusion (BCAO/ABT). Inflammatory gene expression was investigated with real-time RT-polymerase chain reaction at 6, 12, and 24 hours after SHAM, ABT, or BCAO/ABT in brain hippocampal tissue. Naive rats were investigated as reference. RESULTS: ABT alone had no impact on hippocampal inflammatory gene expression, whereas after BCAO/ABT tumor necrosis factor-alpha (10.7 fold at 24 h), interleukin-1ß (2.1 fold at 6 h), interleukin-6 (35.7 fold at 24 h), COX-2 (9.3 fold at 6 h), and inducible nitric oxide synthase (3.4 fold at 24 h) increased compared with SHAM. CONCLUSIONS: ABT by itself did not provoke an inflammatory reaction in the healthy brain. However, in combination with cerebral ischemia the induction of a broad spectrum of inflammatory parameters indicates an inflammatory reaction of the hippocampus beginning after 6 hours and being most pronounced after 24 hours. Therefore, this study shows that cerebral inflammation is not induced by ABT after contact with extracorporal surfaces in rats.

Structural and functional characterization of a human IgG monoclonal antiphospholipid antibody.

Antiphospholipid antibodies (aPL) are likely involved in the pathogenesis of the antiphospholipid syndrome (APS). This study analyzes the structural and functional characteristics of a human monoclonal aPL (HL7G) from the IgG2 subtype with λ light chains generated from a patient with primary APS and recurrent cerebral microemboli. DNA encoding the variable region of heavy and light chains of the antibody was sequenced, analyzed, and compared to HL5B a previously described monoclonal aPL from the same patient. Both antibodies are derived from the same germline genes. HL7G had similar but more extensive somatic mutations in the CDR1 and 2 regions than HL5B, indicating that both antibodies are closely related and derived by a T cell-dependent antigen driven process. In ELISA assays HL7G bound to cardiolipin and several other phospholipid antigens in the absence of protein cofactors. Different from HL5B this aPL bound to ß2-glycoprotein I (ß2GPII). This suggests that reactivity of aPL against ß2GPI is determined by only few specific amino acid exchanges. HL7G was able to induce tissue factor (TF) as one of the procoagulant effects of aPL. Our data suggest that the binding specificity of aPL is only of limited value to predict the biological effect and the pathophysiological impact of the antibodies.

Toll-like receptors play a crucial part in the pathophysiological activity of antiphospholipid antibodies.

The antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombosis, recurrent fetal loss and the presence of a variety of antiphospholipid antibodies (aPL), directed to phospholipids like Cardiolipin and phospholipid binding proteins like ß2-glycoprotein I. Till date, the pathophysiological processes underlying these thrombotic events were still not fully understood. Recent data support the idea that the aPL might act via enhanced cytokine release due to activation of certain Toll-like receptors. The investigation of some of those mechanisms in more detail enlightens the involvement of the intracellular receptors TLR7 and TLR8 in a central point. Using patients' IgG fractions and/or monoclonal aPL, either generated from mouse or from human B-cells for the stimulation experiments of monocytes, endothelial cells or dendritic cells, all these stimuli induced an enhanced expression and secretion of cytokines, especially TNFα, caused by specific regulation or activation of Toll-like receptors. Using specific agonists or inhibitors could confirm the causal connection of these stimulatory effects. This review focuses on the recent developments connecting the binding of aPL with the activity of Toll-like receptors, especially in monocytes, endothelial cells and dendritic cells.

Vitamin D and musculoskeletal health, cardiovascular disease, autoimmunity and cancer: Recommendations for clinical practice.

BACKGROUND: There is increasing evidence that, in addition to the well-known effects on musculoskeletal health, vitamin D status may be related to a number of non-skeletal diseases. An international expert panel formulated recommendations on vitamin D for clinical practice, taking into consideration the best evidence available based on published literature today. In addition, where data were limited to smaller clinical trials or epidemiologic studies, the panel made expert-opinion based recommendations. METHODS: Twenty-five experts from various disciplines (classical clinical applications, cardiology, autoimmunity, and cancer) established draft recommendations during a 2-day meeting. Thereafter, representatives of all disciplines refined the recommendations and related texts, subsequently reviewed by all panelists. For all recommendations, panelists expressed the extent of agreement using a 5-point scale. RESULTS AND CONCLUSION: Recommendations were restricted to clinical practice and concern adult patients with or at risk for fractures, falls, cardiovascular or autoimmune diseases, and cancer. The panel reached substantial agreement about the need for vitamin D supplementation in specific groups of patients in these clinical areas and the need for assessing their 25-hydroxyvitamin D (25(OH)D) serum levels for optimal clinical care. A target range of at least 30 to 40 ng/mL was recommended. As response to treatment varies by environmental factors and starting levels of 25(OH)D, testing may be warranted after at least 3 months of supplementation. An assay measuring both 25(OH)D(2) and 25(OH)D(3) is recommended. Dark-skinned or veiled individuals not exposed much to the sun, elderly and institutionalized individuals may be supplemented (800 IU/day) without baseline testing.

