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.

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.

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.

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.

Nucleic acid recognizing Toll-like receptors and autoimmunity.

The understanding of autoimmune diseases experienced an impressive boost since the Toll-like receptors (TLRs) have been identified as possible key players in autoimmune pathophysiology. Although these receptors recognize a variety of structures derived from viruses, bacteria, and fungi leading to subsequent initiation of the relevant immune responses, recent data support the idea that TLRs are crucial in the induction and perpetuation of certain autoimmune diseases, especially the systemic lupus erythematosus (SLE). In this review, we will summarize recent data on involvement of TLRs in the development of autoimmune diseases. We will focus on TLRs 7, 8, and 9 that were originally identified as receptors specific for bacterial and viral RNA/DNA, but more recent in vitro and in vivo studies have linked these receptors to the detection of host RNA, DNA, and RNA-associated or DNA-associated proteins in the context of autoimmunity.

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.

Toll-like receptors and autoimmunity.

The understanding of autoimmune diseases experienced an impressive boost since the Toll-like receptors (TLRs) have been identified as possible key players in autoimmune pathophysiology. Although these receptors recognize a variety of structures derived from viruses, bacteria and fungi leading to subsequent initiation of the relevant immune responses recent data support the idea that TLRs are crucial in the induction and perpetuation of certain autoimmune diseases, especially the systemic lupus erythematosus (SLE). In this review we will summarize recent data on involvement of TLRs in the development of autoimmune diseases. This review will focus on TLRs 7, 8 and 9 which were originally identified as receptors specific for bacterial and viral RNA/DNA, but more recent in vitro and in vivo studies have linked these receptors to the detection of host RNA, DNA, and RNA- or DNA-associated proteins in the context of autoimmunity.

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.

Are porcine and human TSH receptor antibody measurements comparable?

BACKGROUND: The human TSH receptor antibody (TRAb) assay is reported to show higher sensitivity in comparison to the "classical" porcine TRAb testing. This claim is based on studies comparing porcine TSH receptor (TSH-R) in one type of assay system (non-immobilized TSH-R and PEG to separate bound and free) with recombinant human TSH-R in another type of assay system (TSH-R immobilized on plastic tubes). In this study isotopic TRAb assays both based on the "coated tube" system (second generation) were compared for the first time. MATERIALS AND METHODS: Three hundred and nineteen consecutive undiagnosed patients from Mainz University as well as 72 patients from Dresden University with known Graves' disease were tested in the human TRAb assay and in the porcine TRAb assay. Spearman's coefficient of rank correlation and Chi-square test statistical analysis was performed. RESULTS: In the 319 consecutive patients 35 patients (11.0%) were found positive in the human assay and 36 patients (11.3%) were positive in the porcine assay. Seventy patients with Graves' disease from Dresden were positive in the assay using the porcine TSH-R and 71 out of 72 patients showed positive results in the assay with the recombinant human TSH-R. The Spearman's coefficient of rank correlation for both patient cohorts was r = 0.932 and r = 0.891 (p < 0.001), respectively. There was no significant difference in the clinical assessment regarding the diagnosis of Graves' disease. CONCLUSION: There is no clinically relevant difference in isotopic TRAb second generation assays--independent of the TSH-R used. Both TSH-R assays showed nearly similar TRAb results in consecutive samples as well as in serum samples from patients with Graves' disease.

Different patterns of disease manifestations of parvovirus B19-associated reactive juvenile arthritis and the induction of antiphospholipid-antibodies.

Children with rheumatic oligo- and polyarthritis frequently establish persistent parvovirus B19 infections, which may be associated with the production of antiphospholipid antibodies. Reported in this paper are the data of five girls with polyarticular rheumatic diseases of different types and persistent parvovirus B19 infection associated in four cases with the presence of antibodies against phospholipids. Clinical parameters, virus load, and antiphospholipid-IgG levels were determined during an observation period up to 92 months. In two patients, erythema infectiosum preceded the development of arthritis and B19 viremia persisted. Two other girls showed antibodies against parvoviral structural proteins at time of the manifestation of the rheumatic disease. Subsequent samples also revealed persistent B19 infection. In the fifth patient, parvovirus B19-specific IgG antibodies were detected for the first time after 120 months of progressing disease at an age of 11 1/2 years. Five years later, quantitative polymerase chain reaction (PCR) revealed viral DNA. In a synovial tissue specimen subsequently obtained, parvovirus B19 structural proteins could be detected by immunohistochemistry. Three of five patients recovered completely without severe sequels. One patient is in remission under immunosuppressive therapy. The fifth patient suffers from progressive erosions despite intensive therapeutical efforts. In consequence, parvovirus B 19 should generally be taken into consideration as a trigger of various forms of juvenile arthritis and persistence of infection should be evaluated.