Neuroprotective effect of Fn14 deficiency is associated with induction of the granulocyte-colony stimulating factor (G-CSF) pathway in experimental stroke and enhanced by a pathogenic human antiphospholipid antibody.

Using a transgenic mouse model of ischemic stroke we checked for a possible interaction of antiphospholipid antibodies (aPL) which often cause thromboses as well as central nervous system (CNS) involvement under non-thrombotic conditions and the TWEAK/Fn14 pathway known to be adversely involved in inflammatory and ischemic brain disease. After 7 days, infarct volumes were reduced in Fn14 deficient mice and were further decreased by aPL treatment. This was associated with strongest increase of the endogenous neuroprotective G-CSF/G-CSF receptor system. This unexpected beneficial action of aPL is an example for a non-thrombogenic action and the double-edged nature of aPL.

The roll of Toll-like receptors in the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis, recurrent fetal loss, and the presence of antiphospholipid antibodies (aPL). Recent data support the idea that the thrombotic activity in APS patients is attributed to enhanced cytokine release via activation of certain Toll-like receptors. To investigate these mechanisms more precisely, different experimental approaches were used to investigate this connection in detail. IgG fractions and/or monoclonal aPL, either generated from murine or human B cells were intensely used for stimulation experiments of monocytes, endothelial cells, or dendritic cells. All these stimuli induced an enhanced expression and secretion of cytokines, especially tumor necrosis factor (TNF)-alpha, caused by specific regulation or activation of Toll-like receptors. Using specific agonists or inhibitors could confirm the causal connection of these stimulatory effects. This review focuses on these recent developments, connecting the binding of aPL with the activity of Toll-like receptors, especially in monocytes, endothelial cells, and dendritic cells.

Human antiphospholipid antibodies induce TNFalpha in monocytes via Toll-like receptor 8.

The antiphospholipid syndrome (APS) is characterized by recurrent arterial and/or venous thromboses, pregnancy loss and the presence of antiphospholipid antibodies (aPL). One of the discussed mechanisms of this thrombotic activity in APS patients is attributed to TNFalpha secretion in monocytes after aPL stimulation. To investigate this mechanism in detail, we employed a monoclonal aPL and IgG fractions of APS patients for stimulation of human peripheral monocytes. Stimulation with this monoclonal aPL resulted in an increased expression and secretion of TNFalpha, caused by specific upregulation of TLR8 mRNA and protein expression levels. To confirm the specificity of this finding we could demonstrate that the TNFalpha enhancement could be neutralized by a TLR8-specific inhibitory DNA-oligonucleotide and could be further increased by adding the specific ligands for TLR8. Using APS patients IgG fractions for stimulation of peripheral monocytes revealed a similar TLR8 mRNA elevation and increase in TNFalpha-production. Furthermore the TLR8 expression level in PBMC's of APS patients was as well significantly elevated. It could be demonstrated that the TNFalpha release in monocytes resulting from aPL stimulation was exclusively induced by TLR8 engagement. This could be confirmed in PBMC's of APS patients, hinting that endogenous stimulation of TLR8 in APS patients and consecutive elevation of TNFalpha promotes a proinflammatory environment.

Autoantibody detection using indirect immunofluorescence on HEp-2 cells.

The detection of autoantibodies is an important element in the diagnosis and monitoring of disease progression in patients with autoimmune diseases. In laboratory diagnostic tests for connective tissue and autoimmune liver diseases, indirect immunofluorescence on HEp-2 cells plays a central role in a multistage diagnostic process. Despite the high quality of diagnostics, findings at different laboratories can differ considerably due to a lack of standardization, as well as subjective factors. The present paper formulates recommendations for the standardized processing and interpretation of the HEp-2 cell test for the detection of non-organ-specific (especially antinuclear) antibodies. It provides requirements regarding the diagnostic tests used, instructions for laboratory procedure and evaluation, and recommendations for interpretation. For an optimal laboratory diagnostic process, it is useful to have an informative, tentative clinical diagnosis and an experienced laboratory diagnostician. In addition, the following key elements are recommended: initial screening using indirect immunofluorescence on carefully chosen HEp-2 cells beginning with a serum dilution of 1:80 and evaluation under a microscope with powerful illumination; results from a titer of 1:160 upwards being considered positive; internal laboratory quality control; and standardized interpretation. The aim is to improve diagnostic tests and care of patients with autoimmune diseases as a central concern of the European Autoimmunity Standardization Initiative (EASI).

In vitro effects of antiphospholipid syndrome-IgG fractions and human monoclonal antiphospholipid IgG antibody on human umbilical vein endothelial cells and monocytes.

It has been shown that stimulation of endothelial cells and monocytes by antiphospholipid antibodies leads to a prothrombotic state involving upregulation of tissue factor (TF). We examined the in vitro effects of IgG fractions from patients with antiphospholipid syndrome (APS) and of a beta-2-glycoprotein 1-independent human monoclonal antiphospholipid antibody (HL-5B) on human umbilical vein endothelial cells (HUVEC) in comparison to untreated cell controls and to exposure to monoclonal IgG control antibody. We also examined the effect of recombinant monocyte chemoattractant protein-1 (MCP-1) on peripheral blood monocytes. Stimulation of endothelial cells with APS IgG fractions or HL-5B resulted in time-dependent upregulation of MCP-1 mRNA and protein expression. Stimulation with HL-5B also led to time-dependent upregulation of interleukin (IL)-8 and intracellular adhesion molecule-1 (ICAM-1) mRNA and IL-8 protein expressions. Stimulation of monocytes with recombinant MCP-1 resulted in an upregulation of TF mRNA and TF protein. In conclusion these results might represent a mechanism for antiphospholipid antibody-mediated thrombosis in APS patients.

Optical biosensor-based characterization of anti-double-stranded DNA monoclonal antibodies as possible new standards for laboratory tests.

The serum determination of circulating anti-double-stranded (ds)DNA autoantibodies is a routine measure for the laboratory diagnosis of systemic lupus erythematosus. Since available assays differ substantially and no feasible calibrator is available, the aim of this study was to evaluate a recently introduced surface plasmon resonance (SPR) biosensor chip for binding studies between dsDNA and anti-dsDNA autoantibodies and to demonstrate its usefulness for the characterization of new monoclonal antibody (mAb) standards and standardization of assays. We characterized two human and one murine monoclonal anti-dsDNA antibodies by measuring the kinetic on- and off-rates using the biosensor and calculating functional affinity (avidity) as the ratio of these. Obtained equilibrium dissociation constants were verified by an independent method and inhibition experiments were performed to determine reactivities to DNA of various length and composition. While all mAbs exhibited comparable avidities, which could be confirmed by gel shift experiments, one of them proved to have slower association and dissociation kinetics. This was the only mAb providing positive results in the Farr RIA. In inhibition experiments with ss- and ds-oligonucleotides 10, 24 and 42 bp in length, the mAbs acted substantially different. The study demonstrates how putative standards for the anti-dsDNA determination can be characterized using SPR biosensor technology. Our results suggest that kinetic rate constants seem to be decisive in explaining the behaviour of mAbs. Different reactivities to various DNA species should be taken into account with respect to varying DNA sources in commonly used laboratory assays.

TLR7 and TLR8 ligands and antiphospholipid antibodies show synergistic effects on the induction of IL-1beta and caspase-1 in monocytes and dendritic cells.

TLRs represent the first line of defense against invading pathogens in the innate immune system. Certain cytokines are important mediators and essentially necessary to assure an appropriately regulated immune response. Recent data gave initial evidence that IL-1beta is one of the most relevant members of these regulating cytokines. We investigated the induction of IL-1beta production in monocytes and pDCs stimulated with ligands for TLR7 and TLR8 and with antiphospholipid antibodies (aPL). Using human monocytes and pDCs for stimulation with specific TLR7 and TLR8 ligands such as resiquimod (R848) and single stranded RNA (RNA42) as well as with a human monoclonal aPL HL5B resulted in a specific upregulation of IL-1beta mRNA and protein in these cells. Determination of expression-levels using real-time RT-PCR showed significantly augmented TLR-dependent IL-1beta and caspase-1 expression. This increase could be substantially enhanced by adding the monoclonal aPL HL5B. To demonstrate the direct dependency between TLR stimulation and IL-1beta production, specific TLR inhibitors were applied and the IL-1beta and caspase-1 secretion could be explicitly decreased. The respective protein levels were determined using Western Blot, FACS analysis or ELISA assays. In conclusion we demonstrated that the downstream signaling pathway of TLR7 and TLR8 in monocytes and pDCs after stimulation with specific ligands included not only the secretion of cytokines such as TNFalpha and IL-1beta but as well the activation of necessary regulating proteins like caspase-1. APL seem to enforce this process hinting that endogenous stimulation of TLRs in the Antiphospholipid Syndrome (APS) patients resulted in enhanced secretion of proinflammatory cytokines.

Generation of multifunctional murine monoclonal antibodies specifically directed to the VP1unique region protein of human parvovirus B19.

Little is known about the VP1unique region (VP1u), a part of one major capsid protein of human parvovirus B19 (B19), concerning its involvement in viral replication and infection cycle. Showing a phospholipase A2 (PLA2)-like activity, which is discussed to be necessary for viral release from host cell, its precise function remains unclear. The purpose of this study was to generate multifunctional monoclonal antibodies (mabs) for different applications that may be useful in investigating VP1u's relevance. To establish antiVP1u antibodies, spleen cells from Balb/c mice immunized with purified recombinant viral protein were used for generating antibody-producing hybridoma cell lines. Usability of the antibodies was tested in enzyme-linked immunosorbent assay (ELISA), Western-blot analysis, immunofluorescence and an inhibition assay of enzymatic activity of PLA2. Three hybridoma cell lines secreting mab's specifically directed against the VP1u protein of B19 could be generated and functioned in every screening method used in this study. These antibodies are helpful tools for investigations in B19 research and diagnosis. Furthermore, the antibodies could help in gaining a deeper understanding of VP1u's role in viral replication and infection especially in the importance of its constitutive PLA2-like activity.