Human parvovirus B19 infection and antiphospholipid antibodies.

Erythema infectiosum is the main manifestation of human parvovirus B19 infections. Further B19-related diseases commonly associated with the acute infection are flue-like symptoms, transient aplastic crisis, transient arthralgias, leukopenia and thrombocytopenia, spontaneous abortion and hydrops fetalis in pregnant women. Hepatitis, myocarditis, meningitis, encephalitis as well as pure red cell anemia may occur occasionally. In addition parvovirus B19 infections have been frequently described as cause or trigger of various forms of autoimmune diseases affecting all blood cell lines, joints, connective tissue, uvea, large and small vessels. Molecular mimicry may be one major contribution to the appearance of autoimmune antibodies, f.e. antiphospholipid and antineutrophil cytoplasmic antibodies as well as antinuclear antigens. These mechanisms implicated in the pathogenesis of parvovirus B19 triggered autoimmune diseases, especially focused on the development of antiphospholipid antibodies will be discussed in this short review.

Are antiphospholipid antibodies an essential requirement for an effective immune response to infections?

Antiphospholipid antibodies show a close association to a variety of infections. Recent data implicate that parvovirus B19 may be used as a model-system for studying the interaction of viral infection and the development of these autoantibodies. B19-related diseases commonly associated with the acute infection show flu-like symptoms, transient arthralgias, leukopenia and thrombocytopenia, and, in pregnant women, spontaneous abortion and hydrops fetalis. Hepatitis, myocarditis, meningitis, encephalitis, as well as pure red cell anemia may occur occasionally. In addition, parvovirus B19 infections have been frequently described as the cause or trigger of various forms of autoimmune diseases affecting all blood cell lines, joints, connective tissue, uvea, and large and small vessels. Molecular mimicry may be one major contribution to the appearance of autoimmune antibodies, for example, antiphospholipid and antineutrophil cytoplasmic antibodies as well as antinuclear antigens. These mechanisms implicated in the pathogenesis of parvovirus B19-triggered autoimmune diseases, especially focused on the development of antiphospholipid antibodies, will be discussed in this mini review.

Identification of a peptide mimicking the binding pattern of an antiphospholipid antibody.

Our objective was to characterize monoclonal antiphospholipid antibodies (APL) and identify disease-associated antigens in patients with the antiphospholipid syndrome (APS). We used the monoclonal antibody HL-5B, derived from a patient with APS suffering from multiple ischemic events, to screen a 12-mer peptide phage display library (New England Biolabs, London, England). The identified phage clones were sequenced and the derived consensus peptide was synthesized. The peptide was used to perform competitive inhibition experiments for their ability to inhibit the binding of the monoclonal antibody and of serum antibodies to cardiolipin and phosphatidylserine. Additionally patients and control sera were screened for their binding reactivities to this peptide. Using this 12-mer phage display library the peptide APHKHKASLSIY as consensus peptide for the monoclonal antiphospholipid antibody HL-5B could be identified. In competitive inhibition studies we showed that this peptide is able to inhibit the binding of HL-5B to cardiolipin and phosphatidylserine and furthermore another antiphospholipid antibody used as control was also inhibited in its binding to phospholipids. Using 21 sera from APS patients 67% showed a binding to the peptide in a specific ELISA above the cutoff level, generated with sera from 20 healthy controls. Out of the reactive patients' sera we used two exemplarily to perform inhibition studies. Both sera could be inhibited more than 40% in their binding to cardiolipin in a commercially available antiphospholipid antibody assay (Aescu.diagnostics, Wendelsheim, Germany). The identified peptide APHKHKASLSIY simulates the antigenic structure recognized from a subpopulation of serum antiphospholipid antibodies. This might indicate that the diversity of the antiphospholipid antibodies is limited and only few epitopes or few common structures are responsible for the development of those antibodies. Tests using these epitopes will strongly improve laboratory diagnosis of the APS.

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 antibodies in pediatric patients with prolonged activated partial thromboplastin time during infection.

OBJECTIVE: To investigate the close association between different antiphospholipid antibodies (aPL) caused by infection and their appearance together with a prolonged activated partial thromboplastin time (aPTT). METHODS: Sera from 122 children were evaluated in this study. Thirty-seven children with mild to medium prolonged aPTT (>37.2s) and elevated C-reactive protein (CRP) levels during various forms of infections (group 2), 18 children without infections (group 3) but with mild to medium prolonged aPTT and 13 children with infections (group 4) and with elevated CRP-level as well as a control group (group 1) of 54 patients without any infection and normal aPTT and negative CRP levels were investigated with commercially available ELISA tests (AESKU.Diagnostics, Wendelsheim, Germany) for the presence of antibodies directed against cardiolipin (CL), phosphatidylserine (PS) and beta2-glycoprotein I (beta 2GPI). The cutoff for positive results was defined with the healthy, aged matched control group (group 1) using the mean OD values plus 2 standard deviations. The lupus anticoagulant (dilute Russell's Viper Venom time, dRVVT) and coagulation Factor XII were determined with routine tests (Dade Behring). RESULTS: Detection of at least one antibody to phospholipids was possible in 89.2% of group 2. It could be shown that IgM anti-beta 2GPI antibodies were found in 27 (59.5%) of group 2, but only in 1 (5.6%) of group 3 (p=0.024) and only in 4 (7.4%) of the controls (p=0.014). The presence of IgG-anti-beta 2GPI antibodies showed no significant difference in the different groups. Furthermore, children of groups 2, 3 and 4 had statistically significant higher levels of antibodies against PS IgG and PS IgM than controls. Also, antibodies to CL of the IgG-type were more frequently detected in children of group 2 than in controls (p=0.038). Detection of CL-IgM antibodies did not reach a significant level in the comparison of the different groups. CONCLUSION: During commonly acquired infections elevation of aPL of nearly all types seems to be a common process. Mild prolongation of aPTT might reflect this presence of aPL in the course of the infectious disease. Our data suggest that there exists no differences in specificity in comparison to the "pathogenic" aPL but the presence over time might be the trigger for the autoimmune activity to begin.

Clinical relevance of antiphospholipid antibodies in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies (AMAs), but also with reactivities to other autoantigens. Recent studies showed that antibodies to phospholipids (APAs) represent an important group of autoantibodies identified in patients with PBC. In this study different types of APAs were identified in the sera of patients with PBC and autoimmune hepatitis (AIH) and control subjects. Sera from patients with PBC and AIH were tested for the presence of antibodies directed against cardiolipin (CL), phosphatidylserine (PS), and to beta(2)-glycoprotein I (beta(2)-GPI). Furthermore, an in-house test for antithromboplastin antibodies was performed. Antinuclear antibodies (ANAs) and antimitochondrial antibodies (AMAs) were tested with standard tests. IgM anti-PS antibodies were found in 75% of the 51 PBC patients, but only in 4% of the 48 AIH patients (P < 0.0001). IgM anti-CL antibodies were more frequently detected in AIH than in PBC (75% vs. 89%; P = 0.045). IgM anti-beta(2)-GPI antibodies were observed more frequently in patients with AIH (83%) than with PBC (59%; P = 0.007). The APAs of the IgG type did not differ significantly between the groups of patients. Considering the clinical/laboratory parameters, the levels of alkaline phosphatase (P = 0.017), glutamate pyruvate transaminase (P = 0.035), and glutamate oxalacetate transaminase (P = 0.034) were significantly higher in PBC patients who were positive for IgM anti-PS antibodies than in the anti-PS antibody-negative patients. In conclusion, APAs are present in PBC patients with a higher level of the disease or more intense liver damage than in patients without APAs. Thus IgM anti-PS antibodies represent a new marker of activity in PBC patients.

Generation and characterization of three monoclonal IgM antiphospholipid antibodies recognizing different phospholipid antigens.

Antiphospholipid antibodies (APLs) might be involved in the pathogenesis of the antiphospholipid syndrome (APS). This study analyzes the structural characteristics of monoclonal APLs derived from patients with this disease. Patient-derived B cells were immortalized using Epstein-Barr virus transformation and subsequent fusion to the myeloma cell line CB-F7. APL-producing hybridomas were cloned to obtain cell lines producing monoclonal APL. DNA encoding the variable region of heavy and light chains of the antibodies was sequenced and analyzed regarding their usage within the V-gene family and the existence of somatic hypermutation. Binding patterns of APL to various phospholipids and beta-2-glycoprotein-I (beta2-GPI) were determined using ELISA, with special regard to beta2-GPI dependency. As a result, three APL-producing hybridoma cell lines from patients with APS were established: JGG9, HVA2, and HLC9. APLs were of the IgM isotype and showed different binding patterns toward phospholipids and beta2-GPI. One of them, JGG9, showed extensive somatic hypermutations in both the CDR3 region and a framework region of the heavy chain. JGG9 bound to cardiolipin in the presence of the protein cofactor beta2-GPI. In contrast, the antibodies HVA2 and HLC9 (which also showed somatic hypermutations in the CDR3 region) presented polyreactivity to several phospholipids-cardiolipin, phosphatidyl-serine, -ethanolamine, -inositol, -choline, and sphingomyelin-but not to beta2-GPI. In conclusion, JGG9 presents a high degree of mutation in the CDR3 and framework region resulting from the deletions of nucleotides, and affects amino acid composition. Polyreactivity and the absence of cofactor dependency present HLC9- and HVA2-like natural antibodies that have no contact with any antigen. Nonetheless, these natural antibodies show somatic hypermutation of the heavy chain, indicating antigen-driven maturation. Regarding the possible role of APL in infection, HVA2 in particular may represent a pathogen-maturated antibody showing cross-reactivity between phospholipids and infectious agents. Further experiments are needed to reveal the functional activity of these antibodies.

Parvovirus B19 infection and autoimmune disease.

Human parvovirus B19 infections may cause a widespread benign and self-limiting disease in children and adults, known as erythema infectiosum or fifth disease. A variety of further manifestations are associated with the infection such as arthralgias, arthritis, leukopenia and thrombocytopenia, anemia and vasculitis, spontaneous abortion and hydrops fetalis in pregnant women. Both in children and adults parvovirus B19 infections have been frequently implicated as a cause or trigger of various forms of autoimmune diseases affecting joints, connective tissue and large and small vessels. In addition, autoimmune neutropenia, thrombocytopenia and hemolytic anemia are known as sequelae of B19 infection. The molecular basis of the autoimmune phenomena and resultant pathogenesis is unclear. The involvement of molecular mimicry between cellular and viral proteins, the induction of enhanced cytokine production via the viral transactivator protein NS1 and the phospholipase A2-like activity of the capsid protein VP1 may contribute to the induction of autoimmune reactions. All the known data and the potential mechanisms involved in the pathogenesis will be discussed in this review.

The combination of different antiphospholipid antibody subgroups in the sera of patients with autoimmune diseases is a strong predictor for thrombosis. A retrospective study from a single center.

OBJECTIVE: [corrected] To determine the distribution of different antiphospholipid antibodies (APL-Ab) and their association with thrombosis in patients with autoimmune diseases. METHODS: Clinical data and laboratory features of 30 patients with different autoimmune diseases with positive APL-Ab were retrospectively studied for a period of more than two years. Anti-cardiolipin (aCL), anti-phosphatidylserine (aPS) and anti-beta2-glycoprotein I (abeta2-GPI) antibodies were determined by ELISA. RESULTS: Autoantibodies that target only PS were detected in 53.3% (n = 16) patients, aCL antibodies only were found in one patient (3,3%). In 43.3% (n = 13), aPS were associated with elevated levels of aCL and/or abeta2-GPI antibodies. No thrombotic event occurred in patients with aPS antibodies only compared to 6 patients from the group with different APL-Ab during 808 +/- 92 days of observation. CONCLUSION: The combination of different antiphospholipid antibody subgroups seems to be a predictor for thrombosis. The presence of aPS antibodies without additional aCL or abeta2-GPI is not associated with thrombosis. The measurement of the APL specificities in addition to the aCL antibodies may be important to develop predictive markers for the risk to develop thrombotic events.

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.